Antigen-presenting Machinery Genes Research Articles

TET2 promotes tumor antigen presentation and T cell IFN-γ which is enhanced by vitamin C.

Immune evasion by tumors is promoted by low T cell infiltration, ineffective T cell activity directed against the tumor and reduced tumor antigen presentation. The TET2 DNA dioxygenase gene is frequently mutated in hematopoietic malignancies and loss of TET enzymatic activity is found in a variety of solid tumors. We showed previously that vitamin C (VC), a co-factor of TET2, enhances tumor-associated T cell recruitment and checkpoint inhibitor therapy responses in a TET2-dependent manner. Using single-cell RNA sequencing (scRNA-seq) analysis performed on B16-OVA melanoma tumors, we have shown here that an additional function for TET2 in tumors is to promote expression of certain antigen presentation machinery genes, which is potently enhanced by VC. Consistently, VC promoted antigen presentation in cell-based and tumor assays in a TET2-dependent manner. Quantifying intercellular signaling from the scRNA-seq dataset showed that T cell-derived IFNγ-induced signaling within the tumor and tumor microenvironment requires tumor-associated TET2 expression which is enhanced by VC treatment. Analysis of patient tumor samples indicated that TET activity directly correlates with antigen-presentation gene expression and with patient outcomes. Our results demonstrate the importance of tumor-associated TET2 activity as a critical mediator of tumor immunity which is augmented by high-dose VC therapy.

SMYD3 represses tumor-intrinsic interferon response in HPV-negative squamous cell carcinoma of the head and neck

Cancers often display immune escape, but the mechanisms are incompletely understood. Herein, we identify SMYD3 as a mediator of immune escape in human papilloma virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC), an aggressive disease with poor response to immunotherapy with pembrolizumab. SMYD3 depletion induces upregulation of multiple type I interferon (IFN) response and antigen presentation machinery genes in HNSCC cells. Mechanistically, SMYD3 binds to and regulates the transcription of UHRF1, encoding for a reader of H3K9me3, which binds to H3K9me3-enriched promoters of key immune-related genes, recruits DNMT1, and silences their expression. SMYD3 further maintains the repression of immune-related genes through intragenic deposition of H4K20me3. Invivo, Smyd3 depletion induces influx of CD8+ Tcells and increases sensitivity to anti-programmed death 1 (PD-1) therapy. SMYD3 overexpression is associated with decreased CD8 Tcell infiltration and poor response to neoadjuvant pembrolizumab. These data support combining SMYD3 depletion strategies with checkpoint blockade to overcome anti-PD-1 resistance in HPV-negative HNSCC.

Abstract 3289: Melanoma patients with high and low target expression can benefit from TCR-CD3 bispecifics through direct and indirect mechanisms of tumor control

Abstract Background: ImmTAC molecules are TCR-CD3 bispecific fusion proteins that can redirect and activate polyclonal T cells to kill tumors. Tebentafusp, which targets gp100, is the first ImmTAC to demonstrate an overall survival benefit and is approved for the treatment of HLA-A*02:01 adult patients with unresectable or metastatic uveal melanoma1. Clinical benefit was observed in patients with both high and low gp100 expression2, suggesting direct and indirect mechanisms of tumor control, which we explored using in vitro models. Method: Uveal and cutaneous melanoma cell lines were pulsed with 0-1 µM peptide concentrations or were treated with supernatants from ImmTAC-activated PBMC, with or without blocking antibodies against IFNβ, IFNγ, TNFα, and IL6. Peptide-HLA (pHLA) molecules on tumor cell surface were quantified by microscopy. ImmTAC-redirected T cell activation and killing were measured by ELISPOT, MSD, and xCelligence assays. Antigen presentation machinery (APM) gene expression was measured by qPCR. Linear regression models were used to assess the influence of variables on ImmTAC-mediated T cell activation. Results: Peptide-pulsing of tumor cells showed a dose response relationship between surface pHLA number and ImmTAC-mediated T cell killing (R2= 0.85), which was confirmed by ImmTAC redirected killing assays using tumor lines with differential endogenous expression of pHLA targets. Higher effector: target (E: T) and higher pHLA number are the key factors, by regression analyses, influencing ImmTAC mediated T cell activation. These account for 53% and 29% variability in IFNγ production (p<0.01), respectively. To assess the indirect impact of ImmTAC-activated T cells on tumor cells, cell lines were pre-treated with supernatants. ImmTAC-activated PBMC supernatants reduced tumor growth (2 to 25-fold) and increased tumor death (20 - 80%) in 5 of 8 cell lines tested. Blocking IFNγ restored tumor growth by over 40%, which was further enhanced by additional blocking of IFNβ, TNFα, and IL6. All cell lines significantly upregulated APM genes and became more susceptible to direct ImmTAC-mediated T cell killing (1.4 to 2-fold increase). Conclusions: In vitro, ImmTAC bispecifics kill cancer cells by both direct and indirect mechanisms. Redirection of polyclonal T cells to kill melanoma cells is greatly influenced by surface pHLA number and E:T ratio. ImmTAC-induced cytokines inhibited tumor growth, increased tumor apoptosis and resulted in higher antigen presentation demonstrating the indirect effect of ImmTAC. These observations highlight how ImmTAC-treated melanoma patients could benefit through direct and indirect mechanisms of tumor control, and in part help to explain how patients with low gp100 expression can derive benefit from tebentafusp.

