The ZNF737-CXCL10 axis drives immune exclusion and resistance to anti-PD-1 therapy in bladder cancer.

Brain metastases (BrM) remain an intractable, deadly complication for advanced melanoma patients and efficient therapeutic strategies are desperately needed. The tumor microenvironment (TME) plays an important role in response to therapy. However, studies addressing the contribution of the TME to therapy efficacy for BrM are lacking, mostly due to limited access to human samples and scarcity of appropriate preclinical models. Here, we describe two novel isogenic immunocompetent BrM models generated by intracardiac injection of UV-induced mouse melanoma cell lines, representative of mutant-RAS human melanoma subtypes. We used these models to test immune checkpoint blockade (ICB) therapy and to interrogate the role of the TME in therapeutic efficacy. To evaluate response, we developed and applied a new machine-learning method to quantify metastatic burden. We investigated the TME by high-parametric flow cytometry and single-cell RNA sequencing (scRNA-seq). We showed that the models have distinct metastatic behaviors, with BR1 being mostly brain tropic and BR3 displaying widespread metastases. Notably, BR1 BrM were sensitive to ICB with a better response to anti-PD-L1/anti-CTLA-4 combination therapy as compared to monotherapies. In contrast, BR3 BrM were resistant to both mono- and combination therapies. Interestingly, we found that ICB efficacy on extracranial BR3 metastases is organ-dependent. Characterization of the BrM immune microenvironment before and after treatment revealed dramatic differences between the models. Untreated BR1 BrM showed significant recruitment of T cells, dendritic cells, and natural killer cells, while neutrophils were enriched in untreated ICB-resistant BR3 BrM. Moreover, we uncovered phenotypically distinct microglia populations exclusively present in ICB-sensitive BR1 BrM that positively correlated with T cell infiltration. Consistent with this finding, scRNA-seq showed upregulation of genes encoding for T cell-attracting chemokines and antigen presentation uniquely in the BR1-associated microglia. Post-treatment analysis of the brain TME highlighted beneficial changes induced by ICB in the responsive BR1 model, including increased recruitment of CD8 T cells with an activated phenotype, while a mild recruitment of exhausted T cells was observed in the resistant BR3 model. Altogether, our data emphasize the importance of interrogating the BrM TME to understand therapeutic response. Our unique BrM models, mirroring the diversity of ICB response observed in patients, provide a robust platform for the much-needed mechanistic studies to optimize BrM therapy. Deciphering the contribution of the newly identified BR1 BrM-associated microglia to ICB efficacy will be crucial to the identification of novel therapeutic targets. Citation Format: Amélie Lopès, Jessica Rappaport, Eva Pérez Guijarro, Quanyi Chen, Emily Wu, Isabella Church, April Huang, Jessica Bridge, Sung Chin, Cari Smith, Charli Gruen, Khiem C. Lam, Romina E. Araya, Antonella Sassano, Chi-Ping Day, Glenn Merlino, Romina S. Goldszmid. Interrogating the role of the immune microenvironment in the response of brain metastases to immunotherapy using new preclinical melanoma models. [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 5188.

Background: Irinotecan is a topoisomerase 1 inhibitor pro-drug used to treat gastrointestinal (GI) cancers including as first line for colorectal cancer (CRC) as part of the FOLFIRI plus monoclonal antibody (mAb) regimen, and for pancreatic cancer in the FOLFIRINOX regimen. However, irinotecan causes severe and dose-limiting side effects, including diarrhea and neutropenia. Starpharma has developed a highly optimized polylysine dendrimer enhanced (DEP) nanoparticle conjugate of the irinotecan active metabolite, SN38 (DEP SN38 or DEP irinotecan). DEP SN38 avoids the need for liver conversion of irinotecan to SN38 and achieves preferential tumor targeting. DEP SN38 is in phase 2 clinical investigation as monotherapy or combination therapy in patients with solid tumors (EudraCT no: 2019-001318-40). SN38 is known to enhance the efficacy of immune checkpoint blockade (ICB) via effects on immune cells in the tumor microenvironment. Here, we evaluated the anti-tumor effects of DEP SN38 alone or in combination with either ICB (anti- programmed cell death-1 [PD1] mAb) or poly adenosine diphosphate (ADP) ribose polymerase (PARP) inhibition (olaparib) in mouse models of GI cancer. Methods: HT29 (human CRC) and CAPAN-1 (human pancreatic cancer) cell line xenografts were established subcutaneously in BALB/c nude or NOD-scid-Gamma immunodeficient mice, respectively. Three doses of DEP SN38 (at 5-10mg/kg SN38) or irinotecan (35-80 mg/kg) were administered intravenously (IV) on days 1, 8, 15. Olaparib (50 mg/kg) was delivered by daily IV injection for 5 days/week over 3 weeks. DEP SN38 (IV on days 1, 8, 15 at 18-35 mg/kg SN38) or irinotecan (75 mg/kg) plus rat anti-PD1 mAb therapy or control mAb (intraperitoneally, 100 mg/kg then 50 mg/kg on days 5, 8, 12) was evaluated in both MC38 (C57BL/6) and CT26 (BALB/c) allograft models of CRC. Toleration of drug was bodyweight loss ≤15%. Results: DEP SN38 treatment of HT29 or CAPAN-1 xenografts led to tumor regressions, prolonged control of tumor growth and improved survival. In contrast, conventional irinotecan had minimal effect on tumor growth versus vehicle control. DEP SN38 was well tolerated by mice with minimal bodyweight loss. Administration of anti-PD1 mAb alone confirmed the known modest sensitivity of MC38, and resistance of CT26 syngeneic tumors, to ICB. DEP SN38 plus anti-PD1 mAb, enhanced anti-tumor responses in the MC38 model beyond those seen for each agent alone and also resulted in tumor eradication and prolonged survival. In the CT26 model, anti-PD1 mAb was also augmented by addition of DEP SN38 but not irinotecan alone, despite resistance of CT26 tumors to anti-PD1 mAb alone. The HT29 CRC model was resistant to Olaparib alone. In contrast, anti-tumor effects and prolonged survival were observed with olaparib plus DEP SN38. Conclusions: DEP SN38 demonstrated superior efficacy compared to irinotecan when used in models of GI cancer. The anti-tumor efficacy of both ICB and PARP inhibition was augmented by DEP SN38 in models of CRC, and these combinations should be investigated clinically. Citation Format: Benjamin J Blyth, Brian D Kelly, Michael Giannis, Anne Cargill, Aynaz Seta, