Signaling In Hematopoietic Cells Research Articles

Oncostatin M receptor-dependent signaling assessed by RNA sequencing in mouse hematopoietic stem cells

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines and has been found to have anti-inflammatory and pro-inflammatory properties in various cellular and disease contexts. OSM signals through two receptor complexes, one of which includes OSMRβ. Here, we investigated OSM-OSMRβ signaling in adult mouse hematopoietic stem cells (HSCs) using the conditional Osmrfl/fl mouse model B6;129-Osmrtm1.1Nat/J. We crossed Osmrfl/fl mice to interferon-inducible Mx1-Cre, which is robustly induced in adult HSCs. From these mice, we isolated HSCs by flow cytometry, stimulated with recombinant OSM or vehicle for 1 hour, and assessed gene expression changes in control versus Osmr knockout HSCs by RNA-seq. This data may be utilized to investigate OSMRβ -dependent and -independent OSM signaling as well as the transcriptional effects of an IL-6 family cytokine on mouse HSCs to further define its anti-inflammatory versus pro-inflammatory properties.

Prenatal vitamin D deficiency exposure leads to long-term changes in immune cell proportions

Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We found that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Moreover, further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term offspring also confirm that maternal vitamin D levels in the second trimester significantly affect immune cell proportions in the offspring. These findings imply that the differentiation properties of hematopoiesis act as long-term memories of prenatal vitamin D deficiency exposure in later life.

INOS regulates hematopoietic stem and progenitor cells via mitochondrial signaling and is critical for bone marrow regeneration

Hematopoietic stem cells (HSCs) replenish blood cells under steady state and on demand, that exhibit therapeutic potential for Bone marrow failures and leukemia. Redox signaling plays key role in immune cells and hematopoiesis. However, the role of reactive nitrogen species in hematopoiesis remains unclear and requires further investigation. We investigated the significance of inducible nitric oxide synthase/nitric oxide (iNOS/NO) signaling in hematopoietic stem and progenitor cells (HSPCs) and hematopoiesis under steady-state and stress conditions. HSCs contain low levels of NO and iNOS under normal conditions, but these increase upon bone marrow stress. iNOS-deficient mice showed subtle changes in peripheral blood cells but significant alterations in HSPCs, including increased HSCs and multipotent progenitors. Surprisingly, iNOS-deficient mice displayed heightened susceptibility and delayed recovery of blood progeny following 5-Fluorouracil (5-FU) induced hematopoietic stress. Loss of quiescence and increased mitochondrial stress, indicated by elevated MitoSOX and MMPhi HSCs, were observed in iNOS-deficient mice. Furthermore, pharmacological approaches to mitigate mitochondrial stress rescued 5-FU-induced HSC death. Conversely, iNOS-NO signaling was required for demand-driven mitochondrial activity and proliferation during hematopoietic recovery, as iNOS-deficient mice and NO signaling inhibitors exhibit reduced mitochondrial activity. In conclusion, our study challenges the conventional view of iNOS-derived NO as a cytotoxic molecule and highlights its intriguing role in HSPCs. Together, our findings provide insights into the crucial role of the iNOS–NO–mitochondrial axis in regulating HSPCs and hematopoiesis.

Functional characterization of cooperating MGA mutations in RUNX1::RUNX1T1 acute myeloid leukemia

MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.

MSC Reprogramming and Aberrant Inflammatory Signaling in Myelofibrosis: Insights from Single-Cell RNA-Sequencing of Trephine Bone Marrow Cells