Specific cannabinoids revive adaptive immunity by reversing immune evasion mechanisms in metastatic tumours.

Emerging cancers are sculpted by neo-Darwinian selection for superior growth and survival but minimal immunogenicity; consequently, metastatic cancers often evolve common genetic and epigenetic signatures to elude immune surveillance. Immune subversion by metastatic tumours can be achieved through several mechanisms; one of the most frequently observed involves the loss of expression or mutation of genes composing the MHC-I antigen presentation machinery (APM) that yields tumours invisible to Cytotoxic T lymphocytes, the key component of the adaptive cellular immune response. Fascinating ethnographic and experimental findings indicate that cannabinoids inhibit the growth and progression of several categories of cancer; however, the mechanisms underlying these observations remain clouded in uncertainty. Here, we screened a library of cannabinoid compounds and found molecular selectivity amongst specific cannabinoids, where related molecules such as Δ9-tetrahydrocannabinol, cannabidiol, and cannabigerol can reverse the metastatic immune escape phenotype in vitro by inducing MHC-I cell surface expression in a wide variety of metastatic tumours that subsequently sensitizing tumours to T lymphocyte recognition. Remarkably, H3K27Ac ChIPseq analysis established that cannabigerol and gamma interferon induce overlapping epigenetic signatures and key gene pathways in metastatic tumours related to cellular senescence, as well as APM genes involved in revealing metastatic tumours to the adaptive immune response. Overall, the data suggest that specific cannabinoids may have utility in cancer immunotherapy regimens by overcoming immune escape and augmenting cancer immune surveillance in metastatic disease. Finally, the fundamental discovery of the ability of cannabinoids to alter epigenetic programs may help elucidate many of the pleiotropic medicinal effects of cannabinoids on human physiology.

Clustering by antigen-presenting genes reveals immune landscapes and predicts response to checkpoint immunotherapy

Immune checkpoint blockade (ICB) has demonstrated efficacy by reinvigorating immune cytotoxicity against tumors. However, the mechanisms underlying how ICB induces responses in a subset of patients remain unclear. Using bulk and single-cell transcriptomic cohorts of melanoma patients receiving ICB, we proposed a clustering model based on the expression of an antigen-presenting machinery (APM) signature consisting of 23 genes in a forward-selection manner. We characterized four APM clusters associated with distinct immune characteristics, cancer hallmarks, and patient prognosis in melanoma. The model predicts differential regulation of APM genes during ICB, which shaped ICB responsiveness. Surprisingly, while immunogenically hot tumors with high baseline APM expression prior to treatment are correlated with a better response to ICB than cold tumors with low APM expression, a subset of hot tumors with the highest pre-ICB APM expression fail to upregulate APM expression during treatment. In addition, they undergo immunoediting and display infiltration of exhausted T cells. In comparison, tumors associated with the best patient prognosis demonstrate significant APM upregulation and immune infiltration following ICB. They also show infiltration of tissue-resident memory T cells, shaping prolonged antitumor immunity. Using only pre-treatment transcriptomic data, our model predicts the dynamic APM-mediated tumor-immune interactions in response to ICB and provides insights into the immune escape mechanisms in hot tumors that compromise the ICB efficacy. We highlight the prognostic value of APM expression in predicting immune response in chronic diseases.

Topoisomerase I poison-triggered immune gene activation is markedly reduced in human small-cell lung cancers by impairment of the cGAS/STING pathway

BackgroundCurrent immunotherapy strategies have contrasting clinical results in human lung cancer patients as small-cell lung cancers (SCLC) often show features of immunological cold tumours. Topoisomerase 1 (TOP1) poisons are effective antitumor drugs with good efficacy against lung cancers.MethodsWe used molecular, genetic and bioinformatic approaches to determine the mechanism of micronuclei formation induced by two TOP1 poisons in different human cancer cells, including SCLC cell lines.ResultsTOP1 poisons stimulate similar levels of micronuclei in all tested cell lines but downstream effects can vary markedly. TOP1 poisons increase micronuclei levels with a mechanism involving R-loops as overexpression of RNaseH1 markedly reduces or abolishes both H2AX phosphorylation and micronuclei formation. TOP1 poison-induced micronuclei activate the cGAS/STING pathway leading to increased expression of immune genes in HeLa cells, but not in human SCLC cell lines, mainly due to lack of STING and/or cGAS expression. Moreover, the expression of STING and antigen-presenting machinery genes is generally downregulated in patient tumours of human lung cancer datasets.ConclusionsAltogether, our data reveal an immune signalling mechanism activated by TOP1 poisons, which is often impaired in human SCLC tumours.

