Supplementary Materials for

Preventing chemotherapy-induced myelosuppression by repurposing

the FLT3 inhibitor quizartinib

Samuel J. Taylor, Johanna M. Duyvestyn, Samantha A. Dagger, Emma J. Dishington,

Catherine A. Rinaldi, Oliver M. Dovey, George S. Vassiliou,

Carolyn S. Grove, Wallace Y. Langdon*

*Corresponding author. Email: wally.langdon@uwa.edu.au

Published 9 August 2017, Sci. Transl. Med. 9, eaam8060 (2017)

DOI: 10.1126/scitranslmed.aam8060

This PDF file includes:

Materials and Methods

Fig. S1. Hematopoietic progenitors and stem cells are markedly depleted after a

single intraperitoneal injection of 5-FU.

Fig. S2. Daily dosing with quizartinib for 4 days markedly reduces the number of

hematopoietic progenitors.

Fig. S3. Optimizing quizartinib dosing protects HSCs and progenitor cells from 5-

FU–induced cytotoxicity.

Fig. S4. Imatinib does not induce quiescence of LK or LSK cells nor provide

protection from 5-FU cytotoxicity.

Fig. S5. Crenolanib does not induce quiescence of MPPs nor provide protection

from 5-FU cytotoxicity.

Fig. S6. A quizartinib dose 10 days, but not 7 days, after 5-FU is effective in

inducing quiescence in LSK cells.

Fig. S7. Quizartinib does not induce quiescence of NPM1c/NrasG12D AML cells.

Fig. S8. Analysis of blood from NPM1c/NrasG12D mice shows the development of

AML in mice treated with 10-day cycles of quizartinib priming followed by 5-FU.

www.sciencetranslationalmedicine.org/cgi/content/full/9/402/eaam8060/DC1

Materials and Methods

Mice and transplantation experiments

Male C57BL/6JArc mice and B6.SJL-PtprcaPep3b/BoyJArc (i.e. B6.CD45.1) congenic mice aged 8-

10 weeks were purchased from the Animal Resources Centre (Canning Vale, Western Australia).

The generation of FLT3ITD/ITD mice, which were subsequently found to carry a germline F692L

mutation in FLT3, has been previously described (34, 35). To generate mice carrying both FLT3-

ITD(F692L) and NPM1c mutations, we crossed constitutive FLT3ITD/+(F692L) mice with conditional

NPM1flox-cA mice to generate double heterozygous mice, then crossed to Mx1-Cre transgenic mice to

induce recombination of NPM1flox-cA in HSCs cells (40). The FLT3ITD/+(F692L);NPM1flox-cA;Mx1-

Cre+ mice (hereafter referred to as FLT3-ITD(F692L)/NPM1c mice) rapidly develop AML without

the induction of Cre due to “leakiness” of Cre expression in 2-4% of HSCs/progenitor cells (40). To

generate NPM1flox-cA/+; Mx1-Cre; NrasLSL-G12D/+ mice, we crossed Mx1-Cre; NPM1flox-cA/+ mice with

NrasLSL-G12D mice. The generation and genotyping of NrasLSL-G12D/+ mice have previously been

described (44). To activate the conditional alleles (NPM1cA and NrasG12D) in Mx1-Cre; NPM1flox-cA/+;

NrasLSL-G12D/+ mice, Mx1-Cre was induced by i.p. administration of 200 mg of pIpC 5 times over a

10-day period. These mice are referred to as NPM1c/NrasG12D. AML from FLT3-

ITD(F692L)/Npm1c and NPM1c/NrasG12D mice was transferred to non-irradiated B6.CD45.1 mice

by tail vein injection of up to 2 x106 spleen cells and 5 x105 bone marrow cells from mice that

succumbed to AML. Reconstituted FLT3-ITD(F692L) mice and chimeric WT/FLT3-ITD(F692L)

mice were established by repopulating lethally irradiated (2x 5.5Gy) B6.CD45.1 mice with FLT3-

ITD(F692L) (CD45.2) spleen cells, or a mixture of B6.CD45.1 bone marrow cells and FLT3-

ITD(F692L) (CD45.2) spleen cells, respectively. The reconstituted mice were given at least 6 weeks

to recover after transplantation before analysis commenced.

