inhibiting autophagosome turnover targets therapy resistant ......2018/04/03  · hypoxia in the...

Inhibiting autophagosome turnover

targets therapy resistant leukemia

cells through metabolic disruption

Kaitlyn Dykstra, PhD

Department of Medicine – Leukemia

Lab of Eunice Wang, MD

April 3, 2018

Hematopoiesis

Hematopoietic Stem cell

Myeloid

Progenitor

Lymphoid

Progenitor

Plasma Cell

Megakaryocitic

precursor

RBC precursor

Granulocytic

precursor

T cell /NK

precursor

B cell

precursor

T cell

NK cell

Platelets

Macrophage

RBC

Neutrophils

Slide courtesy

Monica Guzman,

Weill Cornell

Pluripotent Stem cell

Myeloid

Progenitor

Lymphoid

Progenitor

Neutrophils

Plasma Cell

Megakaryocitic

precursor

RBC precursor

Granulocytic

precursor

T cell /NK

precursor

B cell

precursor

T cell

NK cell

Platelets

Macrophage

RBC

Slide courtesy

Monica Guzman,

Weill Cornell

Acute

Myeloid

Leukemia

(AML)

Leukemia

AML Survival Rates Remain Low

Leukemia Subtype 5-year survival

rate

Acute lymphocytic

(ALL)

79.0%

Chronic lymphocytic

(CLL)

68.2%

Acute myeloid

(AML)

26.9%

Chronic myeloid

(CML)

66.9%

SEER Cancer Statistics Review (CSR), 1975-2014 American Cancer Society

CML 14%

Other 9%

ALL 10%

CLL 35%

AML 32%

Estimated New Cases (2018)

60,300 total

Why is AML difficult to cure? • Heterogeneous disease

– 23 different mutated genes

• Compare to CML – Almost all cases are

caused by “Philadelphia chromosome” translocation

– Can be treated with tyrosine kinase inhibitors

The Cancer Genome Atlas Research Network, NEJM, 2013

Why is AML difficult to cure? • AML has an older

patient population – Less tolerant to

chemotherapy

– Less tolerant to bone marrow transplant

• Compared to ALL – Most patients are

pediatric

Sallan, SE. Hematology Am Soc Hematol Educ Program, 2006.

Outcomes are particularly poor in

older adults

Juliusson et al., Blood 119:3890-3899,, 2012 Juliusson et al., Blood 113(18): 4179-4187, 2009

<60

>60

Standard 7+3 induction

chemotherapy

– Treatment has not

changed since the

1970s

– Despite most patients

achieving remission,

relapse inevitably

occurs

Standard of Care for AML

Daunorubicin

or Idarubicin

Cytarabine

(AraC) +

7 days + 3 days

Relapse occurs due to minimal residual

disease including leukemia stem cells in

the bone marrow

Leukemia stem cell (LSC)

AML Blast Minimal residual disease (MRD)

• No signs or symptoms of

disease (remission)

• Disease below the threshold

of diagnostic detection by

microscopy or flow cytometry

How can we eradicate MRD?

Hypoxia in the bone marrow microenvironment

contributes to chemoresistance in AML

Oxygen Level

Cellularity

Portwood et al., Clinical Cancer Research, 2013

O2 levels

• 6% in normal tissues

• 1-3% in bone marrow

Hypoxia contributes to chemoresistance in

AML cell lines

* p<0.05 ** p<0.005 *** p<0.0005 n=3 Live Cells: Annexin V negative/7-AAD negative

How does hypoxia lead to

chemoresistance?

• AraC targets proliferating cells

• Hypoxia reduces cell proliferation

• Is reduced proliferation in hypoxia sufficient to explain AraC resistance?

