Single Cell Deconvolution of Myeloblasts Reveals Depletion of HSCs and Expansion of Inflammatory Granulocyte-Monocyte Progenitors (GMPs) Associated with Adverse Outcomes in Chronic Myelomonocytic Leukemia (CMML)

Myeloblasts are associated with adverse outcomes and define transformation to acute myeloid leukemia in all chronic myeloid neoplasms. Myeloblasts represent hematopoietic stem and progenitor cells (HSPCs) that express CD34, but are never resolved into stem and progenitor subpopulations during clinical evaluation. Therefore, how expansion of myeloblasts reshapes the HSPC compartment and its impact on clinical outcomes remains undefined. To address this important feature of disease progression, we transcriptionally and immunophenotypically mapped CD34 ⁺ HSPCs at single cell resolution for 66 samples from 45 patients with CMML. Single cell-RNA sequencing was performed on 137,578 CD34 ⁺ enriched HSPCs from 39 CMML samples and integrated with 63,672 publicly available CD34 ⁺ normal HSPCs (Fig A). We overlaid each CMML sample on a pseudotime projection of differentiation trajectories from normal samples to establish sample-specific aberrancies in HSPC states. This mapping classified samples into HSPC-biased groups of monocyte (mono)-bias, megakaryocyte erythroid (ME)-bias, and normal-like, respectively enriched for GMP, MEP, and HSC transcriptional signatures (Fig B). These groups were associated with distinct clinical genomic characteristics and were congruent with patient-specific bulk sequencing. For example, ME biased cases had statistically higher hemoglobin and mono-bias cases were associated with adverse survival, inflammatory clinical correlates, and RAS pathway mutations (Fig C). Importantly, we identified significant depletion of HSC across CMML that was most pronounced in the mono-bias group. This was validated by flow cytometry in 26 CD34 ⁺ enriched samples, which showed HSC numbers decreased as myeloblasts expanded and disease progressed (Fig D,E).

The mono-biased group strongly correlated to the fraction of cells that were transcriptionally enriched for cytokine receptor (CR) signaling (cluster 2, Fig F). These cluster 2 cells constituted a subset of GMPs that could be identified by CD120b expression based on COMET analysis (Fig F), were depleted after therapy in sequential samples, and were associated with high CTNNB1 and low IRF8 expression, suggesting that they are self-renewing GMPs as previously reported in murine models (Herault Nature 2017). To validate the clinical relevance of CR signaling in HSPCs, we established a CR high-parameter flow cytometry panel by prioritizing CRs from primary CMML CD34 ⁺ RNA-sequencing data and quantified their expression using PE-conjugated antibodies to screen CR expression and density. This led to a 30-parameter panel that accounted for CR co-expression, spectral overlap, enabled us to both map CRs on HSCs, CMPs, MEPs, and GMPs, and calculate the CR Shannon diversity in 26 CMML and 5 normal controls (Fig G). Patients with CD120b ⁺ GMPs had inferior survival, were associated with higher-risk, proliferative disease, and higher CR diversity (Fig H). Further, increased CR diversity was associated with inferior survival across all HSPC compartments.

Given the expansion of GMPs in mono-biased patients, we hypothesized that prior periods of stress-induced hematopoiesis (SIH) could contribute to the development of this adverse HSPC differentiation trajectory during disease progression. We modeled SIH by performing BMT experiments with NRAS ^Q61R/WT bone marrow cells and controls as RAS mutations were associated with a mono-bias state. These experiments identified a depletion of HSC and expansion of CD120b ⁺ GMPs compared to controls recapitulating the HSPC compartment in human mono-biased cases (Fig I,J). We modeled the impact of SIH in human CMML by chronically treating RAS mutated CMML PDX models with LPS or vehicle and similarly observed HSC depletion and CD120b ⁺ GMP expansion in LPS-treated mice (Fig K,L). Our data suggests that HSC depletion is a characteristic of myeloblast expansion during disease progression. Further, even in a disease with homogenous hematopoietic output (monocytosis), progenitor expansion of HSPCs can occur in three distinct skewed states. The mono-biased state is associated with poor outcomes and can be recapitulated by modeling SIH in CMML. PDX studies are ongoing to validate these results and the effects of SIH on survival. Deconvolution of HSPCs at single cell resolution of other myeloid neoplasms and strategies to mitigate triggers of SIH to prevent the mono-biased state should be explored.

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