The Synergy of Adenosine and Ibrutinib on Platelet

Aggregation in Chronic Lymphocytic Leukemia

Madison Barbara Allerton Hagger

BSc Biological Science, and Conservation and Wildlife Biology

This thesis is presented for the degree of Bachelor of Science Honours, Discipline of

Medical, Molecular and Forensic Sciences

Murdoch University

2021

ii

Declaration

I declare this thesis is my own account of my research and contains as its main content, work

which has not been previously submitted for a degree at any tertiary education institution.

Madison Hagger

iii

Abstract

Introduction:

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults caused by

a gradual clonal expansion of B-cells in the bone marrow. Bruton’s Tyrosine Kinase (BTK) plays

a role in the signalling of immune cells such as B-cells and platelets and has been implicated in

the survival pathways of cancer. The BTK inhibitors, ibrutinib and acalabrutinib, are used to

treat CLL; however, a significant bleeding risk has been observed in patients taking these

inhibitors that is not seen in people with BTK-mutation (X-lined agammaglobulinemia (XLA)).

CLL patients using ibrutinib and acalabrutinib have platelet dysfunction characterised by

impaired response to collagen-related peptide (CRP; a specific glycoprotein VI (GPVI) agonist)

and collagen. A disease factor may potentiate the antiplatelet effects of BTK inhibitors to cause

significant bleeding issues in CLL patients. The CLL microenvironment is characterised by high

levels of CD73-generated adenosine, which plays a role in suppressing immune responses such

as platelet aggregation. Thus, it is hypothesized that adenosine amplifies the effect of ibrutinib

and acalabrutinib to inhibit platelet function, which would explain the associated increased

bleeding risk.

Methods:

Plasma was collected from stable, untreated CLL patients (n = 24) and age-matched healthy

controls (n = 10). Using ELISA and the Malachite green assay, soluble CD73 levels and activity

were determined. For ex vivo platelet activation studies, blood was collected from healthy

volunteers into sodium citrate for whole blood assays (n=2), or ACD anticoagulant (n=6) for

washed platelet tests. Flow cytometry, light transmission aggregometry, and Western blotting

were used to delineate the impact of adenosine on the antiplatelet activities of ibrutinib and

acalabrutinib.

iv

Results:

Soluble CD73 was detected in the plasma of CLL patients, and its activity was confirmed by

measuring phosphate generation from AMP using the Malachite green assay and a selective

CD73 inhibitor. In whole blood platelet activation assays, adenosine potentiated the

antiplatelet effect of ibrutinib and acalabrutinib. In washed platelets, using clinically relevant

concentrations, ibrutinib and acalabrutinib inhibited CRP (P < 0.0001) but not collagen-induced

platelet aggregation as single agents (P > 0.05). When combined with the adenosine derivative,

2-Hexynyladenosine-5'-N-ethylcarboxamide (HENECA), both inhibitors managed to reduce

collagen-induced platelet aggregation, although Ibrutinib’s effect was stronger (P < 0.0001).

The combination of HENECA with ibrutinib or acalabrutinib broadens the antiplatelet activity

of these inhibitors as evidenced by changes in collagen-mediated phosphorylation of the

downstream kinases: ERK, AKT, VASP, BTK and SYK.

Conclusion:

The results of this study provide initial evidence that increased bleeding risk present in the CLL

microenvironment might be caused by the synergy between the BTK inhibitors and high levels

of adenosine. Furthermore, the synergistic effect was more pronounced with ibrutinib rather

than acalabrutinib. Future studies should focus on examining the association between plasma

adenosine levels, CD73 and platelet activation in CLL patients.

v

Table of Contents 1. INTRODUCTION .............................................................................................................................. 1

1.1 BACKGROUND ........................................................................................................................................ 2 1.2 TREATMENTS ......................................................................................................................................... 4 1.3 BRUTON’S TYROSINE KINASE SIGNALLING ................................................................................................... 9 1.4 ADENOSINE IN CANCER .......................................................................................................................... 12 1.5 CD73-GENERATED ADENOSINE IN CANCER ................................................................................................ 14 1.6 CD73-GENERATED ADENOSINE IN HAEMOSTASIS AND THROMBOSIS .............................................................. 16 1.7 AIMS .................................................................................................................................................. 18

2. METHODS ..................................................................................................................................... 20

2.1 MATERIALS AND REAGENTS .................................................................................................................... 21 2.2 PARTICIPANTS ...................................................................................................................................... 21 2.3 PREPARATION OF WASHED HUMAN PLATELETS ........................................................................................... 21 2.4 HUMAN CD73 ELISA ........................................................................................................................... 22 2.5 MALACHITE GREEN PHOSPHATE ASSAY .................................................................................................... 22 2.6 FLOW CYTOMETRY ................................................................................................................................ 23 2.7 PLATELET AGGREGATION ASSAY .............................................................................................................. 23 2.8 WESTERN BLOT AND ANTIBODIES USED ................................................................................................... 23 2.9 STATISTICAL ANALYSIS ........................................................................................................................... 24

3. RESULTS ......................................................................................... ERROR! BOOKMARK NOT DEFINED.

3.1 THE EXPRESSION AND ACTIVITY OF SOLUBLE CD73 IN PLASMA FROM CLL PATIENTS. .......... ERROR! BOOKMARK NOT

DEFINED. 3.2 THE EFFECT OF ADENOSINE ON IBRUTINIB-MEDIATED INHIBITION OF ADP-INDUCED PLATELET ACTIVATION IN WHOLE

BLOOD. ..................................................................................................................................................... 27 3.3 IBRUTINIB INHIBITS CRP-INDUCED PLATELET AGGREGATION, BUT NOT COLLAGEN-INDUCED PLATELET AGGREGATION. ............................................................................................................................................................... 29 3.4 THE ADENOSINE DERIVATIVE HENECA AMPLIFIES THE ANTIPLATELET EFFECTS OF IBRUTINIB AND ACALABRUTINIB. 31 3.5 ADENOSINE ENGAGES SEVERAL SIGNALLING KINASES WHEN COMBINED WITH BTK INHIBITORS. .......................... 33

4. DISCUSSION .................................................................................................................................. 35

5. REFERENCES ................................................................................................................................. 42

6. APPENDICES ................................................................................................................................. 57

APPENDIX A .............................................................................................................................................. 58 The Manufacturer’s Instructions for the Human CD73 ELISA Kit ....................................................... 58

APPENDIX B .............................................................................................................................................. 70 The Manufacturer’s Instructions for the Malachite Green Phosphate Assay Kit ............................... 70

vi

Acknowledgements

Throughout the course of my Honours degree and the writing of this thesis I have received a

great deal of assistance and encouragement.

First, I would like to thank my supervisors, Dr Pat Metharom and Dr Omar Elaskalani. Your

patience and valuable feedback helped me immensely and brought my research to a higher

level. I was able to overcome the challenges this project threw at me due to your guidance and

advice, and I will always be grateful for that.

I would like to acknowledge Associate Professor Murray Adams for his experience, and all his

help with the administrative side of research.

I would like to acknowledge Norbaini Abdol Razak, whose help was invaluable. Thank you for

all your help, and the time you took to teach me vital lab techniques, answer my questions,

and help me figure out errors with the equipment.

I would like to thank my parents, for all their support and encouragement. You have always

believed in me and listened to me vent when I needed to, and I am not sure if I will ever be

able to express in words what that means to me. I would also like to thank my twin sister, who

always listened with a sympathetic ear and who somehow made me laugh no matter how

stressed I was. I would not have been able to do this without you. I would also like to

acknowledge my cousin, Joanne Rowles, who took the time to help me despite being busy with

writing her PhD thesis. Thanks Jo, I will never forget it.

Finally, I would like to thank my friends, Ryan, Erich, Shanice, Perry, and Adam. Thanks for

listening to me spout science jargon at you, and still wanting to hang out with me afterwards.

1

1. Introduction

2

1.1 Background

Cancer is a devastating and complex set of diseases and one of the leading causes of death in

the world. Due to aging populations, the incidence of cancer is increasing in developed

countries, although mortality rates are higher in undeveloped countries due to differences in

lifestyle and healthcare [1]. Despite the abundant research, cancer continues to challenge the

scientific community as it is a complex disease with various forms which require different

treatments. Cancer usually occurs because of a mutation in genes and cell receptors that

causes altered signalling transduction resulting in aberrant cell activity that prevents apoptosis

and promotes cell growth [2]. Normal functions are altered, and cells’ numerous methods of

surviving are used against the individual.

Leukemia is one of the most common causes of cancer-related deaths in Australia, with a

higher incidence in Caucasian persons and males than in other groups [3]. Leukemias are a

complex set of diseases resulting from the neoplastic proliferation of lymphoid or

haemopoietic cells, impairing the immune system and predisposing leukemia patients to

infection [4-6]. Indeed, infection is commonly the first symptom of acute leukemia [6].

Leukemia is commonly caused by mutations originating in a single stem cell, which affect and

alter DNA damage response, cell cycle, NOTCH and NF-kB signalling [5, 7]. The common

mutations found in leukemia are listed and briefly described in Table 1.

It is thought that leukemia cells invade hematopoietic stem cell (HSC) supportive

microenvironments by hijacking vital signalling pathways, thus shifting the equilibrium of

microenvironments to favour expansion of leukemia cells leukemogenesis, and evolution of

chemoresistance [8-10]. There are four broad types of leukemia: acute lymphocytic leukemia

(ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic

myelogenous leukemia (CML). They are differentiated by the stage of maturity at which the

stem cell mutated and how quickly the disease progresses, with the acute forms developing

quickly and killing the patient in a matter of weeks or months while the chronic forms progress

3

slowly and often take years to kill the afflicted. While there are multiple forms of leukemia,

this review will focus predominantly on CLL.

FUNCTIONAL CLASS

MUTATED GENE

ROLE DESCRIPTION REFERENCES

TRANSCRIPTION FACTOR

CEBPA Inherited mutation that disrupts DNA-binding and dimerization. Presence of mutation is characteristic of early myeloid cells, involved in numerous granulocyte-specific genes.

[11-13]

RUNX1 Essential for hematopoietic stem cell (HSC) generation and differentiation, as well as homeostasis. Mutations result in early HSC exhaustion.

[13, 14]

SIGNALING AND KINASE PATHWAY

FLT3 Involved in B and T cell development. Confers a poor prognosis.

[13, 15]

KRAS Involved in differentiation and proliferation. Upregulates GLUT1, thus contributes to the Warburg effect.

[13, 16]

NRAS Mutation causes constitutive activation of the RAS protein. Suggested to inactivate TP53.

[13, 17]

KIT Involved in proliferation and survival. Mutations result in lethal anaemia, HSC defects and mast cell deficiency.

[13, 18]

PTPN11 Regulates the RAS/MAPK signalling pathway. Mutations result in increased oncogenic KRAS activity and downstream signalling in carcinogenesis.

[13, 19]

NF1 Mutations result in proliferative RAS/RAF/MEK signalling

[13]

BIRC3 Inhibits apoptosis. Upregulated in leukemia. [20, 21] DNA METHYLATION AND CHROMATIN MODIFICATION

DNMT3A Responsible for de novo DNA methylation essential for cellular differentiation and heterochromatin formation. Thought to be involved in methylation of tumour suppressor genes.

[13, 22]

IDH1/2 Catalyses the generation of α-ketoglutarate (α-KG). Mutations reduce α-KG and activate the HIF1α pathway.

[13]

TET2 Depletion results in skewed differentiation of hematopoietic precursors and amplification of hematopoietic/progenitor cell renewal.

[13, 23]

ASXL1 Regulates epigenetic marks and transcription. Has a major impact on disease outcome. Associated with aggressive phenotype and poor overall survival.

[13, 24]

TUMOR SUPRESSOR

TP53 Involved in regulation/progression, of the cell cycle, apoptosis, and genomic stability. Usually a missense mutation, results in a loss of function.

[13, 25]

CELL DIFFERENTIATION

NOTCH1 Been implicated in clinical resistance to rituximab. Results in a more aggressive disease.

[20, 26]

Table 1. Common mutations in leukemia and brief descriptions of their normal and oncogenic roles by functional class.

4

CLL is one of the most common types of leukemia, especially among adults, with 15,000 cases

diagnosed every year in the USA alone and comprises 22-30% of leukemia cases worldwide

[27-30]. By the end of 2015, there were 12,930 Australians with CLL, with a higher incidence in

males [31]. CLL is a gradual clonal expansion of mature B-cells in peripheral blood, bone

marrow and secondary lymphoid organs caused by constitutive B-cell receptor signalling and

maintained via overexpression of antiapoptotic protein B-cell lymphoma 2 (BCL2), which

results in a high lymphocyte count in circulating blood [29, 30, 32-34]. The lymphocytes are

small with a fine border of cytoplasm, aggregated chromatin and a dense nucleus lacking

nucleoli [27, 30]. They are regularly accompanied by smudge cells due to the lymphocyte

debris as the peripheral smear is prepared, which is thought to reflect the intrinsic membrane

fragility of CLL cells and cytoskeleton abnormalities, possibly due to a decrease in vimentin [27,

30, 35-37]. The B-cells produced are mature but non-functional, ultimately compromising the

immune system [33]. Granulocytes, which are the major defence against infection, are

overwhelmed by the prevalence of functionally impaired blast cells [6].

Though it is unclear at exactly what stage in lymphocyte maturation the CLL cell originates,

there are thought to be two subsets of CLL: the unmutated type and the mutated type [30].

The unmutated type is believed to originate from a naïve B-cell that had insufficient stimulus

by antigen to form a germinal centre and commonly characterised by ZAP-70 expression as

well as the use of IgHV-69 [30]. This subtype usually has a poorer prognosis than those with

the mutated subtype. The mutated subtype is derived from a memory B-cell that mutated

somatically following stimulation with antigen and antigen selection at the germinal centre

[30]. Gene sequencing studies finding approximately 60 genes that are most often mutated in

CLL, primarily related to cell cycle, response to DNA damage, NOTCH and NF-kB signalling [7,

38, 39]. These mutations occur at a higher frequency in the later stages of CLL, which suggests

these mutations may play a role in disease progression [7, 40]. Because CLL is a relatively slow-

growing cancer, it can take years to progress and consequently can go unnoticed by patients

5

until it is diagnosed during a routine blood exam [33]. It mostly afflicts the elderly, with

approximately 70% of patients diagnosed at 65 years of age or older [41, 42]. Most CLL

patients have a lower platelet count than normal and a reduced platelet response to

adenosine diphosphate (ADP), which is only exacerbated by BTK inhibitor treatment [43].

1.2 Treatments

Treatment therapies are usually withheld until patients show symptoms, as they usually

develop comorbidities that often involve the use of antiplatelet/anticoagulant agents [41, 42].

Methods of treatment of CLL include chemotherapy, bone marrow transplants, adoptive

immunotherapy, targeted therapy, and the use of oral BTK inhibitors, some of which are used

in combination with each other.

Chemotherapy is a standard treatment for cancers, and it has been known to improve

remission duration as well as overall survival rate among most CLL patients; however, relapse

is almost inevitable [28]. Multi-agent combination chemotherapy is used to treat leukemia,

resulting in survival rates ranging from 35-50% [44, 45]. However, as chemotherapy is a type of

cytotoxic therapy, simply intensifying the regimes will do more harm than good, prompting a

need for new, less toxic treatments [44]. Because it is not a targeted therapy and affects fast-

growing cells, cancerous or otherwise, there are a variety of side effects. These include

modification of appetite and behaviour, nausea, abdominal pain, vomiting, spontaneous

bleeding, infection, leg weakness, , loss of hair, oral mucositis, and modification of gut bacteria

[46-48]. These side effects often prolong hospital stays and increase the cost of therapy

through their additional treatment [46]. This, along with the significant chance of relapse, is

the motivation behind the intense research effort into new, more effective treatments for CLL

[28].

