PIM Inhibition By SEL24/MEN1703 Combines Synergistically with Gilteritinib in FLT3-ITD Preclinical Models of Acute Myeloid Leukemia

BACKGROUND. Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer, derived from an uncontrolled clonal proliferation of malignant myeloid blast cells in the bone marrow, transformed by recurrent genetic alterations (Wu M. et al., J. Hematol Oncol, 2018). SEL24/MEN1703 is a first-in-class, oral, type I dual PIM/FMS-like tyrosine kinase 3 (FLT3) inhibitor investigated in the ongoing Ph2 study in patients with AML (DIAMOND-01, ClinicalTrials.gov identifier: NCT03008187) (Czardybon W. et al., Oncotarget 2018). Gilteritinib is a highly potent and selective oral FLT3 inhibitor, FDA-approved for the treatment of relapsed or refractory AML with FLT3 mutations. Although Gilteritinib showed strong single-agent activity in patients with FLT3+ AML (Levis M. and Perl AE., Blood advances, 2020), the development of Gilteritinib resistance limits durability of the response, indicating that novel combination strategies may be clinically important (Zhang LS. et al., Onco Targets and Therapy, 2022). AIM. The main objective of this study is to assess potential synergistic effects in-vitro and in-vivo of the combination of SEL24/MEN1703 and Gilteritinib in AML cell lines with FLT3-ITD and to elucidate the underlying signaling pathways. METHODS.In-vitro cytotoxicity and apoptosis experiments were carried out in FLT3-ITD AML cell lines (MV4-11, MOLM13, MOLM14), testing SEL24/MEN1703 and Gilteritinib either as single agents or in combination for 72h. The combination index was calculated according to the Chou method (Chou TC., Pharmacol Rev, 2006). In-vitro co-culture experiments were performed with FLT3-ITD cell lines MV4-11 and MOLM14 incubated in the presence of human primary bone marrow stromal cells. In MV4-11 and MOLM13 xenograft models, SEL24/MEN1703 and Gilteritinib were administered per OS every day, for 14 and 22 days, respectively. A signaling pathway analysis (pAKT, pERK, pSTAT5, Mcl-1, c-Myc, Bcl-xL, pS6) was also performed through immuno-capillary electrophoresis on tumor nodules collected from MOLM13 xenograft study. RESULTS. The combination treatment was moderately synergistic/additive in both in-vitro cytotoxicity and apoptosis experiments on MV4-11, MOLM13, and MOLM14, at all concentration levels. However, in co-culture experiments, the presence of primary stromal cells resulted in a significantly increased combination index, in tested AML cell lines. In the in-vivo MV4-11 xenograft model, SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg induced a significant anti-tumor activity as single agent treatment, showing 57.2% and 84.1% of tumor volume inhibition (TVI) respectively, whereas the combination treatment SEL24/MEN1703 plus Gilteritinib induced 99.2% of TVI at day 33 (Figure 1). At day 40, when the Gilteritinib schedule was concluded, SEL24/MEN1703 and Gilteritinib as single agents showed 53.1% and 87.6% of TVI respectively, whereas the combo group induced a complete tumor regression (100% of TVI). Moreover, only the combination of SEL24/MEN1703 and Gilteritinib resulted in a delayed tumor regrowth 23.5 days after discontinuation of the treatment. In the MOLM13 model, at day 29, treatment with SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg as a monotherapy induced an antitumor activity with 38.8% and 60.1% of TVI respectively, whereas with the combination treatment resulted in a higher TVI (78.3%), however, no complete tumor regression was observed in this model. In the MOLM13 xenograft model, analysis of downstream signaling pathways of FLT3 and PIM kinases showed that the pS6 effector protein is mainly downregulated in the combination treatment group. CONCLUSIONS. Combination therapy with SEL24/MEN1703 and Gilteritinib is moderately synergistic in-vitro, however, results in robust tumor regressions, including complete responses in in-vivo FLT3-ITD models, at tolerable doses. The higher efficacy of this combination in-vivo and compared to monotherapy could be related to a very potent concomitant inhibition of FLT3 and PIM kinases affecting upregulated FLT3 signaling pathway in-vivo as demonstrated by the reduction of phosphorylation of downstream effector proteins, such as pS6. Overall, these experiments have demonstrated the antitumor potential of concomitant inhibition of FLT3 and PIM kinases which could drive a novel therapeutic strategy in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal