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Arsenic trioxide and sorafenib induce synthetic lethality of FLT3-ITD acute

myeloid leukemia cells

Rui Wang1#*, Ying Li2*, Ping Gong2, Janice Gabrilove1, Samuel Waxman1, and

Yongkui Jing 1, 2

1The Division of Hematology/Oncology, Department of Medicine, The Tisch Cancer Institute, Icahn

School of Medicine at Mount Sinai, New York, USA. 2Department of Pharmacology, Shenyang

Pharmaceutical University, Shenyang, China.

Running title: Targeting FLT3-ITD AML by a novel combination

Correspondence: Yongkui Jing Ph.D., Department of Pharmacology, Shenyang pharmaceutical

University, Liaoning 110016, China; E-mail: [email protected]; Phone: 011-86-24-

23986975;

# Current address: Department of Pathology and Laboratory Medicine, Weill Cornell Medical

College-at Cornell University, New York, USA

*Both authors equally contributed as the first author.

There is no conflict of interest to disclose.

on June 18, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 29, 2018; DOI: 10.1158/1535-7163.MCT-17-0298

http://mct.aacrjournals.org/

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Abstract:

Acute myeloid leukemia (AML) with Fms-related tyrosine kinase 3 internal tandem

duplication (FLT3-ITD) mutation is notoriously hard to treat. We identified two drugs that

together form an effective combination therapy against FLT3-ITD AML. One of the drugs,

Sorafenib, an inhibitor of FLT3-ITD and other kinase activity, produces an impressive but short-

lived remission in FLT3-ITD AML patients. The second, arsenic trioxide (ATO), at

therapeutically achievable concentrations, reduces the level of FLT3-ITD and Mcl-1 proteins, and

induces apoptosis in leukemic cell lines and in primary cells expressing FLT3-ITD. We linked

this relative sensitivity to ATO to low levels of reduced glutathione. While producing

proapoptotic effects, ATO treatment also has an unwanted effect whereby it causes the

accumulation of the phosphorylated (inactive) form of glycogen synthase kinase 3 (GSK-3) a

kinase necessary for apoptosis. When ATO is combined with Sorafenib, GSK-3β is activated,

Mcl-1 is further reduced, and proapoptotic proteins Bak and Bax are activated. Mice xenografted

with FLT3-ITD MOLM13 cell line treated with the Sorafenib/ATO combination have

significantly improved survival. This combination has potential to improve the therapeutic

outcome of FLT3-ITD targeted therapy of AML patients.




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Introduction:

Acute myeloid leukemia (AML) is heterogenous both in molecular abnormality and in response

to therapy. Patients with Fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD)

mutation are the largest subgroup of AML patients with the poorest prognosis (1). FLT3-ITD, present

in ~ 25% of adult AML patients (2-3), is constitutively activated and, by itself, causes

myeloproliferative disorder, while in combination with other oncogenic pathways produces leukemia

(4-5). Several FLT3-ITD kinase inhibitors have been developed and tested in clinical trials (6-9).

They inhibit the activity of FLT3-ITD (10) and produce remissions in some AML patients; the latter

are short-lived ending in an inhibitor-resistant relapse (11-12). A few of the reasons for inhibitor

resistance have been discovered, including secondary mutations in the FLT3-ITD kinase domain (KD)

(13-14). Agents that will target FLT3-ITD with secondary mutations are being developed. Since

multiple mutations of FLT3-ITD have been identified in samples of therapy-resistant patients and cell

lines (14-16), finding inhibitors that will block each of these mutations would require a major effort.

To bypass this problem, we propose the use of agents aimed at elimination of the FLT3-ITD protein

itself, an approach used successfully to treat acute promyelocytic leukemia (APL) with its

pathognomonic fusion protein PML-RAR (17). However, the FLT3-ITD AML is a fatal disease

caused by multiple oncogenes and therefore its treatment will require a combination of drugs. Among

the clinically tested FLT3-ITD inhibitors, Sorafenib, a multi-kinase inhibitor approved for treatment of

several solid tumors (18), is one of the most effective inhibitors of FLT3-ITD AML cells (7, 9, 14, 19-

20). In off-label use, effectiveness of Sorafenib in FLT3-ITD AML patients has been reported (21-23).

Sorafenib improved clinical outcomes in FLT3-ITD AML patients when used prior to, and after bone

marrow transplant (24-26), but the effects were not long-lasting. However, due to its clinical use and

tolerable toxicity, Sorafenib appears to be an excellent candidate for developing combination therapy

for FLT3-ITD AML.

APL with t(15;17) coding PML-RAR fusion protein, a subtype of AML(M3), became a nearly

curable disease due to the development of all trans retinoic acid (ATRA) and arsenic trioxide (ATO),

both targeting PML-RAR fusion protein. Importantly, in about 40% of APL patients, in addition to

the presence of PML-RAR protein, mutant FLT3 are also detected (27). ATO alone or in

combination with ATRA is highly effective in producing long term remissions in APL patients which

also express FLT3-ITD (28-29). In 2 more recent studies the poor response of APL with FLT3-ITD to

ATRA and chemotherapy was improved by addition of ATO in front-line therapy (30-31). These

observations suggest to us that ATO, in addition to its known effect on PML-RAR protein

degradation (32), must also target FLT3-ITD. We previously showed (33) that Sorafenib, at




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concentrations higher than 5 M in combination with ATO is a stronger inducer of apoptosis than each

agent alone and that the combination decreased Mcl-1 and GSH levels in non-FLT3-ITD AML cells.

