Checkpoint Blockade Treatment Research Articles

Identification of Crucial Gene Modules Related to the Efficiency of Anti-PD-1/PD-L1 Therapy and Comprehensive Analyses of a Novel Signature Based on These Modules.

Biomarker development for clinical checkpoint inhibition is still in its early stages. It is critical to determine the cause of the lack of a long-term response in patients after immune checkpoint blockade (ICB) treatment and to develop composite biomarkers or signatures to improve personalized approaches. Three modules that were significantly correlated with the immunotherapeutic response were identified. Stimulatory pathways of cellular immunity, extracellular matrix formation-related pathways, and ATP metabolism-related pathways were enriched. Two distinct transcriptional subtypes were determined. Tumor microenvironment (TME) characteristics were highly correlated with “hot” and “cold” tumors. The ICB score was significantly correlated with clinical characteristics including age, Breslow depth, Clerk level, AJCC stage, and T stage. Meanwhile, a low ICB score is characterized by increased activation of immunity, a higher level of immune infiltration, and immune molecule expression. The ICB score showed a robust ability to predict melanoma prognosis in the discovery, internal validation, and external validation cohorts. In addition, a low ICB score was linked to a higher CR/PR rate in the immunotherapeutic cohort. The ICB score could reflect the pre-existing immune features and the expression pattern of “Cold” versus “Hot” tumors in melanoma patients. Thus, it has the potential to serve as a reliable predictor of melanoma prognosis and response to ICB therapy.

Serine Protease Inhibitor Kazal Type 1, A Potential Biomarker for the Early Detection, Targeting, and Prediction of Response to Immune Checkpoint Blockade Therapies in Hepatocellular Carcinoma.

BackgroundWe aimed to characterize serine protease inhibitor Kazal type 1 (SPINK1) as a gene signature for the early diagnosis, molecular targeting, and prediction of immune checkpoint blockade (ICB) treatment response of hepatocellular carcinoma (HCC).MethodsThe transcriptomics, proteomics, and phenotypic analyses were performed separately or in combination.ResultsWe obtained the following findings on SPINK1. Firstly, in the transcriptomic training dataset, which included 279 stage I and II tumor samples (out of 1,884 stage I–IV HCC specimens) and 259 normal samples, significantly higher area under curve (AUC) values and increased integrated discrimination improvement (IDI) and net reclassification improvement (NRI) were demonstrated for HCC discrimination in SPINK1-associated models compared with those of alpha-fetoprotein (AFP). The calibration of both SPINK1-related curves fitted significantly better than that of AFP. In the two independent transcriptomic validation datasets, which included 201, 103 stage I-II tumor and 192, 169 paired non-tumor specimens, respectively, the obtained results were consistent with the above-described findings. In the proteomic training dataset, which included 98 stage I and II tumor and 165 normal tissue samples, the analyses also revealed better AUCs and increased IDI and NRI in the aforementioned SPINK1-associated settings. A moderate calibration was shown for both SPINK1-associated models relative to the poor results of AFP. Secondly, in the in vitro and/or in vivo murine models, the wet-lab experiments demonstrated that SPINK1 promoted the proliferation, clonal formation, migration, chemoresistance, anti-apoptosis, tumorigenesis, and metastasis of HCC cells, while the anti-SPINK1 antibody inhibited the growth of the cells, suggesting that SPINK1 has “tumor marker” and “targetable” characteristics in the management of HCC. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that SPINK1 was engaged in immunity-related pathways, including T-cell activation. Thirdly, in the transcriptomic analyses of the 368 HCC specimens from The Cancer Genome Atlas (TCGA) cohort, the high abundance of SPINK1 was positively correlated with the high levels of activated tumor-infiltrating CD4+ and CD8+ T lymphocytes and dendritic and natural killer cells, while there were also positive correlations between SPINK1 and immune checkpoints, including PD-1, LAG-3, TIM-3, TIGIT, HAVCR2, and CTLA-4. The ESTIMATE algorithm calculated positive correlations between SPINK1 and the immune and ESTIMATE scores, suggesting a close correlation between SPINK1 and the immunogenic microenvironment within HCC tissues, which may possibly help in predicting the response of patients to ICB therapy.Conclusions SPINK1 could be a potential biomarker for the early detection, targeted therapy, and prediction of ICB treatment response in the management of HCC.

