Increased T-cell Infiltration Research Articles

A computational pipeline for identifying gene targets and signalling pathways in cancer cells to improve lymphocyte infiltration and immune checkpoint therapy efficacy

Tumour-infiltrating lymphocytes (TILs) are crucial for effective immune checkpoint blockade (ICB) therapy in solid tumours. However, ∼70% of these tumours exhibit poor lymphocyte infiltration, rendering ICB therapies less effective. We developed a bioinformatics pipeline integrating multiple previously unconsidered factors or datasets, including tumour cell immune-related pathways, copy number variation (CNV), and single tumour cell sequencing data, as well as tumour mRNA-seq data and patient survival data, to identify targets that can potentially improve T cell infiltration and enhance ICB efficacy. Furthermore, we conducted wet-lab experiments and successfully validated one of the top-identified genes. We applied this pipeline in solid tumours of the Cancer Genome Atlas (TCGA) and identified a set of genes in 18 cancer types that might potentially improve lymphocyte infiltration and ICB efficacy, providing a valuable drug target resource to be further explored. Importantly, we experimentally validated SUN1, which had not been linked to T cell infiltration and ICB therapy previously, but was one of the top-identified gene targets among 3 cancer types based on the pipeline, in a mouse colon cancer syngeneic model. We showed that Sun1 KO could significantly enhance antigen presentation, increase T-cell infiltration, and improve anti-PD1 treatment efficacy. Moreover, with a single-cell multiome analysis, we identified subgene regulatory networks (sub-GRNs) showing Stat proteins play important roles in enhancing the immune-related pathways in Sun1-KO cancer cells. This study not only established a computational pipeline for discovering new gene targets and signalling pathways in cancer cells that block T-cell infiltration, but also provided a gene target pool for further exploration in improving lymphocyte infiltration and ICB efficacy in solid tumours. A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Abatacept decreases renal T-cell infiltration and renal inflammation and ameliorates progressive renal injury in obese Dahl salt-sensitive rats before puberty.

Prepubertal obesity (PPO) is growing at an alarming rate and is now considered a risk factor for renal injury. Recently, we reported that the early development of renal injury in obese Dahl salt-sensitive leptin receptor mutant (SSLepRmutant) rats was associated with increased T-cell infiltration and activation prior to puberty. Therefore, the current study investigated the effect of inhibiting T-cell activation with abatacept on the progression of renal injury in young obese SSLepRmutant rats before puberty. Four-week-old SS and SSLepRmutant rats were treated with IgG or abatacept (1 mg/kg; ip, every other day) for 4 weeks. Abatacept reduced the renal infiltration of T-cells by almost 50% in SSLepRmutant rats. Treatment with abatacept decreased the renal expression of macrophage inflammatory protein-3 alpha (MIP-3α) while increasing IL-4 in SSLepRmutant rats without affecting SS rats. While not having an impact on blood glucose, abatacept reduced hyperinsulinemia and plasma triglycerides in SSLepRmutant rats without affecting SS rats. We did not observe any differences in MAP among the groups. Proteinuria was markedly higher in SSLepRmutant rats versus SS rats throughout the study, and treatment with abatacept decreased proteinuria by about 40% in SSLepRmutant rats without affecting SS rats. We observed significant increases in glomerular and tubular injury and renal fibrosis in SSLepRmutant rats vs SS rats, and chronic treatment with abatacept significantly reduced these renal abnormalities in SSLepRmutant rats. These data suggest that renal T-cell activation contributes to the early progression of renal injury associated with PPO.

