Efficacy Of Immune Checkpoint Blockade Research Articles

Estrogen signaling suppresses tumor-associated tissue eosinophilia to promote breast tumor growth.

Estrogens regulate eosinophilia in asthma and other inflammatory diseases. Further, peripheral eosinophilia and tumor-associated tissue eosinophilia (TATE) predicts a better response to immune checkpoint blockade (ICB) in breast cancer. However, how and if estrogens affect eosinophil biology in tumors and how this influences ICB efficacy has not been determined. Here, we report that estrogens decrease the number of peripheral eosinophils and TATE, and this contributes to increased tumor growth in validated murine models of breast cancer and melanoma. Moreover, estrogen signaling in healthy female mice also suppressed peripheral eosinophil prevalence by decreasing the proliferation and survival of maturing eosinophils. Inhibiting estrogen receptor (ER) signaling decreased tumor growth in an eosinophil-dependent manner. Further, the efficacy of ICBs was increased when administered in combination with anti-estrogens. These findings highlight the importance of ER signaling as a regulator of eosinophil biology and TATE and highlight the potential near-term clinical application of ER modulators to increase ICB efficacy in multiple tumor types.

Immune-modulative nano-gel-nano system for patient-favorable cancer therapy

Current cancer immunotherapies exhibit low response rates attributed to suppressive tumor immune microenvironments (TIMEs). To address these unfavorable TIMEs, supplementation with tumor-associated antigens and stimulation of immune cells at target sites are indispensable for eliciting anti-tumoral immune responses. Previous research has explored the induction of immunotherapy through multiple injections and implants; however, these approaches lack consideration for patient convenience and the implementation of finely tunable immune response control systems to mitigate the side effects of over-inflammatory responses, such as cytokine storms. In this context, we describe a patient-centric nano-gel-nano system capable of sustained generation of tumor-associated antigens and release of adjuvants. This is achieved through the specific delivery of drugs to cancer cells and antigens/adjuvants to immune cells over the long term, maintaining proper concentrations within the tumor site with a single injection. This system demonstrates local immunity against tumors with a single injection, enhances the therapeutic efficacy of immune checkpoint blockades, and induces systemic and memory T cell responses, thus minimizing systemic side effects.

Precise In Situ Delivery of a Photo-enhanceable Inflammasome-Activating Nanovaccine Activates Anti-cancer Immunity.

A variety of state-of-the-art nanovaccines (NVs) combined with immunotherapies have recently been developed to treat malignant tumors, showing promising results. However, immunosuppression in the tumor microenvironment (TME) restrains cytotoxic T cells infiltration and limits the efficacy of immunotherapies in solid tumors. Therefore, tactics for enhancing antigen cross-presentation and reshaping the TME need to be explored to enhance the activity of NV. Here, we developed a photo-enhanceable inflammasome-activating NV (PIN) to achieve precise in situ delivery of a tumor antigen and a hydrophobic small molecule activating the NLRP3-inflammasome pathway. Near-infrared light irradiation promoted PIN accumulation in tumor sites through photo-triggered charge reversal of the nanocarrier. Systematic PIN administration facilitated intratumoral NLRP3 inflammasome activation and antigen cross-presentation in antigen-presenting cells upon light irradiation at tumor sites. Furthermore, PIN treatment triggered immune responses by promoting the production of proinflammatory cytokines and activated of anti-tumor immunity without significant systematic toxicity. Importantly, the PIN enhanced the efficacy of immune checkpoint blockade and supported the establishment of long-term immune memory in mouse models of melanoma and hepatocellular carcinoma. Collectively, this study reports a safe and efficient photoresponsive system for co-delivery of antigens and immune modulators into tumor tissues with promising therapeutic potential.

