Project description:Purpose: to determine the CLL-B cells' interferome Methods: CLL cells from 3 of the patients previously classified as responders were purified and treated with anifrolumab in vitro and RNA was analyzed by RNAseq.
Project description:Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNg) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNg response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models - with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNg sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.
Project description:Mechanistic understanding of how ionizing radiation induces type I interferon signaling and how to amplify this signaling module should help to maximize the efficacy of radiotherapy. In the current study, we report that inhibitors of the DNA damage response kinase ATR can significantly potentiate ionizing radiation-induced innate immune responses. Using a series of mammalian knockout cell lines, we demonstrate that, surprisingly, both the cGAS/STING-dependent DNA-sensing pathway and the MAVS-dependent RNA-sensing pathway are responsible for type I interferon signaling induced by ionizing radiation in the presence or absence of ATR inhibitors. The relative contributions of these two pathways in type I interferon signaling depend on cell type and/or genetic background. We propose that DNA damage-elicited double-strand DNA breaks releases DNA fragments, which may either activate the cGAS/STING-dependent pathway or-especially in the case of AT-rich DNA sequences-be transcribed and initiate MAVS-dependent RNA sensing and signaling. Together, our results suggest the involvement of two distinct pathways in type I interferon signaling upon DNA damage. Moreover, radiation plus ATR inhibition may be a promising new combination therapy against cancer.
Project description:Chronic lymphocytic leukemia (CLL) stereotyped subsets #6 and #8 include cases expressing unmutated B cell receptor immunoglobulin (BcR IG) (U-CLL). Yet, subset #6 (IGHV1-69/IGKV3-20) is less aggressive compared to subset #8 (IGHV4-39/IGKV1(D)-39) which has the highest risk for Richter’s transformation among all CLL. The underlying reasons for this divergent clinical behavior are not fully elucidated. To gain insight into this issue, here we focused on epigenomic signatures and their links with gene expression, particularly investigating genome-wide DNA methylation profiles in subsets #6 and #8 as well as other U-CLL cases not expressing stereotyped BcR IG using the Illumina 450k methylation arrays. Additionally we analysed the methylation profiles of naive and memory B cell subsets from healthy donors and compared them with those of the CLL cases.
Project description:Metastases are the leading cause of death in cancer patients. RhoC, a member of the Rho GTPase family, has been shown to facilitate metastasis of aggressive breast cancer cells by influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. A common phenotypic characteristic of metastatic cells influenced by these mechanisms is dysregulation of cell-cell junctions. Thus, we set out to study how RhoA- and RhoC-GTPase influence the cell-cell junctions in aggressive breast cancers. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM 149 and MDA 231 breast cancer cells results in increased normalization of junctional integrity denoted by junctional protein expression/colocalization. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity compared to wild-type cells as measured by the FITC-Dextran leakage assay, and increased cell-cell adhesion as measured by fluorimetric centrifugation assay. Whole transciptome RNA sequencing demonstrate decreased expression of Type I interferon-stimulated genes in RhoC knockout cells compared to wild-type, and subsequent treatment with interferon-alpha resulted in significant increases in adhesion and decreases in invasiveness of wild-type cells and a dampened response to interferon-alpha stimulation with respect to adhesion and invasiveness in RhoC knockout cells. We delineate a key role of RhoC-GTPase in modulation of junctions and response to interferon, which supports inhibition of RhoC as a potential anti-invasion therapeutic strategy.
Project description:Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNg) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNg response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models – with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNg sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.
Project description:Ionizing radiation promotes cytosolic DNA sensing and consequent antitumor immune responses. But how tumor cell-intrinsic cytosolic DNA sensing is initiated by radiation remains poorly defined. Here, we demonstrated that STING-mediated type I interferon production in tumor cells after radiation relied on the engagement of MLKL-mediated necroptosis, which was elicited by the ZBP1-RIPK3 signaling axis. Physiologically, tumor cell-intrinsic ZBP1-MLKL cascade augmented antitumor immune responses after radiation largely by regulating STING signaling. Mechanistically, ZBP1-MLKL-dependent necroptosis maintained the enrichment of mitochondria DNA inside the cytosol of tumor cells after radiation in a cell-density dependent fashion, contributing to type I interferon responses. In contrast, ablation of caspase-8 unleashed ZBP1-MLKL cascade to gain enhanced cytosolic DNA sensing, and in turn potentiated therapeutic effects of radiation. Thus, our findings uncover an unanticipated mechanism that ZBP1-MLKL-dependent necroptosis drives cytosolic DNA sensing-mediated antitumor immunity after radiation, and provide new strategy to improve radiotherapy by inhibiting caspase-8 cascade.