Pseudoprogression in a patient with metastatic melanoma treated with PD-1 and LAG-3 inhibition.

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.

SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion.

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.

LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells.

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.

Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.

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.

From patient tissue correlates to molecular mechanisms of cancer immune evasion: the emerging role of CD58 and PD-L1 co-regulation via CMTM6.

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.

Neoadjuvant durvalumab plus radiation versus durvalumab alone in stages I-III non-small cell lung cancer: survival outcomes and molecular correlates of a randomized phase II trial.

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.

Smoother: a unified and modular framework for incorporating structural dependency in spatial omics data.

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.

Multi-omic profiling reveals discrepant immunogenic properties and a unique tumor microenvironment among melanoma brain metastases.

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.

Proceedings from the Melanoma Research Foundation Mucosal Melanoma Meeting (December 16, 2022, New York, USA).

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.

Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of melanoma, version 3.0.

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 BRAFV600-mutated disease, management of patients with brain metastases, evaluation of treatment response, special patient populations, patient education, quality of life, and survivorship, among others.

KLRG1 marks tumor-infiltrating CD4 T cell subsets associated with tumor progression and immunotherapy response.

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.

Non-cell-autonomous cancer progression from chromosomal instability.

Chromosomal instability (CIN) is a driver of cancer metastasis1-4, 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.

The CD58-CD2 axis is co-regulated with PD-L1 via CMTM6 and shapes anti-tumor immunity.

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.

Multimodal single-cell and whole-genome sequencing of small, frozen clinical specimens.

Single-cell genomics enables dissection of tumor heterogeneity and molecular underpinnings of drug response at an unprecedented resolution1-11. 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 trial12, 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 diseases13, 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.

B-cells Drive Response to PD-1 Blockade in Glioblastoma Upon Neutralization of TGFß-mediated Immunosuppression.

Immunotherapy has revolutionized cancer treatment but has yet to be translated into brain tumors. Studies in other solid tumors suggest a central role of B-cell immunity in driving immune-checkpoint-blockade efficacy. Using single-cell and single-nuclei transcriptomics of human glioblastoma and melanoma brain metastasis, we found that tumor-associated B-cells have high expression of checkpoint molecules, known to block B-cell-receptor downstream effector function such as plasmablast differentiation and antigen-presentation. We also identified TGFß-1/TGFß receptor-2 interaction as a crucial modulator of B-cell suppression. Treatment of glioblastoma patients with pembrolizumab induced expression of B-cell checkpoint molecules and TGFß-receptor-2. Abrogation of TGFß using different conditional knockouts expanded germinal-center-like intratumoral B-cells, enhancing immune-checkpoint-blockade efficacy. Finally, blocking αVß8 integrin (which controls the release of active TGFß) and PD-1 significantly increased B-cell-dependent animal survival and immunological memory. Our study highlights the importance of intratumoral B-cell immunity and a remodeled approach to boost the effects of immunotherapy against brain tumors.

KLRG1 marks tumor-infiltrating CD4 T cell subsets associated with tumor progression and immunotherapy response.

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 (Tregs) was associated with tumor progression from immune equilibrium to escape, and were also lost in tumors responding to ICB. Validation studies confirmed KLRG1 signatures in human tumorinfiltrating 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 and/or target discovery.

Massively parallel base editing screens to map variant effects on anti-tumor hallmarks of primary human T cells.

Base editing enables generation of single nucleotide variants, but large-scale screening in primary human T cells is limited due to low editing efficiency, among other challenges 1 . Here, we developed a high-throughput approach for high-efficiency and massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale screens editing 102 genes with central functions in T cells and full-length tiling mutagenesis of selected genes, and read out variant effects on hallmarks of T cell anti-tumor immunity, including activation, proliferation, and cytokine production. We discovered a broad landscape of gain- and loss-of-function mutations, including in PIK3CD and its regulatory subunit encoded by PIK3R1, LCK , AKT1, CTLA-4 and JAK1 . We identified variants that affected several (e.g., PIK3CD C416R) or only selected (e.g. LCK Y505C) hallmarks of T cell activity, and functionally validated several hits by probing downstream signaling nodes and testing their impact on T cell polyfunctionality and proliferation. Using primary human T cells in which we engineered a T cell receptor (TCR) specific to a commonly presented tumor testis antigen as a model for cellular immunotherapy, we demonstrate that base edits identified in our screens can tune specific or broad T cell functions and ultimately improve tumor elimination while exerting minimal off-target activity. In summary, we present the first large-scale base editing screen in primary human T cells and provide a framework for scalable and targeted base editing at high efficiency. Coupled with multi-modal phenotypic mapping, we accurately nominate variants that produce a desirable T cell state and leverage these synthetic proteins to improve models of cellular cancer immunotherapies.

Opposing roles of hepatic stellate cell subpopulations in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality worldwide, develops almost exclusively in patients with chronic liver disease and advanced fibrosis1,2. Here we interrogated functions of hepatic stellate cells (HSCs), the main source of liver fibroblasts3, 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.