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The Ras family of small GTPases control diverse signaling pathways through a conserved 'switch' mechanism, which is turned on by the binding of GTP and turned off by GTP hydrolysis. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2'(3')-O-(N-Methylanthraniloyl)-(mant)substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, such as Ras, Rho, and Rab. Using a recently developed nuclear magnetic resonance method that can detect subtle conformational change of proteins, we show here how the kinetics of nucleotide hydrolysis and exchange by 3 small GTPases, alone and in the presence of their cognate GAPs and GEFs, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Rheb is much faster than that of GTP, whereas it is slower with RhoA. On the other hand, the mant tag inhibits TSC2 GAPcatalyzed GTP hydrolysis by Rheb, but promotes RasGAP334-catalyzed GTP hydrolysis by Ras. GEF catalyzed nucleotide exchange, by both Ras and RhoA, was inhibited by the presence of mant. These results indicate that the mant moiety can significantly affect GTPase reaction kinetics in an unpredictable manner, and underscore the importance of validating its use in each system., doi: 10.1139/O09-905 M.T. Mazhab-Jafari, C.B. Marshall, M. Smith, G. Seabrook, V. Stambolic, R. Rottapel, B.G. Neel, and M. Ikura Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, [...]

Abstract Background It is now well established that nearly 20% of human cancers are caused by infectious agents, and the list of human oncogenic pathogens will grow in the future for a variety of cancer types. Whole tumor transcriptome and genome sequencing by next-generation sequencing technologies presents an unparalleled opportunity for pathogen detection and discovery in human tissues but requires development of new genome-wide bioinformatics tools. Results Here we present CaPSID (Computational Pathogen Sequence IDentification), a comprehensive bioinformatics platform for identifying, querying and visualizing both exogenous and endogenous pathogen nucleotide sequences in tumor genomes and transcriptomes. CaPSID includes a scalable, high performance database for data storage and a web application that integrates the genome browser JBrowse. CaPSID also provides useful metrics for sequence analysis of pre-aligned BAM files, such as gene and genome coverage, and is optimized to run efficiently on multiprocessor computers with low memory usage. Conclusions To demonstrate the usefulness and efficiency of CaPSID, we carried out a comprehensive analysis of both a simulated dataset and transcriptome samples from ovarian cancer. CaPSID correctly identified all of the human and pathogen sequences in the simulated dataset, while in the ovarian dataset CaPSID’s predictions were successfully validated in vitro.

Background: Incarceration provides an opportunity for health interventions, including opioid use disorder (OUD) treatment and prevention of opioid-related overdoses post-release. All FDA-approved forms of medication for OUD (MOUD) treatment were mandated in several Massachusetts jails in 2019, with some jails offering extended-release buprenorphine (XR-Bup). Little is known about patient perspectives on and experiences with XR-Bup in carceral settings., Methods: We conducted semi-structured interviews in 2022 with community-dwelling people who received MOUD during a recent incarceration in a Massachusetts jail. We asked participants about their experiences with and perspectives on XR-Bup while in jail. Qualitative data were double-coded deductively and reviewed inductively to identify emergent themes, which were structured using the Theoretical Framework of Acceptability (TFA)., Results: Participants (n = 38) had a mean age of 41.5 years, were 86% male, 84% White, 24% Hispanic, and 95% continued to receive MOUD at the time of their interview, including 11% receiving XR-Bup. Participants who viewed XR-Bup favorably appreciated avoiding the taste of sublingual buprenorphine; avoiding procedural difficulties and indignities associated with daily dosing in carceral settings (e.g., mouth checks, stigmatizing treatment from correctional staff); avoiding daily reminders of their addiction; experiencing less withdrawal; having extra time for other activities, such as work; and reduction of diversion of MOUD within the jail setting. Participants who viewed XR-Bup less favorably preferred to maintain their daily dosing routine; liked daily time out of their housing unit; wanted to know what was "going into my body everyday"; and feared needles and adverse events. Participants also reported that jail clinicians used XR-Bup for patients who were previously caught diverting sublingual buprenorphine, suggesting limited patient participation in decision-making around XR-Bup initiation in some jails., Conclusion: People who received MOUD in Massachusetts jails had both favorable and unfavorable views and experiences with XR-Bup. Understanding these preferences can inform protocols in jails that are considering implementation of XR-Bup treatment., (© 2024. The Author(s).)