Abstract 6250: Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer type in the world, with Human Papilloma virus (HPV)-negative patients having a ~50% recurrence rate and poor median overall survival of ~13 months in the recurrent/metastatic setting. Pembrolizumab, a programmed-death-1 (PD-1) checkpoint inhibitor is active but with a response rate as low as 19%. Our preliminary work has identified SET and MYND Domain containing 3 (SMYD3) as a chromatin modifier that is significantly overexpressed in HPV-negative HNSCC tumor tissues compared to normal epithelium and is associated with low CD8+ T-cell infiltration in HPV-negative HNSCC. Furthermore, we have shown that knockdown of human SMYD3 upregulates the expression of multiple type I IFN response and antigen presentation machinery (APM) genes. This study aims to investigate whether Smyd3 depletion potentiates immune-mediated antitumor efficacy in anti-PD-1 resistant syngeneic mouse models of HPV-negative HNSCC (MOC1), as well as relevant mechanisms. SiRNA-mediated knockdown of Smyd3 in mouse HPV-negative MOC1 cells followed by RNA-seq revealed upregulation of multiple type I IFN response and APM genes in vitro. Multicolor flow cytometry of MOC1 tumors treated with control anti-sense oligonucleotides (ASOs) or Smyd3 ASOs showed that Smyd3 depletion in a syngeneic, heterotopic mouse model (C57BL/6) of flank MOC1 tumors induced intratumoral infiltration of CD8+ T-cells, as well as upregulation of H2-Kb (MHC class I) and mouse Pd-l1 in MOC1 cells. Combined treatment of Smyd3 ASOs with anti-PD-1 induced complete regressions of flank MOC1 tumors in 4/8 treated mice and significant tumor growth restriction in 2/8 mice, while 2/8 tumors “escaped” the treatment effect. Single-cell RNA seq of MOC1 tumors treated with control or Smyd3 ASOs is ongoing to evaluate the effects of Smyd3 depletion on MOC1 cancer cells and immune cell subsets of the tumor microenvironment and to identify potential mechanisms of resistance of Smyd3 ASO + anti-PD-1 combination treatment. Genome-wide mapping of Smyd3 and the repressive mark H4K20me3, which is known to be written by Smyd3, in MOC1 cells using CUT&RUN assays is also ongoing and aims to decipher whether Smyd3 directly binds to and regulates the expression of immune-related genes through H4K20me3. Ex-vivo cytotoxicity assays aim to evaluate whether CRISPR Smyd3 KO in MOC1 cells sensitizes cancer cells to TIL-mediated cytotoxicity. Smyd3 depletion induces an inflamed tumor microenvironment and may potentiate the immune-mediated antitumor efficacy of anti-PD-1 in syngeneic mouse models of HPV-negative HNSCC. This work may lay the biological rationale to combine SMYD3 inhibition with T-cell based immunotherapeutic approaches in HPV-negative HNSCC. Citation Format: Daniel E. Tsai, Yvette Robbins, Angel Huynh, Andrew Sinkoe, Cem Sievers, Madhavi Murali, Xiaolin Luo, Clint T. Allen, Vassiliki Saloura. Investigating the immunomodulatory mechanisms of Smyd3 depletion in HPV-negative head and neck squamous cell carcinoma [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 6250.

