Cell-Type Specific Mechanisms of Hematopoietic Stem Cell (HSC) Expansion Underpin Progressive Disease in Myelodysplastic Syndromes (MDS) and Provide a Rationale for Targeted Therapies

The mechanisms of HMA failure in MDS remain unclear, as recent advances in sequencing approaches did not enable the molecular characterization of the cells that survive therapy and drive resistance and disease progression.

Here, through combined functional and transcriptomic analyses of highly-purified hematopoietic populations isolated from the BM of 132 MDS patients enrolled in clinical trials of single drug HMA therapy, we show that 2 immunophenotypically and molecularly distinct cell types maintain the disease and expand at progression, and we propose therapeutic approaches to overcome MDS evolution.

Unsupervised hierarchical clustering followed by principal component analysis of 101 untreated MDS samples based on the frequency of immunophenotypically defined stem and myeloid progenitor cell populations identified the frequencies of the lymphoid-primed multipotent progenitors (LMPPs) and granulo-monocytic progenitors (GMPs) as the main sources of variation across the samples. Further logistic regression analysis enabled the systematic stratification of the samples in 2 main groups. "CMP pattern" MDS was characterized by the prevalence of common myeloid progenitors (CMPs) in the progenitor compartment while "GMP pattern" MDS was characterized by the increased frequency of GMPs in the progenitors and by increased LMPPs and decreased long-term (LT)-HSC frequencies in the BM (Fig A, B). Functional analysis of the CMPs by immunophenotypic characterization of lineage-primed fractions and colony assays revealed that this population was myeloid-biased only in "CMP pattern" MDS but not in "GMP pattern" MDS, suggesting that 2 distinct hierarchical differentiation routes underlie disease manifestation (Fig C). Further logistic regression analysis of 31 MDS samples from patients with progressive disease showed that whereas "CMP pattern" MDS patients with blast expansion were characterized by a significant increase in the BM frequency of LT-HSCs, "GMP pattern" MDS patients were characterized by the expansion of the LMPPs (Fig D).

To investigate the molecular pathways underlying the 2 different hierarchical patterns of progression, we analyzed the transcriptional profiling of the HSCs that selectively expanded in the 2 groups of MDS patients. RNA-Seq analysis revealed that, compared with those isolated from patients at baseline, LT-HSCs isolated from "CMP pattern" MDS patients with blast expansion had significantly upregulated genes involved in promoting cell proliferation and survival, including the anti-apoptotic regulator BCL2. In contrast, genes involved in the response to TNF-a were significantly upregulated in the LMPPs from "GMP pattern" MDS patients with progressive disease as compared with the LMPPs isolated at baseline (Fig E). Then, we hypothesized that, despite genetic dissimilarities, the HSCs that expanded at progression were addicted to the molecular pathways that were upregulated and that targeting these pathways represented a potential therapeutic strategy to overcome HMA resistance. As proof-of-principle, we treated CD34⁺ cells from "CMP pattern" MDS isolated after HMA failure, co-cultured over a layer of stromal cells, with the BCL2 inhibitor ABT-199 for 72 hours. ABT-199, in combination with 5-azacytidine (AZA), significantly decreased the number of LT-HSCs from MDS patients with progressive disease (Fig F), but did not affect those from MDS patients in whom HMA therapy failed because of persistent MDS. These data suggest that ABT-199 selectively targets the LT-HSCs from "CMP pattern" MDS with progressive disease, which depend on increased levels of BCL2 to support their expansion. Treatment of WT/vav-cre-TET2^L/L BM chimeras with ABT-199 therapy showed selective apoptosis and depletion of the MDS-like LT-HSCs and the concomitant proliferation of the WT LT-HSCs. This suggests that ABT-199 allows selective therapeutic targeting that eliminates abnormal HSCs while restoring normal hematopoiesis (Fig G). Furthermore, ABT-199 treatment of xenografts generated by transplanting the MDS-L cell line in

NSGS mice reduced tumoral burden by depleting the human blast population (Fig H).

Taken together, these findings demonstrate that targeting commonly deregulated pathways in MDS HSCs is a feasible approach to tackling disease progression and provide a rationale for the selective inhibition of BCL2 in "CMP pattern" MDS with progressive disease.

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