Myelofibrosis (MF) results from mutation-induced activation of JAK/STAT signaling in hematopoietic stem, progenitor and mature cells, and features a progressive, fibrosing stromal reaction, splenomegaly, constitutional symptoms and reduced life expectancy. Identification of disrupted cellular pathways within bone marrow (BM) is pivotal for therapeutic breakthrough, but fibrosis severely limits access to stromal elements for single-cell analysis. To overcome this, we utilized collagenase digestion of fresh BM trephine biopsy material surplus to diagnostic needs and profiled isolated cells using scRNAseq. 9 samples were obtained from 8 patients with MF (4, JAK inhibitor naïve; 4, on ruxolitinib); 4 samples were from 4 patients with MF 4, 10, 12 & 12 months after allogeneic transplant; and 7 samples (termed “normal controls”) were from 6 patients who had received an allograft for myelodysplasia or acute leukemia, and 1 who had received CAR-T cells for B-ALL. Normal controls exhibited normal or near-normal blood counts and lacked BM fibrosis. Each sample was subjected to collagenase digestion and flow sorted to isolate non-erythroid hematopoietic (CD45+GlyA-) and microenvironmental (CD45+GlyA-CD71-) cells for single cell RNA sequencing and downstream comparative analysis. 105,231 transcriptomes met quality control criteria (median 4975/sample, range 1036-13277, no significant difference between sample categories) and 43 transcriptional clusters were identified. Compositional analysis identified the non-hematopoietic stromal cell fraction as harboring the biggest differences with, surprisingly, a proportional relative depletion of mesenchymal stromal cells (MSCs) and enrichment of endothelial cells (ECs) in MF vs. normal controls. While transcriptomes of ECs did not differ substantially, there were extensive transcriptional differences between MSCs recovered from MF vs. normal control samples. Subclustering revealed two subsets, MSC1 & MSC2 asymmetrically distributed between MF and normal controls. Intriguingly, MSCs recovered from MF patients after allograft occupied an intermediate position in principal component space indicating that reversion of disease-related MSC transcriptional reprogramming post-allograft occurs over a timescale of months. The reprogrammed transcriptional state of MF MSCs is defined by reduced expression of hematopoietic support factor genes (eg, KITLG, CXCL12, ANGPTL2, NCAM2, CSF1) and increased expression of extracellular matrix (ECM) genes (eg, FN1, COL1A2, COL3A1, DCN, SPARC), features which suggest an explanation for the increased frequency of graft failure following allograft in MF versus other hematologic malignancies. Single-Cell rEgulatory Network Inference and Clustering (SCENIC) indicated transcription factors with either increased (eg, NR2F2, RUNX2, FOXP2) or decreased (eg, CEBPA, FOXC1, and PPARG) transcriptional activity as candidate master transcriptional regulators of the observed MSC reprogramming in MF. Transcriptional change in MSCs correlated strongly with histopathological assessment of BM reticulin. Within the hematopoietic fraction, and agnostic of JAK inhibitor treatment status, comparison of MF vs. control cells unexpectedly revealed significant upregulation of interferon response genes, especially in CD14 + monocytes, but also in neutrophils and their progenitors, centred on expression of interferon-stimulated gene factor 3 (ISGF3) genes IRF9 and STAT1/2. Comparison of MF samples according to ruxolitinib treatment status indicated that ruxolitinib induced downregulation of an NFκB-centered inflammatory program, particularly in neutrophils. Finally, ligand-receptor analysis using NicheNet identified TGFB1, IL1B, and TNF as top predicted ligands for induction of the MF signature in MSCs. While myeloid cells were top producers of these factors, their expression did not vary between MF vs. controls, suggesting other mechanisms, such as prolonged retention of ligand in the ECM, are important. In summary, our comparative scRNA analysis of BM cells from MF patients and controls - the largest reported to date - reveals (i) transcriptional reprogramming of MSCs leading to reduced expression of hematopoietic support factor and increased expression of ECM genes; and (ii) increased expression of interferon and NFkB inflammatory pathways in myeloid cells.

