Search

Abstract Background Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. Results The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. Conclusion Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.

Melanoma brain metastasis is associated with high morbidity and mortality and remains a major clinical challenge. Despite recent successes with combination immune checkpoint inhibitors (ICI) in the treatment of affected patients, the mechanistic underpinnings of T cell entry and response to these drugs in brain metastasis are poorly understood. Using real-time intravital microscopy, Messmer and colleagues identified peritumoral venous vessels (PVVs) as critical sites for T cell entry into brain metastases, a process accelerated by ICI treatment. The expression of intercellular adhesion molecule 1 (ICAM-1) on PVVs was found to be important for T cell recruitment in pre-clinical models and associated with increased T cell infiltration in human brain metastatic lesions. This study highlights PVVs as key vascular entry points for T cells into brain metastases, laying the foundation for enhancing the efficacy of cancer immunotherapies.

Background: Cancer survivors (CS) comprise a particularly high-risk group for both de-novo and recurrent malignancies after solid organ transplantation., Case Presentation: We report a case of relapsed melanoma, presented as metastatic disease seven months after heart transplantation in a patient who had an early-stage melanoma resected 25 years prior. Treatment with a combination of dabrafenib, a BRAF inhibitor, and trametinib, a mitogen-activated protein kinase (MEK) inhibitor resulted in a near-complete metabolic response, without major adverse effects., Conclusion: This case demonstrates the increased risk of recurrence in CS with melanoma, which can persist decades after cancer diagnosis. These patients may be amenable to treatment using modern treatment modalities in oncology., (© 2024. The Author(s).)

Purpose: Sarcoma encompasses a diverse group of cancers that are typically resistant to current therapies, including immune checkpoint blockade (ICB), and underlying mechanisms are poorly understood. The contexture of sarcomas limits generation of high-quality data using cutting-edge molecular profiling methods, such as single-cell RNA-sequencing, thus hampering progress in understanding these understudied cancers., Experimental Design: Here, we demonstrate feasibility of producing multimodal single-cell genomics and whole-genome sequencing data from frozen tissues, profiling 75,716 cell transcriptomes of five undifferentiated pleomorphic sarcoma and three intimal sarcoma samples, including paired specimens from two patients treated with ICB., Results: We find that genomic diversity decreases in patients with response to ICB, and, in unbiased analyses, identify cancer cell programs associated with therapy resistance. Although interactions of tumor-infiltrating T lymphocytes within the tumor ecosystem increase in ICB responders, clonal expansion of CD8+ T cells alone was insufficient to predict drug responses., Conclusions: This study provides a framework for studying rare tumors and identifies salient and treatment-associated cancer cell intrinsic and tumor microenvironmental features in sarcomas., (©2024 The Authors; Published by the American Association for Cancer Research.)

Melanoma represents a critical clinical challenge owing to its unfavorable outcomes. This type of skin cancer exhibits unique adaptability to the brain microenvironment, but its underlying molecular mechanisms are poorly understood. Recent findings have suggested that melanoma brain metastases may share biological processes similar to those found in various neurodegenerative diseases. To further characterize melanoma brain metastasis development, we explore the relationship between the transcriptional profiles of melanoma brain metastases and the neurodegenerative diseases Alzheimer's disease, Parkinson's disease, and multiple sclerosis. We take an in silico approach to unveil a neurodegenerative signature of melanoma brain metastases compared with those of melanoma nonbrain metastasis (53 dysregulated genes were enriched in 11 functional terms, such as associated terms to the extracellular matrix and development) and with those of nontumor-bearing brain controls (195 dysregulated genes, mostly involved in development and cell differentiation, chromatin remodeling and nucleosome organization, and translation). Two genes, ITGA10 and DNAJC6, emerged as key potential markers being dysregulated in both scenarios. Finally, we developed an open-source, user-friendly web tool (https://bioinfo.cipf.es/metafun-mbm/) that allows interactive exploration of the complete results., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromosome-containing micronuclei are a hallmark of aggressive cancers. Micronuclei frequently undergo irreversible collapse, exposing their enclosed chromatin to the cytosol. Micronuclear rupture catalyzes chromosomal rearrangements, epigenetic abnormalities, and inflammation, yet mechanisms safeguarding micronuclear integrity are poorly understood. In this study, we found that mitochondria-derived reactive oxygen species (ROS) disrupt micronuclei by promoting a noncanonical function of charged multivesicular body protein 7 (CHMP7), a scaffolding protein for the membrane repair complex known as endosomal sorting complex required for transport III (ESCRT-III). ROS retained CHMP7 in micronuclei while disrupting its interaction with other ESCRT-III components. ROS-induced cysteine oxidation stimulated CHMP7 oligomerization and binding to the nuclear membrane protein LEMD2, disrupting micronuclear envelopes. Furthermore, this ROS-CHMP7 pathological axis engendered chromosome shattering known to result from micronuclear rupture. It also mediated micronuclear disintegrity under hypoxic conditions, linking tumor hypoxia with downstream processes driving cancer progression.

Pseudoprogression encapsulates a process of temporary radiographic growth followed by subsequent regression of metastatic melanoma lesions in response to immune checkpoint blockade (ICB), such as the combination of anti-programmed cell death protein 1 (PD-1) and anticytotoxic T-lymphocyte-associated antigen 4 therapy. This occurs in approximately 5-10% of ICB-treated patients, but has not yet been described in the context of novel combination therapies. Here, we report a case of an 89-year-old patient with metastatic melanoma to the liver, lung and lymph nodes, who underwent treatment with Opdualag (combining anti-PD-1 nivolumab and anti-lymphocyte-activation gene 3 relatlimab ICBs), and developed pseudoprogression after two cycles of therapy. The patient experienced a radiographic increase in liver metastatic lesion size, but was found to have a subsequent reduction in these lesions. The patient has been on therapy for 18 months without evidence of disease progression and continues to be clinically well-appearing., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Immunotherapies have shown significant promise as an impactful strategy in cancer treatment. However, in glioblastoma multiforme (GBM), the most prevalent primary brain tumor in adults, these therapies have demonstrated lower efficacy than initially anticipated. Consequently, there is an urgent need for strategies to enhance the effectiveness of immune treatments. AURKA has been identified as a potential drug target for GBM treatment. An analysis of the GBM cell transcriptome following AURKA inhibition revealed a potential influence on the immune system. Our research revealed that AURKA influenced PD-L1 levels in various GBM model systems in vitro and in vivo. Disrupting AURKA function genetically led to reduced PD-L1 levels and increased MHC-I expression in both established and patient-derived xenograft GBM cultures. This process involved both transcriptional and non-transcriptional pathways, partly implicating GSK3β. Interfering with AURKA also enhanced NK-cell-mediated elimination of GBM by reducing PD-L1 expression, as evidenced in rescue experiments. Furthermore, using a mouse model that mimics GBM with patient-derived cells demonstrated that Alisertib decreased PD-L1 expression in living organisms. Combination therapy involving anti-PD-1 treatment and Alisertib significantly prolonged overall survival compared to vehicle treatment. These findings suggest that targeting AURKA could have therapeutic implications for modulating the immune environment within GBM cells.

Chromosomal instability (CIN) is a hallmark of cancer that drives metastasis, immune evasion and treatment resistance. CIN results from chromosome mis-segregation events during anaphase, as excessive chromatin is packaged in micronuclei (MN), that can be enumerated to quantify CIN. Despite recent advancements in automation through computer vision and machine learning, the assessment of CIN remains a predominantly manual and time-consuming task, thus hampering important work in the field. Here, we present micronuclAI , a novel pipeline for automated and reliable quantification of MN of varying size, morphology and location from DNA-only stained images. In micronucleAI , single-cell crops are extracted from high-resolution microscopy images with the help of segmentation masks, which are then used to train a convolutional neural network (CNN) to output the number of MN associated with each cell. The pipeline was evaluated against manual single-cell level counts by experts and against routinely used MN ratio within the complete image. The classifier was able to achieve a weighted F1 score of 0.937 on the test dataset and the complete pipeline can achieve close to human-level performance on various datasets derived from multiple human and murine cancer cell lines. The pipeline achieved a root-mean-square deviation (RMSE) value of 0.0041, an R 2 of 0.87 and a Pearson's correlation of 0.938 on images obtained at 10X magnification. We tested the approach in otherwise isogenic cell lines in which we genetically dialed up or down CIN rates, and also on a publicly available image data set (obtained at 100X) and achieved an RMSE value of 0.0159, an R 2 of 0.90, and a Pearson's correlation of 0.951. Given the increasing interest in developing therapies for CIN-driven cancers, this method provides an important, scalable, and rapid approach to quantifying CIN on routinely obtained images. We release a GUI-implementation for easy access and utilization of the pipeline.

Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy., Competing Interests: Declaration of interests EpiCypher is a commercial developer and supplier of reagents and platforms used in this study. All authors affiliated with EpiCypher own shares in (with M.-C.K. also a board member of) EpiCypher Inc. B.I. is a consultant for or received honoraria from Volastra Therapeutics, Johnson & Johnson/Janssen, Novartis, Eisai, AstraZeneca, and Merck and has received research funding to Columbia University from Agenus, Alkermes, Arcus Biosciences, Checkmate Pharmaceuticals, Compugen, Immunocore, and Synthekine. A. Califano is founder, equity holder, and consultant of DarwinHealth Inc., a company that has licensed some of the algorithms used in this manuscript from Columbia University. Columbia University is also an equity holder in DarwinHealth Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. Malat1 localizes to nuclear speckles to couple transcription, splicing and mRNA maturation. In metastatic cells, Malat1 induces WNT ligands, autocrine loops to promote self-renewal and the expression of Serpin protease inhibitors. Through inhibition of caspase-1 and cathepsin G, SERPINB6B prevents gasdermin D-mediated induction of pyroptosis. In this way, SERPINB6B suppresses immunogenic cell death and confers evasion of T cell-mediated tumor lysis of incipient metastatic cells. On-target inhibition of Malat1 using therapeutic antisense nucleotides suppresses metastasis in a SERPINB6B-dependent manner. These results suggest that Malat1-induced expression of SERPINB6B can titrate pyroptosis and immune recognition at metastatic sites. Thus, Malat1 is at the nexus of tumor initiation, reactivation and immune evasion and represents a tractable and clinically relevant drug target., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Immune evasion is a hallmark of cancer, yet the underlying mechanisms are often unknown in many patients. Using single-cell transcriptomics analysis, we previously identified the co-stimulator CD58 as part of a cancer cell-intrinsic immune checkpoint resistance signature in patient melanoma tissue. We subsequently validated CD58 loss as a driver of immune evasion using a patient-derived co-culture model of cancer and cytotoxic tumor-infiltrating lymphocytes in a pooled single-cell perturbation experiment, where we additionally observed concurrent upregulation of PD-L1 protein expression in melanoma cells with CD58 loss. In our most recent study, we uncovered the mechanisms of immune evasion mediated by CD58 loss, including impaired T cell activation and infiltration within tumors, as well as inhibitory signaling by PD-L1 via a shared regulator, CMTM6. Thus, cancer cell-intrinsic reduction of CD58 represents a multi-faceted determinant of immune evasion. Furthermore, its reciprocal interaction with PD-L1 via CMTM6 provides critical insights into how co-inhibitory and co-stimulatory immune cues are regulated., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1-84.5) and 65% (95% CI: 52.5-76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0-80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6-83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence., (© 2023. The Author(s).)

Spatial omics technologies can help identify spatially organized biological processes, but existing computational approaches often overlook structural dependencies in the data. Here, we introduce Smoother, a unified framework that integrates positional information into non-spatial models via modular priors and losses. In simulated and real datasets, Smoother enables accurate data imputation, cell-type deconvolution, and dimensionality reduction with remarkable efficiency. In colorectal cancer, Smoother-guided deconvolution reveals plasma cell and fibroblast subtype localizations linked to tumor microenvironment restructuring. Additionally, joint modeling of spatial and single-cell human prostate data with Smoother allows for spatial mapping of reference populations with significantly reduced ambiguity., (© 2023. The Author(s).)

Melanoma brain metastases (MBM) are clinically challenging to treat and exhibit variable responses to immune checkpoint therapies. Prior research suggests that MBM exhibit poor tumor immune responses and are enriched in oxidative phosphorylation. Here, we report results from a multi-omic analysis of a large, real-world melanoma cohort. MBM exhibited lower interferon-gamma (IFNγ) scores and T cell-inflamed scores compared to primary cutaneous melanoma (PCM) or extracranial metastases (ECM), which was independent of tumor mutational burden. Among MBM, there were fewer computationally inferred immune cell infiltrates, which correlated with lower TNF and IL12B mRNA levels. Ingenuity pathway analysis (IPA) revealed suppression of inflammatory responses and dendritic cell maturation pathways. MBM also demonstrated a higher frequency of pathogenic PTEN mutations and angiogenic signaling. Oxidative phosphorylation (OXPHOS) was enriched in MBM and negatively correlated with NK cell and B cell-associated transcriptomic signatures. Modulating metabolic or angiogenic pathways in MBM may improve responses to immunotherapy in this difficult-to-treat patient subset., (© 2023. The Author(s).)