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, -3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and -3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis., Competing Interests: PP, JM, KZ, DP, SW, YR, MH, RR, BG, RC, TS, FD No competing interests declared, PM Founder of Netris Pharma, (© 2023, Perampalam et al.)

Despite clinical use of immunosuppressive agents, the immunopathogenesis of minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) remains unclear. Src homology 3-binding protein 2 (SH3BP2), a scaffold protein, forms an immune signaling complex (signalosome) with 17 other proteins, including phospholipase Cγ2 (PLCγ2) and Rho-guanine nucleotide exchange factor VAV2 (VAV2). Bioinformatic analysis of human glomerular transcriptome (Nephrotic Syndrome Study Network cohort) revealed upregulated SH3BP2 in MCD and FSGS. The SH3BP2 signalosome score and downstream MyD88, TRIF, and NFATc1 were significantly upregulated in MCD and FSGS. Immune pathway activation scores for Toll-like receptors, cytokine-cytokine receptor, and NOD-like receptors were increased in FSGS. Lower SH3BP2 signalosome score was associated with MCD, higher estimated glomerular filtration rate, and remission. Further work using Sh3bp2KI/KI transgenic mice with a gain-in-function mutation showed ~6-fold and ~25-fold increases in albuminuria at 4 and 12 weeks, respectively. Decreased serum albumin and unchanged serum creatinine were observed at 12 weeks. Sh3bp2KI/KI kidney morphology appeared normal except for increased mesangial cellularity and patchy foot process fusion without electron-dense deposits. SH3BP2 co-immunoprecipitated with PLCγ2 and VAV2 in human podocytes, underscoring the importance of SH3BP2 in immune activation. SH3BP2 and its binding partners may determine the immune activation pathways resulting in podocyte injury leading to loss of the glomerular filtration barrier.

Background: Immune-checkpoint inhibitors (ICI) can lead to immune-related adverse events (irAEs) in a significant proportion of patients. The mechanisms underlying irAEs development are mostly unknown and might involve multiple immune effectors, such as T cells, B cells and autoantibodies (AutoAb)., Methods: We used custom autoantigen (AutoAg) microarrays to profile AutoAb related to irAEs in patients receiving ICI. Plasma was collected before and after ICI from cancer patients participating in two clinical trials (NCT03686202, NCT02644369). A one-time collection was obtained from healthy controls for comparison. Custom arrays with 162 autoAg were used to detect IgG and IgM reactivities. Differences of median fluorescent intensity (MFI) were analyzed with Wilcoxon sign rank test and Kruskal-Wallis test. MFI 500 was used as threshold to define autoAb reactivity., Results: A total of 114 patients and 14 healthy controls were included in this study. irAEs of grade (G) ≥ 2 occurred in 37/114 patients (32%). We observed a greater number of IgG and IgM reactivities in pre-ICI collections from patients versus healthy controls (62 vs 32 p < 0.001). Patients experiencing irAEs G ≥ 2 demonstrated pre-ICI IgG reactivity to a greater number of AutoAg than patients who did not develop irAEs (39 vs 33 p = 0.040). We observed post-treatment increase of IgM reactivities in subjects experiencing irAEs G ≥ 2 (29 vs 35, p = 0.021) and a decrease of IgG levels after steroids (38 vs 28, p = 0.009)., Conclusions: Overall, these results support the potential role of autoAb in irAEs etiology and evolution. A prospective study is ongoing to validate our findings (NCT04107311)., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Objectives: PARPs, which are members of the poly(ADP-ribose) polymerase superfamily, promote tumorigenesis and tumour-associated inflammation and are thus therapeutic targets for several cancers. The aim of the present study is to investigate the mechanistic insight into the roles PARPs for inflammation., Methods: Primary murine macrophages were cultured in the presence or absence of the PARP5 inhibitor NVP-TNKS656 to examine the role of PARP5 for cytokine production., Results: In contrast to the roles of other PARPs for induction of inflammation, we found in the present study that pharmacologic inhibition of PARP5 induces production of inflammatory cytokines in primary murine macrophages. We found that treatment with the PARP5 inhibitor NVP-TNKS656 in macrophages enhanced steady-state and LPS-mediated cytokine production through degradation of IκBα and subsequent nuclear translocation of NF-κB. We also found that pharmacologic inhibition of PARP5 stabilises the adaptor protein 3BP2, a substrate of PARP5, and that accelerated cytokine production induced by PARP5 inhibition was rescued in 3BP2-deleted macrophages. Additionally, we found that LPS increases the expression of 3BP2 and AXIN1, a negative regulator of β-catenin, through suppression of PARP5 transcripts in macrophages, leading to further activation of cytokine production and inhibition of β-catenin-mediated cell proliferation, respectively. Lastly, we found that PARP5 inhibition in macrophages promotes osteoclastogenesis through stabilisation of 3BP2 and AXIN1, leading to activation of SRC and suppression of β-catenin, respectively., Conclusions: Our results show that pharmacologic inhibition of PARP5 against cancers unexpectedly induces adverse autoinflammatory side effects through activation of innate immunity, unlike inhibition of other PARPs.

