Ex Vivo Expansion of Phenotypic and Transcriptomic Chronic Myeloid Leukemia Stem Cells.

Despite decades of research, standard therapies remain ineffective for most leukemias, pushing toward an essential unmet need for targeted drug screens. Moreover, preclinical drug testing is an important consideration for success of clinical trials without affecting non-transformed stem cells. Using the transgenic chronic myeloid leukemia (CML) mouse model, we determine that leukemic stem cells (LSCs) are transcriptionally heterogenous with a preexistent drug-insensitive signature. To test targeting of potentially important pathways, we establish ex vivo expanded LSCs that have long-term engraftment and give rise to multilineage hematopoiesis. Expanded LSCs share transcriptomic signatures with primary LSCs including enrichment in Wnt, JAK-STAT, MAPK, mTOR and transforming growth factor ß signaling pathways. Drug testing on expanded LSCs show that transforming growth factor ß and Wnt inhibitors had significant effects on the viability of LSCs, but not leukemia-exposed healthy HSCs. This platform allows testing of multiple drugs at the same time to identify vulnerabilities of LSCs.

Metabolic alterations mediated by STAT3 promotes drug persistence in CML.

Leukemic stem cells (LSCs) can acquire non-mutational resistance following drug treatment leading to therapeutic failure and relapse. However, oncogene-independent mechanisms of drug persistence in LSCs are incompletely understood, which is the primary focus of this study. We integrated proteomics, transcriptomics, and metabolomics to determine the contribution of STAT3 in promoting metabolic changes in tyrosine kinase inhibitor (TKI) persistent chronic myeloid leukemia (CML) cells. Proteomic and transcriptional differences in TKI persistent CML cells revealed BCR-ABL-independent STAT3 activation in these cells. While knockout of STAT3 inhibited the CML cells from developing drug-persistence, inhibition of STAT3 using a small molecule inhibitor sensitized the persistent CML cells to TKI treatment. Interestingly, given the role of phosphorylated STAT3 as a transcription factor, it localized uniquely to genes regulating metabolic pathways in the TKI-persistent CML stem and progenitor cells. Subsequently, we observed that STAT3 dysregulated mitochondrial metabolism forcing the TKI-persistent CML cells to depend on glycolysis, unlike TKI-sensitive CML cells, which are more reliant on oxidative phosphorylation. Finally, targeting pyruvate kinase M2, a rate-limiting glycolytic enzyme, specifically eradicated the TKI-persistent CML cells. By exploring the role of STAT3 in altering metabolism, we provide critical insight into identifying potential therapeutic targets for eliminating TKI-persistent LSCs.

Bone marrow Tregs mediate stromal cell function and support hematopoiesis via IL-10.

The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis.

Mapping Distinct Bone Marrow Niche Populations and Their Differentiation Paths.

The bone marrow microenvironment is composed of heterogeneous cell populations of non-hematopoietic cells with complex phenotypes and undefined trajectories of maturation. Among them, mesenchymal cells maintain the production of stromal, bone, fat, and cartilage cells. Resolving these unique cellular subsets within the bone marrow remains challenging. Here, we used single-cell RNA sequencing of non-hematopoietic bone marrow cells to define specific subpopulations. Furthermore, by combining computational prediction of the cell state hierarchy with the known expression of key transcription factors, we mapped differentiation paths to the osteocyte, chondrocyte, and adipocyte lineages. Finally, we validated our findings using lineage-specific reporter strains and targeted knockdowns. Our analysis reveals differentiation hierarchies for maturing stromal cells, determines key transcription factors along these trajectories, and provides an understanding of the complexity of the bone marrow microenvironment.