Using FLT3-ITD AML cell lines and primary leukemia cells, we tested apoptosis induction by ATO

and its ability to eliminate FLT3-ITD protein. We also tested the combined effect of ATO with

Sorafenib in FLT3-ITD AML cells in vitro and in vivo. We found that FLT3-ITD AML cells contain

lower levels of GSH and that this property is linked to sensitivity to ATO-induced apoptosis. We also

showed that at therapeutic concentrations, ATO, is capable of degrading FLT3-ITD protein.

Sorafenib, at lower (nM) concentrations, when combined with ATO selectively augments apoptosis of

FLT3-ITD AML cells in vitro, significantly inhibits leukemia growth and prolongs survival of mice

xenografted with FLT3-ITD AML cells. We explored the possible mechanism responsible for the

enhancement of the combination treatment. Our work provides a new approach in which both the

FLT3-ITD protein and its kinase activity are targeted respectively for degradation and inhibition and

induction of synthetic lethality for FLT3-ITD AML therapy.

Materials and methods

Reagents. Arsenic trioxide for injection was obtained from the Pharmacy of Mount Sinai Hospital.

Sorafenib was obtained from LC Laboratories (Woburn, MA, USA). MG132 was purchased from

Sigma-Aldrich Inc (St. Louis, MO, USA). Bortezomib was acquired from Selleck Chemical (Houston,

TX, USA). Antibodies to Bcl-2, β-actin ERK1, FLT3, p-FLT3(Y591/589), HDAC6, Mcl-1 were

purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); to PARP was from BD

Biosciences (San Diego, CA, USA ); to Ac-tubulin, Ac-lysine, Akt, Bim, p-ERK (Thr202/Tyr204),

GSK-3β, p-GSK-3β (Ser9), , p-rpS6 (Ser235/236), and rpS6 were from Cell Signaling Technology, Inc.

(Beverly, MA, USA). Anti-acetylated-HSP90(K243) antibody was obtained from Ruiying

Biotechnology(Suzhou, China) and HSP90 antibody was obtained from Enzo Life Sciences (San

Diego, CA).

Cell lines and treatments. MOLM13 cells were obtained from DSMZ-Deutsche Sammlung von

Mikroorganismen und Zellkulturen GmbH (Braunschweig Germany). THP-1 and MV-4-11 cells were

obtained from American Type Culture Collection (ATCC). These cell lines were cultured in RPMI

1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L-

glutamine, and 10% (v/v) heat-inactivated fetal bovine serum (FBS). 32D cells transfected with or

without FLT3-ITD were provided by Dr. Naoe (Nagoya University Graduate School of Medicine,




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Japan received in 2012) (34). All cell lines were not authenticated by us once received in our

laboratory and mycoplasma was not tested. 32D cells were cultured in RPMI 1640 medium

supplemented with 1 ng/mL IL-3 and 32D/FLT3-ITD cells were cultured in RPMI 1640 medium

without supplemented with IL-3.

Detection of apoptosis and protein levels. Apoptotic cells were determined by the annexin V assay

according to the manufacturer’s instructions (annexin V–FITC Apoptosis Detection Kit (BD

Biosciences). Western blot analysis was used to measure protein levels. All these methods were done

as we previously reported (33, 35).

Analysis of Bak and Bax conformational changes. Cells treated with ATO plus Sorafenib were

harvested, washed in PBS, and suspended in lysis buffer {10 mM HEPES [pH 7.4], 150 mM NaCl, 1%

3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS]} containing protease

inhibitor cocktail. After 30-min incubation at 4ºC, the suspension was centrifuged for 15 min at

14,000 rpm. The obtained supernatant containing 500 µg protein was precleared by 20 µl protein A-

Agarose (Santa Cruz Biotechnology) and then subjected to immunoprecipitation with 3 µg of either

normal mouse IgG (Santa Cruz Biotechnology) or conformation-specific anti-Bak (clone Ab-1,

Calbiochem), anti-active Bax (clone 6A7, Sigma) at 4 ºC for 2 h with gentle rotation. After that, 30 µl

protein A-agarose beads were added and incubated overnight to pull down the protein-antibody

complexes. The bead pellets were then obtained by centrifuging and washed three times using lysis

buffer. The conformational changed Bak and Bax were determined by Western blot analysis using

anti-Bak and anti-Bax polyclonal antibodies (33).

