PD-1 Immune Checkpoint Blockade Research Articles

Abstract PO1-19-08: Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer

Abstract Background: Only a subset of patients with metastatic triple-negative breast cancers (TNBC) demonstrate response to FDA approved PD-1 immune checkpoint blockade (ICB), and few have durable responses. Data suggests that breast cancers have defects in antigen presentation and that antigen presenting cells especially the cDC1 subtype of dendritic cells (DCs) are required for response to ICB. CD40 agonists activate antigen presenting cells including DCs and B cells and repolarize macrophages to an anti-tumor phenotype. Flt3 ligand is a growth factor that increases differentiation and expansion of DCs. We recently demonstrated in pre-clinical TNBC models that the combination of liposomal doxorubicin chemotherapy, a CD40 agonist, and a Flt3 ligand improves outcomes compared to alternate combinations. Methods: This is a single arm phase I pilot study of liposomal doxorubicin, CDX-1140 (CD40 agonist monoclonal antibody), and CDX-301 (recombinant Flt3 ligand) combination therapy in patients with metastatic or unresectable locally advanced metastatic TNBC. Patients will be randomized to 3 lead-in arms (triplet therapy, doublet immunotherapy only, or liposomal doxorubicin only) for one cycle prior to receiving triplet therapy with tissue biopsies done before and after the lead-in treatment. CDX-301 will be given for only two cycles; liposomal-doxorubicin and CDX-1140 will be continued until disease progression or clinically limiting toxicities. Primary endpoint is determination of a recommended phase II dose based on treatment-related adverse events and dose-limiting toxicities. Secondary endpoints include anti-tumor immune response after triplet therapy, after immunotherapy alone, and after liposomal doxorubicin alone; median progression-free survival, overall response rate, duration of response, and clinical benefit rate. Key eligibility criteria are unresectable stage III or stage IV TNBC (ER ≤10%, PR ≤10%, HER2/neu negative), 1st to 3rd line treatment for metastatic disease (1st line patients need to be PD-L1 negative by 22C3 assay), measurable disease by RECIST 1.1 criteria, consent for pre-treatment and on-treatment biopsies of amenable soft tissue tumor lesions, no prior treatment with an anti-CD40 antibody or a Flt3 ligand, no anthracycline treatment in the metastatic setting, no prior progression while on anthracycline-based therapy or within 6 months of completing neoadjuvant chemotherapy, and no history of non-infectious pneumonitis or current pneumonitis. This trial will enroll up to 45 patients across multiple sites (NCT05029999) and is currently open at University of Texas Southwestern Medical Center, Texas Oncology, University of Chicago, University of Texas San Antonio, Sarah Cannon Research Institute, and Johns Hopkins. Citation Format: Sangeetha Reddy, Joyce O'Shaughnessy, Navid Sadeghi, Samira Syed, Cesar Santa-Maria, Virginia Kaklamani, Nan Chen, Denise Yardley, Yisheng Fang, Isaac Chan, Nisha Unni, Sarah Kashanian, Namrata Peswani, Shahbano Shakeel, Meredith Carter, Kelly Kyle, Farjana Fattah, Chul Ahn, Ina Patel, Joshua Gruber, Dawn Klemow, Glenda Delgado, Nicole Sinclair, Michael Yellin, Heather McArthur, Rita Nanda, Suzanne Conzen, Carlos Arteaga. Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-19-08.

Combination of IFN-gamma with STING agonist and PD-1 immune checkpoint blockade: a potential immunotherapy for gastric cancer.

Suppression of the cGAS-STING pathway is an immune escape mechanism in cancer cells. The critical role of this pathway in gastric cancer (GC) is not fully understood. Herein, we evaluated the effect of the interferon-gamma (IFN-gamma), STING agonist, PD-1 immune checkpoint blockade, and their combination on the cGAS-STING pathway in GC. Expression of cGAS and STING in tumor tissue samples and adjacent normal tissue (ANT) biopsies of fifty new GC patients was evaluated by quantitative real-time PCR (qRT-PCR). Moreover, cGAS and STING expression levels were examined in Peripheral Blood Mononuclear Cells (PBMC) samples of forty GC patients and twenty-five healthy subjects. The apoptosis rate of cancer cells was analyzed by Annexin V-FITC/PI. Cell proliferation was measured by the BrdU assay. Also, IFN-β levels were evaluated in the supernatants of the treated groups. The cGAS expression was decreased in patients with distant metastasis. Co-cultures treated with IFN-gamma showed an elevated level of cGAS and STING expressions in PBMC and cancer cells. The rate of apoptosis increased in all the treatment groups. In addition, the rate of proliferation in PBMCs increased in different treated groups. The main role of PBMCs in cytotoxicity was determined by a comparative analysis of the viability of cells treated with all treatments, both with and without PBMCs. The production of IFN-β was elevated in all treated groups. The current study suggests that a combination therapy using IFN-gamma, STING agonist, and anti-PD-1 antibody can provide a promising approach to the treatment of GC.

Abstract 1359: Rewiring CD8+T cell responses to PD-1 immune checkpoint blockade in PDAC by preventing terminal exhaustion via the inhibitory receptor PSGL-1

