1. Preventing chemotherapy-induced myelosuppression by repurposing the FLT3 inhibitor quizartinib
by Samuel J. Taylor, Johanna M. Duyvestyn, Samantha A. Dagger, Emma J. Dishington, Catherine A. Rinaldi, Oliver M. Dovey, George S. Vassiliou, Carolyn S. Grove, and Wallace Y. Langdon
Sci Transl Med
Volume 9(402):eaam8060
August 9, 2017
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

2. Fig. 1. Quizartinib induces a rapid and transient quiescence in MPPs.
Quizartinib induces a rapid and transient quiescence in MPPs. (A) B6 mice were dosed once daily over a 16-day time course with either vehicle (VEH) or quizartinib (QUIZ; 10 mg/kg) and analyzed 24 hours after the final dose. FLT3+ LSK cells (MPPs) were analyzed by flow cytometry for the expression of Ki-67 and cell size [forward light scatter (FSC)], and the percentages of cells within each quadrant are shown. Cells in the top right quadrant are large Ki-67+ cells (highly proliferative cells), and the cells in the lower left quadrant are small Ki-67− cells (G0 quiescent cells). (B) Percentages of Ki-67+ MPPs and (C) large Ki-67+ MPPs at each time point. (D) Cell cycle analysis showing the proportion of MPPs in the S + G2-M phases after 1 or 2 days of vehicle or quizartinib dosing. (E) Amount of FLT3 ligand in the serum over the 16-day time course. Data are from three to four 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, and ****P <
Samuel J. Taylor et al., Sci Transl Med 2017;9:eaam8060
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

3. Fig. 2. A single priming dose of quizartinib protects hematopoietic progenitors from 5-FU–induced death.
A single priming dose of quizartinib protects hematopoietic progenitors from 5-FU–induced death. (A) Numbers of MPPs from B6 mice dosed twice as indicated, with either vehicle or quizartinib (10 mg/kg) 24 hours apart, and an intraperitoneal injection of 5-FU (150 mg/kg) that was given at the time of the second dose. Bone marrow cells were analyzed by flow cytometry 2 days later to identify MPP numbers. (B) Experimental scheme of a 24-hour dosing time course before the mice were injected with 5-FU. Quizartinib or vehicle was administered to B6 mice at 0, 2, 6, 12, 18, or 24 hours before receiving an intraperitoneal (ip) injection of 5-FU. Bone marrow was examined 2 days later by flow cytometry. Fold change in the numbers of MPPs (C), ST-HSCs (D), LT-HSCs (E), and LK cells (F) from quizartinib-primed mice over a 24-hour time course compared to vehicle + 5-FU–treated mice, as represented by dashed line. (G) Experimental scheme for B6 mice dosed with vehicle or quizartinib 6 hours before an intraperitoneal injection of 5-FU. The bone marrow was examined at 2, 4, 6, 8, or 10 days after 5-FU treatment. (H) Representative flow cytometry profiles showing the lineage-negative bone marrow cells from either vehicle- or quizartinib-primed mice at the indicated time points. Gates shown identify the LK (left) and LSK (right) populations. Numbers of (I) LK cells, (J) LSK cells, (K) MPPs, (L) ST-HSCs, (M) LT-HSCs, and (N) total nucleated bone marrow (BM) cells over the time course. The blue shading shows the normal range of cell numbers from two femurs and two tibias from untreated age-matched B6 mice. Data are from three to six 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, and ****P <
Samuel J. Taylor et al., Sci Transl Med 2017;9:eaam8060
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

