C-MYC Augments the Proliferation and Survival of Hematopoietic Stem Cells and Multipotent Progenitors to Drive Myeloproliferative Neoplasms

Human genomic studies have identified frequent MYC amplification and copy number gains in myeloid malignancies, and previous studies have shown that MYC plays important roles in survival of Myeloproliferative Neoplasms (MPN) and Acute Myeloid Leukemia (AML) cells. Notably, our recent studies have shown that MYC impairs myeloid cell differentiation and promotes proliferation of myeloid progenitors and AML cells by controlling genomic methylation. However, it is unclear if increased levels of MYC in hematopoietic stem cells (HSCs) and myeloid progenitors is sufficient to provoke the development of MPN or AML and, if so, how this occurs.

To addresses these questions we generated Mx1-Cre;Rosa26-LSL-MYC transgenic mouse model that inducibly overexpress MYC following polyinosinic:polycytidylic acid (pIpC) injection and Cre-mediated deletion of loxp-stop-loxp cassette. MYC overexpression was confirmed by qRT-PCR and immunoblot. Complete blood counts (CBC) with differential in the Mx1-Cre ^+/-;Rosa26-LSL-MYC ^+/+mice vs. -MYC ^+/-or -wild type (WT) littermate mice at week 23 revealed worsening anemia (Hb, 9.6 vs. 16.3 vs. 15.5g/dL, p<0.0001), lymphopenia (73.2 vs. 84.3 vs. 84.5%, p<0.0001), and monocytosis (7.4 vs. 1.8 vs. 0.9%, p=0.0097). Also, bone marrow (BM) cells from the Mx1-Cre ^+/-;Rosa26-LSL-MYC ^+/+ mice showed increased monocyte- and granulomonocyte-colony forming potential (CFU-M and CFU-GM), but with limited self-renewal capacity ex vivo (i.e., no CFU after 5 serial plating). Further, inducible MYC overexpression promotes expansion of HSCs (Lin ^-Sca-1 ⁺cKit ⁺ [LSK]), multipotent progenitors (MPPs; LSK CD48 ⁺CD150 ^-), common myeloid progenitors (CMPs; Lin ^-Sca1 ^-cKit ⁺), granulocyte-monocyte progenitors (GMPs; Lin ^-Sca-1 ^-cKit ⁺CD34 ⁺FCγR ⁺), and Gr-1/CD11b+ mature myeloid cells, with concomitant reduction of B220+ or CD3+ cells in the BM and spleen. In addition, MYC overexpression provokes splenomegaly (565 vs. 150 vs. 100mg at week 18~22, p<0.0001), extramedullary hematopoiesis with markedly atypical megakaryopoiesis and myeloid preponderance akin to MPN that reduces overall survival (median OS, 157 days vs. not reached vs. not reached, p<0.0001). Collectively, these findings suggest MYC confers enhanced proliferation and survival properties to HSCs and MPPs leading to MPN-like disease.

We have shown MYC oncogenic functions in AML cells requires its suppression of TFEB, an mTORC1 regulated bHLH-LZ transcription factor, and that TFEB functions as a tumor suppressor by inducing IDH1/2-TET2 signaling, thus promoting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) conversion in key genes that drive myeloid differentiation and cell death. Similarly, inducible overexpression of MYC in the Mx1-Cre ^+/-;Rosa26-LSL-MYC ^+/+ mice significantly reduces the expression of Tfeb, Idh1 and Idh2, and 5hmC levels in both c-Kit ⁺ and Cd11b ⁺ BM cells. Further, 4-OHT-mediated silencing of Myc in ex vivo cultured BM cells from the Rosa26-CreER ^T2+/-;Myc ^fl/fl mice impairs myeloid cell proliferation and robustly induces the expression of Tfeb, Idh1, and Idh2 as well as levels of 5hmC. Finally, inducible TFEB expression in normal 32D.3 myeloid progenitor cells impairs cell proliferation and upregulates 5hmC levels, and these responses are partially reversed by treatment with 2-hydroxyglutarate, an oncometabolite that inhibits 5mC-to-5hmC conversion. Collectively, these findings suggest that the MYC-TFEB-IDH1/2 epigenetic circuit plays a pivotal role in promoting myeloid proliferation to drive the malignant transformation of HSCs to the MPN.