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer with increasing incidence and a poor 5-year survival rate of 12%. PDAC tumors are refractory to all currently available treatments, including immune checkpoint blockade (ICB) therapies, and demonstrate limited infiltration of cytotoxic CD8+ T cells that kill tumor cells. There is therefore an urgent, unmet need for novel therapeutic options. Analysis of single-cell RNA-sequencing data of patient PDAC revealed high expression of the novel immune checkpoint, PSGL-1, on infiltrating CD8+ T cells. We hypothesize that PSGL-1-mediated immune suppression is a critical barrier to effective anti-tumor immunity in PDAC. To test this, we utilized a preclinical model of PDAC where orthotopic injection of the KPC.4662 tumor cell line into pancreata recapitulates patient tumor responses with limited T cell infiltrates and uncontrolled tumor growth in C57BL/6 (wildtype) control mice and compared to PSGL-1KO (C57BL/6 background) mice. At endpoint, tumor burden in PSGL-1KO mice was reduced by >50% compared to controls, with 45% of PSGL-1KO animals exhibiting >70% reduction. Total immune infiltration (CD45+) was 2-fold greater in tumors from PSGL-1KO mice, with a significant increase in infiltrating CD3+ T cells. Detailed analyses revealed that infiltrating CD8+ T cells from PSGL-1KO mice were not only more abundant, but also less differentiated towards terminal exhaustion with sustained TCT-1 expression compared to controls. Moreover, PSGL-1KO mice showed protection against metastatic disease. Since our data support a critical role for T cell driven anti-tumor immunity to PDAC, we transiently depleted CD4+ T cells, CD8+ T cells, or both just prior to tumor cell implantation. While depletion of CD8+ T cells did not impede tumor control by PSGL-1KO mice, depletion of all T cells or only CD4+ T cells resulted in the loss of PDAC tumor growth inhibition, demonstrating a key role of CD4+ cells in the response. Given our previous studies demonstrating an intrinsic role for PSGL-1 in development of terminal T cell exhaustion in melanoma, including loss of TCF-1+ CD8+ T cells, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 ICB, which is ineffective as a monotherapy in PDAC patients and mouse models. To test this prediction, PD-1 ICB was therapeutically administered to control and PSGL-1KO mice. While treatment with anti-PD-1 had no effect on the tumor growth in the control animals, PD-1 ICB resulted in complete tumor ablation in 85% of PSGL-1KO animals, demonstrating a profound synergistic effect of PD-1 inhibition in the absence of PSGL-1. From these studies, we conclude that PSGL-1 is a critical inhibitor of T cell anti-tumor responses in PDAC. PSGL-1 therefore represents a novel ICB target with high translational potential for promoting immune responses to PDAC tumors. Citation Format: Jennifer L. Hope, Yijuan Zhang, Hannah A. Hetrick, Gabriele Romano, Sreeja Roy, Michelle Lin, Ashley B. Palete, Swetha Maganti, Dennis C. Otero, Cosimo Commisso, Linda M. Bradley. Rewiring CD8+T cell responses to PD-1 immune checkpoint blockade in PDAC by preventing terminal exhaustion via the inhibitory receptor PSGL-1 [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 1359.

Abstract A051: PSGL-1-deficiency promotes pancreatic ductal adenocarcinoma tumor control and synergy with immune checkpoint blockade

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer with increasing incidence rates while patient prognosis remains poor with a 5-year survival rate of only 12%. PDAC tumors are refractory to all currently available treatments, including existing immune checkpoint blockade (ICB) therapies, and demonstrate limited T cell infiltration. There is a critical need to develop novel approaches to improve patient outcomes. We previously demonstrated that deficiency of P-selectin glycoprotein 1 (PSGL-1) promotes enhanced T cell responses that clear chronic LCMV Cl13 virus infection and inhibit growth of PD-1 blockade resistant melanoma. Here we evaluated if targeting PSGL-1 promotes T cell responses to PDAC tumors to inhibit growth and metastases. KPC.4662 tumor cells were injected orthotopically into the tail of the pancreas of C57BL/6 (wildtype control) and PSGL-1−/− (C57BL/6 background) male or female mice and tumor growth and immune infiltration was evaluated by histology and flow cytometry. At 28 days post-orthotopic tumor cell injection, KPC.4662 tumor burden was reduced by 50% on average (1.1 g vs 0.45g) in PSGL-1−/− mice compared to controls. Total immune infiltration (CD45+) was increased in tumors from PSGL-1−/- mice (10% vs 24%), with a significant increase in CD3+ T cell infiltration. Moreover, fewer mice developed metastases in the peritoneal cavity, liver, and lungs. To assess if PSGL-1−/− mice had the capability to reduce metastatic burden, KPC 4662 tumor cells were injected intravascularly and tumor development in the lungs was assessed on day 17. PSGL-1−/− mice reproducibly developed fewer tumors in the lungs compared to wildtype control mice as determined by histological examination. To address if enhanced control of PDAC tumors was mediated by T cells, we depleted CD4+ and CD8+ T cells immediately prior to orthotopic implantation of KPC.4662 tumor cells. Depletion of T cells resulted in the loss of PDAC tumor growth control in PSGL-1−/− mice. Given our previous studies demonstrating an intrinsic role for PSGL-1 in the development of T cell exhaustion, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 immune checkpoint blockade, a therapy known to be ineffective as a monotherapy in PDAC patients and mouse models. When PD-1 blocking antibody was therapeutically administered on days 10, 12, and 14 post-tumor implantation, 85% of PSGL-1−/− animals demonstrated complete responses. From these studies, we conclude that in the absence of PSGL-1, we observe significantly reduced growth of primary orthotopic PDAC tumors and decreased metastases. These findings are associated with increased CD45+ immune cell infiltration in the tumors and notably functions in a T cell-dependent manner. PSGL-1 therefore represents a novel target for promoting immune responses to PDAC tumors. Citation Format: Jennifer L. Hope, Yijuan Zhang, Hannah A. Faso, Sreeja Roy, Michelle Lin, Ashley B. Palete, Swetha Maganti, Dennis C. Otero, Cosimo Commisso, Linda M. Bradley. PSGL-1-deficiency promotes pancreatic ductal adenocarcinoma tumor control and synergy with immune checkpoint blockade [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 A051.

Carnosic acid nanocluster-based framework combined with PD-1 inhibitors impeded tumorigenesis and enhanced immunotherapy in hepatocellular carcinoma.

Clinically, the immune checkpoint inhibitor anti-PD-1 antibody has shown a certain effect in the treatment of hepatocellular carcinoma (HCC), which is limited to a small number of patients with HCC. This study aims to reveal whether carnosic acid nanocluster-based framework (CA-NBF) has a sensitization effect on anti-PD-1 antibody in the treatment of HCC at the cellular and animal levels. MHCC97H cells were treated with CA-NBF, anti-PD-1 and their combination. The effects of CA-NBF and anti-PD-1 on cell proliferation, cell cycle, apoptosis, invasion, and migration were evaluated by MTT assay, flow cytometry, and scratch test. The effects of CA-NBF and anti-PD-1 on Wnt/β-catenin signaling pathway in MHCC97H cells were detected. A BALB/C nude mouse model of hepatocellular carcinoma was established, and the tumor growth was observed at different time points. The expression of cytotoxic T lymphocyte and helper T lymphocyte markers CD8 and CD4 in tumor tissues was detected by immunohistochemistry. Western blotting was used to detect the Wnt/β-catenin signaling pathway proteins (Wnt-3a, β-catenin, and GSK-3β) level in tumor tissues after CA-NBF and anti-PD-1 treatment. CA-NBF activity was significantly higher than CA, which could prominently reduce the proliferation, migration and invasion of MHCC97H cells and enhance apoptosis by inactivating Wnt/β-catenin signaling pathway. CA-NBF combined with anti-PD-1 antibody further enhanced cell proliferation, migration, invasion and pro-apoptosis but had no significant effect on Wnt/β-catenin signaling pathway. CA-NBF in vivo improved the tumor response to PD1 immune checkpoint blockade in HCC, manifested by reducing tumor size and weight, promoting CD4 and CD8 expression. CA-NBF combined with anti-PD-1 have stronger immunomodulatory and anticancer effects without increasing biological toxicity.

