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Purpose: Mutational data from multiple solid and liquid biospecimens of a single patient are often integrated to track cancer evolution. However, there is no accepted framework to resolve if individual samples from the same individual share variants due to common identity versus coincidence., Experimental Design: Utilizing 8,000 patient tumors from The Cancer Genome Atlas across 33 cancer types, we estimated the background rates of co-occurrence of mutations between discrete pairs of samples across cancers and by cancer type. We developed a mutational profile similarity (MPS) score that uses a large background database to produce confidence estimates that two tumors share a unique, related molecular profile. The MPS algorithm was applied to randomly paired tumor profiles, including patients who underwent repeat solid tumor biopsies sequenced with Memorial Sloan Kettering-IMPACT (n = 53,113). We also evaluated the MPS in sample pairs from single patients with multiple cancers (n = 2,012), as well as patients with plasma and solid tumor variant profiles (n = 884 patients)., Results: In unrelated tumors, nucleotide-specific variants are shared in 1.3% (cancer-type agnostic) and in 10% to 13% (cancer-type specific) of cases. The MPS method contextualized shared variants to specify whether patients had a single cancer versus multiple distinct cancers. When multiple tumors were compared from the same patient and an initial clinicopathologic diagnosis was discordant with molecular findings, the MPS anticipated future diagnosis changes in 28% of examined cases., Conclusions: The use of a novel shared variant framework can provide information to clarify the molecular relationship between compared biospecimens with minimal required input., (©2024 American Association for Cancer Research.)

Purpose: The molecular drivers underlying mucinous tumor pathogenicity are poorly understood. GNAS mutations predict metastatic burden and treatment resistance in mucinous appendiceal adenocarcinoma. We investigated the pan-cancer clinicopathologic relevance of GNAS variants., Methods: We assessed 58,043 patients with Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (IMPACT)-sequenced solid tumors to identify oncogenic variants, including GNAS , associated with mucinous tumor phenotype. We then performed comprehensive molecular analyses to compare GNAS- mutant (mut) and wild-type tumors across cancers. Gene expression patterns associated with GNAS- mut tumors were assessed in a The Cancer Genome Atlas cohort. Associations between GNAS variant status and peritoneal metastasis, first-line systemic therapy response, progression-free survival (PFS), and overall survival (OS) were determined using a propensity-matched subcohort of patients with metastatic disease., Results: Mucinous tumors were enriched for oncogenic GNAS variants. GNAS was mutated in >1% of small bowel, cervical, colorectal, pancreatic, esophagogastric, hepatobiliary, and GI neuroendocrine cancers. Across these cancers, GNAS- mut tumors exhibited a generally conserved C-to-T mutation-high, aneuploidy-low molecular profile with co-occurring prevalent KRAS variants (65% of GNAS-mut tumors) and fewer TP53 alterations. GNAS- mut tumors exhibited recurrently comutated alternative tumor suppressors ( RBM10 , INPPL1 ) and upregulation of MAPK and cell surface modulators. GNAS- mut tumors demonstrate an increased prevalence of peritoneal metastases (odds ratio [OR], 1.7 [95% CI, 1.1 to 2.5]; P = .006), worse response to first-line systemic therapy (OR, 2.2 [95% CI, 1.3 to 3.8]; P = .003), and shorter PFS (median, 5.6 v 7.0 months; P = .047). In a multivariable analysis, GNAS mutated status was independently prognostic of worse OS (hazard ratio, 1.25 [95% CI, 1.01 to 1.56]; adjusted P = .04)., Conclusion: Across the assessed cancers, GNAS- mut tumors exhibit a conserved molecular and clinical phenotype defined by mucinous tumor status, increased peritoneal metastasis, poor response to first-line systemic therapy, and worse survival.