Search

The effects of radiotherapy (RT) on tumor immunity in pancreatic ductal adenocarcinoma (PDAC) are not well understood. To better understand if RT can prime antigen-specific T-cell responses, we analyzed human PDAC tissues and mouse models. In both settings, there was little evidence of RT-induced T-cell priming. Using in vitro systems, we found that tumor–stromal components, including fibroblasts and collagen, cooperate to blunt RT efficacy and impair RT-induced interferon signaling. Focal adhesion kinase (FAK) inhibition rescued RT efficacy in vitro and in vivo, leading to tumor regression, T-cell priming, and enhanced long-term survival in PDAC mouse models. Based on these data, we initiated a clinical trial of defactinib in combination with stereotactic body RT in patients with PDAC (NCT04331041). Analysis of PDAC tissues from these patients showed stromal reprogramming mirroring our findings in genetically engineered mouse models. Finally, the addition of checkpoint immunotherapy to RT and FAK inhibition in animal models led to complete tumor regression and long-term survival. Significance: Checkpoint immunotherapeutics have not been effective in PDAC, even when combined with RT. One possible explanation is that RT fails to prime T-cell responses in PDAC. Here, we show that FAK inhibition allows RT to prime tumor immunity and unlock responsiveness to checkpoint immunotherapy. This article is highlighted in the In This Issue feature, p. 2711

MYSM1 is a chromatin‐binding protein, widely investigated for its functions in haematopoiesis in human and mouse; however, its role in haematologic malignancies remains unexplored. Here, we investigate the cross‐talk between MYSM1 and oncogenic cMYC in the transcriptional regulation of genes encoding ribosomal proteins, and the implications of these mechanisms for cMYC‐driven carcinogenesis. We demonstrate that in cMYC‐driven B cell lymphoma in mouse models, MYSM1‐loss represses ribosomal protein gene expression and protein synthesis. Importantly, the loss of MYSM1 also strongly inhibits cMYC oncogenic activity and protects against B cell lymphoma onset and progression in the mouse models. This advances the understanding of the molecular and transcriptional mechanisms of lymphomagenesis, and suggests MYSM1 as a possible drug target for cMYC‐driven malignancies.

Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of mesenchymal cells supporting tumor progression, whose origin remains to be fully elucidated. Osterix (Osx) is a marker of osteogenic differentiation, expressed in skeletal progenitor stem cells and bone-forming osteoblasts. We report Osx expression in CAFs and by using Osx-cre;TdTomato reporter mice we confirm the presence and pro-tumorigenic function of TdTOSX+ cells in extra-skeletal tumors. Surprisingly, only a minority of TdTOSX+ cells expresses fibroblast and osteogenic markers. The majority of TdTOSX+ cells express the hematopoietic marker CD45, have a genetic and phenotypic profile resembling that of tumor infiltrating myeloid and lymphoid populations, but with higher expression of lymphocytic immune suppressive genes. We find Osx transcript and Osx protein expression early during hematopoiesis, in subsets of hematopoietic stem cells and multipotent progenitor populations. Our results indicate that Osx marks distinct tumor promoting CD45- and CD45+ populations and challenge the dogma that Osx is expressed exclusively in cells of mesenchymal origin.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice., Lysine27-to-methionine mutations in histone H3 genes (H3K27M) occur in a subgroup of gliomas and decrease genome-wide H3K27 trimethylation. Here the authors utilise primary H3K27M tumour lines and isogenic CRISPR-edited controls and show that H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3.

MYSM1 is a chromatin-interacting deubiquitinase recently shown to be essential for hematopoietic stem cell (HSC) function and normal progression of hematopoiesis in both mice and humans. However, it remains unknown whether the loss of function in Mysm1-deficient HSCs is due to the essential role of MYSM1 in establishing the HSC pool during development or due to a continuous requirement for MYSM1 in adult HSCs. In this study we, for the first time, address these questions first, by performing a detailed analysis of hematopoiesis in the fetal livers of Mysm1-knockout mice, and second, by assessing the effects of an inducible Mysm1 ablation on adult HSC functions. Our data indicate that MYSM1 is essential for normal HSC function and progression of hematopoiesis in the fetal liver. Furthermore, the inducible knockout model demonstrates a continuous requirement for MYSM1 to maintain HSC functions and antagonize p53 activation in adult bone marrow. These studies advance our understanding of the role of MYSM1 in HSC biology, and provide new insights into the human hematopoietic failure syndrome resulting from MYSM1 deficiency.

MYSM1 is a chromatin-binding transcriptional cofactor that deubiquitinates histone H2A. Studies of Mysm1-deficient mice have shown that it is essential for hematopoietic stem cell (HSC) function and lymphopoiesis. Human carriers of a rare MYSM1-inactivating mutation display similar lymphopoietic deficiencies. However, the mechanism by which MYSM1 regulates hematopoietic homeostasis remains unclear. Here, we show that Mysm1-deficiency results in p53 protein elevation in many hematopoietic cell types. p53 is a central regulator of cellular stress responses and HSC homeostasis. We thus generated double-knockout mice to assess a potential genetic interaction between Mysm1 and p53 in hematopoiesis. Mysm1(-/-)p53(-/-) mouse characterization showed a full rescue of Mysm1(-/-) developmental and hematopoietic defects. This included restoration of lymphopoiesis, and HSC numbers and functions. These results establish p53 activation as the driving mechanism for hematopoietic abnormalities in Mysm1 deficiency. Our findings may advance the understanding of p53 regulation in hematopoiesis and implicate MYSM1 as a potential p53 cofactor.

