A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin-c-Kit+Sca-1+Flt3+ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1-GFP reporter mice, we identify a transcriptionally and functionally unique Dach1-GFP- subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these 'lymphoid-primed progenitors' (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.

Pax5 loss imposes a reversible differentiation block in B-progenitor acute lymphoblastic leukemia.

Loss-of-function mutations in hematopoietic transcription factors including PAX5 occur in most cases of B-progenitor acute lymphoblastic leukemia (B-ALL), a disease characterized by the accumulation of undifferentiated lymphoblasts. Although PAX5 mutation is a critical driver of B-ALL development in mice and humans, it remains unclear how its loss contributes to leukemogenesis and whether ongoing PAX5 deficiency is required for B-ALL maintenance. Here we used transgenic RNAi to reversibly suppress endogenous Pax5 expression in the hematopoietic compartment of mice, which cooperates with activated signal transducer and activator of transcription 5 (STAT5) to induce B-ALL. In this model, restoring endogenous Pax5 expression in established B-ALL triggers immunophenotypic maturation and durable disease remission by engaging a transcriptional program reminiscent of normal B-cell differentiation. Notably, even brief Pax5 restoration in B-ALL cells causes rapid cell cycle exit and disables their leukemia-initiating capacity. These and similar findings in human B-ALL cell lines establish that Pax5 hypomorphism promotes B-ALL self-renewal by impairing a differentiation program that can be re-engaged despite the presence of additional oncogenic lesions. Our results establish a causal relationship between the hallmark genetic and phenotypic features of B-ALL and suggest that engaging the latent differentiation potential of B-ALL cells may provide new therapeutic entry points.

XIAP discriminates between type I and type II FAS-induced apoptosis.

FAS (also called APO-1 and CD95) and its physiological ligand, FASL, regulate apoptosis of unwanted or dangerous cells, functioning as a guardian against autoimmunity and cancer development. Distinct cell types differ in the mechanisms by which the 'death receptor' FAS triggers their apoptosis. In type I cells, such as lymphocytes, activation of 'effector caspases' by FAS-induced activation of caspase-8 suffices for cell killing, whereas in type II cells, including hepatocytes and pancreatic beta-cells, caspase cascade amplification through caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (BH3 interacting domain death agonist) is essential. Here we show that loss of XIAP (X-chromosome linked inhibitor of apoptosis protein) function by gene targeting or treatment with a second mitochondria-derived activator of caspases (SMAC, also called DIABLO; direct IAP-binding protein with low pI) mimetic drug in mice rendered hepatocytes and beta-cells independent of BID for FAS-induced apoptosis. These results show that XIAP is the critical discriminator between type I and type II apoptosis signalling and suggest that IAP inhibitors should be used with caution in cancer patients with underlying liver conditions.

The pro-apoptotic BH3-only protein Bid is dispensable for development of insulitis and diabetes in the non-obese diabetic mouse.

Type 1 diabetes is caused by death of insulin-producing pancreatic beta cells. Beta-cell apoptosis induced by FasL may be important in type 1 diabetes in humans and in the non-obese diabetic (NOD) mouse model. Deficiency of the pro-apoptotic BH3-only molecule Bid protects beta cells from FasL-induced apoptosis in vitro. We aimed to test the requirement for Bid, and the significance of Bid-dependent FasL-induced beta-cell apoptosis in type 1 diabetes. We backcrossed Bid-deficient mice, produced by homologous recombination and thus without transgene overexpression, onto a NOD genetic background. Genome-wide single nucleotide polymorphism analysis demonstrated that diabetes-related genetic regions were NOD genotype. Transferred beta cell antigen-specific CD8+ T cells proliferated normally in the pancreatic lymph nodes of Bid-deficient mice. Moreover, Bid-deficient NOD mice developed type 1 diabetes and insulitis similarly to wild-type NOD mice. Our data indicate that beta-cell apoptosis in type 1 diabetes can proceed without Fas-induced killing mediated by the BH3-only protein Bid.

