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R.S. Mali et al.
Figure 1 (previous page). Venetoclax combined with quizartinib prolongs survival and reduces tumor burden in FLT3-ITD+ xenograft models. NOD/SCID/IL-2Rγnull (NSG) mice were engrafted with luciferase expressing MV4;11 or Molm13 cells and leukemic cell engraftment was confirmed by bioluminescence imaging (BLI) and treatment began 14 days post-inoculation for the MV4;11 model and 7 days post-inoculation in the Molm13 model. Mice were treated orally with 100 mg/kg vene- toclax, 2.5 mg/kg or 5 mg/kg quizartinib, or the combination once daily for 21 continuous days and overall survival and disease burden was assessed. (A) Kaplan- Meier survival curve for MV4;11-engrafted mice. N=8-10 animals/group and median survival and statistical significance were determined by log-rank test: *P<0.0001 for venetoclax + 2.5 mg/kg quizartinib vs. 2.5 mg/kg quizartinib; **P<0.0001 for venetoclax + 5 mg/kg quizartinib vs. 5 mg/kg quizartinib. (B) Representative BLI for MV4;11-engrafted mice for each group at indicated time point. (C) Quantitation of the BLI signal from MV4;11-engrafted mice in each group at indicated time post-inoculation: *P<0.0001, P=0.0002, P=0.001 and P=0.0004 for venetoclax + 2.5 mg/kg quizartinib vs. 2.5 mg/kg quizartinib for week 3, 4, 5 and 7, respectively, by unpaired t-test for each time point; **P=0.0004, P=0.0247, P<0.0001 and P=0.0001 for venetoclax + 5 mg/kg quizartinib vs. 5 mg/kg quizartinib for week 3, 4, 7 and 8, respectively, by unpaired t-test for each time point. (D) Kaplan-Meier curve for Molm13-engrafted mice. N=10 animals/group and survival and statistical significance were determined by log-rank test: *P<0.0001 for venetoclax + 2.5 mg/kg quizartinib vs. 2.5 mg/kg quizartinib; **P<0.0001 for venetoclax + 5 mg/kg quizartinib vs. 5 mg/kg quizartinib; and ***P<0.0001 for venetoclax + 5 mg/kg quizartinib vs. venetoclax + 2.5 mg/kg quizartinib. (E) Representative BLI for Molm13-engrafted mice at indicated time point. (F) Quantitation of BLI signal from Molm13-engrafted mice at indicated time point post-inoc- ulation: *P<0.0001, P<0.0001 and P=0.0086 for venetoclax + 2.5 mg/kg quizartinib vs. 2.5 mg/kg quizartinib for week 2, 3 and 4, respectively, by unpaired t test for each timepoint; **P<0.0001 for venetoclax + 5 mg/kg quizartinib vs. 5 mg/kg quizartinib for week 2, 3 and 4 by unpaired t-test for each timepoint; and ***P=0.0129 for venetoclax + 5 mg/kg quizartinib vs. venetoclax + 2.5 mg/kg quizartinib at week 5 by unpaired t-test.
proliferation, differentiation, and survival. Notably, FLT3- ITD also activates STAT5, a distinguishing feature from FLT3-TKD and ligand-stimulated wild-type (WT) FLT3.4,5
Intrinsic apoptosis regulates survival through balancing
anti- and pro-apoptotic proteins. The BCL-2 family of
anti-apoptotic proteins includes B-cell lymphoma 2 (BCL-
2), B-cell lymphoma-extra-large (BCL-XL) and myeloid cell
leukemia 1 (MCL-1) that bind and neutralize pro-apoptot-
ic BCL-2 homology 3 (BH3)-only proteins and pro-apop-
totic effector proteins, BCL-2-associated X protein (BAX)
and BCL-2 antagonist/killer (BAK),6 preventing induction
of apoptosis. The development of BH3 mimetic com-
pounds, including venetoclax (ABT-199/GDC-0199), that
specifically target individual BCL-2 family members has
helped clarify dependence of cancer cells on BCL-2,7 BCL-
X 8,9 and/or MCL-110,11 and also revealed mechanisms of L
venetoclax resistance, including upregulation of BCL-XL and/or MCL-1.12
Venetoclax, a highly potent, specific inhibitor of BCL-2, has preclinical and clinical activity across a range of hema- tologic malignancies.13-17 In AML, while venetoclax has limited monotherapy activity,14 venetoclax combined with low-dose cytarabine (LDAC) or hypomethylating agents (HMA) has broad activity across mutation subsets.18,19 In the venetoclax monotherapy trial,14 new FLT3-ITD muta- tions emerged at progression in 4 of 15 patients with ini- tial venetoclax response and 3 of 3 patients with baseline FLT3-ITD mutations showed no measurable reduction in bone marrow (BM) blasts.20 Initial molecular analysis of samples from the venetoclax combination trials revealed that relapse may also be associated with FLT3-ITD muta- tions.21 In 25 patients evaluated at relapse, FLT3-ITD muta- tions expanded in three patients treated with venetoclax plus HMA and new FLT3-ITD mutations were detected in two patients treated with venetoclax plus LDAC. While patient numbers reported in these studies are low, the association of FLT3-ITD mutations with primary resist- ance and emergence of FLT3-ITD mutations at relapse suggests that mutant FLT3 may influence sensitivity to venetoclax and warrants further investigation.