Recent advances in nanotechnology for programmed death ligand 1-targeted cancer theranostics.

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) checkpoint inhibitor-based immunotherapy has provided a unique and potent weapon against cancer in clinical practice. The likelihood of achieving beneficial effects from PD-L1/PD-1 immune checkpoint blockade (ICB) therapy is clinically assessed by detecting PD-L1 expression through invasive tissue biopsies. However, PD-L1 expression is susceptible to tumor heterogeneity and dynamic response to ICB therapy. Moreover, currently, anti-PD-L1 immunotherapy still faces challenges of the low targeting efficiency of antibody drugs and the risk of immune-associated adverse events. To overcome these issues, advanced nanotechnology has been developed for the purpose of quantitative, non-invasive, and dynamic analyses of PD-L1, and to enhance the efficiency of ICB therapy. In this review, we first introduce the nanoprobe-assisted in vitro/in vivo modalities for the selective and sensitive analysis of PD-L1 during the diagnostic and therapeutic process. On the other hand, the feasibility of fabricating diverse functional nanocarriers as smart delivery systems for precisely targeted delivery of PD-L1 immune checkpoint inhibitors and combined therapies is highlighted. Finally, the current challenges are discussed and future perspectives for PD-L1-targeted cancer theranostics in preclinical research and clinical settings are proposed.

Somatic TET2 Mutations Prime the Immune System for Response to Immune Checkpoint Blockade

The driver mutations causing Hematopoietic Stem Cell (HSC) expansion in Clonal Hematopoiesis (CH) are inherited by HSC progeny and in some settings alter the function of mutant immune cells. It is unclear how CH mutations impact immune cell function in response to immune checkpoint blockade (ICB), which is a critical cancer therapy for many solid tumours and hematological cancers. Understanding how CH influences ICB could provide a novel biomarker for patient stratification or personalized therapeutics and may offer insight into immunotherapy mechanisms, yielding novel drug targets to synergistically improve patient outcomes. Somatic mutations inactivating TET2, which is responsible for 5-methylcytosine hydroxylation and demethylation, are one of the most frequent drivers of CH. TET2 inactivation can enhance inflammation from monocytes/macrophages, CD4 + and CD8 + T cells, but its impact on ICB is unknown. Here we sought to define the impact of TET2-mutant CH (TET2-CH) on ICB response. First, we modelled TET2-CH by rescuing lethally irradiated C57BL6/j mice with Tet2-knockout (-KO), Tet2-heterozygous null (-het), or wild type (WT) bone marrow. Mice were subsequently implanted with the syngeneic colorectal adenocarcinoma line MC38 and treated with Programmed Cell Death Protein-1 (PD-1) targeted ICB or Isotype Control (control) antibodies. PD-1 ICB, but not control, treated tumors were significantly smaller in mice with Tet2-het or Tet2-KO versus WT hematopoiesis (Figure 1). There was a trend towards a dose-responsive effect, with more Tet2-het mice responding to PD-1 ICB than wt, and more Tet2-KO mice responding than Tet2-het or wt mice. Therefore, Tet2-mutant leukocytes promote response to PD-1 blockade. PD-1 ICB with targeted cell depletion revealed that the enhanced response in Tet2-het mice requires CD4 + T Cells, CD8 + T Cells and macrophages, but not NK cells. To understand the mechanism through which Tet2-inactivation promotes ICB responses we performed 10X single cell RNA-sequencing on 38,994 CD45 + tumor-infiltrating leukocytes (TILs) from Tet2-het and wt mice treated with PD-1 ICB or control. Tet2-het mutations dramatically changed the TIL landscape with PD-1 blockade by promoting the acquisition of cell fates that drive durable immunotherapy responses including CD8 + effector T cells, CD8 + memory T cells, CD4 + interferon-responsive cells, and macrophages expressing an inflammatory anti-tumour gene signature (Figure 2). In contrast, wt TILs from PD-1 treated tumours were significantly enriched for exhausted CD8 + T cells, CD4 + regulatory T cells, and immunosuppressive macrophages that can promote tumor growth (Figure 2). TIL DNA methylation analysis using Infinium Beadchip arrays revealed promoter hypermethylation in genes defining the immunosuppressive macrophage state. Collectively, this suggests that Tet2-CH reshapes TIL fate following ICB by promoting anti-tumor phenotypes and protecting cells from immunosuppressive states. To investigate the potential clinical relevance of our findings, we retrospectively tested for an association between TET2-CH and immunotherapy outcome in 569 ICB-treated solid tumor patients. CH mutations were detected in publicly-available exome-sequencing data at VAF >0.02 using Mutect2. While CH as a composite of all driver mutations did not correlate with outcome, in multivariate analysis TET2-CH was associated with 6-times greater odds of achieving 6 month clinical benefit in n=205 melanoma patients (95% CI 1.01-66.6). To explore whether TET2-CH might identify patients primed for ICB response, we screened for CH in the prospectively collected baseline PBMCs from 90 solid tumour patients treated in a Phase 2 trial of the PD-1 inhibitor Pembrolizumab (NCT02644369). While underpowered to draw conclusions, we noted that one patient, treated for the skin cancer Merkel Cell Carcinoma, was positive for TET2-CH and responded to ICB, and also survived longer than the cohort median and Merkel Cell Carcinoma median. In summary, our results show that TET2-CH promotes immunotherapy response by shifting TILs from immunosuppressive to anti-tumor fates. Mechanistically, this occurs through lineage-specific promoter hypermethylation and gene silencing. Further study of TET2-CH as a potential biomarker of ICB response is warranted and targeting TET2-regulated pathways may yield novel strategies to improve immunotherapy outcomes.