4. Fig. 3. Quizartinib priming before multiple 5-FU treatments enhances survival and blood recovery and prevents weight loss.
Quizartinib priming before multiple 5-FU treatments enhances survival and blood recovery and prevents weight loss. (A) Experimental scheme. B6 mice were primed with vehicle or quizartinib (10 mg/kg) 6 or 12 hours before an intraperitoneal injection of 5-FU (150 mg/kg). This process was repeated every 7 or 10 days, with mouse survival, blood counts, and weights monitored. (B and C) Kaplan-Meier survival plots of B6 mice primed with vehicle or quizartinib 12 hours before a 5-FU injection. The treatment cycles were repeated every 7 days (B) or 10 days (C), and the arrows indicate each treatment. Another cohort of B6 mice was treated with three 10-day cycles of 5-FU and primed with vehicle or quizartinib 6 hours before 5-FU treatment. The mice were bled every 5 days after the last injection to track numbers of (D) WBCs, (E) lymphocytes, (F) neutrophils, (G) RBCs, and (H) platelets. (I) The weights of mice over the time course were also recorded. One vehicle-primed mouse succumbed 8 days after the third 5-FU injection. Data are from five mice in each treatment group, with blood counts expressed as mean ± SEM. All statistics were calculated using Student’s t tests; *P < 0.05 and **P < 0.01.
Samuel J. Taylor et al., Sci Transl Med 2017;9:eaam8060
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

5. Fig. 4. Quizartinib priming protects hematopoietic progenitors and stem cells from gemcitabine-induced death.
Quizartinib priming protects hematopoietic progenitors and stem cells from gemcitabine-induced death. (A) Experimental scheme. B6 mice were primed with vehicle or quizartinib at 0, 6, and 12 hours, and intraperitoneal injections of gemcitabine were administered at 6, 12, and 24 hours. Bone marrow was analyzed 1 and 3 days after the final gemcitabine injection. (B) Total nucleated bone marrow cell numbers and (C) lineage-negative bone marrow cell numbers at 1 and 3 days. Numbers of (D) LK cells and (E) LSK cells from mice at 1 and 3 days after the final gemcitabine injection. (F) Representative flow cytometry profiles from vehicle or quizartinib plus gemcitabine-treated mice at 1 or 3 days after treatment, displaying the expression of c-Kit and Sca-1 on lineage-negative bone marrow cells. Displayed are the gates that identify LK and LSK populations. Numbers of (G) MPPs, (H) ST-HSCs, and (I) LT-HSCs from mice at 1 and 3 days after the final gemcitabine injection. Data are from four 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, and ****P <
Samuel J. Taylor et al., Sci Transl Med 2017;9:eaam8060
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

6. Fig. 5. Hematopoietic progenitors from FLT3-ITD(F692L) mutant mice dosed with quizartinib are not induced into quiescence or protected from 5-FU cytotoxicity.
Hematopoietic progenitors from FLT3-ITD(F692L) mutant mice dosed with quizartinib are not induced into quiescence or protected from 5-FU cytotoxicity. (A) Percentage of Ki-67+ MPPs from FLT3-ITD(F692L)–transplanted mice dosed daily for 1 or 2 days with vehicle or quizartinib (10 mg/kg). n = 5 per group. (B) Numbers of surviving MPP cells from FLT3-ITD(F692L)–transplanted mice primed with quizartinib (10 mg/kg) at 0, 2, 6, 12, or 18 hours before an injection of 5-FU normalized to vehicle-primed mice at each time point, as shown by the dashed line. n = 5 per time point. (C) Representative flow cytometry profiles of CD11b+ cells from the blood of WT:FLT3-ITD(F692L) chimeric mice, showing the percentages of CD45.1 (WT) versus CD45.2 [FLT3-ITD(F692L)] cells. The mice were bled at the indicated times after the first treatment, and the arrows represent the administration of quizartinib (10 mg/kg; 12-hour priming) + 5-FU (150 mg/kg) at days 0 and 10. (D) Proportions of WT CD11b+ blood cells over the time course (n = 5), where the arrows represent the two treatment cycles. PreB, prebleed. (E) Kaplan-Meier survival curves of WT:FLT3-ITD(F692L) chimeric mice primed with vehicle or quizartinib before 5-FU administration (n = 10 and 5, respectively).
Samuel J. Taylor et al., Sci Transl Med 2017;9:eaam8060
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