Immunoprecipitation (IP) of FLT3 and HSP90. Cells treated and without treatment were harvested,

washed with PBS and lysed using ice-cold NP40 lysis buffer [50 mM Tris-HCl (pH7.5), 150 mM NaCl,

5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na3VO4, 1 mM DTT] for 60 min on ice. Total

protein (400 µg) was first precleared with 20 µl protein A/G plus-agarose (Santa Cruz Biotechnology)

and then subjected to immunoprecipitation with 1-2 µg of either a normal IgG, a FLT3 antibody or a

HSP90 antibody (Enzo Life Sciences) at 4ºC for overnight. Then 20 µl of protein A/G plus-agarose

beads were added and incubated overnight to pull down protein-antibody complexes. The beads were

spun, washed four times with NP-40 lysis buffer, resuspended in 2 x SDS sample buffer (50 mM Tris-

HCl (pH 6.8), 2% SDS, 10% glycerol, 5% β-mercaptoethanol) and heated at 98ºC for 5 min for




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analysis by SDS-polyacrylamide gel electrophoresis and Western blotting analysis using an anti-

HSP90 and an anti-Ac-lysine antibody, respectively.

Measurement of intracellular glutathione (GSH) content. The levels of intracellular GSH were

measured by a monochlorobimane (mBCl) fluorometric method in which mBCl was used as a

sensitive and specific probe to analyze GSH in intact cells (36). Briefly, cells were washed once,

resuspended in 1 mL PBS containing 100 μM mBCl, and maintained at 37ºC in the dark for 30 min

before analysis. The formation of the fluorescent adduct (GS-mBCl) was monitored with a Multi-mode

microplate reader (Spectra Max M5e; Molecular Devices, LLC, Sunnyvale, CA, USA) using excitation

and emission wavelengths of 395 and 482 nm, respectively. The GSH content was calculated as

nanomoles per 106 cells based on a GSH standard curve.

Isolation of patient-derived leukemic blood cells. Leukemic blood cells were obtained from three

peripheral blood samples of AML patients with FLT3-ITD (two samples with FLT3-ITD and NPM1

mutation, and one sample with FLT3-ITD and normal NPM1). The study was conducted in accordance

with International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS)

and that was approved by the Investigation Review Board of Icahn School of Medicine at Mount Sinai.

The written informed consent provided to the patients and signed forms were collect by our colleagues

before collection of the blood. Peripheral blood was collected in heparinized tubes and were subjected

to Ficoll-Hypaque density gradient separation. The leukemic blood cells were washed and resuspended

in RPMI-1640 with 20% FBS at 1 x 107 cells/ml and treated with ATO and/or Sorafenib, and then

subjected for apoptosis analysis after staining with annexin V–FITC and for Western blotting analysis .

In vivo treatment studies. The animal procedures were approved by the Institutional Animal Care

and Use Committee (IACUC) of Icahn School of Medicine at Mount Sinai. Thirty NOD/SCID/IL-2Rg

(NSG) mice (age, 8 weeks) were injected with 2 × 106 log-phase MOLM13 cells subcutaneously in the

right flank (37). Mice with tumors of about 100 mm3 were randomized to six groups of five mice each

and were treated with Sorafenib (40 mg/kg) or ATO (2.5 or 5 mg/kg) or their combinations. A

Cremophor EL/ethanol/water solution (12.5% Cremophor EL/ 12.5% ethanol/ 75%water) containing

Sorafenib was prepared and was administered by oral gavage. ATO in PBS solution was administered

intraperitoneally. Mice were treated for 8 days. Tumors were measured every two days. The tumors in

each mice were dissected at day 9 and weighted. In second experiment, twenty NSG mice (age, 8




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weeks) were injected with 5 × 106 log-phase MOLM13 cells via the lateral tail vein. Five days after

leukemic cell injection, the mice were divided randomly into 4 groups and treated with Sorafenib (20

mg/kg, p.o.), ATO (5 mg/kg, i.p.) alone or their combination daily for five continuous days a week and

kept until the mice deceased. Control mice received the vehicle through the same route and on the

same schedule. Survival times of each group were compared with Kaplan–Meier survival analysis and

the increase of life span (ILS) was calculated as (treated group-control group)/control group x 100%

Statistical analysis

Data were analyzed for statistical significance using student’s t test (Microsoft Excel, Microsoft Corp.,

Redmond, WA, USA). A p-value of <0.05 was considered statistically significant.

Results:

FLT3-ITD AML cell lines contain lower levels of GSH and are sensitive to ATO-induced

apoptosis. Previously we have found that APL cells contained lower levels of GSH and were

sensitive to ATO-induced apoptosis and PML-RAR degradation at therapeutic concentrations (1-2

M) (38). We and other groups also found that other malignant cells with lower GSH levels were

sensitive to ATO-induced apoptosis (39-41). We now compared the GSH content of two FLT3-ITD

expressing cell lines (MV4-11 and MOLM13) to that of a cell line expressing wFLT3 (THP-1), and

found that GSH levels in MV4-11 and in MOLM13 cells were, respectively, 80% and 50% lower

than that in THP-1 cells (Fig. 1A). Apoptosis was induced in a dose-dependent manner by 1-4 M of

ATO in MV4-11 and MOLM13 cells, but was only slightly induced in THP-1 cells (Fig. 1B). To test

whether FLT3-ITD was responsible for the reduction in GSH levels and for the subsequent sensitivity

to ATO we compared parental 32D cells to those transfected with FLT3-ITD (34) and found that the

latter had decreased GSH levels (Fig. 1C). 32D cells expressing FLT3-ITD were more sensitive to

ATO-induced apoptosis than the parental cells at every concentration tested (Fig. 1D). FLT3-ITD is

constitutively active due to autophosphorylation. To test if activation of wtFLT3 causes GSH

reduction and increases sensitivity to ATO, we treated THP-1 cells expressing wtFLT3 with FLT3-

ligand. FLT3-ligand at a concentration of 100 ng/ml decreased the levels of GSH by about 27% (Sup.

