Loss of Function RUNX1 Mutations Restrict Protein Biosynthesis during Pre-Leukemia and MDS Transition but Not after Leukemic Transformation

Runx1, a DNA binding subunit of core binding factors, is found frequently mutated in hematological malignancies. Runx1 mutation can be an early event in leukemogenesis endowing pre-leukemic stem cells with a selective advantage in the bone marrow, and is associated with an unfavorable outcome. In mouse models, loss of function (LOF) Runx1 mutations cause a broad decrease of ribosome biogenesis in hematopoietic stem and progenitor cells (HSPCs) by directly binding to ribosomal related genes essential for protein synthesis, and confers resistance to genotoxic stress (Cai et al. 2015 Cell Stem Cell 17(2):165-77). Paradoxially, leukemia cells generally require higher biosynthetic activity, and AML patients with LOF Runx1 mutations show upregulated ribosome signatures compared with those without Runx1 mutations (Silva et al., 2009 Blood 114:3001-3007). It remains unclear whether RUNX1 plays a role in regulating protein synthesis in leukemogenesis as in normal HSPCs, and if LOF Runx1 mutations are important for leukemia initiation, transformation and/or maintenance. To examine such mechanistic roles of RUNX1 in AML progression, we have used a previously reported MLL-PTD; Mx Cre; Runx1 Flox/Flox (Double mutant -DM) mouse model (Hayashi et al., 2015 Blood 126:303 ) that allows experimental tracking of the step-wise transition of HSPCs from pre-disease stage to a MDS-like stage, prior to full blown AML. Various subpopulations of the HSPCs, including genotypic HSCs, MPP, GMP, CMP, MEP, were isolated from the mice at pre-disease, MDS-like, and AML full-blown stages, and were assayed for protein synthesis rates by O-propargyl puromycin incorporation, DNA synthesis rates by BrdU labeling, and FACS analysis. At the pre-disease state, DM HSCs, as well as all the progenitor populations, had lower protein biosynthesis activity compared with similar populations of wild-type control or MLL-PTD mutant mice, consistent with LOF Runx1 mutations providing stress-resistance and survival advantage. As disease progressed, the DM mice developed MDS-like phenotypes including severe anemia and bone marrow fibrosis, with the HSCs (LSK CD34^-Flt3^- cells) showing increased protein synthesis rate compared with the pre-disease DM mice. Upon the onset of full-blown leukemia, the protein translation rates in all subpopulations of DM HSPCs were significantly faster than the control non-leukemic cells, regardless of the Runx1 mutant status. Importantly, preliminary analyses of two human AML samples found that CD34⁺ cells with LOF Runx1 mutations displayed a similarly enhanced protein synthesis rates than CD34⁺ leukemia bone marrow cells carrying wild type Runx1, as seen in the mouse model. Our results show that at early initiation, LOF Runx1 mutation supresses protein biosynthesis; during transition to MDS, the inhibitory regulation was bypassed in LT-HSCs (LSK CD34^-Flt3^-), suggesting that Runx1-controlled protein translation is involved in the early clonal selection of disease progression. In full-blown leukemia cells including the primitive subpopulations, however the protein synthesis rate appears to become uncoupled from Runx1 regulation possibly due to an activation of compensatory machineries. This study of the role of Runx1 mutation in pre-leukemia cell progression to full blown leukemia raises the question that while some tumor initiating mutations such as LOF Runx1 mutations contribute to the tumor initiation and transformation process, they may not be essential for maintaining certain crucial leukemia cell phenotypes such as protein biosynthesis.