Ferrall‐Fairbanks, Meghan C.

Abstract Background The expansion of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow is universally associated with adverse outcomes and disease progression in all chronic myeloid neoplasms, to include chronic myelomonocytic leukemia (CMML). However, HSPCs are a heterogeneous cellular compartment and the impact of its composition on clinical outcomes remains poorly defined. Single cell RNA-sequencing (scRNA-seq) represents an optimal strategy to annotate cellular composition but no large clinically annotated scRNA-seq datasets are currently available. To address this, we have embarked to generate a clinically annotated 500 patient CMML scRNA-seq atlas from CD34+ enriched BMNCs to establish among the largest clinically and genetically annotated single cell references for any chronic myeloid malignancy. Here we report the interim analysis of 113 clinically annotated cases that demonstrates massive reshaping of HSPC composition that is independently a predictor of overall survival. Methods Single-cell RNA expression profiling was performed on 113 CD34-enriched CMML BMNC samples using 10x Genomics GEM-X Universal 3' Gene Expression v4 (4-plex) chemistry. Libraries were sequenced on NovaSeq S4 flow cells in two runs, and data were processed using Cell Ranger to generate gene expression matrices. A total of 502,659 single cells from 113 samples were processed using Seurat. The upper threshold for gene count was defined as the mean number of detected genes plus two standard deviations data normalization was performed using the log-normalization method. Batch correction and integration were conducted with Harmony to mitigate technical variation across pools. Descriptive statistics were used to tabulate clinical and genetic parameters. KM curves, log-rank test, and cox regression were used to calculate survival, test for statistical differences, and perform multivariate analysis, respectively. Results To derive high resolution cell annotations, we employed the SingleR algorithm using a large hematopoietic reference of over 300,000 single cells (Zhang, X. Nature Immunology, 2024). Using this approach a diverse set of cell types were identified that included HSCs, 23 myeloid progenitors, 18 erythroid progenitors, and 18 lymphoid progenitors. This diverse cellular repertoire displayed massive reshaping relative to the normal CD34 enriched reference including a >53x expansion of multilineage GMPs and a >100x depletion of B-cell progenitors consistent with the known myeloid skewing seen in this disease. To understand whether expansion of specific progenitors was associated with outcomes we iteratively evaluated key progenitor populations and observed that expansions of HSC (HR 0.468 p<0.005) and MEP (HR 0.485, p<0.005) were associated with improved survival while GMP (HR 1.729, p=0.029) expansions were associated with inferior survival when using a cut point derived with a maximally selected ranks statistics approach. To achieve a more comprehensive metric of HSPC composition we explored GMP ratios to include GMP/HSC ratios and GMP/MEP ratios and observed that both were associated with inferior survival in univariate and multivariate when considering clinical and genetic features such as the CPSS-molecular prognostic score and FAB classification. When considering both GMP/HSC and GMP/MEP ratios in a multivariate model only GMP/MEP ratios and CPSS molecular remained independently prognostic. As an orthogonal approach, pseudotime projections were carried out using a reference map from Setty et al., 2019 to calculate differentiation potential and branch probabilities. This approach enabled both cell identity assignment and placement of cells along the hematopoietic differentiation trajectory. Samples were classified as monocyte-biased, MEP-biased, or normal-like, as previously described (Ferrall-Fairbanks et al. 2022) and demonstrated that increased GMP ratios are associated with a monocytic biased trajectory and that this trajectory is also associated with overall survival. Conclusion This study represents an interim analysis of among the largest scRNA-seq cohorts generated in chronic myeloid malignancies. This enabled us, for the first time, to demonstrate that HSPC composition is independently prognostic even when considering modern CMML prognostic models. As this cohort continues to increase, validation of these findings and examining the impact of therapy on HPSC composition in serial samples will be prioritized.

Abstract TET2 mutations (mTET2) are common genetic events in myeloid malignancies and clonal hematopoiesis. These mutations arise in the founding clone and are implicated in many clinical sequelae associated with oncogenic feedforward inflammatory circuits. However, the direct downstream effector of mTET2 responsible for the potentiation of these inflammatory circuits is unknown. To address this, we performed scRNA-seq and scATAC-seq in patients with COVID-19 with and without TET2-mutated clonal hematopoiesis reasoning that inflammation from COVID-19 may highlight critical downstream transcriptional targets of mTET2. Using this approach, we identified metastasis-associated lung adenoma transcript 1 (MALAT1), a therapeutically tractable lncRNA, as a central downstream effector of mTET2 that is both necessary and sufficient to induce the oncogenic proinflammatory features of mTET2 in vivo. We also elucidate the mechanism by which mTET2 upregulate MALAT1 and describe an interaction between MALAT1 and p65, which leads to RNA “shielding” from protein phosphatase 2A dephosphorylation, thus preventing resolution of inflammatory signaling. Significance: This work identifies MALAT1 as a requisite downstream effector of oncogenic feedforward inflammatory circuits necessary for the development of TET2-mutated CH and fulminant myeloid malignancy. We elucidate a novel mechanism by which MALAT1 “shields” p65 from dephosphorylation to potentiate this circuit and nominate MALAT1 inhibition as a future therapeutic strategy.