Abstract A031: Identification of therapeutic vulnerabilities in the subset of pancreatic ductal adenocarcinoma (PDAC) with homologous recombination deficiency

Abstract The genetic landscape of pancreatic ductal adenocarcinoma (PDAC) is characterized by mutations in the KRAS, TP53, CDKN2A and SMAD4 genes alongside multiple low-frequency mutations, which may offer opportunities for precision medicine. The success of poly-ADP-ribose polymerase (PARP) inhibitors in metastatic PDAC characterized by germline mutations in the Breast Cancer (BRCA) genes has led to the hypothesis that mutations in other BRCAness genes may sensitize PDAC to PARP inhibitors. To test this hypothesis, we used KPC mice as a backbone (Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+) to generate models of PDAC driven by the loss of Ataxia Telangiectasia mutated (Atm, KPCA) or Brca1 (KPCB). Loss of Atm or Brca1 resulted in homologous recombination deficiency and genomic instability in vivo. In vitro cultures of KPCA and KPCB PDAC cells had higher numbers of micronuclei and were more radiosensitive than KPC cells. Next, we performed bulk and single-cell RNA sequencing to identify therapeutic vulnerabilities associated with Atm or Brca1 loss. We found that loss of Atm upregulated ATR signaling, suggesting ATR as a therapeutic target in KPCA mice. In agreement, ATR inhibition improved the survival of KPCA mice, with evidence of tumor growth delay. However, though PARP inhibition significantly improved the survival of KPCB mice, with delayed tumor growth, it was ineffective in KPCA mice. In addition, transcriptomic analysis showed an enrichment of immune cells in KPCB PDAC compared to KPC and KPCA PDAC. Notably, we found greater levels of STING signaling, a more diverse and abundant cytokine profile and more CD8 T cells in KPCB PDAC, suggesting sensitivity to immune checkpoint blockade (ICB). However, ICB was ineffective in KPCB mice as a single agent or in combination with PARP inhibition. Based on our transcriptomic data, we hypothesized that boosting antigen priming using a CD40 agonist (CD40a) in combination with FLT3-L may improve ICB efficacy in our mouse models of BRCAness. Immunotherapy consisting of combined ICB, CD40a and FLT-3L had little efficacy in either KPCA or KPCB mice. However, ATR inhibition improved the efficacy of combined ICB, CD40a and FLT-3L in KPCA mice, as indicated by increased CD8 T cell infiltration in the tumor, delayed tumor growth and longer survival. In contrast, we found that PARP inhibition combined with ICB, CD40a and FLT-3L was ineffective in KPCB mice. In summary, we present evidence to suggest that PDAC characterized by BRCAness is not a homogeneous group with respect to sensitivity to PARP inhibition, composition of the tumor microenvironment and response to immunotherapy. We highlight ATM loss as a potential predictive biomarker of ATR inhibitor efficacy and immunotherapy in PDAC. Future directions include the evaluation of inflammation as a therapeutic vulnerability in murine PDAC with loss of Brca1. Ultimately, we aim to inform clinical trials to refine precision medicine of PDAC characterized by BRCAness. Citation Format: Mathias Tesson, Kay Kong, Peter Repsicak, Aldo Bader, Ya-Ching Hsieh, Craig Nourse, Kristina Kirschner, Crispin Miller, Ed Roberts, David Chang, Jen Morton. Identification of therapeutic vulnerabilities in the subset of pancreatic ductal adenocarcinoma (PDAC) with homologous recombination deficiency [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A031.

Anti-4-1BB×PDL1 Bispecific Antibody Reinvigorates Tumor-Specific Exhausted CD8+ T Cells and Enhances the Efficacy of Anti-PD1 Blockade.