Functional Genomic Landscape of Natural Killer Cell Evasion in Multiple Myeloma

Natural killer (NK) cell-based therapies are considered promising future approaches for multiple myeloma (MM) treatment, but immune evasion mechanisms are poorly understood. To determine the mechanisms regulating MM cell response to NK cells, we performed genome-wide (GW) and targeted CRISPR screens in MM cell lines. To further investigate the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed additional pooled CRISPR screens with a single-cell (sc) transcriptome readout using the CROP-seq platform and integrated these findings with data from phenotypic assessment of NK cell response of pooled “DNA-barcoded” cell line including 15 MM cell lines (PRISM platform) and molecular profiling of MM patient samples.Our loss-of-function (LOF) and gain-of-function (GOF) GW-CRISPR screens were performed in the MM1.S, LP1, KMS11 MM lines treated with ex vivo expanded NK cells vs untreated control. The top LOF hits were validated using a focused library of ~600 genes. As expected, LOF of class I HLA /antigen presentation machinery genes, transcriptional regulators of HLA and IFNg pathway genes sensitized MM cells to NK cells, confirming that these pathways represent prominent suppressors of NK cell killing. Moreover, LOF of death receptors or downstream effectors was associated with NK resistance; while LOF of the negative regulators of death receptor signaling (e.g. CFLAR, and XIAP) sensitized to NK cell killing.To mechanistically dissect the transcriptional impact of genes identified as regulators of sensitivity to NK cells, we performed scRNAseq using the CROP-seq platform. Pools of MM1.S and LP1 expressing single-guide (sg)RNAs targeting 31 select hits were co-cultured with NK cells for 24 h or left untreated, followed by scRNA-seq and sgRNA detection, differential gene-expression analysis and patient data correlation.The single cell transcriptional profiling of each CRISPR-based LOF for genes of interest documented that disruption of TRAF2, NFKBIA or NFKBIB enhanced NF-kB signaling and was associated with increased expression of the death receptor FAS, a key mediator of NK cell sensitivity (Figure), and changes in expression of BIRC3, CD70, and CXCL10(the latter may further increase immune reactivity through recruitment of T and NK cells). Interestingly, in our PRISM data, high transcriptional NF-kB signatures and/or presence of TRAF3 mutations (an activator of NF-kB signaling) correlated with decreased NK cell response. Among the individual genes most highly correlated with NK cell resistance was CFLAR, a known NF-kB target gene, and negative regulator of death receptor-mediated apoptosis. In contrast, PTEN and NLRC5 mutations were associated with enhanced NK cell sensitivity in several MM lines tested, consistent with their depletion in the GW LOF CRISPR screens and the HLA I regulatory function of NLRC5.We correlated our findings with patient-derived data from the CoMMpass study. Mutations of NF-kB negative regulators TRAF2 and NFKBIA were associated with increased expression of NF-kB target genes, consistent with our CROP-seq data. NFKBIA mutations were also linked to reduced HLA-E expression in both MM patient and CROP-seq data, suggesting a potential explanation for the NK-sensitizing effect of NFKBIA disruption. NLRC5 mutations were associated with lower HLA-E expression consistent with CROP-seq data, suggesting that, although rare, NLRC5mutations may represent a more NK-sensitive MM subset. TRAF3 alterations occurred both in a distinct TRAF3-altered cluster and in a subset of CoMMpass patients with WHSC1 translocations, consistent with the observations in our PRISM data set. CFLAR expression was enriched in patients with TRAF3 alterations belonging to both of these groups. The aggregate of molecular data from MM patient samples and PRISM or CRISPR-based functional studies preclinically raise the possibility that TRAF3 and WHSC1/t(4;14) alterations may contribute, at least in part through CFLAR, to decreased NK cell response in MM cells.Our data illuminate the complex mechanisms of response to NK cells in MM, highlighting the different effects of distinct molecularly defined subgroups of MM tumor cells with increased susceptibility to NK cell treatment, underlining the potential of such studies as a blueprint for identification of biomarkers individualized use of NK cell-based therapies in MM. [Display omitted] DisclosuresMustjoki: Novartis: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Janpix: Research Funding. Mitsiades: H3 Biomedicine: Research Funding; FIMECS: Consultancy, Honoraria; Adicet Bio: Membership on an entity's Board of Directors or advisory committees; Nurix: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; BMS: Research Funding; Fate Therapeutics: Consultancy, Honoraria; Novartis: Research Funding; Janssen/Johnson & Johnson: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Arch Oncology: Research Funding; Abbvie: Research Funding; Ionis Pharmaceuticals: Consultancy, Honoraria.

Selected Articles from This Issue

Brain metastases are a major cause of morbidity and mortality in patients with RET fusion-positive non-small cell lung cancers. Subbiah and colleagues present safety and efficacy results of the LIBRETTO-001 Phase I/II trial in patients with RET fusion-positive NSCLCs with intracranial metastases. Selpercatinib was well tolerated, achieving a high intracranial response rate, as well as prolonged intracranial duration of response and intracranial progression-free survival. These important trial findings will help to further support selpercatinib as a new standard of care therap. for the primary systemic treatment for RET fusion-positive NSCLC patients with brain metastases.For patients with EGFR-mutant NSCLC, EGFR-TKI treatment has been effective. However, acquired resistance ultimately leads to disease progression, suggesting that TKI targeting resistance mutations, such as osimertinib, be reserved for second-line treatment. Preclinical studies have suggested that double EGFR inhibition by TKI and antibodies may be superior to TKI alone. To assess this strategy, Cortot and colleagues assessed the combination of afatinib plus cetuximab for first-line treatment of EGFR-mutant NSCLC. Although the safety profile was manageable, no benefit of this combination was observed compared to afatinib alone. A shorter PFS was observed to be correlated with mutant EGFR allele frequency. Therefore, this combination does not warrant further consideration, and alternative strategies to delay osimertinib are needed.Clever-1 is a receptor expressed on immunosuppressive macrophages in the tumor microenvironment. To explore its potential as an immunotherapeutic drug target, Virtakoivu and colleagues studied the immune signatures in advanced cancer patients receiving a humanized monoclonal antibody targeting Clever-1 (FP-1305, bexmarilimab). The results show that bexmarilimab activates T cells and drives antitumor immune responses in cold tumors that are not otherwise responsive to immunotherapy. These findings suggest that adaptive immune activation can be achieved by modulating the behavior of macrophages and further support Clever-1 as an immunotherapeutic drug target.Adaptive immunity and protumorigenic inflammation coexist in tumor microenvironments in delicate balance. However, this balance in human specimens has remained understudied. Integrating bulk and single cell RNA sequencing data from urothelial cancers, Wang and colleagues demonstrated that tumor microenvironments with increased features of protumorigenic inflammation relative to adaptive immunity, quantified as the 2IR score, were resistant to PD-1/PD-L1 blockade. Further, the authors identified that single myeloid phagocytic cells with increased expression of proinflammatory genes and decreased expression of antigen presentation machinery genes, inferred to be polarized by IL-1, were key contributors of such tumor microenvironments. Therefore, targeting such myeloid phagocytic cells in urothelial tumors demonstrating increased features of protumorigenic inflammation relative to adaptive immunity may overcome a key mechanism of intrinsic resistance to PD-1/PD-L1 blockade.