7. Fig. 6. Quizartinib priming in combination with 5-FU provides an effective treatment for FLT3-ITD(F692L)/NPM1c–driven AML.
Quizartinib priming in combination with 5-FU provides an effective treatment for FLT3-ITD(F692L)/NPM1c–driven AML. (A) Percentage of Ki-67+ MPPs from naturally bred double-mutant FLT3-ITD(F692L)/NPM1c mice dosed for 2 days with vehicle or quizartinib (30 mg/kg). Bone marrow was analyzed 24 hours after the second dose. n = 3 for each group. (B) Kaplan-Meier survival curves of B6 mice transplanted with FLT3-ITD(F692L)/NPM1c AML cells and dosed daily with either vehicle or quizartinib (10 mg/kg). (C) A second cohort of FLT3-ITD(F692L)/NPM1c–transplanted mice was established to investigate the effectiveness of the quizartinib priming plus 5-FU protocol versus induction chemotherapy of Ara-C and Dox. Displayed are pretreatment percentages of CD45.2+ CD11b+ blood cells and total WBC counts for each test group (n = 5). (D) Kaplan-Meier survival curves of FLT3-ITD(F692L)/NPM1c–transplanted mice from the three test groups. (E) WBC counts and (F) spleen weights from untreated and Ara-C + Dox groups at the time of death. (G) Percentages of CD45.2+ CD11b+ WBCs and (H) total WBC counts from individual mice (M1 to M5) treated with quizartinib + 5-FU over the 176-day time course. (I) Flow cytometry profiles displaying CD45.1+ (WT) versus CD45.2+ [FLT3-ITD(F692L)/NPM1c] LSK cells from the bone marrow of the four surviving quizartinib + 5-FU–treated mice culled at day 176.
Samuel J. Taylor et al., Sci Transl Med 2017;9:eaam8060
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

8. Fig. 7. Quizartinib priming in combination with 5-FU provides an effective treatment for NPM1c/NrasG12D-driven AML.
Quizartinib priming in combination with 5-FU provides an effective treatment for NPM1c/NrasG12D-driven AML. (A) Percentages of Ki-67+ cells within the CD45.2+ (AML) and CD45.1+ (WT) LSK populations from NPM1c/NrasG12D-transplanted mice dosed for 2 days with vehicle or quizartinib (30 mg/kg). n = 5 for each group. ***P < (B) A cohort of NPM1c/NrasG12D-transplanted mice was established to investigate the effectiveness of the quizartinib priming plus 5-FU protocol versus standard induction chemotherapy of Ara-C + Dox. Displayed are pretreatment percentages of CD45.2+ CD11b+ blood cells and WBC counts for each test group (n = 5). (C) Kaplan-Meier survival curves of NPM1c/NrasG12D-transplanted mice from the three test groups. (D) WBC counts and (E) spleen weights from untreated and Ara-C + Dox groups at the time of death. (F) Percentages of CD45.2+ CD11b+ WBCs and (G) WBC counts from individual mice (M1 to M5) treated with quizartinib + 5-FU over the time course. (H) A second cohort of NPM1c/NrasG12D-transplanted B6.CD45.1 mice was established to repeat the examination of quizartinib priming plus 5-FU versus induction chemotherapy. Displayed are pretreatment percentages of CD45.2+ CD11b+ blood cells and total WBC counts for each test group (n = 5). (I) Kaplan-Meier survival curves of NPM1c/NrasG12D-transplanted mice from the three test groups. (J) WBC counts and (K) spleen weights from untreated and Ara-C + Dox groups at the time of death. (L) Percentages of CD45.2+ CD11b+ WBCs and (M) WBC counts from individual mice (M1 to M5) treated with quizartinib and 5-FU over the time course.
Samuel J. Taylor et al., Sci Transl Med 2017;9:eaam8060
Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works