Mucosal melanoma remains a rare cancer with high mortality and a paucity of therapeutic options. This is due in significant part to its low incidence leading to limited patient access to expert care and downstream clinical/basic science data for research interrogation. Clinical challenges such as delayed and at times inaccurate diagnoses, and lack of consensus tumor staging have added to the suboptimal outcomes for these patients. Clinical trials, while promising, have been difficult to activate and accrue. While individual institutions and investigators have attempted to seek solutions to such problems, international, national, and local partnership may provide the keys to more efficient and innovative paths forward. Furthermore, a mucosal melanoma coalition would provide a potential network for patients and caregivers to seek expert opinion and advice. The Melanoma Research Foundation Mucosal Melanoma Meeting (December 16, 2022, New York, USA) highlighted the current clinical challenges faced by patients, providers, and scientists, identified current and future clinical trial investigations in this rare disease space, and aimed to increase national and international collaboration among the mucosal melanoma community in an effort to improve patient outcomes. The included proceedings highlight the clinical challenges of mucosal melanoma, global clinical trial experience, basic science advances in mucosal melanoma, and future directions, including the creation of shared rare tumor registries and enhanced collaborations., (© 2023 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Since the first approval for immune checkpoint inhibitors (ICIs) for the treatment of cutaneous melanoma more than a decade ago, immunotherapy has completely transformed the treatment landscape of this chemotherapy-resistant disease. Combination regimens including ICIs directed against programmed cell death protein 1 (PD-1) with anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) agents or, more recently, anti-lymphocyte-activation gene 3 (LAG-3) agents, have gained regulatory approvals for the treatment of metastatic cutaneous melanoma, with long-term follow-up data suggesting the possibility of cure for some patients with advanced disease. In the resectable setting, adjuvant ICIs prolong recurrence-free survival, and neoadjuvant strategies are an active area of investigation. Other immunotherapy strategies, such as oncolytic virotherapy for injectable cutaneous melanoma and bispecific T-cell engager therapy for HLA-A*02:01 genotype-positive uveal melanoma, are also available to patients. Despite the remarkable efficacy of these regimens for many patients with cutaneous melanoma, traditional immunotherapy biomarkers (ie, programmed death-ligand 1 expression, tumor mutational burden, T-cell infiltrate and/or microsatellite stability) have failed to reliably predict response. Furthermore, ICIs are associated with unique toxicity profiles, particularly for the highly active combination of anti-PD-1 plus anti-CTLA-4 agents. The Society for Immunotherapy of Cancer (SITC) convened a panel of experts to develop this clinical practice guideline on immunotherapy for the treatment of melanoma, including rare subtypes of the disease (eg, uveal, mucosal), with the goal of improving patient care by providing guidance to the oncology community. Drawing from published data and clinical experience, the Expert Panel developed evidence- and consensus-based recommendations for healthcare professionals using immunotherapy to treat melanoma, with topics including therapy selection in the advanced and perioperative settings, intratumoral immunotherapy, when to use immunotherapy for patients with BRAF V600-mutated disease, management of patients with brain metastases, evaluation of treatment response, special patient populations, patient education, quality of life, and survivorship, among others., Competing Interests: Competing interests: CA—Other: Stock Pfizer, Advisory Board: Iovance and Merck, Memorial Sloan Kettering Cancer Center. EIB—Consulting Fees: Nektar, Instilbio, Novartis, Xilio, Sanofi, Merck, and Iovance. DD—IP Rights: US Patent 63/124,231, Compositions and Methods for Treating Cancer, December 11, 2020, US Patent 63/208,719, Compositions and Methods For Determining Responsiveness to Immune Checkpoint Inhibitors (ICI), Increasing Effectiveness of ICI and Treating Cancer, June 9, 2021; Consulting Fees: Checkmate Pharmaceuticals, Finch, Shionogi, Vedanta Biosciences; Fees for Non CE Services: Medical Learning Group (MLG), Clinical Care Options (CCO); Contracted Research: Arcus, Checkmate Pharmaceuticals, CellSight Technologies, Immunocore, Merck, Tesaro/GSK. GTG—Consulting Fees: Bristol Myers Squibb, Regeneron, Genentech, Novartis, Merck, Sapience Therapeutics, Exicure, Eisai, Iovance Biotherapeutics, Lyell Immunopharma, and HUYABIO International; Contracted Research: Exelixis (institutional support), Lucerno Dynamics. OH—Researcher: Arcus, Aduro, Akeso, Amgen, Bioatla, BMS, Cytomx, Exelixis, Roche Genentech, GSK, Immunocore, Idera, Incyte, Iovance, Merck, Moderna, Merck Serono, Nextcure, Novartis, Pfizer, Regeneron, Seattle Gen, Torque, Zelluna; Consultant Advisor Speaker: Alkermes, Amgen, Bactonix, Beigene, Bioatla, BMS, Esai, Roche Genentech, Georiamune, GigaGen, Grit Bio, GSK, Idera, Immunocore, Incyte, Instilbio, IO Biotech, Iovance, Janssen, KSQ, Merck, Moderna, Novartis, Obsidian, Pfizer, Regeneron, Sanofi, Seattle Gen, Tempus, Vial Health Tech, Zelluna; Independent Contractor: Alkermes, Amgen, Bactonix, Beigene, Bioatla, BMS, Esai, Roche Genentech, Georiamune, GigaGen, Grit Bio, GSK, Idera, Immunocore, Incyte, Instilbio, IO Biotech, Iovance, Janssen, KSQ, Merck, Moderna, Novartis, Obsidian, Pfizer, Regeneron, Sanofi, Seattle Gen, Tempus, Vial Health Tech, Zelluna; Publically Traded Stocks: Bactonix. TJH—Contracted Research: Genentech, SkylineDX. BI—Consulting Fees: Johnson & Johnson, Volastra Therapeutics, Merck, AstraZeneca, Eisai and Janssen Pharmaceuticals; research funding to Columbia University from Alkermes, Arcus Biosciences, Checkmate Pharmaceuticals, Compugen, Immunocore, and Synthekine. DBJ—IP Rights: MHC-II for use as immunotherapy biomarker, Abatacept as treatment for irAEs; Consulting Fees: BMS, Catalyst, Iovance, Jansen, Mallinckrodt, Merck, Mosaic ImmunoIngineering, Novartis, Oncosec, Pfizer, Targovax; Contracted Research: BMS, Incyte. RPK—Consulting Fees: Regeneron. JJL—Researcher: AbbVie, Astellas, AstraZeneca, Bristol Myers Squibb, Corvus, Day One, EMD Serono, Fstar, Genmab, Ikena, Immatics, Incyte, Kadmon, KAHR, Macrogenics, Merck, Moderna, Nektar, Next Cure, Numab, Palleon, Pfizer, Replimmune, Rubius, Servier, Scholar Rock, Synlogic, Takeda, Trishula, Tizona, Xencor; Consultant Advisor Speaker: AbbVie, Agenus, Alnylam, Atomwise, Bayer, Bristol Myers Squibb, Castle, Checkmate, Codiak, Crown, Cugene, Curadev, Day One, Eisai, EMD Serono, Endeavor, Flame, G1 Therapeutics, Genentech, Gilead, Glenmark, HotSpot, Kadmon, KSQ, Janssen, Ikena, Inzen, Immatics, Immunocore, Incyte, Instil, IO Biotech, Macrogenics, Merck, Mersana, Nektar, Novartis, Partner, Pfizer, Pioneering Medicines, PsiOxus, Regeneron, Replimmune, Ribon, Roivant, Servier, STINGthera, Synlogic, Synthekine 7 Hills, Affivant, Bright Peak, Exo, Fstar, Inzen, RefleXion, Xilio (stock) Actym, Alphamab Oncology, Arch Oncology, Duke Street Bio, Kanaph, Mavu, NeoTx, Onc.AI, OncoNano, physIQ, Pyxis, Saros, STipe, Tempest, AbbVie, Agenus, Amgen, Immutep, Evaxion. TCM—Consulting Fees: Merck, BMS, GigaGen, OncoSec; Pfizer – Scientific Advisory Board. MJM—Consulting Fees: AstraZeneca, Nektar Therapeutics, Istari Oncology, Immunai, Xilio Therapeutics; Fees for Non CE Services: Bristol Myers Squibb (served as a speaker). ACP—Consulting Fees: Regeneron, BMS, Merck; Fees for Non CE Services: BMS; Contracted Research: Merck, BMS, Iovance, Regeneron, Takeda, Ideaya, Replimune: All payments to institution. KMR—Consulting Fees: BMS, Merck, Eisai, Immunocore. AKSS—Consulting Fees: Novartis, Pfizer, Regeneron, Iovance. Contracted Research: Ascentage, Ideaya, Regeneron, Bristol Myers Squibb, Immunocore, Merck, Nektar, Olatec, Replimune, Seagen. KS—Contracted Research: Trials at Norris Cotton Cancer Center, Syndax, Provectus, OmniSeq, BrightPath Biotherapeutics, Ludwig Institute of Cancer Research, Hlsinn, AbbVie, BMS, OncoSec, Ultimovacs, Altellas, AstraZeneca, Xencor, Exicure, Numab, Checkmate Pharmaceuticals, Natera. RJS—Reseracher: Merck (research funding); Consultant Advisor Speaker: BMS, Merck, Novartis, Pfizer; Other: Faculty for SCION Workshop. JT—Consulting Fees: GSK, Genentech, BMS, Akoya Biosciences, Merck, AstraZeneca, Compugen, Lunaphore; Contracted