Hematopoietic stem cells (HSCs) serve as a life-long reservoir for all blood cell types and are clinically useful for a variety of HSC transplantation-based therapies. Understanding the role of chromatin organization and regulation in HSC homeostasis may provide important insights into HSC development. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multivalent chromatin regulator that possesses 4 nucleosome-binding domains and activates 3 lysine acetyltransferases (KAT6A, KAT6B, and KAT7), suggesting that this protein has the potential to stimulate crosstalk between different chromatin modifications. Here, we investigated the function of BRPF1 in hematopoiesis by selectively deleting its gene in murine blood cells. Brpf1-deficient pups experienced early lethality due to acute bone marrow failure and aplastic anemia. The mutant bone marrow and fetal liver exhibited severe deficiency in HSCs and hematopoietic progenitors, along with elevated reactive oxygen species, senescence, and apoptosis. BRPF1 deficiency also reduced the expression of multipotency genes, including Slamf1, Mecom, Hoxa9, Hlf, Gfi1, Egr, and Gata3. Furthermore, BRPF1 was required for acetylation of histone H3 at lysine 23, a highly abundant but not well-characterized epigenetic mark. These results identify an essential role of the multivalent chromatin regulator BRPF1 in definitive hematopoiesis and illuminate a potentially new avenue for studying epigenetic networks that govern HSC ontogeny.

p53 is a central mediator of cellular stress responses, and its precise regulation is essential for the normal progression of hematopoiesis. MYSM1 is an epigenetic regulator essential for the maintenance of hematopoietic stem cell (HSC) function, hematopoietic progenitor survival, and lymphocyte development. We recently demonstrated that all developmental and hematopoietic phenotypes of Mysm1 deficiency are p53-mediated and rescued in the Mysm1(-/-)p53(-/-) mouse model. However, the mechanisms triggering p53 activation in Mysm1(-/-) HSPCs, and the pathways downstream of p53 driving different aspects of the Mysm1(-/-) phenotype remain unknown. Here we show the transcriptional activation of p53 stress responses in Mysm1(-/-) HSPCs. Mechanistically, we find that the MYSM1 protein associates with p53 and colocalizes to promoters of classical p53-target genes Bbc3/PUMA (p53 upregulated modulator of apoptosis) and Cdkn1a/p21. Furthermore, it antagonizes their p53-driven expression by modulating local histone modifications (H3K27ac and H3K4me3) and p53 recruitment. Using double-knockout mouse models, we establish that PUMA, but not p21, is an important mediator of p53-driven Mysm1(-/-) hematopoietic dysfunction. Specifically, Mysm1(-/-)Puma(-/-) mice show full rescue of multipotent progenitor (MPP) viability, partial rescue of HSC quiescence and function, but persistent lymphopenia. Through transcriptome analysis of Mysm1(-/-)Puma(-/-) MPPs, we demonstrate strong upregulation of other p53-induced mediators of apoptosis and cell-cycle arrest. The full viability of Mysm1(-/-)Puma(-/-) MPPs, despite strong upregulation of many other pro-apoptotic mediators, establishes PUMA as the essential non-redundant effector of p53-induced MPP apoptosis. Furthermore, we identify potential mediators of p53-dependent but PUMA-independent Mysm1(-/-)hematopoietic deficiency phenotypes. Overall, our study provides novel insight into the cell-type-specific roles of p53 and its downstream effectors in hematopoiesis using unique models of p53 hyperactivity induced by endogenous stress. We conclude that MYSM1 is a critical negative regulator of p53 transcriptional programs in hematopoiesis, and that its repression of Bbc3/PUMA expression is essential for MPP survival, and partly contributes to maintaining HSC function.

USP21 is a ubiquitin specific protease that catalyzes protein deubiquitination, however the identification of its physiological substrates remains challenging. USP21 is known to deubiquitinate transcription factor GATA3 and death-domain kinase RIPK1 in vitro, however the in vivo settings where this regulation plays a biologically significant role remain unknown. In order to determine whether USP21 is an essential and non-redundant regulator of GATA3 or RIPK1 activity in vivo, we characterized Usp21-deficient mice, focusing on mouse viability and development, hematopoietic stem cell function, and lymphocyte differentiation. The Usp21-knockout mice were found to be viable and fertile, with no significant dysmorphology, in contrast to the GATA3 and RIPK1 knockout lines that exhibit embryonic or perinatal lethality. Loss of USP21 also had no effect on hematopoietic stem cell function, lymphocyte development, or the responses of antigen presenting cells to TLR and TNFR stimulation. GATA3 levels in hematopoietic stem cells or T lymphocytes remained unchanged. We observed that aged Usp21-knockout mice exhibited spontaneous T cell activation, however this was not linked to altered GATA3 levels in the affected cells. The contrast in the phenotype of the Usp21-knockout line with the previously characterized GATA3 and RIPK1 knockout mice strongly indicates that USP21 is redundant for the regulation of GATA3 and RIPK1 activity during mouse development, in hematopoietic stem cells, and in lymphocyte differentiation. The Usp21-deficient mouse line characterized in this study may serve as a useful tool for the future characterization of USP21 physiological functions.