Mouse experiments were approved and performed in accordance with the guidelines and regulations

of the Animal Ethics Committee at the University of Western Australia (approvals 100/1169 and

100/1413). The University of Western Australia is recognized by the NIH Office of Laboratory

Animal Welfare (#A536-01), and the University of Western Australia Animal Care Services is

accredited by the Association for Assessment and Accreditation of Laboratory Animal Care

(AAALAC) and the National Health and Medical Research Council for compliant animal breeding,

research, and teaching services. All mice for this study were housed under pathogen-free conditions

in microisolator cages at the animal facilities of the University of Western Australia.

Quizartinib, imatinib, and chemotherapeutic drugs

Quizartinib (AC220 hydrochloride salt) was provided by Daiichi-Sankyo Corporation. Mice were

dosed with 10, 30, or 60 mg/kg of quizartinib or vehicle (5% 2-hydroxypropyl-β-cyclodextrin,

Sigma-Aldrich) by oral gavage using 20-gauge, 1.5-inch feeding needles (Braintree Scientific Inc.).

Crenolanib from MedChem Express was prepared in 5% glycerol formal (Sigma-Aldrich). It was

delivered by intra-peritoneal (i.p.) injection at 15 mg/kg. Imatinib (LC laboratories) was prepared in

50% sucrose (to lessen the bitter taste), and mice were dosed by oral gavage with 100 mg/kg or

vehicle once or twice daily. To induce myelosuppression, 5-FU (Sansoz Pty Ltd.) was administered

as a single i.p. injection of 150 mg/kg. Gemcitabine (Hospira Pty Ltd.) was administered by three i.p.

injections of 240 mg/kg over 18 hours. Induction chemotherapy to treat AML consisted of a

combination of cytarabine (Ara-C, Pfizer Australia Pty Ltd.) and doxorubicin (Dox, doxorubicin

HCL, Pfizer Australia Pty Ltd.). Induction therapy involved i.p. delivery of Ara-C at 50 mg/kg and

intravenous delivery of Dox at 1.5 mg/kg for 3 days, followed by Ara-C alone at 50 mg/kg on days 4

and 5 (5 + 3 therapy). All chemotherapeutic drugs were supplied by the Sir Charles Gairdner

Hospital Pharmacy and are registered with the Australian Therapeutics Goods Administration. The

drugs were supplied in the manufacturers’ original containers and were stored within the Pharmacy

Department in closely monitored appropriate conditions before provision for this study.

Analysis of peripheral blood and bone marrow cells

Blood was collected from the tail vein, and differential cell counts determined using a Hemavet

HV950FS blood analyzer (Drew Scientific). Blood smears were examined with an Olympus BX60

microscope, and the images captured using an Olympus DP22 Camera and cellSens Entry Software.

Bone marrow cells were taken from tibias and femurs of each mouse. Red blood cells in the blood

and bone marrow were lysed with an NH4Cl lysis solution before the nucleated hematopoietic cells

were analyzed by flow cytometry (FACS Canto, BD Biosciences) using the monoclonal antibodies

described below. Data were collected using FACSDiva version 6 software (BD Biosciences) and

analyzed using FlowJo 9.3.1 software (Tree Star, Inc.).

Flow cytometry antibodies and procedures

All antibodies were from BD Biosciences, except where noted otherwise. We used antibodies against

CD3-biotin, CD11b-PE-Cy7 and -biotin, CD19-PE, CD48-FITC, CD45.1-FITC, CD45.2-APC, Gr-

1-biotin, and TER119-biotinCD150-PE-Cy7 (Biolegend), c-Kit-APC and -PE (eBioscience), Sca-1-

