Host Antitumor Immune Response Research Articles

Abstract 2655: Unexpected role of the NuRD component, Chd4, in Foxp3+ Treg cells and its relevance to tumor immunotherapy

Abstract We are investigating basic questions like why do Foxp3+ T-regulatory (Treg) cells have at least 3 nuclear complexes involving Hdac1 and Hdac2 (CoREST, Sin3, NuRD), and how can the inhibitory effects of Foxp3+ Tregs on host anti-tumor immune responses be targeted therapeutically? Here, we report on the immune functions of chromodomain-helicase-DNA-binding protein 4 (Chd4), a key enzyme of the nucleosome remodeling and deacetylase complex (NuRD) complex. TCGA analysis showed increased Chd4 expression and Foxp3+ Tregs within lung tumors, leading us to assess the effects of conditional gene deletion within Foxp3+ murine Tregs. Chd4 knockout led to systemic autoimmunity and death of mice within 3 weeks of life and RNAseq studies showed Chd4 loss led to derepression of ~1400 genes and repression of >700 further genes resulting in a Treg signature markedly different from conditional deletion of Hdac1, Hdac2 or Mbd2 in Treg cells. Chd4 is an intrinsically disordered therapeutic target without a defined structure outside its physiological cell environment. To date, no specific inhibitors targeting Chd4 have been developed. We have identified Ch41, a novel and potent inhibitor of Chd4, using a novel cellular target engagement platform. Transcriptomic and mass-spec investigations showed that Ch41 impaired thymic Treg function and iTreg development, led to decreased CNS2 demethylation within the Foxp3 locus, and significantly impaired growth of lung tumors and hepatocellular carcinomas in immunocompetent but not in immunodeficient C57BL/6 mice (p<0.01). Following Chd4 inhibitor therapy, intratumoral conventional T cells had increased activation and cytokine production (IL-2, IFN-g) compared to intratumoral T cells in control tumor-bearing mice but, perhaps unexpectedly, treated mice did not show histologic or biochemical evidence of autoimmunity. We conclude that while tumor associated Tregs have unique properties that include their increased suppressive activity compared to non-tumor associated Treg cells, they are also more susceptible than the latter and conventional T cells to newly developed therapeutic interventions, including targeting of the Chd4 chromodomain of the NuRD complex. Citation Format: Wayne W. Hancock, Liqing Wang, Martina Minisini, Eros di Giorgio, Ivan Babic, Elmar Nurmemmedov. Unexpected role of the NuRD component, Chd4, in Foxp3+ Treg cells and its relevance to tumor immunotherapy [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 2655.

Abstract 4266: Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation

Abstract Lung cancer is the leading cause of cancer related deaths worldwide, with the most prevalent subtype being non-small cell lung cancer (NSCLC). As only a subset of patients exhibits durable clinical responses to current treatments, including targeted therapy and immunotherapy, new treatment options are needed. Integrins (ITGs) are a diverse class of heterodimeric, transmembrane receptors that bind extracellularly to the extracellular matrix (ECM) and intracellularly to the cytoskeleton to integrate signals bi-directionally between the extracellular environment and cell interior. One of 18 α subunits is paired with one of 6 β subunits to create the 24 known αβ ITGs in mammals. ITGs are expressed at low levels in normal tissue and can be upregulated in cancer cells in response to stress signals in the tumor microenvironment (TME). Altered expression of ITGs is associated with enhanced cell proliferation, metastasis, and immune evasion. Targeting of the diverse ITG family members by inhibitors not specific to individual isoforms has obscured understanding of potentially distinct functions of each heterodimer. Through data mining of a genome wide CRISPR screening publicly available at the DepMap portal, we have identified a novel cancer dependency to ITG αvβ5 in approximately 40% of NSCLC cell lines. Using small molecule ITG αv inhibitors, we have mirrored sensitivity of NSCLC cell lines to their ITG αvβ5 CRISPR scores, suggesting that these inhibitors function specifically through ITG αvβ5 in cell lines. RNA sequencing analysis following αvβ5 inhibition in human NSCLC cell lines revealed downregulation of YAP and E2F1 target genes with alterations in gene ontologies associated with cell cycling and cell death in sensitive, but not resistant, lines. This is consistent with the known function of YAP driving gene transcription promoting cell proliferation and survival, and of E2F1 being implicated in regulating the proliferation and self-renewal of cancer stem cell (CSC). Additional changes to immune processes, specifically perturbations to interferon (IFN) pathways, were identified in response to ITG αvβ5 inhibition, suggesting that ITG αvβ5 may play a role in host anti-tumor immune responses. Pharmacological inhibition of ITG αvβ5 also led to a reduction of tumor growth in NSCLC murine models accompanied with increased CD8 T cell activation. These findings support ITG αvβ5 as a unique target of cancer dependency in NSCLC whose inhibition promotes anti-tumor responses through both tumor intrinsic and host immune regulatory mechanisms. Citation Format: Bitta P. Kahangi, William P. Crosson, Ji-Ann Lee, Camelia Dumitras, Tianhao Zhang, Ramin Salehi-Rad, Linh M. Tran, Michael Palazzolo, Dylan Conklin, Steven M. Dubinett, Bin Liu. Evaluation of integrin alpha v beta 5 as a novel target for non-small cell lung cancer via tumor intrinsic and host immune regulation [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 4266.

