An Insertional Mutagenesis Screen for Genes That Cooperate with Mll-AF9 in Leukemogenesis.
Authors: Bergerson Rachel Joy , Collier Lara S , Lugthart Sanne , Allaei Raha , Nixon Molly J , Silverstein Kevin A.T. , Sarver Aaron , Fan Danhua Flora , Lamblin Anne-Francoise J , Wolff Linda , Kersey John H. , O'Sullivan M. Gerard , Delwel Ruud , Kogan Scott C. , Adams David J , Largaespada David 
Published: October 2019

Abstract
Abstract 3962
Poster Board III-898
By altering the activity of specific transcription complexes, the MLL-AF9 fusion oncogene can initiate the process of acute myeloid leukemia (AML) development. However, all the genetic pathways that can cooperate with MLL-AF9 expression to cause full-blown AML are unknown. These pathways will provide therapeutic targets for MLL-AF9-associated AML. Mice with constitutive expression of the Mll-AF9 fusion oncoprotein under the control of the endogenous promoter develop AML but only after a prolonged latency. This model thus provides a system for understanding the evolution of AML initiated by an MLL fusion oncoprotein. We hypothesized that infection with a recombinant Murine Leukemia Virus, abbreviated M4070, could cooperate with MLL-AF9 expression to accelerate the onset of leukemia by causing the secondary mutations required for cancer progression. We bred Mll-AF9 heterozygous males to wild type females, and the offspring were injected at three days of age with M4070 virus (n=211) or were mock infected (n=68). All mice were genotyped and observed for disease progression. Virally infected Mll-AF9/+ mice succumb to disease with a significantly reduced latency period when compared to virally infected wild type (WT) mice (p < .0001) and uninfected Mll-AF9/+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. Histopathology, immunohistochemical staining, analysis of the surface immunophenotype by flow cytometry, and Southern blot analysis of T and B cell receptor rearrangement indicated that infected Mll-AF9/+ animals developed primarily myeloid leukemia (myeloperoxidase positive, Mac1 or Mac1/Gr1 positive, CD3 negative) while infected WT animals developed mostly lymphoid leukemia (CD3 positive, CD4 and/or CD8 positive, myeloperoxidase negative). Retroviral insertion sites were cloned from 167 leukemic tissues from the accelerated leukemia mice using two different restriction enzymes in a shotgun-based, linker-mediated, cloning protocol to identify the genes most frequently mutated in Mll-AF9 positive leukemia. More than 4,100 independent insertions were isolated and 101 common insertion sites (CIS), defined as genomic locations with several proviral insertions from at least 3 mice, were identified. The majority of the CIS harbored proviral insertions in both Mll-AF9/+ and wild type mice, but a subset of CIS were found in only one group or the other. Some of the genes closest to the CIS have been identified as target genes in other proviral screens and some are known cancer genes. We studied a subset of the CIS-associated genes for aberrant expression in leukemic tissues. There was elevated expression of Mn1, and a trend towards increased expression of Bcl11a and Fosb, in our Mll-AF9 murine leukemia samples with proviral insertions nearby these genes. Moreover, elevated expression of MN1, FOSB, and BCL11A has been observed in microarray studies of human patients with AML. We have completed a bone marrow transduction/transplantation experiment to seek functional evidence of cooperation with Mll-AF9. Mice transplanted with Mll-AF9/+ bone marrow that had been transduced with a retrovirus encoding the candidate gene MN1 succumb to myeloid malignancy faster than mice transplanted with wild type bone marrow transduced with MN1, or Mll-AF9/+ bone marrow transduced with a retrovirus encoding just the Green Fluorescent Protein gene. This data suggests that MN1 can cooperate with Mll-AF9 to accelerate myeloid leukemia in a mouse model. We are currently using shRNA knockdown strategies in human cell lines to confirm cooperation of more candidate genes with MLL-AF9 in AML development. Thus, CIS-associated genes from leukemias accelerated by M4070 in Mll-AF9/+ mice may help define important genetic pathways that are altered during progression of AML induced by MLL fusion oncogenes.

Disclosures:
No relevant conflicts of interest to declare.