6

Bone marrow transplants (BMTs) are an effective treatment when the donor is an HLA-

identical sibling, though this efficacy is thought to be associated with the high doses of drugs

and radiation the patients undergo before the transplant [49]. Before the development of

tyrosine kinase inhibitors (TKIs), BMTs were the accepted therapeutic option, as it is

potentially curative for cancers such as CML [50]. However, TKIs have mostly replaced BMTs as

first-line treatment [50]. Although BMTs are an effective treatment, there are some side

effects, such as an increased risk of infection and chronic graft-versus-host disease (GvHD),

where the transferred T cells attack the host tissues and can result in death [50]. Further

research into methods of preventing GvHD while keeping the immune system as intact as

possible to fight off infection would improve prognosis, though immunotherapies may be able

to achieve a similar effect with a reduced if not no risk of transferred T cells attacking the host

[51, 52].

Adoptive immunotherapy is a form of treatment involving the culture and transfer of tumour-

reactive T cells into the patient, thus allowing the immune system to fight cancer [51, 52]. For

a while, it was heavily debated whether the immune system was capable of targeting tumours

[53]. We now have compelling evidence that illustrates the vital role of the immune system in

tumour suppression and its tumour-targeting capabilities [54]. This includes the increased rate

of cancer in immunocompromised people while immunocompetent people occasionally

undergo spontaneous remission, the greater susceptibility of mice with immunological defects

to develop spontaneous or induced tumours compared to wild-type mice, and the

accumulation of immune cells at tumour sites correlating with improved prognosis [53].

However, despite the observed response of immune cells to tumours, the establishment of a

tumour means that cancer managed to evade or overwhelm the immune response. Developing

tumours have a plethora of strategies that promote immune evasion, such as reduction of the

expression of major histocompatibility complex (MHC) or costimulatory proteins resulting in

inadequate immune responses, disruption of natural killer (NK) and natural killer T (NKT) cell

7

recognition, and physical exclusion of immune cells from the site of the tumour [55]. Tumours

can also inhibit inflammatory responses, lose targeted antigens, induce regulatory T cells, or

delete responding T cells [56, 57]. Due to these responses, adoptive immunotherapy has

limited benefits, as the transferred T cells are often deleted before they are able to react [52].

There is still hope for this treatment, as recent studies looking into blocking negative signalling

pathways as well as or instead of providing pro-activation signals has been successful, and has

even been exploited clinically [58-60]. However, before adoptive immunotherapy can reach its

full potential, further research into maximising T cell activation and effector function, as well

as preventing deletion of the tumour-reactive T cells is needed.

CD73 (also known as ecto-5’-nucleotidase) is becoming an increasingly popular target as it is

overexpressed in a variety of cancers and has been found to play a significant role in cancer

cell proliferation, metastasis, invasion, tumour angiogenesis and tumour immune escape [61].

This is because the balance of adenosine 5’-triphosphate (ATP)/adenosine diphosphate (ADP),

adenosine monophosphate (AMP) and adenosine is vital in regulating the progression of

cancer [61, 62]. CD73 is a 70 kDa glycosylphosphatidylinositol (GPI)-anchored cell surface

molecule responsible for hydrolysing AMP into adenosine [61, 62].

Small molecules that directly inhibit tyrosine kinases (TKs) have been investigated as possible

cancer chemotherapeutics, and previous studies have had success with well-studied TKs such

as EGFR and ERBB2 [63-65]. Bruton’s Tyrosine Kinase (BTK) inhibitors such as ibrutinib are also

used. Ibrutinib is a first-in-class, highly potent small molecule that irreversibly and covalently

binds to the Cys481 residue within the ATP binding pocket of BTK [28, 34, 43, 66]. BTK is

important in downstream signalling of immunoreceptor tyrosine-based activation motifs

(ITAM) in hematopoietic cells, and mutations in BTK are associated with reduced B-cell

maturation [67-70]. It has garnered particular interest due to its role in platelet activation

through the effector protein phospholipase Cγ2, which is downstream of the platelet

membrane glycoprotein VI (GPVI) receptor, suggesting that BTK may be needed for multiple

8

platelet activation pathways [67, 71, 72]. Ibrutinib works against CLL cells by directly

preventing cell proliferation, with recent ex vivo studies suggesting that ibrutinib causes

apoptosis of CLL cells, though it is still unclear whether this activity is the main reason behind

ibrutinib’s effectiveness [34, 66]. However, despite ibrutinib’s marked clinical activity, patients

have been known to gain resistance to these drugs through mutation of the BTK gene [34, 73].

The most common mutant of BTK is BTK-C481S, which is induced by ibrutinib treatment, and

causes resistance to ibrutinib by disrupting the covalent binding between BTK and ibrutinib

[66, 73]. Development of other, more effective BTK inhibitors is underway, with some studies

showing promising results, demonstrating the effects of drugs that are able to effectively

inhibit both wild type and mutant forms of BTK with no observed toxicity in AML mouse

models [73]. In addition to drug resistance, the side effects of ibrutinib can also make patients

cease treatment [50].

Side effects of ibrutinib not seen in BTK-deficient patients include diarrhea, rash, atrial

fibrillation, myalgias, arthralgias, ecchymosis and major haemorrhaging as a result of platelet

dysfunction, with a clinical study claiming an increased bleeding risk with an incidence rate of

40% and a significant bleeding incidence of 3-4% [28, 42, 43, 74-76]. These side effects are

likely due to the unspecific binding of ibrutinib to kinases other than BTK, such as epidermal

growth factor receptor (EGFR), T-cell X chromosome kinase (TXK), interleukin-2-inducible T-cell

kinase (ITK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) [28]. A recent

study of CLL patients in the US Veterans Health Administration found that within the follow-up

period after diagnosis of CLL (4.1 years), 9.1% developed a major haemorrhage [42]. The

general pattern of bleeding is consistent with primary haemostatic failure (subcutaneous

bleeding or bruising following minimal trauma). The fact that this bleeding occurs despite

improved platelet counts during ibrutinib treatment suggests that bleeding is a result of

platelet dysfunction [76]. It has also been shown that inhibition of BTK results in a

predisposition for infections, as well as a B-cell dysfunction with a reduction in serum

9

immunoglobin levels [28]. Thus, in some patients, the risk of using therapeutic concentrations

of ibrutinib could outweigh the benefits [28].

Acalabrutinib is another commonly used irreversible BTK inhibitor. It is second-generation and

has improved pharmacological characteristics such as selective binding, preventing it from

binding to similar kinases such as EGFR, TXK, ITK and TEC, a short half-life, favourable plasma

exposure and rapid absorption [28]. Recent studies, however, have suggested that this

selectivity for BTK over TEC is not significant [74, 77]. However, acalabrutinib could possibly

have less of an effect on platelet function due to its binding specificity. Previous studies found

evidence of a possible redundancy in the ITAM- and G protein-coupled receptor signalling

pathways that allow Tec kinase to partially compensate for BTK deletion [67, 78]. In an

uncontrolled, multicentre study by Byrd et al., patients receiving ibrutinib had a reduction of

platelet reactivity at sites of vascular injury [28]. This was not the case in patients treated with

acalabrutinib, which could explain why there were no major bleeding events nor cases of atrial

fibrillation for the duration of the study [28]. However, low grade bleeding, such as petechiae

and contusions, still occurred at a significant rate of incidence [74]. Headaches were also more

common with acalabrutinib treatment than ibrutinib, though the headaches developed during

early treatment and commonly resolved in time [28].

1.3 Bruton’s Tyrosine Kinase Signalling

Tyrosine kinases (TKs) are enzymes capable of transferring phosphates from ATP to tyrosine

residues of specific proteins inside cells, functioning as ‘on’ and ‘off’ switches for a variety of

cell functions [64]. TKs are known to regulate cell proliferation and contribute to cell

sensitivity to apoptotic stimuli [79]. Dysregulation of TKs often results in serious diseases such

as cancer and, consequently, they have been found to be the most common dominant

oncogenes [64]. Several studies have found that inhibitors of a variety of TKs are successful

10

anticancer agents, which demonstrates the significance of this class of proteins in cancer

progression as well as the development of targeted therapies [64].

TKs exist as receptor and non-receptor tyrosine kinases. Receptor tyrosine kinases (RTKs) are

high-affinity surface receptors involved in regulating vital cellular functions, including cell-to-

cell communication, cell growth, motility, differentiation [80]. Non-receptor tyrosine kinases

(nRTKs), unlike RTKs, are cytosolic enzymes [81, 82]. The different families of nRTKs include the

ABL, ACK, CSK, FAK, FES, FRK, JAK, SRC, SYK and TEC kinases [81-84]. They are involved in cell

growth, signal transduction, proliferation, differentiation, adhesion, apoptosis, and migration

and are vital components of the immune system [68, 81, 83, 85].

Bruton’s tyrosine kinase is a tyrosine kinase in the Tec family encoded by the BTK gene in

humans and is vital for downstream signalling of ITAM-coupled receptors in hematopoietic

cells such as B cells, natural killer cells, monocytes, neutrophils and platelets [67, 86]. BTK is

immediately downstream of the B-cell receptor and has been found to be vital for a variety of

cancer cell survival pathways and chemokine mediated homing and adhesion of CLL cells to the

microenvironment, which contributes to cell proliferation and maintenance, and thus is a

promising target for new therapies [28].

BTK plays a crucial role in platelet activation. Platelets are the key player in thrombosis and

haemostasis [87]. BTK is involved in von Willebrand factor (vWF) signalling and collagen-

induced platelet aggregation [67, 88]. Collagen-mediated platelet aggregation occurs when

circulating vWF binds to collagen and undergoes a conformational change. This new shape

allows vWF to bind to the GPIb receptor in platelets, which then allows platelet integrin α2β1 to

bind to collagen [67]. Further platelet recruitment and activation are regulated by GPVI, which

is noncovalently linked to a disulphide-linked homodimer of Fc receptor γ-chains which each

contain an ITAM [67]. When exposed collagen binds to the GPVI, it forms cross-links which

allow Src kinases GPVI-bound Fyn and Lyn to phosphorylate the Fc receptor γ ITAM unit’s

tyrosines [67]. Then the tyrosine kinase SYK binds the phosphorylated ITAMs, enabling Src

11

kinases to phosphorylate SYK in turn and triggering autophosphorylation [67]. The activation of

SYK then triggers the construction and consequent activation of a signalosome composed of

the transmembrane adapter protein LAT, the TEC kinases BTK and TEC in association with the

effector protein PLCγ2, as well as the cytosolic adaptor proteins SLP-76 and Gads [67]. This

signalosome enables the phosphorylation of BTK by SYK and Lyn, which in turn activates PLCγ2

[67, 89]. PLCγ2 then hydrolyses phosphatidylinositol 4,5-bisphosphate into the secondary

messengers 1,4,5-trisphosphate and diacylglycerol, consequently triggering platelet activation

via activation of protein kinase C, synthesis of thromboxane A2, the release of intracellular Ca2+

stores, and the resulting platelet granule secretion [67, 89].

Figure 1 illustrates several other pathways of platelet aggregation that also rely on PLCγ2. As a

result, these pathways may require BTK for activation due to their role in the signalling cascade

[67, 90]. Fibrinogen-induced platelet aggregation through the binding of fibrinogen to the

Figure 1: The signalling pathways of platelet aggregation and activation. Reused from Andrews R.K., Arthur J.F., and Gardiner E.E.’s ‘Targeting GPVI as a novel antithrombotic strategy’ under the creative commons licence (CC BY-NC 3.0) [36].

12

fibrinogen receptor integrin αIIbβ3 activates the Gα protein G13 [67]. G13 then triggers c-Src

activation of SYK, which results in the activation of BTK and phosphatidylinositol 3-kinase to

phosphorylate PLCγ2 [67]. Shear-dependent binding of the platelet membrane complex GPIb-

GPIX-GPVI to vWF induces Lyn to phosphorylate phosphatidylinositol 3-kinase (PI3K), which

also requires BTK to activate PLCγ2 [67]. Thus, BTK is pivotal to proper platelet response and

aggregation, meaning aberrant BTK activity can result in bleeding risks and immune system

dysfunction through its role in B-cell maturation.

In haematological malignancies, BTK plays a vital pathogenic role due to its significance in

hematopoietic signalling [73]. Consequently, BTK has attracted a lot of attention as a possible

target for the treatment of mantle cell lymphoma (MCL) and CLL. Inhibiting BTK has

hematopoietic consequences due to its significant role in the B-cell receptor signalling cascade,

which commonly results in mild bleeding, with a Common Toxicity Criteria (CTC) of grade 1 or

2, usually due to spontaneous bruising or petechiae [66, 88]. However, approximately 8% of

patients experience more severe bleeding, with roughly 5% of patients suffering CTC grade 3

or 4 bleeding, commonly after trauma [88]. Because a large portion of patients (approximately

50%) are undergoing anticoagulant treatment for comorbidities, managing bleeding risk is

vital, as it increases the risk of mortality or a major haemorrhaging event that could lead to

death [88]. A study by Rigg et al. recently found that although inhibition of BTK significantly

reduces GPVI-induced platelet aggregation, activation and spreading, prolonged bleeding has

not been observed in the used bleeding models [67]. Standard of care platelet function assays

like light transmission and multiplate aggregometry could safeguard the use of ibrutinib in CLL

patients [88].

1.4 Adenosine in cancer

In 1929, Alan Drury and Albert Szent-Gyrgyi from the University of Cambridge proposed the

idea that purines not only formed the building blocks for the genetic code and the universal

13

energy currency ATP, but also functioned as extracellular signalling molecules [91, 92]. They

proceeded to investigate by injecting extracts from cardiac tissues intravenously into a whole

animal, after which they observed a transient slowing of the heart rate [91, 93]. Following

several purification steps, it was determined that the biologically active component of the

extract was an adenine compound [93]. Now we have genetic evidence that the abnormal

heart rate induced by the intravascular adenosine injection is mediated via the activation of

adenosine receptors: A1, A2A, A2B, and A3 [91, 94-96]. A1 and A3 receptors trigger a decrease

in intracellular cyclic AMP (cAMP) production and activate PI3K and protein kinase C (PKC),

while A2A and A2B receptors activate adenylate cyclase, which consequently increases

intracellular cAMP levels [96]. Adenosine is an essential metabolite involved in many signalling

pathways that regulate anti-inflammatory response, ischemic pre- and postconditioning,

oxygen supply/demand ratio, trauma, and angiogenesis to maintain haemostasis [62, 97-101].

Under normal physiological conditions, adenosine is constitutively present at nanomolar

concentrations and exerts an immunosuppressive effect to protect tissues against excessive

immunoreactions and initiate tissue repair following injury [96, 100, 102, 103]. This is

necessary to mediate the level of ATP and the consequent proinflammatory responses it

induces, as adenosine is generated via the degradation of ATP catalysed by the concerted

efforts of the enzymes CD39 and CD73 (Figure 2) [101, 102]. Adenosine is produced through

the metabolic processing of ATP to ADP to AMP via CD39, then from AMP to adenosine via

CD73 (Figure 2) [62, 98, 104-107]. It can also be generated intracellularly through the

hydrolysis of AMP by the action of an intracellular 5-nucleotdase or hydrolysis S-adenosyl-

homocysteine [108]. Consequently, ATP is needed to produce adenosine extracellularly. ATP is

predominantly generated via the tricarboxylic acid (TCA) cycle but can also be generated

through the malate-aspartate shuttle (MAS). Most ATP remains in the cell where it can be

used; however extracellular ATP also exists at a concentration of approximately 10 nM under

normal physiological conditions, but spikes as stressed cells or damaged tissue release more

14

ATP [109, 110]. The balance between ATP and adenosine is carefully regulated to ensure

optimal immune response against infection, injury or disease, while simultaneously restricting

collateral tissue or cell damage from extended immune reactions [111, 112].

In cancer, this balance is disturbed by an influx of ATP generated via the tumour

microenvironment (TME) [113, 114]. In response, the expression of the enzymes necessary to

degrade ATP is upregulated, resulting in a significant increase in adenosine concentrations

[102]. Adenosine’s suppressive effect has been shown to promote tumour growth and

significantly impair immunosurveillance, allowing cancer to proliferate undetected [102].

