Andrew J. Lindsay

SUMMARY: IFN-γ is secreted by multiple lymphoid subsets in response to antigen stimulation and can reprogram and prime macrophages epigenetically and transcriptionally to increase responses to inflammatory stimuli such as LPS, IL-1β or TNF-α. IFN-γ-driven M1-like inflammatory macrophage states are shared across human immune-mediated inflammatory diseases (IMIDs) and while IFN-γ is nonredundant for defense to intracellular pathogens it is unclear if this is also the case in IMIDs. To identify additional secreted ligands which could prime and induce M1-like macrophages we screened >600 human proteins in human primary macrophages. Using complementary functional genomics approaches, we discovered that IL-32β induced an M1-like inflammatory state in non-primed and IFN-γ-primed macrophages. IL-32β induced signaling, transcriptional, tolerance, cross-tolerance, and inflammatory responses in macrophages which were MyD88, IRAK1 and Myddosome-dependent. These responses to host IL-32β were similar to yet distinct from, those induced by microbial LPS. IL-32 protein was elevated in serum from patients with severe COVID-19 and IL-32β together with IFN-γ were expressed by T cells and induced a macrophage transcriptional response which was shared by monocytes and macrophages in mild and severe COVID-19.

Outcomes for women with breast cancer have improved dramatically in recent decades. However, many patients present with intrinsic drug resistance and others are initially sensitive to anti-cancer drugs but acquire resistance during the course of their treatment, leading to recurrence and/or metastasis. Drug therapy-induced senescence (TIS) is a form of drug resistance characterised by the induction of cell cycle arrest and the emergence of a senescence-associated secretory phenotype (SASP) that can develop in response to chemo- and targeted- therapies. A wide range of anticancer interventions can lead to cell cycle arrest and SASP induction, by inducing genotoxic stress, hyperactivation of signalling pathways or oxidative stress. TIS can be anti-tumorigenic in the short-term, but pro-tumorigenic in the long-term by creating a pro-inflammatory and immunosuppressive microenvironment. Moreover, the SASP can promote angiogenesis and epithelial-mesenchymal transition in neighbouring cells. In this review, we will describe the characteristics of TIS in breast cancer and detail the changes in phenotype that accompany its induction. We also discuss strategies for targeting senescent cancer cells in order to prevent or delay tumour recurrence.

JRK is a DNA‐binding protein of the pogo superfamily of transposons, which includes the well‐known centromere binding protein B (CENP‐B). Jrk null mice exhibit epilepsy, and growth and reproductive disorders, consistent with its relatively high expression in the brain and reproductive tissues. Human JRK DNA variants and gene expression levels are implicated in cancers and neuropsychiatric disorders. JRK protein modulates β‐catenin–TCF activity but little is known of its cellular functions. Based on its homology to CENP‐B, we determined whether JRK binds centromeric or other satellite DNAs. We show that human JRK binds satellite III DNA, which is abundant at the chromosome 9q12 juxtacentromeric region and on Yq12, both sites of nuclear stress body assembly. Human JRK‐GFP overexpressed in HeLa cells strongly localises to 9q12. Using an anti‐JRK antiserum we show that endogenous JRK co‐localises with a subset of centromeres in non‐stressed cells, and with heat shock factor 1 following heat shock. Knockdown of JRK in HeLa cells proportionately reduces heat shock protein gene expression in heat‐shocked cells. A role for JRK in regulating the heat shock response is consistent with the mouse Jrk null phenotype and suggests that human JRK may act as a modifier of diseases with a cellular stress component.

Purpose: Breast cancer (BC) accounts for roughly 30% of new cancers diagnosed in women each year; thus, this cancer type represents a substantial burden for people and health care systems. Despite the existence of effective therapies to treat BC, drug resistance remains a problem and is a major cause of treatment failure. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. Recent research indicates that inhibition of the endosomal recycling pathway, an intracellular membrane trafficking pathway that returns endocytosed proteins back to the plasma membrane, may be a promising strategy to downregulate clinically relevant cell surface proteins such as HER2 and HER3, and to overcome drug resistance. Methods: To investigate the molecular mechanism of action of an endosomal recycling inhibitor (ERI) called primaquine, we performed a reverse-phase protein array (RPPA) assay using a HER2-positive breast cancer cell line. The RPPA findings were confirmed by Western blot and RT-qPCR in several BC cell lines. Novel drug combinations were tested by MTT cell viability and clonogenic assays. Results: Among the signalling molecules downregulated by ERIs were estrogen receptor-alpha (ER-α) and androgen receptor. We confirmed this finding in other breast cancer cell lines and show that downregulation occurs at the transcriptional level. We also found that ERIs synergise with tamoxifen, a standard-of-care therapy for breast cancer. Discussion: Our data suggest that combining ERIs with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