Acute myeloid leukemia maturation lineage influences residual disease and relapse following differentiation therapy.

Acute myeloid leukemia (AML) is a malignancy of immature progenitor cells. AML differentiation therapies trigger leukemia maturation and can induce remission, but relapse is prevalent and its cellular origin is unclear. Here we describe high resolution analysis of differentiation therapy response and relapse in a mouse AML model. Triggering leukemia differentiation in this model invariably produces two phenotypically distinct mature myeloid lineages in vivo. Leukemia-derived neutrophils dominate the initial wave of leukemia differentiation but clear rapidly and do not contribute to residual disease. In contrast, a therapy-induced population of mature AML-derived eosinophil-like cells persists during remission, often in extramedullary organs. Using genetic approaches we show that restricting therapy-induced leukemia maturation to the short-lived neutrophil lineage markedly reduces relapse rates and can yield cure. These results indicate that relapse can originate from therapy-resistant mature AML cells, and suggest differentiation therapy combined with targeted eradication of mature leukemia-derived lineages may improve disease outcome.

Beta cell apoptosis in diabetes.

Apoptosis of beta cells is a feature of both type 1 and type 2 diabetes as well as loss of islets after transplantation. In type 1 diabetes, beta cells are destroyed by immunological mechanisms. In type 2 diabetes abnormal levels of metabolic factors contribute to beta cell failure and subsequent apoptosis. Loss of beta cells after islet transplantation is due to many factors including the stress associated with islet isolation, primary graft non-function and allogeneic graft rejection. Irrespective of the exact mediators, highly conserved intracellular pathways of apoptosis are triggered. This review will outline the molecular mediators of beta cell apoptosis and the intracellular pathways activated.

Responses against islet antigens in NOD mice are prevented by tolerance to proinsulin but not IGRP.

Type 1 diabetes (T1D) is characterized by immune responses against several autoantigens expressed in pancreatic beta cells. T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) can induce T1D in NOD mice. However, whether immune responses to multiple autoantigens are caused by spreading from one to another or whether they develop independently of each other is unknown. As cytotoxic T cells specific for IGRP were not detected in transgenic NOD mice tolerant to proinsulin, we determined that immune responses against proinsulin are necessary for IGRP-specific T cells to develop. On the other hand, transgenic overexpression of IGRP resulted in loss of intra-islet IGRP-specific T cells but did not protect NOD mice from insulitis or T1D, providing direct evidence that the response against IGRP is downstream of the response to proinsulin. Our results suggest that pathogenic proinsulin-specific immunity in NOD mice subsequently spreads to other antigens such as IGRP.

Interconversion between Tumorigenic and Differentiated States in Acute Myeloid Leukemia.

Tumors are composed of phenotypically heterogeneous cancer cells that often resemble various differentiation states of their lineage of origin. Within this hierarchy, it is thought that an immature subpopulation of tumor-propagating cancer stem cells (CSCs) differentiates into non-tumorigenic progeny, providing a rationale for therapeutic strategies that specifically eradicate CSCs or induce their differentiation. The clinical success of these approaches depends on CSC differentiation being unidirectional rather than reversible, yet this question remains unresolved even in prototypically hierarchical malignancies, such as acute myeloid leukemia (AML). Here, we show in murine and human models of AML that, upon perturbation of endogenous expression of the lineage-determining transcription factor PU.1 or withdrawal of established differentiation therapies, some mature leukemia cells can de-differentiate and reacquire clonogenic and leukemogenic properties. Our results reveal plasticity of CSC maturation in AML, highlighting the need to therapeutically eradicate cancer cells across a range of differentiation states.

Cytotoxic T cell mechanisms of beta cell destruction in non-obese diabetic mice.