ALKBH5-mediated upregulation of CPT1A promotes macrophage fatty acid metabolism and M2 macrophage polarization, facilitating malignant progression of colorectal cancer

m6A modification has been studied in tumors, but its role in host anti-tumor immune response and TAMs polarization remains unclear. The fatty acid oxidation (FAO) process of TAMs is also attracting attention.A co-culture model of colorectal cancer (CRC) cells and macrophages was used to simulate the tumor microenvironment. Expression changes of m6A demethylase genes FTO and ALKBH5 were screened. ALKBH5 was further investigated. Gain-of-function experiments were conducted to study ALKBH5's effects on macrophage M2 polarization, CRC cell viability, proliferation, migration, and more. Me-RIP and Actinomycin D assays were performed to study ALKBH5's influence on CPT1A, the FAO rate-limiting enzyme. AMP, ADP, and ATP content detection, OCR measurement, and ECAR measurement were used to explore ALKBH5's impact on macrophage FAO level. Rescue experiments validated ALKBH5's mechanistic role in macrophage M2 polarization and CRC malignant development.In co-culture, CRC cells enhance macrophage FAO and suppress m6A modification in M2 macrophages. ALKBH5 was selected as the gene for further investigation. ALKBH5 mediates CPT1A upregulation by removing m6A modification, promoting M2 macrophage polarization and facilitating CRC development. These findings indicate that ALKBH5 enhances fatty acid metabolism and M2 polarization of macrophages by upregulating CPT1A, thereby promoting CRC development.

Phase I Trial of Systemic Administration of Vesicular Stomatitis Virus Genetically Engineered to Express NIS and Human Interferon, in Patients with Relapsed or Refractory Multiple Myeloma, Lymphomas, or Histiocytic/Dendritic Cell Neoplasms

Background: Oncolytic virotherapy, an innovative immunomodulatory therapeutic approach, is currently being investigated for its potential in treating relapsed refractory (R/R) hematologic malignancies. This study focuses on the use of an engineered Vesicular Stomatitis Virus called VSV-IFNβ-NIS, a bullet-shaped rhabdovirus which encodes both interferon beta (IFNβ) and sodium iodine symporter (NIS) with particular tumor cell tropism. Viral IFNβ serves as an indicator of viral proliferation and boosts the host's anti-tumor immune response. This is a phase 1 clinical trial with the identifier NCT03017820, wherein patients (pts) with R/R multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) are treated with a single systemic administration of VSV-IFNβ-NIS. Study Design and Methods: Patient Eligibility Criteria: The Mayo Clinic Institutional Review Board approved the protocol. The study includes adult patients with relapsed or refractory multiple myeloma (MM), or T-cell lymphoma (TCL). For MM or plasmacytoma, patients should have <15% plasma cells and a largest diameter of <5 cm. TCL patients should have a mass <5 cm. Study Design: This is a single-center phase 1 trial aiming to determine the maximum tolerated dose of VSV-IFNβ-NIS in patients with R/R MM, and TCL. Secondary objectives included assessing the safety profile and estimating the clinical response rate of VSV-IFNβ-NIS. Correlative objectives involved monitoring viral replication through plasma IFN-β transgene levels and using SPECT/CT imaging with the NIS gene. Viral genomes were measured through quantitative reverse transcription (qRT) polymerase chain reaction (PCR) of VSV RNA, and any virus shedding was examined in body fluids. The tested doses were 5 × 109 TCID50, 1.7 × 1010, 5 × 1010, and 1.7 × 1011 TCID50. The virus was administered via IV infusion over 30 minutes, and patients received 650 mg acetaminophen and 50 mg diphenhydramine hydrochloride before infusion and every 6 hours for 24 hours to manage fever and hypotension. Two expansion cohorts at DL4 have been added (20 additional PTCL, 10 B-cell lymphoma and histiocytic neoplasms) to obtain additional data on tolerability and efficacy and biomarkers of response in these histologies. Safety and Response Assessments: All patients undergo toxicity evaluation, and adverse events (AEs) assessment using the Common Terminology Criteria for Adverse Events (CTCAE) version 4 criteria, except for cytokine release syndrome (CRS) grading, which follows the 2014 Lee criteria. Dose escalation and tolerability are based on the standard 3+3 phase 1 trial design. Ruxolitinib is added from days 2-5 or day -1 to day 9 depending on the arm to mitigate IFNβ toxicity. Response assessment followed the International Myeloma Working Group criteria for MM and the Lugano Classification for NHL and histiocytic neoplasms. Pharmacodynamic and Pharmacokinetic Analyses: For viremia and viral shedding assessment, RNA is isolated from whole blood, mouth rinse, buccal swabs, and urine samples. Quantitative RT-PCR determined VSV-nucleocapsid (N) RNA copy number, and virus shedding is assessed through coculturing samples on Vero cells. Anti-VSV antibodies are evaluated using the standard plaque reduction neutralization assay. Plasma cytokine levels are measured with a 30-plex human cytokine kit, and human IFN-β levels is quantified using an enzyme-linked immunosorbent assay (ELISA) kit. Statistical Analysis: Incidence of dose-limiting toxicities and AEs are tabulated by dose level (DL). AEs are summarized by type, maximum grade, and duration across patients in each DL. Spearman rank correlation tests are used evaluated correlations between laboratory-based measures, and Wilcoxon rank sum tests compared continuous measures between response groups. Response rates will be summarized by DL and disease group, with estimated response rates and corresponding 95% binomial confidence intervals calculated.