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8 + T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: POLE and POLD1 proofreading deficiency (POLE/D1pd) define a rare subtype of ultramutated metastatic colorectal cancer (mCRC; over 100 mut/Mb). Disease-specific data about the activity and efficacy of immune checkpoint inhibitors (ICIs) in POLE/D1pd mCRC are lacking and it is unknown whether outcomes may be different from mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRCs treated with ICIs., Patients and Methods: In this global study, we collected 27 patients with mCRC harboring POLE/D1 mutations leading to proofreading deficiency and treated with anti-programmed cell death-ligand 1 alone +/- anti-cytotoxic T-lymphocyte antigen-4 agents. We collected clinicopathological and genomic characteristics, response, and survival outcomes after ICIs of POLE/D1pd mCRC and compared them with a cohort of 610 dMMR/MSI-H mCRC patients treated with ICIs. Further genomic analyses were carried out in an independent cohort of 7241 CRCs to define POLE and POLD1pd molecular profiles and mutational signatures., Results: POLE/D1pd was associated with younger age, male sex, fewer RAS/BRAF driver mutations, and predominance of right-sided colon cancers. Patients with POLE/D1pd mCRC showed a significantly higher overall response rate (ORR) compared to dMMR/MSI-H mCRC (89% versus 54%; P = 0.01). After a median follow-up of 24.9 months (interquartile range: 11.3-43.0 months), patients with POLE/D1pd showed a significantly superior progression-free survival (PFS) compared to dMMR/MSI-H mCRC [hazard ratio (HR) = 0.24, 95% confidence interval (CI) 0.08-0.74, P = 0.01] and superior overall survival (OS) (HR = 0.38, 95% CI 0.12-1.18, P = 0.09). In multivariable analyses including the type of DNA repair defect, POLE/D1pd was associated with significantly improved PFS (HR = 0.17, 95% CI 0.04-0.69, P = 0.013) and OS (HR = 0.24, 95% CI 0.06-0.98, P = 0.047). Molecular profiling showed that POLE/D1pd tumors have higher tumor mutational burden (TMB). Responses were observed in both subtypes and were associated with the intensity of POLE/D1pd signature., Conclusions: Patients with POLE/D1pd mCRC showed more favorable outcomes compared to dMMR/MSI-H mCRC to treatment with ICIs in terms of tumor response and survival., Competing Interests: Disclosure BR served in a consulting/advisory role and received research funding from Neophore LTD. AM owns stock from Centessa Pharmaceuticals, Deka Biosciences, HotSpot Therapeutics, Marengo Therapeutics, Shattuck Labs; served as consultant or in an advisory role from Adagene, AstraZeneca, BiolineRx, Centessa Pharmaceuticals, Clover Biopharmaceuticals, Deka Biosciences, Depth Charge Therapeutics, EISAI, Grey Wolf Therapeutics, Gritstone Bio, Guidepoint Global, HiFiBiO Therapeutics, Hotspot Therapeutics, ImCheck therapeutics, Innate Pharma, Johnson & Johnson/Janssen, Lytix Biopharma, Medicxi, MSD, Neogene Therapeutics, OSE Immunotherapeutics, Pegaone, Pierre-Fabre, Redx Pharma, Roche, Sanofi, SERVIER, Shattuck Labs, Sotio, Third Rock Ventures; received research funding from Boehringer Ingelheim (Inst), Bristol-Myers Squibb (Inst), MSD (Inst), Transgene (Inst); is an inventor on a patent related to monoclonal antibodies against CD81 (Stanford University); received travel, accommodations, expenses from Sotio; and has other relationship with Elsevier. AC declared research funding from Seagen and GSK and has served in advisory boards of Seagen, Merck, Pfizer, Roche, GSK, Abbvie, Illumina, and Amgen. NHS served in consulting/advisory board: Agenus, Regeneron, Puretech, Novartis, Numab, AstraZeneca, GSK, ABL Bio, Revitope; and received research funding from Roche/Genentech, Pfizer, Merck, BMS, AstraZeneca, Puretech, Immunocore, Regeneron, Agenus. LS served in the scientific Advisory Board for Genor Biopharma, Ltd. AMV reports consulting or advisory role for Roche, Lilly, AstraZeneca, PAIGE; has received research funding from Illumina, Lilly, Verastem, BioMed Valley Discoveries, Bristol-Myers Squibb, Silenseed; and received travel support from Roche. RY has served as an advisor for Pfizer, Mirati Therapeutics, Revolution Medicine, Loxo@Lilly, and Amgen; received a speaker’s honorarium from Zai Lab’ and has received research support to her institution from Pfizer, Boehringer Ingelheim, Mirati Therapeutics, Daiichi-Sankyo, and Boundless Bio. GYK received research funding to institution from AstraZeneca, BMS, CARsgen, Daiichi-Sankyo, I-MAB, Jazz, Merck, Oncolys, Pieris, Triumvira, Zymeworks; served as a consultant for Astellas, AstraZeneca, Bayer, BMS, Daiichi-Sankyo, I-MAB, Jazz, Merck, Pieris, Zymeworks; and travel support from Dava Oncology and I-MAB. CBW has received honoraria/speakers’ fees from