CD8+ T cells are the principal cellular mediators of beta cell destruction in the NOD mouse. Molecular mediators include perforin and granzymes from the cytotoxic granule, Fas ligand and pro-inflammatory cytokines. Our studies in NOD mice have shown that beta cell-specific CD8+ T cells use both the perforin and Fas pathway in vitro. Reducing antigen presentation on beta cells, for example by reducing class I MHC expression by overexpression of SOCS1, protects beta cells in vivo. Perforin deficiency effectively reduces diabetes in NOD mice but in NOD8.3 mice other mechanisms compensate. We have been unable to identify a major role for direct toxicity of cytokines in NOD mice. However, in the LCMV glycoprotein model they may be more important. Deficiency of IL1 or TNF or Fas has a protective effect (greatest for TNF deficiency) but this appears to be due to effects of these cytokines on the immune response rather than on the beta cell. Combinations of interventions, for example, beta cell overexpression of SOCS1 combined with IL1 deficiency may be highly protective. It should be possible to define all the molecular mediators of beta cell destruction, and it may be possible to inhibit at least some of these.

Identification of a Siglec-F+ granulocyte-macrophage progenitor.

In recent years multi-parameter flow cytometry has enabled identification of cells at major stages in myeloid development; from pluripotent hematopoietic stem cells, through populations with increasingly limited developmental potential (common myeloid progenitors and granulocyte-macrophage progenitors), to terminally differentiated mature cells. Myeloid progenitors are heterogeneous, and the surface markers that define transition states from progenitors to mature cells are poorly characterized. Siglec-F is a surface glycoprotein frequently used in combination with IL-5 receptor alpha (IL5Rα) for the identification of murine eosinophils. Here, we describe a CD11b+ Siglec-F+ IL5Rα- myeloid population in the bone marrow of C57BL/6 mice. The CD11b+ Siglec-F+ IL5Rα- cells are retained in eosinophil deficient PHIL mice, and are not expanded upon overexpression of IL-5, indicating that they are upstream or independent of the eosinophil lineage. We show these cells to have GMP-like developmental potential in vitro and in vivo, and to be transcriptionally distinct from the classically described GMP population. The CD11b+ Siglec-F+ IL5Rα- population expands in the bone marrow of Myb mutant mice, which is potentially due to negative transcriptional regulation of Siglec-F by Myb. Lastly, we show that the role of Siglec-F may be, at least in part, to regulate GMP viability.

Granzyme B-mediated death of pancreatic beta-cells requires the proapoptotic BH3-only molecule bid.

Perforin-deficient NOD mice are protected from diabetes, suggesting that cytotoxic granule contents of CD8(+) T-cells have a significant role in killing beta-cells. Despite this, cytotoxic granule effects on human or mouse pancreatic islets have not been reported. We tested the susceptibility of human and mouse islet cells to purified recombinant perforin and granzyme B and measured apoptotic death using a number of assays. Perforin and granzyme B impaired insulin secretion from islet cells, and this was accompanied by cytochrome c release, caspase activation, and DNA fragmentation. Granzyme B-mediated apoptotic changes only occurred in the presence of perforin. When compared with hemopoietic cells, traditionally used as targets to measure cytotoxic T-cell function in vitro, islet cells were relatively resistant to perforin and granzyme B. Inhibition of caspases prevented DNA fragmentation but not cytochrome c release, indicating that mitochondrial disruption due to granzyme B is independent of caspase activation. Consistent with this, islet cells from mice deficient in the BH3-only protein Bid were resistant to cytochrome c release and were protected from apoptosis after exposure to perforin/granzyme B. Our data suggest that Bid cleavage by granzyme B precedes mitochondrial disruption and apoptosis in pancreatic islets.

Conserved IKAROS-regulated genes associated with B-progenitor acute lymphoblastic leukemia outcome.