Cell Death Pathway Regulation by Functional Nanomedicines for Robust Antitumor Immunity.

Cancer immunotherapy has become a mainstream cancer treatment over traditional therapeutic modes. Cancer cells can undergo programmed cell death including ferroptosis, pyroptosis, autophagy, necroptosis, apoptosis and cuproptosis which are find to have intrinsic relationships with host antitumor immune response. However, direct use of cell death inducers or regulators may bring about severe side effects that can also be rapidly excreted and degraded with low therapeutic efficacy. Nanomaterials are able to carry them for long circulation time, high tumor accumulation and controlled release to achieve satisfactory therapeutic effect. Nowadays, a large number of studies have focused on nanomedicines-based strategies through modulating cell death modalities to potentiate antitumor immunity. Herein, immune cell types and their function are first summarized, and state-of-the-art research progresses in nanomedicines mediated cell death pathways (e.g., ferroptosis, pyroptosis, autophagy, necroptosis, apoptosis and cuproptosis) with immune response provocation are highlighted. Subsequently, the conclusion and outlook of potential research focus are discussed.

The Value of Tumor Infiltrating Lymphocytes (TIL) for Predicting the Response to Neoadjuvant Chemotherapy (NAC) in Breast Cancer according to the Molecular Subtypes.

The antitumor host immune response is an important factor in breast cancer, but its role is not fully established. The role of tumor infiltrating lymphocytes (TIL) as an immunological biomarker in breast cancer has been significantly explored in recent years. The number of patients treated with neoadjuvant chemotherapy (NAC) has increased and the identification of a biomarker to predict the probability of pCR (pathological complete response) is a high priority. We evaluated 334 cases of BC treated with NAC followed by surgical resection from 2020-2022 at the Ist Clinic of Oncological Surgery, Oncological Institute "Prof Dr I Chiricuta" Cluj Napoca. Of the above, 122 cases were available for histological evaluation both in pre-NAC biopsy and post-NAC resection tissue. Evaluation of biopsy fragments and resection parts were performed using hematoxylin eosin (H&E). The TIL evaluation took place according to the recommendations of the International TIL Working Group (ITILWG). There was a strong association between elevated levels of pre-NAC TIL. At the same time, there is a statistically significant correlation between stromal TIL and tumor grade, the number of lymph node metastases, the molecular subtype and the number of mitoses (p < 0.005). Intratumoral TIL showed a significant correlation with tumor size, distant metastasis, molecular subtype, number of mitosis, stage and lymph node metastasis (p < 0.005). We also demonstrated that high pre-NAC STIL represents a strong predictive marker for pCR. This study reveals the role of TIL as a predictive biomarker in breast cancer not only for the well-established TNBC (triple negative breast cancer) and HER2+ (Her2 overexpressed) subtypes but also in Luminal A and B molecular subtypes. In this scenario, the evaluation of sTIL as a novel predictive and therapy-predicting factor should become a routinely performed analysis that could guide clinicians when choosing the most appropriate therapy.

The kynurenine pathway presents multi-faceted metabolic vulnerabilities in cancer.

The kynurenine pathway (KP) and associated catabolites play key roles in promoting tumor progression and modulating the host anti-tumor immune response. To date, considerable focus has been on the role of indoleamine 2,3-dioxygenase 1 (IDO1) and its catabolite, kynurenine (Kyn). However, increasing evidence has demonstrated that downstream KP enzymes and their associated metabolite products can also elicit tumor-microenvironment immune suppression. These advancements in our understanding of the tumor promotive role of the KP have led to the conception of novel therapeutic strategies to target the KP pathway for anti-cancer effects and reversal of immune escape. This review aims to 1) highlight the known biological functions of key enzymes in the KP, and 2) provide a comprehensive overview of existing and emerging therapies aimed at targeting discrete enzymes in the KP for anti-cancer treatment.