Hypoxia

Quiescence Metabolic

homeostasis

Therapy resistance

Survival Quiescence

Hypoxia does not reduce the anti-proliferative

or DNA damaging effects of AraC

U n t A ra C U n t A ra C

0

2 5

5 0

% p

-H2

.AX

po

sit

ive

ce

lls

H E L -L u c

n s

2 1 % O 2 1 % O 2

U n t A ra C U n t A ra C

0

5 0 0 0 0 0

1 0 0 0 0 0 0

1 5 0 0 0 0 0

2 0 0 0 0 0 0

2 5 0 0 0 0 0

Liv

e c

ell

s/m

L

H E L -L u c

n s

2 1 % O 2 1 % O 2

Cells/ml (Trypan Blue) DNA Damage (p-H2.AX)

How does hypoxia lead to

chemoresistance? • Hypoxia has previously

been shown to upregulate anti-apoptotic proteins (e.g. Bcl-2) and downregulated pro-apoptotic proteins (e.g. Bax)

• Is reduced chemosensitivity due to changes in expression of these proteins?

Hypoxia

Quiescence Metabolic

homeostasis

Therapy resistance

Survival Quiescence Survival

Expression of anti-apoptotic Bcl-2 and pro-

apoptotic Bax are not affected by hypoxia

Bcl2

Actin

Normoxia Hypoxia Normoxia Hypoxia

Bax

Actin

How does hypoxia lead to

chemoresistance?

• Other survival

pathways altered

under hypoxia

– Autophagy?

Hypoxia

Quiescence Metabolic

homeostasis

Therapy resistance

Survival Survival

Autophagy is a prosurvival process that

is upregulated under hypoxic conditions

Normoxia Hypoxia

LC3-I

LC3-II

p62

actin

- + - + - +

HEL-Luc HL60 MOLM13

Hypoxia

DA

PI/

Cyto

ID

Autophagic flux is upregulated in AML cell

lines after prolonged exposure to hypoxia

2 1 % O 2 1 % O 2

0

5

1 0

1 5

2 0

2 5

3 0

3 5

%C

yto

ID P

os

itiv

e

Ce

lls

*

H E L -L u c H L 6 0 M O L M 1 3

0 .0

0 .2

0 .4

0 .6

Ra

tio

L

C3

BII

/I

N o rm o x ia

H y p o x ia

H E L -L u c H L 6 0 M O L M 1 3

0 .0 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

p6

2 N

orm

ali

ze

d

to N

orm

ox

ia

N o rm o x ia

H y p o x ia

* p<0.05 n=3

Does autophagy drive chemoresistance?

U n t A ra C S p a u 1A ra C

+ S p a u 1

U n t A ra C S p a u 1A ra C

+ S p a u 1

0

2 5

5 0

7 5

1 0 0

%L

ive

Ce

lls

ns ns*

2 1 % O 2 1 % O 2

M O L M 1 3

Autophagosome formation does not

drive chemoresistance under

hypoxia

U n t A ra C M R T A ra C

+ M R T

U n t A ra C M R T A ra C

+ M R T

0

2 5

5 0

7 5

1 0 0

%L

ive

Ce

lls

ns

*

*

2 1 % O 2 1 % O 2

M O L M 1 3

Cyto-ID

MRT68921 (ULK1 inhibitor)

Spautin-1 (Class II PI3K inhibitor)

* p<0.05 n=3

Autophagosome formation does not

drive chemoresistance under

hypoxia

N.S. LC3B-

LC3-I

LC3-II

actin

N.S. Atg7-

Atg7

actin

U n t A ra C U n t A ra C

0

2 5

5 0

7 5

1 0 0

%L

ive

C

ell

s

n o n -s i le n c in g s h R N A

A tg 7 s h R N A

L C 3 B s h R N A

2 1 % O 2 1 % O 2

M O L M 1 3

n s

n s

U n t A ra C U n t A ra C

0

2 5

5 0

7 5

1 0 0

%L

ive

C

ell

s

n o n -s i le n c in g s h R N A

A tg 7 s h R N A

L C 3 B s h R N A

2 1 % O 2 1 % O 2

M O L M 1 3

n s

BafA1 and Chloroquine inhibit autophagosome

turnover through distinct but related mechanisms

Bafilomycin A1 Chloroquine

Vacuolar-type H+-ATPase

inhibitor

• Increases lysosomal pH

Lysosomotropic agent

• Trapped in lysosomes

• Increases lysosomal

pH

Increased lysosomal pH reduces fusion competence.