RNA Methyltransferase FTSJ3 Regulates the Type I Interferon Pathway to Promote Hepatocellular Carcinoma Immune Evasion.

Hepatocellular carcinoma cells use 2'-O-methylation catalyzed by FTSJ3 for immune evasion by suppressing abnormal dsRNA-mediated type I IFN responses, providing a potential target to activate antitumor immunity and enhance immunotherapy efficacy.

Single-cell RNA and T cell receptor (TCR) sequencing in patients with HPV-positive head and neck squamous cell carcinoma (HNSCC) after induction CTLA4 and PD-1 immune checkpoint blockade (ICB).

6056 Background: Combination CTLA-4 and PD-1 ICB results in durable responses in patients with recurrent and metastatic HNSCC and therefore justifies further investigation in the induction or neoadjuvant setting. Methods: We investigated features of the tumor immune microenvironment (TIME) associated with MHR after induction CTLA-4 and PD-1 ICB in patients with newly diagnosed HPV-positive HNSCC. Tumor samples were collected at baseline and on treatment from 31 patients enrolled in a phase 2 trial of a 6-week cycle of CTLA-4 and PD-1 ICB induction followed by dose/volume-adapted IMRT (50-66Gy) concurrent with cycle 2. Tumors were evaluated for histological response and single-cell RNA sequencing (scRNA-seq) and scTCR-seq were performed on the 10X genomics platform. After stringent clustering and annotation of scRNA-seq data, 76,319 CD8+ and 78,622 CD4+ T cells with paired TCR sequence data were evaluable for transcriptomic, TCR repertoire and clonotype differentiation analysis. Results: Percent reduction in tumor viability on histology correlated significantly with reduction in tumor cell proportion in scRNA-seq data (R2= 0.7). Thirteen (48%) of 27 patients with paired samples (missing data due to COVID) had MHR, defined as ≤10% residual tumor viability on ICB treatment. Through TCR tracing, over 100 T cell clonotypes that expanded significantly (p < 0.05, Fisher’s exact) post-ICB were identified. Highly expanded clonotypes resided mainly in exhausted CD8+ T cell clusters and exhibited a tissue resident memory (TRM) phenotype. Examination of their functional states revealed a variety of T cell modulatory trajectories, including reverse transition from exhausted to less exhausted states and from memory to functional states. When compared to patients without MHR, those with MHR had significantly higher ICB-induced TCR clonotype expansion (p = 0.025, Wilcoxon), reductions of activated Tregs (p = 0.003, Wilcoxon) and terminally exhausted CD8+ T cells (p = 0.003, Wilcoxon), and increase of polyfunctional CD8+ T cells (p = 0.014, Wilcoxon). A lasso regression model associated T cell expansion, basal 7-gene TRM and 5-gene effector scores, and on-treatment proliferation score and effector cell proportion with percent reduction in tumor viability (R2 = 0.66). Conclusions: In HPV-HNSCC, induction CTLA4 and PD1 ICB lead to extensive reinvigoration of CD8+ T cell clonotypes with both an exhausted and TRM phenotype, revealing a high degree of plasticity of distinct T cell clones. Paired RNA and TCR profiling facilitated identification of potentially tumor reactive and ICB-responsive T cells and clonotypes and identified several TIME features associated with tumor cell death. Clinical trial information: NCT03799445 .

Dual PD1 and CTLA4 immune checkpoint blockade and hypofractionated radiation in patients with salivary gland cancers.

6011 Background: No systemic therapy standard of care exists for recurrent/metastatic malignancies of the salivary glands (SGC) and immune checkpoint inhibitors (ICIs) have low response rates. Preclinical data in solid tumors suggest syngergistic antitumor effects of ICIs with hypofractionated radiation (XRT). This study explored the safety and activity of nivolumab (N) and ipilimumab (I)with palliative XRT. Methods: This phase I/II open label single arm trial enrolled patients (pts) with incurable SGCs (WHO 2017) with evidence of progression, ECOG 0-1, no prior antiPD1 or CTLA4 directed therapy, RECIST 1.1 measurable disease excluding the XRT site. Pts received N 3mg/kg IV Q 2 weeks x 12 doses followed by 480 mg IV Q4 weeks x 8 doses and I 1 mg/kg IV Q6 weeks x 4 doses. XRT was given to a total dose of 24Gy in 3 fractions every other day over 1 week (wk) and initiated 2 wks after the first dose of N and I. Research blood collection was obtained prior to wks 1, 8 and 16. The primary endpoint was safety and tolerability using CTCAE v. 4, secondary endpoints were objective response rates (ORR) by RECIST 1.1 criteria in non-radiated sites of measurable disease, overall survival (OS) and progression free survival (PFS). The study was approved by the FHCC IRB and registered on clinicaltrials.gov (NCT03749460). Results: Between 4/2019 and 5/2022, 20 pts were enrolled, the median age was 58 (range 27-77) years, 10 (50%) were male, 12 (60%) had ECOG 0, and 7 (35%) were Asian. Most common histologies were adenoid cystic 9 (45%) and salivary duct 4 (20%), 15 (75%) had no prior systemic therapy. Ten (50%) had both local and distant disease, 14 (70%) had commercial NGS testing, all had TMB < 10mut/Mb, MSI-stable tumors. All patients completed XRT with the most common XRT site being the lung 13(65%) and bone 7(35%). The median number of N doses received was 12 (range 3-20) and I doses 4 (range 1-4). Enrollment was halted until first 6 pts were assessed for dose limiting toxicities during initial 12 wks of treatment, none were observed. Among all pts enrolled, 5 (20%) Grade 3 AEs were observed: adrenal insufficiency, hypokalemia , lung infection, hypotension, and mania. No grade 4/5 toxicities were observed. One pt was not evaluable for RECIST 1.1 due to rapid disease progression: partial responses were observed in 4 (20%: 2 pts with salivary duct, 1 acinic cell and 1 adenoid cystic) with a median duration of 15.5 months (mos) (range 6-16 mos), stable disease in 6 (30%) all lasting 6 mos or greater, and progressive disease in 9 (45%). With a median follow-up of 16 mos, median OS was 25 mos (95% CI: [1.56, 2.59]) and median PFS was 7.2 mos (95% CI: [0.21, 1.56]). Exploratory correlative peripheral blood analysis is ongoing and will be reported. Conclusions: Nivolumab/ipilimumab and palliative XRT results in low rates of severe toxicities, modest ORR but durable ORR/SD. Further work is necessary to explore predictors for response. Clinical trial information: NCT03749460 .

