The novel hypomethylating agent NTX-301 reprograms epigenetic and Hippo signaling pathways and exhibits pre-clinical activity in venetoclax-resistant and TP53-mutant AML.
TL;DR
Data suggest that NTX-301 exerts potent anti-leukemia activities superior to 5-AZA and synergizes with VEN in VEN-resistant and TP53-mutant AML, in part by suppressing DNMT1 and inducing DNA damage responses and apoptosis, by inducing p53 signaling and demethylating LATS1/2, thus activating Hippo signaling.
Abstract
Background
Hypomethylating agent (HMA) and the BCL-2 inhibitor venetoclax (VEN) combinations have evolved into frontline therapies for patients with acute myeloid leukemia (AML), yielding high response rates. However, most patients ultimately relapse, particularly those with TP53 mutations. We investigated mechanisms of action and therapeutic efficacy of NTX-301, a next-generation HMA. Methods used include flow cytometry-based cell viability assays, Western blot, reverse-phase protein arrays, RNA-sequencing, CyTOF single-cell mass cytometry, and methylation profiling in various therapy-resistant AML models.
Results
We demonstrate that NTX-301 exhibits superior efficacy compared to 5-azacytidine (5-AZA) in 5-AZA or VEN-resistant AML. It synergizes with VEN in VEN- or VEN/HMA-resistant and TP53-mutant AML blasts and stem/progenitor cells (combination index<1). NTX-301 inhibits DNMT1 and increases p73, caspase-8/activated caspase-8 levels in TP53-WT and TP53-mutant AML and activates p53 signaling. It extends survival (≥45%) in both, xenograft and PDX models. Methylation profiling revealed that NTX-301 is a more targeted HMA compared to 5-AZA, enabling suppression of functionally enriched genes/pathways. Pathway analysis of 954 commonly hypomethylated genes showed profoundly greater enrichment of Hippo signaling in NTX-301-treated compared to 5-AZA-treated cells, and enrichment of insulin signaling, VEGF pathway, and cell cycle selectively in NTX-301- but not in 5-AZA-treated cells. NTX-301-mediated Hippo signaling was validated at protein levels.
Conclusion
Data suggest that NTX-301 exerts potent anti-leukemia activities superior to 5-AZA and synergizes with VEN in VEN-resistant and TP53-mutant AML, in part by suppressing DNMT1 and inducing DNA damage responses and apoptosis, by inducing p53 signaling and demethylating LATS1/2, thus activating Hippo signaling.