U n t A ra C B a fA 1 A ra C

+ B a fA 1

U n t A ra C B a fA 1 A ra C

+ B a fA 1

0

2 5

5 0

7 5

1 0 0

%L

ive

Ce

lls

ns

*

*

2 1 % O 2 1 % O 2

M O L M 1 3

Inhibiting autophagosome turnover overcomes

hypoxia induced chemoresistance

U n t A ra C C Q A ra C

+ C Q

U n t A ra C C Q A ra C

+ C Q

0

2 5

5 0

7 5

1 0 0

%L

ive

Ce

lls

ns

*

*

2 1 % O 2 1 % O 2

M O L M 1 3

Bafilomycin A1 Chloroquine

* p<0.05 n=3

Relapse occurs due to minimal residual

disease (MRD) and leukemia stem cells

(LSCs) in the bone marrow

Leukemia stem cell (LSC)

AML Blast

Can late stage autophagy inhibitors

target leukemia stem cells?

LSC Characteristics

• CD34+/CD38-/Lin-

– not exclusively

• Preferentially localized in

hypoxic bone marrow

• Quiescent

• Increased autophagy

CB CD34+ BM CD34+

Figure 2: Basal level of LC3 II in normal CB CD34+ cells and primary AML samples. Representative single confocal sections of

cells expressing LC3 II (green), LAMP2 (red) and DAPI (blue). A:CD34+CB; B: normal BM;

C: CD34+CD38- cell; D:AML12; E:AML18; F: AML7.

A B C

D E F

Figure 2: Basal level of LC3 II in normal CB CD34+ cells and primary AML samples. Representative single confocal sections of

cells expressing LC3 II (green), LAMP2 (red) and DAPI (blue). A:CD34+CB; B: normal BM;

C: CD34+CD38- cell; D:AML12; E:AML18; F: AML7.

A B C

D E F AML (CD34+CD38-)

Figure 2: Basal level of LC3 II in normal CB CD34+ cells and primary AML samples. Representative single confocal sections of

cells expressing LC3 II (green), LAMP2 (red) and DAPI (blue). A:CD34+CB; B: normal BM;

C: CD34+CD38- cell; D:AML12; E:AML18; F: AML7.

A B C

D E F

Neng Yang, Guzman Lab LC3: green / Lysosome: red

Functional definition for LSC

Guzman Lab

Leukemia stem cell

Leukemia progenitor

Colony forming assays (CFU)

Functional definition for LSC

Guzman Lab

Leukemia stem cell

Leukemia progenitor

Xenotransplantation in sub-lethally irradiated immunodeficient mice

Late stage autophagy inhibitors inhibit

primary AML colony formation

1st

PTL AraC CLQ NH4CL Baf A1 3-MA0

20

40

60

80

100

120

FA1441

FG11452

AML032508

GJ1545

AML022806

AML072110

CP1215

FL1473

CF

U re

lativ

e to

UT

2nd

PTL AraC CLQ NH4CL Baf A1 3-MA0

20

40

60

80

100

FA1441

FG11452

AML032508

GJ1545

AML022806

AML072110

CP1215

FL1473

CF

U re

lative

to

UT

Neng Yang, Guzman Lab

Bafilomycin A1 selectively eradicates

leukemia stem cells

Neng Yang, Guzman Lab

4 weeks

AML

Primary AML

BafA1

(1mg/kg; 2X/week)

4 weeks

Why do late stage autophagy

inhibitors target LSCs?

• Inhibiting OXPHOS has

been demonstrated to

target leukemia stem cells

and therapy resistant cells

• Do late stage autophagy

inhibitors modulate

OXPHOS?