TTYH3, a potential prognosis biomarker associated with immune infiltration and immunotherapy response in lung cancer

PurposeThe recognition of new diagnostic and prognostic biological markers for lung cancer is an essential and eager study. It’s shown that ion channels play important roles in regulating various cellular processes and have been suggested to be associated with patient survival. However, tweety family member 3 (TTYH3), as a maxi-Cl- channel, its role in lung cancer remains elusive. MethodsThe expression, diagnostic and prognostic efficacy of TTYH3 were analyzed by public databases and clinical samples. Cell functional experiments were used to explore the effects of TTYH3 on cell viability. GO and KEGG enrichment analysis revealed underlying pathways that TTYH3 and its co-expressed genes were enriched in. TIMER, TIDE and R language analyses were used to detect the correlation between TTYH3 and immune infiltration cell and immunotherapy response. ResultsTTYH3 was up-regulated in lung cancer tissues compared to normal tissues and possessed a prominent diagnostic and prognostic value. TTYH3 knockdown significantly inhibited the proliferation of lung cancer cells. Enrichment analyses showed that TTYH3 and its co-expressed genes were mainly involved in immune related signaling pathways. Further investigation clarified that TTYH3 had a positive correlation with the infiltration of TAMs, Treg infiltration as well as T cell exhaustion and high TTYH3 expression indicated worse immunotherapy response and shorter survival after immune checkpoint blockade treatment. ConclusionThis study not only revealed the diagnostic and prognostic value of TTYH3 but also provided TTYH3-based estimation of immunotherapy response for lung cancer patients, which might provide new strategies like anti-TTYH3 combined with immune therapy for the treatment of lung cancer.

CGAS-STING signaling encourages immune cell overcoming of fibroblast barricades in pancreatic cancer

Immune checkpoint blockade (ICB) treatment improves the prognosis of several types of solid tumors, however, responsiveness to ICB therapy remains low in pancreatic ductal adenocarcinoma (PDACs), which has a rich tumor microenvironment (TME). The TME is composed of various stromal cells, including cancer-associated fibroblasts (CAFs), which contribute to the establishment of an immunosuppressive microenvironment. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is an innate immune pathway that results in the upregulation of immune cell recruiting-cytokines and anti-tumor efficacy. In this study, we aimed to investigate the impact of cGAS-STING expression and the presence of CAFs upon immune cell infiltration in PDACs. cGAS and STING co-expressing PDAC cases showed favorable survival, with many cytotoxic CD8 + T cell infiltrations from the stromal component adjacent to the cancer cells toward cancer cells, but not in cGAS-STING signaling defected PDAC cases. The signatures of tumor-restrain CAFs were expressed in tumors with cGAS-STING signaling. Finally, transwell co-culture experiments demonstrated that immune cell infiltration was impeded by the presence of CAFs, but not by activation of cGAS-STING signaling. In conclusion, pro-infiltration signals, such as cGAS-STING, and characterization of CAFs are crucial in defeating CAF barricades and encouraging immune cell infiltration in PDACs.

Melanoma-Specific Clinical Outcomes of Inpatient Immune Checkpoint Blockade Treatment.

BackgroundLittle is known about patient outcomes with advanced melanoma following inpatient initiation or continuation of immune checkpoint blockade (ICB).Methods and ResultsWe conducted a single institution retrospective case series of advanced melanoma patients who initiated ICB as an inpatient (initial inpatient cohort, n = 9), or continued ICB as an inpatient after previously starting as an outpatient (outpatient then inpatient cohort, n = 5). One patient had a partial response to ICB initiated as an inpatient, but ultimately died of melanoma after 13.5 months. Median overall survival for initial inpatient cohort was 1.0 month (95% CI: 0.2-11.2), and 1.4 months (95% CI: 0.4-58.0) for the outpatient then inpatient cohort. Three patients were alive >6 months after inpatient ICB administration.ConclusionDespite overall poor outcomes, some patients may benefit from inpatient ICB. This study provides additional information for clinicians to appropriately counsel patients on expectations following inpatient ICB.