Amgen, Bayer, BMS, Chugai, Celgene, Falk, GSK, MSD, Merck, Janssen, Ipsen, Roche, Servier, SIRTeX, Taiho; served on advisory boards for Amgen, Bayer, BMS, Celgene, Incyte, Janssen, MSD, Servier, Shire/Baxalta, Rafael Pharmaceuticals, RedHill, Roche; has received travel support by Bayer, Celgene, Janssen, RedHill, Roche, Servier, Taiho; has received research grants (institutional) by Roche; serves as an officer for the European Society of Medical Oncology (ESMO) and Arbeitsgemeinschaft internistische Onkologie (AIO) and on expert committees for the German Cancer Aid (Deutsche Krebshilfe) and German Cancer Society (Deutsche Krebsgesellschaft); is a member of the EU Commission expert group: Mission Board for Cancer. BD and ABS are full time employees of Foundation Medicine and own stock and or stock options in Roche Holdings, Inc. CC served as expert testimony for Amgen; received honoraria as invited speaker for Bayer, Merck Serono, Servier; participated in advisory board for MSD, Nordic Pharma, Pierre-Fabre, Roche, Takeda; reports institutional financial interest for Bayer as Coordinating PI, Hutchinson as Local PI, Merck as Coordinating PI, Roche as Coordinating PI, Seagen as Local PI, Servier as Coordinating PI. SL reports research funding (to institution) from Amgen, Astellas, AstraZeneca, Bayer, Bristol-Myers Squibb, Daichii Sankyo, Hutchinson, Incyte, Merck Serono, Mirati, MSD, Pfizer, Roche, Servier; personal honoraria as invited speaker from Amgen, Bristol-Myers Squibb, Incyte, GSK, Lilly, Merck Serono, MSD, Pierre-Fabre, Roche, Servier; participation in advisory board for Amgen, Astellas, Astra Zeneca, Bayer, Bristol-Myers Squibb, Daiichi-Sankyo, GSK, Incyte, Lilly, Merck Serono, MSD, Servier, Takeda. FP reports institutional research grants from BMS, Incyte, Agenus, Amgen, Lilly and AstraZeneca; and personal fees from BMS, MSD, Amgen, Merck Serono, Pierre-Fabre, Servier, Bayer, Takeda, Astellas, Johnson&Johnson, Rottapharm, Ipsen, AstraZeneca, GSK, Daiichi-Sankyo, and Seagen. All other authors have declared no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: To compare X-ray and MRI as diagnostic tests of active Charcot neuro-osteoarthropathy (CNO) in diabetes., Research Design and Methods: X-rays and MRI scans of 48 participants were rated for severity of fracture (0 = no fracture, 1 = fracture, 2 = collapse/fragmentation), and for absence/presence of bone marrow edema (BME) on MRI and absence/presence of bone injury on X-ray. The agreement between modalities was assessed with tests for symmetry, marginal homogeneity, and κ-coefficients., Results: X-ray underscored MRI in grading fractures in the metatarsals (P = 0.05) and tarsals (P < 0.001) and reported as normal 79% of the bones with BME. The agreement between X-ray and MRI for grading severity of fracture was moderate to substantial (κ = 0.53; P < 0.001) and for detecting bone injury, slight to fair (κ = 0.17; P < 0.001)., Conclusions: The significant underperformance of X-ray in the assessment of the hot, swollen foot in diabetes should be considered when confirming or refuting the diagnosis of active CNO., (© 2023 by the American Diabetes Association.)

N6-methyladenosine is a highly dynamic, abundant mRNA modification which is an excellent potential mechanism for fine tuning gene expression. Plants adapt to their surrounding light and temperature environment using complex gene regulatory networks. The role of m 6 A in controlling gene expression in response to variable environmental conditions has so far been unexplored. Here, we map the transcriptome-wide m 6 A landscape under various light and temperature environments. Identified m 6 A-modifications show a highly specific spatial distribution along transcripts with enrichment occurring in 5'UTR regions and around transcriptional end sites. We show that the position of m 6 A modifications on transcripts might influence cellular transcript localization and the presence of m 6 A-modifications is associated with alternative polyadenylation, a process which results in multiple RNA isoforms with varying 3'UTR lengths. RNA with m 6 A-modifications exhibit a higher preference for shorter 3'UTRs. These shorter 3'UTR regions might directly influence transcript abundance and localization by including or excluding cis-regulatory elements. We propose that environmental stimuli might change the m 6 A landscape of plants as one possible way of fine tuning gene regulation through alternative polyadenylation and transcript localization.

