The Pan-FLT3/BTK Multi-Kinase Inhibitor CG '806 Induces AML Killing in FLT-Mutant and Wild Type Cells, and Exerts Synergistic Pro-Apoptotic Effects with Concomitant Targeting of Anti-Apoptotic Bcl-2 and/or Mcl-1

Internal tandem duplication (ITD) or point mutations of the Fms-like tyrosine kinase 3 (FLT3) are present in approximately 30% of patients with acute myeloid leukemia (AML) (Thiede et al., 2002). The ITD mutation is associated with very poor prognosis (Kottaridis et al., 2003) and additional acquired mutations of FLT3, including the D835 or the "gatekeeper" F691 mutation, have been identified in patients who developed resistance to currently used FLT3 inhibitors (Man et al., 2012; Smith et al., 2012). In addition, the upregulation of FLT3 ligand (FL) resulting from conventional chemotherapy, or bone marrow (BM) microenvironmental-mediated protection of AML cells, are also associated with resistance to FLT3-targeted therapy (Sato et al., 2011).

CG'806 is a first-in-class inhibitor that demonstrates sub-nanomolar activity against FLT3, Bruton's tyrosine kinase (BTK), aurora kinases, and certain other kinase family clusters in cell-free systems. We evaluated the anti-leukemia effect of CG'806 in human and murine leukemia cell lines harboring FLT3-ITD mutations and determined induced cell growth inhibition and apoptosis at sub-nanomolar to low nanomolar levels. Importantly, CG'806 also exerted pronounced anti-leukemia activity against cells with D835 TKD and FLT3-ITD plus F691L, Y842D or D835 dual mutations. CG'806 triggered marked apoptosis in vitro primary AML patient samples with and without FLT3-ITD mutations. Mechanistically, CG'806 demonstrated profound suppression of phospho-FLT3, -BTK and -aurora kinases, and their downstream signaling pathways (Zhang et al., AACR Hematological Malignancies, 2017). In addition, CG'806 completely abrogated protection mediated by FL or BM stromal cells in FLT3 ITD mutated MOLM14 cells, while quizartinib and sorafenib activity was markedly reduced in co-culture with mesenchymal stromal cells (MSCs). In a Baf3-ITD cell-engrafted murine leukemia model, CG'806 (delivered orally Qdx5 each week using a primitive formulation) significantly reduced leukemia burden, eliminated leukemic blasts, and extended survival in a dose-dependent manner versus the (vehicle) control.

Interestingly, CG'806 did not modulate anti-apoptotic proteins Bcl-2 levels, but upregulated anti-apoptotic Mcl-1, albeit marked cleavage of caspase-3 and PARP was observed in murine Baf3-FLT3-ITD mutated cells. Hence, we tested combinations of CG'806 plus the Bcl-2 antagonist ABT199, the Mcl-1 inhibitor A1210477, or both. All combinations showed marked synergistic pro-apoptotic effects in both FLT3 wild type (WT) and mutated leukemic cells, including those cells harboring dual or "gatekeeper" mutations (e.g., CIs were 0.41 ± 0.04, 0.26 ± 0.04, 0.59 ± 0.09 and 0.90 ± 0.02 in FLT3 WT, ITD, ITD+F691L and ITD+D835Y cells, respectively, in triple combo of CG'806, ABT199 and A1210477).

In summary, CG'806 was effective in both FLT3 WT and mutant AML cells, including those harboring dual ITD plus D835/F691 mutations. CG'806 also abrogated AML cell protection mediated by FL or BM stromal cells. Concomitant blockade of FLT3 and Bcl-2 or Mcl-1 synergistically induced pro-apoptotic effects in both FLT3 WT and mutant leukemic cells including dual mutant cells. CG'806 is under development for the treatment of patients with FLT3 WT and FLT3-mutated AML.

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