Preclinical activity of ziftomenib and CAR T cell immunotherapy in KMT2A -rearranged (R) acute leukemias

Introduction: Many patients with KMT2A -R acute leukemias experience chemoresistance and/or subsequent relapse despite intensive frontline therapies. While CD19 chimeric antigen receptor T cells (CD19CART) induce initial remission in most children with relapsed/refractory B-acute lymphoblastic leukemia (ALL), post-CD19CART relapse occurs in ~50% of patients, many of whom experience CD19 antigen loss and/or lineage switch (LS) to fatal acute myeloid leukemia (AML). To address this unmet need, we previously developed CAR T cells targeting the FLT3 receptor (FLT3CART) and reported potent anti-leukemia activity in preclinical models of wild-type FLT3-overexpressing KMT2A -R ALL and LS AML. In the current study, we tested the hypothesis that co-targeting of a highly-expressed cell surface antigen with CARTs and critical intracellular biology with the selective menin inhibitor (MENi) ziftomenib in KMT2A -R ALL would have therapeutic synergy and ameliorate resistance mechanisms of single agent therapies. Experimental procedures: Human KMT2A -R and non-R ALL cell lines were co-incubated in vitro with FLT3CART or CD19CART +/- ziftomenib in viability assays. CART-induced IFN-gamma production was measured via ELISA. Patient-derived xenograft (PDX) models of infant, pediatric, and young adult non- KMT2A -R (n=1), KMT2A -R ALL (n=6), and KMT2A -R LS AML (n=1) were treated intravenously with saline, mock T cells, FLT3CART, or CD19CART +/- ziftomenib daily per os as previously described (Loftus Haematologica 2021, Falkenstein Blood 2022, Niswander Haematologica 2023). Human CART and leukemia cells were quantified weekly via flow cytometry (FC) analysis of murine peripheral blood and in end-study bone marrow and/or spleens. In vivo IFN-gamma production was measured weekly in murine plasma. Results: Ziftomenib monotherapy induced robust inhibition of leukemia proliferation in vitro and in vivo in all tested KMT2A -R ALL or LS AML models. Importantly, ziftomenib did not impair CART functionality , as assessed by cytokine production and FC immunophenotyping of T cell subsets and activation/exhaustion markers. Ziftomenib co-therapy significantly augmented in vivo FLT3CART or CD19CART activity in most KMT2A -R ALL and the LS AML PDX models. Paradoxically, in vivo IFN-gamma production in murine blood and CART cells in end-study spleens were decreased in all ziftomenib + CART co-treated KMT2A -R ALL or LS AML PDX models versus FLT3CART or CD19CART alone, which we posit was due to rapid cytotoxic efficacy of ziftomenib against target antigen-expressing leukemia cells necessary for CART activation and expansion. Conclusions: Our preclinical data strongly support combinatorial efficacy of the MENi ziftomenib and FLT3CART or CD19CART immunotherapy in KMT2A -R ALL that may overcome or even prevent subsequent LS relapse. Studies in additional KMT2A -R ALL and LS AML PDX models are ongoing to validate and extend these results.