To overcome the limited efficacy of immune checkpoint blockade, there is a need to find novel cancer immunotherapeutic strategies for the optimal treatment of cancer. The novel anti-4-1BB×PDL1 bispecific antibody-ABL503 (also known as TJ-L14B)-was designed to simultaneously target PDL1 and 4-1BB and demonstrated strong antitumor T-cell responses without considerable toxicity. In this study, we investigated the mechanisms by which the combination of ABL503 and anti-PD1 blockade affected the reinvigoration of exhausted tumor-infiltrating CD8+ T cells (CD8+ TIL) and antitumor efficacy. Single-cell suspensions of hepatocellular carcinoma and ovarian cancer tissues from treatment-naïve patients were used for immunophenotyping of CD8+ TILs and in vitro functional assays. Humanized hPD1/hPDL1/h4-1BB triple-knock-in mice were used to evaluate the effects of ABL503 and anti-PD1 blockade in vivo. We observed that ABL503 successfully restored the functions of 4-1BB+ exhausted CD8+ TILs, which were enriched for tumor-specific T cells but unresponsive to anti-PD1 blockade. Importantly, compared with anti-PD1 blockade alone, the combination of ABL503 and anti-PD1 blockade further enhanced the functional restoration of human CD8+ TILs in vitro. Consistently, the combination of ABL503 with anti-PD1 in vivo significantly alleviated tumor growth and induced enhanced infiltration and activation of CD8+ TILs. ABL503, a PDL1 and 4-1BB dual-targeting bispecific antibody, elicits pronounced additive tumor growth inhibition, with increased infiltration and functionality of exhausted CD8+ T cells, which in turn enhances the anticancer effects of anti-PD1 blockade. These promising findings suggest that ABL503 (TJ-L14B) in combination with PD1 inhibitors will likely further enhance therapeutic benefit in clinical trials. See related commentary by Molero-Glez et al., p. 3971.

Deciphering the impact of aggregated autophagy-related genes TUBA1B and HSP90AA1 on colorectal cancer evolution: a single-cell sequencing study of the tumor microenvironment

BackgroundColorectal cancer (CRC) is the third most prevalent cancer worldwide, with the tumor microenvironment (TME) playing a crucial role in its progression. Aggregated autophagy (AA) has been recognized as a factor that exacerbates CRC progression. This study aims to study the relationship between aggregated autophagy and CRC using single-cell sequencing techniques. Our goal is to explain the heterogeneity of the TME and to explore the potential for targeted personalized therapies.ObjectiveTo study the role of AA in CRC, we employed single-cell sequencing to discern distinct subpopulations within the TME. These subpopulations were characterized by their autophagy levels and further analyzed to identify specific biological processes and marker genes.ResultsOur study revealed significant correlations between immune factors and both clinical and biological characteristics of the tumor microenvironment (TME), particularly in cells expressing TUBA1B and HSP90AA1. These immune factors were associated with T cell depletion, a reduction in protective factors, diminished efficacy of immune checkpoint blockade (ICB), and enhanced migration of cancer-associated fibroblasts (CAFs), resulting in pronounced inflammation. In vitro experiments showd that silencing TUBA1B and HSP90AA1 using siRNA (Si-TUBA1B and Si-HSP90AA1) significantly reduced the expression of IL-6, IL-7, CXCL1, and CXCL2 and inhibition of tumor cell growth in Caco-2 and Colo-205 cell lines. This reduction led to a substantial alleviation of chronic inflammation and highlighted the heterogeneous nature of the TME.ConclusionThis study marks an initial foray into understanding how AA-associated processes may potentiate the TME and weaken immune function. Our findings provide insights into the complex dynamics of the TME and highlight potential targets for therapeutic intervention, suggesting a key role for AA in the advancement of colorectal cancer.

Mitochondrial rewiring with small-molecule drug-free nanoassemblies unleashes anticancer immunity

The immunosuppressive tumor microenvironment (TME) remains a major obstacle to tumor control and causes suboptimal responses to immune checkpoint blockade (ICB) therapy. Thus, developing feasible therapeutic strategies that trigger inflammatory responses in the TME could improve the ICB efficacy. Mitochondria play an essential role in inflammation regulation and tumor immunogenicity induction. Herein, we report the discovery and characterization of a class of small molecules that can recapitulate aqueous self-assembly behavior, specifically target cellular organelles (e.g., mitochondria), and invigorate tumor cell immunogenicity. Mechanistically, this nanoassembly platform dynamically rewires mitochondria, induces endoplasmic reticulum stress, and causes apoptosis/paraptosis-associated immunogenic cell death. After treatment, stressed and dying tumor cells can act as prophylactic or therapeutic cancer vaccines. In preclinical mouse models of cancers with intrinsic or acquired resistance to PD-1 blockade, the local administration of nanoassemblies inflames the immunologically silent TME and synergizes with ICB therapy, generating potent antitumor immunity. This chemically programmed small-molecule immune enhancer acts distinctly from regular cytotoxic therapeutics and offers a promising strategy for synchronous and dynamic tailoring of innate immunity to achieve traceless cancer therapy and overcome immunosuppression in cancers.

