Ktx-1001, a potent, selective MMSET/NSD2 inhibitor, enhances immunomodulatory and immune-directed therapies in preclinical models of multiple myeloma

Abstract Background: In t(4;14) multiple myeloma (MM), the histone methyl transferase NSD2 is dysregulated due to translocation of the IgH super-enhancer, resulting in its overexpression and globally elevated levels of histone 3 lysine 36 dimethylation (H3K36me2). KTX-1001 is an oral, first-in-class, selective inhibitor of NSD2 currently under evaluation in a Phase 1 clinical trial for patients with relapsed/refractory MM (NCT05651932, Bories ASH 2024). This study highlights the potential of KTX-1001 to synergize with immunomodulatory drugs (IMiDs/CELMoDs) and immune-directed therapies, including T-cell engagers, underscoring its value in enhancing anti-tumor immune responses. Methods: MM cell lines harboring t(4;14) translocation and resistant to standard therapies were treated with escalating concentrations of KTX-1001 in combination with pomalidomide (POM), mezigdomide (MEZI), anti-CD38 monoclonal antibodies (daratumumab, [DARA], isatuximab, [ISA]), and T-cell engagers (teclistamab (TEC, anti-BCMA) and talquetamab (TALQ, anti-GPRC5D). IMiD-resistant H929-POMR cells were evaluated for POM and MEZI combinations. For immune-directed therapies, healthy donor PBMCs were cocultured with MM cell lines (XG26: t(4;14); XG12: high NSD2, non-t(4;14)). Cell viability was assessed using CellTiter-Glo or flow cytometry, and combination effects were quantified via Bliss synergy scoring. Results: Combining KTX-1001 with POM (5nM-100nM) synergistically reduced viability in H929-POMR cells, with pronounced effects observed even at the lowest tested doses (500nM KTX-1001 + 5nM POM). With 5 nM POM alone cell viability was 84% vs 57%, 38%, and 33% with addition of KTX-1001 at 0.5uM, 1uM, or 2.5 uM, respectively. In the same model, the combination of KTX-1001 with MEZI (0.1nM-100nM) also synergistically decreased cell viability at all doses tested. Notably, the combination of the lowest doses tested, 500 nM KTX-1001 with 0.1 nM MEZI, resulted in >90% reduction in cell viability. In immune-based co-cultures, pre-treatment of XG-26 cells with KTX-1001 increased TEC-mediated cell lysis when co-cultured with healthy donor PBMCs for 24 hours (TEC alone: 33% lysis vs plus KTX-1001 0.5 um: 52%) and induced a dose-dependent activation of early (CD69+) CD4+ and CD8+ T cells (~10% increase with TEC+KTX vs TEC alone). With TALQ, KTX-1001 modestly increased lysis in XG-26 cells at the highest tested doses (0.1 ug/mL TALQ + 1uM KTX-1001). In the non-t(4;14) XG12 cell line, the combination of KTX-1001 (1 uM) with TALQ (0.1 ug/mL) resulted in increased MM cell lysis (~5% over TALQ alone) and slight increases in activation of early CD4+ and CD8+ T cells. Conclusion: These preclinicalfindings supportthe therapeutic potential of combining KTX-1001 with immunomodulatory and immune-directed therapies in MM. KTX-1001 demonstrated synergistic reductions in cell viability in IMiD-resistant t(4;14) cell lines when combined with POM or MEZI. In immune-based co-culture assays, pre-treatment with KTX-1001 enhanced cytotoxicity induced by T-cell engagers, TEC and TALQ in t(4;14) MM cell lines. Notably, synergy between KTX-1001 and TALQ was also observed in high NSD2 non-t(4;14) MM cells, suggesting broader applicability beyond translocation-defined subsets.