Genetic alterations disrupting the transcription factor IKZF1 (encoding IKAROS) are associated with poor outcome in B lineage acute lymphoblastic leukemia (B-ALL) and occur in >70% of the high-risk BCR-ABL1+ (Ph+) and Ph-like disease subtypes. To examine IKAROS function in this context, we have developed novel mouse models allowing reversible RNAi-based control of Ikaros expression in established B-ALL in vivo. Notably, leukemias driven by combined BCR-ABL1 expression and Ikaros suppression rapidly regress when endogenous Ikaros is restored, causing sustained disease remission or ablation. Comparison of transcriptional profiles accompanying dynamic Ikaros perturbation in murine B-ALL in vivo with two independent human B-ALL cohorts identified nine evolutionarily conserved IKAROS-repressed genes. Notably, high expression of six of these genes is associated with inferior event-free survival in both patient cohorts. Among them are EMP1, which was recently implicated in B-ALL proliferation and prednisolone resistance, and the novel target CTNND1, encoding P120-catenin. We demonstrate that elevated Ctnnd1 expression contributes to maintenance of murine B-ALL cells with compromised Ikaros function. These results suggest that IKZF1 alterations in B-ALL leads to induction of multiple genes associated with proliferation and treatment resistance, identifying potential new therapeutic targets for high-risk disease.

Glucose induces pancreatic islet cell apoptosis that requires the BH3-only proteins Bim and Puma and multi-BH domain protein Bax.

OBJECTIVE: High concentrations of circulating glucose are believed to contribute to defective insulin secretion and beta-cell function in diabetes and at least some of this effect appears to be caused by glucose-induced beta-cell apoptosis. In mammalian cells, apoptotic cell death is controlled by the interplay of proapoptotic and antiapoptotic members of the Bcl-2 family. We investigated the apoptotic pathway induced in mouse pancreatic islet cells after exposure to high concentrations of the reducing sugars ribose and glucose as a model of beta-cell death due to long-term metabolic stress. RESEARCH DESIGN AND METHODS: Islets isolated from mice lacking molecules implicated in cell death pathways were exposed to high concentrations of glucose or ribose. Apoptosis was measured by analysis of DNA fragmentation and release of mitochondrial cytochrome c. RESULTS: Deficiency of interleukin-1 receptors or Fas did not diminish apoptosis, making involvement of inflammatory cytokine receptor or death receptor signaling in glucose-induced apoptosis unlikely. In contrast, overexpression of the prosurvival protein Bcl-2 or deficiency of the apoptosis initiating BH3-only proteins Bim or Puma, or the downstream apoptosis effector Bax, markedly reduced glucose- or ribose-induced killing of islets. Loss of other BH3-only proteins Bid or Noxa, or the Bax-related effector Bak, had no impact on glucose-induced apoptosis. CONCLUSIONS: These results implicate the Bcl-2 regulated apoptotic pathway in glucose-induced islet cell killing and indicate points in the pathway at which interventional strategies can be designed.

Interferon signalling in pancreatic beta cells.

Type 1 diabetes results from apoptotic destruction of insulin-producing beta cells by a range of effector molecules produced by immune cells that infiltrate pancreatic islets. Interferons are found within the inflammatory infiltrate of islets during progression to type 1 diabetes. Interferons can promote the action of effector cells that induce beta cell death. They can also act directly on islet cells to induce gene expression, and together with other cytokines they can cause beta cell death. Because of their pleiotropic nature, it was proposed that this family of cytokines may be involved in type 1 diabetes development. In the non-obese diabetic mouse model, interventions have been made at multiple points in the signalling pathways of interferons. This review aims to construct a clear picture of the outcomes of these interventions to determine how interferons are involved in the pathogenesis of type 1 diabetes.

Islet beta-cells deficient in Bcl-xL develop but are abnormally sensitive to apoptotic stimuli.