Modification of proteins by ADP-ribose (PARsylation) is catalyzed by the poly(ADP-ribose) polymerase (PARP) family of enzymes exemplified by PARP1, which controls chromatin organization and DNA repair. Additionally, PARsylation induces ubiquitylation and proteasomal degradation of its substrates because PARsylation creates a recognition site for E3-ubiquitin ligase. The steady-state levels of the adaptor protein SH3-domain binding protein 2 (3BP2) is negatively regulated by tankyrase (PARP5), which coordinates ubiquitylation of 3BP2 by the E3-ligase ring finger protein 146 (RNF146). 3BP2 missense mutations uncouple 3BP2 from tankyrase-mediated negative regulation and cause Cherubism, an autosomal dominant autoinflammatory disorder associated with craniofacial dysmorphia. In this review, we summarize the diverse biological processes, including bone dynamics, metabolism, and Toll-like receptor (TLR) signaling controlled by tankyrase-mediated PARsylation of 3BP2, and highlight the therapeutic potential of this pathway., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Insomnia is highly prevalent among persons with chronic pain. Although cognitive behavioral therapy for insomnia is recommended as first-line treatment for insomnia, it is underutilized. We tested the feasibility of a potentially scalable alternative - Brief Behavioral Therapy for Insomnia (BBTI) for former National Football League (NFL) players, a group with a high prevalence of chronic pain. We assessed changes in sleep, pain, and psychological health., Methods: Single-arm clinical trial of an adapted telephone-delivered BBTI intervention in 40 former NFL players with insomnia. We collected data on changes in sleep, pain, and psychological health outcomes., Results: Among former players (30% racial/ethnic minorities), BBTI was both acceptable and feasible. BBTI was associated with improvements in sleep disturbance (primary exploratory sleep outcome, mean T-score change -6.2, 95% CI: -7.6, -4.8), sleep-related impairment (mean T-score change -5.7, 95% CI: -7.9, -3.5) and insomnia severity (mean change -5.3, 95% CI: -6.8, -3.5) post-intervention. Improvements were maintained at 2-months. BBTI was also associated with improvements in pain interference and intensity, but not psychological health., Conclusion: An adapted telephone-delivered BBTI is acceptable and feasible among retired players with a range of insomnia symptoms and shows promise for improving sleep and pain. These data support the need for future trials assessing BBTI's effect on both sleep and pain outcomes.

Introduction: Release from incarceration is a high-risk period for opioid overdose. Concern about COVID-19 spread in jails led to early releases; it is unknown whether pandemic era releases of persons with opioid use disorder (OUD) contributed to increases in community overdose rates., Methods: Observational data compared overdose rates three months after release among jailed persons with OUD released before (9/1/2019-3/9/2020) and during the pandemic (3/10/2020-8/10/2020) from seven jails in Massachusetts. Data on overdoses come from the Massachusetts Ambulance Trip Record Information System and Registry of Vital Records Death Certificate file. Other information came from jail administrative data. Logistic models regressed overdose on release period, controlling for MOUD received, county of release, race/ethnicity, sex, age, and prior overdose., Results: Pandemic releases with OUD had a higher risk of fatal overdose (adjusted odds ratio [aOR] 3.06; 95% CI, 1.49 to 6.26); 20 persons released with OUD (1.3%) experienced a fatal overdose within three months of release, versus 14 (0.5%) pre-pandemic. MOUD had no detectable relationship with overdose mortality. Pandemic release did not impact non-fatal overdose rates (aOR 0.84; 95% CI 0.60 to 1.18), though in-jail methadone treatment was protective (aOR 0.34; 95% CI 0.18 to 0.67)., Conclusions: Persons with OUD released from jail during the pandemic experienced higher overdose mortality compared to pre-pandemic, but the number of deaths was small. They did not experience significantly different rates of non-fatal overdose. Early jail releases during the pandemic were unlikely to explain much, if any, of the observed increase in community overdoses in Massachusetts., Competing Interests: No conflict declared., (© 2023 The Authors. Published by Elsevier B.V.)