Fig. 1A), but did not enhance ATO-induced apoptosis in THP-1 cells (Sup. Fig. 1B). These data

suggest that FLT3-ITD might be the factor which not only lowers GSH levels but also renders those

cells more sensitive to ATO-induced apoptosis.

ATO induces degradation of FLT3-ITD. We found that treatment with ATO (at 1 and 2 μM)




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reduced the levels of FLT3-ITD and, to a lesser extent, the phosphorylated FLT3-ITD protein in

MOLM13 cells (Fig. 2A), but had only a minimal effect on wtFLT3 in THP-1 cells (Fig. 2C). In

32D cells transfected with FLT3-ITD, ATO treatment reduced both total and auto-phosphorylated

FLT3-ITD (Fig. 2B). Since FLT3-ITD AML cells contain lower levels of GSH, we tested if depleting

GSH enhances ATO-induced degradation of wtFLT3 and apoptosis in THP-1 cells. We pretreated

THP-1 cells with buthionine sulfoximine (BSO), which inhibited GSH synthesis and depleted GSH

in most cancer cells (40), and found that in BSO pre-treated cells 1 μM of ATO was indeed able to

decrease the levels of the wtFLT3 protein and to induce apoptosis based on PARP cleavage (Fig. 2D).

These data support the notion that activated FLT3-ITD sensitizes cells to ATO, at least in part,

through lowering GSH levels. It has been shown that FLT3-ITD is a client protein of HSP90 and that

the levels of FLT3-ITD protein are decreased in cells treated with HSP90 inhibitors (42-43). The

affinity of HSP90 for client proteins is regulated by acetylation through HDAC6 (44). ATO has been

reported to inhibit HDAC6 activity in myeloma cells (45). We found that in MOLM13 cells treated

with ATO HDAC6 activity was decreased, as judged by the increase in its substrate (acetylated α-

tubulin), and this was associated with the decrease in the levels of HDAC6 protein (Fig.2A). The

FLT3-ITD bound HSP90 was decreased and the acetylated HSP90 level was increased in MOLM13

cells treated with ATO at 1 and 2 M as determined by immunoprecipitation assay (Fig. 2E). It has

been reported that after inhibition of HSP90, FLT3-ITD undergoes proteasomal degradation (46) but

also it reported that the proteasome inhibitors induce FLT3-ITD degradation through autophagy-

mediated pathway (47). We found that ATO-induced FLT3-ITD degradation was not blocked by

proteasome inhibitors Boretzomib and MG132 but that treatment with either one of these inhibitors

induced the microtubule-associated protein 1A/1B-light chain 3 (LC3) cleavage, a marker of

autophagy, suggesting that FLT3-ITD degradation is linked to autophagy after treatments with both

Boretzomib and MG132 (Fig. 2F). Our data suggest that ATO treatment interrupts the axis of

HDAC6/HSP90/FLT3-ITD leading to FLT3-ITD degradation.

ATO and Sorafenib regulate the levels of phosphorylated-GSK3 and BimEL differently in

FLT3-ITD AML cells. Previously we found (33) that the mechanism through which ATO induced

apoptosis in APL-NB4 cells involved repression of Mcl-1 protein by GSK3β activation (inhibition of

its phosphorylation). We observed a similar reduction in Mcl-1 protein level in MOLM13 cells

treated with ATO, but rather than being decreased, the phosphorylated GSK3 levels were elevated

(Fig. 3A). However, we observed that ATO treatment of MOLM13 cells inhibited ERK and rpS6

phosphorylation and decreased Akt and BH3-only BimEL protein (Fig. 3A). These data suggest that




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an Akt-independent inactivation (phosphorylation) of GSK3 occurs in ATO-treated FLT3-ITD AML

cells that may attenuate ATO-induced apoptosis. Sorafenib is one of FLT3-ITD inhibitors used off-

label in AML patients. At lower concentrations of Sorafenib FLT3-ITD AML cells are more

responsive to apoptosis induction than other AML cells (48). We tested the effect of Sorafenib on

Mcl-1, phosphorylated GSK3 and Bim levels. Sorafenib at 50 nM was sufficient to induce

apoptosis and to decrease Mcl-1 protein level and FLT3-ITD phosphorylation. It also reduced p-

GSK3 levels and increased BimEL (Fig. 3B). This change coincided with PARP cleavage (Fig. 3B).

Sorafenib is a multi-kinase inhibitor which also inhibits the FLT3-ITD kinase. To test if the reduction

in GSK3 phosphorylation is through inhibition of FLT3-ITD, we tested the levels of p-FLT3-ITD

and p-GSK3 in MOLM13 cells treated with AC220, a specific inhibitor of FLT3-ITD (10). Like

Sorafenib (Fig. 3B), AC220 inhibited the phosphorylation of FLT3-ITD and GSK3 and induced

apoptosis in MOLM13 cells (Sup. Fig. 2A). These data suggest that, at low concentrations, Sorafenib

leads to GSK3 activation (de-phosphorylation) through inhibition of FLT3-ITD activity in FLT3-

ITD AML cells.