PD-1 immune checkpoint blockade can synergize with PSGL-1 inhibition to reinvigorate exhausted T cells

Abstract P-selectin glycoprotein ligand-1 (PSGL-1) is an immune checkpoint regulator that is highly expressed on T cells. PSGL-1- deficient mice infected with chronic lymphocytic choriomeningitis virus (LCMV) were shown to control chronic viral infection due to increased effector function by anti-viral T cells. However, the cell-intrinsic role of PSGL-1 expression on CD4 +and CD8 +T cell exhaustion is not fully known. Using adoptive transfers of WT or PSGL-1-deficient TCR transgenic CD4 +and CD8 +T cells in mice infected with chronic LCMV, we characterized the differentiation and effector response. We found increased expansion and effector function by PSGL-1-deficient T cells early during infection, however, at later stages of infection, PSGL-1-deficient T cells were functionally exhausted. We found that exhausted CD4 +and CD8 +PSGL-1-deficient T cells were reinvigorated more effectively than WT cells after PD-L1 blockade due to their increased proliferation, cytokine production, and accumulation. We also observed increased responses of CD8 +PSGL-1-deficient T cells in melanoma tumors. Our findings showed that PSGL-1 expression was required for the maintenance of both CD8 +and CD4 +exhausted T cells and that PSGL-1 deletion synergized with PD-1 blockade to reinvigorate these cells. These findings highlight an important cell-intrinsic role for PSGL-1 expression in the maintenance and reinvigoration of exhausted T cells. T32 AI 141346 R01 AI13723

Abstract 1838: Immunomodulatory effects of curcumin monoglucuronide on PD1 immune checkpoint blockade in mouse Pten-null prostate cancer

Abstract Prostate cancer is notoriously resistant to immune checkpoint blockade (ICB) immunotherapy due to its immunosuppressive tumor microenvironment (TME). Thus, reprograming the TME could improve antitumor immune responses to ICB. Curcumin is a naturally derived compound with chemopreventive and immunomodulatory properties, however, its potential is limited due to its poor bioavailability. Curcumin monoglucuronide (CMG) is a water-soluble prodrug with improved bioavailability over free-form of curcumin. We previously demonstrated the immunomodulatory potential of CMG in a transgenic mouse model of Pten-null prostate cancer, here we examine its ability to improve the antitumor immune response to ICB of the programmed death cell protein 1 (PD1). Thirty-two-week-old PSA-Cre/Ptenf/f knockout (KO) mice were treated with anti-PD1 (aPD1) blocking antibody, CMG alone and in combination for 17 days weeks. Antitumor responses were determined by prostate weight, histology, and cancer proliferation and apoptosis by quantitative immunohistochemistry (qIHC). Immune responses were assessed by focused panel qRT-PCR, qIHC, and flow cytometric (FC) analysis. Systemic immune responses were assessed by FC analysis if peripheral blood and secondary lymphoid organs. Short-term dosing of aPD1 alone or in combination did not affect tumor burden as assessed by a reduction of prostate weight, however, histological analysis revealed that mice treated with CMG/aPD1 exhibited an increase in the proportion of edematous dilated glands and a non-significant reduction of solid tumor. Mice treated with CMG/aPD1 showed a significant reduction of s cancer cell proliferation (Ki67) and mice treated with CMG/aPD1 showed a 1.5-fold increase in cancer cell apoptosis. Gene expression analysis indicated upregulation of genes associated with NKT, NK and dendritic cells with combination therapy. FC and qIHC analysis indicated that T cell activation and T cell cytotoxicity was improved when CMG was administered together with aPD-1. Systemically, treatments with PD-L1 alone or in combination with CMG were associated with increased circulating myeloid-derived suppressor cells (MDSCs). Our findings provide preclinical evidence for the antitumor and immune modulatory activity of CMG in mouse Pten-deficient prostate cancer and supports further study to evaluate long-term therapy and explore additional treatment combinations. Citation Format: Yurie Kura, Marco A. De Velasco, Kazuko Sakai, Naomi Ando, Noriko Sako, Kazutoshi Fujita, Eri Banno, Yasunori Mori, Mamoru Hashimoto, Masahiro Nozawa, Kazuhiro Yoshimura, Hideaki Kakeya, Kazuto Nishio, Hirotsugu Uemura. Immunomodulatory effects of curcumin monoglucuronide on PD1 immune checkpoint blockade in mouse Pten-null prostate 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 1838.

Abstract 5745: Proposing the best MET inhibitor to improve anti-PD-1 efficacy in HCC

Abstract Despite recently approved immunotherapy combinations, Hepatocellular carcinoma (HCC) remains among the most therapeutically intractable cancers with a 5-year survival rate of only 18%. Underlying chronic liver disease due to hepatitis B/C infection, alcohol abuse, hemochromatosis, or nonalcoholic steatohepatitis promotes HCC development. Current therapeutic strategies include surgery, radiotherapy and multikinase inhibitors alone or with immune-checkpoint blockade (ICB). In particular, the receptor tyrosine kinase MET and its ligand hepatocyte growth factor (HGF) are known drivers of HCC through promotion of tumor growth, survival, tissue invasion, and angiogenesis. Previously, our collaborators showed that the limited clinical benefit of MET kinase inhibition was partially a result of induced upregulation PD-L1 leading to local T cell suppression. We hypothesized that combining MET inhibition with blockade of the PD-1 immune checkpoint would overcome the limitations of each individual approach and act synergistically to promote HCC tumor regression. We first validated that both MET selective (Capmatinib and Tivantinib) and non-selective (Cabozantinib) inhibitors all induced PD-L1 on HCC cell lines in vitro. Next, we compared Capmatinib and Cabozantinib alone and with PD-1 ICB in vivo in the ICB-sensitive Hepa1-6 and HCA-1 models of HCC. In HCA-1, statistically significant benefit of the MET inhibitor and αPD-1 combination was evident in limitation of tumor growth and extension of survival. In each case, the Capmatinib combination trended toward better outcomes with PD-1 blockade. Mechanistically, the Capmatinib and αPD-1 combination decreased tumor Treg cells while promoting increased accumulation of activated, non-exhausted, proliferating CD8 T cells. Central memory CD8 T cell frequencies were increased by the combination during the effector phase, and combination treated animals were better protected against subsequent rechallenge than those cured by αPD-1 alone. In the myeloid compartment, M1 macrophage frequencies were increased while PMN-MDSC both decreased in frequency and in Arginase 1 levels. Finally, we studied a serially-passaged, αPD-1 refractory DEN HCC model. Despite lack of efficacy of either component therapy, the MET inhibitor and αPD-1 combination significantly extended survival and inhibited tumor growth. As in the αPD-1 sensitive setting, Capmatinib appeared to offer the most therapeutic benefit with αPD-1. Ongoing studies are focused on characterizing changes mediated by the combination therapy which confer aPD-1 sensitivity to the otherwise refractory DEN HCC tumor microenvironment. Molecular studies are focused on understanding the superior ICB potentiating capacity of Type I MET inhibitors versus the other classes. Given the clinical availability of numerous MET and PDL-1 inhibitors, we hope to inform near-term optimization of MET and αPD-1 clinical trial design for HCC. Citation Format: Ricardo A. de Azevedo, Broderick Turner, Priyamvada Jayaprakash, Krithikaa Rajkumar Bhanu, Anupallavi Srinivasamani, Brittany Morrow, Michelle Winkler, Shweta Mahendra Hedge, Arthur Liu, Ravaen Slay, Michael A. Curran. Proposing the best MET inhibitor to improve anti-PD-1 efficacy in HCC. [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 5745.

