Cellular Barcoding Reveals That the Adult Hematopoietic Stem Cell Niche Is Clonaly and Transcriptionally Remodeled by Acute Leukemia

Cellular Barcoding Reveals That the Adult Hematopoietic Stem Cell Niche Is Clonaly and Transcriptionally Remodeled by Acute Leukemia

Leonard I. Zon¹

¹Boston Children’s Hospital, Harvard Medical School, Harvard University, Boston MA USA

Adult hematopoietic stem cells (HSCs) reside niches that provide key regulatory signals for their maintenance and function. Leukemia is associated with alterations of the niche that may support tumor growth. Mutation or activation of the MYC oncogene family is a frequent event leading to the induction of leukemogenesis. We developed a new zebrafish model of acute erythroid leukemia (AEL) by driving overexpression of human CMYC under the blood specific promotor draculin (drl). Flow cytometry analyses of drl:CMYC adult marrows demonstrated a significant expansion of the progenitor compartment (fc = 4.8; p < 0.000001) accompanied by a significant decrease of mature blood cell types compared to controls (erythroid, lymphoid and myeloid, fc = -4.5, -3.3 and -6.5 ; p < 0.000001). Bulk RNA-Sequencing of drl:CMYC marrows revealed a significant upregulation of the erythroid master regulator gata1a (fc = 1.4, p = 0.01) and fetal hemoglobins hbbe1.1 and hbbe1.2 (fc = 4.7 and 2.9, respectively; p = 0.0004). Transplantation of drl:CMYC marrows resulted in robust engraftment and disease propagation upon primary (7 engrafted / 7 transplanted recipients) and secondary (17/18) transplants. We used GESTALT (Genome Editing of Synthetic Target Arrays for Lineage Tracing) to uniquely barcode single cells using the CRISPR-CAS9 system during zebrafish embryonic development. We induced the first round of barcoding via microinjection at the single-cell stage and the second round of barcoding by heat-shock at 28 hours post-fertilization, the time of HSC birth. We also injected these GESTALT embryos with drl:CMYC to induce AEL. This way, we were able to uniquely barcode HSCs and their niche and induce AEL to perform clone tracing. We analyzed the number of marrow and peripheral blood HSC clones and observed that leukemic marrows have half the number of clones compared to controls (p = 0.008) indicative of a clonal expansion of the disease. We performed barcode and single-cell transcriptome profiling of flk1:GFP+ niche endothelial cells from adult AEL and control marrows. We found no significant change in the number of endothelial cells clones but identified a novel AEL specific venous endothelial subpopulation significantly upregulating 99 genes (fc > 1; p < 0.05), including apelin, cxcr4a, esm1, apoa1b and angpt2a suggestive of active vascular remodeling and angiogenesis likely supporting leukemogenesis. We sorted cxcl12a:dsRed⁺ niche stromal cells and found that AEL marrows have significantly reduced stromal cell clones compared to controls (fc = -2.1, p = 0.02). Examination of the repartition of stromal clones in leukemic marrows revealed a selective clonal expansion leading to stromal clones representing > 20% of the stromal compartment. We hypothesized that AEL supports the expansion of a subset of stromal cells to promote disease progression. We profiled the transcriptome of 3,263 cxcl12a:dsRed⁺ stromal cells from AEL and control marrows and identified three main subpopulations: cxcl12a abundant reticular cells (CARCs), lepr⁺ mesenchymal stromal cells (MSCs) and alcam⁺ MSCs. AEL marrows displayed an increased fraction of lepr⁺ MSCs (66% versus 24% in control marrows) demonstrating that AEL selectively amplifies lepr⁺ MSCs. Together our data support a model in which leukemia induces clonal and transcriptional remodeling of the HSC niche to promote disease progression.

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