Adenosine accumulates in the TME due to hypoxia and the related metabolic stresses and cell

damage, which causes an increase in extracellular adenosine and signalling through adenosine

receptors [101-103, 115]. Hypoxia and its transcription factors hypoxia-induced factor 1a/2a

(HIF-1a/2a) increase adenosine concentrations and cell response to adenosine by reducing the

expression of adenosine kinase, which is responsible for catalysing the phosphorylation of

adenosine into adenosine monophosphate (AMP) and increasing the expression of the

adenosine receptors A2A and A2B (Figure 2) [102]. It increases the expression of CD39 and

CD73, directly influencing adenosine levels in the microenvironment by increasing the cells’

capacity to generate adenosine [102].

1.5 CD73-generated adenosine in cancer

CD73 has recently been investigated as a possible target for treatments of haematological

malignancies and cancers, such as leukemia, due to its involvement in the adenosine pathway

and the consequent effect it has on tumour progression and metastasis [116]. CD73 (also

known as ecto-5’-nucleotidase) is a molecule found anchored to the cell surface by GPI and is

responsible for converting AMP to adenosine [62, 98, 104-106]. CD73 is highly expressed on

the vascular endothelium, though it is found on the surface of many cell types and even has a

soluble form upon shedding of the GPI anchor [97, 104, 105, 117]. In addition to its

15

immunosuppressive effects via its mediation of adenosine concentrations, studies have

revealed that CD73 has a role in cancer proliferation, invasion, metastasis, and differentiation

[118-120]. It induces cancer cell invasion and migration through the release of matrix

metalloproteinases (MMPs) that degrades the extracellular matrix (ECM) [118]. CD73

promotes proliferation and differentiation by potentiating EGFR/AKT and VEGF/AKT pathways

[118]. Because of its involvement in a multitude of cancer pathways, CD73 has become a

popular biomarker and a prominent target for cancer therapies [97, 118, 121].

Many cancers, both with solid tumours and without, create hypoxic TMEs [122, 123]. Hypoxia

refers to a condition in which oxygen is limited which is governed by the HIF1α transcription

factor. This factor is known to influence signalling pathways important to leukemia

proliferation and maintenance [122]. Under hypoxic conditions, extracellular ATP and ADP are

accumulated, and CD73 expression is increased via HIF1- α, resulting in the production of

adenosine [1-3]. Adenosine is a signalling molecule that acts through adenosine receptors.

Platelets express the adenosine receptors A2A and A2B. Through binding to A2A, adenosine

can dampen platelet activation. The association between adenosine and platelet activation in

vivo has been previously reported in cardiovascular diseases [7, 124]. However, to our

knowledge, there has been no study to explore the role of CD73-generated adenosine in

haemostasis in cancer.

CD73 is generally associated with a poor prognosis in most cancer patients and is found

overexpressed in a variety of cancers, especially hypoxic cancerous tissues, though some

studies have produced conflicting results where high CD73 indicates a good prognosis in

bladder cancer [118, 119, 121]. Adenosine produced by CD73 can be either taken up by the

cells to replenish the nucleotide pool, or it induces immunosuppression and anti-inflammatory

effects [118, 120, 121, 125]. In CLL cells, CD73 is expressed in proliferation centres and is

characterised by high enzymatic activity [118, 125]. CD73-generated adenosine activates type

1 purinergic A2A receptors that are also constitutively active in CLL cells and thus exacerbates

16

proliferation of neoplastic cells [125]. Activation of adenosine receptors results in an increase

of cAMP in the cytoplasm, ultimately reducing drug-induced apoptosis in CLL cells [125].

Moreover, extracellular adenosine generated as a response to TME hypoxia is known to play a

pivotal role in the inhibition of anticancer immune responses [118]. Recent studies have shown

that downregulation of extracellular adenosine generated by tumours via oxygen

supplementation significantly improved the cytotoxic capacity of both NK and T cells, further

demonstrating the importance of adenosine in cancer survival [118]. Therefore, targeting the

adenosinergic pathway, in which CD73 is vital, has the potential to be an incredibly effective

cancer treatment with the ability to improve adoptive immunotherapy and therefore enhance

survival rates [118].

1.6 CD73-generated adenosine in haemostasis and thrombosis

Normally, the concentration of ATP in the intracellular space is in the millimolar range (1-10

mM); however, injury, damage from chemotherapy or radiotherapy, inflammation or hypoxia

can compromise membrane integrity and result in a severe increase in ATP concentration [98,

117]. This triggers the initiation of the haemostatic process. Damage to the endothelium

exposes circulating platelets to subendothelial surfaces and serine proteases associated with

the coagulation cascade [126]. Platelets are activated via two independent pathways: the

collagen-dependent pathways and the thrombin-dependent pathways [126]. Vascular injury

results in exposure of circulating platelets to collagen-bound vWF and thrombin generation.

Through cleaving PAR1 receptor in platelets, thrombin initiates more platelet activation.

Activated platelets release a myriad of biological factors (e.g. thromboxane A2, serotonin, ATP

and ADP), which attract and activate more platelets [126].

ADP binds to P2Y12 receptor which cause platelets to release their dense granules, further

increasing the level of ATP and ADP [98, 106, 117]. This results in continued activation and

recruitment of platelets, which forms a thrombus at the site of vascular injury, thus

maintaining haemostasis by preventing blood loss [106, 126]. CD39/CD73 are primarily

17

responsible for degrading levels of ATP and ADP into adenosine to prevent uncontrolled

clotting and thrombosis by inhibiting platelet aggregation [127]. This was demonstrated in a

2004 study where CD73-/- mice were found to have shorter bleeding time after tail tip

resection than the wild-type mice, suggesting that the absence of CD73 prevents inhibition of

platelet aggregation [62].

Adenosine is also rapidly released into the extracellular space upon cell damage and metabolic

stress [115]. In this way, it has a dual effect on homeostasis by not only inducing organ and

vascular protective immune responses but also acting as an alarm molecule that warns the

surrounding tissue of injury in a paracrine and autocrine manner [115]. Which effect adenosine

triggers mostly depends on the concentration of adenosine, as the different adenosine

receptors have varying affinities, resulting in different adenosine concentrations required for

their activation [128]. Consequently, adenosine plays a vital role in homeostasis and

haemostasis, and therefore the enzymes responsible for adenosine generation are also

essential to maintain this balance.

Thrombosis occurs when a clot forms within a blood vessel and blocks it, which can be fatal

depending on where the blockage occurs. Adenosine and its analogues have been shown to

inhibit platelet aggregation via the generation of cAMP through the activation of the

adenosine receptor A2A (Figure 2) [62, 98]. Through this signalling pathway, adenosine can

inhibit platelet activation and thus induce an antithrombotic effect [62, 98]. ADP activates the

platelet receptors P2Y1 and P2Y12 to gather more platelets and activate them via degranulation

induced by activation of αIIbβ3, resulting in aggregation and the formation of a clot (Figure 1).

The aggregation will continue until ATP and ADP are degraded, meaning enzymes such as CD39

and CD73 are vital for not only haemostasis but for preventing thrombosis as well [106].

Indeed, the generation of AMP and adenosine has been shown to prevent thrombosis via

limiting the size of the haemostatic plug through the mediation of platelet aggregation and

18

recruitment [105, 129]. Knockout of CD73 was found to reduce the time to thrombosis in mice,

illustrating this effect in vivo [106].

1.7 Aims Firstly, considering the significant increase in bleeding risk during BTK inhibitor treatment that

is not found in BTK-deficient people and therefore cannot be explained by the inhibition of

BTK, this study aimed to investigate the cause of the increased bleeding risk in CLL patients.

Secondly, as previous studies found that only a third of CLL patients’ B-cells express CD73, this

study aimed to determine whether there was a soluble form of CD73 in the plasma of CLL

patients at higher concentrations than in healthy people. Thirdly, we sought to investigate the

effect of BTK inhibitors with and without adenosine on platelet activation and aggregation in

response to different platelet agonists. Fourthly, this study aimed to elucidate the signalling

pathways triggered in platelets after exposure to both BTK inhibitors and adenosine. Lastly, it

Figure 2: Purinergic signalling and the generation of adenosine. ATP can be released into the induce a pro-inflammatory response by binding to P2 receptors, or it can be converted to adenosine by the rapid processing of CD39 and CD73 and bind to adenosine receptors which mostly trigger anti-inflammatory or immunoregulatory effects. Extracellular adenosine can also be transported into targets cells through adenosine transporters or can be processed further into inosine. Figure reused from de Leve, Wirsdörfer, and Jendrossek’s ‘The CD73/Ado System – A New Player in RT Induced Adverse Effects’, 2019, under the Creative Commons License CC BY 4.0 [43].

19

is hypothesized that there is a synergy between the elevated levels of adenosine characteristic

of CLL and the BTK inhibitors that results in the inhibition of platelet aggregation. Investigation

of the platelet activity in response to clinically relevant doses of ibrutinib and acalabrutinib and

the agonists ADP, collagen, and CRP supported this hypothesis.

20

2. Methods

21

2.1 Materials and reagents

Collagen was obtained from Helena Labs (Melbourne, Australia), and Collagen-related peptide

(CRP) was a kind gift from Professor Elizabeth Gardiner, Australian National University.

Ibrutinib and acalabrutinib were obtained from Apexbio (Houston, USA). 2-Hexynyl-5'-N-

ethylcarboxamidoadenosine (HENECA) was obtained from Abcam (Cambridge, UK). The

antibodies used for Western blotting were all rabbit antibodies, specific for p-SYK (Tyr 352), p-

BTK (Tyr 223), p-Plcγ2 (Tyr 759), p-AKT (Ser 473), p-VASP (Ser 239), p-ERK1/2 (Thr 202/ Tyr 204)

and α-actinin, and were all purchased from Cell Signalling Technology (Danvers, USA).

2.2 Participants

This study was approved by the Curtin University Human Research Ethics Committee

(HRE2020-0384). Healthy, age-matched volunteers (n = 10) recruited from Curtin Health

Innovation Research Institute were used as controls. Plasma was collected from stable,

untreated CLL patients (n = 24).

2.3 Preparation of washed human platelets

Blood was obtained from healthy donors and collected in a 50 mL syringe containing acid-

citrate-dextrose (ACD) with informed consent as required by the Curtin University Human

Research Ethics Committee (HRE2020-0384). After collection, washed platelets were prepared

as previously described [130]. Briefly, blood was centrifuged for 20 minutes at 150 x g, after

which the platelet rich plasma (PRP) fraction was carefully collected and then centrifuged for

10 minutes at 800 x g. The supernatant was disposed of and the platelet pellet was

resuspended in CGS buffer (33.33 mM glucose, 123.2 mM NaCl and 14.7 mM trisodium citrate,

pH 7) and then centrifuged for another 10 minutes at 800 x g. This was repeated for a total of

three washes. Prostaglandin E1 (PGE1, 1 µM) was added before each centrifugation to prevent

22

early activation of the platelets. The platelets were then resuspended in calcium free Tyrode

buffer (5.5 mM glucose, 12 mM NaHCO3, 0.49 mM MgCl2, 2.6 mM KCl, 5 mM HEPES, 0.36 mM

NaH2PO4 and 138 mM NaCl, pH 7.4) at a concentration of 1 x 109/mL. Prior to experimentation,

platelets were supplemented with 1.8 mM CaCl2 to provide a better environment for platelet

activation for the aggregation assay.

2.4 Human CD73 ELISA

Human blood from CLL patients (n = 24) and age-matched healthy controls (n = 10) was

collected into 3.2% sodium citrate Vacutainer tubes and plasma was prepared by centrifuging

the samples for 15 minutes at 1,500 x g. Samples were then aliquoted and stored at -20˚C until

further analysis. The levels of human CD73 was measured using the Human CD73 ELISA Kit

NT5E (Abcam, Cambridge, UK). CD73 concentrations in the samples were calculated from a

standard curve generated at 450 nm using a plate reader, as described in the manufacturer’s

protocol (Appendix A).

2.5 Malachite Green Phosphate Assay

Plasma from 5 CLL patients was examined for soluble CD73 (sCD73) activity. Plasma was

preincubated with PBS or CD73 inhibitor AB680 (2 nM). The CD73 substrate AMP (100 µM) or

PBS was added, and the plasma was further incubated for 1 h at 37˚C. Levels of free

phosphate in samples were measured using the Malachite Green Phosphate assay kit MAK307

(Sigma-Aldrich, St. Louis, USA) following the manufacturer’s instructions (Appendix B).

23

2.6 Flow Cytometry

Human blood was collected from healthy donors into sodium citrate, then diluted 1:20 in

Tyrode HEPES buffer. The diluted blood was then incubated with either DMSO or ibrutinib (0.5

µM) for 15 minutes at 37˚C, after which adenosine (10 µM) or vehicle was added and left to

incubate for a further 5 minutes. ADP (2 µM) or PBS were then added to the relevant reactions

and left to incubate for a further 15 minutes. The samples were stained with the antibodies PE-

CD62 (P-selectin) and FITC-PAC1 (active-form αIIbβ3). The reactions were left to incubate in

the dark for another 15 minutes, then 1 mL of ice-cold 1% PFA/PBS was added. Samples were

analysed using a BD LSRFortessa™ Flow Cytometer.

2.7 Platelet Aggregation Assay

Washed platelets at a concentration of 3 x 108/mL were incubated in Tyrode-HEPES buffer

supplemented with 1.8 mM CaCl2 with either PBS, inhibitors, or inhibitors in combination with

HENECA for 1 hour. Where HENECA was used, the reactions were incubated for 55 minutes,

then HENECA was added 5 minutes before the agonist (either collagen or CRP), after which the

platelet aggregation was measured. Platelet aggregation was recorded for at least six minutes

using a light transmission aggregometer (Model 700 Aggregometer, Chrono-Log Corporation,

Havertown, USA) at 37˚C with constant stirring at 1200 rpm.

2.8 Western Blot and Antibodies Used

After aggregation assaying, the reactions were lysed using Laemmli sample buffer in

combination with Protease/Phosphatase Inhibitor Cocktail (Cell Signalling Technology,

Danvers, USA) and β-mercaptoethanol. The samples were then heated at 95˚C for five

minutes. Forty-five microlitres of the samples were loaded in each lane and separated using

sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After separation, the protein was

24

transferred to a polyvinylidene difluoride (PVDF) protein blotting membrane by submerging it

in transfer buffer at 40 volts for two hours. Non-specific binding was then blocked by covering

the membrane with 5% non-fat powdered milk in Tris-buffered saline with 0.1% Tween 20

(TBS-T) at room temperature for one hour. After washing the membrane with TBS-T 3 times

for five minutes each, the relevant antibody (at a dilution of 1:1000) was added to the

membrane, which was then left to incubate overnight at 4˚C. The secondary antibody,

horseradish peroxidase-conjugated anti-rabbit antibody at a dilution of 1:20,000 (Jackson

ImmunoResearch, West Grove, USA), was used to detect the primary antibody. After the

secondary antibody was left to incubate for 3 hours at room temperature, Amersham ECL

Western Blotting Detection Reagent (GE Healthcare Life Sciences, Chicago, USA) was used to

develop the membrane, and the resulting chemiluminescence was then recorded using the

ChemiDoc imaging system (Bio-Rad, Hercules, USA).

2.9 Statistical Analysis

Each experiment was conducted at least 3 times, and GraphPad PRISM 9 software was used to

analyse the data using One-Way ANOVA to examine statistical differences between the means.

Results are expressed as the mean ± standard error. Any differences found were considered

statistically significant at P < 0.05.

25

3. Results

26

3.1 The expression and activity of soluble CD73 in plasma from CLL patients.

Previous studies have examined the expression of membrane-bound CD73 on B-cells in CLL

patients and found that one-third of CLL patients express CD73 on B-cells. However, it is

unknown whether a soluble form of CD73 also exists in the plasma of CLL patients. Therefore,

we sought to examine the levels of soluble CD73 expression and activity in CLL patients and

determine whether there were any significant differences between soluble CD73 in CLL

patients and healthy volunteers.

Figure 3. The expression and activity of soluble CD73 in plasma from CLL patients. A) Plasma was collected from citrated blood obtained from CLL patients (n=24) and age-matched healthy controls (n=10). Soluble CD73 was measured using Human CD73 ELISA Kit (ABCAM) following the manufacturer’s instructions. B) Plasma from 5 CLL patients were examined for sCD73 activity. Samples were chosen to include patients with the highest and lowest sCD73 level. CD73 activity was measured using Malachite Green Phosphate assay (Sigma-Aldrich) following manufacturer’s instructions. AMP was used as the CD73 substrate. Phosphate generation was reduced when the plasma was preincubated with the CD73 inhibitor AB680 (2 nM). CD73i: CD73 inhibitor AB680, OD: optical density.

