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

Dustin T Rae,H Joachim Deeg,Jonah D Hocum,Grant D Trobridge,Victor Bii

doi:10.18632/oncotarget.5133

Dustin T Rae, H Joachim Deeg + Show 3 more

Open Access

https://doi.org/10.18632/oncotarget.5133

Copy DOI

Abstract

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.

Highlights

Acute myeloid leukemia (AML) is a clonal disorder arising from a heterogeneous population of pre-leukemic and leukemic cells
Our strategy was to engineer a genotoxic shuttle vector rescue plasmid that expressed the D171N mutant to identify genes that may collaborative via insertional mutagenesis
With the gene alterations recurring in many AML patients, and differential expression between high and low risk AML, we investigated whether the genes identified could stratify patients for overall survival and disease free www.impactjournals.com/oncotarget survival (Fig. 6)

Summary

Introduction

Acute myeloid leukemia (AML) is a clonal disorder arising from a heterogeneous population of pre-leukemic and leukemic cells. Investigations of the molecular mechanisms that mediate AML are confounded by tumor heterogeneity, which makes it difficult to identify the causative mutation amongst the multiple background or passenger mutations accumulated in cancerous cells. Several driver genes have been identified in myeloid leukemogenesis, many mutations and potential collaborating mutations have yet to be identified. The RUNX1 (AML1) mutant (D171N) occurs in 40% of myelodysplastic syndromes (MDS), with 30%–40%. Identifying the genes that collaborate with the D171N mutation is important as these genes may have prognostic potential to improve outcomes for MDS/AML patients. With molecular testing entering mainstream clinical practice, identifying new driver genes and contributing factors may improve the ability to screen and diagnose patients [3]

Objectives

Methods

Results

Conclusion