In situ enzymatic peptide-based nanomedicine with combined effects for enhanced tumor radio-immunotherapy

Despite the combination of radiotherapy and immunotherapy in clinical practice have shown promising synergistic anti-tumor effect, the immunosuppressive tumor microenvironment (TME) remains to be one of stumbling block impeding the therapeutic efficacy of radio-immunotherapy. In this study, in situ self-assembled peptide-based drug delivery systems with multiple effects were constructed for enhancing tumor radio-immunotherapy. The two peptide-drug conjugates could co-assembled into peptide-based nanomedicine on the cancer cell membrane under enzyme catalysis and continuously release small molecular active drugs in the cytoplasm, which combined action lead YAP phosphorylation and nuclear transfer failure. By simutaneously suppressing glycolysis and reactivating the dysfunctional Hippo pathway, the peptide-based nanomedicine also remitted the immunosuppressive TME by reducing lactate levels and the expression of immunosuppressive factors, respectively. As a result, the pro-tumorigenic M2 phenotype of tumor-associated macrophages (TAMs) was decreased and the anti-tumorigenic M1 phenotype of TAMs was increased. Such a dual intervention strategy from cellular metabolic level and gene transcription level not only inhibited tumor cell proliferation but also reversed the immunosuppressive TME, thereby augmenting the combined efficacy of radiotherapy and immune checkpoint blockade (ICB) therapy. The work is anticipated to contribute novel insights into anti-cancer drug targeted delivery and tumor multi-modal synergistic therapy.

Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint blockade

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity, research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast, cancer cell-intrinsic PD-1 functional regulation is not well understood. Here, we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-β triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling, increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer, and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors.

Probiotics functionalized with a gallium-polyphenol network modulate the intratumor microbiota and promote anti-tumor immune responses in pancreatic cancer

The intratumor microbiome imbalance in pancreatic cancer promotes a tolerogenic immune response and triggers immunotherapy resistance. Here we show that Lactobacillus rhamnosus GG probiotics, outfitted with a gallium-polyphenol network (LGG@Ga-poly), bolster immunotherapy in pancreatic cancer by modulating microbiota-immune interactions. Upon oral administration, LGG@Ga-poly targets pancreatic tumors specifically, and selectively eradicates tumor-promoting Proteobacteria and microbiota-derived lipopolysaccharides through a gallium-facilitated disruption of bacterial iron respiration. This elimination of intratumor microbiota impedes the activation of tumoral Toll-like receptors, thus reducing immunosuppressive PD-L1 and interleukin-1β expression by tumor cells, diminishing immunotolerant myeloid populations, and improving the infiltration of cytotoxic T lymphocytes in tumors. Moreover, LGG@Ga-poly hampers pancreatic tumor growth in both preventive and therapeutic contexts, and amplifies the antitumor efficacy of immune checkpoint blockade in preclinical cancer models in female mice. Overall, we offer evidence that thoughtfully designed biomaterials targeting intratumor microbiota can efficaciously augment immunotherapy for the challenging pancreatic cancer.

Microbiota-induced plastic T cells enhance immune control of antigen-sharing tumors.

