Beyond the Barrier: Unraveling the Mechanisms of Immunotherapy Resistance.

Immune checkpoint blockade (ICB) induces a remarkable and durable response in a subset of cancer patients. However, most patients exhibit either primary or acquired resistance to ICB. This resistance arises from a complex interplay of diverse dynamic mechanisms within the tumor microenvironment (TME). These mechanisms include genetic, epigenetic, and metabolic alterations that prevent T cell trafficking to the tumor site, induce immune cell dysfunction, interfere with antigen presentation, drive heightened expression of coinhibitory molecules, and promote tumor survival after immune attack. The TME worsens ICB resistance through the formation of immunosuppressive networks via immune inhibition, regulatory metabolites, and abnormal resource consumption. Finally, patient lifestyle factors, including obesity and microbiome composition, influence ICB resistance. Understanding the heterogeneity of cellular, molecular, and environmental factors contributing to ICB resistance is crucial to develop targeted therapeutic interventions that enhance the clinical response. This comprehensive overview highlights key mechanisms of ICB resistance that may be clinically translatable.

A pan-cancer analysis of the microbiome in metastatic cancer.

Microbial communities are resident to multiple niches of the human body and are important modulators of the host immune system and responses to anticancer therapies. Recent studies have shown that complex microbial communities are present within primary tumors. To investigate the presence and relevance of the microbiome in metastases, we integrated mapping and assembly-based metagenomics, genomics, transcriptomics, and clinical data of 4,160 metastatic tumor biopsies. We identified organ-specific tropisms of microbes, enrichments of anaerobic bacteria in hypoxic tumors, associations between microbial diversity and tumor-infiltrating neutrophils, and the association of Fusobacterium with resistance to immune checkpoint blockade (ICB) in lung cancer. Furthermore, longitudinal tumor sampling revealed temporal evolution of the microbial communities and identified bacteria depleted upon ICB. Together, we generated a pan-cancer resource of the metastatic tumor microbiome that may contribute to advancing treatment strategies.

Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

Anti-LAG-3 boosts CD8 T cell effector function.

LAG-3 is the third immune checkpoint pathway successfully targeted for cancer therapy. Although ineffective as a monotherapy, combination of LAG-3 and PD-1 blockade improves survival from advanced melanoma. In this issue of Cell, two studies in mice and a human clinical trial provide insights on LAG-3 in immune regulation.

Microbial metabolite enhances immunotherapy efficacy by modulating T cell stemness in pan-cancer.

The immune checkpoint blockade (ICB) response in human cancers is closely linked to the gut microbiota. Here, we report that the abundance of commensal Lactobacillus johnsonii is positively correlated with the responsiveness of ICB. Supplementation with Lactobacillus johnsonii or tryptophan-derived metabolite indole-3-propionic acid (IPA) enhances the efficacy of CD8+ T cell-mediated αPD-1 immunotherapy. Mechanistically, Lactobacillus johnsonii collaborates with Clostridium sporogenes to produce IPA. IPA modulates the stemness program of CD8+ T cells and facilitates the generation of progenitor exhausted CD8+ T cells (Tpex) by increasing H3K27 acetylation at the super-enhancer region of Tcf7. IPA improves ICB responsiveness at the pan-cancer level, including melanoma, breast cancer, and colorectal cancer. Collectively, our findings identify a microbial metabolite-immune regulatory pathway and suggest a potential microbial-based adjuvant approach to improve the responsiveness of immunotherapy.

Custom scoring based on ecological topology of gut microbiota associated with cancer immunotherapy outcome.

The gut microbiota influences the clinical responses of cancer patients to immunecheckpoint inhibitors (ICIs). However, there is no consensus definition of detrimental dysbiosis. Based on metagenomics (MG) sequencing of 245 non-small cell lung cancer (NSCLC) patient feces, we constructed species-level co-abundance networks that were clustered into species-interacting groups (SIGs) correlating with overall survival. Thirty-seven and forty-five MG species (MGSs) were associated with resistance (SIG1) and response (SIG2) to ICIs, respectively. When combined with the quantification of Akkermansia species, this procedure allowed a person-based calculation of a topological score (TOPOSCORE) that was validated in an additional 254 NSCLC patients and in 216 genitourinary cancer patients. Finally, this TOPOSCORE was translated into a 21-bacterial probe set-based qPCR scoring that was validated in a prospective cohort of NSCLC patients as well as in colorectal and melanoma patients. This approach could represent a dynamic diagnosis tool for intestinal dysbiosis to guide personalized microbiota-centered interventions.

Blockade of LAG-3 and PD-1 leads to co-expression of cytotoxic and exhaustion gene modules in CD8+ T cells to promote antitumor immunity.

Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8+ T cell receptor signaling and altered CD8+ T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8+ T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8+ T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38+TIM3+CD8+ T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies.

ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of anti-tumor immunity.

Clinical trials have identified ARID1A mutations as enriched among patients who respond favorably to immune checkpoint blockade (ICB) in several solid tumor types independent of microsatellite instability. We show that ARID1A loss in murine models is sufficient to induce anti-tumor immune phenotypes observed in ARID1A mutant human cancers, including increased CD8+ T cell infiltration and cytolytic activity. ARID1A-deficient cancers upregulated an interferon (IFN) gene expression signature, the ARID1A-IFN signature, associated with increased R-loops and cytosolic single-stranded DNA (ssDNA). Overexpression of the R-loop resolving enzyme, RNASEH2B, or cytosolic DNase, TREX1, in ARID1A-deficient cells prevented cytosolic ssDNA accumulation and ARID1A-IFN gene upregulation. Further, the ARID1A-IFN signature and anti-tumor immunity were driven by STING-dependent type I IFN signaling, which was required for improved responsiveness of ARID1A mutant tumors to ICB treatment. These findings define a molecular mechanism underlying anti-tumor immunity in ARID1A mutant cancers.

Immune checkpoint therapy-current perspectives and future directions.

Immune checkpoint therapy (ICT) has dramatically altered clinical outcomes for cancer patients and conferred durable clinical benefits, including cure in a subset of patients. Varying response rates across tumor types and the need for predictive biomarkers to optimize patient selection to maximize efficacy and minimize toxicities prompted efforts to unravel immune and non-immune factors regulating the responses to ICT. This review highlights the biology of anti-tumor immunity underlying response and resistance to ICT, discusses efforts to address the current challenges with ICT, and outlines strategies to guide the development of subsequent clinical trials and combinatorial efforts with ICT.

Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

Immune checkpoint blockade provokes resident memory T cells to eliminate head and neck cancer.

Immune checkpoint blockade is effective in treating many human cancers. In this issue of Cell, Luoma et al. show that tissue-resident memory T cells in head and neck cancers rapidly respond to immune checkpoint blockade, and they identify specific CD8+ T cells in pretreatment blood that predict pathologic tumor regression.

The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes.

Blocking PD-1/PD-L1 signaling transforms cancer therapy and is assumed to unleash exhausted tumor-reactive CD8+ T cells in the tumor microenvironment (TME). However, recent studies have also indicated that the systemic tumor-reactive CD8+ T cells may respond to PD-1/PD-L1 immunotherapy. These discrepancies highlight the importance of further defining tumor-specific CD8+ T cell responders to PD-1/PD-L1 blockade. Here, using multiple preclinical tumor models, we revealed that a subset of tumor-specific CD8+ cells in the tumor draining lymph nodes (TdLNs) was not functionally exhausted but exhibited canonical memory characteristics. TdLN-derived tumor-specific memory (TTSM) cells established memory-associated epigenetic program early during tumorigenesis. More importantly, TdLN-TTSM cells exhibited superior anti-tumor therapeutic efficacy after adoptive transfer and were characterized as bona fide responders to PD-1/PD-L1 blockade. These findings highlight that TdLN-TTSM cells could be harnessed to potentiate anti-tumor immunotherapy.

A method for predicting drugs that can boost the efficacy of immune checkpoint blockade.

Combination therapy is a promising therapeutic strategy to enhance the efficacy of immune checkpoint blockade (ICB); however, predicting drugs for effective combination is challenging. Here we developed a general data-driven method called CM-Drug for screening compounds that can boost ICB treatment efficacy based on core and minor gene sets identified between responsive and nonresponsive samples in ICB therapy. The CM-Drug method was validated using melanoma and lung cancer mouse models, with combined therapeutic efficacy demonstrated in eight of nine predicted compounds. Among these compounds, taltirelin had the strongest synergistic effect. Mechanistic analysis and experimental verification demonstrated that taltirelin can stimulate CD8+ T cells and is mediated by the induction of thyroid-stimulating hormone. This study provides an effective and general method for predicting and evaluating drugs for combination therapy and identifies candidate compounds for future ICB combination therapy.

Circadian control of tumor immunosuppression affects efficacy of immune checkpoint blockade.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

Breaking NGF-TrkA immunosuppression in melanoma sensitizes immunotherapy for durable memory T cell protection.

Melanoma cells, deriving from neuroectodermal melanocytes, may exploit the nervous system's immune privilege for growth. Here we show that nerve growth factor (NGF) has both melanoma cell intrinsic and extrinsic immunosuppressive functions. Autocrine NGF engages tropomyosin receptor kinase A (TrkA) on melanoma cells to desensitize interferon γ signaling, leading to T and natural killer cell exclusion. In effector T cells that upregulate surface TrkA expression upon T cell receptor activation, paracrine NGF dampens T cell receptor signaling and effector function. Inhibiting NGF, either through genetic modification or with the tropomyosin receptor kinase inhibitor larotrectinib, renders melanomas susceptible to immune checkpoint blockade therapy and fosters long-term immunity by activating memory T cells with low affinity. These results identify the NGF-TrkA axis as an important suppressor of anti-tumor immunity and suggest larotrectinib might be repurposed for immune sensitization. Moreover, by enlisting low-affinity T cells, anti-NGF reduces acquired resistance to immune checkpoint blockade and prevents melanoma recurrence.