Abstract 6127: Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals

Abstract Background: Approximately 50% of high-grade serous ovarian carcinomas (HGSOC) are homologous recombination deficient (HRD). HRD is associated with increased T-cell infiltration and improved survival. This study aimed to describe the distribution of HRD features and cytotoxic T-cell infiltration by gene expression subtype among Black HGSOC cases. Methods: We included Black HGSOC cases from the African American Cancer Epidemiology Study and the North Carolina Ovarian Cancer Study. We used RNASeq data to assign gene expression subtypes (n=160), and whole exome sequencing data from matched blood and tumor formalin-fixed paraffin-embedded specimens (n=206) to characterize HRD features. We defined HRD features as (1) germline variants and somatic mutations in HRD genes, and (2) HRD-associated signatures (SBS3 and ID6) that we obtained from de novo mutational signature analysis for single-base pair substitution, insertion, and deletion. We estimated the proportion of intratumoral cytotoxic T cells using multiplex immunofluorescence (n=125). Results: The subtype distribution in our study was 39% immunoreactive, 29% mesenchymal 27% proliferative, and 4% differentiated. Germline variants and somatic mutations in any HRD gene were observed in 27% and 23% of cases, respectively, and at least one of these was observed in 42% of cases. Pathogenic germline variants and somatic mutations were observed in 7% and 3% of cases, respectively. The prevalence of germline or somatic variants by gene was 16% for BRCA1, 14% for BRCA2, 11% for ATM, 5% for RAD51, and 2% each for BRIP1, PALB2, and BARD1. We observed a COSMIC HRD signature in 43% of cases, with 25% SBS3 only, 5% ID6 only, and 13% with both signatures. The prevalence of any HRD gene variant or signature was higher in immunoreactive cases (52% and 59%, respectively) than in mesenchymal (28% and 43%) or proliferative (44% and 28%) cases. Germline HRD variants were more common in the proliferative subtype (44%) compared with immunoreactive (25%) and mesenchymal (17%). In contrast, somatic HRD mutations were more common in the immunoreactive subtype (33%), compared with proliferative (16%) and mesenchymal (15%). This was due to a higher occurrence of BRCA1 somatic mutations in the immunoreactive subtype (25%) compared with proliferative (9%) and mesenchymal (8%). A higher proportion of cytotoxic T-cells (≥1% vs <1%) was also observed in the immunoreactive subtype (44%) compared with mesenchymal (31%) and proliferative (11%). Conclusion: We observed differences in the distribution of HRD features and cytotoxic T-cells by gene expression subtype. In particular, in the immunoreactive subtype, the most common gene expression subtype in Black HGSOC cases, we observed a higher occurrence of any HRD feature, somatic BRCA1 mutations, and cytotoxic T cell infiltration than in any of the other gene expression subtypes. Citation Format: Katherine Lawson-Michod, Mollie E. Barnard, Natalie Davidson, Lindsay J. Collin, Courtney Johnson, Lucas A. Salas, Casey Greene, Jeffrey R. Marks, Lauren Peres, Joellen M. Schildkraut, Jennifer A. Doherty. Characterization of germline and somatic homologous recombination deficiency features in high-grade serous ovarian cancer among Black individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6127.

Abstract 6516: Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound

Abstract Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, located in the brainstem, and traditionally thought to be immune-excluded with no significant infiltration by cytotoxic T-cells, and therefore not amenable to immunotherapies. Focused ultrasound technology (FUS) for drug delivery is being investigated for DIPG in both preclinical and clinical trial settings. In this study, we sought to study the effects of FUS and radiation therapy (RT) on immune cell infiltration in DIPG tumors to assess their ability to alter the immune microenvironment and potentially enhance combination immunotherapies. Specifically, we generated single-cell RNAseq profiles for 27,780 cells from syngeneic orthotopic DIPG tumors (KPAP cell line containing H3K27M mutation, ATRX and P53 loss, and PDGFRA overexpression) after 14 days of tumor growth across the following treatment arms: vehicle control, FUS, and RT (n=2 each group). FUS was applied by 4-point sonication around the tumor with a pressure of 0.6 Mpa at days 8 and 11 post tumor implantation (PTI). RT was administered in fractions of 3 Gy daily from days 8 to 11 PTI (12Gy total). We leveraged a set of algorithms for network-based inference of regulatory protein activity to infer a set of context-matched gene regulatory networks from which protein activity can be inferred by expression of downstream targets. By this approach, we identified distinct clusters of immune cells segregating by cell type as inferred by singleR and activity of known lineage markers. Strikingly, we found that both RT and FUS significantly depleted a TGFb-expressing population of immune-suppressive macrophages as compared to vehicle controls. Importantly, FUS only additionally increased T-cell infiltration into the tumor micro-environment over three-fold as compared to vehicle controls, which was not observed in response to RT. Further protein validation is underway. This has significant implications for therapeutic combinations of FUS-mediated drug delivery with immune checkpoint inhibitors in DIPG, such that in addition to drug delivery, FUS-mediated immune modulatory effects may provide more proinflammatory response to enhance immunotherapy. Citation Format: Erin White, Hong-Jian Wei, Ester Calvo Fernández, Hanna Minns, Jovana Pavisic, Robyn Gartrell, Cheng-Chia Wu, Andrea Califano, Aleksandar Obradovic. Single-cell protein activity inference reveals lymphocytic immune infiltration in diffuse intrinsic pontine glioma induced by focused ultrasound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6516.

