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Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease-initiating leukemia stem cells (LSC). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSC. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSC. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSC but is not essential for normal human hematopoietic stem and progenitor cell function. In order to elucidate the molecular mechanisms by which SIRT3 is essential in LSC we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support OXPHOS and ATP production in human LSC. Further, we discovered two approaches to further sensitize LSC to SIRT3 inhibition. First, we found that LSC tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSC to YC8-02 and potentiates LSC death. Second, SIRT3 inhibition sensitizes LSC to the BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.

Single-cell proteomics can provide insights into the molecular basis for cellular heterogeneity. Here, the authors develop a multiplexed single-cell proteomics and computational workflow, and show that their strategy captures the cellular hierarchies in an Acute Myeloid Leukemia culture model.

Molecular heterogeneity of acute myeloid leukaemia (AML) across patients is a major challenge for prognosis and therapy. Here, the authors show that NPM1 mutated AML is a heterogeneous class, consisting of two subtypes which exhibit distinct molecular characteristics, differentiation state, patient survival and drug response.

Summary: Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells.

Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34+ blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34+CD38- phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34+ hematopoietic stem cells from peripheral blood stem cell grafts and CD34+ blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34+ blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.

Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.

A deep proteomics analysis was conducted on a primary acute myeloid leukemia culture system to identify potential protein targets regulated by miR-126. Leukemia cells were transduced either with an empty control lentivirus or one containing the sequence for miR-126, and resulting cells were analyzed using ultra-high performance liquid chromatography (UHPLC) coupled with high resolution mass spectrometry. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD001994. The proteomics data and statistical analysis described in this article is associated with a research article, “miR-126 regulates distinct self-renewal outcomes in normal and malignant hematopoietic stem cells” (Lechman et al., 2016) [1], and serves as a resource for researchers working in the field of microRNAs and their regulation of protein levels.

Background Diamond-Blackfan anemia is a fatal congenital anemia characterized by a specific disruption in erythroid progenitor cell development. Approximately 25% of patients have mutations in the ribosomal protein RPS19 suggesting that Diamond-Blackfan anemia may be caused by a defect in ribosome biogenesis and translation. However, it is unclear how these defects specifically disrupt early erythropoiesis. Recent studies have shown that the retroviral receptor/heme exporter FLVCR1 is critical for early erythropoiesis. FLVCR1 null mice, despite dying in utero and having reduced myeloid and lymphoid cell growth, show a disruption in early erythropoiesis and have craniofacial and limb deformities similar to those found in some Diamond-Blackfan anemia patients.Design and Methods In this study, we recapitulated the Diamond-Blackfan anemia hematologic features of reduced erythropoiesis but normal myelopoiesis by disrupting FLVCR1 in human hematopoietic stem cells.Results We found that CD71high cells, which are enriched for immature erythroid cells, from Diamond-Blackfan anemia patients negative for RPS19 gene mutations express alternatively spliced isoforms of FLVCR1 transcript which encode proteins whose expression and function are disrupted. More importantly, our results suggest alternative splicing of FLVCR1 is significantly enhanced in Diamond-Blackfan anemia immature erythroid cells. Furthermore, we also observed enhanced FLVCR1 alternative splicing and a dramatic reduction of FLVCR1 protein expression in RPS19 down-regulated human K562 cells, which were used as a model to represent RPS19 gene mutated Diamond-Blackfan anemia.Conclusions Taken together, our results suggest enhanced alternative splicing of FLVCR1 transcripts and subsequent FLVCR1 insufficiency as an additional contributing factor to the erythropoietic defect observed in Diamond-Blackfan anemia.

Abstract The hematopoietic system is a distributed tissue that consists of functionally distinct cell types continuously produced through hematopoietic stem cell (HSC) differentiation. Combining genomic and phenotypic data with high‐content experiments, we have built a directional cell–cell communication network between 12 cell types isolated from human umbilical cord blood. Network structure analysis revealed that ligand production is cell type dependent, whereas ligand binding is promiscuous. Consequently, additional control strategies such as cell frequency modulation and compartmentalization were needed to achieve specificity in HSC fate regulation. Incorporating the in vitro effects (quiescence, self‐renewal, proliferation, or differentiation) of 27 HSC binding ligands into the topology of the cell–cell communication network allowed coding of cell type‐dependent feedback regulation of HSC fate. Pathway enrichment analysis identified intracellular regulatory motifs enriched in these cell type‐ and ligand‐coupled responses. This study uncovers cellular mechanisms of hematopoietic cell feedback in HSC fate regulation, provides insight into the design principles of the human hematopoietic system, and serves as a foundation for the analysis of intercellular regulation in multicellular systems.

Xenografting primary human solid tumor tissue into immunodeficient mice is a widely used tool in studies of human cancer biology; however, care must be taken to prove that the tumors obtained recapitulate parent tissue. We xenografted primary human hepatocellular carcinoma (HCC) tumor fragments or bulk tumor cell suspensions into immunodeficient mice. We unexpectedly observed that 11 of 21 xenografts generated from 16 independent patient samples resembled lymphoid neoplasms rather than HCC. Immunohistochemistry and flow cytometry analyses revealed that the lymphoid neoplasms were comprised of cells expressing human CD45 and CD19/20, consistent with human B lymphocytes. In situ hybridization was strongly positive for Epstein-Barr virus (EBV) encoded RNA. Genomic analysis revealed unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements in each B-cell neoplasm. These data demonstrate that the lymphoid neoplasms were EBV-associated human B-cell lymphomas. Analogous to EBV-associated lymphoproliferative disorders in immunocompromised humans, the human lymphomas in these HCC xenografts likely developed from reactivation of latent EBV in intratumoral passenger B lymphocytes following their xenotransplantation into immunodeficient recipient mice. Given the high prevalence of latent EBV infection in humans and the universal presence of B lymphocytes in solid tumors, this potentially confounding process represents an important pitfall of human solid tumor xenografting. This phenomenon can be recognized and avoided by routine phenotyping of primary tumors and xenografts with human leukocyte markers, and provides a compelling biological rationale for exclusion of these cells from human solid tumor xenotransplantation assays.