Development of PLM-102, a novel dual inhibitor of FLT3 and RET as a new therapeutic option for acute myeloid leukemia.

Abstract

e15103 Background: Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of all acute myeloid leukemia (AML) cases, with the internal tandem duplication (ITD) representing the most common type of FLT3 mutation (FLT3-ITD; approximately 25% of all AML cases). Although several FLT3 inhibitors have been developed, occurrence of secondary TKD mutations of FLT3 such as FLT3/D835Y and FLT3/F691L causes the acquired resistance to the current FLT3 inhibitors and eventually become a key area of unmet medical needs. Here, we have revealed that PLM-102, a novel, orally active FLT3 and RET dual inhibitor, has a potential to overcome the acquired resistance to current FLT3 inhibitors. Methods: 1. Kinase assay- Biochemical assays for FLT3 (WT and D835Y) and RET (WT and Mutants) were performed according to ADP-Glo kinase assay protocol(Promega). 2. Cell proliferation and Apoptosis- Human leukemia cell line MV4-11 and MOLM-14 were purchased from ATCC and DSMZ. Cells were seeded at a density of 2 X 103 cells per well and treated with the indicated concentrations of inhibitors for 72 hours at 37°C. Cell viability was determined by an Alamar Blue assay (Bio-Rad). Caspase-3/7 activity was measured by using the Caspase-Glo 3/7 assay (Promega). 3. Western blot analysis- Immunoblotting using MOLM-14 cells was performed using anti-phospho-FLT3 (Cell Signaling Technology #3461) and anti-FLT3 antibody (Cell Signaling Technology #3462). 4. In vivo mouse models- The MV4-11 and MOLM-14 cells are implanted into the subcutaneous space of the left flank of the mice. Resulting tumors are monitored by calipering twice weekly. Treatment started after randomization when tumor volumes had reached a size of approximately 100-150 mm3. For statistical analysis, analysis of variance (ANOVA) was performed using Prism 9.0 to examine statistical differences. Results: In compared to FDA-approved Gilteritinib, PLM-102 showed stronger sub-nanomolar IC50 values in FLT3 kinases regardless of wild-type, ITD, TKD and ITD/TKD mutants in both kinase- and cell-based assays. PLM-102 inhibited phosphorylation of FLT3 and its downstream signaling pathways, and induced apoptosis as evidenced by PARP-cleavage and caspase-3 activation. Moreover, PLM-102 showed an excellent anti-tumor activity in mouse xenograft models implanted with MV4-11 and MOLM-14 AML cells. Conclusions: Taken together, PLM-102 showing potent anti-cancer activity against various in vitro and in vivo AML models could be developed as a valuable agent overcoming the acquired resistance to the current FLT3 inhibitor.