Abstract 3271: Multiomic meta-analysis of differential response to PD-1 and CTLA-4 blockade in metastatic melanoma

Abstract Background: While CTLA-4 and PD-1 immune-checkpoint blockade (ICB) have revolutionized melanoma treatment, these treatments have different mechanisms, response rates, and toxicities. Differential predictors of response are poorly characterized. Combination ICB has the highest response rates but also much higher toxicity, and which patients to treat with single agent vs. combination ICB is controversial. In this study, we set out to understand the differential biology of anti-CTLA-4 and anti-PD-1 by integrating transcriptomic and clinical characterization from large cohorts of patients with metastatic melanoma treated with anti-CTLA-4, anti-PD-1, or combination blockade. Methods: We performed a standardized meta-analysis of all available cohorts of ICB-treated (anti-PD-1 alone, anti-CTLA-4 alone, and combination) melanoma patients with sequencing of pretreatment tumor and clinical annotations (n=572; 268 evaluated post quality-control filtering, cutaneous). Raw data in the form of FASTQs were obtained for all cohorts, and data were reprocessed using standardized pipelines. Immune infiltrate was characterized using single-sample gene set enrichment analysis (ssGSEA) scores from 18 immune cell type signatures, and GSEA was performed on differentially expressed genes between responders and non-responders for each treatment cohort accounting for differences in immune infiltrate. To dissect the cell types and drivers of nominated signatures, we analyzed scRNAseq from a separate cohort of pretreatment metastatic melanoma. Results: We found that immune infiltrate was strongly predictive of response to combination blockade (OR=6.16, p<0.05) but had a weaker signal in the monotherapy context (anti-PD-1 OR=1.42, n.s.; anti-CTLA-4 OR=1.10, n.s.). Among samples with a high degree of immune-infiltrate treated with either anti-CTLA-4 and anti-PD-1, non-responders were enriched in hypoxia (avg. NES=-1.38, FDR<0.05) and epithelial-mesenchymal transition (avg. NES=-2.32, FDR<0.05) gene signatures. Preliminary analysis of the single cell cohort shows a specific population of myeloid cells (MW p<0.05) and exhausted CD8+ T cells (MW p<0.05) increased in pretreatment hypoxic tumors. Conclusion: High rates of response to combination in patients with high levels of immune infiltrate suggests that immune high patients may differentially benefit from combination blockade. Identifying features of non-responding patients stratified by immune infiltrate can lead to new biological insights into ICB. Citation Format: Amy Y. Huang, Natalie Vokes, Cora Ricker, Tyler Aprati, Emily Robitschek, Jiekun Yang, Li-Lun Ho, Kyriakitsa Galani, Kelly P. Burke, Giuseppe Tarantino, Jiajia Chen, Arlene H. Sharpe, Eliezer M. Van Allen, Manolis Kellis, Genevieve M. Boland, David Liu. Multiomic meta-analysis of differential response to PD-1 and CTLA-4 blockade in metastatic melanoma [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 3271.

Abstract 2835: Fibroblast activation protein (FAP)-targeted radiotherapy increases tumor CD8+ T cell infiltration and enhances response to PD-1 immune checkpoint blockade

Abstract Background: FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high expression in tumors but limited expression in normal tissues. FAP-2286 is a FAP-targeted radiopharmaceutical in clinical development for multiple solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes for imaging and therapeutic use. Preclinically, we evaluated the potential modulation of the immune response and antitumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the β-emitting radionuclide lutetium-177 (177Lu) as a monotherapy and in combination with a programmed cell death protein 1-targeting antibody (anti-PD-1). Methods: C57BL/6 mice bearing MCA205 fibrosarcoma mouse FAP-expressing tumors (MCA205-mFAP) were treated with 177Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of 177Lu-FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses. Results: 177Lu-FAP-2287 accumulated in MCA205-mFAP tumors with 8.2 %ID/g uptake at 3 hours post-injection (pi), which was maintained at 2.9 %ID/g by 24 hours pi. Treatment with 177Lu-FAP-2287 demonstrated significant tumor growth inhibition (TGI, [74%; 267 mm3; P < 0.01]) vs vehicle control (750 mm3). Significant TGI was also observed from anti-PD-1 (57%; 373 mm³; P < 0.01) and the combination (92%; 145 mm³; P < 0.001) vs vehicle control. No significant differences in TGI were observed between the three treatment groups. In flow cytometry analysis of tumors, 177Lu-FAP-2287 increased CD8+ T cell infiltration by 2-fold on day 8 pi, alone or in combination with anti-PD-1 (16.9% and 18.0% of leukocytes, respectively), compared with 7.5% for vehicle control (P < 0.01). In contrast, anti-PD-1 alone had a nonsignificant increase in CD8+ T cell infiltration of 10.8% vs vehicle control (P > 0.05). On day 13 pi, the combination had durable higher levels of CD8+ T cells, with 16.3% of leukocytes vs 5.0% for vehicle control (P < 0.01). Monotherapy 177Lu-FAP-2287 also displayed a trend towards higher levels of CD8+ T cells at Day 13 vs vehicle (11.9% of leukocytes; P > 0.05). The increase in CD8+ T cells was accompanied by changes in co-stimulatory molecules such as CD86, resulting in greater activation by antigen-presenting cells. Additional immune cell characterization will be presented. Conclusions: FAP-targeted radiopharmaceutical enhances PD-1-antibody-mediated TGI by increasing recruitment of tumor-infiltrating CD8+ T cells, which is enhanced and maintained in the presence of anti-PD-1. These findings provide a rationale for clinical studies of combined 177Lu-FAP-2286 radiotherapy and immune checkpoint blockade in FAP-positive tumors. Citation Format: Dirk Zboralski, Aileen Höhne, Esben Christensen, Matthias Paschke, Liliane Robillard, Andrew D. Simmons, Frank Osterkamp, Thomas C. Harding, Minh Nguyen. Fibroblast activation protein (FAP)-targeted radiotherapy increases tumor CD8+ T cell infiltration and enhances response to PD-1 immune checkpoint blockade [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 2835.