Abstract 2117: Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) causes approximately 10,000 deaths annually in the United States, with human-papilloma-virus (HPV)-negative patients having a particularly poor prognosis. Pembrolizumab, a programmed-death-1 (PD-1) antibody, was recently approved as a first-line therapy for patients with relapsed disease, however response rates are as low as 19%, underscoring the need for an improved understanding of immunotherapy resistance mechanisms. We have previously shown that HPV-negative HNSCC tumors with higher mRNA levels of SET and MYND Domain Containing 3 (SMYD3), a chromatin modifier known to trimethylated H3K4 (H3K4me3), have significantly lower mRNA levels of CD8 and CD8+ T-cell attracting chemokines, and that SMYD3 depletion in HNSCC cells in vitro leads to upregulation of CD8+ T-cell attracting chemokines. The goal of this work was to elucidate epigenetic mechanisms of immune evasion mediated by SMYD3 in HPV-negative HNSCC. Methods: siRNA- mediated SMYD3 knockdown was performed for 3 days to investigate immuno-modulatory epigenetic mechanisms mediated by SMYD3. RT-qPCR for immune-related genes, Western blotting for histone marks, ChIP-sequencing for H3K4me3, ChIP-qPCRs to determine genomic interactions of SMYD3 and ATAC-sequencing were performed. SMYD3 and CD8 immunohistochemical staining (IHC) of normal squamous, dysplastic epithelium and HPV-negative HNSCC tumors was also performed. Results: siRNA mediated knockdown of SMYD3 resulted in enrichment of inflammatory pathways and upregulation of type 1 interferon response and antigen presentation machinery genes in HNSCC cell lines. Immunoblotting for histone marks showed no reduction in H3K4me3 following SMYD3 depletion for 3 days. ChIP-sequencing for H3K4me3 showed differential distribution pattern with an increase of H3K4me3 peaks on immune-related genes, and a decrease of H3K4me3 on UHRF1, a reader of the repressive histone mark H3K9me3. RNA-seq, RT-PCR and ChIP-qPCR showed that SMYD3 may regulate the transcription of UHRF1 by binding to its promoter. SMYD3 depletion also led to decreased protein levels of UHRF1. ChIP for UHRF1 and H3K9me3 showed that UHRF1 binds directly on the promoters of CXCL9 and CXCL10 which are enriched for H3K9me3. IHC on Normal Squamous Epithelium and Squamous Cell Carcinoma tissues of increasing cancer stages showed increase in SMYD3 levels with the higher stage of cancer, and an inverse correlation with CD8+ T cell infiltration within the tumors. Conclusion: This is the first study reporting that the chromatin modifier SMYD3 may attenuate type I IFN responses in HPV-negative HNSCC by regulating the expression of a major epigenetic regulator, UHRF1, which may silence the expression of immune-related genes through H3K9me3. Citation Format: Nupur Nigam, Benjamin Jonah Bernard, Kyunghee Burkitt, Sohyoung Kim, Yvette Robbins, Richard L. Bennett, Jonathan D. Licht, Vassiliki Saloura. Immunomodulatory functions of SMYD3 in HPV-negative head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2117.

Neoantigen load as a prognostic and predictive marker for stage II/III non-small cell lung cancer in Chinese patients.

BackgroundThe prognosis for patients with stage II/III non–small cell lung cancer (NSCLC) is unsatisfactory, even after complete tumor resection and adjuvant chemotherapy. Here, we assessed the prognostic and predictive value of immunogenomic signatures for stage II/III NSCLC in Chinese patients.MethodsA total of 91 paired resected stage II/III NSCLC and normal tissues, including 47 squamous cell lung carcinomas (SCC) and 44 lung adenocarcinomas (ADC), were collected and analyzed using whole exome sequencing (WES) to identify immunogenomic signatures for association with clinicopathological variables and disease‐free survival (DFS).ResultsHigher neoantigen load (NAL, >2 neoantigens/Mb) exhibited better DFS for SCC patients (p = 0.021) but not ADC patients. A benefit from adjuvant chemotherapy was correlated with lower NAL (≤2 neoantigens/Mb) (p = 0.009). However, tumor mutation burden (TMB), mutations of individual gene, oncogene pathways, and antigen presentation machinery genes, and human leukocyte antigen (HLA)‐I number and HLA‐I loss of heterozygosity (LOH) had no prognostic or predictive value for DFS of SCC or ADC patients.ConclusionsNAL is a useful biomarker for lung SCC prognosis and prediction of chemotherapy responses in Chinese patients. The predictive value of NAL for adjuvant immunotherapy should be further explored in patients with resected NSCLC.