Abstract 4173: Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR

Abstract Cancer immunotherapy, especially those targeting the immune checkpoint PD-1 and PD-L1 has emerged as a revolutionizing treatment modality. Immune checkpoint inhibitors (ICIs)-mediated anti-tumor response depends on T cell capability of recognizing and killing tumor cells. ‘Hot’ tumors, which are characterized by the T cell infiltration, show a better response rate to immune checkpoint blockade (ICB) treatment. In contrast, ‘Cold’ tumors have a particularly low objective response rate (ORR) to ICB. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to turn immunologically cold tumors into hot tumors. The RNA-binding protein HuR is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs. In this study, we show the direct binding of HuR protein to PD-L1 mRNA in multiple types of cells by Ribonucleoprotein immunoprecipitation (RNP-IP) and further validated by RNA pulldown assay. We also show that PD-L1 mRNA decay is greatly expedited in HuR-knockout cells, which underlines the stabilization function of HuR on PD-L1 transcripts. Immunoblotting results demonstrate that PD-L1 protein expression level is decreased in HuR-knockout cells while up-regulated in HuR-proficient cells in comparison with control. These data together clarify HuR is engaged in PD-L1 post-transcription regulation. Based on this mechanism, we develop a combination strategy using HuR inhibitors and anti-PD1 antibodies to overcome the limitation by regulating the tumor immune microenvironment. KH-200 is a compound that disrupts the transportation of HuR from nuclear to cytoplasm. To test the combination strategy in vivo, we established the EMT6 syngeneic breast cancer model. After 4-week treatment, the combination group shows great effects in inhibiting tumor growth and prolonging survival. A similar combination effect was further observed in Lewis lung syngeneic lung cancer model. In conclusion, we preliminarily explored the role of HuR in tumor immune evasion. The method using HuR inhibitors as a combination in immunotherapy shows effectiveness in immunologically cold tumor treatment and may have reference significance for clinical cancer therapy. Citation Format: Qi Zhang, Yang Zhe, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Improve anti-PD-1 immunotherapy response in immunologically cold tumors by harnessing RNA-binding protein HuR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4173.

Abstract 662: Late CTLA-4 Ig treatment improves antitumor efficacy of immunotherapy

Abstract The promising approach of combining immune checkpoint therapies such as anti-CTLA-4 and anti-PD-1 increases antitumor response and overall survival rate relative to single treatments. However, it also increases the frequency and severity of immune-related adverse events (irAEs), such as cardiotoxicity. Our previous results showed that CTLA-4 Ig (abatacept), an inhibitor of T cell costimulation through CD28, can reverse those irAEs in patients with cancer, however, its effect on the antitumor response remains unclear. In the B16F10 melanoma model, we injected mice with CTLA-4 Ig antibody when: 1) mice were first treated with anti-CTLA-4, anti-PD-1, or combination (early time point) and 2) the immunotherapy was finished (late time point). We demonstrated that CTLA-4 Ig at the early time point compromised the antitumor efficacy of the immunotherapy. In contrast, the antitumor efficacy of the immunotherapy was improved if mice were treated with CTLA-4 Ig at the late time point. We also found that the antitumor response induced by CTLA-4 Ig (late) was dependent on CD80/86. Interestingly, the frequency of ICOS+ Foxp3+ Tregs was significantly reduced by CTLA-4 Ig at the late time point. Thus, we treated B16F10 tumors in Foxp3DTR mice with anti-CTLA-4 and then with CTLA-4 Ig (late). The antitumor tumor efficacy was similar between mice treated with or without Diphtheria toxin (DT) at the late time point. This suggests that the improved antitumor efficacy of CTLA-4 Ig at the late time point was Treg dependent. Collectively, we show that CTLA-4 Ig treatment has a differential role throughout the time course of immunotherapy treatments, and this work suggests a potential combination treatment strategy with checkpoint blockade and late CTLA-4 Ig treatment. Citation Format: Stephen Mok, James P. Allison. Late CTLA-4 Ig treatment improves antitumor efficacy of immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 662.

Abstract 1302: Priming tumors using pH sensitizers potentiates the anti-tumor T cell response induced by immune checkpoint blockade therapy

Abstract While immune checkpoint blockade (ICB) treatments such as nivolumab and ipilimumab have spelled hope for many cancer patients, not all patients benefit from this form of therapy. One mechanism by which tumors suppress the anti-tumor T cell activity that results from ICB is through decreasing extracellular tumor pH (pHe), a function inherent to tumor cells due to the Warburg effect. We thus hypothesize that increasing pHe prior to ICB therapy potentiates the T cell-mediated anti-tumor effect of ICB treatment. To undergo this study, we first screened a panel of six inhibitors termed pH sensitizers, and found that esomeprazole significantly reduced the proton efflux rate (PER) of 4T1, but not B16-F10 cells. Furthermore, esomeprazole did not significantly reduce cell viability of either 4T1 or B16-F10 cells, and did not significantly inhibit T cell activity. Moving forward, our next objectives are to investigate the effects of esomeprazole and the combination of esomeprazole and ICB on tumor-bearing mice. Assessment of the tumor growth in response to esomeprazole and ICB and ex vivo interrogation of the immune cell infiltrate into the tumors will elucidate the efficacy of the treatment combination in terms of tumor control and anti-tumor immunity. Modification of pHe by esomeprazole will be examined by acidoCEST MRI, which will also provide insights as to the ideal pHe parameters for optimal ICB therapy in patients. Citation Format: Renee Chin, Jorge de la Cerda, William Schuler, Mark D. Pagel. Priming tumors using pH sensitizers potentiates the anti-tumor T cell response induced by immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1302.