Research: BMS, Akoya Biosciences; Other: Akoya Biosciences; Other Details: Patent pending for multiplexing imaging strategy; Ownership Interest Less Than 5 Per Cent: Akoya Biosciences. HAT—Consulting Fees: Genentech, BMS, Novartis, Merck, Eisai, Karyopharm, Boxer Capital, Iovance, Jazz Pharmaceuticals, Medicenna; Contracted Research: Genentech, BMS, Novartis, Merck, GSK, EMD Serono, Eisai, Dragonfly Therapeutics, RAPT Therapeutics. JKT—Nothing to Disclose. CV—Nothing to Disclose. SAW—Consulting Fees: Lyell; Contracted Research: research funds to institution from BMS and Apexigen. MKW—Consulting Fees: Merck, Pfizer, Bristol Myers Squibb, Regeneron, EMD-Serono, ExiCure, Castle Biosciences, Adagene. SITC Staff—CG, AK, NL, SM-W—Nothing to Disclose., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Current methods for biomarker discovery and target identification in immuno-oncology rely on static snapshots of tumor immunity. To thoroughly characterize the temporal nature of antitumor immune responses, we developed a 34-parameter spectral flow cytometry panel and performed high-throughput analyses in critical contexts. We leveraged two distinct preclinical models that recapitulate cancer immunoediting (NPK-C1) and immune checkpoint blockade (ICB) response (MC38), respectively, and profiled multiple relevant tissues at and around key inflection points of immune surveillance and escape and/or ICB response. Machine learning-driven data analysis revealed a pattern of KLRG1 expression that uniquely identified intratumoral effector CD4 T cell populations that constitutively associate with tumor burden across tumor models, and are lost in tumors undergoing regression in response to ICB. Similarly, a Helios - KLRG1 + subset of tumor-infiltrating regulatory T cells was associated with tumor progression from immune equilibrium to escape and was also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumor-infiltrating CD4 T cells associate with disease progression in renal cancer. These findings nominate KLRG1 + CD4 T cell populations as subsets for further investigation in cancer immunity and demonstrate the utility of longitudinal spectral flow profiling as an engine of dynamic biomarker discovery., Competing Interests: Competing interests: BI has received consulting fees from Volastra Therapeutics, Merck, AstraZeneca and Janssen Pharmaceuticals and has received research funding to Columbia University from Alkermes, Arcus Biosciences, Checkmate Pharmaceuticals, Compugen, Immunocore, and Synthekine. CGD is a coinventor on patents licensed from JHU to BMS and Janssen, has served as a paid consultant to AZ Medimmune, BMS, Pfizer, Roche, Sanofi Aventis, Genentech, Merck, and Janssen, has received sponsored research funding to his institution from BMS IIoN and Janssen, and is a current employee of Janssen., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chromosomal instability (CIN) is a driver of cancer metastasis 1-4 , yet the extent to which this effect depends on the immune system remains unknown. Using ContactTracing-a newly developed, validated and benchmarked tool to infer the nature and conditional dependence of cell-cell interactions from single-cell transcriptomic data-we show that CIN-induced chronic activation of the cGAS-STING pathway promotes downstream signal re-wiring in cancer cells, leading to a pro-metastatic tumour microenvironment. This re-wiring is manifested by type I interferon tachyphylaxis selectively downstream of STING and a corresponding increase in cancer cell-derived endoplasmic reticulum (ER) stress response. Reversal of CIN, depletion of cancer cell STING or inhibition of ER stress response signalling abrogates CIN-dependent effects on the tumour microenvironment and suppresses metastasis in immune competent, but not severely immune compromised, settings. Treatment with STING inhibitors reduces CIN-driven metastasis in melanoma, breast and colorectal cancers in a manner dependent on tumour cell-intrinsic STING. Finally, we show that CIN and pervasive cGAS activation in micronuclei are associated with ER stress signalling, immune suppression and metastasis in human triple-negative breast cancer, highlighting a viable strategy to identify and therapeutically intervene in tumours spurred by CIN-induced inflammation., (© 2023. The Author(s).)