Therapies that harness the immune system to target and eliminate tumor cells have revolutionized cancer care. Immune checkpoint blockade (ICB), which boosts the anti-tumor immune response by inhibiting negative regulators of T cell activation1-3, is remarkably successful in a subset of cancer patients, yet a significant proportion do not respond to treatment, emphasizing the need to understand factors influencing the therapeutic efficacy of ICB4-9. The gut microbiota, consisting of trillions of microorganisms residing in the gastrointestinal tract, has emerged as a critical determinant of immune function and response to cancer immunotherapy, with multiple studies demonstrating association of microbiota composition with clinical response10-16. However, a mechanistic understanding of how gut commensal bacteria influence the efficacy of ICB remains elusive. Here we utilized a gut commensal microorganism, segmented filamentous bacteria (SFB), which induces an antigen-specific Th17 cell effector program17, to investigate how colonization with it affects the efficacy of ICB in restraining distal growth of tumors sharing antigen with SFB. We find that anti-PD-1 treatment effectively inhibits the growth of implanted SFB antigen-expressing melanoma only if mice are colonized with SFB. Through T cell receptor clonal lineage tracing, fate mapping, and peptide-MHC tetramer staining, we identify tumor-associated SFB-specific Th1-like cells derived from the homeostatic Th17 cells induced by SFB colonization in the small intestine lamina propria. These gut-educated ex-Th17 cells produce high levels of the pro-inflammatory cytokines IFN-γ and TNF-α, and promote expansion and effector functions of CD8+ tumor-infiltrating cytotoxic lymphocytes, thereby controlling tumor growth. A better understanding of how distinct intestinal commensal microbes can promote T cell plasticity-dependent responses against antigen-sharing tumors may allow for the design of novel cancer immunotherapeutic strategies.

YAP1 Inhibition Induces Phenotype Switching of Cancer-Associated Fibroblasts to Tumor Suppressive in Prostate Cancer.

Prostate cancer (PCa) rarely responds to immune-checkpoint blockade (ICB) therapies. Cancer-associated fibroblasts (CAFs) are critical components of the immunologically "cold" tumor microenvironment and are considered a promising target to enhance the immunotherapy response. In this study, we aimed to reveal the mechanisms regulating CAF plasticity to identify potential strategies to switch CAFs from pro-tumorigenic to anti-tumor phenotypes and enhance ICB efficacy in PCa. Integration of four PCa single-cell RNA-sequencing datasets defined pro-tumorigenic and anti-tumor CAFs, and RNA-seq, flow cytometry, and a PCa organoid model demonstrated the functions of two CAF subtypes. Extracellular matrix-associated CAFs (ECM-CAF) promoted collagen deposition and cancer cell progression, and lymphocyte-associated CAFs (Lym-CAF) exhibited an anti-tumor phenotype and induced the infiltration and activation of CD8+ T cells. YAP1 activity regulated the ECM-CAF phenotype, and YAP1 silencing promoted switching to Lym-CAFs. NF-κB p65 was the core transcription factor in the Lym-CAF subset, and YAP1 inhibited nuclear translocation of p65. Selective depletion of YAP1 in ECM-CAFs in vivo promoted CD8+ T-cell infiltration and activation and enhanced the therapeutic effects of anti- PD-1 treatment in PCa. Overall, this study revealed a mechanism regulating CAF identity in PCa and highlighted a therapeutic strategy for altering the CAF subtype to suppress tumor growth and increase sensitivity to ICB.

Bi-specific antibody engagers for cancer immunotherapy.

Bispecific antibody engagers are fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains (VHH kappa ) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumor activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. In the MC38 mouse model of colorectal carcinoma, the anti-CTLA-4 VHH-VHH kappa conjugate eradicates tumors and reduces the number of intratumoral regulatory T cells. The anti-PD-L1 VHH-VHH kappa conjugate is less effective in the MC38 model, whilst still outperforming an antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHH kappa conjugate was strongly enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

Diet restriction enhances the effect of immune checkpoint block by inhibiting the intratumoralmTORC1/B7-H3 axis.

