Targeting alternative transcription/translation of RUNX1-ETO fusion gene

Loyola University Chicago, Maywood IL

Investigators

Abstract

? DESCRIPTION (provided by applicant): Leukemia fusion genes, the fusion products of chromosomal translocations, are detected in >55% leukemia patients. Leukemic cells develop addiction to and reliance upon leukemia fusion proteins, the protein products of fusion genes, for their proliferation and survival. There is considerable speculation that targeted inactivation f leukemia fusion proteins is a most effective strategy for anti-leukemia therapy. We conjecture that inhibiting the production of fusion proteins by repressing the transcription and/or translatio of fusion genes represents a novel and perhaps more effective treatment for leukemia. We want to use leukemia cells that are RUNX-ETO+ (a fusion gene in which the ETO gene has been fused to the RUNX1 gene due to t(8;21)) as a model system to test such a hypothesis because the promoters for RUNX1 and the 5'UTRs for RUNX1 transcripts have been well documented. RUNX1 has two promoters, P1 and P2, which produce transcripts which have two distinct 5'UTRs. P1 transcripts have a <450bp 5'UTR the translation of which is regulated by a cap-dependent mechanism, while P2 transcripts have a ~1.6kb 5'UTR the translation of which is regulated by a cap-independent IRES-mediated mechanism. Interestingly, the expression of RUNX1 in hematopoietic stem/ progenitor cells (HSPC) is primarily controlled by P1, while the expression of RUNX1-ETO in leukemia cells is controlled by P2. Such use of alternative promoter and translational entities by RUNX1-fusion genes in leukemia cells provide us with an opportunity to treat RUNX1-fusion gene+ leukemia by specifically inhibiting the transcription/translation of the fusion genes with fewer effects on normal HSPC. Many inhibitors of translation have been used clinically to treat leukemia, but with limited success. All such inhibitors were designed to repress cap-dependent translation; inhibitors for cap-independent translation have not yet been identified. We want to: 1) develop morpholino antisense oligonucleotides (MPOs) to specifically inhibit RUNX- ETO by targeting the translation of P2-transcripts; 2) identify chemicals that can repress P2-controlled RUNX1-ETO expression and IRES-mediated RUNX1-ETO translation in leukemia cells by a large-scale screening of a small molecule library. To do so, we generated an RE-GFP reporter cell line by fusing a GFP to the last exon of RUNX1-ETO fusion gene in Kasumi-1 cells. The intensity of GFP in such reporter cells reliably reflects the levels of RUNX1-ETO protein. Using this reporter cell line, we will evaluate the efficiency of MPOs in order to identify the most efficient MPOs for potential clinical translation and screen a small molecule library in order to identify small molecules that can specifically repress the production of RUNX1-ETO protein in leukemia cells. The candidates identified in such screening will further be evaluated to determine whether they can repress the growth of RUNX1-fusion gene+ leukemia cells without affecting the growth of normal HSPC. We also want to determine whether the candidates inhibit the production of RUNX1-ETO by repressing transcription or translation of fusion gene.