Abstract 5311: KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity

Abstract Several anti-CD40 agonist antibodies have been evaluated in clinical trials based on their preclinical antitumor activity via generation of antitumor innate and adaptive immunity through activation of antigen-presenting cells. However, conventional monospecific anti-CD40 agonist antibodies have faced limitations in terms of safety margins and clinical efficacy. To address this problem, we developed KK2269, a novel bispecific antibody (REGULGENT™ format) that binds to both CD40 and epithelial cell adhesion molecule (EpCAM), which is highly expressed in several types of tumors. KK2269 demonstrated EpCAM-dependent activation of CD40 signals both in vitro and in vivo, confirming that it is a tumor-targeting CD40 agonist. Liver metastasis is common in EpCAM-positive cancers, including colon, stomach, and lung cancers. Intrahepatic tumors can attenuate the efficacy of immune checkpoint inhibitors due to the liver tumor-specific suppressive immune milieu. Although CD40 agonists are expected to overcome the immunosuppressive environment, non-tumor-targeting CD40 agonists cause hepatotoxicity. In the present study, we used a syngeneic intrahepatic tumor model to examine whether KK2269 exhibits antitumor efficacy without causing hepatotoxicity. Firstly, we confirmed that KK2269 administration did not increase aspartate aminotransferase (AST) or cause pathological abnormalities in the liver despite Il12b induction in the EpCAM-positive tumor. Additionally, gene expression analysis of peritumoral normal liver revealed that KK2269 did not induce inflammatory cytokines. In contrast, a conventional anti-CD40 agonist antibody increased AST, caused pathological abnormalities, and upregulated inflammatory cytokines in peritumoral normal liver, as reported in the clinical setting. Secondly, the antitumor effect of KK2269 in combination with docetaxel was evaluated. The combination therapy synergistically reduced intrahepatic tumor volume, and the effect persisted even 21 days after the administration. Gene expression analysis revealed a lower immune infiltration in the intrahepatic tumor versus subcutaneous tumor and that KK2269 in combination with docetaxel increased T-cell infiltration and activation even in the immunosuppressive intrahepatic tumor. In summary, our study demonstrated that KK2269 activated dendritic cells in an EpCAM-positive tumor in the liver but did not affect peritumoral normal liver tissues. In addition, KK2269 in combination with docetaxel showed potent antitumor effect accompanied by T-cell infiltration and activation against the intrahepatic tumor, which has a lower immune infiltration. These results suggest that the combination therapy of KK2269 and docetaxel is promising for metastatic EpCAM-positive tumors. Citation Format: Yoshiki Sumitomo, Kyoko Kawasaki, Yuta Tezuka, Masato Saito, Akari Yao, Munetoshi Ando. KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5311.

Interplay between ATRX and IDH1 mutations governs innate immune responses in diffuse gliomas

Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in ATRX, defining molecular alterations in IDH-mutant astrocytomas, have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this, we generated ATRX-deficient glioma models in the presence and absence of the IDH1R132H mutation. ATRX-deficient glioma cells are sensitive to dsRNA-based innate immune agonism and exhibit impaired lethality and increased T-cell infiltration in vivo. However, the presence of IDH1R132H dampens baseline expression of key innate immune genes and cytokines in a manner restored by genetic and pharmacological IDH1R132H inhibition. IDH1R132H co-expression does not interfere with the ATRX deficiency-mediated sensitivity to dsRNA. Thus, ATRX loss primes cells for recognition of dsRNA, while IDH1R132H reversibly masks this priming. This work reveals innate immunity as a therapeutic vulnerability of astrocytomas.

Abstract C023: PTEN/STAT3 pathway in cancer-associated fibroblasts in Pancreatic Cancer

Abstract Pancreatic cancer is a devastating disease with a high mortality rate with a five-year survival rate of 12%. Recent studies have shed light on the crucial role of cancer-associated fibroblasts (CAFs) in shaping the tumor microenvironment. Our group demonstrated PTEN loss occurs in CAFs in ~25% of PDAC patient samples, which leads to increased activation of signaling pathways in CAFs, including the STAT3 pathway. Conditional deletion of STAT3 in CAFs in a dual flpO/cre recombinase PDAC model correlated with increased survival, decreased M2-like macrophages and increased T-cell infiltration. In the present study we aimed to test the hypothesis that direct interactions between PTEN and STAT3 in cancer CAFs contributes to an immuno-suppressive tumor microenvironment in pancreatic ductal carcinoma (PDAC). To address this hypothesis, we utilized CAFS isolated from the dual recombinase PDAC model. Proximity ligation assays with these CAFS demonstrated that PTEN-STAT3 are in a complex in the cell cytosol that translocate to the nucleus upon IL6 treatment. This finding was validated by co-immunoprecipitation experiments. ChIP-seq analysis of CAFs with PTEN and pSTAT3/STAT3 antibodies revealed a significant overlap in PTEN and pSTAT3/STAT3 occupation of gene promoters. Integrative analysis of PTEN-STAT3 bound genes with corresponding RNA-seq data revealed that the PTEN/STAT3 complex targets cytokines and ligands that directly affect T-cell function, contributing to an immune suppressive microenvironment. Analysis of human PDAC samples support the relationship between STAT3 activation and T-cell activity. Identifying PTEN/STAT3 targets in CAFs may provide new avenues for overcoming the anti-tumor immune responses in PDAC. Citation Format: Samaneh Saberi, Cameron Bumbleburg, Victoria Jordan, Julia E. Lefler, Sudarshana Sharma, Michael C. Ostrowski. PTEN/STAT3 pathway in cancer-associated fibroblasts in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C023.

