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R.S. Mali et al.
quizartinib treatment increased BIM:BCL-2 complexes in FLT3-ITD+ cell lines (Figure 7B), and co-treatment of cells with quizartinib and venetoclax caused greater reduction in BCL-XL and MCL-1 protein expression in FLT3-ITD+ cell lines (Figure 7C) compared to single agents. Therefore, FLT3-ITD inhibition switched survival dependence to BCL-2 in vitro, providing mechanistic rationale for the syn- ergistic activity of quizartinib and venetoclax.
BCL-XL and MCL-1 expression is maintained in vitro by multiple FLT3-ITD downstream pathways
In order to determine the FLT3-ITD signaling pathways regulating expression of BCL-2, BCL-XL and MCL-1, selec- tive kinase inhibitors pictisilib (PI3K), cobimetinib (MEK) or ruxolitinib (JAK) were utilized. BCL-XL and BCL-2 pro- tein were unchanged by any treatment in the FLT3-ITD+ cell lines while PI3K inhibition slightly reduced MCL-1 protein across all cell lines and JAK inhibition reduced MCL-1 protein only in FLT3-ITD+ cell lines (Online Supplementary Figure S8A-B). In order to pharmacologically test dependence on these pathways, Bliss analysis was performed. PI3K and BCL-2 co-inhibition was the most synergistic in the FLT3-ITD+ cell lines followed by MEK and BCL-2 co-inhibition. Although ruxolitinib reduced MCL-1 protein expression, the combination was not syn- ergistic in FLT3-ITD+ cell lines (Online Supplementary Figure S8C). All Bliss sums ranked less than quizartinib plus venetoclax. The reduced synergy and inability of individual downstream pathways to modulate BCL-XL and MCL-1 protein support directly targeting FLT3-ITD rather than downstream effectors in combination with venetoclax. Together, the data presented here demonstrat- ed that the combination of quizartinib and venetoclax tar- gets all three anti-apoptotic proteins through indirect reg- ulation of BCL-XL and MCL-1 by FLT3-ITD inhibition and potent inhibition of BCL-2 by venetoclax.
Discussion
Although minimally active as a monotherapy, veneto- clax combined with HMA or LDAC is efficacious in AML patients who are unfit for high intensity chemotherapy across diverse mutation subsets.18,19 Given this activity, there is clinical rationale for developing combination regi- mens that maximize efficacy while minimizing toxicity. With this in mind, we provide strong mechanistic ration- ale for a combination regimen of FLT3-ITD inhibition and venetoclax to be tested clinically.
FLT3 inhibitors became standard of care in FLT3-mutant AML with midostaurin plus chemotherapy in frontline AML and gilteritinib monotherapy in relapsed/refractory AML receiving Food and Drug Administration approval. However, duration of response is often short-lived and patients can relapse with aggressive disease. Therefore, combining FLT3 inhibitors with agents that potently induce apoptosis has the potential to achieve more durable responses for FLT3-ITD+ patients. Combination activity of midostaurin or gilteritinib and venetoclax in preclinical FLT3-ITD+ models has recently been described.31 However, the role of BCL-XL in FLT3-ITD+ cells or the contribution of FLT3-ITD downstream path- ways in mediating BCL-2 family dependence was not explored in depth. Here we have demonstrated that BCL- 2, BCL-XL and MCL-1 all promote survival of FLT3-ITD+
cells in vivo and that targeting of single pathways down- stream of FLT3-ITD is insufficient to modulate BCL-XL and MCL-1. We have provided a more thorough investi- gation of BCL-2 family dependencies in vitro and in vivo and demonstrate robust anti-leukemia activity of venetoclax combined with FLT3 inhibitors in FLT3-ITD+ preclinical AML models. The data presented here demon- strated that FLT3-ITD+ inhibition indirectly modulated BCL-XL and MCL-1 expression while venetoclax potently inhibited BCL-2 resulting in simultaneous targeting of all three anti-apoptotic proteins. Importantly, our studies uti- lized clinically achievable concentrations of inhibitors and demonstrated in vivo anti-tumor activity in models of established leukemia.
We identified that inhibition of individual signaling pathways downstream of FLT3-ITD was insufficient in modulating anti-apoptotic proteins, suggesting that multi- ple downstream pathways need to be simultaneously inhibited to induce apoptosis. Such functional co-opera- tion has been described for BCR-ABL, where RAS, STAT5 and PI3K individually contribute to survival and provide overlapping anti-apoptotic signals.38 Potently targeting FLT3-ITD presents an opportunity to inhibit multiple downstream pathways to indirectly reduce BCL-XL and MCL-1 expression and switch survival dependence to BCL-2. This switch in survival dependence sensitizes mutant cells to venetoclax treatment as demonstrated by enhanced anti-tumor activity of quizartinib plus veneto- clax compared to monotherapies. While mechanisms of resistance to the combination of FLT3-ITD inhibition and venetoclax were not explored here, resistance to FLT3 inhibitors is well-described and includes acquired inhibitor-resistant point mutations,39 suboptimal drug con- centrations in the BM, bypass of FLT3-ITD signaling,40 and alteration of apoptotic proteins.41 Additionally, mutations in BCL-2 have been identified in chronic lymphocytic leukemia patients following relapse as a mechanism of venetoclax resistance.42,43 Based on the indirect regulation of BCL-XL and MCL-1 by FLT3-ITD signaling described here, potential resistance mechanisms to the combination of venetoclax and FLT3-ITD inhibition could include inhibitor-resistant point mutations that interfere with FLT3-ITD signaling inhibition or activating mutations in parallel signaling pathways, both of which would likely alter BCL-2 family expression. Investigation into resist- ance mechanisms is the focus of future work.
Importantly, venetoclax plus quizartinib combination activity was demonstrated in vivo in FLT3-ITD heterozy- gous (Molm13) and homozygous (MV4;11) preclinical models. Superior long-term survival and disease free state was observed in the MV4;11 xenograft model, which could reflect stronger FLT3-ITD addiction in the homozy- gous model as described by Pratz et al.44 or result from dif- ferent secondary mutations in the models. Combination activity was also demonstrated in a PDX model (Online Supplementary Figure S3A) with co-occurring FLT3/DNMT3A/NPM1 mutations, a subset associated with heavy disease burden and poor event free survival compared to non-triplet mutant patients.45,46 Notably, gilteritinib improved survival in FLT3/DNMT3A/NPM1 mutant patients compared to chemotherapy47 and our data indicate venetoclax may further enhance gilteritinib activ- ity in this subset. While additional studies are required to parse out biological differences between FLT3-ITD het- erozygous and homozygous mutations and the role of co-
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haematologica | 2021; 106(4)