Abstract 5096: Characteristics of tumor immune microenvironment in patients carrying comutations of DNA damage response pathways in esophageal cancer

Abstract Background: Immunotherapy exhibits promising clinical efficacy in treating unresectable esophageal cancer, and several studies have demonstrated that both comutations in DNA damage response (DDR) pathways and tumor immune microenvironment(TIME) could serve as potential biomarkers for immune checkpoint blockade (ICB) treatment. However, the associtation between DDR comutations and TIME remains unclear. Methods: Between April 2020 and September 2020, NGS and TIME analysis of tumor tissue from 22 patients with esophageal cancer were performed by 3D Medicines, Inc., a College of American Pathologists (CAP)-accredited and Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. We retrospectively investigated the association between genomic alterations and immune microenvironment features. Among eight DDR pathways, comutations of check point factors(CPF) plus base excision repair(BER), homologous recombination repair(HRR), mismatch repair(MMR) or Fanconi anemia(FA) were defined as "DDR co-mut+", while mutation of CPF or comutations of CPF plus nucleotide excision repair (NER) were defined as “DDR co-mut-”. In addition, the relationship between comutations of DDR pathways and tumor mutational burden (TMB) was also analyzed. Results: 22 patients with esophageal cancer were included for analysis. 16 of 22 patients (72.7%) were male and the median age was 62 years old(range, 52 to 73 years). Based on the definition of DDR co-mut+ and DDR co-mut-, 13 patients were devided into DDR co-mut+ group, and 9 patients belonged to DDR co-mut- group. Gene alterations and TIME analysis revealed that patients with DDR co-mut+ showed a higher abundance of CD56dim natural killer cells in both tumor(p=0.0161) and stroma(p=0.028) compared with DDR co-mut- patients. No differences were observed in the densities of CD8+ T cells, M1-TAMs, and M2-TAMs between DDR co-mut+ and DDR co-mut- patients. TMB analysis displayed slightly higher in DDR co-mut+ group with no significant difference. Conclusions: Patients with DDR co-mut+ showed better infiltration of CD56dim NK cells in tumor and stroma in esophageal cancer. Combination of DDR comutations with TIME features may be a more promising biomarker to guide immunotherapy. Keywords: esophageal cancer; DNA damage response pathway; tumor immune microenvironment; tumor mutational burden Citation Format: Hao Chen, Xihua Xia, Junling Zhang, Yanan Chen, Mengli Huang. Characteristics of tumor immune microenvironment in patients carrying comutations of DNA damage response pathways in esophageal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5096.

Abstract 2482: Evaluating immune checkpoint blockade treatment efficacy via [89Zr]-CD4 and [89Zr]-CD8 PET imaging in breast cancer mouse models