Viale et al., Cancer Research, 2015

Basal respiration

Maximal Respiration

Normoxia Untreated

Hypoxia Untreated

Normoxia 5nM BafA1

Hypoxia 5nM BafA1

Late stage autophagy inhibitors block OXPHOS

under hypoxia in AML cells

* p<0.05 ** p<0.005 *** p<0.0005 n=3

Late stage autophagy inhibitors block OXPHOS

under hypoxia in AML cells

Normoxia Untreated

Hypoxia Untreated

Normoxia 50µM CQ

Hypoxia 50µM CQ * p<0.05 ** p<0.005 *** p<0.0005 n=3

Basal respiration

Maximal Respiration

Late stage autophagy inhibitors block OXPHOS

in BafA1 sensitive primary AML samples

Normoxia Untreated

Hypoxia Untreated

Normoxia 25nM BafA1

Hypoxia 25nM BafA1

Patient Sample 1 Patient Sample 2 Patient Sample 3

Late stage autophagy inhibitors block OXPHOS

in BafA1 sensitive primary AML samples

Normoxia Untreated

Hypoxia Untreated

Normoxia 25nM BafA1

Hypoxia 25nM BafA1

Patient Sample 1

0

25

50

75

100

Unt 25nM Baf Unt 25nM Baf

Normoxia Hypoxia

%A

po

pto

tic

Patient Sample 1

n=1

Late stage autophagy inhibitors block OXPHOS

in BafA1 sensitive primary AML samples

Normoxia Untreated

Hypoxia Untreated

Normoxia 25nM BafA1

Hypoxia 25nM BafA1

Patient Sample 3

0

25

50

75

100

Unt 25nM Baf Unt 25nM Baf

Normoxia Hypoxia

%A

po

pto

tic

Patient Sample 3

n=1

Early stage autophagy inhibitors do not

modulate OXPHOS

* p<0.05 ** p<0.005 *** p<0.0005 n=3

OXPHOS inhibition by late stage autophagy

inhibitors is not due to decreases in

mitochondrial mass

MitoTracker MitoTracker

Unstained

Hypoxia Untreated

Hypoxia 5nM BafA1

Hypoxia 25µM CQ

Late stage autophagy inhibitors induce

mitochondrial ROS production

0

1

2

3

4

5

6

7

8

9

10

Untreated 25nMBafA1

50nMBafA1

50uM CQ 100uM CQ Untreated 25nMBafA1

50nMBafA1

50uM CQ 100uM CQ

Normoxia Hypoxia

Rel

ativ

e M

ito

SO

X M

FI

4h

8h

24h

n=2

BafA1 reduces glycolytic function in hypoxia

Normoxia Untreated

Hypoxia Untreated

Normoxia 5nM BafA1

Hypoxia 5nM BafA1

Glycolysis

Glycolytic Capacity

* p<0.05 ** p<0.005 *** p<0.0005 n=3

Chloroquine does not upregulated glycolytic

function in hypoxia

Glycolysis

Glycolytic Capacity

Normoxia Untreated

Hypoxia Untreated

Normoxia 50uM CQ

Hypoxia 50uM CQ * p<0.05 ** p<0.005 *** p<0.0005 n=3

Conclusions

• Hypoxia reduces chemosensitivity in

AML cell lines and increases

autophagic flux.

• Inhibiting the late stages of autophagy

overcomes both hypoxia induced

chemoresistance and eradicates LSCs

• Late stage autophagy inhibitors disrupt

both mitochondrial respiration and

glycolysis under hypoxia

Hypoxia

Glycolysis

Chemoresistance

Late Stage AI

OXPHOS

Leukemia Stem

Cell Maintenance

Future directions • Test BafA1 and chloroquine in

combination with AraC and in an MRD

model of AML

• Elucidate the connection between

blocking autophagosome

turnover/lysosomal disruption and

metabolism – Mitochondrial ROS

– Ca2+

• Identify new inhibitors with better

specificity/distribution – Bone marrow targeted nanoparticles

Acknowledgements Wang Lab

Eunice Wang, MD

Matthew Johnson

Scott Portwood, MS, MBA

Amanda Przespolewski, DO

Dirkje Hanekamp, MS

Megan Johnson

Weill Cornell

Monica Guzman, PhD

Neng Yang, PhD

NCI Cancer Center Support Grant

5P30 CA001656

- Flow and Image Cytometry

Shared Resource

- Immune Analysis Facility

Roswell Park Alliance Foundation

Jacquie Hirsch Leukemia Research Fund