Light is a crucial exogenous signal sensed by cryptochrome (CRY) blue light receptors to modulate growth and the circadian clock in plants and animals. However, how CRYs interpret light quantity to regulate growth in plants remains poorly understood. Furthermore, CRY2 protein levels and activity are tightly regulated in light to fine-tune hypocotyl growth; however, details of the mechanisms that explain precise control of CRY2 levels are not fully understood. We show that in Arabidopsis, UBP12 and UBP13 deubiquitinases physically interact with CRY2 in light. UBP12/13 negatively regulates CRY2 by promoting its ubiquitination and turnover to modulate hypocotyl growth. Growth and development were explicitly affected in blue light when UBP12/13 were disrupted or overexpressed, indicating their role alongside CRY2. UBP12/13 also interacted with and stabilized COP1, which is partially required for CRY2 turnover. Our combined genetic and molecular data support a mechanistic model in which UBP12/13 interact with CRY2 and COP1, leading to the stabilization of COP1. Stabilized COP1 then promotes the ubiquitination and degradation of CRY2 under blue light. Despite decades of studies on deubiquitinases, the knowledge of how their activity is regulated is limited. Our study provides insight into how exogenous signals and ligands, along with their receptors, regulate deubiquitinase activity by protein-protein interaction. Collectively, our results provide a framework of cryptochromes and deubiquitinases to detect and interpret light signals to control plant growth at the most appropriate time., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The Arabidopsis COP1/SPA complex is a key repressor of photomorphogenesis that suppresses light signaling in the dark. Both COP1 and SPA proteins are essential components of this complex. Although COP1 also exists in humans, SPA genes are specific to the green lineage. To elucidate the evolution of SPA genes we analyzed SPA functions in the moss Physcomitrella patens by characterizing knockout mutants in the two Physcomitrella SPA genes PpSPAa and PpSPAb. Light-grown PpspaAB double mutants exhibit smaller gametophores than the wild-type. In the dark, PpspaAB mutant gametophores show enhanced continuation of growth but etiolate normally. Gravitropism in the dark is reduced in PpspaAB mutant protonemata. The expression of light-regulated genes is mostly not constitutive in PpspaAB mutants. PpSPA and PpCOP1 interact; PpCOP1 also interacts with the transcription factor PpHY5 and, indeed, PpHY5 is destabilized in dark-grown Physcomitrella. Degradation of PpHY5 in darkness, however, does not require PpSPAa and PpSPAb. The data suggest that COP1/SPA-mediated light signaling is only partially conserved between Arabidopsis and Physcomitrella. Whereas COP1/SPA interaction and HY5 degradation in darkness is conserved, the role of SPA proteins appears to have diverged. PpSPA genes, unlike their Arabidopsis counterparts, are only required to suppress a subset of light responses in darkness., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The COP1/SPA complex is an E3 ubiquitin ligase that acts as a key repressor of photomorphogenesis in dark-grown plants. While both COP1 and the four SPA proteins contain coiled-coil and WD-repeat domains, SPA proteins differ from COP1 in carrying an N-terminal kinase-like domain that is not present in COP1. Here, we have analyzed the effects of deletions and missense mutations in the N-terminus of SPA1 when expressed in a spa quadruple mutant background devoid of any other SPA proteins. Deletion of the large N-terminus of SPA1 severely impaired SPA1 activity in transgenic plants with respect to seedling etiolation, leaf expansion and flowering time. This ΔN SPA1 protein showed a strongly reduced affinity for COP1 in vitro and in vivo, indicating that the N-terminus contributes to COP1/SPA complex formation. Deletion of only the highly conserved 95 amino acids of the kinase-like domain did not severely affect SPA1 function nor interactions with COP1 or cryptochromes. In contrast, missense mutations in this part of the kinase-like domain severely abrogated SPA1 function, suggesting an overriding negative effect of these mutations on SPA1 activity. We therefore hypothesize that the sequence of the kinase-like domain has been conserved during evolution because it carries structural information important for the activity of SPA1 in darkness. The N-terminus of SPA1 was not essential for light responsiveness of seedlings, suggesting that photoreceptors can inhibit the COP1/SPA complex in the absence of the SPA1 N-terminal domain. Together, these results uncover an important, but complex role of the SPA1 N-terminus in the suppression of photomorphogenesis., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Diaphanous-related formins are eukaryotic actin nucleation factors regulated by an autoinhibitory interaction between the N-terminal RhoGTPase-binding domain (mDiaN) and the C-terminal Diaphanous-autoregulatory domain (DAD). Although the activation of formins by Rho proteins is well characterized, its inactivation is only marginally understood. Recently, liprin-α3 was shown to interact with mDia1. Overexpression of liprin-α3 resulted in a reduction of the cellular actin filament content. The molecular mechanisms of how liprin-α3 exerts this effect and counteracts mDia1 activation by RhoA are unknown. Here, we functionally and structurally define a minimal liprin-α3 core region, sufficient to recapitulate the liprin-α3 determined mDia1-respective cellular functions. We show that liprin-α3 alters the interaction kinetics and thermodynamics of mDiaN with RhoA·GTP and DAD. RhoA displaces liprin-α3 allosterically, whereas DAD competes with liprin-α3 for a highly overlapping binding site on mDiaN. Liprin-α3 regulates actin polymerization by lowering the regulatory potency of RhoA and DAD on mDiaN. We present a model of a mechanistically unexplored and new aspect of mDiaN regulation by liprin-α3., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)