Summary Prostate cancer is the second most frequent cancer diagnosed in men, and accounts for one-fifth of cancer associated deaths worldwide. Despite the availability of effective prostate cancer therapies, if it is not cured by radical local treatment, progression to drug resistant metastatic prostate cancer is inevitable. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. We have recently published research demonstrating that targeting the endosomal recycling pathway, a membrane transport pathway that recycles internalised cell surface proteins back to the plasma membrane, may be a novel means to downregulate clinically relevant cell surface proteins and potentially overcome drug resistance. A reverse phase protein array (RPPA) assay of breast cancer cells treated with an endosomal recycling inhibitor identified the androgen receptor (AR) as one of the top downregulated proteins. We confirmed that endosomal recycling inhibitors also downregulated AR in prostate cancer cells and show that this occurs at the transcriptional level. We also found that endosomal recycling inhibitors synergise with enzalutamide, a standard-of-care therapy for prostate cancer. Our data suggest that combining recycling inhibitors with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

Prostate cancer is the second most frequent cancer diagnosed in men, and accounts for one-fifth of cancer associated deaths worldwide. Despite the availability of effective prostate cancer therapies, if it is not cured by radical local treatment, progression to drug resistant metastatic prostate cancer is inevitable. Therefore, new drugs and treatment regimens are urgently required to overcome resistance. We have recently published research demonstrating that targeting the endosomal recycling pathway, a membrane transport pathway that recycles internalised cell surface proteins back to the plasma membrane, may be a novel means to downregulate clinically relevant cell surface proteins and potentially overcome drug resistance. A reverse phase protein array (RPPA) assay of breast cancer cells treated with an endosomal recycling inhibitor identified the androgen receptor (AR) as one of the top downregulated proteins. We confirmed that endosomal recycling inhibitors also downregulated AR in prostate cancer cells and show that this occurs at the transcriptional level. We also found that endosomal recycling inhibitors synergise with enzalutamide, a standard-of-care therapy for prostate cancer. Our data suggest that combining recycling inhibitors with hormone receptor antagonists may enhance their efficacy and reduce the emergence of drug resistance.

Purpose Glioblastoma (GBM) is the most common and most aggressive brain cancer in adults. Approximately 250,000 new cases are diagnosed worldwide per year, and its incidence is rising due to our aging population. The standard treatment is brutal and has not changed in 20 years, and more than 85% of patients will die within two years of their diagnosis. There is thus an urgent need to identify new drug targets and develop novel therapeutic strategies that will increase survival and improve quality of life. Methods Using publicly available genomics, transcriptomics and proteomics datasets, we show that key regulators of endosomal recycling are dysregulated in GBM and are linked to survival outcomes. Results We report here that an intracellular membrane trafficking pathway called the endosomal recycling pathway represents a novel target that could be exploited for the development of small molecule inhibitors to treat people with GBM. We report an 8-gene endosomal recycling prognostic signature that can distinguish low-risk from high-risk GBM and potentially identify tumours that may benefit from endosomal recycling inhibitors. Conclusion This study presents the first systematic analysis of the endosomal recycling pathway in glioblastoma and proposes that it is a promising target for the development of novel therapies and therapeutic strategies that could improve the outcomes for patients with glioblastoma.

SMAUG1 is a human RNA-binding protein that is known to be dysregulated in a wide range of diseases. It is evolutionarily conserved and has been shown to form condensates containing translationally repressed RNAs. This indicates that condensation is central to proper SMAUG1 function; however, the factors governing condensation are largely unknown. In this work, we show that SMAUG1 drives the formation of liquid-like condensates in cells through its non-conventional C-terminal prion-like disordered region. We use biochemical assays to show that this liquid-liquid phase separation is independent of RNA binding and does not depend on other large, disordered regions that potentially harbor several binding sites for partner proteins. Using a combination of computational predictions, structural modeling, in vitro and in cell measurements, we also show that SMAUG1-driven condensation is negatively regulated by direct interactions with members of the 14-3-3 protein family. These interactions are mediated by four distinct phospho-regulated short linear motifs embedded in the disordered regions of SMAUG1, working synergistically. Interactions between SMAUG1 and 14-3-3 proteins drive the dissolution of condensates, alter the dynamics of the condensed state, and are likely to be intertwined with currently unknown regulatory mechanisms. Our results provide information on how SMAUG1 phase separation is regulated and the first known instance of 14-3-3 proteins being able to completely dissolve condensates by directly interacting with a phase separation driver, which might be a general mechanism in cells to regulate biological condensation. Graphical Highlights: SMAUG1 is a human RNA-binding protein capable of condensation with unknown regulation A prion-like domain of SMAUG1 drives condensation via liquid-liquid phase separation SMAUG1 interacts with 14-3-3 proteins via four phospho-regulated short linear motifs 14-3-3 interactions change the dynamics of SMAUG1 condensates, promoting their dissolution This is the first described regulatory mechanism for SMAUG1-driven condensation