Combination of ATO and Sorafenib enhances apoptosis in FLT3-ITD AML cell lines and

primary AML cells. We treated MOLM13 cells with a combination of 0.5-1 μM of ATO and 25-100

nM of Sorafenib and found that these combinations significantly enhanced apoptosis (Fig. 4A). The

combination indexes were analyzed using Compusyn software showing CIs of less than 1, indicating

synergy (Fig. 4B). In comparison to the effect of the individual drugs, the combination of 1 μM of

ATO with 25 nM of Sorafenib increased PARP cleavage, further reduced Mcl-1 level (Fig. 4C).

Sorafenib treatment alone shifts the balance of FLT3-ITD toward the heavier, glycosylated mature

form, known to localize to the plasma membrane (49). Both forms were decreased by ATO treatment

(Fig. 4C). Although ATO alone increased the levels of p-GSK3, Sorafenib in combination with

ATO decreased the levels of p-GSK3 (Fig. 4C). The finding that ATO together with AC220 also

increases apoptosis in MOLM13 cells, while reducing levels of p-GSK3 (Sup. Fig. 2B) suggests

that GSK3 is activated in the combination treatments and contributed to the enhanced apoptosis

induction. Mcl-1 is an antiapoptotic protein that blocks Bak activation and that Bim is a proapoptotic

protein that activates Bax (50). Immunoprecipitation analysis revealed that Sorafenib was more

effective than ATO in activating both Bak and Bax and that enhanced Bak activation, not Bax

activation, was observed in the combination treatment (Fig . 4D). To test the role of GSK3 in this

combination treatment, GSK3 inhibitor SB216763 was used. SB216763 inhibited both Bax and




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Bak activation in Sorafenib/ATO treated MOLM13 cells with more profoundly on Bax activation

(Fig. 4E) and reduced apoptotic cells from 63% to 28% (Sup. Fig. 3). Previously we found that

Sorafenib enhanced ATO-induced apoptosis and GSK3 activation by depleting GSH in HL-60 cells

(33). This required high concentrations of Sorafenib. We measured the levels of GSH in either

Sorafenib or AC220 treated cells, and found that both decreased GSH levels at concentrations which

inhibited FLT3-ITD phosphorylation (Sup. Fig. 4). However, Sorafenib at 25 nM and AC220 at 2

nM, the concentrations sufficient to enhance ATO-induced apoptosis, did not significantly decrease

the levels of GSH (Sup. Fig. 4). These data suggest that FLT3-ITD activity regulates GSH levels, but

that the apoptosis induced by the two drug combination might not involve GSH depletion. We

propose that the enhanced apoptosis induction by this combination results from that 1) ATO and

sorafenib inhibit FLT3-ITD through different mechanisms which release repression on GSK3; 2)

ATO treatment causes a feedback inactivation of GSK3 which is overcome by Sorafenib; 3) GSK3

activation plays an important role in the activation of Bax/Bak and apoptosis induction (Fig. 4F).

To examine whether apoptosis is enhanced by the combination therapy in primary leukemic

cells, three blood samples from patients with FLT3-ITD leukemia were used to test for the levels of

FLT3-ITD and apoptosis after treatment with ATO alone and in combination with Sorafenib.

Concentration higher than 2.5 M of Sorafenib alone was required to induce apoptosis in these cells.

The sensitivity to ATO was equal to that of MOLM13 (Fig. 5A); at 1 M and 2 M ATO decreased

the FLT3-ITD, Mcl-1 and Akt levels (Fig. 5B). Importantly, addition of ATO augmented Sorafenib-

induced apoptosis (Fig. 5A). These data indicate that ATO is able to decrease FLT3-ITD and Mcl-1

proteins in primary cells and enhances Sorafenib-induced apoptosis at a clinically achievable

concentration.

ATO enhances Sorafenib effect in NSG mice xenografted with FLT3-ITD MOLM13 cells. We

tested the effect of treatment with Sorafenib alone (40 mg/kg); ATO alone (2.5 mg/kg); ATO alone (5

mg/kg); and the combinations of the two (ATO 2.5 mg/kg and Sorafenib 40 mg/kg) and (ATO 5

mg/kg and Sorafenib 40 mg/kg) on growth of MOLM13 cells inoculated s.c. into NSG mice.

Treatment was initiated at day 5 after cell inoculation and lasted for 8 days. Sorafenib (p.o.) alone or

ATO (i.p.) alone reduced tumor size by ~50% (Fig. 6A). The combination of 40 mg/kg of Sorafenib

with 5 mg/kg of ATO produced a profoundly (~90%) and significantly reduced tumor size without

obvious toxic effect as determined by the maintenance of body weight (Fig. 6A and Sup. Fig. 5). We

inoculated MOLM13 cells i.v. through tail vein and treated the mice with Sorafenib (20 mg/kg, p.o.)

alone, ATO (5 mg/kg, i.p.) alone or with the combination. Treatment was initiated at day 5 after cell




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inoculation and lasted until mice were deceased. These mice survived for an average of 17 days

without treatment, ATO alone had a minor effect, Sorafenib prolonged significantly the survival of

the leukemia mice (an average of 34 days) while the combination extended the survival to an average

of 47 days (Fig. 6B, 6C).

