Deconstructing The Clonal Advantage And Clonal Stability Of 5q-Candidate Genes In Del(5q) Mds On A Single Cell Level

Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) acquire a clonal advantage in the bone marrow and out-compete normal hematopoiesis. A critical, yet unsolved question remains: how does genetic haploinsufficiency in del(5q) cells contribute to the clonal advantage of HSPCs? We investigated the role of haploinsufficiency for three candidate genes in the common deleted region on chromosome 5 (Csnk1a1, Egr1 and Apc) in direct competition with each other and wild-type (wt) cells on a single cell level by employing a novel lentiviral genetic barcoding strategy. We introduced genotype and cell-specific barcodes into HSPCs from murine models haploinsufficient for Csnk1a1, Egr1 or Apc. Barcoded HSPCs were sort-purified, genotypes mixed and subsequently competitively transplanted into lethally irradiated mice and re-transplanted after 16 weeks in a secondary transplant. The barcoded progeny was reliably recovered from peripheral blood and relative contribution of the barcoded clones to differentiated blood lineages was followed over 32 weeks. Despite heterogeneity in clonal evolution among the mice, all haploinsufficient clones had the potential to outcompete wt clones (3 of 5 mice in the primary transplant, 3 out of 4 mice in the secondary transplant). Csnk1a1 haploinsufficient clones showed the largest clonal abundance and clonal persistence. Expansion of oligoclonal Csnk1a1 haploinsufficient HSPCs was further enhanced in the secondary transplant in all mice. Egr1 haploinsufficient clones showed potential for prominent oligoclonal expansion in one mouse, but decreased in abundance in all secondary transplants. Apc haploinsufficient clones showed persistence but not expansion in 3 out of 5 mice. These results were validated by conventional competitive transplants, which demonstrated that Csnk1a1 and Egr1 haploinsufficient cells achieved the highest advantage over wt hematopoiesis in the primary transplant and more enhanced in the secondary transplant. Since Csnk1a1 regulates β-catenin protein stability, we hypothesized that the clonal expansion of Csnk1a1 haploinsufficient HSPCs is dependent on β-catenin levels. We performed a second genetic barcoding competitive transplant, comparing Csnk1a1-/+ HSPC directly to double haploinsufficient Csnk1a1-/+/Ctnnb1-/+ (β-catenin encoding) HSPCs. We included additional double haploinsufficient mutants Csnk1a1-/+/Apc-/+ and Csnk1a1-/+/Egr1-/+. Results showed pronounced expansion of Csnk1a1-/+ clones, while Csnk1a1-/+/Ctnnb1-/+ clones were outcompeted over time, suggesting that the advantage of Csnk1a1-/+ clones is β-catenin dependent. Csnk1a1-/+/Egr1-/+ and Csnk1a1-/+/Apc-/+ clones were less advantageous than Csnk1a1-/+ clones. To further investigate the mechanism of clonal fitness in Csnk1a1-/+ haploinsufficient HSPCs, we performed droplet based single cell RNA sequencing of Csnk1a1-/+ and wt Lin-Sca1+cKit+ (LSK) HSPCs. Csnk1a1 -/+ LSK were characterized by a higher fraction of cells expressing cell cycle genes compared to wt cells. In line, transcriptional alterations in the most primitive HSCs suggest that the clonal advantage is conveyed by canonical Wnt signaling activating downstream targets such E2F proteins. Csnk1a1-/+ haploinsufficient multipotent progenitors and myeloid/lymphoid primed progenitors expressed marked upregulation of metabolic pathways, mitochondrial respiration, cell cycle and differentiation, ubiquitination/proteasome system and deregulation of ribosome biogenesis. In conclusion, we demonstrate using a novel genetic barcoding approach in a competitive transplant setting that Csnk1a1-/+ haploinsufficient HSPCs have the potential for oligoclonal expansion and clonal persistence. Wnt/β-catenin signaling plays a central role in the clonal expansion. Interestingly, in Csnk1a1 haploinsufficiency the HSC state is preserved and the increased proliferation and metabolic activation are hallmark features of differentiating progenitor cells at MPP stage, increasing with cell cycle activation, thus ensuring clonal stability and preventing HSC exhaustion over time. Disclosures Brümmendorf: University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; Merck: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Ebert:Celgene: Research Funding; Deerfield: Research Funding; Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute..