Experimental hematology最新文献

Hematopoietic stem cells (HSCs) are a population of tissue-specific stem cells that reside in the bone marrow of adult mammals, where they self-renew and continuously regenerate the adult hematopoietic lineages over the life of the individual. Prominence as a stem cell model and clinical usefulness have driven interest in understanding the physiologic processes that lead to the specification of HSCs during embryonic development. High-efficiency directed differentiation of HSCs by the instruction of defined progenitor cells using sequentially defined instructive molecules and conditions remains impossible, indicating that comprehensive knowledge of the complete set of precursor intermediate identities and required inductive inputs remains incompletely understood. Recently, interest in the molecular and cellular microenvironment where HSCs are specified from endothelial precursors—the “specification niche”—has increased. Here we review recent progress in understanding these niche spaces across vertebrate phyla, as well as how a better characterization of the origin and molecular phenotypes of the niche cell populations has helped inform and complicate previous understanding of signaling required for HSC emergence and maturation.

Regenerative erythropoiesis is critical for the recovery from surgery, chemotherapy, bone marrow transplantation and infection. We identified a new erythroid progenitor with colony-forming unit-erythroid (CFU-E) activity, which we named stress CFU-E (sCFU-E). sCFU-E cells are targets of erythropoietin (Epo) and its receptor EpoR, are only expanded in erythroid stress, and are essential for the recovery of erythrocyte numbers in regenerative erythropoiesis. Interestingly, in myeloproliferative neoplasms (MPN), sCFU-E are hijacked by the oncogenic JAK2 mutant, JAK2(V617F), to drive constitutive EpoR signaling and overproduction of erythrocytes.

Mechanistically, Epo promotes sCFU-E expansion through the JAK2-STAT5 pathway by inducing the expression of IRS2, thereby engaging pro-growth signaling from the IGF1 receptor (IGF1R). Inhibition of IGF1R/IRS2 signaling impairs sCFU-E cell growth, whereas exogenous IRS2 expression rescues cell growth in sCFU-E expressing truncated EpoR with defective STAT5 activation. Inability to expand sCFU-E cells by truncated EpoR protects against JAK2(V617F)-driven erythrocytosis in mice. In samples from MPN patients, the number of sCFU-E-like cells increases, and inhibition of IGR1R/IRS2 signaling blocks Epo-hypersensitive erythroid cell colony formation. Moreover, metabolomics analyses showed that sCFU-E accumulates high levels of ascorbate (vitamin C), and ascorbate accelerates sCFU-E differentiation independent of its function as an antioxidant. Epo regulates sCFU-E differentiation by inducing the expression of SLC23A2, an ascorbate transporter.

Our discovery and analysis of a novel stress-specific erythroid progenitor cell population, which connects regenerative erythropoiesis with pathogenic erythrocytosis, could offer valuable insights for developing new treatments for both anemia and MPN.

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Mutations in the tumour suppressor TP53 are common in many cancers, including aggressive blood cancers, and confer poor responses to chemotherapy. Newer BH3-mimetic drugs, such as the BCL-2 inhibitor Venetoclax, were postulated to be effective therapy for TP53 mutant blood cancers since these drugs initiate apoptosis downstream of TP53 and therefore should function agnostic of TP53 status. However recent data from our lab and others indicate wild-type TP53 is required for maximal cancer cell killing by BH3-mimetic drugs.

Using pre-clinical models of several blood cancers and CRISPR/Cas9 approaches, we interrogated the role of TP53 in the apoptotic response to BH3-mimetic drugs. We found that TP53 is not needed for BH3-mimetics to induce apoptosis via mitochondrial outer membrane permeabilization (MOMP). However, TP53 becomes activated downstream of MOMP, leading to induction of the pro-apoptotic BH3-only proteins and a second wave of apoptosis that reinforces killing of the cancer cells. Blood cancers with mutant TP53 cannot induce this enforcing wave of apoptosis and are therefore more likely to survive and contribute to relapse.

Through these analyses we identified an alternative complementary pathway to activate apoptosis using STING agonist drugs. We found that STING agonists could induce BH3-only protein expression in a TP53-independent manner, boosting the pro-apoptotic signal. Combining STING agonists with BH3-mimetic drugs led to highly effective killing of mouse B cell lymphomas, human NK/T cell lymphomas and patient-derived Acute Myeloid Leukemia blasts, even those that were mutated for TP53. Since STING agonists are already in clinical trials to induce anti-tumour immunity, we anticipate repurposing them to boost apoptosis alongside BH3-mimetic drugs in clinical trials for blood cancer patients would be effective and relatively straight forward.

Aberrant expression of cytokine receptor-like factor 2 (CRLF2) occurs in 5–15% of B-cell acute lymphoblastic leukaemia (B-ALL) and is associated with poor outcomes.

Approximately 50% of CRLF2+ B-ALLs also harbor activating mutations in JAK2. Coexpression of CRLF2 and mutant JAK2 results in constitutive STAT5 activation, and factor-independent transformation of B cell progenitors. The current consensus is that JAK/STAT activation is the hallmark of CRLF2 B-ALL, however JAK2 inhibitors such as Ruxolitinib have shown limited efficacy in this leukemia. We have shown that some CRLF2+ B-ALLs lacking JAK2 mutations instead harbor activating mutations in the RAS-ERK pathway (e.g. KRAS-G12D). Using single-cell sequencing of matched diagnosis and relapse patient samples, we show that in patients with both STAT and ERK activating lesions, these mutations are present in competing clones which fluctuate during disease progression. However, it remains unknown how subclonal mutations alter the signalling properties and drug responses of CRLF2+ leukemias. To investigate this, we established murine models expressing the human CRLF2 receptor complex and common JAK2 and RAS pathway mutations. Using phospho-proteomics, and high throughput drug screening we show for the first time that the combination of CRLF2 with RAS mutations activates distinct signalling networks, compared to CRLF2 combined with mutant JAK2, and that this drives unique drug dependencies that can be therapeutically leveraged. To investigate subclonal dynamics in vivo, we use advanced imaging approaches to visualise how distinct sublones engage bone marrow niche structures during development, and under pressure of chemotherapy. This work reveals novel insights into the importance of subclonal mutations on the biology of CRLF2+ B-ALL.

The first haematopoietic stem and progenitor cells (HSPCs) in the embryo arise through a process known as endothelial-to-haematopoietic transition (EHT). In a subset of endothelial cells referred to as haemogenic endothelium (HE), the endothelial transcriptional programme is gradually replaced by a haematopoietic one, promoting haematopoietic commitment and ultimately EHT. This process is critically dependent on the transcription factor RUNX1. There is currently limited knowledge on the transcriptional regulation and downstream function of RUNX1 during human EHT. Here, using an in vitro human induced pluripotent stem cell (hiPSC) differentiation model, we identified five candidate EHT RUNX1 enhancers, characterised by H3K27ac and open chromatin, one of which is only accessible in HE and four are accessible in haematopoietic cells. Through gene regulatory network (GRN) analysis, performed on joint single-cell chromatin accessibility and gene expression profiling data, we identified a set of candidate upstream RUNX1 activators and repressors. These included known RUNX1 regulators (e.g. GATA2, MEIS1, EPAS1) as well as potentially novel ones. To identify the downstream target genes of RUNX1, we profiled RUNX1-binding sites genome-wide in hiPSC-derived HE, where most of these sites were not acetylated and were associated with endothelial genes, suggesting RUNX1 might directly repress the endothelial programme. Together, our data are expected to improve our understanding of the regulatory mechanisms underlying human EHT.

Driver mutations in classical myeloproliferative neoplasms (MPNs) (eg. JAK2V617F ) must be initiated and maintained in the haematopoietic stem cell (HSC) pool. Leukaemic transformation to post-MPN AML is characterised by additional genetic lesions and mutations in TP53 are predictive of poor outcomes. Interferon alpha (IFNa) can drive HSC cell cycle entry preferentially in Jak2V617F HSCs resulting in reduced self-renewal capacity. Clinically, IFNa therapy can achieve long-term reductions in JAK2V617F allelic burden. However, the impact of additional mutations on IFNa responses in MPN and transformation to AML remains unclear.

We have generated a single cell RNA sequencing pipeline to monitor the genetic and transcriptional heterogeneity of the HSPC compartment of MPN patients during AML transformation. Transformation is linked with a loss of HSPC hierarchy and devolution to a dominant multipotent progenitor (MPP) state. Using these data to inform parallel murine studies, we demonstrate that haematopoietic expression of Jak2V617F with Trp53-loss is sufficient to drive a fully penetrant leukemia preceded by a distinct MPN disease phase in mice. The resulting AML exhibits a dominant lineage-biased MPP that has leukaemia initiating activity in secondary recipients.

IFNa is still able to induce haematological responses in Jak2V617F MPN with Trp53-loss. However, Trp53-loss also provides a selective advantage for HSCs in the context of chronic exposure to IFNa. Surprisingly, the effects of IFNa on reduced stem cell function are retained in the absence of p53 and chronic administration of IFNa is sufficient to delay leukaemic transformation. Furthermore, IFNa therapy is also effective at preventing disease progression in an established Jak2V617F AML with Trp53-loss. These findings have important implications for the treatment of MPN with TP53 mutations.

Clonal hematopoiesis is common in solid tumour patients, who frequently have loss of function mutations in TET2. TET2 restricts innate and adaptive immunity, so we hypothesized that TET2-mutant clonal hematopoiesis (TET2-CH) is associated with immunotherapy response. To test this hypothesis, syngeneic colorectal cancer-bearing mice with Tet2-heterozygous null (Tet2-het) or wild type hematopoiesis were treated with anti-PD-1 immunotherapy. Treatment responses were greater and tumors were smaller in Tet2-het mice. The Tet2-effect required phagocytes, CD4, and CD8 T cells, but not NK cells. scRNA-seq revealed how Tet2-mutations reshape the tumor-infiltrating cell (TIL) landscape with immunotherapy by inducing anti-tumour states and restricting pro-tumour cell states. Tet2-mutant monocytes upregulated T cell costimulatory genesets and we found enhanced communication between Tet2-het antigen presenting and T cells. Combined sc-genotyping and RNA-seq of primary TET2-CH patient leukocytes showed that, like mouse TILs, human TET2-mutant monocytes upregulated costimulatory and inflammatory programs associated with immunotherapy response. TET2-mutant CD8 T cells were rare but strikingly enriched for memory programs and TCR signaling, yet suppressed an exhaustion signature. Melanoma patient RNA-seq showed TET2-CH+ tumours are enriched for antigen presentation/costimulation and T cell memory versus exhaustion. TET2-CH+ melanomas also had increased immune infiltrate, T cells and dendritic cells, and re-analysis of 200 immunotherapy-treated melanoma patients showed those with TET2-CH were 6-fold more likely to benefit from immunotherapy. Therefore, across mouse tumours, human leukocytes and tumours, somatic TET2-mutations activate transcriptional programs in myeloid and T cells associated with anti-tumour immunity, which correlate with enhanced immunotherapy response in melanoma.

Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal, however the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.

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