PE-Cy7 (eBioscience), B220-biotin, FLT3-PE (eBioscience), . Cells incubated with biotinylated

antibodies were treated with streptavidin conjugated with APC-Cy7 (BD Biosciences). Multipotent

progenitors (MPPs) were defined as FLT3+ LSK cells or CD48+ CD150- LSK cells, and short-term

and long-term hematopoietic cells (ST-, LT-HSCs) were defined as CD48- CD150- LSK and CD48-

CD150+ LSK cells, respectively. Ki-67 expression was determined by examining bone marrow cells

fixed with Cytofix/Cytoperm (BD Biosciences), permeabilized with Perm/Wash buffer (BD

Biosciences), and stained with either anti-Ki-67-FITC antibodies or FITC-labeled isotype control

antibodies (BD Biosciences). Cell cycle analysis was performed by permeabilizing bone marrow

cells with a solution of phosphate buffered saline containing 0.1% saponin (Sigma-Aldrich, S7900),

5% newborn calf serum, and 250 mg/ml sodium azide. DNA was stained by adding 7-AAD (BD

Biosciences) to a final concentration of 2.5 µg/ml. Cells were left at 4˚C for two hours and room

temperature for 30 min before analysis by flow cytometry. For Ki-67 and cell cycle analyses, the

bone marrow cells were first stained with fluorescence-tagged antibodies described above in order to

identify HSCs and progenitor populations, before fixing and permeabilization.

Supplementary Figures:

Fig. S1. Hematopoietic progenitors and stem cells are markedly depleted after a single

intraperitoneal injection of 5-FU. B6 mice were left untreated or injected with 150 mg/kg of 5-FU,

and bone marrow cells were examined by flow cytometry after 1 or 2 days. (A) Representative c-Kit

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Fig. S1. Hematopoietic progenitors and stem cells are markedly depleted following a single intraperitoneal

injection of 5-FU. B6 mice were left untreated or injected with 150 mg/kg of 5-FU, and bone marrow cells were

examined by flow cytometry after 1 or 2 days. (A) Representative c-Kit and Sca-1 flow cytometry profiles of

lineage negative bone marrow cells (upper panels), and CD48 and CD150 profiles of LSK cells (lower panels), from

an untreated mouse, and mice examined 1 or 2 days after 5-FU. (B) Gating strategies for lineage negative and LSK

bone marrow cells. (C) Numbers of LK cells, (D) LSK cells, (E) MPPs, (F) ST-HSCs and (G) LT-HSCs after 1 or

2 days of 5-FU exposure compared to untreated mice. The numbers represent cells isolated from 2 femurs and 2

tibias per mouse. (H) The proportions of MPPs, ST-HSCs and LT-HSCs from untreated mice and mice 1 or 2 days

after the 5-FU injection. The greater susceptibility of MPPs to 5-FU cytotoxicity compared to LT-HSCs is clearly

evident by day 2. Data are from 3 mice at each time-point for each group, and the graphs are expressed as mean

± SEM. All statistics were calculated using Student’s t-tests, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

and Sca-1 flow cytometry profiles of lineage negative bone marrow cells (upper panels), and CD48

and CD150 profiles of LSK cells (lower panels) from an untreated mouse and mice examined 1 or 2

days after 5-FU. (B) Gating strategies for lineage negative and LSK bone marrow cells. (C) Numbers

of LK cells, (D) LSK cells, (E) MPPs, (F) ST-HSCs, and (G) LT-HSCs after 1 or 2 days of 5-FU

exposure compared to untreated mice. The numbers represent cells isolated from 2 femurs and 2

tibias per mouse. (H) The proportions of MPPs, ST-HSCs, and LT-HSCs from untreated mice and

mice 1 or 2 days after the 5-FU injection. Data are from 3 mice at each time point for each group and

are presented as mean ± SEM. All statistics were calculated using Student’s t-tests, *p<0.05,

**p<0.01, ***p<0.001, ****p<0.0001.

Fig. S2. Daily dosing with quizartinib for 4 days markedly reduces the number of

hematopoietic progenitors. B6 mice were dosed daily for 1, 2, or 4 days with vehicle or 10 mg/kg

of quizartinib, and bone marrow cells were analyzed by flow cytometry 24 hours after dosing. (A)