27

No significant difference was found in sCD73 level between CLL patients and age-matched

healthy controls. CD73 activity in patients with high sCD73 was reduced by the CD73 inhibitor,

as indicated by the decrease in phosphate generation (Figure 3B). A similar trend was observed

in patients with low sCD73.

3.2 The effect of adenosine on Ibrutinib-mediated inhibition of ADP-induced platelet activation in whole blood.

In the CLL microenvironment, ADP is generated at a higher concentration than is normal. ADP

is known to induce platelet activation, and activation consequently causes platelets to release

their dense granules, further increasing the level of ADP. This should trigger sustained platelet

aggregation; however, this is not observed in ibrutinib treated CLL patients. Thus, the level of

platelet activation was examined via the measurement of the platelet activation markers

CD62P and PAC1 to determine whether ibrutinib inhibited platelet activation. Levels of PAC1

and CD62P were slightly reduced in response to ibrutinib alone; however, there was a more

reduction in both CD62P and PAC1 when ibrutinib and adenosine were combined (Figure 4A).

By itself, ibrutinib lowered the median level of PAC1 but not CD62P (Figure 4B). When

combined with adenosine, the median levels of both PAC1 and CD62P were reduced (Figure

4B). A similar reduction was observed in both PAC1 and CD62P in response to adenosine alone

(Figure 4B).

28

Figure 4: Preliminary data showing the effect of adenosine on Ibrutinib-mediated inhibition of ADP-induced platelet activation in whole blood. Blood was collected from healthy volunteers in sodium citrate, then diluted 1:20 in Tyrode-HEPES buffer. Diluted blood was then incubated with Ibrutinib (0.5 µM) for 15 min at 37˚C, after that adenosine (10 µM) or vehicle was added for 5 minutes. ADP (2 µM) or PBS was then added, and the blood was further incubated for 15 minutes at 37˚C. The samples were stained with PE-CD62P (P-selectin) and FITC-PAC1 (active-form αIIbβ3) for 15 minutes in the dark, then washed in stain buffer (BD) and fixed in ice-cold 1% PFA in PBS and kept at 4˚C. Samples were examined using BD LSRFortessa™ Flow Cytometer. Platelets were identified by their small size using forward scatter area (FSC-A), side scatter area (SSC-A) and then plotted between the two activation markers CD62P (Y-axis) and PAC1 (X-axis). B) The columns represent the median of PE-CD62P and FITC-PAC1 fluorescence intensity as measured using FlowJo (BD). The data are a representative sample of two independent experiments with similar results.

29

3.3 Ibrutinib inhibits CRP-induced platelet aggregation but not collagen-induced platelet aggregation.

A few studies have examined the impact of BTK inhibitors on the aggregation of washed

platelets before; however, these used higher concentrations than was clinically relevant [131,

132]. Using the concentration that platelets in CLL patients are exposed to is vital in

understanding what is causing the dysfunction of platelets in these people. Thus, the effect of

low clinically relevant concentrations (10-50 nM) of the inhibitors on platelet aggregation was

investigated.

The representative collagen traces for ibrutinib, acalabrutinib and the vehicle showed no

significant inhibition of aggregation, though ibrutinib appeared to inhibit some platelet

aggregation compared to acalabrutinib and vehicle (Figure 5C). In the representative CRP

traces, however, ibrutinib inhibited platelet aggregation significantly, having a similar amount

of aggregation as resting platelets even in response to the high concentration of CRP (Figure

5A-B). Figure 5D shows that ibrutinib was found to significantly inhibit CRP-induced platelet

aggregation, reducing aggregation by 70.06% (± 9.4, n = 6). Acalabrutinib, on the other hand,

was unable to inhibit aggregation significantly, only reducing aggregation by 5.86% (± 5.26, n =

6). The phosphorylation of BTK was completely inhibited in the presence of ibrutinib in

response to both CRP and collagen (Figure 5E). Acalabrutinib, on the other hand, partially

inhibited BTK phosphorylation in response to collagen but not CRP. Ibrutinib consistently

inhibited the phosphorylation of PLCγ2 in response to CRP and collagen. While acalabrutinib

effect was only noticeable on collagen-induced PLCγ2 phosphorylation.

30

Figure 5. Ibrutinib inhibits CRP- but not collagen-induced platelet aggregation. (A-C) Washed platelets (WP) were incubated with clinically relevant doses of ibrutinib (10 nM) or acalabrutinib (20 or 50 nM) for 1 hour at 37°C before the addition of the agonist; A) CRP (10 µg/ml) B) CRP (50 µg/ml) C) collagen (10 µg/ml). All traces were representative of 8 repeated experiments. D) Bar graphs depict % of platelet aggregation treated with CRP (50 µg/ml) alone or with ibrutinib (10 nM) or acalabrutinib (50 nM). Percentage platelet aggregation was measured at 6 min n=4-5, ****P < 0.0001. E) Ibrutinib and acalabrutinb decreases CRP and collagen-mediated phosphorylation of PLCγ and BTK. WP (3 × 108/mL) were incubated with ibrutinib or acalabrutinib for 1h at 37˚C. The aggregation reaction was set up using Chrono-log aggregometer and CRP (50 μg/mL), Collagen (10 μg/mL), or vehicle (PBS) was added. The reaction was stopped by Laemmli sample buffer supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signalling Technology) and β-mercaptoethanol. Forty-five μl of total cell lysate was then analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for PLCγ Y759 and p-BTK Y223. Equal loading was verified by α-actinin. The immunoblots are representative of 3 independent experiments.

31

3.4 The adenosine derivative HENECA amplifies the antiplatelet effects of ibrutinib and acalabrutinib.

Having determined that clinically relevant concentrations of BTK inhibitors on their own did

not inhibit collagen-induced platelet aggregation, it was determined that ibrutinib on its own is

not responsible for the bleeding risk observed. Considering this bleeding risk is specific to CLL

patients treated with ibrutinib, it was thought that a characteristic of the disease (such as

adenosine, which is found in higher concentrations than in healthy people) interacts with

ibrutinib and amplifies its effect. Therefore, this study sought to examine the effect of

adenosine on the inhibition of collagen-induced platelet aggregation via ibrutinib and

acalabrutinib.

Light transmission aggregometry found that the ibrutinib only trace showed some inhibition of

collagen-induced platelet aggregation. However, the ibrutinib and HENECA trace stayed

around the level of resting platelets. This demonstrated a significant reduction in aggregation

(Figure 6A). Acalabrutinib alone was unable to inhibit collagen-induced platelet aggregation,

with the acalabrutinib trace following the same pattern as the vehicle trace; however, when

combined with HENECA, there was a significant decrease in platelet aggregation (Figure 6B).

The trace in Figure 6C showed a significant reduction in collagen-induced platelet aggregation

in response to HENECA. Resting platelets remained at a constant level of inactivity, and the

aggregation of collagen alone was continuous and was like the other vehicle traces. As shown

in Figure 6D, ibrutinib and acalabrutinib by themselves did not inhibit platelet aggregation in

response to collagen, with a mean aggregation of 104% (± 5.98, n = 8) and 114.8% (± 1.95, n =

6), respectively. However, aggregation was significantly reduced by each drug when combined

with the adenosine derivative HENECA, with ibrutinib and HENECA inhibiting aggregation by

76.51% (± 5.03, n = 6), while acalabrutinib in combination with HENECA inhibited aggregation

by 24.84% (± 5.89, n = 6). HENECA by itself was also found to cause a significant reduction in

32

platelet aggregation (35.68% ± 10.97, n = 6). The photo demonstrates the striking differences

in aggregation that can even be detected by observation with the naked eye.

Figure 6. The adenosine derivative HENECA amplifies the antiplatelet effects of ibrutinib and acalabtrutinib. (A-B) WP were incubated with clinically relevant doses of ibrutinib (10 nM) or acalabrutinib (20 nM) for 1 hour at 37°C. HENECA (10 µM) or vehicle (buffer) was added, before the addition of collagen (10 µg/ml). C) Aggregation traces showing the impact of HENECA as a single agent on collagen-induced platelet aggregation. D) Bar graphs depict % of platelet aggregation treated with collagen (10 µg/ml) ± ibrutinib (10 nM) or acalabrutinib (20 nM) ± HENECA. Percentage platelet aggregation was measured at 6 min n=5-7, ****P < 0.0001. E) Aggregation tubes showing platelet aggregation in response to collagen + ibrutinib or acalabrutinib ± HENECA. Aggregates of platelets significantly disappear when HENECA is added to either ibrutinib or acalabrutinib.

Coll 10 µg/ml

Ibru/HENECA Ibru 10 nM Acala 20 nM Acala/HENECA

E

33

3.5 Adenosine engages several signalling kinases when combined with BTK inhibitors.

Adenosine appeared to amplify the inhibitive effect of both ibrutinib and acalabrutinib on

collagen-induced platelet aggregation. The signalling pathways inhibited by the BTK inhibitors,

particularly ibrutinib, and adenosine were thought to inhibit the alternate signalling pathways

of platelet aggregation, thus causing this significant bleeding risk phenotype in CLL patients.

Therefore, this study measured the phosphorylation of some of the major signalling molecules

involved in these pathways.

HENECA alone showed only a slight reduction in p-VASP in response to collagen. In contrast,

phosphorylation of AKT remained at the level of collagen alone in response to the inhibitors.

This change was stopped by the addition of HENECA (Figure 7). ERK was found to be

phosphorylated in all tests save for the control; however, there was some inhibition with

ibrutinib in combination with HENECA. BTK phosphorylation was entirely stopped by the tests

with ibrutinib, and significantly reduced by acalabrutinib, though the addition of HENECA

improved the inhibition caused by acalabrutinib even further. HENECA alone also reduced BTK

phosphorylation, however not as much as the BTK inhibitors by themselves or in combination

with HENECA (Figure 7). Ibrutinib inhibited phosphorylation of SYK slightly, and ibrutinib with

HENECA inhibited it significantly, while the other tests had a level of SYK phosphorylation like

that induced by collagen alone.

34

Figure 7. Adenosine engages several signalling kinases when combined with BTK inhibitors. WP (3 × 108/mL) were incubated with ibrutinib or acalabrutinib for 1h at 37˚C. The adenosine derivative HENECA (10 µM) was then added. The aggregation reaction was set up using Chrono-log aggregometer and collagen (10 μg/mL) or vehicle (PBS) was added. The reaction was stopped by Laemmli sample buffer supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signalling Technology) and β-mercaptoethanol. Forty-five μl of total cell lysate was then analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotted for p-AKT Y473, p-VASP S239, p-ERK1/2, p-BTK Y223 and p-SYK Y352. Equal loading was verified by α-actinin. The immunoblots are representative sample of 3 independent experiments.

35

4. Discussion

36

BTK inhibitors such as ibrutinib and acalabrutinib are used to treat CLL because of BTK’s role in

B-cell signalling and proliferation [66]. BTK is also crucial in the signalling and function of

hematopoietic cells such as platelets [67, 86]. Platelets play a vital role in haemostasis and

thrombosis [87, 133, 134]. Numerous studies have observed a significant bleeding risk

associated with patients using ibrutinib and acalabrutinib [28, 42, 43, 74]. BTK is downstream

of the platelet GPVI receptor. However, patients with a genetic defect in GPVI or BTK are not

known to present with significant bleeding issues such as those seen in CLL patients using BTK

inhibitors. Therefore, this study hypothesized that a CLL related factor might synergise with

BTK inhibitors to cause broad inhibition of platelet activation, and consequently, increased risk

of bleeding. CD73-generated adenosine is enriched in the CLL microenvironment and is a

known inhibitor of platelet function. This study explored the impact of the interaction of BTK

inhibitors and adenosine on platelet function via light transmission aggregometry, ELISA,

malachite green assay, flow cytometry and Western blotting [28, 43, 88].

First, we examined the presence of soluble CD73 in the plasma of CLL patients. As shown in

figure 3, a soluble functionally active form of CD73 was present in CLL patients, though

expression appeared to be lower in the CLL group than in healthy individuals. This coincides

with previous reports of elevated sCD73 levels in cancer patients compared to healthy controls

[135]. No other studies to date have measured levels of sCD73 in CLL specifically, though many

have investigated the level of sCD73 in other cancers and found a significant increase [136-

138]. Because of this, sCD73 has been proposed to be a prognostic marker [138, 139]. It is

important to note that CLL cells are characterised by hypoxia, increased expression of CD39

and production of ATP/ADP. Therefore, the production of adenosine will depend on not only

the expression of CD73 but also the increased amounts of the substrate (ATP/ADP CD39

AMP), which is higher in CLL compared to healthy controls [125].

Next, we examined the impact of adenosine on the antiplatelet activity of ibrutinib in whole

blood. Since this experiment was carried out in whole blood, we used 0.5 µM of ibrutinib,

37

which corresponds to maximum plasma concentration in CLL patients [140]. The preliminary

data indicate that adenosine can potentiate the antiplatelet effect of ibrutinib. A previous

study of ibrutinib on platelet aggregation in whole blood from CLL patients by Lipsky et al.

found that ibrutinib improved platelet activation in response to ADP, contradicting the partial

inhibition observed in this study [141]. However, this discrepancy may be because the whole

blood from this study was collected from healthy volunteers instead of CLL patients and that

the sample size was so small (n = 2). CLL patients have higher expression of CD39 and CD73 in

both soluble and membrane-bound forms, which affects the concentration of ADP in the

blood, which could cause the difference in the results of this study and Lipsky et al.’s [125].

Other studies have reported no significant impact on ADP-induced aggregation, most likely

because of BTK lacking a role in G protein-coupled receptor signalling in response to ADP [142].

Adenosine has a short half-life in blood; therefore, future work should focus on replicating this

experiment using either CD73, a stable derivative of adenosine, or blood from CLL patients

with a high level of sCD73.

We next examined the effect of ibrutinib and acalabrutinib as single agents, and in

combination with adenosine, on platelet aggregation. This study shows, for the first time, that

clinically relevant nanomolar concentrations of ibrutinib and acalabrutinib inhibit GPVI-

induced platelet aggregation. This is consistent with previous clinical data demonstrating

reduced CRP-induced platelet aggregation in ibrutinib treated CLL patients, and the data from

this study showing similar inhibition in healthy donors as well (Figure 3) [132]. However, a

recent study by Nicholson et al. found that ibrutinib delayed but did not inhibit CRP-induced

platelet activation, thus demonstrating a lack of ibrutinib effect on GPVI-mediated platelet

aggregation in CLL patients [131]. The concentrations used in the Nicholson et al. study,

however, were chosen on the basis that the concentrations of “free”, unbound maximum

plasma concentrations for ibrutinib and acalabrutinib in CLL patients are 0.5 and 1.3 µM,

respectively, as was described in previous studies [132]. These concentrations are unlikely, as

38

it is well known that at physiological pH ibrutinib and acalabrutinib are basically insoluble in

water and readily bind to the plasma proteins, making 97% biologically inactive [66]. Thus, only

the unbound concentration of the inhibitors is pharmacologically active and therefore suitable

for plasma free in vitro studies [143]. The study by Nicholson et al. based their inhibitor

concentrations on a pharmacokinetic study of ibrutinib conducted by Advani et al. that stated

the total plasma concentration of ibrutinib was 150 ng/mL, or 0.34 µM [144]. The total plasma

concentration of acalabrutinib, according to a study by Byrd et al., was found to be 700 ng/ml

or 0.7 µM [28]. Both total plasma concentrations are suitable for platelet studies in PRP, but

not washed platelets, as only 3% is biologically active due to the binding of 97% to the plasma

proteins.

In CLL patients, it was found that the free unbound maximum plasma concentrations of

ibrutinib and acalabrutinib achieved at a steady-state was in the low nanomolar range, and

these concentrations (≤ 50 nM) have been shown to successfully inhibit in vitro and in vivo BTK

signalling in cancer cells [89, 145-147]. However, there is a significant gap in knowledge in BTK

inhibition in different cell types. Low nanomolar concentration of BTK inhibitors has been

shown to inhibit BTK in cancer cells but not in platelets [148]. This discrepancy can be

explained from a few points. First, incubation time does affect the potency of inhibition.

