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Supplementary Data from In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients’ erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV.Significance:Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV.This article is highlighted in the In This Issue feature, p. 265

Supplementary Table from In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target

International audience; The persistence of cancer cells resistant to therapy remains a major clinical challenge. In triple-negative breast cancer, resistance to chemotherapy results in the highest recurrence risk among breast cancer subtypes. The drug-tolerant state seems largely defined by nongenetic features, but the underlying mechanisms are poorly understood. Here, by monitoring epigenomes, transcriptomes and lineages with single-cell resolution, we show that the repressive histone mark H3K27me3 (trimethylation of histone H3 at lysine 27) regulates cell fate at the onset of chemotherapy. We report that a persister expression program is primed with both H3K4me3 (trimethylation of histone H3 at lysine 4) and H3K27me3 in unchallenged cells, with H3K27me3 being the lock to its transcriptional activation. We further demonstrate that depleting H3K27me3 enhances the potential of cancer cells to tolerate chemotherapy. Conversely, preventing H3K27me3 demethylation simultaneously to chemotherapy inhibits the transition to a drug-tolerant state, and delays tumor recurrence in vivo. Our results highlight how chromatin landscapes shape the potential of cancer cells to respond to initial therapy.

SUMMARYFollowing infection, hematopoietic stem and progenitor cells (HSPCs) support immunity by increasing the rate of innate immune cell production but the metabolic cues that guide this process are unknown. To address this question, we developed MetaFate, a method to trace the metabolic expression state and developmental fate of single cellsin vivo. Using MetaFate we identified a gene expression program of metabolic enzymes and transporters that confers differences in myeloid differentiation potential in a subset of HSPCs that express CD62L. Using single-cell metabolic profiling, we confirmed that CD62Lhighmyeloid-biased HSPCs have an increased dependency on oxidative phosphorylation and glucose metabolism. Importantly, metabolism actively regulates immune-cell production, with overexpression of the glucose-6-phosphate dehydrogenase enzyme of the pentose phosphate pathway skewing MPP output from B-lymphocytes towards the myeloid lineages, and expansion of CD62LhighHSPCs occurring to support emergency myelopoiesis. Collectively, our data reveal the metabolic cues that instruct innate immune cell development, highlighting a key role for the pentose phosphate pathway. More broadly, our results show that HSPC metabolism can be manipulated to alter the cellular composition of the immune system.

SummaryTriple-negative breast cancer is associated with the worst prognosis and the highest risk of recurrence among all breast cancer subtypes1. Residual disease, formed by cancer cells persistent to chemotherapy, remains one of the major clinical challenges towards full cure2,3. There is now consensus that non-genetic processes contribute to chemoresistance in various tumor types, notably through the initial emergence of a reversible chemotolerant state4–6. Understanding non-genetic tumor evolution stands now as a prerequisite for the design of relevant combinatorial approaches to delay recurrence. Here we show that the repressive histone mark H3K27me3 is a determinant of cell fate under chemotherapy exposure, monitoring epigenomes, transcriptomes and lineage with single-cell resolution. We identify a reservoir of persister basal cells with EMT markers and activated TGF-β pathway leading to multiple chemoresistance phenotypes. We demonstrate that, in unchallenged cells, H3K27 methylation is a lock to the expression program of persister cells. Promoters are primed with both H3K4me3 and H3K27me3, and removing H3K27me3 is sufficient for their transcriptional activation. Leveraging lineage barcoding, we show that depleting H3K27me3 alters tumor cell fate under chemotherapy insult – a wider variety of tumor cells tolerate chemotherapy. Our results highlight how chromatin landscapes shape the potential of unchallenged cancer cells to respond to therapeutic stress.

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients’ erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV. Significance: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265

The cytokine erythropoietin (EPO) is a potent inducer of erythrocyte development and one of the most prescribed biopharmaceuticals. The action of EPO on erythroid progenitor cells is well established, but its direct action on hematopoietic stem and progenitor cells (HSPCs) is still debated. Here, using cellular barcoding, we traced the differentiation of hundreds of single murine HSPCs, after ex vivo EPO-exposure and transplantation, in five different hematopoietic cell lineages, and observed the transient occurrence of high-output Myeloid-Erythroid-megaKaryocyte (MEK)-biased and Myeloid-B-cell-Dendritic cell (MBDC)-biased clones. Single-cell RNA sequencing (ScRNAseq) analysis of ex vivo EPO-exposed HSPCs revealed that EPO induced the upregulation of erythroid associated genes in a subset of HSPCs, overlapping with multipotent progenitor (MPP) 1 and MPP2. Transplantation of Barcoded EPO-exposed-MPP2 confirmed their enrichment in Myeloid-Erythroid-biased clones. Collectively, our data show that EPO does act directly on MPP independent of the niche, and modulates fate by remodeling the clonal composition of the MPP pool.