Pharmacological depletion of RNA splicing factor RBM39 by indisulam synergizes with PARP inhibitors in high-grade serous ovarian carcinoma.

Ovarian high-grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer with limited therapeutic options and a poor prognosis. In recent years, poly-ADP ribose polymerase (PARP) inhibitors have demonstrated significant clinical benefits, especially in patients with BRCA1/2 mutations. However, acquired drug resistance and relapse is a major challenge. Indisulam (E7070) has been identified as a molecular glue that brings together splicing factor RBM39 and DCAF15 E3 ubiquitin ligase resulting in polyubiquitination, degradation, and subsequent RNA splicing defects. In this work, we demonstrate that the loss of RBM39 induces splicing defects in key DNA damage repair genes in ovarian cancer, leading to increased sensitivity to cisplatin and various PARP inhibitors. The addition of indisulam also improved olaparib response in mice bearing PARP inhibitor-resistant tumors. These findings demonstrate that combining RBM39 degraders and PARP inhibitors is a promising therapeutic approach to improve PARP inhibitor response in ovarian HGSC.

You and me versus the rest of the world: the effects of affiliative motivation and ingroup partner status on social tuning.

Bandura argues that individuals are more likely to engage in social learning when they identify with a social model and when they are motivated or rewarded. Therefore, in the present work, we investigate how these two key factors, perceived similarity and affiliative motivation, influence the extent to which individuals engage in social tuning or align their views with an interaction partner-especially if their partner's attitudes differ from the larger social group. Experiment 1 (170 participants) explored the role of perceived similarity through group membership when needing to work collaboratively with a collaboration partner whose climate change beliefs differed from a larger social group. Experiment 2 (115 participants) directly manipulated affiliative motivation (i.e., length of interaction time) along with perceived similarity (i.e., Greek Life membership) to explore if these factors influenced social tuning of drinking attitudes and behaviors. Experiments 3 (69 participants) and 4 (93 participants) replicated Experiment 2 and examined whether tuning occurred for explicit and implicit attitudes towards weight (negative views Experiment 3 and positive views Experiment 4). Results indicate that when individuals experience high affiliative motivation, they are more likely to engage in social tuning of explicit and implicit attitudes when their interaction partner belongs to their ingroup rather than their outgroup. These findings are consistent with the tenets of Social Learning Theory, Shared Reality Theory, and the affiliative social tuning hypothesis.

TLR7 activation at epithelial barriers promotes emergency myelopoiesis and lung antiviral immunity.

Monocytes are heterogeneous innate effector leukocytes generated in the bone marrow and released into circulation in a CCR2-dependent manner. During infection or inflammation, myelopoiesis is modulated to rapidly meet the demand for more effector cells. Danger signals from peripheral tissues can influence this process. Herein we demonstrate that repetitive TLR7 stimulation via the epithelial barriers drove a potent emergency bone marrow monocyte response in mice. This process was unique to TLR7 activation and occurred independently of the canonical CCR2 and CX3CR1 axes or prototypical cytokines. The monocytes egressing the bone marrow had an immature Ly6C-high profile and differentiated into vascular Ly6C-low monocytes and tissue macrophages in multiple organs. They displayed a blunted cytokine response to further TLR7 stimulation and reduced lung viral load after RSV and influenza virus infection. These data provide insights into the emergency myelopoiesis likely to occur in response to the encounter of single-stranded RNA viruses at barrier sites.

Dual G9A/EZH2 Inhibition Stimulates Antitumor Immune Response in Ovarian High-Grade Serous Carcinoma.

Ovarian high-grade serous carcinoma (HGSC) prognosis correlates directly with presence of intratumoral lymphocytes. However, cancer immunotherapy has yet to achieve meaningful survival benefit in patients with HGSC. Epigenetic silencing of immunostimulatory genes is implicated in immune evasion in HGSC and re-expression of these genes could promote tumor immune clearance. We discovered that simultaneous inhibition of the histone methyltransferases G9A and EZH2 activates the CXCL10-CXCR3 axis and increases homing of intratumoral effector lymphocytes and natural killer cells while suppressing tumor-promoting FoxP3+ CD4 T cells. The dual G9A/EZH2 inhibitor HKMTI-1-005 induced chromatin changes that resulted in the transcriptional activation of immunostimulatory gene networks, including the re-expression of elements of the ERV-K endogenous retroviral family. Importantly, treatment with HKMTI-1-005 improved the survival of mice bearing Trp53-/- null ID8 ovarian tumors and resulted in tumor burden reduction. These results indicate that inhibiting G9A and EZH2 in ovarian cancer alters the immune microenvironment and reduces tumor growth and therefore positions dual inhibition of G9A/EZH2 as a strategy for clinical development.

Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation.

Defective silencing of retrotransposable elements has been linked to inflammageing, cancer and autoimmune diseases. However, the underlying mechanisms are only partially understood. Here we implicate the histone H3.3 chaperone Daxx, a retrotransposable element repressor inactivated in myeloid leukaemia and other neoplasms, in protection from inflammatory disease. Loss of Daxx alters the chromatin landscape, H3.3 distribution and histone marks of haematopoietic progenitors, leading to engagement of a Pu.1-dependent transcriptional programme for myelopoiesis at the expense of B-cell differentiation. This causes neutrophilia and inflammation, predisposing mice to develop an autoinflammatory skin disease. While these molecular and phenotypic perturbations are in part reverted in animals lacking both Pu.1 and Daxx, haematopoietic progenitors in these mice show unique chromatin and transcriptome alterations, suggesting an interaction between these two pathways. Overall, our findings implicate retrotransposable element silencing in haematopoiesis and suggest a cross-talk between the H3.3 loading machinery and the pioneer transcription factor Pu.1.