Longer incubation times have been noticed in cancer cells studies compared to platelets (≥ 30

minutes in cancer cells, a few minutes in platelets) [149]. Another reason could be the

variation of the presence and activity of drug membrane transporters across cell types, which

affects the drug’s ability to fully occupy its intracellular target via changes in the drug

concentration [150, 151].

A study by Levade et al. reported that ibrutinib dose-dependently (0, 0.015, 0.025, 0.05, 0.5, 1

µM) inhibited collagen-induced platelet aggregation [132]. The results of this study suggest

that ibrutinib, at a low, clinically relevant concentration, does not significantly inhibit collagen-

induced platelet aggregation as a single agent (Figure 6) [132]. Previous studies have shown

39

that CLL patients have impaired platelet aggregation in response to collagen, which is

exacerbated by ibrutinib [76, 141]. Washed platelets obtained from healthy volunteers were

not inhibited by ibrutinib alone; however, significantly inhibited collagen-induced platelet

aggregation in the presence of HENECA (Figure 6). HENECA by itself also managed to inhibit

collagen-induced aggregation to a degree, but when combined with ibrutinib inhibited even

more. This explains the impaired platelet function of CLL before and after treatment with

ibrutinib. Levels of adenosine are elevated in CLL due to the increased expression of CD39 and

CD73. The high concentration of adenosine interacts with ibrutinib and results in further

decrement reported previously [76, 141]. This investigation demonstrated that this could occur

even at low nanomolar concentrations of ibrutinib.

This study found that ibrutinib, as well as acalabrutinib, still inhibited collagen-induced BTK

phosphorylation, even though platelet aggregation was not significantly inhibited (Figure 7).

This suggests signalling redundancy allowing continued downstream signalling of the GPVI

pathway, most likely relying on another Tec family kinase, TEC, to regulate the

phosphorylation of PLCγ2 and maintain platelet function [78]. Despite ibrutinib being known to

also inhibit TEC in an off-target manner, at low nanomolar concentrations, there is not enough

TEC inhibition to overcome the signalling redundancy. Acalabrutinib, which is more specific for

BTK, had no significant impact on platelet aggregation but still inhibited BTK phosphorylation,

though not as thoroughly as ibrutinib due to its lower potency [131]. This can be seen in

figures 6A, 6B and 7, where ibrutinib inhibits platelet aggregation (but not significantly) while

acalabrutinib reaches normal levels of platelet aggregation.

This study used the incubation time (1 hour) from previous BTK studies in cancer cells and

exposed washed platelets to low clinically relevant inhibitor concentrations. Under these

conditions, ibrutinib and acalabrutinib inhibited GPVI-mediated platelet aggregation (Figure 5).

Ibrutinib, but not acalabrutinib, continued to inhibit platelet aggregation at a high

40

concentration of CRP (50 µg/mL). This supports the known potency of ibrutinib over

acalabrutinib [28, 74, 131].

In ibrutinib-treated patients, collagen-induced platelet aggregation is inhibited, despite this

study demonstrating that clinically relevant concentrations of ibrutinib do not affect collagen-

induced platelet aggregation [75, 76]. The contradiction between these two in vitro results

could be due to a disease factor. This factor could synergise with ibrutinib and acalabrutinib to

amplify their inhibitive effects to overcome collagen-induced platelet aggregation. This study

was designed on the suspicion that adenosine was this disease factor, as multiple studies have

described the CLL microenvironment as rich in adenosine, thought to be a result of high levels

of abnormally active or expressed CD39 and CD73 [2, 3, 118, 120, 121, 125]. Our results clearly

show that the adenosine derivative HENECA amplified the antiplatelet effects of ibrutinib and

acalabrutinib to cause significant inhibition of collagen-induced platelet aggregation.

Activation of the A2A receptor by HENECA increased p-VASP, which is known to keep platelet

in resting form. HENECA also potentiated ibrutinib and acalabrutinib mediated inhibition of

AKT and ERK phosphorylation. The data clearly show that the presence of adenosine engages

more signalling kinases beyond GPVI/BTK. This result supports our hypothesis that a disease-

related factor may potentiate the antiplatelet spectrum of ibrutinib and acalabrutinib to cause

broad inhibition of platelet function.

Recently, an inverse relationship between plasma adenosine level and platelet activation was

found [106, 152, 153]. However, this relationship has yet to be examined in cancer. In this

study, adenosine was found to reduce phosphorylation of AKT and increase phosphorylation of

VASP in response to collagen-induced platelet aggregation, most likely through the activation

of the A2A receptor (Figure 5). This amplified the effect of ibrutinib and acalabrutinib (to a

lesser extent), with a significant change in phosphorylation of AKT, ERK, BTK, VASP and SYK

being observed in comparison to the BTK inhibitors and adenosine as single agents (Figure 5).

41

This synergy enabled ibrutinib to inhibit collagen-induced platelet aggregation, and inhibited

aggregation more than adenosine as a single agent.

The present study shows a novel interaction between adenosine and BTK inhibitors that

significantly inhibits platelet aggregation. This interaction could be clinically relevant to CLL

patients. However, several limitations arose during the execution of the study. First, the

sample size is not powerful enough to detect a correlation between CD73, plasma adenosine

level, and platelet function. To be able to confidently draw conclusions and overcome the large

amount of variation between individuals, the sample size should be increased. This would also

make the sampled population more representative of the target population. The pilot data

generated in this study could provide the basis for sample size calculations to occur and

predict the number of patients and controls needed for a more extensive study. Second, the

study only examined the in vitro effects of ibrutinib and acalabrutinib on washed platelets and

whole blood and would benefit from further investigation in vivo. Nonetheless, this

investigation provides insight into the bleeding risk found in ibrutinib and acalabrutinib treated

CLL patients.

In conclusion, the results of this study suggest that the significant bleeding risk found in CLL

patients being treated by BTK inhibitors, but not observed in BTK-deficient people, is most

likely caused by the high levels of adenosine in the CLL microenvironment. The high levels of

adenosine synergise with the BTK inhibitors, particularly ibrutinib, and prevents platelet

aggregation, resulting in bleeding events. Future work should examine the correlation

between CD73 (soluble and membrane-bound), plasma adenosine level, platelet activation,

and bleeding issues in CLL patients. Moreover, the impact of CD73 and adenosine on platelet

function in the presence or absence of BTK inhibitors can be further studied in transgenic mice

of CLL. Overall, these studies will yield new insight into how an immune regulatory pathway

(CD39/CD73-adenosine) may be utilised as a biomarker and therapeutic target in CLL.

42

5. References

1. Torre, L.A.M., et al., Global cancer statistics, 2012. Ca : a Cancer Journal for Clinicians,

2015. 65(2): p. 87.

2. Nylund, G., et al., P2Y2- and P2Y4 purinergic receptors are over-expressed in human

colon cancer. Auton Autacoid Pharmacol, 2007. 27(2): p. 79-84.

3. Wiedmeier, J.E., et al., Mortality and glycemic control among patients with acute and

chronic myeloid leukemia and diabetes: a case–control study. Future Science OA, 2021.

7(1): p. FSO639.

4. Juliusson, G. and R. Hough, Leukemia. Prog Tumor Res, 2016. 43: p. 87-100.

5. Bain, B.J., Leukaemia Diagnosis. 2017, Hoboken, UNITED KINGDOM: John Wiley &

Sons, Incorporated.

6. Hofmann, W.-k., M. Stauch, and K. Höffken, Impaired granulocytic function in patients

with acute leukaemia: only partial normalisation after successful remission-inducing

treatment. Journal of Cancer Research & Clinical Oncology, 1998. 124(2): p. 113-116.

7. Kleinstern, G., et al., Tumor mutational load predicts time to first treatment in chronic

lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis beyond the CLL

international prognostic index. American Journal of Hematology, 2020. 95(8): p. 906-

917.

8. Ishikawa, F., et al., Chemotherapy-resistant human AML stem cells home to and

engraft within the bone-marrow endosteal region. Nature Biotechnology, 2007. 25: p.

1315+.

9. Lane, S.W., D.T. Scadden, and D.G. Gilliland, The leukemic stem cell niche: current

concepts and therapeutic opportunities. Blood, 2009. 114(6): p. 1150-1157.

10. Colmone, A., et al., Leukemic Cells Create Bone Marrow Niches That Disrupt the

Behavior of Normal Hematopoietic Progenitor Cells. Science, 2008. 322(5909): p. 1861-

1865.

43

11. Smith, M.L., et al., Mutation of CEBPA in Familial Acute Myeloid Leukemia. New

England Journal of Medicine, 2004. 351(23): p. 2403-2407.

12. Csaba, B., et al., Germ-line GATA2 p.THR354MET mutation in familial myelodysplastic

syndrome with acquired monosomy 7 and ASXL1 mutation demonstrating rapid onset

and poor survival. Haematologica, 2012. 97(6): p. 890-894.

13. DiNardo, C.D. and J.E. Cortes, Mutations in AML: prognostic and therapeutic

implications. Hematology. American Society of Hematology. Education Program, 2016.

2016(1): p. 348-355.

14. Jacob, B., et al., Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5

activation in leukemogenesis. Blood, 2010. 115(8): p. 1610-1620.

15. Daver, N., et al., Targeting FLT3 mutations in AML: review of current knowledge and

evidence. Leukemia, 2019. 33(2): p. 299-312.

16. Yun, J., et al., Glucose Deprivation Contributes to the Development of KRAS Pathway

Mutations in Tumor Cells. Science, 2009. 325(5947): p. 1555-1559.

17. Fedorenko, I.V., G.T. Gibney, and K.S.M. Smalley, NRAS mutant melanoma: biological

behavior and future strategies for therapeutic management. Oncogene, 2013. 32(25):

p. 3009-3018.

18. Edling, C.E. and B. Hallberg, c-Kit—A hematopoietic cell essential receptor tyrosine

kinase. The International Journal of Biochemistry & Cell Biology, 2007. 39(11): p. 1995-

1998.

19. Ruess, D.A., et al., Mutant KRAS-driven cancers depend on PTPN11/SHP2 phosphatase.

Nature Medicine, 2018. 24: p. 954.

20. Pozzo, F., et al., NOTCH1 mutations associate with low CD20 level in chronic

lymphocytic leukemia: evidence for a NOTCH1 mutation-driven epigenetic

dysregulation. Leukemia, 2016. 30(1): p. 182-189.

21. Lee, S.-H. and C. Mayr, Gain of Additional BIRC3 Protein Functions through 3ʹ-UTR-

Mediated Protein Complex Formation. Molecular Cell, 2019. 74(4): p. 701-712.e9.

44

22. Xie, S., et al., Cloning, expression and chromosome locations of the human DNMT3

gene family. Gene, 1999. 236(1): p. 87-95.

23. Moran-Crusio, K., et al., Tet2 Loss Leads to Increased Hematopoietic Stem Cell Self-

Renewal and Myeloid Transformation. Cancer Cell, 2011. 20(1): p. 11-24.

24. Gelsi-Boyer, V., et al., Mutations in ASXL1 are associated with poor prognosis across

the spectrum of malignant myeloid diseases. Journal of Hematology & Oncology, 2012.

5(1): p. 12.

25. Olivier, M., M. Hollstein, and P. Hainaut, TP53 mutations in human cancers: origins,

consequences, and clinical use. Cold Spring Harbor perspectives in biology, 2010. 2(1):

p. a001008-a001008.

26. Puente, X.S., et al., Non-coding recurrent mutations in chronic lymphocytic leukaemia.

Nature, 2015. 526(7574): p. 519-524.

27. Nabhan, C. and S.T. Rosen, Chronic lymphocytic leukemia: a clinical review. Jama, 2014.

312(21): p. 2265-76.

28. Byrd, J.C., et al., Acalabrutinib (ACP-196) in Relapsed Chronic Lymphocytic Leukemia.

The New England Journal of Medicine, 2016. 374(4): p. 323-332.

29. CHIORAZZI, N., K. HATZI, and E. ALBESIANO, B-Cell Chronic Lymphocytic Leukemia, a

Clonal Disease of B Lymphocytes with Receptors that Vary in Specificity for

(Auto)antigens. Annals of the New York Academy of Sciences, 2005. 1062(1): p. 1-12.

30. Saba, H.I. and G. Mufti, Advances in Malignant Hematology. 2011, Hoboken, UNITED

KINGDOM: John Wiley & Sons, Incorporated.

31. Health, A.I.o. and Welfare, Cancer data in Australia. 2020, AIHW: Canberra.

32. Wiestner, A., Ibrutinib and Venetoclax — Doubling Down on CLL. The New England

Journal of Medicine, 2019. 380(22): p. 2169-2171.

33. Redaelli, A., et al., The clinical and epidemiological burden of chronic lymphocytic

leukaemia. European Journal of Cancer Care, 2004. 13(3): p. 279-287.

45

34. Cheng, S., et al., BTK inhibition targets in vivo CLL proliferation through its effects on B-

cell receptor signaling activity. Leukemia, 2014. 28(3): p. 649-57.

35. Nowakowski, G.S., et al., Using Smudge Cells on Routine Blood Smears to Predict

Clinical Outcome in Chronic Lymphocytic Leukemia: A Universally Available Prognostic

Test. Mayo Clinic Proceedings, 2007. 82(4): p. 449-453.

36. Nowakowski, G.S., et al., Percentage of smudge cells on routine blood smear predicts

survival in chronic lymphocytic leukemia. J Clin Oncol, 2009. 27(11): p. 1844-9.

37. Johansson, P., et al., Percentage of smudge cells determined on routine blood smears is

a novel prognostic factor in chronic lymphocytic leukemia. Leuk Res, 2010. 34(7): p.

892-8.

38. Puente, X.S., et al., Non-coding recurrent mutations in chronic lymphocytic leukaemia.

Nature, 2015. 526(7574): p. 519-24.

39. Landau, D.A., et al., Mutations driving CLL and their evolution in progression and

relapse. Nature, 2015. 526(7574): p. 525-30.

40. Barrio, S., et al., Genomic characterization of high-count MBL cases indicates that early

detection of driver mutations and subclonal expansion are predictors of adverse clinical

outcome. Leukemia, 2017. 31(1): p. 170-176.

41. Barrientos, J.C., et al., Characterization of Atrial Fibrillation and Bleeding Risk Factors in

Patients with Chronic Lymphocytic Leukemia (CLL): A Population-Based Retrospective

Cohort Study of Administrative Medical Claims Data in the United States (US). Blood,

2015. 126(23): p. 3301-3301.

42. Georgantopoulos, P., et al., Major hemorrhage in chronic lymphocytic leukemia

patients in the US Veterans Health Administration system in the pre-ibrutinib era:

Incidence and risk factors. Cancer Medicine, 2019. 8(5): p. 2233-2240.

43. Dmitrieva, E.A., et al., Platelet function and bleeding in chronic lymphocytic leukemia

and mantle cell lymphoma patients on ibrutinib. Journal of Thrombosis and

Haemostasis, 2020. 18(10): p. 2672-2684.

46

44. Kantarjian, H., et al., Results of inotuzumab ozogamicin, a CD22 monoclonal antibody,

in refractory and relapsed acute lymphocytic leukemia. Cancer, 2013. 119(15): p. 2728-

2736.

45. Heyfets, A. and E. Flescher, Cooperative cytotoxicity of methyl jasmonate with anti-

cancer drugs and 2-deoxy-d-glucose. Cancer Letters, 2007. 250(2): p. 300-310.

46. Graul-Conroy, A., E.J. Hicks, and W.E. Fahl, Equivalent chemotherapy efficacy against

leukemia in mice treated with topical vasoconstrictors to prevent cancer therapy side

effects. International Journal of Cancer, 2016. 138(12): p. 3011-3019.

47. Reyna-Figueroa, J., et al., Probiotic Supplementation Decreases Chemotherapy-induced

Gastrointestinal Side Effects in Patients With Acute Leukemia. J Pediatr Hematol Oncol,

2019. 41(6): p. 468-472.