Intratumoral administration of the immunologic adjuvant AS01B in combination with autologous CD1c (BDCA-1)+/CD141 (BDCA-3)+ myeloid dendritic cells plus ipilimumab and intravenous nivolumab in patients with refractory advanced melanoma

BackgroundPatients with advanced melanoma who progress after treatment with immune checkpoint-inhibitors (ICI) and BRAF-/MEK-inhibitors (if BRAFV600 mutated) have no remaining effective treatment options. The presence of CD1c (BDCA-1)+ and CD141...

RALDH1 Inhibition Shows Immunotherapeutic Efficacy in Hepatocellular Carcinoma.

Globally, hepatocellular carcinoma (HCC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related death. We previously identified an immune evasion pathway whereby tumor cells produce retinoic acid (RA) to promote differentiation of intratumoral monocytes into protumor macrophages. Retinaldehyde dehydrogenase 1 (RALDH1), RALDH2, and RALDH3 are the three isozymes that catalyze RA biosynthesis. In this study, we have identified RALDH1 as the key driver of RA production in HCC and demonstrated the efficacy of RALDH1-selective inhibitors (Raldh1-INH) in suppressing RA production by HCC cells. Raldh1-INH restrained tumor growth in multiple mouse models of HCC by reducing the number and tumor-supporting functions of intratumoral macrophages as well as increasing T-cell infiltration and activation within tumors. Raldh1-INH also displayed favorable pharmacokinetic, pharmacodynamic, and toxicity profiles in mice thereby establishing them as promising new drug candidates for HCC immunotherapy.

The Combination of Nivolumab and CC-486 Is Active in Hodgkin Lymphoma Refractory to PD-1 Blockade

Introduction: PD1 blockade is highly active in patients (pts) with relapsed/refractory (RR) Hodgkin lymphoma (HL); however, most patients ultimately progress on therapy, and HL that is refractory to PD1 blockade remains an unmet need. Furthermore, as PD-1 directed therapies are being administered in earlier lines, it is anticipated that pts with RR HL will be increasingly refractory to PD-1 directed therapies. Hypomethylating agents (HMA) have been shown to increase T-cell infiltration and upregulate PD-1 and CTLA-4 expression in tumors, as well as suppress myeloid-derived suppressor cells. We hypothesized that HMA might restore the sentivity of HL to PD-1 directed therapies. Therefore, we conducted a phase I study evaluating the safety and efficacy of nivolumab and CC-486, an oral HMA, in pts with RR HL refractory to PD-1 blockade. Methods:Adult pts with RR HL who had progressed after at least 1 prior line of therapy and were refractory to PD-1 therapy were enrolled to receive oral CC-486 and nivolumab IV in 28-day cycles. PD-1 refractoriness was defined as progressive disease (PD) as best response, or PD while receiving or within 12 weeks of the last anti-PD-1 directed therapy. Pts were treated in a dose-finding cohort at 2 dose levels (DL) using a Rolling 6 design and then in a dose expansion cohort at the recommended phase 2 dose (RP2D). In DL1, CC-486 was administered at 200 mg PO daily on days 1-7, and in DL2 CC-486 was dosed at 300 mg PO daily on days 1-7; nivolumab 480 mg IV was given on day 8 in both DLs. Treatment could be continued for up to 2 years. Primary endpoints were determination of the RP2D and overall response rate (ORR) defined as the proportion of patients with a complete response (CR) or partial response (PR). Responses were assessed by investigators using PET-CT according to the 2014 Lugano Classification. Results: As of July 7, 2023, 21 pts have been enrolled including 3 at DL1 and 18 at DL2 (RP2D). At baseline, 57% were male with a median age of 40 years (range 25-71), 95% had stage III-IV disease, and 71% had primary refractory disease, defined as lack of a complete remission (CR) or relapse within 3 months of frontline treatment. The median number of prior therapies was 6 (range 2-14), and 90% had prior brentuximab vedotin (BV). 76% of pts had received pembrolizumab, 67% had received nivolumab, and 43% had received both agents previously. 52% had undergone autologous stem cell transplant and 5% had had prior allogeneic stem cell transplant. Baseline characteristics are shown in Table 1. The median number of cycles was 7 (range: 3-17). Among 19 pts evaluable for response (2 pending), ORR was 63% and CR rate was 10%. An additional 32% had SD as their best response; only one pt had PD as the best response. The median follow-up time was 9 months (range 2.8-16.3), and 71% of pts remain on treatment with an estimated median PFS of 11.3 months (95% CI: 7.1-N/A). 6 pts stopped therapy, including 4 for disease progression, 1 at treating physician's discretion and 1 due to patient preference. The most common adverse events (AEs), any grade (Gr), were nausea (86%), diarrhea (71%), vomiting (48%), headache (33%), fatigue (29%), neutropenia (19%), and anemia (19%). Only two Gr 3+ AEs were recorded: one patient experienced Gr 3 anemia and another Gr 4 hypercalcemia. Notably, the hypercalcemia fully resolved with supportive care and did not recur with subsequent cycles. The only immune-related AE was grade 1 hypophysitis in a single pt. Conclusions: Nivolumab combined with CC-486 is tolerable and elicits a high response rate in a PD-1 refractory cohort. At this time, the study has been amended to enroll 10 more patients at DL2 to provide a better estimation of the efficacy.