Epithelial ovarian cancer is the most lethal gynecological malignancy, owing notably to its high rate of therapy-resistant recurrence in spite of good initial response to chemotherapy. Although poly(ADP-ribose) polymerase inhibitors (PARPi) have shown promise for ovarian cancer treatment, extended therapy usually leads to acquired PARPi resistance. Here we explored a novel therapeutic option to counter this phenomenon, combining PARPi and inhibitors of nicotinamide phosphoribosyltransferase (NAMPT). Cell-based models of acquired PARPi resistance were created through an in vitro selection procedure. Using resistant cells, xenograft tumors were grown in immunodeficient mice, while organoid models were generated from primary patient tumor samples. Intrinsically PARPi-resistant cell lines were also selected for analysis. Our results show that treatment with NAMPT inhibitors effectively sensitized all in vitro models to PARPi. Adding nicotinamide mononucleotide, the resulting NAMPT metabolite, abrogated the therapy-induced cell growth inhibition, demonstrating the specificity of the synergy. Treatment with olaparib (PARPi) and daporinad (NAMPT inhibitor) depleted intracellular NAD+ , induced double-strand DNA breaks, and promoted apoptosis as monitored by caspase-3 cleavage. The two drugs were also synergistic in mouse xenograft models and clinically relevant patient-derived organoids. Therefore, in the context of PARPi resistance, NAMPT inhibition could offer a promising new option for ovarian cancer patients., (© 2023. The Author(s).)

Ubiquitin-mediated proteasomal degradation is a post-transcriptional protein modification that is comprised of various components including the 76-amino acid protein ubiquitin (Ub), Ub-activating enzyme (E1), Ub-conjugating enzyme (E2), ubiquitin ligase (E3), deubiquitinating enzyme (DUB) and proteasome. We and others have recently provided genetic evidence showing that E3-ubiquitin ligases are associated with bone metabolism, the immune system and inflammation through ubiquitylation and subsequent degradation of their substrates. Dysregulation of the E3-ubiquitin ligase RNF146-mediated degradation of the adaptor protein 3BP2 (SH3 domain-binding protein 2) causes cherubism, an autosomal dominant disorder associated with severe inflammatory craniofacial dysmorphia syndrome in children. In this review, on the basis of our discoveries in cherubism, we summarize new insights into the roles of E3-ubiquitin ligases in the development of human disorders caused by an abnormal osteoimmune system by highlighting recent genetic evidence obtained in both human and animal model studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Asano, Matsumoto, Wada and Rottapel.)