Contribution of Antigen-Processing Machinery Genetic Polymorphisms to Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic and recurrent inflammatory dermatosis. We recently described an association of the C allele of the single nucleotide polymorphism (SNP) rs26618 in the ERAP1 gene and a synergism of ERAP1 and ERAP2 effects on AD risk. Here, we examined whether polymorphisms of other antigen-presenting machinery genes encoding immunoproteasome components LMP2 and LMP7 and peptide transporter components TAP1 and TAP2 may also affect susceptibility to AD or its outcome. We found that the LMP7 rs2071543*T allele decreased disease risk by about 1.5-fold (odds ratio 0.66, 95% confidence interval 0.44–0.99). On the other hand, the LMP2 rs1351383*C allele reduced the mean age at diagnosis from 23 to 15 years (p < 0.001). Similarly, the TAP1 rs1135216*C allele decreased the mean age at diagnosis from almost 20 to 14 years (p = 0.033). The results are discussed in light of other reports on the role of these polymorphisms in human disease.

Integrative molecular characterization of sarcomatoid and rhabdoid renal cell carcinoma

Sarcomatoid and rhabdoid (S/R) renal cell carcinoma (RCC) are highly aggressive tumors with limited molecular and clinical characterization. Emerging evidence suggests immune checkpoint inhibitors (ICI) are particularly effective for these tumors, although the biological basis for this property is largely unknown. Here, we evaluate multiple clinical trial and real-world cohorts of S/R RCC to characterize their molecular features, clinical outcomes, and immunologic characteristics. We find that S/R RCC tumors harbor distinctive molecular features that may account for their aggressive behavior, including BAP1 mutations, CDKN2A deletions, and increased expression of MYC transcriptional programs. We show that these tumors are highly responsive to ICI and that they exhibit an immune-inflamed phenotype characterized by immune activation, increased cytotoxic immune infiltration, upregulation of antigen presentation machinery genes, and PD-L1 expression. Our findings build on prior work and shed light on the molecular drivers of aggressivity and responsiveness to ICI of S/R RCC.

Abstract PO041: Landscape of molecular events regulating tumor cell responses to natural killer cells

Abstract Natural killer (NK) cells exhibit potent activity in pre-clinical models of diverse hematologic malignancies and solid tumors and infusion of high numbers of NK cells, either autologous or allogeneic, after their ex vivo expansion and activation, has been feasible and safe in clinical studies. To systematically define molecular features in human tumor cells which determine their degree of sensitivity to human allogeneic NK cells, we quantified the NK cell responsiveness of hundreds of molecularly-annotated “DNA-barcoded” solid tumor cell lines in multiplexed format (PRISM; Profiling Relative Inhibition Simultaneously in Mixtures approach), correlating cytotoxicity scores for each cell line with the CCLE transcriptional data (RNA-seq), to reveal genes that are associated with resistance or sensitivity to NK cells. In addition, we applied genome-scale CRISPR-based gene editing screens in several solid tumor cell lines to interrogate, at a functional level, which genes regulate tumor cell response to NK cells. Based on these orthogonal studies, NK sensitive tumor cells tend to exhibit high levels of the NK cell-activating ligand B7-H6 (NCR3LG1); low levels of the inhibitory ligand HLA-E; microsatellite instability (MSI) status; high transcriptional signature for chromatin remodeling complexes and low antigen presentation machinery genes. Treatment with an HDAC inhibitor reduced the sensitivity of SW620 colon cancer cells, increased antigen presentation machinery, including HLA-E, and reduced B7-H6. Importantly, we observe that transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor resistance in clinical samples. Strikingly, comprehensive analysis of the CCLE transcriptional signatures revealed that cell lines with mesenchymal-like program tend to be more sensitive to NK cells treatment, compared with cell lines of epithelial-like program. Indeed, mesenchymal tumors tend to have lower expression of antigen presentation machinery in both CCLE and TCGA, suggesting a link between these two machieneries. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies. Citation Format: Michal Sheffer, Emily Lowry, Nicky Beelen, Minasri Borah, Suha Naffar-Abu Amara, Chris C. Mader, Jennifer Roth, Aviad Tsherniak, Olga Dashevsky, Sara Gandolfi, Samantha Bender, Jordan Bryan, Cong Zhu, Li Wang, Ricardo De-Matos Simoes, Channing Yu, Yiguo Hu, Olli Dufva, Marios Giannakis, Todd Golub, Rizwan Romee, Satu Mustjoki, Aedin C. Culhane, Lotte Wieten, Constantine S. Mitsiades. Landscape of molecular events regulating tumor cell responses to natural killer cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO041.