Oncolytic virus Ad-TD-nsIL-12 inhibits glioma growth and reprograms the tumor immune microenvironment

AimsGliomas are the most common central nervous system malignancies, with limited therapeutic options and poor prognosis, which are primarily attributed to the “immune desert” microenvironment. Previously, we constructed a three-gene-deleted oncolytic adenovirus (Ad-TD) loaded with non-secreting interleukin-12 (nsIL-12), which could be amplified in tumor cells and induce immunity to suppress tumors. However, the effects of this oncolytic virus on gliomas and their immune microenvironment remain unclear. There is an urgent need for further research. Materials and methodsWe constructed a Syrian hamster brain tumor model and demonstrated the efficacy and mechanism of the novel oncolytic virus in treating brain tumors through a series of in vitro and in vivo experiments. We investigated the efficacy and safety (the number of hamsters in each group is either 5 or 10) of the oncolytic virus treatment in Syrian hamsters using a virus-treated group, a control virus-treated group, and a blank control group. Key findingsIn vitro assays showed that Ad-TD-nsIL-12 could specifically proliferate in brain tumor cells which induce tumor cell apoptosis and intracellular expression of interleukin (IL)-12. Moreover, in vivo experiments demonstrated that Ad-TD-nsIL-12 could effectively inhibit the progression of brain tumors and prolong survival. Ad-TD-nsIL-12 significantly enhanced T-cell infiltration in the brain tumor microenvironment. SignificanceAd-TD-nsIL-12 can inhibit glioma progression and increase T-cell infiltration in the tumor tissue, particularly infiltration by cytotoxic T cells (CD8+). Ad-TD-nsIL-12 can amplify and produce IL-12, inducing anti-glioma immune responses to inhibit tumor progression.

Targeting GAS6/AXL signaling improves the response to immunotherapy by restoring the anti-immunogenic tumor microenvironment in gastric cancer

AimsImmunotherapy has shown remarkable effects on several malignancies; however, its impact on gastric cancers has been limited. Therefore, a novel strategy to overcome resistance to immunotherapy is required. In this study, we compared the gene expression profiles of two murine GC cell lines that exhibited different effects on tumor immunity. The functions of specific genes related to negative tumor immunity and the impact of a specific inhibitor were evaluated in syngeneic GC mouse models. Materials and methodsRT-PCR and Western blotting validated Gas6 and AXL expression in murine cell lines. RT-PCR compared YTN16 and YTN3 GC cell's impact on T cell activation. AXL, the receptor for GAS6 in YTN16, was validated by western blotting. Gas6 was inhibited in YTN16 cells using shRNA, and then the gene expression pattern, effects to T cell activation, and tumor growth were assessed. YTN16 cells were injected into mice and treated with CCB-3233, anti-PD-1 antibody, or both. Immunohistochemistry and flow cytometry evaluated tumor-infiltrating immune cells. Key findingsYTN16 cells expressed more Gas6 and had reduced T cell activation compared to YTN3 cells. AXL activation was higher in YTN16. CCB-3233 reduced AXL phosphorylation. Knocking down Gas6 in YTN16 reduced immunosuppression-related genes and increased tumor-infiltrating T cells. Combined CCB-3233 and anti-PD-1 treatment reduced tumor growth and increased T-cell infiltration. Human GC data revealed a negative correlation between GAS6 and immune activation-related genes. SignificanceThe GAS6/AXL pathway contributes to immunotherapy resistance in GC. Targeting this pathway may be a novel therapeutic strategy.