CD8 + T cell proliferation and differentiation into effector and memory states are high-energy processes associated with changes in cellular metabolism. CD28-mediated costimulation of T cells activates the PI3K/AKT/mammalian target of rapamycin signaling pathway and induces eukaryotic translation initiation factor 4E-dependent translation through the derepression by 4E-BP1 and 4E-BP2. In this study, we demonstrate that 4E-BP1/2 proteins are required for optimum proliferation of mouse CD8 + T cells and the development of an antiviral effector function. We show that translation of genes encoding mitochondrial biogenesis is impaired in T cells derived from 4E-BP1/2-deficient mice. Our findings demonstrate an unanticipated role for 4E-BPs in regulating a metabolic program that is required for cell growth and biosynthesis during the early stages of CD8 + T cell expansion., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Drug repurposing is an attractive option for oncology drug development. Itraconazole is an antifungal ergosterol synthesis inhibitor that has pleiotropic actions including cholesterol antagonism, inhibition of Hedgehog and mTOR pathways. We tested a panel of 28 epithelial ovarian cancer (EOC) cell lines with itraconazole to define its spectrum of activity. To identify synthetic lethality in combination with itraconazole, a whole-genome drop-out genome-scale clustered regularly interspaced short palindromic repeats sensitivity screen in two cell lines (TOV1946 and OVCAR5) was performed. On this basis, we conducted a phase I dose-escalation study assessing the combination of itraconazole and hydroxychloroquine in patients with platinum refractory EOC (NCT03081702). We identified a wide spectrum of sensitivity to itraconazole across the EOC cell lines. Pathway analysis showed significant involvement of lysosomal compartments, the trans-golgi network and late endosomes/lysosomes; similar pathways are phenocopied by the autophagy inhibitor, chloroquine. We then demonstrated that the combination of itraconazole and chloroquine displayed Bliss defined synergy in EOC cancer cell lines. Furthermore, there was an association of cytotoxic synergy with the ability to induce functional lysosome dysfunction, by chloroquine. Within the clinical trial, 11 patients received at least one cycle of itraconazole and hydroxychloroquine. Treatment was safe and feasible with the recommended phase II dose of 300 and 600 mg twice daily, respectively. No objective responses were detected. Pharmacodynamic measurements on serial biopsies demonstrated limited pharmacodynamic impact. In vitro , itraconazole and chloroquine have synergistic activity and exert a potent antitumor effect by affecting lysosomal function. The drug combination had no clinical antitumor activity in dose escalation., Significance: The combination of the antifungal drug itraconazole with antimalarial drug hydroxychloroquine leads to a cytotoxic lysosomal dysfunction, supporting the rational for further research on lysosomal targeting in ovarian cancer., Competing Interests: S. Marastoni reports a Canadian patent number 3,116,081 pending. A. Madariaga reports personal fees from Clovis and AstraZeneca outside the submitted work. Z.J. Li reports a Canadian patent number to CA3,116,081 pending. I. Colombo reports personal fees from MSD; other from GSK, AstraZeneca, MSD, Bayer, and Oasmia outside the submitted work. J.P. Bruce reports other from Bowhead Health outside the submitted work. M. Koritzinsky reports other from The Princess Margaret Cancer Foundation during the conduct of the study; in addition, M. Koritzinsky has a Canadian patent number number 3,116,081 pending. B.G. Wouters reports grants from Ontario Institute for Cancer Research, Canadian Institutes for Cancer Research, and Princess Margaret Cancer Foundation during the conduct of the study; other from Northern Biologics outside the submitted work; in addition, B.G. Wouters has a Canadian patent number 3,116,081 issued. A.M. Oza is PI and on clinical trial steer- ing committees for trials with Clovis, GSK, AstraZeneca—all uncompensated. A.M. Oza is uncompensated CEO of Ozmosis Research, a Not For Profit Clin- ical Trials Management company associated with UHN. A.M. Joshua reports non-financial support from Mayne Pharma during the conduct of the study; other from Pricilium outside the submitted work; in addition, A.M. Joshua has a Canadian patent number 3,116,081 pending. S. Lheureux reports grants from OICR - TRI Ovarian Cancer during the conduct of the study; grants and personal fees from AstraZeneca, GSK; personal fees from Eisai, Merck, Shattuck Labs; grants from Roche, and outside the submitted work. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Dysregulation of Toll-like receptor (TLR) signaling contributes to the pathogenesis of autoimmune diseases. Here, we provide genetic evidence that tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, negatively regulates TLR2 signaling. We show that mice lacking tankyrase in myeloid cells developed severe systemic inflammation with high serum inflammatory cytokine levels. We provide mechanistic evidence that tankyrase deficiency resulted in tyrosine phosphorylation and activation of TLR2 and show that phosphorylation of tyrosine 647 within the TIR domain by SRC and SYK kinases was critical for TLR2 stabilization and signaling. Last, we show that the elevated cytokine production and inflammation observed in mice lacking tankyrase in myeloid cells were dependent on the adaptor protein 3BP2, which is required for SRC and SYK activation. These data demonstrate that tankyrase provides a checkpoint on the TLR-mediated innate immune response.