48. Sitaresmi, M.N., et al., Chemotherapy-Related Side Effects in Childhood Acute

Lymphoblastic Leukemia in Indonesia: Parental Perceptions. Journal of Pediatric

Oncology Nursing, 2009. 26(4): p. 198-207.

49. Gale, R. and R. Champlin, HOW DOES BONE-MARROW TRANSPLANTATION CURE

LEUKAEMIA? The Lancet, 1984. 324(8393): p. 28-30.

50. Wu, J., et al., Late mortality after bone marrow transplant for chronic myelogenous

leukemia in the context of prior tyrosine kinase inhibitor exposure: A Blood or Marrow

Transplant Survivor Study (BMTSS) report. Cancer, 2019. 125(22): p. 4033-4042.

51. Rosenberg, S.A., et al., Adoptive cell transfer: a clinical path to effective cancer

immunotherapy. Nature reviews. Cancer, 2008. 8(4): p. 299-308.

52. Berrien-Elliott, M.M., et al., Durable Adoptive Immunotherapy for Leukemia Produced

by Manipulation of Multiple Regulatory Pathways of CD8⁺ T-Cell

Tolerance. Cancer Research, 2013. 73(2): p. 605-616.

53. Blattman, J.N. and P.D. Greenberg, Cancer Immunotherapy: A Treatment for the

Masses. Science, 2004. 305(5681): p. 200-205.

47

54. Shankaran, V., et al., IFNγ and lymphocytes prevent primary tumour development and

shape tumour immunogenicity. Nature, 2001. 410(6832): p. 1107-1111.

55. Groh, V., et al., Tumour-derived soluble MIC ligands impair expression of NKG2D and T-

cell activation. Nature, 2002. 419(6908): p. 734-738.

56. Staveley-O’Carroll, K., et al., Induction of antigen-specific T cell anergy: An early event

in the course of tumor progression. Proceedings of the National Academy of Sciences,

1998. 95(3): p. 1178-1183.

57. Lee, P.P., et al., Characterization of circulating T cells specific for tumor-associated

antigens in melanoma patients. Nature Medicine, 1999. 5(6): p. 677-685.

58. Ansell, S.M., et al., Phase I Study of Ipilimumab, an Anti–CTLA-4 Monoclonal Antibody,

in Patients with Relapsed and Refractory B-Cell Non–Hodgkin Lymphoma. Clinical

Cancer Research, 2009. 15(20): p. 6446-6453.

59. Carthon, B.C., et al., Preoperative CTLA-4 Blockade: Tolerability and Immune

Monitoring in the Setting of a Presurgical Clinical Trial. Clinical Cancer Research, 2010.

16(10): p. 2861-2871.

60. Phan, G.Q., et al., Cancer regression and autoimmunity induced by cytotoxic T

lymphocyte-associated antigen 4 blockade in patients with metastatic melanoma.

Proceedings of the National Academy of Sciences, 2003. 100(14): p. 8372-8377.

61. Gao, Z.-w., K. Dong, and H.-z. Zhang, The Roles of CD73 in Cancer. BioMed Research

International, 2014. 2014: p. 460654.

62. Koszalka, P., et al., Targeted Disruption of CD73 Ecto-5-Nucleotidase Alters

Thromboregulation and Augments Vascular Inflammatory Response. Circulation

Research, 2004. 95(8): p. 814-821.

63. Kris, M.G., et al., Efficacy of Gefitinib, an Inhibitor of the Epidermal Growth Factor

Receptor Tyrosine Kinase, in Symptomatic Patients With Non–Small Cell Lung CancerA

Randomized Trial. JAMA, 2003. 290(16): p. 2149-2158.

48

64. Eifert, C., et al., A novel isoform of the B cell tyrosine kinase BTK protects breast cancer

cells from apoptosis. Genes, Chromosomes and Cancer, 2013. 52(10): p. 961-975.

65. Shepard, H.M., C.M. Brdlik, and H. Schreiber, Signal integration: a framework for

understanding the efficacy of therapeutics targeting the human EGFR family. J Clin

Invest, 2008. 118(11): p. 3574-81.

66. Korycka-Wołowiec, A., D. Wołowiec, and T. Robak, Pharmacodynamic considerations

of small molecule targeted therapy for treating B-cell malignancies in the elderly.

Expert Opinion on Drug Metabolism & Toxicology, 2015. 11(9): p. 1371-1391.

67. Rigg, R.A., et al., Oral administration of Bruton's tyrosine kinase inhibitors impairs

GPVI-mediated platelet function. American Journal of Physiology-Cell Physiology, 2016.

310(5): p. C373-C380.

68. Bradshaw, J.M., The Src, Syk, and Tec family kinases: Distinct types of molecular

switches. Cellular Signalling, 2010. 22(8): p. 1175-1184.

69. López-Herrera, G., et al., Brutonˈs tyrosine kinase—an integral protein of B cell

development that also has an essential role in the innate immune system. Journal of

Leukocyte Biology, 2014. 95(2): p. 243-250.

70. Rawlings, D.J., et al., Activation of BTK by a Phosphorylation Mechanism Initiated by

SRC Family Kinases. Science, 1996. 271(5250): p. 822-825.

71. Quek, L.S., J. Bolen, and S.P. Watson, A role for Bruton's tyrosine kinase (Btk) in platelet

activation by collagen. Current Biology, 1998. 8(20): p. 1137-S1.

72. WATSON, S.P., et al., GPVI and integrin αIIbβ3 signaling in platelets. Journal of

Thrombosis and Haemostasis, 2005. 3(8): p. 1752-1762.

73. Zhang, H., et al., Abstract 794: CG'806, a first-in-class pan-FLT3/pan-BTK inhibitor,

targets multiple pathways to kill diverse subtypes of acute myeloid leukemia and B-cell

malignancy in vitro. Cancer Research, 2018. 78(13 Supplement): p. 794-794.

74. Series, J., et al., Differences and similarities in the effects of ibrutinib and acalabrutinib

on platelet functions. Haematologica, 2019. 104(11): p. 2292-2299.

49

75. Wiedower, E., et al., Unusual, spontaneous aneurysm formation in a patient being

treated with ibrutinib for chronic lymphocytic leukemia. Therapeutic Advances in

Hematology, 2016. 7(4): p. 231-232.

76. Kamel, S., et al., Ibrutinib inhibits collagen-mediated but not ADP-mediated platelet

aggregation. Leukemia, 2015. 29: p. 783+.

77. Chen, J., et al., The effect of Bruton's tyrosine kinase (BTK) inhibitors on collagen-

induced platelet aggregation, BTK, and tyrosine kinase expressed in hepatocellular

carcinoma (TEC). Eur J Haematol, 2018.

78. Atkinson, B.T., W. Ellmeier, and S.P. Watson, Tec regulates platelet activation by GPVI

in the absence of Btk. Blood, 2003. 102(10): p. 3592-3599.

79. Paul, M.K. and A.K. Mukhopadhyay, Tyrosine kinase - Role and significance in Cancer.

International journal of medical sciences, 2004. 1(2): p. 101-115.

80. Du, Z. and C.M. Lovly, Mechanisms of receptor tyrosine kinase activation in cancer.

Molecular Cancer, 2018. 17(1): p. 58.

81. Angelis, D., T.D. Fontánez-Nieves, and M. Delivoria-Papadopoulos, The Role of Src

Kinase in the Caspase-1 Pathway After Hypoxia in the Brain of Newborn Piglets.

Neurochemical Research, 2014. 39(11): p. 2118-2126.

82. Delong, L. and A. Mamorska-Dyga, Syk inhibitors in clinical development for

hematological malignancies. Journal of Hematology & Oncology, 2017. 10.

83. Boucheron, N. and W. Ellmeier, The Role of Tec Family Kinases in the Regulation of T-

helper-cell Differentiation. International Reviews of Immunology, 2012. 31(2): p. 133-

154.

84. Neet, K. and T. Hunter, Vertebrate non-receptor protein–tyrosine kinase families.

Genes to Cells, 1996. 1(2): p. 147-169.

85. Weiss, A. and D.R. Littman, Signal transduction by lymphocyte antigen receptors. Cell,

1994. 76(2): p. 263-274.

50

86. Clark, J.C., et al., Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but

not the Proximal Signalling Events. Thromb Haemost, 2019. 119(7): p. 1124-1137.

87. Elaskalani, O., et al., The Role of Platelet-Derived ADP and ATP in Promoting Pancreatic

Cancer Cell Survival and Gemcitabine Resistance. Cancers, 2017. 9(10): p. 142.

88. Kazianka, L., et al., Ristocetin-induced platelet aggregation for monitoring of bleeding

tendency in CLL treated with ibrutinib. Leukemia, 2017. 31: p. 1117.

89. Honigberg, L.A., et al., The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell

activation and is efficacious in models of autoimmune disease and B-cell malignancy.

Proceedings of the National Academy of Sciences of the United States of America,

2010. 107(29): p. 13075-13080.

90. Andrews, R.K., J.F. Arthur, and E.E. Gardiner Targeting GPVI as a novel antithrombotic

strategy. Journal of blood medicine, 2014. 5, 59-68 DOI: 10.2147/jbm.s39220.

91. Eltzschig, H.K., Extracellular adenosine signaling in molecular medicine. Journal of

Molecular Medicine, 2013. 91(2): p. 141-6.

92. Zheng, D.-W., et al., Photo-Powered Artificial Organelles for ATP Generation and Life-

Sustainment. Advanced Materials, 2018. 30(52): p. 1805038.

93. Drury, A.N. and A. Szent-Györgyi, The physiological activity of adenine compounds with

especial reference to their action upon the mammalian heart. The Journal of

physiology, 1929. 68(3): p. 213-237.

94. Koeppen, M., T. Eckle, and H.K. Eltzschig, Selective deletion of the A1 adenosine

receptor abolishes heart-rate slowing effects of intravascular adenosine in vivo. PLoS

One, 2009. 4(8): p. e6784.

95. Eltzschig, H.K., Adenosine: an old drug newly discovered. Anesthesiology, 2009. 111(4):

p. 904-915.

96. Ghiringhelli, F., et al., Production of Adenosine by Ectonucleotidases: A Key Factor in

Tumor Immunoescape. Journal of Biomedicine and Biotechnology, 2012. 2012: p.

473712.

51

97. Heuts, D.P.H.M., et al., Crystal Structure of a Soluble Form of Human CD73 with Ecto-

5ʹ-Nucleotidase Activity. ChemBioChem, 2012. 13(16): p. 2384-2391.

98. Johnston-Cox, H.A. and K. Ravid, Adenosine and blood platelets. Purinergic signalling,

2011. 7(3): p. 357-365.

99. Antonioli, L., et al., CD39 and CD73 in immunity and inflammation. Trends in Molecular

Medicine, 2013. 19(6): p. 355-367.

100. Fredholm, B.B., Adenosine, an endogenous distress signal, modulates tissue damage

and repair. Cell Death and Differentiation, 2007. 14(7): p. 1315-23.

101. Karmouty-Quintana, H., Y. Xia, and M.R. Blackburn, Adenosine signaling during acute

and chronic disease states. Journal of Molecular Medicine, 2013. 91(2): p. 173-181.

102. Allard, B., et al., Immunosuppressive activities of adenosine in cancer. Current Opinion

in Pharmacology, 2016. 29: p. 7-16.

103. Sitkovsky, M. and D. Lukashev, Regulation of immune cells by local-tissue oxygen

tension: HIF1α and adenosine receptors. Nature Reviews Immunology, 2005. 5(9): p.

712-721.

104. Hart, S., et al., GPCR-induced migration of breast carcinoma cells depends on both

EGFR signal transactivation and EGFR-independent pathways. Biological Chemistry,

2005. 386(9): p. 845.

105. Colgan, S.P., et al., Physiological roles for ecto-5’-nucleotidase (CD73). Purinergic

Signalling, 2006. 2(2): p. 351.

106. Covarrubias, R., et al., Role of the CD39/CD73 Purinergic Pathway in Modulating

Arterial Thrombosis in Mice. Arteriosclerosis, Thrombosis, and Vascular Biology, 2016.

36(9): p. 1809-1820.

107. de Leve, S., F. Wirsdörfer, and V. Jendrossek, The CD73/Ado System—A New Player in

RT Induced Adverse Late Effects. Cancers, 2019. 11(10): p. 1578.

108. Gessi, S., et al., Adenosine receptors and cancer. Biochimica et Biophysica Acta (BBA) -

Biomembranes, 2011. 1808(5): p. 1400-1412.

52

109. Trautmann, A., Extracellular ATP in the immune system: more than just a "danger

signal". Sci Signal, 2009. 2(56): p. pe6.

110. Dosch, M., et al., Mechanisms of ATP Release by Inflammatory Cells. International

journal of molecular sciences, 2018. 19(4): p. 1222.

111. Alam, M.S., et al., Extracellular adenosine generation in the regulation of pro-

inflammatory responses and pathogen colonization. Biomolecules, 2015. 5(2): p. 775-

792.

112. Montalban del Barrio, I., et al., Adenosine-generating ovarian cancer cells attract

myeloid cells which differentiate into adenosine-generating tumor associated

macrophages - a self-amplifying, CD39- and CD73-dependent mechanism for tumor

immune escape. Journal for ImmunoTherapy of Cancer, 2016. 4.

113. Moreno-Sánchez, R., et al., Who controls the ATP supply in cancer cells? Biochemistry

lessons to understand cancer energy metabolism. The International Journal of

Biochemistry & Cell Biology, 2014. 50: p. 10-23.

114. Rodríguez-Enríquez, S., M.E. Torres-Márquez, and R. Moreno-Sánchez, Substrate

Oxidation and ATP Supply in AS-30D Hepatoma Cells. Archives of Biochemistry and

Biophysics, 2000. 375(1): p. 21-30.

115. Hasko, G., et al., Adenosine receptors: therapeutic aspects for inflammatory and

immune diseases. Nature Reviews Drug Discovery, 2008. 7: p. 759+.

116. Hemminki, K. and Y. Jiang, Risks among siblings and twins for childhood acute lymphoid

leukaemia: results from the Swedish Family-Cancer Database. Leukemia, 2002. 16: p.

297+.

117. Allard, B., et al., The ectonucleotidases CD39 and CD73: Novel checkpoint inhibitor

targets. Immunological Reviews, 2017. 276(1): p. 121-144.

118. Wang, J., et al., Purinergic targeting enhances immunotherapy of CD73⁺

solid tumors with piggyBac-engineered chimeric antigen receptor natural killer cells.

Journal for Immunotherapy of Cancer, 2018. 6.

53

119. Chambers, A.M. and S. Matosevic, Immunometabolic Dysfunction of Natural Killer Cells

Mediated by the Hypoxia-CD73 Axis in Solid Tumors. Frontiers in Molecular

Biosciences, 2019: p. NA.

120. Yang, H., et al., CD73, Tumor Plasticity and Immune Evasion in Solid Cancers. Cancers,

2021. 13(2): p. 177.

121. Wettstein, M.S., et al., CD73 Predicts Favorable Prognosis in Patients with Nonmuscle-

Invasive Urothelial Bladder Cancer. Disease Markers, 2015. 2015: p. 785461.

122. Deynoux, M. and N. Sunter, Hypoxia and Hypoxia-Inducible Factors in Leukemias.

Frontiers in Oncology, 2016.

123. Wilson, W.R. and M.P. Hay, Targeting hypoxia in cancer therapy. Nature Reviews

Cancer, 2011. 11: p. 393+.

124. Nowell, P.C., Discovery of the Philadelphia chromosome: a personal perspective. The

Journal of clinical investigation, 2007. 117(8): p. 2033-2035.

125. Serra, S., et al., CD73-generated extracellular adenosine in chronic lymphocytic

leukemia creates local conditions counteracting drug-induced cell death. Blood, 2011.

118(23): p. 6141-6152.

126. Deaglio, S. and S.C. Robson, Ectonucleotidases as regulators of purinergic signaling in

thrombosis, inflammation, and immunity. Advances in pharmacology (San Diego,

Calif.), 2011. 61: p. 301-332.

127. Zukowska, P., et al., Deletion of CD73 in mice leads to aortic valve dysfunction.

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2017. 1863(6): p.