Regulation of Metabolic Homeostasis By TRAF6 Contributes to the Leukemia Progression

TNF receptor associated factor 6 (TRAF6) is an E3 ubiquitin ligase that mediates innate immune signaling that converges on key mediators of Toll-like receptor and interleukin-1 signaling. Dysregulated TRAF6 expression and innate immune pathway activation is implicated in the pathogenesis of various myeloid malignancies. For instance, increased TRAF6-mediated signaling in hematopoietic stem and progenitor cells (HSPCs) results in myelodysplastic syndrome-like cellular features, such as the functional impairment of HSPCs. On the other hand, our previous study indicated that loss of TRAF6 induces the leukemic transformation of pre-leukemic cells ( Muto et al. Cell Stem Cell). These paradoxical observations suggests that TRAF6 acts as both an oncogene and tumor suppressor gene in hematopoietic cells in a disease type- and context-dependent manner. However, the direct consequence of TRAF6 loss in AML cells, rather than in pre-leukemic cells, has not yet been explored. Therefore, the role of TRAF6 in the pathogenesis of AML remains obscure. In this study, we found elevated level of TRAF6 expression in AML cells, and determined the effect of TRAF6 loss on the cellular and molecular features of AML cells. We proposed a novel role of TRAF6 as a regulator of metabolic homeostasis, which is important for the progression of AML. To determine the requirement of TRAF6 in AML, we first evaluated cell the effect of TRAF6 loss in AML cell lines on proliferative capacities. Multiple types of human AML cell lines showed significant growth impairment upon TRAF6-knockdown. Furthermore, transplantation of TRAF6-deficient HSPCs expressing the leukemic oncogene MLL-AF9 to the recipient mice resulted in suppression of overt leukemia. These findings confirm that loss of TRAF6 suppresses leukemic cell function in vitro and in vivo. To assess the molecular mechanism of inhibitory effect of TRAF6 loss in AML, we next performed gene expression analysis using human AML database and TRAF6-knockdown AML cell lines. Interestingly, we found that reduced levels of TRAF6 in AML is associated with changes in the expression of genes involved in mitochondria-related processes. Our extensive in vitro validation experiments including extracellular flux assay and metabolome analysis confirmed that TRAF6 loss in AML cells induces dynamic changes in metabolites and the impairment of mitochondrial function. To identify the mechanism of metabolic alterations by TRAF6 loss in leukemia, we compared the upregulated genes in TRAF6-knockdown AML cells with the 130 previously identified essential genes for AML cell survival in vitro and in vivo ( Yamauchi et al. Cancer Cell). We focused on O-GlcNAc transferase ( OGT) as a gene associated in mitochondrial function and AML cell survival. Importantly, TRAF6 gene expression in human AML patient samples was positively correlated with the expression of OGT. To confirm the importance of OGT in the regulation of mitochondrial function mediated by TRAF6 in leukemia, we utilized the inhibitor of O-GlcNAcase (OGA), a remover of O-GlcNAc from its substrates. The treatment with MK8719 (OGA inhibitor) mildly, but significantly, restored the number of TRAF6 knockdown AML cells, which was correlated with the changes in mitochondrial function, indicating that O-GlcNAc modification regulated by TRAF6 is important for AML progression. In summary, We provide evidence for the oncogenic function of TRAF6 in leukemia, and shed light on the novel TRAF6/OGT/O-GlcNAc axis that regulates the metabolic reprogramming required for leukemogenesis.

Genetic Deletion or Pharmacologic Inhibition of PTPN11 Impedes the Development and Progression of Myeloproliferative Neoplasms Induced By JAK2V617F and MPLW515L Mutants

