Gene expression change after inactivation of ARHGAP45 by CRISPR-Cas9 and/or treatment of MBQ-167

GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) function as negative and positive regulators of small GTPases, orchestrating GDP/GTP-bound binary activity switches. Yet their functional significance in hematopoietic malignancies remains largely uncharted. In our study, guided by single-cell transcriptomic analysis and functional CRISPR screens in primary AML specimens and their derived models, we uncover an unexpected and selective oncogenic role for ARHGAP45 in AML. ARHGAP45 is upregulated in malignant cells while notably absent in healthy counterparts. Depletion of ARHGAP45 impedes AML growth and triggers myeloid differentiation but is dispensable for normal CD34+ hematopoietic stem/progenitor cells (HSPC) development. Using CRISPR-mediated epistasis screens, we further show that the ARHGEF1-RhoA-PKN1 axis is responsible for the ARHGAP45 phenotype. In addition, we further found that ARHGAP45 prevents RhoA overactivation, loss of cell polarity, and lineage commitment. On the other hand, our screens also unveil the synergistic effect of combinatorial ARHGAP45 and CDC42 inhibition, which we further validate in vitro and in vivo. Collectively, our studies reveal that ARHGAP45 is indispensable for leukemia stemness and aggressiveness and rationalize therapeutic strategies for modulating GAP and GTPase activity in AML.