Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease.

Thrombopoietin (Tpo), which binds to its specific receptor, the Mpl protein, is the major cytokine regulator of megakaryopoiesis and circulating platelet number. Tpo binding to Mpl triggers activation of Janus kinase 2 (Jak2) and phosphorylation of the receptor, as well as activation of several intracellular signalling cascades that mediate cellular responses. Three tyrosine (Y) residues in the C-terminal region of the Mpl intracellular domain have been implicated as sites of phosphorylation required for regulation of major Tpo-stimulated signalling pathways: Mpl-Y565, Mpl-Y599 and Mpl-Y604. Here, we have introduced mutations in the mouse germline and report a consistent physiological requirement for Mpl-Y599, mutation of which resulted in thrombocytopenia, deficient megakaryopoiesis, low hematopoietic stem cell (HSC) number and function, and attenuated responses to myelosuppression. We further show that in models of myeloproliferative neoplasms (MPN), where Mpl is required for pathogenesis, thrombocytosis was dependent on intact Mpl-Y599. In contrast, Mpl-Y565 was required for negative regulation of Tpo responses; mutation of this residue resulted in excess megakaryopoiesis at steady-state and in response to myelosuppression, and exacerbated thrombocytosis associated with MPN.

ERG and c-MYC regulate a critical gene network in BCR::ABL1-driven B cell acute lymphoblastic leukemia.

Philadelphia chromosome-positive B cell acute lymphoblastic leukemia (B-ALL), characterized by the BCR::ABL1 fusion gene, remains a poor prognosis cancer needing new therapeutic approaches. Transcriptomic profiling identified up-regulation of oncogenic transcription factors ERG and c-MYC in BCR::ABL1 B-ALL with ERG and c-MYC required for BCR::ABL1 B-ALL in murine and human models. Profiling of ERG- and c-MYC-dependent gene expression and analysis of ChIP-seq data established ERG and c-MYC coordinate a regulatory network in BCR::ABL1 B-ALL that controls expression of genes involved in several biological processes. Prominent was control of ribosome biogenesis, including expression of RNA polymerase I (POL I) subunits, the importance of which was validated by inhibition of BCR::ABL1 cells by POL I inhibitors, including CX-5461, that prevents promoter recruitment and transcription initiation by POL I. Our results reveal an essential ERG- and c-MYC-dependent transcriptional network involved in regulation of metabolic and ribosome biogenesis pathways in BCR::ABL1 B-ALL, from which previously unidentified vulnerabilities and therapeutic targets may emerge.

The histone acetyltransferase KAT6B is required for hematopoietic stem cell development and function.

The histone lysine acetyltransferase KAT6B (MYST4, MORF, QKF) is the target of recurrent chromosomal translocations causing hematological malignancies with poor prognosis. Using Kat6b germline deletion and overexpression in mice, we determined the role of KAT6B in the hematopoietic system. We found that KAT6B sustained the fetal hematopoietic stem cell pool but did not affect viability or differentiation. KAT6B was essential for normal levels of histone H3 lysine 9 (H3K9) acetylation but not for a previously proposed target, H3K23. Compound heterozygosity of Kat6b and the closely related gene, Kat6a, abolished hematopoietic reconstitution after transplantation. KAT6B and KAT6A cooperatively promoted transcription of genes regulating hematopoiesis, including the Hoxa cluster, Pbx1, Meis1, Gata family, Erg, and Flt3. In conclusion, we identified the hematopoietic processes requiring Kat6b and showed that KAT6B and KAT6A synergistically promoted HSC development, function, and transcription. Our findings are pertinent to current clinical trials testing KAT6A/B inhibitors as cancer therapeutics.

Multidimensional biomarker predicts disease control in response to immunotherapy in recurrent or metastatic head and neck squamous-cell carcinoma.

