A novel retroviral mutagenesis screen identifies prognostic genes in RUNX1 mediated myeloid leukemogenesis

Using a novel retroviral shuttle vector approach we identified genes that collaborate with a patient derived RUNX1 (AML1) mutant. RUNX1 mutations occurs in 40% of myelodysplastic syndromes (MDS). MDS are a group of hematopoietic stem cell disorders that are characterized by dysplasia that often progress to acute myeloid leukemia (AML). Our goal was to identify genes dysregulated by vector-mediated genotoxicity that may collaborate with the RUNX1 mutant (D171N). D171N expressing cells have a survival and engraftment disadvantage and require additional genetic lesions to survive and persist. By dysregulating genes near the integrated vector provirus, the shuttle vector can promote transformation of D171N cells and tag the nearby genes that collaborate with D171N. In our approach, a gammaretroviral shuttle vector that expresses D171N is used to transduce CD105⁺, Sca-1⁺ mouse bone marrow. Mutagenized cells are expanded in liquid culture and vector integration sites from surviving cells are then identified using a retroviral shuttle vector approach. We repeatedly recovered integrated vector proviruses near genes (Itpkb, Ccdc12, and Nbeal2). To assess the prognostic significance of the genes identified we examined differential expression, overall survival, and relapse free survival of AML patients with alteration in the genes identified using The Cancer Genome Atlas (TCGA) AML data set. We found that ITPKB functions as an independent factor for poor prognoses and RUNX1 mutations in conjunction with ITPKB, CCDC12, and NBEAL2 have prognostic potential in AML.