TRIB2 Transformed GMP is the Myeloid Leukaemia Initiating Cell

serine/threonine pseudokinases. When ectopically expressed in hematopoietic stem and progenitor cell (HSPC) enriched bone marrow cells, TRIB2 was shown to induce acute myeloid leukemia (AML) in a murine transplant model dependent on C/EBPα degradation, showing it to be a potent myeloid oncogene. The cell of origin or leukemia initiating cell (LIC) refers to the cell from which a specific leukemia normally arises, and it is hypothesised that the LIC may influence the progression, disease phenotype and response to therapy. It remains unclear whether the LIC is a HSC or a more committed progenitor cell in TRIB2-driven AML. Aims: Our current study focuses on identifying the LIC in TRIB2-driven AML and characterizing its role in disease potency and maintenance. Methods: FACS sorted CD45.2+ stem and progenitor cell populations (HSC, MPP, CMP, GMP and MEP) transduced with a lentiviral vector encoding TRIB2 were cultured in methocult supplemented with cytokines that support myeloid cell growth and differentiation. Following the first plating (P1), GFP+ cells were sorted and serially replated. Cells which still formed colonies by the third replating (P3) implied acquisition of self-renewal ability and increased proliferation characteristic of myeloid transformation. These CD45.2+ cells from P3 were transplanted into sublethally irradiated CD45.1+ C57BL/6 recipient mice and chimeric animals were monitored for 1 year. Chemoresistance experiments were performed on the bulk bone marrow population (CD45.2+ cells >95%) from the TRIB2 AML mice. Cells were treated with a range of concentrations of daunorubicin (DNR), followed by trypan blue cell counts to assess viability. Results: Our study identified that while lentivirally transduced TRIB2 can transform all stem and progenitor cell populations of the hematopoietic system with variable efficiencies in vitro, the GMP subpopulation was identified as the LIC of TRIB2-driven AML. TRIB2 transformed GMP cells generated a more potent AML with complete penetrance and shortened latency compared to all other HSPC populations analysed. Indeed, phenotypically different diseases were propagated from TRIB2 expression in the HSC and GMP, with the former having a weakly penetrant, longer latency AML with a mixed lineage phenotype, whereas the later was a dominantly myeloid disease phenotype with a short latency. We next addressed the chemotherapeutic response of TRIB2 positive AML cells. We show that GMP-TRIB2 AML and bulk TRIB2 AML cells are chemoresistant. TRIB2 overexpression decreases DNR induced apoptosis, and knockdown of TRIB2 expression in AML cells leads to an increase in apoptotic gene expression. Our studies illustrate that TRIB2 expression is key in mediating the anti-apoptotic signals following DNR treatment. Summary/Conclusions:We identify the GMP as the LIC in TRIB2 driven AML. Our findings are further supported by our previous work showing that degradation of C/EBPa is required for TRIB2-driven AML and the GMP population expresses the highest level of C/EBPa in hematopoiesis. We provide evidence for TRIB2 role in chemoresistance, and that the TRIB2 LIC is a highly chemoresistant cell. Our findings provide insight into the molecular events contributing to AML, and provide potential for novel avenues for therapeutic targeting.