PURPOSE: Anti-PD-1 therapy provides clinical benefit in 40-50% of patients with relapsed and/or metastatic head and neck squamous cell carcinoma (RM-HNSCC). Selection of anti- PD-1 therapy is typically based on patient PD-L1 immunohistochemistry (IHC) which has low specificity for predicting disease control. Therefore, there is a critical need for a clinical biomarker that will predict clinical benefit to anti-PD-1 treatment with high specificity. METHODS: Clinical treatment and outcomes data for 103 RM-HNSCC patients were paired with RNA-sequencing data from formalin-fixed patient samples. Using logistic regression methods, we developed a novel biomarker classifier based on expression patterns in the tumor immune microenvironment to predict disease control with monotherapy PD-1 inhibitors (pembrolizumab and nivolumab). The performance of the biomarker was internally validated using out-of-bag methods. RESULTS: The biomarker significantly predicted disease control (65% in predicted non-progressors vs. 17% in predicted progressors, p < 0.001) and was significantly correlated with overall survival (OS; p = 0.004). In addition, the biomarker outperformed PD-L1 IHC across numerous metrics including sensitivity (0.79 vs 0.64, respectively; p = 0.005) and specificity (0.70 vs 0.61, respectively; p = 0.009). CONCLUSION: This novel assay uses tumor immune microenvironment expression data to predict disease control and OS with high sensitivity and specificity in patients with RM-HNSCC treated with anti-PD-1 monotherapy.

Overexpression of Lmo2 initiates T-lymphoblastic leukemia via impaired thymocyte competition.

Cell competition has recently emerged as an important tumor suppressor mechanism in the thymus that inhibits autonomous thymic maintenance. Here, we show that the oncogenic transcription factor Lmo2 causes autonomous thymic maintenance in transgenic mice by inhibiting early T cell differentiation. This autonomous thymic maintenance results in the development of self-renewing preleukemic stem cells (pre-LSCs) and subsequent leukemogenesis, both of which are profoundly inhibited by restoration of thymic competition or expression of the antiapoptotic factor BCL2. Genomic analyses revealed the presence of Notch1 mutations in pre-LSCs before subsequent loss of tumor suppressors promotes the transition to overt leukemogenesis. These studies demonstrate a critical role for impaired cell competition in the development of pre-LSCs in a transgenic mouse model of T cell acute lymphoblastic leukemia (T-ALL), implying that this process plays a role in the ontogeny of human T-ALL.

The SOCS1 KIR and SH2 domain are both required for suppression of cytokine signaling in vivo.

Suppressor Of Cytokine Signaling (SOCS) 1 is a critical negative regulator of cytokine signaling and required to protect against an excessive inflammatory response. Genetic deletion of Socs1 results in unrestrained cytokine signaling and neonatal lethality, characterised by an inflammatory immune infiltrate in multiple organs. Overexpression and structural studies have suggested that the SOCS1 kinase inhibitory region (KIR) and Src homology 2 (SH2) domain are important for interaction with and inhibition of the receptor-associated JAK1, JAK2 and TYK2 tyrosine kinases, which initiate downstream signaling. To investigate the role of the KIR and SH2 domain in SOCS1 function, we independently mutated key conserved residues in each domain and analysed the impact on cytokine signaling, and the in vivo impact on SOCS1 function. Mutation of the SOCS1-KIR or SH2 domain had no impact on the integrity of the SOCS box complex, however, mutation within the phosphotyrosine binding pocket of the SOCS1-SH2 domain specifically disrupted SOCS1 interaction with phosphorylated JAK1. In contrast, mutation of the KIR did not affect the interaction with JAK1, but did prevent SOCS1 inhibition of JAK1 autophosphorylation. In human and mouse cell lines, both mutants impacted the ability of SOCS1 to restrain cytokine signaling, and crucially, Socs1-R105A and Socs1-F59A mice displayed a neonatal lethality and excessive inflammatory phenotype similar to Socs1-null mice. This study defines a critical and non-redundant role for both the KIR and SH2 domain in endogenous SOCS1 function.

Iron homeostasis governs erythroid phenotype in polycythemia vera.

Polycythemia vera (PV) is a myeloproliferative neoplasm driven by activating mutations in JAK2 that result in unrestrained erythrocyte production, increasing patients' hematocrit and hemoglobin concentrations, placing them at risk of life-threatening thrombotic events. Our genome-wide association study of 440 PV cases and 403 351 controls using UK Biobank data showed that single nucleotide polymorphisms in HFE known to cause hemochromatosis are highly associated with PV diagnosis, linking iron regulation to PV. Analysis of the FinnGen dataset independently confirmed overrepresentation of homozygous HFE variants in patients with PV. HFE influences the expression of hepcidin, the master regulator of systemic iron homeostasis. Through genetic dissection of mouse models of PV, we show that the PV erythroid phenotype is directly linked to hepcidin expression: endogenous hepcidin upregulation alleviates erythroid disease whereas hepcidin ablation worsens it. Furthermore, we demonstrate that in PV, hepcidin is not regulated by expanded erythropoiesis but is likely governed by inflammatory cytokines signaling via GP130-coupled receptors. These findings have important implications for understanding the pathophysiology of PV and offer new therapeutic strategies for this disease.