Numbers of LK cells, (B) MPPs defined as FLT3+ LSK cells, (C) MPPs defined as CD48+ CD150-

LSK cells, (D) ST-HSCs, and (E) LT-HSCs. Data are from 3 mice at each time point for each group

and are presented as mean ± SEM. All statistics were calculated using Student’s t-tests, *p<0.05,

**p<0.01, ***p<0.001.

B CA

ED

Fig. S2. Daily dosing with quizartinib for 4 days significantly reduces the number of hematopoietic pro-

genitors. B6 mice were dosed daily for 1, 2 or 4 days with vehicle or 10 mg/kg of quizartinib, and bone

marrow cells were analyzed by flow cytometry 24 hours after dosing. (A) Numbers of LK cells, (B) MPPs

defined as FLT3+ LSK cells, (C) MPPs defined as CD48+ CD150- LSK cells, (D) ST-HSCs and (E)

LT-HSCs. MPPs defined as CD48+ CD150- LSK cells were examined since it is possible that the decrease

in the numbers of MPPs defined as FLT3+ LSK cells could be due to a loss of FLT3 expression, rather

than a loss of cells. We found a significant loss of CD48+ CD150- LSK cells after 4 days of quizartinib

dosing, and therefore conclude that loss of FLT3+ LSK cells is predominantly due to cell loss rather than a

loss of FLT3 expression. Data are from 3 mice at each time-point for each group, and the graphs are

expressed as mean ± SEM. All statistics were calculated using Student’s t-tests, *p<0.05, **p<0.01,

***p<0.001.

Fig. S3. Optimizing quizartinib dosing protects HSCs and progenitor cells from 5-FU–induced

cytotoxicity.

(A) Representative flow cytometry profiles of lineage negative bone marrow cells from B6 mice

primed with 10 mg/kg quizartinib 0-24 hours before an injection of 150 mg/kg 5-FU. (B-F) B6 mice

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Fig. S3. Optimizing quizartinib’s protection of hematopoietic stem and progenitor cells (A) Representative flow

cytometry profiles of lineage negative bone marrow from B6 mice primed with 10 mg/kg quizartinib 0-24

hours before an injection of 150 mg/kg 5-FU. (B-F) B6 mice were dosed with vehicle or 10, 30 or 60

mg/kg of quizartinib and injected with 150 mg/kg of 5-FU 6 hours later . Bone marrow was analysed 2 days

after 5-FU by flow cytometry. (B) Numbers of LK cells, (C) LSK cells, (D) MPPs, i.e. CD48+ CD150-

LSK cells, (E) ST-HSCs and (F) LT-HSCs. The numbers represent cells isolated from 2 femurs and 2 tibias

per mouse. Data are from 3 mice for each group, and the graphs are expressed as mean ± SEM. All statis-

tics were calculated using Student’s t-tests, *p<0.05.

were dosed with vehicle or 10, 30, or 60 mg/kg of quizartinib and injected with 150 mg/kg of 5-FU 6

hours later. Bone marrow was analyzed 2 days after 5-FU by flow cytometry. (B) Numbers of LK

cells, (C) LSK cells, (D) MPPs defined as CD48+ CD150- LSK cells, (E) ST-HSCs, and (F) LT-

HSCs. The numbers represent cells isolated from 2 femurs and 2 tibias per mouse. Data are from 3

mice at each time point for each group and are presented as mean ± SEM. All statistics were

calculated using Student’s t-tests, *p<0.05.