IMMU-15. INTERPLAY BETWEEN ATRXAND IDH1 MUTATIONS GOVERNS INNATE IMMUNE RESPONSES IN DIFFUSE GLIOMAS

Abstract Stimulating the innate immune system has been explored as a therapeutic option for the treatment of gliomas. Inactivating mutations in alpha-thalassemia retardation X-linked (ATRX) are defining molecular alterations in IDH-mutant astrocytomas and have been implicated in dysfunctional immune signaling. However, little is known about the interplay between ATRX loss and IDH mutation on innate immunity. To explore this interaction, we used both CRISPR and the RCAS/tva system to generate murine models of ATRX-deficient glioma, in the presence and absence of the IDH1 R132H mutation. We found that ATRX-deficient glioma cells were exquisitely sensitive to dsRNA-based innate immune agonism, displaying increases in inflammatory gene expression and profound cytokine release in vitro, while mice bearing ATRX KO tumors displayed increased T-cell infiltration and improved survival. The presence of IDH1 R132H dampened baseline expression of key innate immune genes and cytokines, with genetic and pharmacological inhibition of IDH1 R132H partially rescuing this suppression. Despite the suppression observed at baseline, IDH1 R132H expression did not interfere with the ATRX KO-mediated sensitivity to dsRNA agonism. These results demonstrate that ATRX loss primes cells for recognition of and activation against dsRNA, while IDH1 R132H reversibly masks this priming. Thus, this study suggests that innate immunity is a potential therapeutic vulnerability of astrocytoma. Further studies are in progress to validate the influence of ATRX loss on immune signaling and tumor-immune coevolution within human tumors.

ONCOS-102 plus pemetrexed and platinum chemotherapy in malignant pleural mesothelioma: a randomized phase 2 study investigating clinical outcomes and the tumor microenvironment

BackgroundONCOS-102, an oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor, can alter the tumor microenvironment to an immunostimulatory state. Combining ONCOS-102 with standard-of-care chemotherapy for malignant pleural mesothelioma (MPM) may improve treatment...

HER2Δ16 Engages ENPP1 to Promote an Immune-Cold Microenvironment in Breast Cancer.

The tumor-immune microenvironment (TIME) is a critical determinant of therapeutic response. However, the mechanisms regulating its modulation are not fully understood. HER2Δ16, an oncogenic splice variant of the HER2, has been implicated in breast cancer and other tumor types as a driver of tumorigenesis and metastasis. Nevertheless, the underlying mechanisms of HER2Δ16-mediated oncogenicity remain poorly understood. Here, we show that HER2∆16 expression is not exclusive to the clinically HER2+ subtype and associates with a poor clinical outcome in breast cancer. To understand how HER2 variants modulated the tumor microenvironment, we generated transgenic mouse models expressing either proto-oncogenic HER2 or HER2Δ16 in the mammary epithelium. We found that HER2∆16 tumors were immune cold, characterized by low immune infiltrate and an altered cytokine profile. Using an epithelial cell surface proteomic approach, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) as a functional regulator of the immune cold microenvironment. We generated a knock-in model of HER2Δ16 under the endogenous promoter to understand the role of Enpp1 in aggressive HER2+ breast cancer. Knockdown of Enpp1 in HER2Δ16-derived tumor cells resulted in decreased tumor growth, which correlated with increased T-cell infiltration. These findings suggest that HER2Δ16-dependent Enpp1 activation associates with aggressive HER2+ breast cancer through its immune modulatory function. Our study provides a better understanding of the mechanisms underlying HER2Δ16-mediated oncogenicity and highlights ENPP1 as a potential therapeutic target in aggressive HER2+ breast cancer.

Granular analysis of individual immune-related gene expression in a randomized phase I/II study of the oncolytic adenovirus, ONCOS-102, in combination with pemetrexed/cisplatin (P/C) in patients (pts) with unresectable malignant pleural mesothelioma (MPM).