Abstract Objective: Immune checkpoint blockades have shown a great promise in cancer therapy. However, as the overall response rate varies, there is a profound unmet need to monitor the treatment efficacy and predict therapeutic responders. Methods: In vivo blocking and biodistribution studies were performed to validate the specificity of [89Zr]-mouse-CD4 and [89Zr]-mouse-CD8. 4T1 and MMTV-HER2 mouse models of breast cancer (N=80 per model) were imaged on Days 0, 2, and 6 during treatment (saline, anti-PD1, anti-CTLA4, or combinational therapies) and evaluated for long-term changes in tumor response. Therapeutic responders were determined through the thresholding of tumor mass at the end of study. Intratumoral and splenic CD4+ and CD8+ cells were characterized in vivo via [89Zr]-mouse-CD4 and [89Zr]-mouse-CD8 positron emission tomography (PET) imaging during immunotherapy treatment. An additional of mice (N=16) were euthanized on day 7 post treatment for biological validation studies. Autoradiography and immunofluorescence staining (CD8 and CD4) were performed to validate the biological accuracy of [89Zr]-mouse-CD4 and [89Zr]-mouse-CD8 PET imaging. Results: Splenic CD4 and CD8 signal showed three-fold and two-fold decreased in the blocking group compared to non-blocking group, respectively (p<0.01). In 4T1 model, the changes of intratumoral CD4 SUVmean (the mean value of standard uptake value) from day 0 to day 6 significantly increased in responder group with anti-PD1 treatment (p<0.01). The baseline intratumoral CD8 SUVmean showed no significant difference between responders and non-responders; inversely, the changes from day 0 to day 6 increased in MMTV-HER2 (p=0.02) and 4T1 model (p=0.07) with combinational therapies. Spleen masses of responders decreased with anti-CTLA4 treatment (p<0.05) and for all treatments (p=0.06), in 4T1 model only. Terminal tumor mass and spleen mass were positively correlated in 4T1 model (r=0.26, p=0.02). In 4T1 model, splenic CD4 SUVmean on day 6 was negatively correlated with terminal spleen mass (r=-0.36, p=0.02) and tumor mass (r=0.33, p=0.04). PET, autoradiography, and immunofluorescence staining showed strong correlations when comparing in vivo imaging to ex vivo validations (r>0.75, p<0.01). Conclusion: [89Zr]-CD4 and [89Zr]-CD8 PET imaging can accurately evaluate CD4+ and CD8+ cell populations in vivo. Biomarkers for predicting immunotherapy response varies with different targeted drugs and tumor models. Characterizing immune cell population during immune checkpoint blockade treatment via advanced PET imaging can provide understanding of immunological alterations, monitor immunotherapy response and may potentially help in clinical decision making. Citation Format: Yun Lu, Hailey Houson, Alessandro Mascioni, Fang Jia, Patrick N. Song, Tiara Napier, Amer M. Mansur, Carlos A. Gallegos, Benjamin M. Larimer, Suzanne E. Lapi, Anna G. Sorace. Evaluating immune checkpoint blockade treatment efficacy via [89Zr]-CD4 and [89Zr]-CD8 PET imaging in breast cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2482.

Abstract 2731: Longitudinally modeling anti-neoantigen immunity under paradigms of immunodominance

Abstract Neoantigen clonal architecture is known to influence the efficacy of immunotherapy. The immunodominance of clonal neoantigens over a canvas of heterogeneous subclonal neoantigens is particularly challenging to recapitulate in experimental models. We developed a computational model of neoantigen evolution that longitudinally tracks the “presentome” - that is, a cell’s aggregate neoantigen repertoire and HLA status - of simulated non-small cell lung cancer (NSCLC) prior to and during treatment with checkpoint blockade. Using neoantigen immunogenicity parameters from published reports, we first recapitulated the presentome of early-stage NSCLC. Upon simulating checkpoint blockade treatment of advanced NSCLC, we found salient correlates and consequences of response, including a profound clonal collapse among completely responding tumors. Our work could be adapted to longitudinally model the neoantigen architecture of other cancers and modalities of immunotherapy, ultimately informing cognizant strategies for future therapeutic development. Citation Format: Tim Qi, Benjamin Vincent, Yanguang Cao. Longitudinally modeling anti-neoantigen immunity under paradigms of immunodominance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2731.

Identification and Verification of Immune Subtype-Related lncRNAs in Clear Cell Renal Cell Carcinoma.

BackgroundAccording to clinical study results, immune checkpoint blockade (ICB) treatment enhances the survival outcome of patients with clear cell renal cell carcinoma (ccRCC). Previous research has divided ccRCC patients into immune subtypes with distinct ICB response rates. However, the study on the association between lncRNAs and ccRCC immune subtypes is lacking.MethodsDifferentially expressed lncRNAs/mRNAs between two major immune subgroups were calculated. A weighted gene co-expression network analysis (WGCNA) was conducted to establish the lncRNA-mRNA co-expression network and select the key lncRNAs. Then, prognostic lncRNAs were selected from the network by the bioinformatics method. Next, the risk-score was estimated by lncRNA expression and their coefficients. Finally, a nomogram based on lncRNAs and clinical parameters was created to predict the prognosis of ccRCC.ResultsLncRNAs and mRNAs associated with ccRCC immune subtypes were identified. The lncRNAs and mRNAs from a gene module closely linked to the immune subtype were used to construct a network. The KEGG pathways enriched in the network were related to immune system activation processes. These 8 lncRNAs (AL365361.1, LINC01934, AC090152.1, PCED1B-AS1, LINC00426, AC007728.2, AC243829.4, and LINC00158) were found to be positively correlated with immune cells of the tumor microenvironment. The C-index of the nomogram was 0.777, and the calibration curve data suggests that the nomogram has a high degree of discriminating capacity.ConclusionIn summary, we discovered core lncRNAs linked with immune subtypes and created corresponding lncRNA–mRNA networks. These lncRNAs are anticipated to have predictive significance for ccRCC and may provide insight into novel biomarkers for the disease.