Pancreatic Cancer Chemotherapy Is Potentiated by Induction of Tertiary Lymphoid Structures in Mice.

BACKGROUND AND AIMS: The presence of tertiary lymphoid structures (TLSs) may confer survival benefit to patients with pancreatic ductal adenocarcinoma (PDAC), in an otherwise immunologically inert malignancy. Yet, the precise role in PDAC has not been elucidated. Here, we aim to investigate the structure and role of TLSs in human and murine pancreatic cancer. METHODS: Multicolor immunofluorescence and immunohistochemistry were used to fully characterize TLSs in human and murine (transgenic [KPC (KrasG12D, p53R172H, Pdx-1-Cre)] and orthotopic) pancreatic cancer. An orthotopic murine model was developed to study the development of TLSs and the effect of the combined chemotherapy and immunotherapy on tumor growth. RESULTS: Mature, functional TLSs are not ubiquitous in human PDAC and KPC murine cancers and are absent in the orthotopic murine model. TLS formation can be induced in the orthotopic model of PDAC after intratumoral injection of lymphoid chemokines (CXCL13/CCL21). Coadministration of systemic chemotherapy (gemcitabine) and intratumoral lymphoid chemokines into orthotopic tumors altered immune cell infiltration ,facilitating TLS induction and potentiating antitumor activity of chemotherapy. This resulted in significant tumor reduction, an effect not achieved by either treatment alone. Antitumor activity seen after TLS induction is associated with B cell-mediated dendritic cell activation. CONCLUSIONS: This study provides supportive evidence that TLS induction may potentiate the antitumor activity of chemotherapy in a murine model of PDAC. A detailed understanding of TLS kinetics and their induction, owing to multiple host and tumor factors, may help design personalized therapies harnessing the potential of immune-oncology.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response. This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 µL basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFNγ and TNFα) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFNγ+, TNFα+ and CD107a+ CD4+ and CD8+ T cells was quantified. This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Discrepancies in the Tumor Microenvironment of Spontaneous and Orthotopic Murine Models of Pancreatic Cancer Uncover a New Immunostimulatory Phenotype for B Cells.

B cells are salient features of pancreatic ductal adenocarcinoma (PDAC) tumors, yet their role in this disease remains controversial. Murine studies have indicated a protumoral role for B cells, whereas clinical data show tumor-infiltrating B cells are a positive prognostic factor, both in PDAC and other cancers. This disparity needs to be clarified in order to develop effective immunotherapies. In this study, we provide new evidence that reconcile human and mouse data and highlight the importance of using relevant preclinical tumor models when assessing B cell function. We compared B cell infiltration and activation in both a genetic model of murine PDAC (KPC mouse) and an injectable orthotopic model. A pronounced B cell infiltrate was only observed in KPC tumors and correlated with T cell infiltration, mirroring human disease. In contrast, orthotopic tumors exhibited a relative paucity of B cells. Accordingly, KPC-derived B cells displayed markers of B cell activation (germinal center entry, B cell memory, and plasma cell differentiation) accompanied by significant intratumoral immunoglobulin deposition, a feature markedly weaker in orthotopic tumors. Tumor immunoglobulins, however, did not appear to form immune complexes. Furthermore, in contrast to the current paradigm that tumor B cells are immunosuppressive, when assessed as a bulk population, intratumoral B cells upregulated several proinflammatory and immunostimulatory genes, a distinctly different phenotype to that of splenic-derived B cells; further highlighting the importance of studying tumor-infiltrating B cells over B cells from secondary lymphoid organs. In agreement with the current literature, genetic deletion of B cells (µMT mice) resulted in reduced orthotopic tumor growth, however, this was not recapitulated by treatment with B-cell-depleting anti-CD20 antibody and, more importantly, was not observed in anti-CD20-treated KPC mice. This suggests the result from B cell deficient mice might be caused by their altered immune system, rather than lack of B cells. Therefore, our data indicate B cells do not favor tumor progression. In conclusion, our analysis of relevant preclinical models shows B cells to be active members of the tumor microenvironment, producing immunostimulatory factors that might support the adaptive antitumor immune response, as suggested by human PDAC studies.

A Subset of CCL25-Induced Gut-Homing T Cells Affects Intestinal Immunity to Infection and Cancer.

Protective immunity relies upon differentiation of T cells into the appropriate subtype required to clear infections and efficient effector T cell localization to antigen-rich tissue. Recent studies have highlighted the role played by subpopulations of tissue-resident memory (TRM) T lymphocytes in the protection from invading pathogens. The intestinal mucosa and associated lymphoid tissue are densely populated by a variety of resident lymphocyte populations, including αß and γδ CD8+ intraepithelial T lymphocytes (IELs) and CD4+ T cells. While the development of intestinal γδ CD8+ IELs has been extensively investigated, the origin and function of intestinal CD4+ T cells have not been clarified. We report that CCR9 signals delivered during naïve T cell priming promote the differentiation of a population of α4ß7+ IFN-γ-producing memory CD4+ T cells, which displays a TRM molecular signature, preferentially localizes to the gastrointestinal (GI) tract and associated lymphoid tissue and cannot be mobilized by remote antigenic challenge. We further show that this population shapes the immune microenvironment of GI tissue, thus affecting effector immunity in infection and cancer.