Anti-leukemic efficacy of a novel inhibitor for KMT2A-r/FLT3-driven AML via multi-kinase targeting and synergy with menin inhibition Free

Introduction: Novel tyrosine kinase inhibitors (TKIs) are needed for combination therapy with menin inhibitors (menin-i) for KMT2A-rearranged (KMT2A-r) AML, particularly for patients with concurrent FLT3 mutations. While menin-i show promise in targeting KMT2A-r leukemias, preclinical and early clinical data suggest that combining them with other targeted therapies, like TKIs, can overcome resistance mechanisms and enhance anti-leukemic effects.

Objectives: We have developed and characterized a new class of potent kinase inhibitors, that exhibit nanomolar potency against FLT3-ITD, FLT3-TKD and FLT3-wt in biochemical/cellular assays in vitro and/or in vivo (Gucký et al., 2018, PMID: 29672049; EU patent: EP3601287; US patent: US11028087B2). The lead compound LGR3922 (referred to as compound 7d in Gucký et al., 2018) also demonstrates high inhibitory activity (>90% inhibition at 10 nM) against several additional kinases, established or emerging therapeutic targets in AML: MAP kinase-interacting serine/threonine kinase 2 (MNK2), Src family kinases (SFKs), Ca²⁺/calmodulin-dependent protein kinase II-γ (CaMKIIγ), salt-inducible kinases (SIK), and Cdc2-like kinase 1 (CLK1).

We investigated the preclinical efficacy and underlying molecular mechanism of LGR3922 in models of KMT2A-r/FLT3-driven AML. Given that the kinases selectively inhibited by LGR3922 in vitro and in vivo are among the critical effectors of leukemogenesis in KMT2A-r AML, we hypothesized that LGR3922 and menin-i may exhibit a strong potentiation of antileukemic efficacy when combined.

Methods: Cytotoxicity and proliferation assays were performed in KMT2A-r/FLT3mut AML cell lines (MV4-11, MOLM-13, HB11;19) and murine Mll-ENL-transformed Meer cells (Takacova et al., 2012, PMID: 22516260). Antileukemic efficacy was further evaluated in xenograft models, Mll-ENL knock-in-based AML NOD.SCID mouse model and primary AML patient samples. Phosphorylation status of key targets, including FLT3, SFKs, eIF4E via MNK2, CaMKIIγ, HDAC4 via SIK, and SR proteins via CLK1, as well as total MEF2C levels, were assessed by immunoblotting. Synergistic activity with the FDA-approved menin-i revumenib was examined using proliferation assays, immunoblotting, flow cytometry, and qRT-PCR analyses.

Results: LGR3922 demonstrated potent inhibition of oncogenic FLT3 and additional target kinases, resulting in suppressed proliferation and enhanced apoptosis in vitro. In vivo, a single 10 mg/kg dose of LGR3922 induced inhibition of phosphorylated FLT3 (p-FLT3), STAT5 (p-STAT5), ERK1/2 (p-ERK1/2), the MNK2–p-eIF4E axis, CaMKIIγ, the SIK–HDAC4 pathway, CLK1–SR proteins, and total MEF2C levels. Repeated dosing led to significant tumor regression or complete leukemia eradication along with stimulation of erythropoiesis. In an Mll-ENL knock-in-based NOD.SCID mouse model, LGR3922 significantly decreased the Mll-ENL leukemic allelic burden compared to midostaurin. In primary AML samples, LGR3922 reduced above mentioned kinase signaling pathways, colony cellularity and selectively eliminated KMT2A-r/FLT3mut cells, though MEF2C - a menin-i-sensitive gene - was slightly upregulated. These data suggested that suppression of KMT2A-r/FLT3 oncogenic kinase axes with LGR3922 may pre-sensitize leukemia cells to menin-i. Indeed, the combined treatment with LGR3922 and revumenib synergistically suppressed proliferation in KMT2A-r/FLT3mut AML cell lines and Meer cells, outperforming single-agent treatments. In Meer cells, it synergized in downregulation of Meis1, Hoxa9, and Mef2c transcripts and MEF2C protein, and in significant increase in differentiation monitored by CD11b expression.

Conclusions: LGR3922 represents a promising candidate for targeted therapy in KMT2A-r/FLT3mut AML, offering a unique multi-kinase mechanism that disrupts key signaling pathways not targeted by current therapies. Demonstrated in vivo efficacy, favorable toxicity profile, and synergistic activity with menin-i support its further development and testing. Since one of the common side effects of menin-i therapy is anemia, the observed erythropoiesis-stimulating effect of LGR3922 as an unexpected side effect could be advantageous for therapeutic exploration of the compound. Our data also underscore the therapeutic potential of concurrently targeting KMT2A-r-associated oncogenic kinase dependencies and menin-KMT2A-r transcriptional targets.

Grant support: GA23-05462S, GA24-11730S, JG_2023_016, IGA UPOL LF_2025_003, PROPOST, LX22NPO5102.