Oncogene Cooperativity Analysis Reveals a Novel Set of Genes That Regulate the In Vivo Growth and Survival of Leukemia Stem Cells

Abstract 553

In order to increase our understanding of key biological properties governing the development of leukemia stem cells (LSCs), we employed a novel gene identification strategy based on cooperation between initiating oncogenes. Previous studies have demonstrated that genes whose expression is regulated in a synergistic manner as a consequence of two cooperating oncogenes (termed “cooperativity response genes”, or CRGs) are highly enriched for activity in tumor formation. Further, in contrast to the thousands of genes identified by differential expression analyses of normal vs. leukemic cell populations, CRGs represent a much smaller subset of targets; thereby, providing a defined set of genes to investigate. We adapted the CRG strategy to identify synergistically regulated genes in primitive leukemic cells. Using a mouse model of myeloid blast crisis leukemia induced through the cooperation of BCR-ABL and NUP98-HOXA9, we performed genome-wide transcriptional profiling comparing hematopoietic cells expressing each translocation alone or in combination. Using this system, we were able to model the genetic alterations induced as normal cells progressed towards LSC transformation, identifying 72 CRGs (50 aberrantly up-regulated and 22 down-regulated) with potential importance in leukemia development. To investigate the relevance of these CRGs in leukemia biology, an RNAi screen approach was employed. Primary leukemic progenitors were purified and transduced with a custom lentiviral RNAi library and subsequently transplanted into recipient animals to assess the engraftment potential upon perturbation of the individual CRGs. Our findings demonstrate that knock-down of expression in 35 of 50 (70%) leukemia CRGs reduced in vivo growth of primitive leukemia, a finding that was independently validated through single gene perturbation of several genes that scored in the RNAi screen (GJB3, EphA3, PMP22, Serinc2, SerpinB2, and CP). In particular, serpinB2, a gene that scored strongly in the RNAi analysis, was shown to directly effect the frequency of LSC in vivo. Given that the cooperative gene signature represented genes with many distinct cellular functions, we hypothesized that the CRG expression profile represents a key regulatory network in leukemia survival. To investigate our hypothesis we utilized the Broad Institute's Connectivity Map (CMAP) to identify pharmacological compounds with the ability to modulate multiple CRGs simultaneously. This analysis revealed that both Tyrophostin AG-825 (AG825) and 4-hydroxy-2-nonenol (4HNE) were predicted to reverse the gene expression induced as a consequence of leukemic transformation. To test the effect of these agents as selective toxicants to leukemia, we treated both normal and leukemia murine bone marrow cells with each compound. Both bulk and phenotypically primitive leukemic cells were eradicated in dose-responsive fashion upon treatment with either AG825 or 4HNE, while normal cells showed significantly reduced sensitivity. Progenitor function as measured by colony forming assays also showed a selective reduction in leukemia colony formation, suggesting that both these compounds are toxic to the majority of leukemic cell types. Interestingly, similar results were obtained when human normal and leukemic bone marrow specimens were treated with both drugs, suggesting the CRG signature represents an important class of genes with conserved function across species. To determine the level of conservation of the leukemia CRG signature between murine and human leukemia, we profiled eight normal and leukemic patient specimens for expression of the CRG signature. Of the 39 evaluable human CRG orthologs, 13 showed similar expression trends in human leukemia samples relative to normal controls. Intriguingly, both AG825 and 4HNE were predicted to inhibit this 13-gene signature by the CMAP database, suggesting that the compounds may act through these genes to influence leukemia cell death. Taken together, our findings demonstrate the importance of cooperative gene regulation in leukemogenesis and provide a novel platform for future research toward more effective therapeutic strategies to treat leukemia.