Dendritic Cell Vaccination in Non-Small Cell Lung Cancer: Remodeling the Tumor Immune Microenvironment.

Non-small-cell lung cancer (NSCLC) remains one of the leading causes of death worldwide. While NSCLCs possess antigens that can potentially elicit T cell responses, defective tumor antigen presentation and T cell activation hinder host anti-tumor immune responses. The NSCLC tumor microenvironment (TME) is composed of cellular and soluble mediators that can promote or combat tumor growth. The composition of the TME plays a critical role in promoting tumorigenesis and dictating anti-tumor immune responses to immunotherapy. Dendritic cells (DCs) are critical immune cells that activate anti-tumor T cell responses and sustain effector responses. DC vaccination is a promising cellular immunotherapy that has the potential to facilitate anti-tumor immune responses and transform the composition of the NSCLC TME via tumor antigen presentation and cell-cell communication. Here, we will review the features of the NSCLC TME with an emphasis on the immune cell phenotypes that directly interact with DCs. Additionally, we will summarize the major preclinical and clinical approaches for DC vaccine generation and examine how effective DC vaccination can transform the NSCLC TME toward a state of sustained anti-tumor immune signaling.

Cancer Cell Membrane-Coated Nanoparticle Co-loaded with Photosensitizer and Toll-like Receptor 7 Agonist for the Enhancement of Combined Tumor Immunotherapy.

Tumor immunotherapy has shown considerable therapeutic potential in the past few years, but the clinical response rate of immunotherapy is less than 20%. Encountering the high heterogeneity of tumors, it will be a general trend to apply combined therapy for cancer treatment. Photodynamic therapy (PDT) transiently kills tumor cells by producing reactive oxygen species (ROS), while residual tumor cells are prone to metastasis, leading to tumor recurrence. In combination with tumor immunotherapy, it is hoped to awaken the host immune system and eradicate residual tumor cells. Herein, cancer cell membrane-coated nanoparticles as a platform to combine PDT, TLR7 agonist, and tumor antigen for the enhancement of tumor therapeutic efficacy are designed. The final biomimetic nanoparticles (CCMV/LTNPs) can specifically kill tumor cells through PDT, while strong host antitumor immune responses are elicited to eliminate residue tumor cells under the help of immune adjuvant and tumor antigen from the cancer cell membrane. In summary, a photoimmunotherapy strategy is designed that synergistically enhances the tumor therapeutic effects by killing tumor cells through PDT and activating host antitumor immune responses through the co-delivery of adjuvant and tumor antigen, which may offer a promising strategy for clinical immunotherapy in the future.

Tumoral P2Y2 receptor modulates tumor growth and host anti-tumor immune responses in a syngeneic murine model of oral cancer.

Head and neck squamous cell carcinomas (HNSCCs) are a heterogenous group of tumors and among the top 10 most common cancers and they arise from the epithelial tissues of the mucosal surfaces of the oral cavity, oropharynx, and larynx. Aberrant purinergic signaling has been associated with various cancer types. Here, we studied the role ofthe P2Y2 purinergic receptor (P2Y2R) in the context of oral cancer. We utilized bioinformatics analysis of deposited datasets to examine purinome gene expression in HNSCC tumors and cells lines and functionally characterized nucleotide-induced P2 receptor signaling in human FaDu and Cal27 and murine MOC2 oral cancer cell lines. Utilizing tumorigenesis assays with wild-type or P2ry2 knockout MOC2 cells we evaluated the role of P2Y2Rs in tumor growth and the host anti-tumor immune responses. Our data demonstrate that human and murine oral cancer cell lines express numerous P2 receptors, with the P2Y2R being highly expressed. Using syngeneic tumor graftsin wild-type mice, we observed that MOC2 tumors expressing P2Y2R were larger than P2Y2R-/- tumors. Wild-type MOC2 tumors contained a lower population of tumor-infiltrating CD11b+F4/80+ macrophages and CD3+cells, which were revealed to be CD3+CD4+IFNγ+ T cells, compared to P2Y2R-/- tumors. These results were mirrored when utilizing P2Y2R-/- mice, indicating that the changes in MOC2 tumor growth and to the host anti-tumor immune response were independent of host derived P2Y2Rs. Results suggest that targeted suppression of the P2Y2R in HNSCC cells in vivo, rather than systemic P2Y2R antagonism, may be a more effective treatment strategy for HNSCCs.

Thrombin Cleavage of Osteopontin and the Host Anti-Tumor Immune Response.