Fig. S4. Imatinib does not induce quiescence of LK or LSK cells nor provide protection from 5-

FU cytotoxicity. (A) Mice were dosed twice daily with vehicle or 100 mg/kg of imatinib for 2 or 4

days before analysis of BM cells by flow cytometry 24 hours later. The proliferative activity of LK

and LSK cells was determined by the percentage of Ki-67+ cells. The data are from 7 mice at day 2

and 6 mice at day 4, from 2 separate experiments. (B) Representative Ki-67 and forward light scatter

(FSC) flow cytometry profiles of LK and LSK cells after two and four days of dosing with vehicle or

Day 2 Day 4

LK cells LK cells LSK cellsLSK cells

Vehicle Vehicle

Imatinib Imatinib

Ki-67

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imatinib. The gates show percentages of Ki-67 positive and negative cells, which are further divided

by cell size (FSC). (C) Mice were dosed with vehicle or 100 mg/kg of imatinib before an i.p.

injection of 150 mg/kg of 5-FU either 6 or 12 hours later. The numbers of LK and LSK cells in the

bone marrow from two tibias and femurs were determined by flow cytometry two days after

treatment. The data are from 5 mice in each group and from two experiments, and no significant

difference in cell numbers was seen between the treatment groups. The data are presented as mean ±

SEM.

Vehicle Crenolanib

Vehicle Crenolanib Quizartinib

Ki-67

FS

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Quizartinib

FLT3

One Day of Treatment

Two Days of Treatment

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Vehicle + 5-FU Crenolanib + 5-FU Quizartinib + 5-FU

0.31% 0.23% 0.36% 0.33% 10.1% 2.13%

Sca-1

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Fig. S5. Crenolanib does not induce quiescence of MPPs nor provide protection from 5-FU

cytotoxicity. (A) Representative Ki-67 vs. FSC flow cytometry profiles of MPPs from B6 mice

injected twice daily for one day with either vehicle or 15 mg/kg of crenolanib. The bone marrow was

analyzed 24 hours later by flow cytometry to quantify Ki-67 in small and large MPPs. (B)

Cumulative data showing a significant increase in Ki-67+ MPPs from crenolanib-treated mice

compared to vehicle-treated mice. N=7 for each group, p<0.01. (C) Representative Ki-67 v FSC flow

cytometry profiles of MPPs from B6 mice injected twice daily for two days with either vehicle or 15

mg/kg of crenolanib, or once daily for two days with 30 mg/kg of quizartinib. The bone marrow was

analyzed 24 hours later. (D) Cumulative data showing a significant increase in Ki-67+ MPPs from

crenolanib-treated mice compared to vehicle-treated mice. N=3 for each group, p<0.0001. (E) FLT3

expression on LSK cells from mice treated for two days with vehicle, crenolanib, or quizartinib and

examined 24 hours after the last treatment. The reduction in the proportion of FLT3+ cells is evident

in the inhibitor-treated mice. (F) Quantification of FLT3 ligand in the serum from mice treated for

two days with vehicle (twice daily), crenolanib (twice daily), or quizartinib (once daily). Serum was

taken 24 hours after the final treatment. N=7 for vehicle and crenolanib groups, and N=3 for

quizartinib group. Graphs are presented as mean ± SEM, and statistics were calculated using

Student’s t-tests. **p<0.01, ****p<0.0001. (G) Representative c-Kit vs. Sca-1 flow cytometry

profiles of lineage negative bone marrow cells from mice injected twice over 12 hours with either

vehicle or 15 mg/kg of crenolanib, or dosed once with 30 mg/kg of quizartinib. All mice were

injected with 150 mg/kg of 5-FU 6 hours after the final treatment, and the bone marrow analyzed 4

days later. (H) Numbers of LK cells, LSK cells, and MPPs from two tibias and femurs. N=4 for

vehicle and quizartinib-treated mice and n=3 for crenolanib-treated mice.

Fig. S6. A quizartinib dose 10 days, but not 7 days, after 5-FU is effective in inducing

quiescence in LSK cells. B6 mice were injected with 150 mg/kg of 5-FU, or left untreated, and 7 or

10 days later were dosed with vehicle or 10 mg/kg of quizartinib. The LSK population was examined

18 hours after quizartinib dosing to identify cells that were (A) Ki-67+ or (B) in S+G2/M phase of the

cell cycle. (C) Serum concentration of FLT3 ligand in mice from each treatment group. The large

increase in FLT3 ligand at day 7 correlates with the loss of quizartinib-induced quiescence. Data are

from 3-5 mice for each group, and are shown as mean ± SEM. All statistics were calculated using

Student’s t-tests, *p<0.05, ****p<0.0001.