Myeloproliferative neoplasms (MPN) including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are clonal myeloid malignancies derived from mutated hematopoietic stem cells. The JAK2V617F mutation was found in ~95% cases of PV and 50-60% patients with ET and MF. Mutations in MPL and CALR were also detected in ET and MF. The JAK inhibitors, Ruxolitinib and Fedratinib, can reduce splenomegaly and alleviate constitutional symptoms but they are not sufficient to induce remission of MPN. So, there is an unmet need to identify new therapeutic targets and therapies for MPN/MF. The non-receptor protein tyrosine phosphatase 11 (PTPN11), which positively regulates hematopoietic cell signaling, is constitutively hyperphosphorylated in MPN patient cells and in hematopoietic cell lines expressing MPN driver mutants. So, we hypothesize that PTPN11 plays an important role in MPNs. To examine the role of PTPN11 in JAK2V617F-induced MPN, we generated inducible PTPN11-deficient heterozygous JAK2V617F mice by crossing PTPN11 floxed mouse with Mx1Cre and JAK2V617F knock-in mice. While expression of heterozygous JAK2V617F (JAK2 VF/+) induced a PV-like MPN characterized by increased WBC, neutrophil, RBC, hemoglobin and platelet counts in the peripheral blood and enlargement of spleen size, deletion of PTPN11 normalized the blood counts and spleen size in JAK2 VF/+ mice. PTPN11 deletion significantly reduced the expansion of hematopoietic stem/progenitors, erythroid, megakaryocytic, and granulocytic cells in the bone marrow (BM) and spleens of JAK2 VF/+ mice. Furthermore, deletion of PTPN11 abrogated Epo-independent CFU-colonies, a hallmark feature of PV disease, in the BM and spleens of JAK2 VF/+ mice. We also investigated the role of PTPN11 in the development of MF using homozygous JAK2V617F and MPLW515L mouse models. We found that deletion of PTPN11 significantly reduced WBC, neutrophil and platelet counts, normalized spleen size and abrogated BM fibrosis in JAK2V617F and MPLW515L mouse models. These results suggest that PTPN11 plays an important role in the development and progression of PV and MF. Data from our PTPN11 genetic deletion study led us to investigate the effects of PTPN11 inhibition against MPN/MF. We tested the efficacy of an allosteric PTPN11 inhibitor SHP099 in MPN hematopoietic cell lines and mouse models of MPN/MF. We observed that treatment of SHP099 significantly reduced proliferation of hematopoietic cells expressing JAK2V617F and MPLW515L. SHP099 treatment also significantly inhibited hematopoietic progenitor colony growth in PV and MF patient CD34+ cells. Next, we tested the in vivo efficacy of SHP099 against PV using heterozygous JAK2V617F mice. Treatment of SHP099 significantly inhibited the increase in WBC, neutrophil and RBC counts and markedly reduced splenomegaly in JAK2 VF/+ mice. SHP099 treatment also significantly reduced hematopoietic stem/progenitors and myeloid precursors in the BM and spleens of JAK2 VF/+ mice. Furthermore, SHP099 treatment abrogated Epo-independent CFU-E colony formation in the BM and spleens of JAK2 VF/+ mice. We also tested the efficacy of SHP099 alone or in combination with ruxolitinib against myelofibrosis using homozygous JAK2V617F (JAK2 VF/VF) and MPLW515L mouse models. SHP099 treatment alone significantly reduced WBC and neutrophil counts and markedly reduced splenomegaly in JAK2 VF/VF and MPLW515L mice. Combined treatment of SHP099 and ruxolitinib resulted in greater reduction of blood cell counts and spleen size in JAK2 VF/VF and MPLW515Lmice. Histopathologic analyses showed that SHP099 treatment significantly reduced BM fibrosis in JAK2 VF/VF and MPLW515L mice while combined treatment of SHP099and ruxolitinib resulted in greater inhibition of BM fibrosis in these mice. To gain insights into the mechanism of inhibition of MPN/MF by PTPN11 deletion or inhibition, we performed RNA-seq analysis on purified LSK cells from JAK2 VF/+ and PTPN11deleted-JAK2 VF/+ mice as well as from JAK2 VF/VF mice treated with ruxolitinib and SHP099/ruxolitinib. RNA-seq data analysis revealed that genes related to MYC targets, ribosome biogenesis and translation were significantly down-regulated by PTPN11 deletion or SHP099/ruxolitinib combination treatment. Overall, our results suggest that inhibition of PTPN11 alone or in combination with JAK2 inhibition might be useful for treatment of PV and MF.

Autonomous IL-36R signaling in neutrophils activates potent antitumor effector functions.