Osteopontin (OPN) is a multi-functional protein that is involved in various cellular processes such as cell adhesion, migration, and signaling. There is a single conserved thrombin cleavage site in OPN that, when cleaved, yields two fragments with different properties from full-length OPN. In cancer, OPN has tumor-promoting activity and plays a role in tumor growth and metastasis. High levels of OPN expression in cancer cells and tumor tissue are found in various types of cancer, including breast, lung, prostate, ovarian, colorectal, and pancreatic cancer, and are associated with poor prognosis and decreased survival rates. OPN promotes tumor progression and invasion by stimulating cell proliferation and angiogenesis and also facilitates the metastasis of cancer cells to other parts of the body by promoting cell adhesion and migration. Furthermore, OPN contributes to immune evasion by inhibiting the activity of immune cells. Thrombin cleavage of OPN initiates OPN's tumor-promoting activity, and thrombin cleavage fragments of OPN down-regulate the host immune anti-tumor response.

βhCG mediates immune suppression through upregulation of CD11b+ Gr1+ myeloid derived suppressor cells, CD206+ M2 macrophages, and CD4+ FOXP3+ regulatory T-cells in BRCA1 deficient breast cancers.

BRCA1 mutation is reported in about 70% of all triple negative breast cancers (TNBC), while BRCA1 defect due to promoter hypermethylation is seen in about 30%-60% of sporadic breast cancers. Although PARP inhibitors and platinum-based chemotherapy are used to treat these cancers, more efficient therapeutic approaches are required to overcome the resistance to treatment. Our previous findings have reported elevated βhCG expression but not αhCG in BRCA1 deficient breast cancers. As βhCG causes immune suppression in pregnancy, this study explored the immunomodulatory effect of βhCG in BRCA1mutated/deficient TNBC. We observed that Th1, Th2, and Th17 cytokines are upregulated in the presence of βhCG in BRCA1 defective cancers. In NOD-SCID and syngeneic mouse models, βhCG increases the frequency of Myeloid-derived suppressor cells in tumour tissues and contributes to macrophage reprogramming from antitumor M1 to pro-tumour M2 phenotype. βhCG reduces the CD4+ T-cell infiltration while increasing the density of CD4+ CD25+ FOXP3+ regulatory T-cell in BRCA1 deficient tumour tissues. In contrast, xenograft tumours with βhCG knocked down TNBC cells did not show these immune suppressive effects. We have also shown that βhCG upregulates pro-tumorigenic markers arginase1(Arg1), inducible nitric oxide synthase, PD-L1/PD-1, and NFκB in BRCA1 defective tumours. Thus, for the first time, this study proves that βhCG suppresses the host antitumor immune response and contributes to tumour progression in BRCA1 deficient tumours. This study will help develop new immunotherapeutic approaches for treating BRCA1 defective TNBC by regulating βhCG.

SIRPα blockade improves the antitumor immunity of radiotherapy in colorectal cancer

High-dose hypofractionated radiotherapy (HRT) is an important anticancer treatment modality that activates antitumor host immune responses. However, HRT for oligometastases of colorectal cancer (CRC) has shown frustrating results in the clinic. As part of immune evasion, myeloid cells express signal regulatory protein α (SIRPα) to inhibit phagocytosis by phagocytes in the tumor microenvironment (TME). We postulated that SIRPα blockade enhances HRT by alleviating the inhibitory action of SIRPα on phagocytes. We demonstrated that SIRPα on myeloid cells was upregulated in the TME after HRT. When SIRPα blockade was administered with HRT, we observed superior antitumor responses compared with anti-SIRPα or HRT alone. When anti-SIRPα was administered to local HRT, the TME could become a tumoricidal niche that was heavily infiltrated by activated CD8+ T cells, but with limited myeloid-derived suppressor cells and tumor-associated macrophages. While CD8+ T cells were required for the effectiveness of the anti-SIRPα + HRT combination. The triple therapy with anti-SIRPα + HRT + anti-PD-1 had superior antitumor responses compared with the combination of any two therapies and established a strong and long-lasting adaptive immunological memory. Collectively, SIRPα blockade provides a novel way to overcome HRT resistance in oligometastatic CRC patients. Our results herein provide a valuable cancer treatment strategy that has the potential to be translated into clinical practice.

ISG20 stimulates anti-tumor immunity via a double-stranded RNA-induced interferon response in ovarian cancer.

