Functional Complementation between β-Catenin and Hoxa9 Sustains HSC Self-Renewal in a Prmt1-Dependent Manner

Self-renewal is a fundamental property essential for both normal and malignant hematopoietic stem cells (HSCs). While we and others have previously shown that activation of Hoxa9 or β-catenin enhances HSC self-renewal, its inactivation has modest impacts on adult HSCs and their malignant counterparts (Cobas et al., 2004; Lawrence et al., 2005; Siriboonpiputtana et al., 2017; Smith et al., 2011; So et al., 2004; Zhao et al., 2007), suggesting the presence of complementary pathways capable of mediating self-renewal in the absence of either protein. However, simultaneous inactivation of other key Hox genes involved in hematopoietic self-renewal including Hoxb3/b4 did not yield additional hematopoietic phenotypes in the Hoxa9 knockout (KO) background (Magnusson et al., 2007). Similarly, suppression of γ-catenin in β-catenin KO mice did not result in additional hematopoietic defects, and exhibited largely normal hematopoietic functions (Koch et al., 2008). Therefore, the alternative pathways that support hematopoietic self-renewal upon inactivation of Hoxa9 or β-catenin remain largely unknown.

Our recent studies examining the regulation of posterior Hoxa loci reveals a key function of long non-coding RNA, HOTTIP in protecting Hoxa9 gene expression and uncovers a co-regulation of canonical Wnt signalling pathways by HOTTIP in HSCs, providing a molecular link between these two previously unrelated pathways. This finding is also consistent with our recent report demonstrating their functional complementation in AML stem cells, where suppression of Hoxa9 sensitizes HSC-derived AML stem cells to β-catenin inhibition and ablates their transformation ability, suggesting a novel crosstalk between β-catenin and Hoxa9in mediating hematopoietic self-renewal. To this end, the current study developed and characterized a novel β-catenin^fl/fl Hoxa9^-/-Rosa-CreER mouse model. In contrast to single Hoxa9 or β-catenin inactivation where a mild hematopoietic phenotype has been reported in adult HSCs, we found that double inactivation of Hoxa9 and β-catenin resulted in severe hematopoietic defects. In vitroclonogenic assays revealed that bone marrow cells harbouring combined inactivation of Hoxa9/β-catenin generated markedly reduced myeloid colony numbers (>9-fold reduction compared to either single KO alone) of which mature CFU-G (50%) and CFU-E (50%) were the predominant composition. Colonies generated from isolated LSK (Lin^-c-kit⁺Sca1⁺) populations were equivalent in quantity but devoid of multipotential progenitors (CFU-GEMM) in contrast to the diverse colony composition of control and single KO cells. Transplantation of β-catenin^fl/flHoxa9^-/-bone marrow to lethally irradiated recipient mice followed by β-catenin inactivation (4 days tamoxifen treatment) resulted in defects in all HSC and progenitor compartments compared to single KO alone at all time points measured (3wk, 6wk and 12 wk post tamoxifen treatment). Immunophenotypic analysis revealed that the defect originated as early as the LT-HSC stage with a drastic 7-fold reduction in LT-HSCs (Lin^-c-kit⁺Sca1⁺CD150⁺CD48^-) and 3-fold reduction in ST-HSCs (Lin^-c-kit⁺Sca1⁺CD150^-CD48^-). Consistent with a stem cell defect, we also observed significant reductions in downstream myeloid progenitor populations including common myeloid progenitor (CMP) (2-fold), granulocyte-monocyte progenitor (GMP) (2-fold) and megakaryocyte and erythrocyte progenitors (MEP) (7-fold). Mechanistically, Hoxa9 and β-catenin co-regulate expression of Prmt1, a key epigenetic regulator with multifaceted functions in mediating RNA splicing and DNA damage response in hematopoietic cells. To further investigate the role of Prmt1 in hematopoietic development, we generated a novel Prmt1 KO model where Prmt1 can be conditionally inactivated in HSCs. Deletion of Prmt1 alone phenocopied simultaneous inactivation of β-catenin and Hoxa9, resulting in severe reductions in LT-HSC (3.5-fold), ST-HSC (4-fold) and downstream hematopoietic progenitor populations (CMP 4.5-fold, GMP 3.5-fold and MEP 4-fold). Together these data suggest that the posterior Hoxa loci and canonical Wnt pathways are developmentally regulated in a complementary manner as a safeguard mechanism to allow efficient hematopoietic self-renewal, which is largely dependent on intact Prmt1 functions.