While the rapid advancement of immunotherapies has revolutionized cancer treatment, only a small fraction of patients derive clinical benefit. Eradication of large, established tumors appears to depend on engaging and activating both innate and adaptive immune system components to mount a rigorous and comprehensive immune response. Identifying such agents is a high unmet medical need, because they are sparse in the therapeutic landscape of cancer treatment. Here, we report that IL-36 cytokine can engage both innate and adaptive immunity to remodel an immune-suppressive tumor microenvironment (TME) and mediate potent antitumor immune responses via signaling in host hematopoietic cells. Mechanistically, IL-36 signaling modulates neutrophils in a cell-intrinsic manner to greatly enhance not only their ability to directly kill tumor cells but also promote T and NK cell responses. Thus, while poor prognostic outcomes are typically associated with neutrophil enrichment in the TME, our results highlight the pleiotropic effects of IL-36 and its therapeutic potential to modify tumor-infiltrating neutrophils into potent effector cells and engage both the innate and adaptive immune system to achieve durable antitumor responses in solid tumors.

SRSF2-P95H decreases JAK/STAT signaling in hematopoietic cells and delays myelofibrosis development in mice.

Heterozygous mutation targeting proline 95 in Serine/Arginine-rich Splicing Factor 2 (SRSF2) is associated with V617F mutation in Janus Activated Kinase 2 (JAK2) in some myeloproliferative neoplasms (MPNs), most commonly primary myelofibrosis. To explore the interaction of Srsf2P95H with Jak2V617F, we generated Cre-inducible knock-in mice expressing these mutants under control of the stem cell leukemia (Scl) gene promoter. In transplantation experiments, Srsf2P95H unexpectedly delayed myelofibrosis induced by Jak2V617F and decreased TGFβ1 serum level. Srsf2P95H reduced the competitiveness of transplanted Jak2V617F hematopoietic stem cells while preventing their exhaustion. RNA sequencing of sorted megakaryocytes identified an increased number of splicing events when the two mutations were combined. Focusing on JAK/STAT pathway, Jak2 exon 14 skipping was promoted by Srsf2P95H, an event detected in patients with JAK2V617F and SRSF2P95 co-mutation. The skipping event generates a truncated inactive JAK2 protein. Accordingly, Srsf2P95H delays myelofibrosis induced by the thrombopoietin receptor agonist Romiplostim in Jak2 wild-type animals. These results unveil JAK2 exon 14 skipping promotion as a strategy to reduce JAK/STAT signaling in pathological conditions.