Resistance to apoptosis can be achieved through onco- genic signaling or transcriptional regulation that alter expression of apoptotic proteins. RAS-MAPK, PI3K and the Janus kinase (JAK)-STAT pathways can all regulate anti- and pro-apoptotic proteins.22-24 As such, STAT5, a known tran- scriptional regulator of BCL-XL and BCL-2,25 can also regu- late MCL-1 expression in FLT3-ITD+ cells.26 Given that FLT3-ITD regulates multiple survival pathways and is linked to increased BCL-X 27-30 and MCL-126,29 expression,
scientific rationale to investigate BH3 mimetics combined with FLT3-ITD inhibition. Ma et al. described combination activity of venetoclax and the FLT3 inhibitors midostaurin and gilteritinib in FLT3-ITD+ AML models.31 However, direct comparison of BCL-2, BCL-XL and MCL-1 inhibition in reducing survival when combined with FLT3-ITD inhibi- tion has not been investigated, nor has the role of individual signaling pathways downstream of FLT3-ITD in regulating BCL-2 family members. Therefore, we aimed to thorough- ly investigate the combined efficacy of FLT3-ITD and BCL- 2 family inhibition using potent and selective pharmacolog- ic inhibitors in pre-clinical models of FLT3-ITD+ AML. We describe herein the superiority of targeting BCL-2, rather than BCL-XL or MCL-1, in combination with FLT3 inhibi- tion in FLT3-ITD+ AML. Given that venetoclax is presently being tested in AML clinical trials, our preclinical data pro- vides a strong mechanistic rational for further evaluation of venetoclax combinations with FLT3-ITD inhibitors for the treatment of FLT3-ITD+ patients.
Methods
Orthotopic cell line xenograft models
Cell line xenograft studies were approved by Genentech's Institutional Animal Care and Use Committee (IACUC) and adhere to the Eighth Edition of the Guide for the Care and Use of Laboratory Animals (NRC 2011). NOD/SCID/IL-2Rγnull (NSG) mice (Jackson Laboratory, Bar Harbor, ME) were housed in auto- claved individually ventilated cages. Eight to 10 week old mice were pre-conditioned with busulfan (20 mg/kg; Sigma) by intraperitoneal administration 24hours prior to cell line inocula- tion. Luciferase-positive AML cell lines (2x106 cells) were suspend- ed in Hanks' balanced salt solution (HBSS) and injected via tail vein. Engraftment of leukemic cells and disease burden was deter- mined by bioluminescence imaging (BLI). Equally engrafted mice were grouped out based on BLI at 7 (Molm13) or 14 days (MV4;11) post inoculation and treated as described for 21 contin- uous days. Animals were monitored for signs of disease progres- sion and euthanized at first measurement of greater than 20% weight loss or when reaching any humane endpoint.
Primary patient-derived xenograft models
Primary patient samples were collected and utilized at the M.D. Anderson Cancer Center (MDACC). All patients gave informed consent in accord with the Declaration of Helsinki under Institutional Review Board-approved protocols. Studies were approved by the MDACC IACUC and adhere to the Eighth Edition of the Guide for the Care and Use of Laboratory Animals (NRC 2011). Seven week old NSG mice were pre-conditioned by
L
which can promote venetoclax resistance, there is strong
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haematologica | 2021; 106(4)