Background & Aims: N6-methyladenosine (m 6 A) governs the fate of RNAs through m 6 A readers. Colorectal cancer (CRC) exhibits aberrant m 6 A modifications and expression of m 6 A regulators. However, how m 6 A readers interpret oncogenic m 6 A methylome to promote malignant transformation remains to be illustrated., Methods: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m 6 A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m 6 A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP)., Results: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m 6 A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo., Conclusions: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m 6 A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Reports of severe COVID-19 being associated with thrombosis, antiphospholipid antibodies (APLA), and antiphospholipid syndrome have yielded disparate conclusions. Studies comparing patients with COVID-19 with contemporaneous controls of similar severity are lacking., Methods: 22 COVID-19 + and 20 COVID-19 - patients with respiratory failure admitted to intensive care were studied longitudinally. Demographic and clinical data were obtained from the day of admission. APLA testing included anticardiolipin (aCL), anti-β2glycoprotien 1 (β2GP1), antidomain 1 β2GP1 and antiphosphatidyl serine/prothrombin complex. Antinuclear antibodies (ANAs) were detected by immunofluorescence and antibodies to cytokines by a commercially available multiplexed array. Analysis of variance was used for continuous variables and Fisher's exact test was used for categorical variables with α=0.05 and the false discovery rate at q=0.05., Results: APLAs were predominantly IgG aCL (48%), followed by IgM (21%) in all patients, with a tendency towards higher frequency among the COVID-19 + . aCL was not associated with surrogate markers of thrombosis but IgG aCL was strongly associated with worse disease severity and higher ANA titres regardless of COVID-19 status. An association between aCL and anticytokine autoantibodies tended to be higher among the COVID-19 + ., Conclusions: Positive APLA serology was associated with more severe disease regardless of COVID-19 status., Trial Registration Number: NCT04747782., Competing Interests: Competing interests: MJF is the Director of MitogenDx. MJF is a consultant for and received speaking honoraria from Inova Diagnostics Inc (San Diego, California, USA) and Werfen International (Barcelona, Spain). All the other authors have no disclosures to declare., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Limited data are available on the safety and efficacy of immune checkpoint inhibitors (ICI) in patients with preexisting autoimmune diseases (PAD)., Methods: Retrospective study of patients with PAD referred for rheumatologic evaluation prior to starting or during immunotherapy between January 2013 and July 2019 from 10 academic sites across Canada. Data were extracted by chart review using a standardized form., Results: Twenty-seven patients with PAD on ICI therapy were identified. The most common PADs were rheumatoid arthritis (30%), psoriasis/psoriatic arthritis (30%), inflammatory bowel disease (IBD, 15%) and axial spondyloarthritis (11%), and the most frequently observed cancers were lung cancer and melanoma. All patients received anti-PD-1 therapies, and 2 received additional sequential anti-CTLA-4 therapy. PAD exacerbations occurred in 52% over a median (IQR) follow-up of 11.0 (6.0-17.5) months, with 14% being severe, 57% requiring corticosteroids, 50% requiring immunosuppression and 14% requiring ICI discontinuation. Flares were generally more frequent and severe in patients who previously required more intensive immunosuppression (i.e., biologics). Flares occurred despite background immunosuppression at the time of ICI initiation. In patients with preexisting psoriasis, IBD and axial spondyloarthritis, rheumatic immune-related adverse events (irAEs), mostly polyarthritis and tenosynovitis, were frequently observed. Tumor progression was not associated with exposure to immunosuppressive drugs before or after ICI initiation and was numerically less frequent in patients with irAEs., Conclusion: PAD exacerbations in the context of ICI treatment are common, although generally mild, and occur despite background immunosuppression. Exacerbations are more frequent and severe in patients on more intensive immunosuppressive therapies pre-immunotherapy., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Rab11 GTPase proteins are required for cytokinesis, ciliogenesis, and lumenogenesis. Rab11a is critical for apical delivery of podocalyxin (PODXL) during lumen formation in epithelial cells. SH3BP5 and SH3BP5L are guanine nucleotide exchange factors (GEFs) for Rab11. We show that SH3BP5 and SH3BP5L are required for activation of Rab11a and cyst lumen formation. Using proximity-dependent biotin identification (BioID) interaction proteomics, we have identified SH3BP5 and its paralogue SH3BP5L as new substrates of the poly-ADP-ribose polymerase Tankyrase and the E3 ligase RNF146. We provide data demonstrating that epithelial polarity via cyst lumen formation is governed by Tankyrase, which inhibits Rab11a activation through the suppression of SH3BP5 and SH3BP5L. RNF146 reduces Tankyrase protein abundance and restores Rab11a activation and lumen formation. Thus, Rab11a activation is controlled by a signaling pathway composed of the sequential inhibition of SH3BP5 paralogues by Tankyrase, which is itself suppressed by RNF146., (© 2021 Chandrakumar et al.)