Immune checkpoint blockade therapy has demonstrated significant therapeutic efficacy in certain cancer types; however, the impact of dietary restriction remains scarcely reported in this context. This study aimed to investigate the influence of dietary restriction on anti-PDL-1 therapy and the interplay of immune cells within this context. Using an anti-PDL-1 regimen combined with dietary restrictions, tumor progression was assessed in LLC-bearing mice. Flow cytometry was employed to analyze immune cell infiltration and differentiation levels within the tumor microenvironment. The expression of mTORC1/B7-H3 in tumors subjected to dietary restriction was also examined. LLC tumors with elevated B7-H3 expression were validated in mice to determine its inhibitory effect on immune cell proliferation and differentiation. A CD3/B7-H3 chimeric antibody was developed for therapeutic intervention in B7-H3 overexpressing tumors, with subsequent T cell responses assessed through flow cytometry. Dietary restriction potentiated the effect of anti-PDL1 therapy by suppressing the intratumorally mTORC1/B7-H3 axis. In vivo experiments demonstrated that elevated B7-H3 expression in tumors reduced infiltration and activation of CD8 + T cells within the tumor, while it did not affect tumor-infiltrating Tregs. In vitro studies revealed that high B7-H3 expression influenced the proliferation and activation of CD8 + T cells within a Coculture system. The constructed CD3/B7-H3 chimeric antibody prominently activated TCR within B7-H3 overexpressing tumors and impeded tumor progression. The findings suggest that dietary restriction enhances the efficacy of immune checkpoint blockade by modulating the intratumoral mTORC1/B7-H3 axis.

Dual-Responsive Epigenetic Inhibitor Nanoprodrug Combined with Oncolytic Virus Synergistically Boost Cancer Immunotherapy by Igniting Gasdermin E-Mediated Pyroptosis.

Cancer immunotherapy has emerged as a promising approach for the treatment of various cancers. However, the immunosuppressive tumor microenvironment (TME) limits the efficacy of current immunotherapies. In this study, we designed a dual-responsive DNA methyltransferase inhibitor nanoprodrug ACNPs for combination therapy with oncolytic herpes simplex virus (oHSV). We found that the epigenetic inhibitor 5-Azacytidine (5-Aza) upregulated gasdermin E (GSDME) expression at the gene level, whereas the oHSV decreased the ubiquitination and degradation of GSDME to elevate its levels. Based on these observations, we further discovered that ACNPs and oHSV synergistically enhanced GSDME-mediated pyroptosis. Additionally, the combination therapy of ACNPs and oHSV effectively inhibited tumor growth, remodeled the immunosuppressive TME, and improved the efficacy of immune checkpoint blockade (ICB) therapy. These results demonstrate the potential to overcome immunosuppression through synergistic combinations, offering a promising approach for cancer immunotherapy.

Antibiotic use attenuates response to immune checkpoint blockade in urothelial carcinoma via inhibiting CD74-MIF/COPA: revealing cross-talk between anti-bacterial immunity and anti-tumor immunity through a tumor marker prognostic study.

Immune checkpoint blockade (ICB) has emerged as a promising therapy for both resectable urothelial carcinoma (UC) patients preparing for radical surgery and unresectable UC patients, whereas the objective response rate of ICB remains unsatisfactory due to various factors. Antibiotic (ATB) use can influence intra-tumoral bacteria, which may further reduce ICB efficacy. The study aims to evaluate the effects of ATB use on prognosis and response in UC patients undergoing ICB, and explore potential molecular mechanisms of ATBs and intra-tumoral bacteria impacting UC immune microenvironment. Pooled analyses, synthesizing evidence from 12 studies and 3496 UC patients with ICB treatment, was conducted via a meta-analysis. In addition, single-cell RNA and single-cell microbiome data were analyzed based on eight UC samples and 63185 single cells. Bulk RNA sequencing and clinical data from a single-arm, multi-center, atezolizumab-treated, phase 2 trial, IMvigor210, were used for validation. The study is registered at PROSPERO (XXX) and at Research Registry (XXX). ATB use exhibited worse overall survival (HR=1.46, 95%CI=[1.20, 1.77], P<0.001, heterogeneity I²=51%) and lower objective response (OR=0.43, 95%CI=[0.27, 0.68], P<0.001, heterogeneity I²=0%) in UC patients receiving ICB. Single-cell transcriptome and single-cell microbiome analyses identified the presence of intra-tumoral bacteria was obviously related to elevated anti-bacterial immune functions; and anti-bacterial immunity was positively correlated to anti-tumor immunity in UC immune microenvironment. Intra-tumoral bacteria could up-regulate CD74-MIF/COPA signaling of immune cells and activation of CD74-MIF/COPA mediated the promotion of T cell anti-tumor function induced by anti-bacterial immune cells. UC patients with higher CD74-MIF/COPA signaling carried better overall survival (HR=1.60, 95%CI=[1.19, 2.15], P=0.002) in IMvigor210 immunotherapy cohort. ATB use reduces overall survival and objective response to ICB in UC patients. Anti-bacterial immune cell functions induced by intra-cellular bacteria in UC microenvironment might up-regulate the function of anti-tumor T immune cells via activating CD74-MIF/COPA, whereas ATB could inhibit the above process through killing intra-cellular bacteria and result in poorer clinical benefit of ICB. The use of ATB should be considered carefully during neoadjuvant immunotherapy period for resectable UC patients preparing for radical surgery and during immunotherapy period for unresectable UC patients.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Tumor microenvironment RNA test to predict immunotherapy outcomes in advanced gastric cancer: The TIMES001 trial.