Abstract OT2-17-01: Phase 1 trial of anthracycline chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer

Abstract Background: Only a subset of patients with metastatic triple-negative breast cancers (TNBC) demonstrate response to FDA approved PD-1 immune checkpoint blockade (ICB), and few have durable responses. Data suggests that breast cancers have defects in antigen presentation and that antigen presenting cells especially the DC1 subtype of dendritic cells (DCs) are required for response to ICB. CD40 agonists activate antigen presenting cells including DCs and B cells and also repolarize macrophages to an anti-tumor phenotype. Flt3 ligand is a growth factor that increases differentiation and expansion of DCs. We recently demonstrated in pre-clinical TNBC models that the combination of liposomal-doxorubicin chemotherapy, a CD40 agonist, and a Flt3 ligand improves outcomes compared to alternate combinations. Methods: This is a single arm phase I pilot study of liposomal-doxorubicin, CDX-1140 (CD40 agonist), and CDX-301 (Flt3 ligand) combination therapy in patients with metastatic or unresectable locally advanced metastatic TNBC. Patients will be randomized to 3 lead-in arms (triplet therapy, doublet immunotherapy only, or liposomal-doxorubicin only) for 1 cycle prior to receiving triplet therapy with fresh tissue biopsies before and after the lead-in treatment. CDX-301 will be discontinued after 2 cycles; liposomal-doxorubicin and CDX-1140 will be continued until disease progression or clinically limiting toxicities. Primary endpoint is determination of a recommended phase 2 dose based on treatment-related adverse events including dose-limiting toxicities. Secondary endpoints include anti-tumor immune response after triplet therapy, after immunotherapy alone, and after liposomal-doxorubicin alone; median progression-free survival, overall response rate, duration of response, and clinical benefit rate. Key eligibility criteria are unresectable stage III or stage IV TNBC (ER ≤10%, PR ≤10%, HER2/neu negative), 1st to 3rd line metastatic treatment setting (1st line patients need to be PD-L1 negative by 22C3 assay), measurable disease by RECIST 1.1 criteria, consent for pre-treatment and on-treatment biopsies of amenable soft tissue tumor lesions, no prior treatment with an anti-CD40 antibody or a Flt3 ligand, no anthracycline treatment in the metastatic setting, no prior progression while on anthracycline-based therapy or within 6 months of completing neoadjuvant chemotherapy, and no history of non-infectious pneumonitis or current pneumonitis. This trial will enroll up to 45 patients across multiple sites (NCT05029999). Citation Format: Sangeetha Reddy, Meredith Carter, Isaac Chan, Nisha Unni, Namrata Peswani, Dawn Klemow, Samira Syed, Shahbano Shakeel, Farjana Fattah, Chul Ahn, Yisheng Fang, Heather McArthur, Nicole Sinclair, Michael Yellin, Denise Yardley, Nan Chen, Joyce O’Shaughnessy, Rita Nanda, Suzanne D. Conzen, Carlos Arteaga. Phase 1 trial of anthracycline chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer [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 OT2-17-01.

Virotherapy combined with anti-PD-1 transiently reshapes the tumor immune environment and induces anti-tumor immunity in a preclinical PDAC model.

Pancreatic ductal adenocarcinoma (PDAC) is largely refractory to cancer immunotherapy with PD-1 immune checkpoint blockade (ICB). Oncolytic virotherapy has been shown to synergize with ICB. In this work, we investigated the combination of anti-PD-1 and oncolytic measles vaccine in an immunocompetent transplantable PDAC mouse model. We characterized tumor-infiltrating T cells by immunohistochemistry, flow cytometry and T cell receptor sequencing. Further, we performed gene expression profiling of tumor samples at baseline, after treatment, and when tumors progressed. Moreover, we analyzed systemic anti-tumor and anti-viral immunity. Combination treatment significantly prolonged survival compared to monotherapies. Tumor-infiltrating immune cells were increased after virotherapy. Gene expression profiling revealed a unique, but transient signature of immune activation after combination treatment. However, systemic anti-tumor immunity was induced by virotherapy and remained detectable even when tumors progressed. Anti-PD-1 treatment did not impact anti-viral immunity. Our results indicate that combined virotherapy and ICB induces anti-tumor immunity and reshapes the tumor immune environment. However, further refinement of this approach may be required to develop its full potential and achieve durable efficacy.

Combination of GD2-directed bispecific trifunctional antibody therapy with Pd-1 immune checkpoint blockade induces anti-neuroblastoma immunity in a syngeneic mouse model.