A CUntreated 7 Days 10 Days

Ki-67

Days post 5-FU

Untreated

Day 7 post 5-FU

Day 10 post 5-FU

VEHQUIZ

Cell cycle

B

Fig. S6. A quizartinib dose 10 days after 5-FU, but not 7 days after 5-FU, is effective in inducing

quiescence in LSK cells. B6 mice were injected with 150 mg/kg of 5-FU, or left untreated, and 7 or

10 days later were dosed with vehicle, or 10 mg/kg of quizartinib. The LSK population was examined

18 hours after quizartinib dosing to identify cells that were (A) Ki-67+ or (B) in S+G2/M phase of

the cell cycle. (C) Serum levels of FLT3 ligand from mice in each treatment group. The large increase

in FLT3 ligand at day 7 correlates with the loss of quizartinib-induced quiescence. Data are from 3-5

mice for each group, and the graphs are expressed as mean ± SEM. All statistics were calculated using

Student’s t-tests, *p<0.05, ****p<0.0001.

Fig. S7. Quizartinib does not induce quiescence of NPM1c/NrasG12D AML cells. (A)

Representative flow cytometry profiles from WT B6.CD45.1 mice that were transplanted with

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Quizartinib: 2 daysVehicle: 2 days

B Untreated Ara-C + Dox

Fig. S7. Quizartinib does not induce quiescence of NPM1c/NrasG12DAML cells. (A) Representative flow

cytometry profiles from WT B6.CD45.1 mice that were transplanted with NPM1c/NrasG12D AML cells and

dosed for 2 days with either vehicle (left panels) or 30 mg/kg quizartinib (right panels). Displayed are line -

age negative bone marrow cells gated on the LSK population, and then gated on CD45.2+ (AML cells)

and CD45.2- (WT cells). These two populations were examined for Ki-67 expression, highlighting the resist -

ance of the AML cells to quizartinib-induced quiescence. The numbers in each gate are expressed as per-

centages. (B) Blood films from an untreated mouse (left panels), and a mouse treated with cytarabine and

doxorubicin (right panels). Blood from each mouse was collected at the time of death. Scale bar = 50 μ

m (upper panels) and 20 μm (lower panels).

NPM1c/NrasG12D AML cells and dosed for 2 days with either vehicle (left panels) or 30 mg/kg

quizartinib (right panels). Displayed are lineage negative bone marrow cells gated on the LSK

population, and then gated on CD45.2+ (AML cells) and CD45.2- (WT cells). These two populations

were examined for Ki-67 expression. The numbers in each gate are expressed as percentages. (B)

Blood films from an untreated mouse (left panels), and a mouse treated with cytarabine and

doxorubicin (right panels). Blood from each mouse was collected at the time of death. Scale bar = 50

µm (upper panels) and 20 μm (lower panels).

Fig S8. Analysis of blood from NPM1c/NrasG12D mice shows the development of AML in mice

treated with 10-day cycles of quizartinib priming followed by 5-FU. Flow cytometry profiles and

blood films from two cohorts of B6.CD45.1 mice transplanted with NPM1c/NrasG12D AML cells,

and treated by quizartinib priming and 5-FU. The flow cytometry profiles of blood taken at the

indicated times after transplantation show the expression of CD45.1 (WT cells) vs CD45.2 (AML

cells) on CD11b+ gated WBCs. The red arrows indicate each treatment point. The treatment for the

mice in (A) involved four 10-day cycles on days 15, 25, 35, and 45 after transplantation, and this was

repeated on days 82, 92, 102, and 112. (B) Blood films from the surviving 3 mice at day 214 post-

transplantation. The numbers of WBCs from these mice are shown above the images. The treatment

for the mice in (C) involved 6 consecutive 10-day cycles starting on day 15 after transplantation. (D)

Blood films from 3 mice at day 113 post-transplantation. The numbers of WBCs from these mice are

shown above the images. Scale bars = 50 μm.