Discussion

FLT3-ITD AML remains a very serious, incurable illness with an urgent need for new effective

treatments. The current approaches to inhibit FLT3-ITD kinase activity with several inhibitors,

although clinically effective, produce only transient responses due to the development of resistance

(51). We explored the possibility of targeting FLT3-ITD protein for degradation as a therapy. We

found that at clinically achievable concentrations of 1-2 M, ATO treatment caused degradation of

FLT3-ITD protein and induced apoptosis associated with the down-regulation of Mcl-1 in cell lines

and primary cells with FLT3-ITD (Fig. 3A, 5B). Previously we found that APL cells were relatively

sensitive to ATO-induced apoptosis due to lower levels of GSH than that in other types of AML cells

(38). We found that the sensitivity to ATO in FLT3-ITD cells was also partially defined by the lower

level of GSH (Fig. 1A, 1B). Forced expression of FLT3-ITD reduced the levels of GSH and sensitized

32D cells to ATO-induced apoptosis (Fig. 1C, 1D). We found that, by decreasing the level of HDAC6

protein, ATO inhibited HDAC6 activity (Fig. 2A), led to increased acetylation of HSP90, and

dissociated from FLT3-ITD (Fig. 2E). This is a new mechanism of FLT3-ITD degradation by ATO in

which by interrupting the interaction between FLT3-ITD and HSP90. The sensitivity of FLT3-ITD to

ATO-induced degradation depends on the lower levels of GSH.

Mcl-1 is the most critical anti-apoptotic protein for acute myeloid leukemia survival (52).

FLT3-ITD has been shown to up-regulate Mcl-1 (53) while decreasing the pro-apoptotic BH3-only

protein Bim (54). Bim is crucial for apoptosis induction because of its ability to bind all anti-apoptotic

Bcl-2 family members (55) and to directly activate Bax (56). Both Mcl-1 and Bim are being

considered as targets in leukemia therapy (57-58). Both Mcl-1 and Bim are regulated by GSK3. Bim

is stabilized while Mcl-1 is destabilized by phosphorylation with GSK3. The activity of GSK3 is

also controlled by phosphorylation; the phosphorylated GSK3 is the inactive form. We found that in

ATO treated MOLM13 cells phosphorylated GSK-3 levels were elevated while Bim levels were

reduced even with decreased levels of Mcl-1 (Fig. 3A). These data suggest that ATO reduces Mcl-1

through a GSK3β-independent mechanism and that GSK3 inactivation may play a negative role in

ATO induced apoptosis probably due to destabilization of Bim, a scenario that needs to be overcome.




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Among the inhibitors of FLT3-ITD kinase, Sorafenib was reported to inhibit Mcl-1 and upregulate

Bim in AML cells (48). We found that in FLT3-ITD AML cells Sorafenib, at a low concentration (50

nM), decreased the levels of Mcl-1 and phosphorylated GSK3 while increasing the levels of Bim (Fig.

3B). When combined with ATO, Sorafenib was able to block the ATO-induced GSK3 phosphorylation,

further reduced Mcl-1 level and increased Bim level (Fig. 4C), leading to activation of both Bak and

Bax (Fig, 4D). We found that activation of GSK3 (de-phosphorylation) by Sorefenib was associated

with inhibition of FLT3-ITD activity (Fig. 3B). Inhibition of FLT3-ITD by a specific inhibitor AC220

(Supp. Fig 2A), also enhances ATO-induced apoptosis in MOLM13 cells (Sup. Fig. 2B). GSK3

inhibitor SB216763 significantly attenuated ATO/Sorafenib-induced apoptosis and Bax/Bak activation

(Fig. 4E and Sup. Fig. 3), suggesting that a GSK3 to Bax/Bak activation cascade contributes to the

apoptosis induction by these drugs combination (Fig. 4F). ATO in combination with Sorafenib at lower

concentrations selectively induced apoptosis only in FLT3-ITD AML cells, but not in wtFLT3 THP-1

cells (Fig. 2B), suggesting that ATO and Sorafenib combination would most likely have limited toxicity

in normal tissues. However, the effect of this combination on wtFLT3 patients samples and normal

CD34+ cells should be tested and compared in the future studies. The relative selectivity of this

combination depends on the presence of FLT3-ITD and the lower levels of intracellular GSH, which is

regulated by FLT3-ITD (Fig. 1). Therefore, the presence of FLT3-ITD not only provides a target for

Sorafenib but also creates a sensitive condition for effective ATO treatment. The combination of ATO

and Sorafenib could be translated into a successful combination therapy for FLT3-ITD AML patients.

Acknowledgments:

This work was partly supported by The Samuel Waxman Cancer Research Foundation to Y. Jing. We

thank Dr. Liliana Ossowski (Icahn School of Medicine at Mount Sinai) for editing of this manuscript.