Clinical trials support the efficacy of immune checkpoint blockades (ICBs) plus chemotherapy in a subset of patients with metastatic gastric cancer (mGC). To identify the determinants of response, we developed a TMEscore model to assess tumor microenvironment (TME), which was previously proven to be a biomarker for ICBs. A reference database of TMEscore assays was established using PCR assay kits containing 30 TME genes. This multi-center prospective clinical trial (NCT#04850716) included patients with mGC who were administered ICB combined with chemotherapy as a first-line regimen. Eighty-six tumor samples extracted from five medical centers before treatment were used to estimate the TMEscore, PD-L1 (CPS), and mismatch repair deficiency. The objective response rate (ORR) and median PFS of the cohort were 31.4% and six months. Enhanced ORR was observed in TMEscore-high mGC patients (ORR= 59%). The survival analysis demonstrated that high TMEscore was significantly associated with a more favorable PFS and OS. Moreover, TMEscore was found to be a predictive biomarker that surpassed MSI and CPS (AUC= 0.873, 0.511, and 0.524, respectively). By integrating the TMEscore and clinical variables, the fused model further enhances the predictive efficiency and translational application in a clinical setting. This prospective clinical study indicates that the TMEscore assay is a robust biomarker for screening patients with mGC who may derive survival benefits from ICB plus chemotherapy. Guangdong Basic and Applied Basic Research Foundation (2023A1515011214), Science and Technology Program of Guangzhou (202206080011), and Guangzhou Science and Technology Project (2023A03J0722 and 2023A04J2357).

PIKfyve controls dendritic cell function and tumor immunity.

The modern armamentarium for cancer treatment includes immunotherapy and targeted therapy, such as protein kinase inhibitors. However, the mechanisms that allow cancer-targeting drugs to effectively mobilize dendritic cells (DCs) and affect immunotherapy are poorly understood. Here, we report that among shared gene targets of clinically relevant protein kinase inhibitors, high PIKFYVE expression was least predictive of complete response in patients who received immune checkpoint blockade (ICB). In immune cells, high PIKFYVE expression in DCs was associated with worse response to ICB. Genetic and pharmacological studies demonstrated that PIKfyve ablation enhanced DC function via selectively altering the alternate/non-canonical NF-κB pathway. Both loss of Pikfyve in DCs and treatment with apilimod, a potent and specific PIKfyve inhibitor, restrained tumor growth, enhanced DC-dependent T cell immunity, and potentiated ICB efficacy in tumor-bearing mouse models. Furthermore, the combination of a vaccine adjuvant and apilimod reduced tumor progression in vivo. Thus, PIKfyve negatively controls DCs, and PIKfyve inhibition has promise for cancer immunotherapy and vaccine treatment strategies.