Augmentation of endogenous double-stranded RNA (dsRNA) has become a promising strategy for activating anti-tumor immunity through induction of type I interferon (IFN) in the treatment of ovarian carcinoma. However, the underlying regulatory mechanisms of dsRNA in ovarian carcinoma remain elusive. From The Cancer Genome Atlas (TCGA), we downloaded RNA expression profiles and clinical data of patients with ovarian carcinoma. Using the consensus clustering method, patients can be classified by their expression level of core interferon-stimulated genes (ISGs): IFN signatures high and IFN signatures low. The IFN signatures high group had a good prognosis. Gene set enrichment analysis (GSEA) showed that differentially expressed genes (DEGs) were primarily associated with anti-foreign immune responses. Based on results from protein-protein interaction (PPI) networks and survival analysis, ISG20 was identified as a key gene involved in host anti-tumor immune response. Further, elevated ISG20 expression in ovarian cancer cells led to increased IFN-β production. The elevated interferon improved the immunogenicity of tumor cells and generated chemokines that attract immune cells to infiltrate the area. Upon overexpression of ISG20, endogenous dsRNA accumulated in the cell and stimulated IFN-β production through the Retinoic acid-inducible gene I (RIG-I)-mediated dsRNA sense pathway. The accumulation of dsRNA was associated with the ribonuclease activity of ISG20. This study suggests that targeting ISG20 is a potential immune therapeutic approach to treat ovarian cancer.

Effect of homologous recombination deficient (HRD) breast cancers on a distinct immune marker phenotype by comprehensive genomic and immune profiling (CGIP).

1029 Background: Preclinical evidence suggests that breast cancer with features of genomic instability may upregulate the host antitumor immune response by producing neoantigens through DNA damage and increasing interferon production through the stimulator of interferon genes (STING) pathway. In this study, we evaluated the association between features of genomic instability and immune response in a real-world breast cancer population. Methods: We analyzed retrospective comprehensive genomic and immune profiling (CGIP) results from 529 breast tumors tested in the real-world clinical setting. We defined the HRD phenotype as tumor with any single nucleotide variants (SNV), indels, copy number variations (CNV) or fusions in the following genes: ARID1A, ATM, ATRX, BAP1, BARD1, BLM, BRCA1/2, BRIP1, CHEK1/2, FANCA, MRE11A, NBN, PALB2, RAD50 and RAD51. Otherwise, they were considered HR-proficient (HRP). PD-L1 expression (CPS positive ≥10) and TMB (high ≥10 Mut/Mb) were determined using IHC and DNA sequencing, respectively. mRNA expression signatures of tumor inflammation (TIGS, strong/moderate/weak), cell proliferation (CP, high/moderate/poor) and cancer testis antigen burden (CTAB, high/low) were determined by RNA-sequencing from a 395-gene panel. We used over-representation and proportion analysis using chi-squared test to determine association of HRD to immune correlates. Results: Among 529 cases, the median patient age was 63.2 years (25.5-93.5). The majority of patients were female (519, 98%), and the most common tumor histology was invasive ductal carcinoma (287, 54%). A total of 405 (77%) and 124 (23%) of patients had HRD and HRP phenotypes, respectively. A greater proportion of HRD tumors (16%) had higher TMB compared to HRP (5.6%, p=0.003). HRP tumors showed a relatively low CP index, whereas HRD tumors were associated with a moderate CP index score (p = 0.007). HRD phenotype was associated with a significantly higher proportion of weakly inflamed tumors, as represented by lower TIGS scores (p=0.007). No significant difference in PD-L1 or CTAB was found between HRD and HRP phenotypes. Conclusions: Breast tumors with mutations in the HR genes demonstrated greater TMB and moderate cellular proliferation index of both tumor and immune cells, which suggest susceptibility to immune checkpoint inhibitors. Interestingly, this cohort lacked elevated markers of immune infiltration (TIGS), indicating a mechanism of potential tumor immune evasion. These results suggest that CGIP may allow for more informed treatment decisions in HRD breast cancers. Furthermore, assessment of HR status and immunotherapy susceptibility may support clinical trial selections for therapies targeting the complex interplay of genomic and immune components of breast cancer (e.g., a combination of PARP inhibitor and immunotherapy).

Immunostimulatory effects of nanosecond pulsed electric fields

Abstract Intense pulsed electric fields (PEF) are increasingly employed for tumor ablation, including inoperable tumors, bleeding metastases, and tumors unresponsive to alternative treatments. PEF disrupt the membrane barrier of tumor cells leading to irreversible changes in homeostasis and cell death. Using different tumor models, we found that treatment with nanosecond duration pulsed electric fields (nsPEF) enhance the host antitumor immune response. The strongest effect was measured in mice bearing CT26 colon carcinoma tumors where the local treatment with nsPEF protected 78% of the animals from a second tumor cell challenge. We hypothesize that nsPEF-treated tumors act as an in-situ cancer vaccine by initiating innate immunity while molding and sustaining adaptive immunity. Indeed, we provide evidence that the damage created by these short electrical stimuli is sensed by the innate immune platform known as inflammasome. Upon assembly, the inflammasome induces proinflammatory cytokine processing and a form of cell death known as pyroptosis. We found that nsPEF triggered pyroptosis in macrophages accompanied by caspase-1 activation and release of IL-1β. Concurrently, to sustain adaptive immunity, nsPEF in tumor cells activate persistent ER-stress accompanied by phosphorylation of translation initiation factor 2α (eIF2α): a biomarker of immunogenic cell death. Tumor antigen-release from nsPEF-treated tumor cells comes with the release of damage-associated molecular patterns such as ATP, HMGB1 and calreticulin. Understanding and manipulating the cross talk between innate and adaptive immunity after nsPEF is expected to improve the immunotherapeutic response triggered by these treatments. Pulse Biosciences, Inc.

