The Fms-like receptor tyrosine kinase 3 (FLT3) is expressed in approximately 90% of acute myeloid leukemia (AML), almost all B-lineage acute lymphoblastic leukemia (B-ALL), and some other hematologic malignancy subtype patients. Approximately one third of AML patients harbor constitutive activating internal tandem duplication in FLT3 (FLT3-ITD), which is associated with very poor prognosis. These observations have made FLT3 an attractive drug target. As more potent FLT3 inhibitors are developed, additional acquired point mutations have been identified, commonly at activation loop residue D835 and “gatekeeper” residue F691, which are related to drug resistance and relapse. CG′806, a first-in-class small molecule multikinase inhibitor against FLT3 and Bruton's tyrosine kinase (BTK), is under development as a next-generation agent for the treatment of FLT3-driven AML. Work from our collaborative laboratories has demonstrated that CG′806 retains full activity against Ba/F3 cells housing FLT3-ITD and/or point mutations, including D835G, D835Y, D835H, and F691L (abstract submitted to 2017 AACR Hematologic Malignancy Meeting). Herein we evaluated the potency of CG′806 on various hematologic malignancy cell lines and patient primary bone marrow specimens.

Primary patient mononuclear cells were derived from 172 patients diagnosed with AML (n=82), myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPN, n=15), ALL (n=17), and chronic lymphocytic leukemia (CLL, n=58). Sensitivity to CG′806 was evaluated across a range of concentrations after 72-hour treatment. IC50s were calculated as a measure of drug sensitivity and compared to nonproprietary competitive agents in AML and CLL, including the bromodomain inhibitors OTX-015 and JQ-1 and the FLT3 inhibitor quizartinib. The inhibitory effect of CG′806 on FLT3 signaling was evaluated in MV4-11 cells by Western blotting.

Across the four general subtypes of hematologic malignancies in the dataset, there is broad sensitivity to CG′806 with 59% (48/82) AML, 29% (5/17) ALL, 53% (8/15) MDS/MPN, and 40% (23/58) CLL cases exhibiting an IC50 of less than 0.1 μM. CG′806 demonstrated median IC50s of 0.07 μM and 0.22 μM against primary AML and CLL cells, respectively. FLT3 mutational status is known for 38 of the tested AML patient samples. Among those, the FLT3-ITD+ AML samples tended to have enhanced sensitivity to CG′806 (median IC50 = 0.02 μM, n=8) as compared to the FLT3-WT samples (median IC50 = 0.12 μM, n=30). CG′806 also exerted potent picomolar to low nanomolar IC50 antiproliferative activity against human AML, B-ALL, mantle cell lymphoma, Burkitt's lymphoma, and diffuse large B-cell lymphoma cell lines. As compared to the FLT3 inhibitor quizartinib, CG′806 at a concentration of 500 pM completely inhibited phosphorylation of FLT3 and STAT5 in MV4-11 cells upon 1 hour treatment, whereas quizartinib only partially inhibited their phosphorylation.

CG′806 exhibits broad and potent activity against AML patient samples, as well as other hematologic malignancy subtypes. The median IC50 data for CG′806 indicates greater potency relative to the tested nonproprietary agents across AML and CLL. Preliminary analyses reveal a trend of greater sensitivity to CG′806 in FLT3 mutant AML cases compared with FLT3 wild type; however, ongoing accrual of additional patient samples will be required to sufficiently power a statistical association of CG'806 sensitivity with FLT3 mutational status. In summary, preclinical analyses of CG′806 against primary hematologic malignancy patient samples and cultured cell lines show evidence of broad drug activity in AML and other disease subtypes and support further development of this agent for hematologic malignancies.

Citation Format: Stephen E. Kurtz, Beth Wilmot, Shannon McWeeney, Avanish Vellanki, Andrea Local, Khalid Benbatoul, Peter Folger, Susan Sheng, Hongying Zhang, Stephen B. Howell, William G. Rice, Brian J. Druker, Jeffrey W. Tyner. CG′806, a first-in-class FLT3/BTK inhibitor, exhibits potent activity against AML patient samples with mutant or wild type FLT3, as well as other hematologic malignancy subtypes [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 44.