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Venetoclax enhances FLT3-ITD inhibition in AML
blocked additional pro-survival nodes. Treatments were well tolerated based on minimal changes in body weight (Online Supplementary Figure S4). These data underscore the need to target all three BCL-2 family members to robustly induce FLT3-ITD+ leukemia cell death in vivo.
Maximum efficacy is demonstrated in vitro when simultaneously inhibiting BCL-2, BCL-XL and MCL-1 in FLT3-ITD+ cell lines
In order to further dissect the combination of venetoclax and quizartinib, survival and signaling pathways were pharmacologically investigated in vitro. FLT3-ITD+ and FLT3-WT (HL60 and OCI-AML3) cell lines were treated in a dose matrix with quizartinib and venetoclax and ana- lyzed by the Bliss independence model34 to determine syn- ergy. Bliss scores added within a cell line are referred to as a Bliss sum and > 100 was considered highly synergistic. Only the FLT3-ITD+ cell lines showed positive Bliss scores, indicating the combination is synergistic in vitro (Figure 5A; Online Supplementary Figure S5A). Confirming the Bliss analysis, co-treatment with low dose quizartinib and venetoclax reduced proliferation and increased apop- tosis in the FLT3-ITD+ cell lines (Figure 5B; Online Supplementary Figure S5B). Venetoclax combined with sorafenib or midostaurin also reduced viability (Online Supplementary Figure S5C) while no combination effect was observed in FLT3-WT cell lines (Online Supplementary Figure S5B-C) indicating FLT3-ITD-specific response. In order to pharmacologically assess the contribution of MCL-1 and BCL-XL in vitro, AMG 176 and A1331852 (BCL-XL specific)9 were utilized in combination with quizartinib. Bliss analysis demonstrated that quizartinib combined with AMG 176 or A1331852 was not synergis- tic in vitro (Figure 5C).
In order to understand the dependence of FLT3-ITD+ cell lines on BCL-2, BCL-XL and MCL-1 in vitro, combina- tions of venetoclax, navitoclax (BCL-2/BCL-XL), A1331852 (BCL-XL) and AMG 176 (MCL-1) were tested. In both cell lines, co-inhibition of BCL-2, BCL-XL and MCL-1 (AMG 176 + navitoclax) was as synergistic as quizartinib plus venetoclax highlighting that all three proteins mediated survival. However, differential responses between cell lines were observed for other combinations. For the Molm13 cell line, venetoclax plus A1331852 (BCL-XL) or AMG 176 (MCL-1) showed comparable or more synergy than quizartinib plus venetoclax, respectively and AMG 176 plus A1331852 (MCL-1 and BCL-XL inhibition) was least synergistic (Figure 5D; Online Supplementary Figure S6A). These data suggest that BCL-2 may play an impor- tant survival role in Molm 13 cells and needs to be co-tar- geted. The combination of navitoclax plus AMG 176 was less effective than venetoclax plus AMG 176, which could be a result of the reduced potency of navitoclax toward BCL-2 compared to venetoclax.9 This is reflected by the combination activity observed at lower doses of veneto- clax (<4 nM) when combined with AMG 176 compared to navitoclax plus AMG 176 (Online Supplementary Figure S6A).
For the MV4;11 cell line, synergy scores were similar for venetoclax plus AMG 176 (MCL-1) and AMG 176 plus A1331852 (MCL-1 and BCL-XL inhibition) indicating that MCL-1 inhibition is synergistic with BCL-2 or BCL-X9 inhibition in vitro (Figure 5D; Online Supplementary Figure S6A). Interestingly, in vitro venetoclax plus AMG 176 and navitoclax plus AMG 176 responses were similar whereas
in vivo navitoclax plus AMG 176 was superior to veneto- clax plus AMG 176 (Figure 4B). Assessment of protein expression in cells grown in vitro versus in vivo revealed increased BCL-XL expression and decreased BCL-2 expres- sion in vivo (Online Supplementary Figure S6B) which could account for the greater dependence on BCL-XL for survival in vivo. Venetoclax plus A1331852 (BCL-XL) was not syner- gistic in vitro (Figure 5D), consistent with minimal navito- clax activity in vivo (Figure 4B). These data further demon- strate the need for simultaneous inhibition of BCL-2, BCL- XL and MCL-1 in order to achieve maximal anti-tumor activity in FLT3-ITD+ models as each of these proteins appear to mediate survival.
BCL-XL and MCL-1 expression is regulated by FLT3-ITD
in vitro
Given that BCL-XL and MCL-1 are described to be downstream of FLT3-ITD, we evaluated the effect of FLT3 inhibition on protein expression. Quizartinib treatment resulted in kinase inhibition only in FLT3-ITD+ cell lines as confirmed by reduced phosphorylation of downstream effectors ERK, AKT and STAT5 (Figure 6A). While protein levels remained unchanged in FLT3-WT cells, quizartinib reduced BCL-XL protein by 20-40% and MCL-1 protein by 60-80%, but did not alter BCL-2 expression in FLT3-ITD+ cell lines (Figure 6B; Online Supplementary Figure S7A). Sorafenib and midostaurin also reduced MCL-1 protein in the FLT3-ITD+ cell lines (Online Supplementary Figure S7B), confirming FLT3-ITD specificity and consistent with reported data.31 A shorter treatment period demonstrated maximum MCL-1 downregulation as early as 8 hours post FLT3-ITD inhibition (Figure 6C; Online Supplementary Figure S7C) reflective of the short half-life for MCL-1 (~2 hours).35 Assessment of gene expression showed minimal decrease in mRNA expression following quizartinib treat- ment (Online Supplementary Figure S7D), suggesting post- transcriptional regulation. Further, proteasome (MG132) but not caspase (Z-VAD-FMK) inhibition rescued loss of MCL-1 protein induced by quizartinib (Online Supplementary Figure S7E). Given the long half-life of BCL-XL (~20 hours36) and toxicity associated with MG132, BCL-XL protein changes were not captured. However, these data support that BCL-XL and MCL-1 are indirectly regulated post-transcriptionally downstream of FLT3-ITD.
FLT3-ITD+ cells become BCL-2 dependent following quizartinib treatment
In order to determine if FLT3-ITD inhibition altered sur- vival dependence, dynamic BH3 profiling was utilized. MV4;11 cells were pre-treated with quizartinib or sorafenib followed by treatment with BH3 domain mimetic peptides and assessed for mitochondrial cytochrome c (Cytc) loss.37 FLT3-ITD inhibition primed cells toward apoptosis as determined by increased Cytc loss when co-treated with low-dose BIM peptide (0.01 mM) compared to vehicle-treated (Figure 7A). FLT3- ITD inhibition increased Cytc loss when co-treated with BAD, MS-1 and FS-1 peptides, suggesting dependence on BCL-2/BCL-XL, MCL-1 and BCL2-related protein A1 (A1) proteins, respectively, with BAD (BCL-2/BCL-XL) peptide showing greatest activity (Figure 7A). Co-treatment with HRK peptide (BCL-XL) demonstrated little activity, while venetoclax combined with FLT3-ITD inhibition resulted in Cytc loss similar to BAD peptide confirming shifted sur- vival dependency to BCL-2 in vitro (Figure 7A). Further,
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