Abstract 1311: Inter- and intra-tumor heterogeneity of genetic and immune profiles in hereditary clear cell renal cell carcinoma

Abstract [Background] Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome attributed to germline VHL mutation. Patients with this disease are at risk of developing multiple independent clear cell renal carcinomas (ccRCCs), which offers a unique opportunity to analyze inter- and intra-tumor heterogeneity of genetic and immune profiles in the same patient. [Method] We selected seven patients with clinically diagnosed VHL disease who underwent one or two surgeries, in each of which two to five ccRCC tumors (total, n = 25) were removed. We harvested one to six regions from each tumor (total, n = 48), and performed whole-exome sequencing on them, together with normal blood (n = 7), cyst (n = 3), and adjacent normal renal tissue (n = 7) specimens from these patients. In addition, we carried out RNA-sequencing (for 20 tumors and 3 adjacent tissues) and digital multiplexed gene expression analysis using the NanoString nCounter system with Human Immunology V2 panel (for 36 tumors and 5 adjacent tissues) to clarify their immune profiles. [Result] Germline VHL mutations were confirmed in all seven patients. A total of 1,141 non-silent somatic mutations (8-44 mutations per patient) were detected, including 22 mutations affecting previously reported driver genes (such as BAP1, PBRM1, PTEN, PIK3CA, and SETD2). In consistent with previous reports, tumors arising in this context are clonally independent and harbor distinct secondary events, including mutations and copy number alterations (as exemplified by loss of heterozygosity of 3p). By contrast, multiple specimens from the same tumor showed almost the same genetic alterations, although several subclonal mutations were observed. Unsupervised hierarchical clustering of gene expression data discovered two robust subsets with distinct immune-related signatures. Particularly, the subset with high immune signature (n = 29) showed increased antigen presentation machinery genes (B2M, TAP1 and TAP2) and cytotoxic T lymphocyte genes (CD8, PRF1, and GZMA) than that with low immune signature (n = 19), suggesting a higher cytolytic activity in the former subset. Pathway analysis revealed the enrichment of cytokine interaction, JAK-STAT signaling and Toll-like receptor signaling. Interestingly, specimens from the same tumor invariably showed a similar immune signature, whereas different tumors frequently exhibit different immune signatures despite the identical immune background, suggesting a greater difference of immune status across tumors than within a tumor. [Conclusion] This study delineates inter- and intra-tumor heterogeneity of genetic and immune profiles in hereditary clear cell renal cell carcinoma. Our findings demonstrate more profound inter-tumor than intra-tumor heterogeneity in immune status, suggesting that tumor factors have a greater impact on immune microenvironment than host factors. Citation Format: Mariko Tabata, Yusuke Sato, Yasunori Kogure, Marni B. McClure, Yuji Kumade, Yuki Saito, Sumito Shingaki, Kota Yoshifuji, Junji Koya, Kazushi Yoshida, Takashi Kohno, Kenichi Chiba, Ai Okada, Yuichi Shiraishi, Haruki Kume, Keisuke Kataoka. Inter- and intra-tumor heterogeneity of genetic and immune profiles in hereditary clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1311.

Tumor immune microenvironment characterization in clear cell renal cell carcinoma identifies prognostic and immunotherapeutically relevant messenger RNA signatures.

BackgroundTumor-infiltrating immune cells have been linked to prognosis and response to immunotherapy; however, the levels of distinct immune cell subsets and the signals that draw them into a tumor, such as the expression of antigen presenting machinery genes, remain poorly characterized. Here, we employ a gene expression-based computational method to profile the infiltration levels of 24 immune cell populations in 19 cancer types.ResultsWe compare cancer types using an immune infiltration score and a T cell infiltration score and find that clear cell renal cell carcinoma (ccRCC) is among the highest for both scores. Using immune infiltration profiles as well as transcriptomic and proteomic datasets, we characterize three groups of ccRCC tumors: T cell enriched, heterogeneously infiltrated, and non-infiltrated. We observe that the immunogenicity of ccRCC tumors cannot be explained by mutation load or neo-antigen load, but is highly correlated with MHC class I antigen presenting machinery expression (APM). We explore the prognostic value of distinct T cell subsets and show in two cohorts that Th17 cells and CD8+ T/Treg ratio are associated with improved survival, whereas Th2 cells and Tregs are associated with negative outcomes. Investigation of the association of immune infiltration patterns with the subclonal architecture of tumors shows that both APM and T cell levels are negatively associated with subclone number.ConclusionsOur analysis sheds light on the immune infiltration patterns of 19 human cancers and unravels mRNA signatures with prognostic utility and immunotherapeutic biomarker potential in ccRCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1092-z) contains supplementary material, which is available to authorized users.

The absence of HLA class I expression in non-small cell lung cancer correlates with the tumor tissue structure and the pattern of T cell infiltration.

We wanted to analyze whether tumor HLA class I (HLA-I) expression influences the pattern of the immune cell infiltration and stromal cell reaction in the tumor microenvironment. Tumor tissues obtained from 57 patients diagnosed with lung carcinomas were analyzed for HLA expression and leukocyte infiltration. 28 patients out of the 57 were completely negative for HLA-I expression (49.1%) or showed a selective HLA-A locus downregulation (three patients, 5.2%). In 26 out of 57 tumors (47.8%) we detected a positive HLA-I expression but with a percentage of HLA-I negative cells between 10 and 25%. The HLA-I negative phenotype was produced by a combination of HLA haplotype loss and a transcriptional downregulation of β2-microglobulin (β2-m) and LMP2 and LMP7 antigen presentation machinery genes. The analysis and localization of different immune cell populations revealed the presence of two major and reproducible patterns. One pattern, which we designated "immune-permissive tumor microenvironment (TME)," was characterized by positive tumor HLA-I expression, intratumoral infiltration with cytotoxic T-CD8+ cells, M1-inflammatory type macrophages, and a diffuse pattern of FAP+ cancer-associated fibroblasts. In contrast, another pattern defined as "non-immune-permissive TME" was found in HLA-I negative tumors with strong stromal-matrix interaction, T-CD8+ cells surrounding tumor nests, a dense layer of FAP+ fibroblasts and M2/repair-type macrophages. In conclusion, this study revealed marked differences between HLA class I-positive and negative tumors related to tissue structure, the composition of leukocyte infiltration and stromal response in the tumor microenvironment.