Glycolytic and fatty acid oxidation genes affect the treatment and prognosis of liver cancer.

BACKGROUNDMetabolic reprogramming is a feature of tumour cells and is essential to support their rapid proliferation. The glycolytic activity of liver cancer cells is significantly higher than that of normal liver cells, and the rapidly proliferating tumour cells are powered by aerobic glycolysis. Lipid metabolism reprogramming enables tumour cells to meet their needs for highly proliferative growth and is an important driving force for the development of hepatocellular carcinoma (HCC).AIMTo explore the influence of different metabolic subtypes of HCC and analyse their significance in guiding prognosis and treatment based on the molecular mechanism of glycolysis and fatty acid oxidation (FAO).METHODSBy downloading related data from public databases including the Cancer Genome Atlas (TCGA), the Molecular Signatures Database, and International Cancer Genome Consortium, we utilised unsupervised consensus clustering to divide TCGA Liver Hepatocellular Carcinoma samples into four metabolic subgroups and compared single nucleotide polymorphism, copy number variation, tumour microenvironment, and Genomics of Drug Sensitivity in Cancer and Tumour Immune Dysfunction and Exclusion between different metabolites. The differences and causes of survival and the clinical characteristics between them were analysed, and a prognostic model was established based on glycolysis and FAO genes. Combined with the clinical features, a Norman diagram was created to compare the pros and cons of each model.RESULTSIn the four metabolic subgroups, with the increase in glycolytic expression, the median survival of patients showed the worst results, while FAO showed the best. When comparing the follow-up analysis of each group, we considered that the differences between them might be related to reactive oxygen species, somatic copy number variation of key genes, and immune microenvironment. It was also found that the FAO group and the low-risk group had better efficacy and response to immune checkpoint blockade treatment and anti-tumour drugs.CONCLUSIONThere are obvious differences in genes, chromosomes, and clinical characteristics between metabolic subgroups. The establishment of a prognostic model could predict patient prognosis and guide clinical treatment.

Rejuvenating hepatic tumor microenvironment immunity with a phosphatidylserine-targeting small molecule drug conjugate

We report the design, synthesis and evaluation of a class of phosphatidylserine-targeting zinc (II) dipicolylamine drug conjugates and show that conjugate 16b elicits immune cell infiltration and remodels the “cold” hepatic tumor microenvironment to the inflamed “hot” tumor. Structure-property relationship study via linker modifications and subsequent pharmacokinetics profiling were carried out to improve the solubility and stability of the conjugates in vivo. In a spontaneous hepatocellular carcinoma mouse model, we showed that conjugate 16b exhibited better antitumor efficacy than sorafenib. In particular, significant increase of CD8+ T cell infiltration and granzyme B level was observed, providing insights in sensitizing tumors from intrinsic immune suppressive microenvironment. Evaluation of tumor inflammation-related mRNA expression profile revealed that conjugate 16b, through inductions of key gene expressions including STAT1, CXCL9, CCL5, and PD-L1, rejuvenated tumor microenvironment with enhancement in T cell-, macrophage-, NK cell-, chemokines and cytokines’- functions. Our study establishes that an apoptosis-targeting theranostic enables enrichment of multifaceted immune cells into the tumor mass, which provides potential therapeutic strategies in the combination with immune checkpoint blockade treatment.