Proinflammatory microenvironment promotes lymphoma progression in mice with high megakaryocyte and TPO levels.

Platelets have been shown to enhance the survival of lymphoma cell lines. However, it remains unclear whether they play a role in lymphoma. Here, we investigated the potential role of platelets and/or megakaryocytes in the progression of Eµ-myc lymphoma. Eµ-myc tumor cells were transplanted into recipient wild-type (WT) control, Mpl-/-, or TpoTg mice, which exhibited normal, low, and high platelet and megakaryocyte counts, respectively. TpoTg mice that underwent transplantation exhibited enhanced lymphoma progression with increased white blood cell (WBC) counts, spleen and lymph node weights, and enhanced liver infiltration when compared with WT mice. Conversely, tumor-bearing Mpl-/- mice had reduced WBC counts, lymph node weights, and less liver infiltration than WT mice. Using an Mpl-deficient thrombocytopenic immunocompromised mouse model, our results were confirmed using the human non-Hodgkin lymphoma GRANTA cell line. Although we found that platelets and platelet-released molecules supported Eµ-myc tumor cell survival in vitro, pharmacological inhibition of platelet function or anticoagulation in WT mice transplanted with Eµ-myc did not improve disease outcome. Furthermore, transient platelet depletion or sustained Bcl-xL-dependent thrombocytopenia did not alter lymphoma progression. Cytokine analysis of the bone marrow fluid microenvironment revealed increased levels of the proinflammatory molecule interleukin 1 in TpoTg mice, whereas these levels were lower in Mpl-/- mice. Moreover, RNA sequencing of blood-resident Eµ-myc lymphoma cells from TpoTg and WT mice after tumor transplantation revealed the upregulation of hallmark gene sets associated with an inflammatory response in TpoTg mice. We propose that the proinflammatory microenvironment in TpoTg mice promotes lymphoma progression.

SOCS2 regulation of growth hormone signaling requires a canonical interaction with phosphotyrosine.

Suppressor of cytokine signaling (SOCS) 2 is the critical negative regulator of growth hormone (GH) and prolactin signaling. Mice lacking SOCS2 display gigantism with increased body weight and length, and an enhanced response to GH treatment. Here, we characterized mice carrying a germ-line R96C mutation within the SOCS2-SH2 domain, which disrupts the ability of SOCS2 to interact with tyrosine-phosphorylated targets. Socs2R96C/R96C mice displayed a similar increase in growth as previously observed in SOCS2 null (Socs2-/-) mice, with a proportional increase in body and organ weight, and bone length. Embryonic fibroblasts isolated from Socs2R96C/R96C and Socs2-/- mice also showed a comparable increase in phosphorylation of STAT5 following GH stimulation, indicating the critical role of phosphotyrosine binding in SOCS2 function.

Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance.

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal.

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac), and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used 2 complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1-null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow 2 to 6 weeks after Hbo1 deletion. Hbo1-deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors. The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-, and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1, and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

Altered microbiota by a high-fat diet accelerates lethal myeloid hematopoiesis associated with systemic SOCS3 deficiency.

The suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling required to prevent excessive cellular responses. In particular, SOCS3 is involved in the regulation of metabolic syndromes, such as obesity and diabetes, by suppressing leptin and insulin signals. SOCS3 also suppresses the inflammatory response associated with metabolic stress, but this specific role remains undefined. Wild-type mice on a high-fat diet (HFD) exhibited only fatty liver, whereas systemic deletion of SOCS3 resulted in excessive myeloid hematopoiesis and hepatic inflammation. In addition, depletion of the gut microbiota resulted in considerable improvement in excess granulopoiesis and splenomegaly, halting the progression of systemic inflammation in SOCS3KO mice on the HFD. This result suggests that intestinal dysbiosis is involved in inflammation associated with SOCS3KO. Although contributing to diet-induced obesity and fatty liver, SOCS3 is nevertheless critical to suppress excess myeloid hematopoiesis and severe systemic inflammation associated with intestinal dysbiosis on HFD.

