A genetic screen in Drosophila uncovers the multifaceted properties of the NUP98-HOXA9 oncogene.

Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 to the homeodomain of the transcription factor HOXA9. The mechanisms enabling NA9-induced leukemia are poorly understood. Here, we conducted a genetic screen in Drosophila for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, we discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, our work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets.

Human NUP98-HOXA9 promotes hyperplastic growth of hematopoietic tissues in Drosophila.

Acute myeloid leukemia (AML) is a complex malignancy with poor prognosis. Several genetic lesions can lead to the disease. One of these corresponds to the NUP98-HOXA9 (NA9) translocation that fuses sequences encoding the N-terminal part of NUP98 to those encoding the DNA-binding domain of HOXA9. Despite several studies, the mechanism underlying NA9 ability to induce leukemia is still unclear. To bridge this gap, we sought to functionally dissect NA9 activity using Drosophila. For this, we generated transgenic NA9 fly lines and expressed the oncoprotein during larval hematopoiesis. This markedly enhanced cell proliferation and tissue growth, but did not alter cell fate specification. Moreover, reminiscent to NA9 activity in mammals, strong cooperation was observed between NA9 and the MEIS homolog HTH. Genetic characterization of NA9-induced phenotypes suggested interference with PVR (Flt1-4 RTK homolog) signaling, which is similar to functional interactions observed in mammals between Flt3 and HOXA9 in leukemia. Finally, NA9 expression was also found to induce non-cell autonomous effects, raising the possibility that its leukemia-inducing activity also relies on this property. Together, our work suggests that NA9 ability to induce blood cell expansion is evolutionarily conserved. The amenability of NA9 activity to a genetically-tractable system should facilitate unraveling its molecular underpinnings.

Inhibitors that stabilize a closed RAF kinase domain conformation induce dimerization.

RAF kinases have a prominent role in cancer. Their mode of activation is complex but critically requires dimerization of their kinase domains. Unexpectedly, several ATP-competitive RAF inhibitors were recently found to promote dimerization and transactivation of RAF kinases in a RAS-dependent manner and, as a result, undesirably stimulate RAS/ERK pathway-mediated cell growth. The mechanism by which these inhibitors induce RAF kinase domain dimerization remains unclear. Here we describe bioluminescence resonance energy transfer-based biosensors for the extended RAF family that enable the detection of RAF dimerization in living cells. Notably, we demonstrate the utility of these tools for profiling kinase inhibitors that selectively modulate RAF dimerization and for probing structural determinants of RAF dimerization in vivo. Our findings, which seem generalizable to other kinase families allosterically regulated by kinase domain dimerization, suggest a model whereby ATP-competitive inhibitors mediate RAF dimerization by stabilizing a rigid closed conformation of the kinase domain.