The cell-autonomous balance of immune-inhibitory and -stimulatory signals is a critical process in cancer immune evasion. Using patient-derived co-cultures, humanized mouse models, and single-cell RNA-sequencing of patient melanomas biopsied before and on immune checkpoint blockade, we find that intact cancer cell-intrinsic expression of CD58 and ligation to CD2 is required for anti-tumor immunity and is predictive of treatment response. Defects in this axis promote immune evasion through diminished T cell activation, impaired intratumoral T cell infiltration and proliferation, and concurrently increased PD-L1 protein stabilization. Through CRISPR-Cas9 and proteomics screens, we identify and validate CMTM6 as critical for CD58 stability and upregulation of PD-L1 upon CD58 loss. Competition between CD58 and PD-L1 for CMTM6 binding determines their rate of endosomal recycling over lysosomal degradation. Overall, we describe an underappreciated yet critical axis of cancer immunity and provide a molecular basis for how cancer cells balance immune inhibitory and stimulatory cues., Competing Interests: Declaration of interests B.I. is a consultant for or received honoraria from Volastra Therapeutics, Johnson & Johnson/Janssen, Novartis, Eisai, AstraZeneca and Merck, and has received research funding to Columbia University from Agenus, Alkermes, Arcus Biosciences, Checkmate Pharmaceuticals, Compugen, Immunocore, and Synthekine. K.W.W. serves on the scientific advisory board of T-Scan Therapeutics, SQZ Biotech, Nextechinvest and receives sponsored research funding from Novartis. He is a co-founder of Immunitas, a biotech company. None of these represent a conflict of interest pertaining to the presented work. P.H., J.C.M., and B.I. filed a patent pertaining to the presented work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [ 64 Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

Single-cell genomics enables dissection of tumor heterogeneity and molecular underpinnings of drug response at an unprecedented resolution 1-11 . However, broad clinical application of these methods remains challenging, due to several practical and preanalytical challenges that are incompatible with typical clinical care workflows, namely the need for relatively large, fresh tissue inputs. In the present study, we show that multimodal, single-nucleus (sn)RNA/T cell receptor (TCR) sequencing, spatial transcriptomics and whole-genome sequencing (WGS) are feasible from small, frozen tissues that approximate routinely collected clinical specimens (for example, core needle biopsies). Compared with data from sample-matched fresh tissue, we find a similar quality in the biological outputs of snRNA/TCR-seq data, while reducing artifactual signals and compositional biases introduced by fresh tissue processing. Profiling sequentially collected melanoma samples from a patient treated in the KEYNOTE-001 trial 12 , we resolved cellular, genomic, spatial and clonotype dynamics that represent molecular patterns of heterogeneous intralesional evolution during anti-programmed cell death protein 1 therapy. To demonstrate applicability to banked biospecimens of rare diseases 13 , we generated a single-cell atlas of uveal melanoma liver metastasis with matched WGS data. These results show that single-cell genomics from archival, clinical specimens is feasible and provides a framework for translating these methods more broadly to the clinical arena., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

How cancer-associated chromatin abnormalities shape tumor-immune interaction remains incompletely understood. Recent studies have linked DNA hypomethylation and de-repression of retrotransposons to anti-tumor immunity through the induction of interferon response. Here, we report that inactivation of the histone H3K36 methyltransferase NSD1, which is frequently found in squamous cell carcinomas (SCCs) and induces DNA hypomethylation, unexpectedly results in diminished tumor immune infiltration. In syngeneic and genetically engineered mouse models of head and neck SCCs, NSD1-deficient tumors exhibit immune exclusion and reduced interferon response despite high retrotransposon expression. Mechanistically, NSD1 loss results in silencing of innate immunity genes, including the type III interferon receptor IFNLR1, through depletion of H3K36 di-methylation (H3K36me2) and gain of H3K27 tri-methylation (H3K27me3). Inhibition of EZH2 restores immune infiltration and impairs the growth of Nsd1-mutant tumors. Thus, our work uncovers a druggable chromatin cross talk that regulates the viral mimicry response and enables immune evasion of DNA hypomethylated tumors., Competing Interests: Declaration of interests B.I. is a paid consultant for Volastra Therapeutics., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis 1,2 . Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts 3 , during hepatocarcinogenesis. Genetic depletion, activation or inhibition of HSCs in mouse models of HCC revealed their overall tumour-promoting role. HSCs were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analyses of mouse and human HSC subpopulations by single-cell RNA sequencing together with genetic ablation of subpopulation-enriched mediators revealed dual functions of HSCs in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSCs, protected against hepatocyte death and HCC development. By contrast, type I collagen, enriched in activated myofibroblastic HSCs, promoted proliferation and tumour development through increased stiffness and TAZ activation in pretumoural hepatocytes and through activation of discoidin domain receptor 1 in established tumours. An increased HSC imbalance between cytokine-producing HSCs and myofibroblastic HSCs during liver disease progression was associated with increased HCC risk in patients. In summary, the dynamic shift in HSC subpopulations and their mediators during chronic liver disease is associated with a switch from HCC protection to HCC promotion., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Uveal melanoma (UM) is associated with poor outcomes in the metastatic setting and harbors activating mutations resulting in upregulation of MAPK signaling in almost all cases. The efficacy of selumetinib, an oral allosteric inhibitor of MEK1/2, was limited when administered at a continual dosing schedule of 75 mg BID. Preclinical studies demonstrate that intermittent MEK inhibition reduces compensatory pathway activation and promotes T cell activation. We hypothesized that intermittent dosing of selumetinib would reduce toxicity, allow for the administration of increased doses, and achieve more complete pathway inhibition, thus resulting in improved antitumor activity., Methods: We conducted a phase Ib trial of selumetinib using an intermittent dosing schedule in patients with metastatic UM. The primary objective was to estimate the maximum tolerated dose (MTD) and assess safety and tolerability. Secondary objectives included assessment of the overall response rate (RR), progression-free survival (PFS) and overall survival (OS). Tumor biopsies were collected at baseline, on day 3 (on treatment), and between days 11-14 (off treatment) from 9 patients for pharmacodynamic (PD) assessments., Results: 29 patients were enrolled and received at least one dose of selumetinib across 4 dose levels (DL; DL1: 100 mg BID; DL2: 125 mg BID; DL3: 150 mg BID; DL4: 175 mg BID). All patients experienced a treatment-related adverse event (TRAE), with 5/29 (17%) developing a grade 3 or higher TRAE. Five dose limiting toxicities (DLT) were observed: 2/20 in DL2, 2/5 in DL3, 1/1 in DL4. The estimated MTD was 150 mg BID (DL3), with an estimated probability of toxicity of 29% (90% probability interval 16%-44%). No responses were observed; 11/29 patients achieved a best response of stable disease (SD). The median PFS and OS were 1.8 months (95% CI 1.7, 4.5) and 7.1 months (95% CI 5.3, 11.5). PD analysis demonstrated at least partial pathway inhibition in all samples at day 3, with reactivation between days 11-14 in 7 of those cases., Conclusions: We identified 150 mg BID as the MTD of intermittent selumetinib, representing a 100% increase over the continuous dose MTD (75 mg BID). However, no significant clinical efficacy was observed using this dosing schedule., Competing Interests: SK reports honoraria from Castle Biosciences. SP reports institutional clinical trial support from Bristol Myers Squibb, Foghorn Therapeutics, Ideaya Biosciences, InxMed, Lvgen, Novartis, Provectus Biopharmaceuticals, Seagen, Syntrix Bio, TriSalus Life Sciences and consulting fees from BMS, Delcath Systens, Advance Knowledge in Healthcare, Cardinal Health, Immunocore, Novartis, and TriSalus Life Sciences. AS reports advisory/personal fees from Bristol-Myers Squibb (BMS), Immunocore, Novartis, and research funding from Pfizer, BMS, Immunocore, Novartis, Targovax, Polaris, Checkmate Pharmaceuticals, Foghorn Therapeutics, Linneaus Therapeutics, and Prelude Therapeutics. GS reports stock or other ownership interests in GenCirq, Bionaut Labs, and January Therapeutics, advisory/consulting/personal fees from Bionaut Labs, Ellipses Pharma, GenCirq, Epizyme, Array BioPharma, Apexigen, Oncogenuity, OnCusp Therapeutics, Concarlo, Shanghai Pharma, Astex Pharmaceuticals, January Therapeutics, Sellas Life Sciences, PureTech Health, and Killys Therapeutics, research funding from Astex Pharmaceuticals, Incyte, Calithera Biosciences, Lilly, Daiichi Sankyo, Fortress Biotech, Karyopharm Therapeutics, Oxford Biotherapeutics, TopAlliance Biosciences, Adaptimmune, Springworks Therapeutics, and TRACON Pharma. RC reports consulting fees from Alkermes, Bristol Myers Squibb, Castle Biosciences, Delcath Systems, Eisai, Jiangsu Hengrui Pharmaceuticals, Ideaya Biosciences, Immunocore, InxMed, Iovance Biotherapeutics, Merck, Novartis, OncoSec Medical, Pierre Fabre, PureTech Health, Regeneron, Sanofi Genzyme, Sorrento Therapeutics, Trisalus and advisory board fees from Aura Biosciences, Chimeron, and Rgenix. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2022 Khan, Patel, Shoushtari, Ambrosini, Cremers, Lee, Franks, Singh-Kandah, Hernandez, Sender, Vuolo, Nesson, Mundi, Izar, Schwartz and Carvajal.)