Growth Factor-Dependent Activation of a MYC-Induced Latent AML Program in Human Hematopoietic Cells

Background: Current evidence suggests that genetic and epigenetic abnormalities drive the development of human Acute Myeloid Leukemias (AMLs). However, whether these are sufficient to establish a permanent, self-sustaining AML population, and the potential role of shared perturbed downstream pathways is unknown. We hypothesized that a modest upregulated expression of MYC might play such a role given its commonly increased expression in many AML patients' cells. To test this hypothesis, we assessed the dynamics and types of cells produced in sublethally irradiated NOD-Rag1-/--IL2Rγc-/-(NRG) mice transgenically producing human IL3, GM-CSF and SCF (NRG-3GS mice) following their transplantation with freshly isolated subsets of normal CD34+ cord blood (CB) cells that were first lentivirally transduced with a human MYC cDNA. Results: FACS and Western blot analyses indicated this produced a 2 to 5-fold increase in MYC mRNA and protein levels in MYC-transduced CD34+ CB cells, and 21/22 NRG-3GS mice injected with ≥6,500 of these cells developed a fatal human AML population within 7 weeks. Histological analysis of their bone marrow and spleen cells showed both contained a prominent human CD123+CD33+CD15±CD34-CD14-CD19-CD3- blast population. Additional limiting dilution transplants showed that both the CD34+CD38- cells (enriched for hematopoietic stem cells) and the more differentiated CD34+ GMPs were similarly highly susceptible (at frequencies of 1/14 and 1/46, respectively) and, in both cases, generated progeny that could initiate serially transplantable leukemias with the same phenotypic and transcriptomic features. Comparison to normal CB cells indicated these most closely resembled GMPs, and comparison to pediatric AML patient samples indicated a similarity to myelomonocytic leukemias with enhanced MYC expression. Interestingly, 14 sublethally irradiated NRG mice (the parental strain not producing human 3GS) transplanted with matched aliquots of CD34+ MYC-transduced cells regenerated a normal spectrum of CD19+ lymphoid cells, CD14+ and CD15+ GM cells and readily detectable CD34+ cells for up to 32 weeks of follow-up with no evidence of leukemogenesis. However, transfer of these regenerated human cells into secondary NRG-3GS mice, even after this extended period, enabled their rapid production of a lethal human AML in all 5 mice tested. In contrast, matched aliquots transplanted into 5 NRG recipients produced declining grafts of normal cells. This finding was then exploited to determine which growth factors were responsible for activating the AML program by transplanting NRG mice with CD34+ CB cells transduced with MYC and just a single growth factor, or all 3 as a positive control. In this set of experiments, a lethal human AML was obtained when MYC was paired with human IL3 or GM-CSF (or all 3 together), but not with SCF (or no growth factors). Conclusion: We report here a new in vivo model of MYC-induced human myeloid leukemogenesis that produces a serially transplantable AML closely resembling human pediatric myelomonocytic leukemias with elevated MYC expression. The rapidity, consistency, and high frequency of this transformation process obtained by transducing late granulopoietic as well as early types of normal human CD34+ progenitor cells makes this system highly attractive for future mechanistic and therapeutic testing experiments. The discovery that MYC deregulation alone generates a stable "latent program" that can be rapidly activated by exposure to exogenous growth factors typical of inflammatory states also raises intriguing questions about the potential role of such events in the genesis of AML populations that arise in patients. Disclosures Beer: Karus therapeutics Ltd.: Employment.