Loss of RBMS1 promotes anti-tumor immunity through enabling PD-L1 checkpoint blockade in triple-negative breast cancer

Immunotherapy has been widely utilized in multiple tumors, however, its efficacy in the treatment of triple-negative breast cancers (TNBC) is still being challenged. Meanwhile, functions and mechanisms of RNA binding proteins in regulating immunotherapy for TNBC remain largely elusive. Here we reported that the RNA binding protein RBMS1 is prevalent among immune-cold TNBC. Through a systematic shRNA-mediated screen, we found depletion of RBMS1 significantly reduced the level of programmed death ligand 1 (PD-L1) in TNBC. Clinically, RBMS1 was increased in breast cancer and its level was positively correlated to that of PD-L1. RBMS1 ablation stimulated cytotoxic T cell mediated anti-tumor immunity. Mechanistically, RBMS1 regulated the mRNA stability of B4GALT1, a newly identified glycosyltransferase of PD-L1. Depletion of RBMS1 destabilized the mRNA of B4GALT1, inhibited the glycosylation of PD-L1 and promoted the ubiquitination and subsequent degradation of PD-L1. Importantly, combination of RBMS1 depletion with CTLA4 immune checkpoint blockade or CAR-T treatment enhanced anti-tumor T-cell immunity both in vitro and in vivo. Together, our findings provided a new immunotherapeutic strategy against TNBC by targeting the immunosuppressive RBMS1.

Harness RNA‐binding protein HuR to boost Anti‐PD‐1 Immunotherapy in immunologically cold tumors

Cancer immunotherapy, especially those targeting the immune checkpoint PD‐1 and PD‐L1 has emerged as a revolutionizing treatment modality. Immune checkpoint inhibitors (ICIs)‐mediated anti‐tumor response depends on T cell capability of recognizing and killing tumor cells. ‘Hot’ tumors, which are characterized by the T cell infiltration, show a better response rate to immune checkpoint blockade (ICB) treatment. In contrast, ‘Cold’ tumors have a particularly low objective response rate (ORR) to ICB. In consideration of the durable and impressive responses observed in ‘hot’ tumors, it will be valuable to make an endeavor to turn immunologically cold tumors into hot tumors. The RNA‐binding protein HuR is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs.In this study, we show the direct binding of HuR protein to PD‐L1 mRNA in multiple types of cells by Ribonucleoprotein immunoprecipitation (RNP‐IP) and further validated by RNA pulldown assay. We also show that PD‐L1 mRNA decay is greatly expedited in HuR‐knockout cells, which underlines the stabilization function of HuR on PD‐L1 transcripts. Immunoblotting results demonstrate that PD‐L1 protein expression level is decreased in HuR‐knockout cells while up‐regulated in HuR‐proficient cells in comparison with control. These data together clarify HuR is engaged in PD‐L1 post‐transcription regulation.Based on this mechanism, we develop a combination strategy using HuR inhibitors and anti‐PD1 antibody to overcome the limitation by regulating the tumor immune microenvironment.KH‐200 is a compound that disrupts the transportation of HuR from nuclear to cytoplasm. To test the combination strategy in vivo, we established the EMT6 syngeneic breast cancer model. After 4‐week treatment, the combination group shows great effects in inhibiting tumor growth and prolonging survival. The similar combination effect was further observed in Lewis lung syngeneic lung cancer model.In conclusion, we preliminarily explored the role of HuR in tumor immune evasion. The method using HuR inhibitors as a combination in immunotherapy shows an effective in immunologically cold tumor treatment and may have reference significance for clinical cancer therapy.

Glycolipid-loaded nanoparticle immunotherapy cooperates with checkpoint inhibitors to harness iNKT cells for tumor control

Abstract Invariant natural killer T cells (iNKT) have a well-documented role in direct and indirect killing of tumor cells through their release of proinflammatory cytokines and cytotoxic compounds. To improve outcomes in poorly immunogenic cancers while avoiding NKT cell anergy, we induced anti-tumor responses by activating NKT cells with polymeric nanoparticles containing a low dose of a glycolipid adjuvant, α-Galactosyl-Ceramide (aGalCer), and a model tumor neoantigen, ovalbumin (OVA). Prophylactic vaccination of mice with aGalCer-OVA nanoP before inoculation with B16F10-OVA melanoma cells reduced tumor growth rate and increased overall survival compared to mice immunized with control nanoparticles. In mice with an established B16 tumor, therapeutic administration of aGalCer-OVA nanoP provided significantly greater anti-tumor effects, reduced tumor growth rate and increased survival, compared to control nanoparticles. Therapeutic treatment with aGalCer-OVA nanoP, but not control nanoPs, also induced significant infiltration of iNKT, NK, and antigen-specific CD8 T cells in the tumor, and enhanced production of granzyme B and perforin by CD8 T cells. Furthermore, immune checkpoint blockade treatment (anti-CTLA-4 + anti-PD-1) enhanced the effectiveness of aGalCer-OVA nanoP, leading to clearance of established tumors nearly 50% of the time. These results provide strong evidence that activation of iNKT cells via nanoparticles containing a glycolipid adjuvant and tumor antigen is a viable anti-cancer strategy that partners well with other clinically used immunotherapies. Supported by grants from NIH (T32 AI138944) and the CTSA (TL1 TR002467)