Deficiency of metabolic regulator PKM2 activates the pentose phosphate pathway and generates TCF1+ progenitor CD8+ T cells to improve immunotherapy.

TCF1high progenitor CD8+ T cells mediate the efficacy of immunotherapy; however, the mechanisms that govern their generation and maintenance are poorly understood. Here, we show that targeting glycolysis through deletion of pyruvate kinase muscle 2 (PKM2) results in elevated pentose phosphate pathway (PPP) activity, leading to enrichment of a TCF1high progenitor-exhausted-like phenotype and increased responsiveness to PD-1 blockade in vivo. PKM2KO CD8+ T cells showed reduced glycolytic flux, accumulation of glycolytic intermediates and PPP metabolites and increased PPP cycling as determined by 1,2-13C glucose carbon tracing. Small molecule agonism of the PPP without acute glycolytic impairment skewed CD8+ T cells toward a TCF1high population, generated a unique transcriptional landscape and adoptive transfer of agonist-treated CD8+ T cells enhanced tumor control in mice in combination with PD-1 blockade and promoted tumor killing in patient-derived tumor organoids. Our study demonstrates a new metabolic reprogramming that contributes to a progenitor-like T cell state promoting immunotherapy efficacy.

Understanding and treating the inflammatory adverse events of cancer immunotherapy.

During the past decade, immunotherapies have made a major impact on the treatment of diverse types of cancer. Inflammatory toxicities are not only a major concern for Food and Drug Administration (FDA)-approved checkpoint blockade and chimeric antigen receptor (CAR) T cell therapies, but also limit the development and use of combination therapies. Fundamentally, these adverse events highlight the intricate balance of pro- and anti-inflammatory pathways that regulate protective immune responses. Here, we discuss the cellular and molecular mechanisms of inflammatory adverse events, current approaches to treatment, as well as opportunities for the design of immunotherapies that limit such inflammatory toxicities while preserving anti-tumor efficacy.

Strength in numbers: predicting response to checkpoint inhibitors from large clinical datasets.

The advent of immune checkpoint blockers for cancer therapy has spawned great interest in identifying molecular features reflecting the complexity of tumor immunity, which can subsequently be leveraged as predictive biomarkers. In a thorough big-data approach analyzing the largest series of homogenized molecular and clinical datasets, Litchfield et al. identified a set of genomic biomarkers that identifies immunotherapy responders across cancer types.

FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells.

Repetitive elements (REs) compose ∼50% of the human genome and are normally transcriptionally silenced, although the mechanism has remained elusive. Through an RNAi screen, we identified FBXO44 as an essential repressor of REs in cancer cells. FBXO44 bound H3K9me3-modified nucleosomes at the replication fork and recruited SUV39H1, CRL4, and Mi-2/NuRD to transcriptionally silence REs post-DNA replication. FBXO44/SUV39H1 inhibition reactivated REs, leading to DNA replication stress and stimulation of MAVS/STING antiviral pathways and interferon (IFN) signaling in cancer cells to promote decreased tumorigenicity, increased immunogenicity, and enhanced immunotherapy response. FBXO44 expression inversely correlated with replication stress, antiviral pathways, IFN signaling, and cytotoxic T cell infiltration in human cancers, while a FBXO44-immune gene signature correlated with improved immunotherapy response in cancer patients. FBXO44/SUV39H1 were dispensable in normal cells. Collectively, FBXO44/SUV39H1 are crucial repressors of RE transcription, and their inhibition selectively induces DNA replication stress and viral mimicry in cancer cells.

Hallmarks of response, resistance, and toxicity to immune checkpoint blockade.

Unprecedented advances have been made in cancer treatment with the use of immune checkpoint blockade (ICB). However, responses are limited to a subset of patients, and immune-related adverse events (irAEs) can be problematic, requiring treatment discontinuation. Iterative insights into factors intrinsic and extrinsic to the host that impact ICB response and toxicity are critically needed. Our understanding of the impact of host-intrinsic factors (such as the host genome, epigenome, and immunity) has evolved substantially over the past decade, with greater insights on these factors and on tumor and immune co-evolution. Additionally, we are beginning to understand the impact of acute and cumulative exposures-both internal and external to the host (i.e., the exposome)-on host physiology and response to treatment. Together these represent the current day hallmarks of response, resistance, and toxicity to ICB. Opportunities built on these hallmarks are duly warranted.