HMX3 is a critical vulnerability in MECOM-negative KMT2A::MLLT3 acute myelomonocytic leukemia.

KMT2A::MLLT3 acute myelomonocytic leukemia (AML) comes in two clinically and biologically different subtypes. One is characterized by inferior outcome, older age, and MECOM oncogene expression. The other is mainly observed in children and young adults, associates with better clinical outcome, but lacks MECOM. To identify cell fate determining transcription factors downstream of KMT2A::MLLT3, we applied a bioinformatic algorithm that integrates gene and enhancer expression from primary MECOM-positive and -negative KMT2A::MLLT3 AML samples. This identified MECOM to be most influential in the MECOM-positive group, while neuronal transcription factor HMX3 was most influential in the MECOM-negative group. In large AML cohorts, HMX3 expression associated with a unique gene expression profile, younger age (p < 0.002) and KMT2A-rearranged and KAT6A-CREBBP leukemia (p < 0.00001). HMX3 was not expressed in other major genetic risk groups and healthy blood cells. RNA-sequencing analyses following forced HMX3 expression in healthy CD34+ cells and its silencing in KMT2A::MLT3 cells showed that HMX3 drives cancer-associated E2F and MYC gene programs (p < 0.001). HMX3 expression in healthy CD34+ cells blocked monocytic but not granulocytic colony formation. Strikingly, HMX3 silencing in KMT2A::MLLT3 patient cells resulted in cell cycle arrest, monocytic differentiation and apoptosis. Thus, the neuronal transcription factor HMX3 is a leukemia-specific vulnerability in KMT2A::MLLT3 AML.