Activity of transcription factors is normally regulated through interaction with other transcription factors, chromatin remodeling proteins and transcriptional co-activators. In distinction to these well-established transcriptional controls of gene expression, we have uncovered a unique activation model of transcription factors between tyrosine kinase ABL and RUNX2, an osteoblastic master transcription factor, for cancer invasion. We show that ABL directly binds to, phosphorylates, and activates RUNX2 through its SH2 domain in a kinase activity-dependent manner and that the complex formation of these proteins is required for expression of its target gene MMP13. Additionally, we show that the RUNX2 transcriptional activity is dependent on the number of its tyrosine residues that are phosphorylated by ABL. In addition to regulation of RUNX2 activity, we show that ABL transcriptionally enhances RUNX2 expression through activation of the bone morphogenetic protein (BMP)-SMAD pathway. Lastly, we show that ABL expression in highly metastatic breast cancer MDA-MB231 cells is associated with their invasive capacity and that ABL-mediated invasion is abolished by depletion of endogenous RUNX2 or MMP13. Our genetic and biochemical evidence obtained in this study contributes to a mechanistic insight linking ABL-mediated phosphorylation and activation of RUNX2 to induction of MMP13, which underlies a fundamental invasive capacity in cancer and is different from the previously described model of transcriptional activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 He, Matsumoto, Asano, Yamamura, Katsuyama, La Rose, Tomonobu, Komalasari, Sakaguchi, Rottapel and Wada.)

Bone is a highly dynamic organ that undergoes remodeling equally regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. To clarify the regulation of osteoblastogenesis, primary murine osteoblasts are required for an in vitro study. Primary osteoblasts are isolated from neonatal calvariae through digestion with collagenase. However, the number of cells collected from one pup is not sufficient for further in vitro experiments, leading to an increase in the use of euthanized pups. We hypothesized that the viscosity of digested calvariae and digestion solution supplemented with collagenase results in cell clumping and reduction of isolated cells from bones. We simply added Benzonase, a genetically engineered endonuclease that shears all forms of DNAs/RNAs, in order to reduce nucleic acid-mediated viscosity. We found that addition of Benzonase increased the number of collected osteoblasts by three fold compared to that without Benzonase through reduction of viscosity. Additionally, Benzonase has no effect on cellular identity and function. The new osteoblast isolation protocol with Benzonase minimizes the number of neonatal pups required for an in vitro study and expands the concept that isolation of other populations of cells including osteocytes that are difficult to be purified could be modified by Benzonase.

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.

Ovarian cancer (OC) is the most deadly gynecological malignancy, with unmet clinical need for new therapeutic approaches. The relaxin peptide is a pleiotropic hormone with reproductive functions in the ovary. Relaxin induces cell growth in several types of cancer, but the role of relaxin in OC is poorly understood. Here, using cell lines and xenograft models, we demonstrate that relaxin and its associated GPCR RXFP1 form an autocrine signaling loop essential for OC in vivo tumorigenesis, cell proliferation, and viability. We determined that relaxin signaling activates expression of prooncogenic pathways, including RHO, MAPK, Wnt, and Notch. We found that relaxin is detectable in patient-derived OC tumors, ascites, and serum. Further, inflammatory cytokines IL-6 and TNF-α activated transcription of relaxin via recruitment of STAT3 and NF-κB to the proximal promoter, initiating an autocrine feedback loop that potentiated expression. Inhibition of RXFP1 or relaxin increased cisplatin sensitivity of OC cell lines and abrogated in vivo tumor formation. Finally, we demonstrate that a relaxin-neutralizing antibody reduced OC cell viability and sensitized cells to cisplatin. Collectively, these data identify the relaxin/RXFP1 autocrine loop as a therapeutic vulnerability in OC.