Microrna-142 improves IL1RAP CAR-T cell activity in Acute Myeloid Leukemia

Abstract

Abstract Chimeric antigen receptor (CAR) T cells have shown promise in hematologic malignancies but face major challenges in acute myeloid leukemia (AML), including antigen heterogeneity, immune escape, and a suppressive tumor microenvironment that limits T cell persistence and efficacy. We recently reported that IL1RAP is highly expressed on human AML blasts, but not on normal hematopoietic stem cells (HSCs). Therefore, we generated IL1RAP CAR T cells and un-transduced T (UTD-T) or IL1RAP-mut CAR-T as controls. IL1RAP CAR-T, and not UTD-T, co-cultured with IL1RAPhigh AML cell lines or primary bulk or CD34+ blasts showed significant increase in cell cycling, cytokine production, and E:T-ratio-dependent tumor lysis, compared with CAR-T co-cultured with IL1RAPlow K562 cells or healthy donor HSCs. To test the antileukemic activity of IL1RAP CAR-T in vivo, we treated Luci/GFP-expressing MOLM13 cell line-derived xenograft (CDX) with one dose of IL1RAP CAR-T or UTD-T on day 3. IL1RAP CAR-T-treated mice demonstrated significantly lower tumor burden by bioluminescence imaging and longer survival than UTD-T-treated or untreated controls (median survival: 48.5 vs 30 vs 30.5 days; p = 0.01). Similar results were observed using IL1RAP-mut CAR-T as control. Importantly, no signs of systemic toxicity were observed. Given that IL1RAP CAR-T rapidly decreased over time, to test if longer exposure to IL1RAP CAR-T resulted in a more potent antileukemic effect, we treated the mice with two doses of T cells on days 3 and 20 post-AML transplant, respectively. CDX treated with two doses of CAR-T showed significantly lower tumor burden and longer survival than UTD-T-treated or untreated controls (median: 96 vs 35 vs 34 days, p = 0.002) and appeared to live longer than those treated with only one dose, with one mouse achieving leukemia-free survival beyond 200 days. These results prompted us to seek strategies that could prolong persistence and AML exposure to functional CAR T cells. We recently reported that a cytokine-driven reduction in miR-142 impairs T cell differentiation, activation and antileukemic surveillance in BC CML (Nat Commun, 2025). In AML patients and MllPTD/wt/Flt3ITD/ITD mice, T cells showed reduced miR-142 levels, increased apoptosis, and diminished cytokine production. Transplantation of AML blasts into wild-type (wt) mice led to acquired miR-142 loss in host T cells. To measure the antileukemic activity of miR-142 deficit T cells, we co-transplanted LSCs and miR-142−/− or miR-142+/+ T cells into NSG recipients which lack T cells and observed fewer T cells, more leukemic blasts, and shorter survival in recipients of LSC/miR-142−/− T than those of LSC/miR-142+/+ T (median: 51 vs 59 days, p=0.0013). Reduced T cells, increased leukemic blasts, and shorter survival were also observed in Mir142−/− vs Mir142+/+(median: 42 vs 50.5 days, p=0.0004), and T-specific miR-142 KO (Mir142flox(f)/f Lck-cre+) vs wt (median: 53.5 vs 63 days, p=0.0087) recipients transplanted with murine AML blasts as well as in NSGS recipients of human AML blasts/miR-142 KD T vs those of AML blasts/miR-142 wt-T (median: 32 vs 41 days, p=0.0017). Treatment with synthetic M-miR-142 for 3 weeks restored T cell numbers and function and significantly improved survival in both AML murine and patient-derived xenograft (PDX) models (median: 43 vs 38 days, p = 0.0006; 60 vs 51 days, p = 0.0002).Thus, we postulated that loss of miR-142 in leukemic BM niche could also affect the fitness and antileukemic activity of IL1RAP CAR T cells in AML. Therefore, we treated exhausted IL1RAP CAR T cells (cultured >1 month) with M-miR-142 (2 μM). Compared with scrambled control (SCR), M-miR-142 treatment reduced apoptosis, lowered PD-1, increased CAR T persistence, and enhanced AML killing. Metabolic analysis of CAR T showed that M-miR-142 significantly increased glycolysis and oxidative phosphorylation, suggesting restoration of the metabolic switch required for T cell activation. In AML PDX mice treated with IL1RAP CAR T cells on day 7 and M-miR-142 beginning on day 10 for 3 weeks, we observed a higher frequency of BM CAR T cells, lower leukemia burden, and longer survival compared to CAR T+SCR or M-miR-142 treated controls (median: 78 vs 51 vs 28 days; p = 0.0018). Similar results were observed in two additional PDX models. No toxicity was observed in any cohort. Collectively, these results support the therapeutic potential of M-miR-142 to enhance IL1RAP CAR T cell efficacy in AML.