e20536 Background: ONCOS-102 is an oncolytic adenovirus expressing granulocyte-macrophage colony stimulating factor (GM-CSF; Ad5/3-D24-GMCSF) that stimulates immune responses and modulates the tumor microenvironment, both as a monotherapy and in combination with programmed cell death protein 1 (PD-1) blockade. We present granular analyses of individual immune-related gene expression in response to ONCOS-102 in a randomized phase I/II study in MPM. Methods: Following a safety run-in (n = 6), 25 pts were randomized to receive ONCOS-102 (3 x 1011 virus particles in 2.5 mL) intratumorally under CT or US guidance on Days 1, 4, 8, 36, 78 and 120, plus six cycles of P/C starting on Day 22 (ONCOS+P/C), or six cycles of P/C only (control). Both treatment-naïve (1L) and previously treated patients were enrolled. Survival was followed up to 30 months. Tumor biopsies were collected at baseline and Day 36, and immunofluorescence for immune cell subsets, RNASeq and qPCR were performed. Results: In the pooled population (n = 31), median overall survival (OS) was 16.6 vs 18.3 months for ONCOS-102 + P/C vs control arms ( P> 0.05). In 1L patients (n = 17) median OS was 20.3 vs 13.5 months ( P> 0.05), and 30-month OS rate was 34.1% vs 0. A significant increase in tumour-infiltrating immune cells was observed (baseline to Day 36) with ONCOS-102 + P/C (n = 20) but not control (n = 11) using immunofluorescence and transcriptome analysis. CD8+, GranzymeB+ CD8+ cells, and CD8+:regulatory T-cell and M1:M2 macrophage ratios increased in ONCOS-102-treated pts (P≤0.016). A difference in tumor immune-infiltration was observed in ONCOS-102 + P/C pts stratified by survival at Month 18: while deregulation of complement pathways and inflammatory response was seen at baseline, at Day 36 adaptive immune response and T-cell activation were observed in pts who were alive but not in those deceased. Additionally, GM-CSF protein, likely derived from ONCOS-102, was transiently present in higher concentration at Days 4 and 8 in 18-month survivors vs deceased pts. Based on a previously described predictive inflammatory gene signature for treatment with checkpoint inhibitors in mesothelioma, a binomial model assessing baseline transcriptome levels demonstrated 89% accuracy in predicting 18-month survival. Conclusions: ONCOS-102 caused increased T-cell infiltration and upregulated immune response-related gene expression in tumor lesions. A trend for prolonged OS was observed in the 1L setting, only. Consistent with observations in PD-1 resistant melanoma, ONCOS-102-induced immune response in the tumor is hallmarked by a high prevalence of cytotoxic T-cells and depolarization of macrophages, which may support addition of checkpoint inhibitors to the combination of ONCOS-102 plus P/C in MPM. Clinical trial information: NCT02879669 .

T cell-Mediated Development of Stromal Fibroblasts with an Immune-Enhancing Chemokine Profile.

Stromal fibroblasts reside in inflammatory tissues that are characterized by either immune suppression or activation. Whether and how fibroblasts adapt to these contrasting microenvironments remains unknown. Cancer-associated fibroblasts (CAF) mediate immune quiescence by producing the chemokine CXCL12, which coats cancer cells to suppress T-cell infiltration. We examined whether CAFs can also adopt an immune-promoting chemokine profile. Single-cell RNA sequencing of CAFs from mouse pancreatic adenocarcinomas identified a subpopulation of CAFs with decreased expression of Cxcl12 and increased expression of the T cell-attracting chemokine Cxcl9 in association with T-cell infiltration. TNFα and IFNγ containing conditioned media from activated CD8+ T cells converted stromal fibroblasts from a CXCL12+/CXCL9- immune-suppressive phenotype into a CXCL12-/CXCL9+ immune-activating phenotype. Recombinant IFNγ and TNFα acted together to augment CXCL9 expression, whereas TNFα alone suppressed CXCL12 expression. This coordinated chemokine switch led to increased T-cell infiltration in an in vitro chemotaxis assay. Our study demonstrates that CAFs have a phenotypic plasticity that allows their adaptation to contrasting immune tissue microenvironments.

Supramolecular Filament Hydrogel as a Universal Immunomodulator Carrier for Immunotherapy Combinations.

A major challenge of cancer immunotherapy is to develop delivery strategies that can effectively and safely augment the immune system's antitumor response. Here, we report on the design and synthesis of a peptide-based supramolecular filament (SF) hydrogel as a universal carrier for localized delivery of three immunomodulating agents of distinct action mechanisms and different molecular weights, including an aPD1 antibody, an IL15 cytokine, and a STING agonist (CDA). We show that in situ hydrogelation can be triggered to occur upon intratumoral injection of SF solutions containing each of aPD1, IL15, or CDA. The formed hydrogel serves as a scaffold depot for sustained and MMP-2-responsive release of immunotherapeutic agents, achieving enhanced antitumor activities and reduced side effects. When administered in combination, the aPD1/IL15 or aPD1/CDA hydrogel led to substantially increased T-cell infiltration and prevented the development of adaptive immune resistance induced by IL15 or CDA alone. These immunotherapy combinations resulted in complete regression of established large GL-261 tumors in all mice and elicited a protective long-acting and systemic antitumor immunity to prevent tumor recurrence while eradicating distant tumors. We believe this SF hydrogel offers a simple yet generalizable strategy for local delivery of diverse immunomodulators for enhanced antitumoral response and improved treatment outcomes.