The Role of the Intestinal Microbiome in Antibiotic Associated Neutropenia

Prolonged antibiotic administration has been associated with bone marrow suppression in approximately 10-15% of patients. The mechanisms that underlie the association between prolonged antibiotic therapy and bone marrow suppression, including neutropenia, remain poorly understood. Our recent studies in murine models demonstrated that prolonged broad-spectrum antibiotics suppress hematopoiesis by depletion of the intestinal microbiota. Furthermore, oral administration of the microbial metabolites nucleotide-binding oligomerization domain-containing protein 1 ligand (NOD1L) and desaminotyrosine (DAT) was sufficient to restore normal hematopoiesis in antibiotic-treated mice. Additionally, Type I IFN signaling in hematopoietic cells was noted to be critical for the intestinal microbiota to promote hematopoiesis, as administration of IFNα during antibiotic therapy rescued normal hematopoiesis. These findings support a paradigm in which products of the intestinal microbiota enter the bloodstream and travel to the bone marrow where they promote the production of growth cytokines that support normal hematopoiesis. To elucidate whether changes in the microbiome similar to those observed in mice also occur in humans treated with prolonged courses of antibiotics, we carried out a multicenter non-treatment, non-intervention study. We enrolled patients aged 0-17 years receiving long-term (>2 weeks) intravenous (IV) or oral (PO) antibiotic courses across two tertiary children's hospitals. We excluded patients with history of abnormal blood counts, bone marrow failure, gastrointestinal infection / syndromes / abnormalities, immunodeficiency, malignancy, severe malnutrition, use of prophylactic antibiotics or use of antibiotics immediately before admission. Patients noted to develop neutropenia while receiving antibiotics were enrolled in the retrospective arm. A stool sample was collected at 2 timepoints: at the start and at the completion of antibiotics for the prospective arm of the study; at the onset of neutropenia while on antibiotics and at resolution of neutropenia after completion of antibiotics for the retrospective arm. Here, we report an interim analysis performed for an initial cohort of 5 patients with antibiotic-associated neutropenia and 10 controls matched for age and race/ethnicity. For clinical data analysis, we assessed continuous variables using parametric and non-parametric tests when appropriate while using χ2 or Fisher's exact tests for categorical variables. Stool samples were sent for 16s rDNA sequencing to determine whether there are significant changes in the composition (individual microbial taxa) and diversity (both alpha and beta) of the gut microbiome between groups; as well as for untargeted metabolomic profiling to assess whether changes in the relative abundance of specific microbiome-related metabolites, including but not limited to NOD1L and DAT, are associated with the development of neutropenia with prolonged antibiotic treatment. Analyses of these data are currently ongoing. Neutropenia occurred in 9.4% of our prospective patient samples (3 out of 32 patients). Median age was 13y (range 6 mo. - 17y) for the neutropenic group and 11y (range 1-15y) for the control group. Forty percent of patients were female in both groups (Table 1). Total length of IV antibiotic and ICU admission were positively correlated with neutropenia in this cohort (Table 2). To our knowledge, this is the first clinical study linking changes in the intestinal microbiota with antibiotic associated neutropenia. The incidence of antibiotic-associated neutropenia was comparable to what has been previously reported. The analysis of stool samples from this cohort through 16sDNA sequencing and untargeted metabolomic profiling will outline specific changes in microbiome load, diversity, as well as specific taxa and metabolites potentially related to the development of neutropenia. Altogether, we hope to utilize an improved understanding of the molecular pathways, mediators and receptors by which the microbiome sustains normal hematopoiesis to prevent and treat antibiotic-associated bone marrow suppression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Quantitative MRI Identifies Heterogeneous Bone Marrow Treatment Responses in a Mouse Model of Myelofibrosis

Myeloproliferative neoplasms (MPNs), a class of blood cancers, typically arise from mutations in the thrombopoietin receptor (MPL), JAK2, or calreticulin that constitutively activate JAK/STAT signaling in hematopoietic stem and progenitor cells. Myelofibrosis (MF), the most aggressive of the MPNs, can arise as a primary neoplasm or secondary to other MPNs. MF is characterized by progressive fibrosis of bone marrow (BM), destruction of the hematopoietic niche, and eventual BM failure. Recently, the focus of treatment in MF has shifted toward restoration of healthy BM architecture and a functional hematopoietic niche rather than focusing solely on alleviating symptoms. Accomplishing this paradigm shift in drug development and therapy requires new approaches to monitor spatial and temporal changes in BM in patients during treatment. We present a quantitative magnetic resonance imaging (MRI) approach to analyze key pathologic changes in BM in a mouse model of MPN driven by MPLW515L. To evaluate disease extent, progression, and response to therapy in mice, we use anatomical MRI for spleen volume and three quantitative MRI metrics for BM: 1) apparent diffusion coefficient (ADC), 2) proton density fat fraction (PDFF), and 3) magnetization transfer ratio (MTR). PDFF and ADC assess the fat content and water movement of the BM and are useful in identifying cellularity changes such as hypercellularity in MPNs and MF. MTR probes changes in macromolecular structure, such as those observed with increasing fibrosis in MF. We used quantitative MRI to measure response to treatment in BM and change in spleen volume in response to three drugs: ruxolitinib (JAK1/2 inhibitor), fedratinib (JAK2 inhibitor), and navitoclax (BCL-2/BCL-xL inhibitor). Within 10 days after starting treatment, diseased spleen volumes in the ruxolitinib group (~3.5x healthy volume) had the greatest average reduction in spleen volume measured by MRI (-73.5% ± 11.4%) followed by the fedratinib group (-52.7% ± 16.7%). Interestingly, the spleens of every ruxolitinib-treated mouse displayed consistent and prolonged spleen volume reductions, while only a subset of mice in the other treatment groups (~70% fedratinib mice; ~33% navitoclax mice) demonstrated reductions in spleen volume. Despite the robust spleen response in ruxolitinib-treated mice, only some of the mice (~60%) exhibited sustained changes in BM ADC, MTR, and PDFF signals expected with reversion to healthy BM (decreased ADC, decreased MTR, and increased PDFF). In mice treated with fedratinib or navitoclax, even fewer mice (~40% and ~33% respectively) showed reversion toward healthy BM. Importantly, we observed spatial heterogeneity of BM response within the tibia of single mice, where proximal and distal tibia regions often responded disparately (~49% of cases). Because improving BM cellularity and fibrosis is increasingly the target of therapy in MPNs and in MF specifically, there is a distinct need for better techniques for longitudinally evaluating variations in treatment efficacy in living subjects. In this study, we demonstrate that the combination of three quantitative MRI metrics (ADC, PDFF, and MTR) can detect the impact of therapy on the BM in a preclinical mouse model of MPN/MF. Interestingly, we identified pronounced heterogeneity at multiple levels during treatment: 1) differences in changes in spleen volume versus BM; 2) disparate treatment responses of mice within a treatment group; and 3) contrasting regional treatment responses of the BM within a single mouse. Because these quantitative MRI metrics are directly translatable to clinical medicine and can identify variations in treatment efficacy in BM, both between and within subjects, this study sets the stage for future drug development and analysis of therapeutic interventions on the BM of patients with MF.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Axin2/Conductin Is Required for Normal Haematopoiesis and T Lymphopoiesis.

