Oncogene Withdrawal Selectively Alters Phosphoprotein States and Shifts Differentiation Status In Myeloid Leukemia Subpopulations

Abstract 3160

While oncogene addiction is a well-documented phenomenon, the molecular mechanisms by which oncogene withdrawal triggers cell death are poorly understood. Interrogation of this phenomenon in a manipulatable murine model, coupled to concomitant analysis of human AML samples, could elucidate this phenomenon for therapeutic applications. In order to decipher these molecular mechanisms, we employ a murine model harboring a tetracycline repressible, activated NRAS (NRAS^G12V) transgene along with an MLL/AF9 transgene to induce AML development. Primary leukemia cells are then transplanted into SCID mice and, upon development of full-blown leukemia, NRAS^G12V transgene expression is repressed with doxycycline. Previous work has shown that repression of NRAS^G12V in this model leads to widespread apoptosis of the leukemia cells (Kim et al. Blood 2009). To analyze the kinetics of this response, we found that the tumor burden, as assayed by the white blood cell (WBC) count, declines by 60 hours of doxycycline treatment. NRAS^G12V message levels are undetectable by 12 hours while protein expression begins to decline after 48 hours. To dissect the signaling network directing the apoptotic response, the phosphorylation status of critical signaling intermediates was analyzed by flow cytometry at time points from 48–96 hours post-doxycycline treatment. This analysis revealed numerous modifications in known NRAS effectors including loss of phosphoErk1/2, phosphoSTAT3, and phosphop38. These alterations correlate with immunophenotypic cell surfaces markers. Whereas leukemia cells expressing mature myeloid markers (Mac1+) do not exhibit alterations in any of the phosphoproteins tested, only Mac1- leukemia cells show meaningful changes in RAS-activated signaling molecules. Whether mouse leukemic stem cells reside in the Mac1+ fraction or in the less differentiated subpopulation remains unclear. These findings suggest that subpopulations of leukemia cells exhibit differential vulnerabilities to oncogene addiction. Furthermore, these studies also reveal that oncogene withdrawal leads to a reduction of the Mac1+Gr1- population and an enrichment of the Mac1-Gr1- and Mac1+Gr1+ populations. Therefore, in addition to effecting apoptosis, oncogene withdrawal leads to alterations in the differentiation status of the leukemia, which could alter the self-renewal capacity of these cells. Using these results, we have designed an extensive antibody panel and are currently using a CyTOF mass spectrometer, a new technology that allows us to perform 30 dimensional measurements to profile immunophenotypic markers and phosphoprotein states with single cell resolution. Simultaneous, high-dimensional single cell profiling of signaling states enables integrative network analysis of these data and as such will allow us to discern pathway dependencies and regulatory relationships that traditional low dimensional flow cytometry cannot (Sachs et al. Science 2005, Sachs et al. IEEE Eng Med Biol Soc 2009). This approach will provide a novel and powerful method to elucidate the critical pathways and leukemic subpopulations that define the response to oncogene withdrawal. Furthermore, these findings will be compared to a concomitant study of 30-dimensional measurements of human AML samples which could facilitate harnessing the oncogene addiction phenomenon in therapeutic applications.