Sequential treatment with a TNFR2 agonist and a TNFR1 antagonist improves outcomes in a humanized mouse model for MS

TNF signaling is an essential regulator of cellular homeostasis. Through its two receptors TNFR1 and TNFR2, soluble versus membrane-bound TNF enable cell death or survival in a variety of cell types. TNF-TNFRs signaling orchestrates important biological functions such as inflammation, neuronal activity as well as tissue de- and regeneration. TNF-TNFRs signaling is a therapeutic target for neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer’s disease (AD), but animal and clinical studies yielded conflicting findings. Here, we ask whether a sequential modulation of TNFR1 and TNFR2 signaling is beneficial in experimental autoimmune encephalomyelitis (EAE), an experimental mouse model that recapitulates inflammatory and demyelinating aspects of MS. To this end, human TNFR1 antagonist and TNFR2 agonist were administered peripherally at different stages of disease development in TNFR-humanized mice. We found that stimulating TNFR2 before onset of symptoms leads to improved response to anti-TNFR1 therapeutic treatment. This sequential treatment was more effective in decreasing paralysis symptoms and demyelination, when compared to single treatments. Interestingly, the frequency of the different immune cell subsets is unaffected by TNFR modulation. Nevertheless, treatment with only a TNFR1 antagonist increases T-cell infiltration in the central nervous system (CNS) and B-cell cuffing at the perivascular sites, whereas a TNFR2 agonist promotes Treg CNS accumulation. Our findings highlight the complicated nature of TNF signaling which requires a timely balance of selective activation and inhibition of TNFRs in order to exert therapeutic effects in the context of CNS autoimmunity.

Abstract CT206: A phase I study of binimetinib, a MEK inhibitor, in combination with pembrolizumab in patients with advanced non-small cell lung cancer (NSCLC)

Abstract Introduction: MEK inhibition combined with PD-1 axis inhibition may achieve a greater clinical response than either inhibitor alone due to increased T-cell infiltration of tumours. We combined binimetinib with pembrolizumab in patients with stage IV advanced non-small cell lung cancer (NSCLC) and PD-L1 tumor proportion score>=50%. Methods: A 3 + 3 dose escalation design was used. Binimetinib at a dose level 1 (DL1; 45 mg) or dose level -1 (DL-1; 30 mg) twice daily orally continuously was given with pembrolizumab 200 mg IV q 21 days. The primary objective was to define the recommended phase II dose (RP2D) of the combination. Secondary outcomes included safety of the combination and response (RECIST 1.1) with a planned Phase Ib expansion in patients with RAS/RAF/MEK dysregulated tumours via next generation gene sequencing. Genomic markers are being explored in tissue and plasma. Results: Eleven patients (3 DL1, 8 DL-1) were enrolled with the following mutations: 7 with KRAS (3 G12C, 2 G12D, 1 G12V, 1 G12A), 2 BRAF (1 G409A, 1 V600E) and 1 STK11 which acts as a tumor suppressor gene encoding for LKB1. Two of 3 patients at DL1 experienced dose limiting toxicity (DLT) including grade 3 elevated amylase, grade 3 diarrhea, grade 4 elevated lipase and severe grade 2 rash requiring dose reduction. Of 8 patients treated at DL-1, 1 progressed in cycle 1, another was noncompliant with treatment. Of the remaining 6 patients, 1 experienced DLT with grade 3 rash with inability to administer binimetinib for >75% of cycle 1. The most common toxicities across all cycles (n=11 patients) were: rash (82%), diarrhea (36%) and pruritis (36%). Nine patients were evaluated for response with partial response in 3 (33%), stable disease in 4 (44%) and progressive disease in 2 (22%). All 3 responding patients had RAS or RAF alterations (KRASG12C, KRASG12V, BRAFV600E) while the patient with STK11 mutant disease had early disease progression. Conclusion: The RP2D of the combination in patients with advanced NSCLC is binimetinib 30 mg BID plus pembrolizumab 200 mg IV q21 days. Updated data from cohort expansion including response and molecular correlates of treatment response and resistance will be presented. Citation Format: Christopher F. Theriau, Jamie Feng, Lawson Eng, Frances A. Shepherd, Mary DeCarolis, Vivian Glenns, Swati Kulkarni, Rachel VanderMeer, Urszula Zurawska-Fortin, Desiree Hao, Lisa Le, Isabel Wozniczka, Aida Al-Kindy, Danny Xie, Tong Zhang, Vasanth Subramanian, Tracy Stockley, Penelope A. Bradbury, Geoffrey Liu, Natasha B. Leighl. A phase I study of binimetinib, a MEK inhibitor, in combination with pembrolizumab in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT206.