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells.

T cells show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to provide a renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains an important challenge. Here, we report an efficient serum- and feeder-free system for differentiating human PSCs into hematopoietic progenitors and T cells. This fully defined approach allowed us to study the impact of individual proteins on blood emergence and differentiation. Providing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition enhanced downstream progenitor T cell output by ~80-fold. These two proteins synergized to activate notch signaling in nascent hematopoietic stem and progenitor cells, and VCAM1 additionally promoted an inflammatory transcriptional program. We also established optimized medium formulations that enabled efficient and chemically defined maturation of functional CD8αβ+, CD4−, CD3+, TCRαβ+ T cells with a diverse TCR repertoire.

New-targeted therapy for leukemia based on Endoplasmic Reticulum Stress

The unfolded protein response (UPR) is an evolutionarily conserved adaptive pathway, which is activated by the stress of the endoplasmic reticulum (ER). ER stress often occurs due to the high protein demand in cells and protein folding errors in several diseases, such as different cancers and autoimmune diseases. UPR is mediated by three primary arms called inositol-requiring enzyme-1α (IRE1α), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6α (ATF6α). Given that homeostasis in protein synthesis is frequently deregulated in cancers, UPR plays a critical role in controlling survival and cell death. In addition,, resistance to apoptosis is mediated by the pro-survival mechanism of ER stress in cancer cells. Recent evidence highlighted the deregulation of UPR signaling in hematopoietic stem cells (HSCs) and leukemic cells, so that targeting UPR-driven pro-survival pathways may present new therapeutic benefits in leukemia. In this review article, we aim to provide an updated knowledge on the role of UPR as a novel therapeutic target in leukemia. We first define the different types of leukemia and their challenges with current treatments, and then explore the contribution of UPR to leukemia pathogenesis and treatment. Finally, UPR targeting strategies in pre-clinical and clinical trials of patients with leukemia will be presented.

No time to die? Intrinsic apoptosis signaling in hematopoietic stem and progenitor cells and therapeutic implications.