Differential requirement for the Polycomb repressor complex 2 in dendritic cell and tissue-resident myeloid cell homeostasis.

Dendritic cells (DCs) and macrophages are at the forefront of immune responses, modifying their transcriptional programs in response to their tissue environment or immunological challenge. Posttranslational modifications of histones, such as histone H3 lysine-27 trimethylation (H3K27me3) by the Polycomb repressive complex 2 (PRC2), are tightly associated with epigenetic regulation of gene expression. To explore whether H3K27me3 is involved in either the establishment or function of the mononuclear phagocyte system, we selectively deleted core components of PRC2, either EZH2 or SUZ12, in CD11c-expressing myeloid cells. Unexpectedly, EZH2 deficiency neither prevented the deposition and maintenance of H3K27me3 in DCs nor hindered DC/macrophage homeostasis. In contrast, SUZ12 deficiency markedly impaired the capacity of DCs and macrophages to maintain H3K27me3. SUZ12 ablation induced a rapid loss of the alveolar macrophage and Langerhans cell networks under both steady state and inflammatory conditions because these cells could no longer proliferate to facilitate their self-renewal. Despite the reduced H3K27me3, DC development and function were unaffected by SUZ12 ablation, suggesting that PRC2-mediated gene repression was dispensable for DC homeostasis. Thus, the role of SUZ12 highlights the fundamentally different homeostatic mechanisms used by tissue-resident myeloid cells versus DCs.

Proteomic analyses reveal that immune integrins are major targets for regulation by Membrane-Associated Ring-CH (MARCH) proteins MARCH2, 3, 4 and 9.

MARCH proteins are membrane-associated Ring-CH E3 ubiquitin ligases that dampen immune responses by downregulating cell surface expression of major histocompatibility complexes I and II as well as immune co-stimulatory receptors. We recently showed that MARCH2,3,4 and 9 also downregulate cell surface expression of the inflammatory cytokine receptor for interleukin-6 (IL6Rα). Here we use over-expression of these MARCH proteins in the M1 myeloid leukaemia cell line and cell surface proteomic analyses to globally analyse other potential targets of these proteins. A large range of cell surface proteins regulated by more than one MARCH protein in addition to several MARCH protein-specific cell surface targets were identified most of which were downregulated by MARCH expression. Prominent among these were several integrin complexes associated with immune cell homing, adhesion and migration. Integrin α4ß1 (VLA4 or VCAM-1 receptor) was downregulated only by MARCH2 and we showed that in MARCH2 knockout mice, Integrin α4 was upregulated specifically in mature B-lymphocytes and this was accompanied by decreased numbers of B-cells in the spleen.

The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis.

Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.

T-ALL can evolve to oncogene independence.

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

Necroptosis is dispensable for the development of inflammation-associated or sporadic colon cancer in mice.

Chronic inflammation of the large intestine is associated with an increased risk of developing colorectal cancer (CRC), the second most common cause of cancer-related deaths worldwide. Necroptosis has emerged as a form of lytic programmed cell death that, distinct from apoptosis, triggers an inflammatory response. Dysregulation of necroptosis has been linked to multiple chronic inflammatory diseases, including inflammatory bowel disease and cancer. Here, we used murine models of acute colitis, colitis-associated CRC, sporadic CRC, and spontaneous intestinal tumorigenesis to investigate the role of necroptosis in these gastrointestinal pathologies. In the Dextran Sodium Sulfate-induced acute colitis model, in some experiments, mice lacking the terminal necroptosis effector protein, MLKL, or its activator RIPK3, exhibited greater weight loss compared to wild-type mice, consistent with some earlier reports. However, the magnitude of weight loss and accompanying inflammatory pathology upon Mlkl deletion varied substantially between independent repeats. Such variation provides a possible explanation for conflicting literature reports. Furthermore, contrary to earlier reports, we observed that genetic deletion of MLKL had no impact on colon cancer development using several mouse models. Collectively, these data do not support an obligate role for necroptosis in inflammation or cancer within the gastrointestinal tract.

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.