CG '806, a First-in-Class Pan-FLT3/BTK Inhibitor, Exhibits Potent Growth Inhibition As a Single Agent and in Combination with a BET Bromodomain Inhibitor or a Bcl2 Inhibitor on Primary AML and CLL Patient Samples

Introduction: Acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) are 2 of the most common adult hematologic malignancies. Cytogenetic and deep-sequencing studies of AML have revealed a complex and heterogeneous disease, with nearly 2,000 somatically mutated genes observed across the TCGA cohort of 200 patients, where nearly 200 of these somatic mutations were observed recurrently. Although the standard of care for AML has remained unchanged for decades, the development of FLT3 inhibitors represents a focused attempt at new therapies, as gain-of-function point mutation or internal tandem duplication (ITD) of FLT3 occur in ~30% of AML patients. Numerous FLT3 inhibitors have been developed with several showing transient clinical benefit, but responses have only lasted 3-4 months. Similarly, a majority of CLL patients develop resistance to treatment and remain incurable with current therapies. Recent insights into the importance of B-cell receptor-mediated kinase signaling pathways have resulted in new therapeutic strategies for CLL patients, including agents that target the kinases PI3K, SYK, and BTK. These agents have shown clinical activity, but responses have not been uniform, and the majority of patients eventually develop drug resistance. Thus, both AML and CLL are in urgent need of effective therapies that provide more durable clinical responses, which will most certainly involve novel drug combinations.

CG'806 is a first-in-class small molecule multi-kinase inhibitor against wild type (WT) and mutant forms of the FLT3 and Bruton's tyrosine kinase (BTK) enzymes. We previously demonstrated that CG'806 has inhibitory activity against Ba/F3 cells expressing FLT3-ITD or point mutations, including D835G, D835Y, D835H, and F691L. We also evaluated the potency of CG'806 on various hematologic malignancy cell lines and patient primary bone marrow specimens. CG'806 exhibits broad and potent activity against primary patient samples over a range of hematologic malignancy subtypes, including AML, CLL, myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN), and acute lymphoblastic leukemia (ALL). Preliminary analyses reveal a trend of greater sensitivity to CG'806 in FLT3-mutant AML cases compared with wild-type FLT3, although ongoing accrual of additional patient samples will be required to sufficiently power a statistical association of CG'806 sensitivity with FLT3 mutational status.

Methods: We used an ex vivo drug sensitivity assay to determine the activity of CG'806 as a single agent and in combination with the BET bromodomain inhibitor OTX-015 or the Bcl2 inhibitor venetoclax on freshly isolated primary patient samples. Combinations were tested at fixed, equimolar ratios over the same dose range. After a 3-day ex vivo culture, cell viability was assessed using a colorimetric, tetrazolium-based MTS assay and IC₅₀ values calculated. Sensitivity to CG'806 was evaluated across a range of concentrations after 72-hour treatment. IC₅₀ was calculated as a measure of drug sensitivity. The inhibitory effect of CG'806 on FLT3 signaling was evaluated in MV4-11 cells by immunoblotting.

Results: Across the 4 general subtypes of hematologic malignancies in the dataset with patient samples, there was broad sensitivity to CG'806, with 55% (90/164) AML, 48% (46/96) CLL, 22% (6/27) ALL, and 53% (14/26) MDS/MPN cases exhibiting an IC₅₀ <0.1 μM. CG'806 demonstrated median IC₅₀ values of 0.07 μM and 0.14 μM against primary AML and CLL cells, respectively. CG'806 also exerted potent picomolar to low-nanomolar IC₅₀ anti-proliferative activity against human AML, B-ALL, mantle-cell lymphoma, Burkitt's lymphoma, and diffuse large B-cell lymphoma cell lines. CG'806 in combination with OTX-015 demonstrated median IC₅₀ values of 0.02 μM and 0.04 μM against primary AML and CLL cells, respectively. CG'806 in combination with venetoclax demonstrated median IC₅₀ values of 0.02 μM and 0.01 μM against primary AML and CLL cells, respectively.

Conclusions: In summary, CG'806 has potent inhibitory activity on primary patient samples and cultured cell lines, showing evidence of broad drug activity in AML and other disease subtypes to support further development of this agent for hematologic malignancies. We also identified positive interaction of CG'806 with a BET inhibitor and a Bcl2 inhibitor, suggesting these classes of drugs are potential combination partners for CG'806.