Stratification From Heterogeneity of the Cell-Death Signal Enables Prognosis Prediction and Immune Microenvironment Characterization in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the primary subtype of esophageal cancer (EC) characterized by a high incidence rate and extremely poor prognosis worldwide. Previous studies suggested that the specific cell death signal was linked to different immune subtypes in multiple cancers, while a comprehensive investigation on ESCC is to be performed yet. In the current study, we dissected different cell death signals in ESCC tumors and then integrated that functional information to stratify ESCC patients into different immunogenic cell death (ICD) subtypes. By systematically analyzing the transcriptomes of 857 patients and proteomic profile of 124 patients, we found that the signals of necroptosis, pyroptosis, and ferroptosis are positively associated with activated immunity in ESCC. We identified two ICD pattern terms, namely, ICD-high and ICD-low subtypes that positively correlated to both progression-free survival and overall survival. In addition, cell fraction deconvolution analysis revealed that more infiltrated leukocytes were enriched in ICD-high types, especially antigen-presenting cells, such as dendritic cells and macrophages. With the XGBoost algorithm, we further developed a 14-gene signature which can simplify the subtyping for allocating new samples, by which we validated the prognosis value of the signature and proved that the ICD score scheme could serve as a promising biomarker for stratifying patients with immunotherapy in several immune checkpoint blockade treatment cohorts. Collectively, we successfully constructed the ICD scheme, which enables predicting of the prognosis or immunotherapy efficacy in ESCC patients and uncovered the critical interplay between cell death signals and immune status in ESCC.

Concurrent delivery of immune checkpoint blockade modulates T cell dynamics to enhance neoantigen vaccine-generated antitumor immunity.

Neoantigen vaccines aiming to induce tumor-specific T cell responses have achieved promising anti-tumor effects in early clinical trials. However, the underlying mechanism regarding response or resistance to this treatment remains unclear. Here, we observe that neoantigen vaccine-generated T cells can synergize with immune checkpoint blockade (ICB) for effective tumor control. Specifically, we performed single-cell sequencing on over 100,000 T cells and uncovered that combined therapy induces an antigen-specific CD8 T cell population with active chemokine signaling (Cxcr3+/Ccl5+), lower co-inhibitory receptor expression (Lag3−/Havcr2−) and higher cytotoxicity (Fasl+/Gzma+). Furthermore, the generation of neoantigen-specific T cells in the draining lymph node is required for combination treatment. Signature genes of this unique population are associated with T cell clonal frequency and better survival in humans. Our study profiles the dynamics of tumor infiltrated T cells during neoantigen vaccine and ICB treatments, high-dimensionally identifies neoantigen-reactive T cell signatures for future development of therapeutic strategies.

Integrative Genomic Profiling Uncovers Therapeutic Targets of Acral Melanoma in Asian Populations.

Acral melanoma is the major subtype of melanoma seen in Asian patients with melanoma and is featured by its insidious onset and poor prognosis. The genomic study that elucidates driving mutational events is fundamental to the development of gene-targeted therapy. However, research on genomic profiles of acral melanoma in Asian patients is still sparse. We carried out whole-exome sequencing (WES) on 60 acral melanoma lesions (with 55 primary samples involved), targeted deep sequencing in a validation cohort of 48 cases, RNA sequencing in 37 acral melanoma samples (all from the 60 undergoing WES), and FISH in 233 acral melanoma specimens (54 of the 60 undergoing WES included). All the specimens were derived from Asian populations. BRAF, NRAS, and KIT were discerned as significantly mutated genes (SMG) in acral melanoma. The detected COSMIC signature 3 related to DNA damage repair, along with the high genomic instability score, implied corresponding pathogenesis of acral melanoma. Moreover, the copy number gains of EP300 were associated with the response of acral melanoma to targeted therapy of A485 (a p300 inhibitor) and immune checkpoint blockade treatment. In addition, the temporal order in mutational processes of the samples was reconstructed, and copy-number alterations were identified as early mutational events. Our study provided a detailed view of genomic instability, potential therapeutic targets, and intratumoral heterogeneity of acral melanoma, which might fuel the development of personalized strategies for treating acral melanoma in Asian populations.