Molecular Analysis and In Vivo Efficacy Studies on a Novel Chemical-Series of FLT3 Inhibitors in Human FLT3-ITD AML

Approximately 25% of all acute myeloid leukemias (AMLs) contain an internal tandem duplication (ITD) in the juxtamembranal region of FLT3. Even with standard chemotherapies, FLT3-ITD AML is associated with poor prognosis, with a five-year survival rate of approximately 15% as compared to 40% for wild-type FLT3 AML. Therefore, FLT3 inhibitors have been developed as a targeted treatment approach. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming, with few complete remissions and a high rate of relapse and resistance. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse in FLT3-ITD AML patients.

Through chemical and structure-activity relationship studies, we have identified a novel class of FLT3 tyrosine kinase inhibitors. We have previously shown that our current lead compound (CTDS-004) has excellent selectivity against the kinase activity of FLT3-ITD and FLT-ITD D835Y at a subnanomolar concentration (IC50 <5 x 10^-10 M). Additionally, CTDS-004 potently inhibits proliferation and progenitor function, and induces apoptosis of FLT3-ITD positive human AML cell lines in comparison to the second generation FLT3 inhibitors, AC220 and Crenolanib. Importantly, CTDS-004 exhibits minimal effects on the function and viability of normal human CD34+ BM cells. The most important distinction between CTDS-004 and current FLT3-ITD inhibitors is that CTDS-004 retains its efficacy and prevents subclonal recovery of FLT3-ITD AML cells that have become refractory to AC220 or Crenolanib treatment. To gain insight into the mechanistic basis for the superior response of FLT3-TID AML to CTDS-004 as compared to AC220, we performed RNA sequencing of MLL-AF9 FLT3-ITD cells after 6 and 12 hours of treatment with AC220 or CTDS-004 (IC10: 0.3 and 0.1 nM, respectively). Although both compounds target FLT3-ITD, there were striking distinctions in gene expression changes after treatment with AC220 and CTDS-004, highlighting the differences in the biological impact and cytotoxic effect of these compounds.

Our lead compound has promising pharmacokinetic and pharmacodynamics properties in vivo, therefore, we tested CTDS-004 in a xenograft model of human AML. NRGS mice (NOD.Cg-Rag1^tm1MomIL2rg^tm1WjlTg[CMV-IL3,CSF2,KITLG]1EAv/J) were transplanted with human MLL-AF9 FLT3-ITD leukemic cells (2 x 10⁵ cells/mouse) via tail vein injection. Starting on day 10 post-engraftment, the mice were treated with CTDS-004 (30 mg/kg; n=5), AC220 (15 mg/kg; n=5), or PBS (n=5) via intraperitoneal injection on a cycle of five days of daily injection followed by two days of rest for the duration of the experiment. Kaplan-Meier survival analysis revealed that CTDS-004 is extremely effective at prolonging survival in the human FLT3-ITD AML xenograft model. There was a significant increase in median survival for CTDS-004 and AC220 treatment groups compared to the PBS control group (Median survival post-xenograft: PBS: 28 days, AC220: 54 days, CTDS-004: 53 days; p=0.0002). Taken together, these findings suggest our novel FLT3 inhibitor shows promise for the treatment of FLT3-ITD positive AML, and particularly for patients that have intrinsic and/or acquired resistance to FLT3 tyrosine kinase inhibitors.