OBJECTIVE: Bcl-xL is an antiapoptotic member of the Bcl-2 family of proteins and a potent regulator of cell death. We investigated the importance of Bcl-xL for beta-cells by deleting the Bcl-x gene specifically in beta-cells and analyzing their survival in vivo and in culture. RESEARCH DESIGN AND METHODS: Islets with beta-cells lacking the Bcl-x gene were assessed in vivo by histology and by treatment of mice with low-dose streptozotocin (STZ). Islets were isolated by collagenase digestion and treated in culture with the apoptosis inducers staurosporine, thapsigargin, gamma-irradiation, proinflammatory cytokines, or Fas ligand. Cell death was assessed by flow cytometric analysis of subgenomic DNA. RESULTS: Bcl-xL-deficient beta-cells developed but were abnormally sensitive to apoptosis induced in vivo by low-dose STZ. Although a small proportion of beta-cells still expressed Bcl-xL, these did not have a survival advantage over their Bcl-xL-deficient neighbors. Islets appeared normal after collagenase isolation and whole-islet culture. They were, however, abnormally sensitive in culture to a number of different apoptotic stimuli including cytotoxic drugs, proinflammatory cytokines, and Fas ligand. CONCLUSIONS: Bcl-xL expression in beta-cells is dispensible during islet development in the mouse. Bcl-xL is, however, an important regulator of beta-cell death under conditions of synchronous stress. Bcl-xL expression at physiological levels may partially protect beta-cells from apoptotic stimuli, including apoptosis because of mediators implicated in type 1 diabetes and death or degeneration of transplanted islets.

Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells.

OBJECTIVE: Apoptosis of pancreatic beta-cells is critical in both diabetes development and failure of islet transplantation. The role in these processes of pro- and antiapoptotic Bcl-2 family proteins, which regulate apoptosis by controlling mitochondrial integrity, remains poorly understood. We investigated the role of the BH3-only protein Bid and the multi-BH domain proapoptotic Bax and Bak, as well as prosurvival Bcl-2, in beta-cell apoptosis. RESEARCH DESIGN AND METHODS: We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-alpha, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, gamma-radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry. RESULTS: Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand-induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF-alpha plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor-induced apoptosis. CONCLUSIONS: These results demonstrate that Bid is essential for death receptor-induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which beta-cells are destroyed.

Perforin and Fas induced by IFNgamma and TNFalpha mediate beta cell death by OT-I CTL.

Direct interaction between auto-reactive CTL and specific peptide-MHC class I complexes on pancreatic beta cells is critical in mediating beta cell destruction in type I diabetes. We used mice with genetic modifications in three major pathways used by CTL, perforin, Fas and pro-inflammatory cytokines to assess the relative contribution of these mechanisms to beta cell death. In vitro-activated ovalbumin (OVA)-specific CTL, from OT-I TCR-transgenic mice, specifically killed transgenic beta cells expressing OVA (from RIP-mOVA mice) in a 16-h cytotoxicity assay. Perforin-deficient CTL had a reduced ability to kill OVA-expressing islets in vitro (22.1 +/- 3.8%) compared with wild-type CTL (71.4 +/- 4.6%). Fas-deficient islets were only slightly protected from wild-type CTL but were completely protected from the residual killing observed with perforin-deficient CTL. Residual cytotoxicity in perforin-deficient CTL was also prevented by overexpression of SOCS-1, which blocks multiple cytokine signaling pathways. It was also prevented by pre-incubation with anti-tumor necrosis factor-alpha (anti-TNFalpha) antibody or by blocking IFNgamma responsiveness through expressing a dominant negative IFNgamma receptor. Perforin-deficient CTL produced IFNgamma and TNFalpha that was shown to directly induce islet Fas expression during the assays. This suggests that Fas-deficiency, SOCS-1 overexpression and blockade of IFNgamma and TNFalpha all protect beta cells from residual cytotoxicity of perforin-deficient CTL by blocking Fas upregulation. These findings indicate that wild-type CTL destroy antigen-expressing islets via a perforin-dependent mechanism. However, in the absence of perforin, the Fas/FasL pathway provides an alternative mechanism dependent on islet cell Fas upregulation by cytokines IFNgamma and TNFalpha.