Dysregulated apoptosis contributes to the pathogenesis of many hematologic malignancies. BH3-mimetics, antagonists of antiapoptotic BCL-2 proteins, represent novel, and promising cancer drugs. While the acute myelosuppressive effects of Venetoclax, the first Food and Drug Administration approved BCL-2 inhibitor, are fairly well described, little is known about side effects of novel BH3-mimetics and effects of chronic Venetoclax treatment. Highly relevant publications focused on the effects of acute and chronic Venetoclax therapy, with focus on cell-type specific adaptive mechanisms, the emergence of clonal hematopoiesis, and the selection of BAX-mutated hematopoietic cells in patients treated with Venetoclax for a long period. Important advances were made in understanding primary and secondary Venetoclax resistance and prediction of Venetoclax response. Combination therapies of BH3-mimetics targeting different BCL-2 proteins are highly anticipated. However, human stem and progenitors require both MCL-1 and BCL-XL for survival, and serious myelosuppressive effects of combined MCL-1/BCL-XL inhibition can be expected. Long-term studies are indispensable to profile the chronic side effects of Venetoclax and novel BH3-mimetics and better balance their risk vs. benefit in cancer therapy. Combination therapies will be powerful, but potentially limited by severe myelosuppression. For precision medicine, a better knowledge of BCL-2 proteins in the healthy and diseased hematopoietic system is required.

The bacterial microbiota regulates normal hematopoiesis via metabolite-induced type 1 interferon signaling

AbstractAntibiotic therapy, especially when administered long term, is associated with adverse hematologic effects such as cytopenia. Signals from the intestinal microbiota are critical to maintain normal hematopoiesis, and antibiotics can cause bone marrow suppression through depletion of the microbiota. We reported previously that STAT1 signaling is necessary for microbiota-dependent hematopoiesis, but the precise mechanisms by which the gut microbiota signals to the host bone marrow to regulate hematopoiesis remain undefined. We sought to identify the cell type(s) through which STAT1 promotes microbiota-mediated hematopoiesis and to elucidate which upstream signaling pathways trigger STAT1 signaling. Using conditional knockout and chimeric mice, we found that the microbiota induced STAT1 signaling in non-myeloid hematopoietic cells to support hematopoiesis and that STAT1 signaling was specifically dependent on type I interferons (IFNs). Indeed, basal type I IFN signaling was reduced in hematopoietic progenitor cells with antibiotic treatment. In addition, we discovered that oral administration of a commensal-derived product, NOD1 ligand, rescues the hematopoietic defects induced by antibiotics in mice. Using metabolomics, we identified additional microbially produced candidates that can stimulate type I IFN signaling to potentially rescue the hematopoietic defects induced by antibiotics, including phosphatidylcholine and γ-glutamylalanine. Overall, our studies define a signaling pathway through which microbiota promotes normal hematopoiesis and identify microbial metabolites that may serve as therapeutic agents to ameliorate antibiotic-induced bone marrow suppression and cytopenia.

PI3 kinase isoform p110δ is more important than p110α in KIT signaling in hematopoietic cells

PI3 kinases are important for KIT signaling and KIT mutants mediated cell transformation. In order to know the difference of PI3 kinase isoforms p110α and p110δ in the signaling of wild-type KIT and the often occurred KIT mutation D816V in hematopoietic malignancy mastocytosis, the predominant PI3 kinase isoform p110δ in hematopoietic tissues was knocked out in hematopoietic cells. We found that loss of p110δ expression dramatically inhibits PI3 kinase activation mediated by both wild-type KIT and KIT/D816V. By over expression of p110α in p110δ knock out cells, wild-type KIT mediated PI3 kinase activation was not changed while over expression of p110δ increased PI3 kinase activation. Similarly, in KIT/D816V expressing cells without p110δ expression, over expression of p110δ but not p110α restored PI3 kinase activation. In agreement with the signaling results, cell proliferation, cell survival and cell cycle assay further showed that over expression of p110δ but not p110α in p110δ knock out cells increases both wild-type KIT and KIT/ D816V mediated cell survival and proliferation. These results suggested that p110δ plays a more important role than p110α in KIT signaling and KIT mutant mediated cell transformation in hematopoietic cells.

Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell–derived compartments

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α-dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.