Abstract 5181: The role of Neuropilin-1 signaling in CD4 conventional T cell immune function in non-small cell lung cancer

Abstract Despite advances in therapy, lung cancer remains the leading cause of cancer related deaths. In non-small cell lung cancer (NSCLC), a subset of tumor-infiltrating CD4 T cells express Neuropilin-1 (NRP1), a transmembrane protein that binds to multiple ligands with diverse functions in tumor and immune cells. Recent studies reveal that NRP1 can be induced upon T cell receptor stimulation and that NRP1 signaling promotes increased stabilization and function of CD4+ FoxP3+ regulatory T cells (Tregs), while signaling on CD8 T cells results in inhibition of CD8 T cell migration and cytotoxicity; therefore, NRP1 is implicated as a newly defined immune checkpoint protein. The functional role of NRP1 in CD4+ FoxP3- T conventional cells (CD4 Tconv) and the ligands involved are largely unexplored. We have found that human lung cancer malignant pleural effusions (MPE) from a subset of metastatic lung cancer patients contain both NRP1+ and NRP1- CD4 Tconv cells, providing a relevant clinical setting to study the function of NRP1 in human T cells. Our preliminary studies reveal that MPE-derived NRP1+ CD4 Tconv cells have altered expression of CD25, suggestive of a more activated state as compared to that of NRP1- CD4 Tconv cells. Additionally, we have observed disproportionate cell death of NRP1+ CD4 Tconv cells that can be partially rescued with anti-NRP1 monoclonal antibodies (mAbs). Surviving NRP1+ cells are more likely to express PD-1 and secrete effector cytokines. In our murine NSCLC model carrying key driver mutations KrasG12D P53−/− Lkb1−/− and higher tumor mutational burden (KPL-3M), analysis of tumor-infiltrating CD4 Tconv cells confirm that NRP1+ CD4 T cells are preferentially found within tumors and with higher PD-1 expression as compared to NRP1- cells. Furthermore, treatment with anti-NRP1 mAbs confers anti-tumor efficacy in this model, indicating that NRP1 is a functionally important inhibitor of host anti-tumor immune responses. These data suggest that CD4 Tconv cells expressing NRP1 are activated but prone to cell death, warranting further investigation into its potential as a therapeutic target for NSCLC. Citation Format: Bitta P. Kahangi, Raymond J. Lim, Jensen Abascal, William P. Crosson, Edgar Perez Reyes, Camelia Dumitras, Linh M. Tran, Kostyantyn Krysan, Ramin Salehi-Rad, Steven M. Dubinett, Bin Liu. The role of Neuropilin-1 signaling in CD4 conventional T cell immune function in non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5181.

The role of viruses in cancer development versus cancer therapy: An oncological perspective.

Despite great medical advances, oncological research is still looking for novel therapeutic approaches due to the limitation of conventional therapeutic agents. Virotherapy is one of these new emerging therapeutic approaches that attract attention with their widespread applications. Virotherapy use lives oncolytic viruses or genetically engineered viruses that selectively infect the tumor cells, replicate, and disrupt the cancerous cells that also induce their anticancer activity by stimulating the host antitumor immune response. Moreover, viruses are widely used as target delivery vectors for specifically delivering different genes, therapeutic agents, and immune-stimulating agents. In addition to having antitumor activity by themselves in combination with conventional therapeutic agents like immune therapy and chemotherapy, Virotherapy agents also elicit promising outcomes. Therefore, in addition to their promising result in monotherapy use, virotherapy agents can also be used in combination with conventional cancer therapy, epigenetic modulators, and even microRNAs without any cross-resistance, which allows the patient not to be deprived of her routine medicine. Still, this combination therapy reduces the adverse effect of the conventional therapies. All together suggest that virotherapy agents as novel potential agents in the field of cancer therapy.