1464-1472.

128. Fredholm, B.B., et al., Aspects of the general biology of adenosine A2A signaling.

Progress in Neurobiology, 2007. 83(5): p. 263-276.

129. Marcus, A.J., et al., The endothelial cell ecto-ADPase responsible for inhibition of

platelet function is CD39. J Clin Invest, 1997. 99(6): p. 1351-60.

54

130. Elaskalani, O., N.B. Abdol Razak, and P. Metharom, Neutrophil extracellular traps

induce aggregation of washed human platelets independently of extracellular DNA and

histones. Cell Communication and Signaling, 2018. 16(1): p. 24.

131. Nicolson, P.L.R., et al., Inhibition of Btk by Btk-specific concentrations of ibrutinib and

acalabrutinib delays but does not block platelet aggregation mediated by glycoprotein

VI. Haematologica, 2018. 103(12): p. 2097-2108.

132. Levade, M., et al., Ibrutinib treatment affects collagen and von Willebrand factor-

dependent platelet functions. Blood, 2014. 124(26): p. 3991-3995.

133. Klaeske, K., et al., Device-induced platelet dysfunction in patients after left ventricular

assist device implantation. Journal of Thrombosis and Haemostasis. n/a(n/a).

134. Sambrano, G.R., et al., Role of thrombin signalling in platelets in haemostasis and

thrombosis. Nature, 2001. 413: p. 74+.

135. Ghalamfarsa, G., et al., CD73 as a potential opportunity for cancer immunotherapy.

Expert Opinion on Therapeutic Targets, 2019. 23(2): p. 127-142.

136. Jiang, T., et al., Comprehensive evaluation of NT5E/CD73 expression and its prognostic

significance in distinct types of cancers. BMC Cancer, 2018. 18.

137. Muñóz-Godínez, R., et al., Detection of CD39 and a Highly Glycosylated Isoform of

Soluble CD73 in the Plasma of Patients with Cervical Cancer: Correlation with Disease

Progression. Mediators of Inflammation, 2020. 2020.

138. He, Y., et al., NMR-Based Assay for the Ex Vivo Determination of Soluble CD73 Activity

in Serum. Anal Chem, 2020. 92(21): p. 14501-14508.

139. Huang, Q., et al., Levels and enzyme activity of CD73 in primary samples from cancer

patients. Cancer Research, 2015. 75(15 Supplement): p. 1538-1538.

140. Administration, T.G. Australian Public Assessment Reports-Extract from the Clinical

Evaluation Report for Ibrutinib Proprietary Product Name: Imbruvica Sponsor: Janssen-

Cilag Pty Ltd. 2014 21/08/2020]; Available from:

https://www.tga.gov.au/sites/default/files/auspar-ibrutinib-160202-cer.pdf.

55

141. Andrew, H.L., et al., Incidence and risk factors of bleeding-related adverse events in

patients with chronic lymphocytic leukemia treated with ibrutinib. Haematologica,

2015. 100(12): p. 1571-1578.

142. von Hundelshausen, P. and W. Siess, Bleeding by Bruton Tyrosine Kinase-Inhibitors:

Dependency on Drug Type and Disease. Cancers, 2021. 13(5): p. 1103.

143. Liston, D.R. and M. Davis, Clinically Relevant Concentrations of Anticancer Drugs: A

Guide for Nonclinical Studies. Clinical Cancer Research, 2017. 23(14): p. 3489-3498.

144. Advani, R.H., et al., Bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) has significant

activity in patients with relapsed/refractory B-cell malignancies. J Clin Oncol, 2013.

31(1): p. 88-94.

145. RESEARCH, C.F.D.E.A., Ibrutinib; CLINICAL PHARMACOLOGY AND BIOPHARMACEUTICS

REVIEW(S). FDA, 2013(205552Orig1s000): p. 16.

146. RESEARCH, C.F.D.E.A., Acalabrutinib; MULTI-DISCIPLINE REVIEW. FDA,

2017(210259Orig1s000): p. 68.

147. Woyach, J.A., et al., Resistance Mechanisms for the Bruton's Tyrosine Kinase Inhibitor

Ibrutinib. The New England Journal of Medicine, 2014. 370(24): p. 2286-2294.

148. Wang, X., et al., Bruton's Tyrosine Kinase Inhibitors Prevent Therapeutic Escape in

Breast Cancer Cells. Molecular Cancer Therapeutics, 2016. 15(9): p. 2198-2208.

149. Denzinger, V., et al., Optimizing Platelet GPVI Inhibition versus Haemostatic

Impairment by the Btk Inhibitors Ibrutinib, Acalabrutinib, ONO/GS-4059, BGB-3111 and

Evobrutinib. Thromb Haemost, 2019. 119(3): p. 397-406.

150. Yim, D.S., Potency and plasma protein binding of drugs in vitro-a potentially misleading

pair for predicting in vivo efficacious concentrations in humans. Korean Journal of

Physiology & Pharmacology, 2019. 23(4): p. 231-+.

151. Zhang, H., et al., The BTK Inhibitor Ibrutinib (PCI-32765) Overcomes Paclitaxel

Resistance in ABCB1- and ABCC10-Overexpressing Cells and Tumors. Molecular Cancer

Therapeutics, 2017. 16(6): p. 1021-1030.

56

152. Agarwal, K.C., et al., Platelet-activating factor (PAF)-induced platelet aggregation

modulation by plasma adenosine and methylxanthines. Biochemical Pharmacology,

1994. 48(10): p. 1909-1916.

153. Matsubara, S. and I. Sato, Plasma adenosine levels and P-selectin expression on

platelets in preeclampsia. Obstet Gynecol, 2001. 98(2): p. 354-5.

57

6. Appendices

58

Appendix A The Manufacturer’s Instructions for the Human CD73 ELISA Kit

Version 5e Last updated 15 April 2021

ab213761 –

Human CD73 ELISA Kit

(NT5E)

For the quantitative detection of Human CD73 in cell culture supernatants, cell lysates, serum and plasma (heparin, EDTA).

This product is for research use only and is not intended for diagnostic use.

Copyright © 2020 Abcam. All rights reserved Table of Contents

6. OVERVIEW 53

7. PROTOCOL SUMMARY 53

8. PRECAUTIONS 54

59

9. STORAGE AND STABILITY 54

10. LIMITATIONS 54

11. MATERIALS SUPPLIED 54

12. MATERIALS REQUIRED, NOT SUPPLIED 55

13. TECHNICAL HINTS 55

14. REAGENT PREPARATION 56

10.STANDARD PREPARATION 57

11.SAMPLE PREPARATION 57

12.ASSAY PROCEDURE 58

13.CALCULATIONS 59

14.TYPICAL DATA 60

15.TYPICAL SAMPLE VALUES 60

16.TROUBLESHOOTING 61

17.NOTES 61

Copyright © 2020 Abcam. All rights reserved

53

1. Overview

The Human CD73 Enzyme-Linked Immunosorbent Assay (ELISA) kit

(NT5E) (ab213761) is designed for the quantitative measurement of Human CD73 in cell culture supernatants, cell lysates, serum and plasma (heparin, EDTA).

The ELISA kit is based on standard sandwich enzyme-linked immunosorbent assay technology. A polyclonal antibody from sheep specific for CD73/NT5E has been pre-coated onto 96-well plates. Standards (Expression system for standard: CHO; Immunogen sequence: W27-K547) and test samples are added to the wells, a biotinylated detection polyclonal antibody from sheep specific for CD73/NT5E is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with PBS or TBS buffer. HRP substrate TMB is used to visualize HRP enzymatic reaction. TMB is catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic TMB Stop Solution. The density of yellow is proportional to the Human CD73/NT5E amount of sample captured in plate.

5'-nucleotidase (5'-NT), also known as ecto-5'-nucleotidase or CD73 (Cluster of Differentiation 73), is an enzyme that in humans is encoded by the NT5E gene. Ecto-5-prime-nucleotidase catalyzes the conversion at neutral pH of purine 5-prime mononucleotides to nucleosides, the preferred substrate being AMP. The enzyme consists of a dimer of 2 identical 70-kD subunits bound by a glycosyl phosphatidyl inositol linkage to the external face of the plasma membrane. The enzyme is used as a marker of lymphocyte differentiation. Consequently, a deficiency of NT5 occurs in a variety of immunodeficiency diseases. Other forms of 5-prime nucleotidase exist in the cytoplasm and lysosomes and can be distinguished from ecto-NT5 by their substrate affinities, requirement for divalent magnesium ion, activation by ATP, and inhibition by inorganic phosphate. Rare allelic variants are associated with a syndrome of adult-onset calcification of joints and arteries (CALJA) affecting the iliac, femoral, and tibial arteries reducing circulation in the legs and the joints of the hands and feet causing pain.

6. Protocol Summary

Prepare all reagents, samples, and standards as instructed

Add 100 µL standard or sample to appropriate wells Incubate at 37°C for 90

minutes

Discard plate content. Do not wash.

Add 100 µL biotinylated Antibody into all wells Incubate at 37°C for 60 minutes

Wash each well three times with 300 µL 0.01M PBS (or TBS)

Add 100 µL ABC working solution Incubate at 37°C for 30 minutes

Wash each well five times with 300 µL 0.01M PBS (or TBS)

Add 90 μL of prepared TMB

54

Incubate at 37°C in dark for 15-20 minutes

Add 100 µL TMB Stop Solution and read OD at 450 nm within 30 minutes

7. Precautions

Please read these instructions carefully prior to beginning the ELISA assay. All kit components have been formulated and quality control tested to function

successfully as a kit. We understand that, occasionally, experimental protocols might need to be modified to

meet unique experimental circumstances. However, we cannot guarantee the performance of the product outside the conditions detailed in this protocol booklet.

Reagents should be treated as possible mutagens and should be handled with care and disposed of properly. Please review the Safety Datasheet (SDS) provided with the product for information on the specific components.

Observe good laboratory practices. Gloves, lab coat, and protective eyewear should always be worn. Never pipette by mouth. Do not eat, drink, or smoke in the laboratory areas.

All biological materials should be treated as potentially hazardous and handled as such. They should be disposed of in accordance with established safety procedures.

8. Storage and Stability

Store ELISA kit at -20ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Materials Supplied section. Aliquot components in working volumes before storing at the recommended temperature.

9. Limitations

ELISA kit intended for research use only. Not for use in diagnostic procedures.

Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted.

10. Materials Supplied

Item Quantity

Storage Condition

(Before prep)

Storage Condition

(After prep)

Anti-Human CD73 coated Microplate (12 x 8 wells)

1 x 96 well plate -20ºC -20ºC

Lyophilized recombinant Human CD73 standard 2 x 1 vial -20ºC -20ºC

Biotinylated anti- Human CD73 antibody 100 µL -20ºC -20ºC

Avidin-Biotin-Peroxidase Complex (ABC) 100 µL -20ºC -20ºC

Sample Diluent Buffer 30 mL -20ºC -20ºC

Antibody diluent buffer 12 mL -20ºC -20ºC

55

ABC Diluent Buffer 12 mL -20ºC -20ºC

TMB Color Developing Agent 10 mL -20ºC -20ºC

TMB Stop Solution 10 mL -20ºC -20ºC

Adhesive Plate Seal 4 -20ºC -20ºC

11. Materials Required, Not Supplied

These materials are not included in the kit, but will be required to successfully perform this assay:

Microplate reader capable of measuring absorbance at 450 nm. Automated plate washer. Multi- and single-channel pipettes. Clean tubes and Eppendorf tubes. Washing buffer (neutral 0.01M PBS or 0.01M TBS). Preparation of 0.01M TBS: Add 1.2 g Tris, 8.5 g NaCl; 450 μL of purified acetic acid or 700

μL of concentrated hydrochloric acid to 1,000 mL distilled water and adjust pH to 7.2~7.6. Finally, adjust the total volume to 1 L.

Preparation of 0.01 M PBS: Add 8.5 g sodium chloride, 1.4 g Na2HPO4 and 0.2 g NaH2PO4 to 1,000 mL distilled water and adjust pH to 7.2~7.6. Finally, adjust the total volume to 1 L.

12. Technical Hints

Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers.

Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample,

standard and reagent additions.

Ensure plates are properly sealed or covered during incubation steps. Don’t let the 96-well plate dry, for a dry plate will inactivate active components on plate.

Complete removal of all solutions and buffers during wash steps is necessary to minimize background.

All samples should be mixed thoroughly and gently. Avoid multiple freeze/thaw of samples. When generating positive control samples, it is advisable to change pipette tips after each

step.

Before using the kit, spin tubes and bring down all components to the bottom of tubes. In order to avoid marginal effect of plate incubation due to temperature difference

(reaction may be stronger in the marginal wells), it is suggested that the diluted ABC and TMB solution will be pre-warmed in 37ºC for 30 minutes before using.

To avoid high background always add samples or standards to the well before the addition of the antibody cocktail.

This kit is sold based on number of tests. A ‘test’ simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets

56

your requirements. Please contact our Technical Support staff with any questions.

13. Reagent Preparation

Equilibrate all reagents to room temperature (18-25°C) prior to use. The kit contains enough reagents for 96 wells.

Prepare only as much reagent as is needed on the day of the experiment. 9.1 Anti-human CD73 coated Microplate (12 x 8 wells) One plate of 96

wells. Ready to use. Store at -20ºC.

9.2 Lyophilized recombinant Human CD73 standard (2 x 10 ng) 9.2.1 CD73 standard solution should be prepared no more than

2 hours prior to the experiment. Two tubes of CD73 standard (2 x 10 ng) are included in each kit. Use one tube for each experiment.

9.2.2 Add 1 mL sample diluent buffer into one tube to create 10,000 pg/mL of Human CD73 stock solution. Keep the tube at room temperature for 10 minutes and mix thoroughly.

9.3 Biotinylated anti- Human CD73 antibody The solution should be prepared no more than 2 hours prior to the experiment.

9.3.1 The total volume should be: 100 µL/well x (the number of wells). (Allowing 100 µL – 200 µL more than total volume)

9.3.2 Biotinylated anti-Human CD73 antibody should be diluted in 1:100 with the antibody diluent buffer and mixed thoroughly. (i.e. Add 1 μL Biotinylated anti-Human CD73 antibody to 99 μL antibody diluent buffer.)

9.4 Avidin-Biotin-Peroxidase Complex (ABC) Before use, briefly centrifuge the tubes in case any of the contents are trapped in the lid or sticking to the tube walls. The solution should be prepared no more than 1 hour prior to the experiment.

9.4.1 The total volume should be: 100 µL/well x (the number of wells). (Allowing 100 µL - 200 µL more than total volume) 9.4.2 Avidin- Biotin-Peroxidase Complex (ABC) should be diluted in 1:100 with the ABC dilution buffer and mixed thoroughly. (i.e. Add 1 μL ABC to 99 μL ABC diluent buffer.)

9.5 Sample diluent buffer 30 mL. Ready to use. Store at -20ºC.

9.6 Antibody diluent buffer 12 mL. Ready to use. Store at -20ºC.

9.7 ABC diluent buffer 12 mL. Ready to use. Store at -20ºC.

9.8 TMB 10 mL. Ready to use. Store at -20ºC.

9.9 TMB Stop Solution 10 mL. Ready to use. Store at -20ºC.

57

10.Standard Preparation

10.1 Add 1000 µL of the above 10,000 pg/mL CD73 stock solution into a tube and mix thoroughly.

10.2 To prepare standards, label 7 Eppendorf tubes with 5,000 pg/mL, 2500 pg/mL, 1250 pg/mL, 312 pg/mL, 156 pg/mL and 0 pg/mL respectively.

10.3 Aliquot 300 µL of the sample diluent buffer into each tube.

10.4 Add 300 µL of the above 10,000 pg/mL CD73 solution into 2nd tube and mix.

10.5 Transfer 300 µL from 2nd tube to 3rd tube and mix. Transfer 300 µL from 3rd tube to 4th tube and mix, and so on.

Tube # Volume to dilute Volume of

diluent

Concentratio n

(pg/mL)

1 1000 μL of 10,000 pg/mL

stock solution 0 μL 10,000

2 300 μL of 10,000 pg/mL stock

solution 300 μL 5,000

3 300 μL of tube #2 300 μL 2500

4 300 μL of tube #3 300 μL 1250

5 300 μL of tube #4 300 μL 625

6 300 μL of tube #5 300 μL 312

7 300 μL of tube #6 300 μL 156

8 0 μL 300 μL 0

Note: The standard solutions are best used within 2 hours. The

10,000 pg/mL standard solution should be stored at 4°C for up to 12 hours, or at -20°C for up to 48 hours. Avoid repeated freeze-thaw cycles.