13

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17

Figure legends:

Figure 1. FLT3-ITD AML cells contain lower levels of GSH and are more sensitive to ATO-

induced apoptosis than wild type FLT3 AML cells. A, Comparison of intracellular GSH levels of

MV4-11 (FLT3-ITD), MOLM13 (FLT3-ITD) and THP-1 (wild type FLT3) cells. ** p < 0.01,

comparing to THP-1 cells. B, Apoptosis induced by ATO in MOLM13, MV4-11 and THP-1 cells.

Cells treated with ATO for 24 h were examined for apoptosis using Annexin V staining and FACS. C,

Intracellular GSH levels of 32D cells (parental) or transfected with FLT3-ITD. D, ATO-induced

apoptosis in 32D cells (parental) or transfected with FLT3-ITD. Cells treated with ATO for 24 h were

stained with Annexin V and analyzed by FACS for apoptosis. ** p< 0.01 (32D/FLT3-ITD compared to

parental 32D cells).

Figure 2. ATO induced degradation of FLT3-ITD, but not wtFLT3, associated with lower levels

of GSH and inhibition of HDAC6. A, MOLM13 cells were treated with ATO at the indicated

concentrations for 24 h and the protein levels of FLT3-ITD, HDAC6, Ac--tubulin were determined by

Western blot analysis. B, 32D/FLT3-ITD cells were treated with ATO for 24 h and FLT3-ITD and

phosphorylated FLT3-ITD were measured. C, THP-1 cells were treated with ATO at the indicated

concentrations for 24 h and the levels of FLT3 were determined. D, THP-1 cells were pretreated with or

without 200 μM of BSO for 4 h and then with 1 μM of ATO for 24 h. The levels of FLT3 and cleaved

PARP were determined. E, The interaction of HSP90 with FLT3-ITD (left panel) and acetylated HSP90

(right panel) were determined in MOLM13 cells treated with ATO for 24 h using immunoprecipitation

and Western blot analysis. F, MOLM13 cells were pretreated with 1 M of ATO for 16 h then in

combination with 50 nM of bortezomib (Bor) or 5M of MG132 for 8 h and the levels of FLT3-ITD

and autophagy marker LC3 were determined.

Figure 3. Different effects of ATO and Sorafenib (Sor) on the levels of Mcl-1, BimEL and

phosphorylated GSK3β in MOLM13 cells. MOML13 cells were treated with ATO (A) or Sorafenib

(B) at the indicated concentrations for 24 h. The protein levels of the antiapoptotic proteins, ERK and

Akt and their downstream products were determined using Western blot analysis.

Figure 4. Synergistic apoptosis induction by ATO in combination with Sorafenib in MOLM13

cells linked to activation of Bak, Bax and GSK3. A, Apoptotic cells in MOLM13 cells treated for




18

24 h with Sorafenib (Sor) alone at the indicated concentrations or combined with 0.5M or 1μM of

ATO. B, The median dose effect was analyzed using CompouSyn software to determine synergy. CI

values of less than 1.0 (horizontal line) corresponds to a synergistic interaction. Fa on the x-axis denotes

the fraction affected. C, Western blot analyses of FLT3-ITD, Mcl-1 and Bim, phosphorylated GSK3

and PARP cleavage in MOLM13 cells treated for 24h with Sorafenib, ATO or their combination. D,

Activated Bak and Bax in MOLM13 cells treated with ATO, Sorafenib, and their combination for 24 h

were detected by immunoprecipitation and Western blot analysis. E, Effect of GSK3 inhibitor

SB216763 on the levels of activated Bax and Bak in MOLM13 cells treated with ATO/Sorafenib for

24 h. MOLM13 cells were pretreated with SB216763 for 4 h and then with ATO/Sorafenib at the

indicated concentrations for 24 h. The activated Bak and Bax were determined by immunoprecipation

and Western blot analysis. F, The potential working model of Sorafenib in combination with ATO to

enhance apoptosis induction in FLT3-ITD AML cells.

Figure 5. Sorafenib/ATO combination enhances apoptosis in primary FLT3-ITD AML cells. A,

Primary FLT3-ITD leukemia cells from 3-AML patients were treated for 24 h with 5 µM of Sorafenib, 1

or 2 µM of ATO or their combination. Apoptotic cells were detected with Annexin V-FITC using flow

cytometry. ** p<0.01 (combination treated group compared to single agent treatment). B, Western

blot analysis of FLT3-ITD, Mcl-1, Akt, and GAPDH in samples 1 and 2.

Figure 6. Sorafenib/ATO combination significantly inhibit growth of MOLM13 cells in NSG mice.

A, Tumor weights of MOLM13 cells grown in mice inoculated s.c. and treated daily for 8 days with

PBS, Sorafenib 40 mg/kg, ATO 2.5 mg/kg and 5 mg/kg as well as the combinations. * p<0.05;

**p<0.01; ***p<0.001 compared to control group. ##, p<0.01 compared to single agent treatment. B &

C, Survival of mice inoculated with MOLM13 cells through the tail vein and treated with Sorafenib (20

mg/kg), ATO (5 mg/kg) and the combination and kept until the mice decreased. Kaplan-Meyer survival

plot (B) and the average survival times of each group and an increase in life span (ILS) were calculated

(C). ##p<0.01 compared to Control group; *p<0.05 compared to Sorafenib alone group.