Despite advances in treating high-risk neuroblastoma, 50-60% of patients still suffer relapse, necessitating new treatment options. Bispecific trifunctional antibodies (trAbs) are a promising new class of immunotherapy. TrAbs are heterodimeric IgG-like molecules that bind CD3 and a tumor-associated antigen simultaneously, whereby inducing a TCR-independent anti-cancer T cell response. Moreover, via their functional Fc region they recruit and activate cells of the innate immune system like antigen-presenting cells potentially enhancing induction of adaptive tumor-specific immune responses. We used the SUREK trAb, which is bispecific for GD2 and murine Cd3. Tumor-blind trAb and the monoclonal ch14.18 antibody were used as controls. A co-culture model of murine dendritic cells (DCs), T cells and a neuroblastoma cell line was established to evaluate the cytotoxic effect and the T cell effector function in vitro. Expression of immune checkpoint molecules on tumor-infiltrating T cells and the induction of an anti-neuroblastoma immune response using a combination of whole cell vaccination and trAb therapy was investigated in a syngeneic immunocompetent neuroblastoma mouse model (NXS2 in A/J background). Finally, vaccinated mice were assessed for the presence of neuroblastoma-directed antibodies. We show that SUREK trAb-mediated effective killing of NXS2 cells in vitro was strictly dependent on the combined presence of DCs and T cells. Using a syngeneic neuroblastoma mouse model, we showed that vaccination with irradiated tumor cells combined with SUREK trAb treatment significantly prolonged survival of tumor challenged mice and partially prevent tumor outgrowth compared to tumor vaccination alone. Treatment led to upregulation of programmed cell death protein 1 (Pd-1) on tumor infiltrating T cells and combination with anti-Pd-1 checkpoint inhibition enhanced the NXS2-directed humoral immune response. Here, we provide first preclinical evidence that a tumor vaccination combined with SUREK trAb therapy induces an endogenous anti-neuroblastoma immune response reducing tumor recurrence. Furthermore, a combination with anti-Pd-1 immune checkpoint blockade might even further improve this promising immunotherapeutic concept in order to prevent relapse in high-risk neuroblastoma patients.

Abstract B018: Novel therapeutics for targeting the aberrant nuclear export machinery in colorectal cancer

Abstract XPO1 is a nuclear export receptor responsible for exporting many key proteins critical for cell survival from the nucleus to the cytoplasm. Recently, we identified a highly statistically significant hotspot mutation in XPO1 R749Q occurring in patients with colorectal cancer. Out of 96 patients identified to have XPO1 R749Q mutations, 27 (28%) were in colorectal cancer (CRC). 95% of XPO1 R749Q mutant CRC were tumor mutation burden-high (TMB-H; >10 mut/Mb) and 0 were mis-match repair deficient (dMMR)/micro satellite instability-high (MSI-H). Co-mutation analysis showed that 95% of XPO1 R749Q CRC were POLE mutated, which was significantly enriched over XPO1 WT tumors. To investigate whether XPO1 R749Q might play a role in CRC biology, we have generated two different isogenic colorectal cancer cell lines bearing the XPO1 R749Q mutation (HCT116 and LS174T) using CRISPR-CAS9 to better understand the role of this aberrant XPO1 mutation on nuclear export and tumorigenesis. Immunofluorescence (IF) and Stochastic Optical Reconstruction Microscopy (STORM) super-resolution experiments showed that R749Q mutant cells had significantly increased localization of XPO1 in the cytoplasm of the XPO1 R749Q mutant cells when compared to wildtype (XPO1 WT) cells, particularly at the edge of the nuclear pores of the XPO1 R749Q mutant cells suggesting increased nuclear export. Mass spectrometry analysis of nuclear and cytoplasmic fractionated proteins confirmed that XPO1 R749Q mutant cells had increased export of proteins from the nucleus compared to XPO1 WT cells. Structural modeling using published structures of XPO1 bound to Ran-GTP predicted that XPO1 R749Q mutation increased affinity of the regulatory H9-loop of XPO1 to Ran, thus favoring increased shuttling and retention in the cytoplasmic compartment. Therefore, we hypothesize that XPO1 R749Q may represent a novel mechanism of nuclear export alteration involved with kinetic tuning of global transport. Despite the increased nuclear export function, XPO1 R749Q cells did not show a proliferative advantage. We thus set out to test whether XPO1 R749Q might be enriched in cancer as an adaptive response to chemotherapy treatment. Using a chemical compound library of >200 FDA-approved cancer therapies we observed a strong therapeutic resistance of XPO1 R749Q cells relative to XPO1 WT cells, specifically to chemotherapies used in the treatment of colon cancer, such as irinotecan. However, these cells remained sensitive to treatment with the XPO1 inhibitor selinexor. Mice xenografted with XPO1 R749Q mutant HCT116 cells showed moderate tumor response to selinexor or irinotecan monotherapies but prolonged tumor responses to combination therapy. Recent clinical data has shown POLE mutations to be a biomarker for response to PD-1 immune checkpoint blockade, especially in CRC. Since our genetic data identify POLE mutations commonly co-occurring with XPO1 R749Q in colon cancer, we are planning to test the combination of PD-1 inhibitors and selinexor to target both these mutations, as well as CRC tumors without these mutations. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Monika Chojnacka, Gabriel Gaidosh, Jumana Afaghani, Maurizio Affer, Jenna Zabroski, Jacob Jahn, Vindhya Nawaratne, Ryan Notti, Ramiro Verdun, Daniel Bilbao, Josean Rodriguez, Justin Taylor. Novel therapeutics for targeting the aberrant nuclear export machinery in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B018.

Myeloid-derived itaconate suppresses cytotoxic CD8+ T cells and promotes tumour growth.

The tumour microenvironment possesses mechanisms that suppress anti-tumour immunity. Itaconate is a metabolite produced from the Krebs cycle intermediate cis-aconitate by the activity of immune-responsive gene 1 (IRG1). While it is known to be immune modulatory, the role of itaconate in anti-tumour immunity is unclear. Here, we demonstrate that myeloid-derived suppressor cells (MDSCs) secrete itaconate that can be taken up by CD8+ T cells and suppress their proliferation, cytokine production and cytolytic activity. Metabolite profiling, stable-isotope tracing and metabolite supplementation studies indicated that itaconate suppressed the biosynthesis of aspartate and serine/glycine in CD8+ T cells to attenuate their proliferation and function. Host deletion of Irg1 in female mice bearing allografted tumours resulted in decreased tumour growth, inhibited the immune-suppressive activities of MDSCs, promoted anti-tumour immunity of CD8+ T cells and enhanced the anti-tumour activity of anti-PD-1 antibody treatment. Furthermore, we found a significant negative correlation between IRG1 expression and response to PD-1 immune checkpoint blockade in patients with melanoma. Our findings not only reveal a previously unknown role of itaconate as an immune checkpoint metabolite secreted from MDSCs to suppress CD8+ T cells, but also establish IRG1 as a myeloid-selective target in immunometabolism whose inhibition promotes anti-tumour immunity and enhances the efficacy of immune checkpoint protein blockade.