Abstract P3-06-04: Treatment of Metastatic Breast Cancer with Multipotent Oncolytic/Helper Adenovirus CAdVEC

Abstract Background: Metastatic breast cancer (MBC) which causes significant morbidity and mortality worldwide is in need of more effective treatment regimens. In combination with chemotherapy, anti-PD1 antibody pembrolizumab has been shown to prolong progression-free survival (PFS) of patients with triple-negative subtype MBC (TN-MBC), however, its efficacy remains low for the other 80% of patients with MBC. MBC’s heterogenous pattern of immune infiltration and expression make it challenging to treat with single immunotherapeutic agents such as pembrolizumab and successful immunotherapy must therefore target multiple pathways. To augment antitumor host immune responses during treatment, studies have examined adjunct agents such as “oncolytic” adenovirus (OAds), which are vectors that preferentially replicate in and lyse tumor cells leading to the activation of host immunity. OAds have been tested in a myriad of clinical trials with the hope to enhance host immune activation but those trials have shown limited successes. Methods: To overcome the multiple immunogenic barriers in solid tumors, our group developed a binary oncolytic/helper-dependent adeno-immunotherapy (CAdVEC). The first generation CAdVEC (CAdTrio) contains an OAd and a “helper-dependent” adenovirus (HDAd) that produces immunostimulant molecules including interleukin (IL)-12p70 and anti-PD-L1 antibody. Based on successful results using animal models, a first-in-human Phase 1 study with CAdTrio was conducted among patients with all solid tumors (NCT03740256). Four patients with MBC were enrolled in the virus dose-escalation phase of the trial and received an intra-tumor injection of CAdTrio. Given the novelty of this binary agent, starting dose of CAdTrio was more than 2-logs lower than that used in other OAd trials. Three patients received dose level (DL) 1 and one patient received DL2. All patients also received pembrolizumab 6 weeks after the virus injection. The primary endpoint for this phase I dose escalation was dose-limiting toxicities (DLT). Secondary outcomes included overall response rates (ORR), disease control rate (DCR), PFS, overall survival (OS), and treatment-related adverse events. Results: No patients developed DLT. The most common toxicities were fever, fatigue and pain around the injection site, but none were greater than grade 2. No significant elevation in liver enzymes were observed. Three of the four patients had partial response (PR). One patient progressed after ten weeks of stable disease and passed away. The three patients with PR received pembrolizumab within 7 weeks of CAdVEC injection. Analysis of injected tissues prior to pembrolizumab treatment showed that CAdTrio repolarized the tumor microenvironment toward immune activity by increasing the number of infiltrating Th1 immune cells, leading to responses in some treated tumors and even in one distant metastasis, demonstrating the potent systemic immune response to local CAdTrio treatment in patients with MBC. Conclusions: Our study demonstrated that intra-tumor injection with CAdTrio was safe and effective in patients with MBC but the significance of the results was limited by the small sample size. An MBC dedicated phase II trial is planned to be conducted to fully evaluate the efficacy and safety of CAdVEC treatment and to further elucidate mechanisms of resistance/sensitivity among patients with MBC. Citation Format: Natalie Chen, Daniel Wang, Caroline E. Porter, Amanda Rosewell Shaw, Catherine R. Robertson, Mae L. Woods, Ya Xu, Greyson Biegert, Alphi Kuriakose, Tao Wang, Bambi J. Grilley, Helen Heslop, Malcolm K. Brenner, Masataka Suzuki, Bora Lim. Treatment of Metastatic Breast Cancer with Multipotent Oncolytic/Helper Adenovirus CAdVEC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-04.

Exploring the Interactions of Oncolytic Viral Therapy and Immunotherapy of Anti-CTLA-4 for Malignant Melanoma Mice Model.

Oncolytic ability to direct target and lyse tumor cells makes oncolytic virus therapy (OVT) a promising approach to treating cancer. Despite its therapeutic potential to stimulate anti-tumor immune responses, it also has immunosuppressive effects. The efficacy of OVTs as monotherapies can be enhanced by appropriate adjuvant therapy such as anti-CTLA-4. In this paper, we propose a mathematical model to explore the interactions of combined therapy of oncolytic viruses and a checkpoint inhibitor, anti-CTLA-4. The model incorporates both the susceptible and infected tumor populations, natural killer cell population, virus population, tumor-specific immune populations, virus-specific immune populations, tumor suppressive cytokine IFN-g, and the effect of immune checkpoint inhibitor CTLA-4. In particular, we distinguish the tumor-specific immune abilities of CD8+ T, NK cells, and CD4+ T cells and describe the destructive ability of cytokine on tumor cells as well as the inhibitory capacity of CTLA-4 on various components. Our model is validated through the experimental results. We also investigate various dosing strategies to improve treatment outcomes. Our study reveals that tumor killing rate by cytokines, cytokine decay rate, and tumor growth rate play important roles on both the OVT monotherapy and the combination therapy. Moreover, parameters related to CD8+ T cell killing have a large impact on treatment outcomes with OVT alone, whereas parameters associated with IFN-g strongly influence treatment responses for the combined therapy. We also found that virus killing by NK cells may halt the desired spread of OVs and enhance the probability of tumor escape during the treatment. Our study reveals that it is the activation of host anti-tumor immune system responses rather than its direct destruction of the tumor cells plays a major biological function of the combined therapy.