Combined Therapy With BRD4 Antagonist and FLT3 Inhibitor Exerts Synergistic Activity Against Cultured and Primary AML Blast Progenitors Expressing FLT-ITD
Authors: Rodriguez Melissa , Fiskus Warren , Sharma Sunil , Qi Jun , Valenta John A , Schaub Leasha J , Shah Bhavin , Devaraj Santhana , Iyer Swaminathan Padmanabhan , Bradner James E , Bhalla Kapil N. 
Published: October 2019

Abstract
Mutations in FLT3 are detected in approximately 30% of AML and are associated with poor overall survival. Although first (PKC412, sorafenib and CEP701) and second generation (AC220) FLT3 tyrosine kinase inhibitors (TKI) induce remissions, resistance-causing gatekeeper (F691I/L), activation loop (AL) mutations (D835V/Y/F) or compound FLT3-ITD/F691I AL mutations are known to impair the in vitro and in vivo activity of the FLT3-TKIs. The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., MYC and BCL-2. BRD4 antagonists JQ1 and I-BET151 disrupt the binding of the bromodomain of BRD4 to acetylated lysines on histone proteins, thereby inhibiting expressions of c-MYC and BCL-2 and inducing apoptosis of AML cells. Based on this, we evaluated the in vitro and in vivo activity of JQ1 and FLT3 antagonists AC220 and ponatinib against cultured mouse lymphoid (Ba/F3/FLT3-ITD), as well as human cultured (MOLM13 and MV4-11) and primary AML blast progenitor cells (BPCs) expressing FLT3-ITD. JQ1, but not its inactive enantiomer R-JQ1, potently induced apoptosis of not only Ba/F3/FLT3-ITD but also of Ba/F3/FLT3-ITD expressing the highly FLT3 TKI-resistant mutations F691L and D835V (IC50 values for JQ1 were 697, 1588 and 909 nM, in the three cell lines, respectively). This was associated with attenuation of c-MYC, but the induction of BIM levels. Both JQ1 and I-BET151 dose-dependently induced apoptosis of MOLM13 and MV4-11 cells, as well as of primary AML BPCs expressing FLT3-ITD. Concomitantly, JQ1 treatment attenuated c-MYC, BCL2 and CDK6, while inducing p21, p27, BIM and cleaved PARP levels. JQ1 and I-BET151 did not induce apoptosis of CD34+ normal bone marrow progenitor cells. Following engraftment of NOD/SCID mice with MOLM13 cell xenograft, treatment with JQ1 (50 mg/kg, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, daily x 5 days per week x 3 weeks), versus treatment with vehicle control, significantly improved survival of the mice (p< 0.05), without causing any toxicity. This was associated with the in vivo attenuation of c-MYC and BCL-2 levels in the harvested AML cells from the mice. Co-treatment with JQ1 or I-BET151 and FLT3 antagonist AC220 or ponatinib synergistically induced apoptosis of MOLM13 and MV4-11 cells. This was associated with greater reduction in the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. Knockdown of BRD4 by treatment with specific shRNA phenocopied the effects of JQ1 and sensitized MOLM13 cells to ponatinib and AC220. As compared to each agent alone, treatment with JQ1 and ponatinib or AC220 also induced more apoptosis of primary AML BPCs expressing FLT3-ITD, associated again with greater reduction of the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. We also determined the effects of JQ1 against MOLM13/TKIR cells that were generated under the continuous selection pressure of FLT3 TKI, and exhibited > 10 fold resistance to ponatinib but > 50 fold resistance to AC220. Importantly, as compared to the parental MOLM13, the MOLM13/TKIR cells were markedly more sensitive to JQ1-induced apoptosis (p< 0.001). Additionally, co-treatment with JQ1 and ponatinib but not AC220 synergistically induced apoptosis of MOLM13/TKIR cells. Supporting our previous findings (Blood. 2005;105:1768) that FLT3-ITD is a heat shock protein (hsp) 90 client-oncoprotein, the non-geldanamycin hsp90 inhibitor AUY922 was equally effective in inducing apoptosis of MOLM13 versus MOLM13/TKIR cells. Collectively, these findings demonstrate that BRD4 antagonist exhibits potent activity against cultured and primary AML cells expressing FLT-3-ITD, as well as against cellular models of FLT3 with gate-keeper and activation loop mutations. These findings also highlight the novel and synergistic activity of the combination of BRD4 antagonist and AC220 or ponatinib against AML BPCs expressing FLT3-ITD, and support the rationale for testing ponatinib and BRD4 antagonist against TKI-refractory AML expressing FLT3-ITD.

Disclosures:
No relevant conflicts of interest to declare.