Abstract 569: MicroRNA-mediated resistance to anti-PD1 therapy in lung cancer

Abstract Immunotherapies targeting programmed cell death receptor-1 (PD-1) have shown some clinical success, validating the role of immune modulation in the treatment of cancer. However, only 18% of non-small cell lung cancer patients have responded to anti-PD1 treatment thus far, the remaining 82% have not. This raises fundamental questions about mechanisms of resistance to anti-PD1 therapy and potential strategies to overcome resistance. Previously, our group generated an anti-PD-1-resistant preclinical tumor model after sequential in vivo passage of the murine lung cancer cell line 344SQ (p53R172HΔg/+KrasLA1/+) into syngeneic mice, previously treated with anti-mouse PD-1 antibodies. In this study, we analyzed global gene expression of microRNAs and correlated with our previous mRNA microarray data to validate potential target candidates in order to identify mechanisms of resistance to anti-PD1 therapy. For the microRNA microarray studies, 344SQ_P (parental) or 344SQ_R (resistant) cells were inoculated into the right flank of 129Sv/ev mice (female mice, 12-14 weeks old, 5 mice per group). Anti-PD1 or control IgG (10mg/kg) antibodies were given on days 4 and 7 after tumor inoculation. On day 11, tumor tissues were collected and immediately frozen in liquid nitrogen. RNAs from three independent biologic replicates per group were used for GeneChip miRNA 4.0 Array (Affymetrix). Significant differences in microRNA gene expression levels between groups were defined as an adjusted P value &amp;lt;0.05 and absolute fold change of &amp;gt; 2. Heatmaps were created by using the heatmap.2 function in the gplots R package. Our previous studies demonstrated that major histocompatibility complex (MHC) class I and II and antigen presentation pathway, were significantly downregulated in the anti-PD-1-resistant tumors compared with parental tumors. Here, we found that microRNAs significantly upregulated in the anti-PD1 resistant model directly regulates MHC class I and II and antigen presentation machinery genes, including B2M, CIITA, PSMB8, PSMB8, TAP2 and TAPBP, promoting resistance to anti-PD1 therapy. Because activated CD8+ tumor-infiltrating lymphocytes must bind to MHC I proteins to kill target tumor cells, one way in which tumors can evade destruction by the immune system is through downregulating expression of MHC I on their surfaces. Therefore, our study identified upregulation of microRNAs that modulates the antigen presentation pathway as an underlying mechanism by which some tumors do not respond to anti-PD1 therapy. Our future goal is to validate these findings on our ongoing clinical studies. Citation Format: Maria A. Cortez, Xiaohong Wang, Cristina Ivan, Jonathan E. Schoenhals, Sharareh Niknam, Ailin Li, David Valdecanas, James P. Allison, Padmanee Sharma, Willem W. Overwijk, Daniel Gomez, Joey Y. Chang, Stephen Hahn, George A. Calin, James W. Welsh. MicroRNA-mediated resistance to anti-PD1 therapy in lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 569.

Induction of MHC class I molecule cell surface expression and epigenetic activation of antigen‐processing machinery components in a murine model for human papilloma virus 16‐associated tumours

Epigenetic events play an important role in tumour progression and also contribute to escape of the tumour from immune surveillance. In this study, we investigated the up-regulation of major histocompatibility complex (MHC) class I surface expression on tumour cells by epigenetic mechanisms using a murine tumour cell line expressing human E6 and E7 human papilloma virus 16 (HPV16) oncogenes and deficient in MHC class I expression, as a result of impaired antigen-presenting machinery (APM). Treatment of the cells with the histone deacetylase inhibitor Trichostatin A, either alone or in combination with the DNA demethylating agent 5-azacytidine, induced surface re-expression of MHC class I molecules. Consequently, the treated cells became susceptible to lysis by specific cytotoxic T lymphocytes. Further analysis revealed that epigenetic induction of MHC class I surface expression was associated with the up-regulation of APM genes [transporter associated with antigen processing 1 (TAP-1), TAP-2, low-molecular-mass protein 2 (LMP-2) and LMP-7]. The results demonstrate that expression of the genes involved in APM are modulated by epigenetic mechanisms and suggest that agents modifying DNA methylation and/or histone acetylation have the potential to change the effectiveness of antitumour immune responses and therapeutically may have an impact on immunological output.