0

5

10

15

20

25

30

35

40

45

50

0 1 2

An

nexin

V-p

osit

ive c

ells (

%)

ATO Concetration (μM)

32D

32D/FLT3-ITD

0

5

10

15

20

25

30

35

THP-1 MV-4-11 MOLM13

GS

H c

on

ten

t

(nm

ol/

10

6 c

ells)

**

**

B A

C D

**

**

Fig. 1

0

10

20

30

40

50

60

70

80

90

0 1 2 4

An

nexin

V-p

osit

ive c

ells

(%)

ATO Concentration (μM)

THP-1

MV-4-11

MOLM13

**

0

5

10

15

20

25

32D 32D/FLT3-ITD

GS

H C

on

ten

ts

(nm

ol/

10

6 c

ells)




Fig. 2

ATO 1μM ̶ + ̶ +

BSO

FLT3

D

B

FLT3-ITD

p-FLT3-ITD

32D /FLT3-ITD

ATO (mM) 0 0.5 1 2

GAPDH

GAPDH

p-FLT3

PARP

C THP-1

ATO (mM) 0 0.5 1 2

FLT3

GAPDH

p-FLT3

ATO (mM) 0 0.5 1 2

MOLM13

FLT3-ITD

Ac-α-Tubulin

HDAC6

GAPDH

A

ATO (mM) 0 0.5 1 2

IP: FLT3

HSP90 IB:

Input

FLT3-ITD

HSP90

E

F

LC3

Actin

FLT3-ITD

ATO 1μM ─ + ─ +

Bor 50 nM ─ ─ + +

p-FLT3-ITD

ATO 1μM ─ + ─ +

MG132 5 μ M ─ ─ + +

FLT3-ITD

Actin

LC3

THP-1

IP: HSP90

HSP90 Input

IB: Ac-Lysine

ATO (mM) 0 0.5 1 2

HSP90 IB:

FLT3-ITD IB:




Fig. 3

p-ERK(1/2)

Mcl-1

p-S6

PARP

p-GSK3β

Bcl-2

BimEL

Akt

GAPDH

ATO (mM) 0 0.5 1 2 3 4

A

FLT3-ITD

p-FlT3-ITD

ERK(1/2)

p-Akt

GSK3β

S6

Sor (nM) 0 25 50 100 200

FLT3-ITD

p-GSK3β

PARP

p-S6

Mcl-1

Bcl-2

BimEL

p-ERK1/2

GAPDH

B

p-FlT3-ITD

ERK(1/2)

p-Akt

Akt

GSK3β

S6




A

Fig. 4

B

ATO 1 μM

IB:Bak poly

IP: Bak(Ab-1)

Sorafenib 25 nM - - + + IgG

- + - +

ATO 1 μM

IB:Bax poly

IP: Bax(6A7)

Sorafenib 25 nM - - + + IgG

- + - +

D

IB:Bak poly

IP: Bak(Ab-1)

- - + +

- - + +

ATO 1 μM

Sorafenib 25 nM

SB216763 5 μM - + - + IgG

IB:Bax poly

IP: Bax(6A7)

- - + + - - + +

ATO 1 μM

Sorafenib 25 nM

SB216763 5 μM - + - + IgG

E

C

FLT3-ITD

Sor 25 nM ̶ ̶ + +

ATO 1 μM ̶ + ̶ +

PARP

Mcl-1

BimEL

GAPDH

p-GSK3β

Bak

Bax

p-FLT3

GSK3β

0

20

40

60

80

100

0 25 50 100

Sor Concentration (nM)

Ap

op

toti

c ce

lls

(%)

Sor alone

plus ATO 0.5μM

plus ATO 1μM

Bax/Bak

Sorafenib ATO FLT3-ITD

GSK3b

Apoptosis

F




B

0

10

20

30

40

50

60

70

80

An

nex

in V

-posi

tive

cell

s (%

)

AML#1

AML#2

AML#3

16.6%

24.5%

29.1%

54.7%

36.2%

66.6%

**

**

**

**

ATO (μM) — 1 — 1 2 2

Sor (μM) — — 5 5 — 5

AML#1 AML#2

FLT3-ITD

Mcl-1

Akt

GAPDH

ATO (μM) 0 1 2 0 1 2

A

Fig. 5 on June 18, 2020. © 2018 American Association for Cancer Research. mct.aacrjournals.org Downloaded from



0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Tu

mo

r W

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t (g

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***

*

##

***

##

** **

ATO (mg/kg) 0 0 2.5 2.5 5 5

Sor (mg/kg) 0 40 0 40 0 40

A B

Treatment No. of mice Survival \Time (d) ILS (%)

Control 5 18.2±1.8 0

ATO 5 21.8±0.8 8.8

Sorafenib 5 35.4±0.9## 94.5

ATO+Sorafenib 5 47.8±1.8## ⃰ 162.6

C

Fig. 6

D a y s

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Published OnlineFirst June 29, 2018.Mol Cancer Ther Rui Wang, Ying Li, Ping Gong, et al. FLT3-ITD acute myeloid leukemia cellsArsenic trioxide and sorafenib induce synthetic lethality of

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