11.Sample Preparation

Store samples to be assayed within 24 hours at 4°C. For long-term storage, aliquot and freeze samples at -20°C. Avoid repeated freeze-thaw cycles.

Cell lysates: After sufficient splitting, there should be no obvious cell sediment. Centrifuge cell lysates at approximately 10,000 x g for 5 minutes. Collect the cell lysate supernatants to go ahead.

Serum: Allow the serum to clot in a serum separator tube (about 4 hours) at room temperature. Centrifuge at approximately 1,000 x g for 15 minutes. Analyze the serum immediately or aliquot and store samples at -20°C.

Cell culture supernatant: Remove particulates by centrifugation, assay immediately or aliquot and store samples at -20°C.

58

Plasma: Collect plasma using heparin or EDTA as an anticoagulant. Centrifuge for 15 minutes at 1,500 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C.

It is recommended to estimate the concentration of the target protein in the sample and select a proper dilution factor so that the diluted target protein concentration falls near the middle of the linear regime in the standard curve. Dilute the sample using the provided diluent buffer. The following is a guideline for sample dilution. Several trials may be necessary in practice. The sample must be well mixed with the diluents buffer.

High target protein concentration (20,000 pg/mL200,000 pg/mL). The working dilution is 1:100. i.e. Add 1 μL sample into 99 μL sample diluent buffer.

Medium target protein concentration (2,000 pg/mL- 20,000 pg/mL). The working dilution is 1:10. i.e. Add 10 μL sample into 90 μL sample diluent buffer.

Low target protein concentration (31.2 pg/mL-2,000 pg/mL). The working dilution is 1:2. i.e. Add 50 μL sample to 50 μL sample diluent buffer.

Very Low target protein concentration (0 pg/mL-31.2 pg/mL). No dilution necessary, or the working dilution is 1:2.

12.Assay Procedure

It is recommended to assay all standards, controls and samples in duplicate.

The ABC working solution and TMB color developing agent must be kept warm at 37°C for 30 minutes before use. When diluting samples and reagents, they must be mixed completely and evenly. Standard CD73 detection curve should be prepared for each experiment. The user will decide sample dilution fold by crude estimation of CD73 amount in samples.

12.1 Aliquot 100 µL per well of the 10,000 pg/mL , 5,000 pg/mL, 2500 pg/mL, 1250 pg/mL, 625pg/mL, 312 pg/mL, 156 pg/mL and 0 pg/mL. Human CD73 standard solutions into the precoated 96-well plate.

12.2 Add 100 µL of the sample diluent buffer into the control well (Zero well). 12.3 Add 100 µL of each properly diluted sample of Human cell culture supernatants,

cell lysates, tissue homogenates, serum or plasma (heparin, EDTA) to each empty well. See “Sample

Preparation” above for details. It is recommended that each Human CD73 standard solution and each sample be measured in duplicate.

12.4 Seal the plate with a new adhesive cover provided and incubate at 37°C for 90 minutes.

12.5 Remove the cover, discard plate content, and blot the plate onto paper towels or other absorbent material. Do NOT let the wells completely dry at any time.

12.6 Add 100 µL of biotinylated anti-Human CD73 antibody working solution into each well, seal the plate with a new adhesive cover provided and incubate at 37°C for 60 minutes.

12.7 Wash plate 3 times with 0.01M TBS or 0.01M PBS; and each time let washing buffer stay in the wells for 1 minute. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (Plate Washing Method: Discard the solution in the plate without touching the side walls. Blot the plate

59

onto paper towels or other absorbent material. Soak each well with at least 300 µL PBS or TBS buffer for 1~2 minutes. Repeat this process two additional times for a total of three washes. Note: For automated washing, aspirate all wells and wash three times with PBS or TBS buffer, overfilling wells with PBS

or TBS buffer. Blot the plate onto paper towels or other absorbent material.)

12.8 Add 100 µL of prepared ABC working solution into each well, seal the plate with a new adhesive cover provided and incubate at 37°C for 30 minutes.

12.9 Wash plate 5 times with 0.01M TBS or 0.01M PBS; and each time let washing buffer stay in the wells for 1-2 minutes. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (See Step 12.7 for plate washing method.)

12.10 Add 90 μL of prepared TMB color developing agent into each well, seal the plate with a new adhesive cover and incubate at 37°C in dark for 15-20 minutes.

Note: For reference only, the optimal incubation time should be determined by end user. And the shades of blue can be seen in the wells with the four most concentrated Human CD73 standard solutions; the other wells show no obvious color.

12.11 Add 100 µL of prepared TMB Stop Solution into each well. The color changes into yellow immediately.

12.12 Read the O.D. absorbance at 450 nm in a microplate reader within 30 minutes after adding the TMB Stop Solution.

13.Calculations

The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The Human CD73 concentration of the samples can be interpolated from the standard curve.

(the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero well).

Note: if the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.

60

14.Typical data

Typical standard curve – Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.

Sample Human CD73

(pg/mL) O.D.

1 0 0.055

2 156 0.125

3 312 0.169

4 625 0.299

5 1,250 0.545

6 2,500 0.956

7 5,000 1.437

8 10,000 2.064

Figure 1. Human CD73 ELISA Kit (NT5E) (ab213761) Standard Curve.

15.Typical sample values

Sensitivity – The biological sensitivity of the assay is <10 pg/mL. The range is 156 pg/mL – 10,000 pg/mL.

Precision – Intra-assay precision: (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.

Sample Number of measures

Mean (pg/mL)

Standard Deviatio

n CV%

1 16 230 15.41 6.7

61

2 16 1,941 79.58 4.1

3 16 5,361 387.29 7.2

Inter-assay precision: (Precision between assays) Three samples of known concentration were tested in separate assays to assess interassay precision.

Sample Number of assays

Mean (pg/mL)

Standard Deviatio

n CV%

1 24 222 18.64 8.4

2 24 1,912 101.33 5.3

3 24 4,929 359.81 7.3

Specificity: Natural and recombinant Human CD73.

Cross-reactivity: There is no detectable cross-reactivity with other relevant proteins.

16.Troubleshooting Problem Cause Solution

Poor standard curve

Inaccurate Pipetting Check Pipettes

Improper standard dilution

Prior to opening, briefly spin the stock standard tube and

dissolve the powder thoroughly by gentle

mixing

Low Signal

Incubation times too brief

Ensure sufficient incubation times

standard/sample incubation

Inadequate reagent volumes or

improper dilution

Check Pipettes and ensure correct

preparation

Incubation times with TMB too brief

Ensure sufficient incubation time until blue color develops

prior addition of TMB Stop

Solution

Plate is insufficiently washed

Review manual for proper wash technique. If using a plate

washer, check all Large CV ports for obstructions.

Contaminated wash buffer Prepare fresh wash buffer

Low sensitivity Improper storage of the ELISA kit

All components 4°C. Keep TMB substrate solution

protected from light.

17.Notes

69

ab213761 Human CD73 ELISA Kit (NT5E) 19 ab213761 Human CD73 ELISA Kit (NT5E) 20 ab213761 Human CD73 ELISA Kit (NT5E) 21 Technical Support

Copyright © 2020 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.

For all technical or commercial enquiries please go to:

www.abcam.com/contactus www.abcam.cn/contactus (China)

www.abcam.co.jp/contactus (Japan)

Copyright © 2020 Abcam. All rights reserved

70

Appendix B The Manufacturer’s Instructions for the Malachite Green Phosphate Assay Kit

Malachite Green Phosphate Assay Kit

Catalog Number MAK307

Storage Temperature 2–8 C

TECHNICAL BULLETIN

71

Product Description The Malachite Green Phosphate Assay Kit is based on quantification of the green complex formed between Malachite Green, molybdate, and free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600–660 nm) or on a plate reader.

This kit may be used to measure the liberation of free orthophosphate in the following applications:

Phosphatase Assays: liberation of phosphate from peptide, protein, or small molecule substrate.

Lipase Assays: liberation of phosphate from phospholipids

Nucleoside Triphosphatase Assays: liberation of phosphate from nucleoside triphosphates (ATP,

GTP, TTP, CTP etc).

Quantitation of phosphate in phospholipids, proteins and DNAs, etc.

Drug Discovery: high-throughput screen for phosphatase inhibitors.

The non-radioactive colorimetric assay kit has been optimized to offer superior sensitivity and prolonged shelf life. The assay is simple and fast, involving a single addition step for phosphate determination. Assays can be executed in tubes, cuvettes, or multiwell plates. The assays can be conveniently performed in 96 and 384 well plates for high-throughput screening of enzyme inhibitors.

Key Features:

Reagent is very stable: Due to the innovative formulation, no precipitation of reagent occurs. Therefore no filtration of reagent is needed prior to assays, as is often required with other commercial kits.

High sensitivity and wide detection range: detection of as little of 1.6 pmoles of phosphate

and useful range between 0.02–40 M phosphate.

Fast and convenient: homogeneous “mix-andmeasure” assay allows quantitation of free phosphate within 30 minutes.

Compatible with routine laboratory and HTS formats: assays can be performed in tubes,

cuvettes, or microplates, on spectrophotometers and plate readers.

72

Robust and amenable to HTS: Z factors of 0.7–0.9 are observed in 96 and 384 well plates.

Can be readily automated on HTS liquid handling systems.

Components The kit is sufficient for 2,500 assays in 96 well plates.

Reagent A 50 mL

Catalog Number MAK307A

Reagent B 1 mL

Catalog Number MAK307B

1 mM Phosphate Standard 1 mL

Catalog Number MAK307C

Reagents and Equipment Required but Not Provided.

96 well flat-bottom plate – It is recommended to use clear plates for colorimetric assays.

Spectrophotometric multiwell plate reader.

Precautions and Disclaimer This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

2

Preparation Instructions Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Working Reagent: Each 96 well plate assay requires

20 L of Working Reagent. Prepare a sufficient volume of Working Reagent by mixing 100 volumes of Reagent A and 1 volume of Reagent B (e.g., 5 mL of

Reagent A and 50 L of Reagent B). Working Reagent is stable for at least 1 day at room temperature. Notes: The reagent must be brought to room temperature before use. Before each

73

assay, it is important to check that all enzyme preparations and assay buffers do not contain free phosphate. This can be conveniently done by adding 20 L of the Working Reagent to 80 L of sample solution.

The blank OD values at 620 nm should be 0.2. If the OD readings are 0.2, check water phosphate level.

Double distilled water usually has OD readings 0.1.

Lab detergents may contain high levels of phosphate. Make sure labware is free from contaminating phosphate after thorough washes.

Storage/Stability The kit is shipped at ambient temperature. Store all components at 2–8 C upon receiving.

Procedure All samples and standards should be run in duplicate.

Assay Reaction 1. Preparation of phosphate standards. Prepare a Premix solution containing 40 M phosphate by

pipetting 40 L of the 1 mM phosphate standard into 960 L of ultrapure water or enzyme reaction buffer. Number the tubes (see Figure 1).

Figure 1. Preparation of Standards

# Premix + water Final Vol ( L)

Phosphate Conc ( M)

pmoles Phosphate in

50 L 1 200 L + 0 L 200 40 2,000 2 160 L + 40 L 200 32 1,600 3 120 L + 80 L 200 24 1,200 4 80 L + 120 L 200 16 800 5 60 L + 140 L 200 12 600 6 40 L + 160 L 200 8 400 7 20 L + 180 L 200 4 200 8 0 L + 200 L 200 0 0

2. Transfer 80 L of test samples into separate wells of the plate. Notes: In the case of enzyme reactions, the reaction may be terminated by adding a specific inhibitor, or can be stopped directly by the addition of the Working Reagent. Dilution of reaction mixture may be necessary prior to the assay.

74

Because any exogenous free phosphate would interfere with the assay, it is important to ensure the protein preparation, the reaction buffer, and labware employed in the assay do not contain free phosphate. This can be conveniently checked by adding the Working Reagent to the buffer and measuring the color formation.

Precipitation may occur at high concentrations of phosphate ( 100 M), or in the presence of high concentrations of proteins and metals. If precipitation occurs, perform a serial dilution of sample with ultrapure water, run the assay, and determine the dilution factor from wells with no precipitation. Repeat assays using diluted samples.

For ATPase or GTPase assays, the ATP or GTP concentration should be 0.25 mM. If the reaction mixture contains 0.25 mM ATP or GTP, dilute samples with ultrapure water. For example, if the ATPase reaction contained 1 mM ATP, at the end of reaction, dilute reaction mixture 4-fold with water prior to the assay.

3. Add 20 L of Working Reagent to each well. Mix gently by tapping the plate. 4. Incubate for 30 minutes at room temperature for color development. 5. Measure absorbance at 600–660 nm (620 nm) on a plate reader.

For assays in 384 well plates, the procedures are the same, except the volume of the standard and sample solution should be 40 L and that of the Working Reagent should be 10 L.

Results Calculation Plot OD620 nm versus phosphate standard concentrations. Determine sample phosphate concentrations from the standard curve.

3

References 1. Guérette, D. et al., Molecular evolution of type VI intermediate filament proteins. BMC

Evolutionary Biology 2007, 7, 164 (2007). 2. Green, M.L. et al., Ethylene glycol induces hyperoxaluria without metabolic acidosis in rats.

Am. J. Physiol. Renal Physiol., 289, F536–F543 (2005). 3. Saran, D. et al., Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate

formation activities catalyzed by an RNA enzyme. Nucleic Acids Research, 34(11), 3201–3208 (2006).

75

4. Adkins, M.W. et al., Chromatin disassembly from the PHO5 promoter Is essential for the recruitment of the general transcription machinery and coactivators. Mol. Cell. Biol., 27, 6372–6382 (2007).

1

4

Troubleshooting Guide

Problem Possible Cause Suggested Solution

Assay not working

Cold assay buffer Assay Buffer must be at room temperature

Omission of step in procedure Refer and follow Technical Bulletin precisely

Plate reader at incorrect wavelength Check filter settings of instrument

Type of 96 well plate used For colorimetric assays, use clear plates

Samples with erratic readings

Samples prepared in different buffer Use the Assay Buffer provided or refer to Technical Bulletin for instructions

Cell/Tissue culture samples were incompletely homogenized

Repeat the sample homogenization, increasing the length and extent of homogenization step.

Samples used after multiple freeze-thaw cycles Aliquot and freeze samples if samples will be used multiple times

Presence of interfering substance in the sample If possible, dilute sample further

Use of old or inappropriately stored samples Use fresh samples and store correctly until use

Lower/higher readings in samples and standards

Improperly thawed components Thaw all components completely and mix gently before use

Use of expired kit or improperly stored reagents Check the expiration date and store the components appropriately

Allowing the reagents to sit for extended times on ice Prepare fresh Reaction Mix before use

Incorrect incubation times or temperatures Refer to Technical Bulletin and verify correct incubation times and temperatures

Incorrect volumes used Use calibrated pipettes and aliquot correctly

Non-linear standard curve

Use of partially thawed components Thaw and resuspend all components before preparing the reaction mix

Pipetting errors in preparation of standards Avoid pipetting small volumes

Pipetting errors in the Reaction Mix Prepare a Reaction Mix whenever possible

Air bubbles formed in well Pipette gently against the wall of the plate well

Standard stock is at incorrect concentration Refer to the standard dilution instructions in the Technical Bulletin

Calculation errors Recheck calculations after referring to Technical Bulletin

Substituting reagents from older kits/lots Use fresh components from the same kit

Unanticipated results

Samples measured at incorrect wavelength Check the equipment and filter settings

Samples contain interfering substances If possible, dilute sample further

Sample readings above/below the linear range Concentrate or dilute samples so readings are in the linear range

SJ,JAC,MAM 03/16-1

2

2016 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered i the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.