Momelotinib-mediated transcriptional reprogramming induces synthetic lethal interaction with calcineurin-NFAT signaling

Abstract Despite notable advances in the development of targeted therapies for acute myeloid leukemia (AML), the emergence of treatment resistance remains a formidable clinical challenge. The majority of patients experience relapse within six to nine months. Stroma and microenvironment-derived growth factor signaling converges on key survival pathways, JAK-STAT, MAPK, and IKK-NFκB, which collectively drive leukemic cell persistence resulting to full blown resistance. Consequently, the combined inhibition of FLT3, MAPK, and JAK1/2 have shown effective leukemic suppression that paved the way for the clinical evaluation of combined JAK and FLT3 inhibition. Despite the noted efficacy of Jak2 and FLT3 inhibitor combination in vivo, it is not curative, and all treated mice eventually relapsed. Strategies aimed at greater disease eradication and suppression of resistance are needed. We reasoned that novel drug-induced dependencies unique to each drug can be exploited to develop more effective therapeutic approaches. We performed a whole genome CRISPR-Cas9 dropout screen to identify drug-induced dependencies (DID) caused by momelotinib, gilteritinib, and venetoclax treatment. This chemical-genetic screen identified the genes regulating replication, transcription, splicing, ribosome biogenesis, translation, and DNA damage/repair as common dependencies across all drugs. Consistent with prior findings, gilteritinib significantly depleted genes involved in DNA and purine synthesis, while the loss of P53 and BAX conferred resistance to both gilteritinib and venetoclax. In contrast to other treatments, momelotinib treatment uniquely depleted genes involved in cytokine signaling and the calcineurin-NFATC pathway. Functional studies employing CRISPR-Cas9–mediated gene deletion and cDNA overexpression confirmed that momelotinib treatment induces a selective dependency on calcineurin-NFAT signaling for survival. Using a high-risk murine AML model (Flt3ITD/ITD:Tet2-/-), which is refractory to existing targeted therapies, the combination of momelotinib and calcineurin inhibitors (such as cyclosporine or tacrolimus) resulted in a curative response. Similarly, in patient-derived xenograft (PDX) models that were transplanted with primary chemoresistant AML specimens, mice treated with momelotinib and calcineurin inhibitor-based regimens also exhibited a curative response. Mechanistically, the polypharmacological activity of momelotinib, by effectively blocking JAK-STAT and IKK-NF-κB activity while partially suppressing ERK-AP1 signaling, drives transcriptional reprogramming that enforces greater reliance on calcineurin-NFAT–mediated survival. Integrative analyses combining RNA-seq, ATAC-seq, and CUT&RUN profiling confirmed increased NFAT transcriptional activity at key survival gene loci following momelotinib treatment. Importantly, the combination of momelotinib with calcineurin inhibitors exhibited a robust therapeutic window, with selective targeting of leukemic cells while sparing normal hematopoiesis. This selectivity is likely due to the context-dependent interaction of AP-1, NF-κB, STATs, and NFAT transcription factors in leukemic cells compared to normal cells. Given the profound anti-leukemic efficacy, durable responses, and notable therapeutic window, these findings provide a compelling rationale for clinical evaluation of momelotinib and calcineurin inhibitor combinations in AML.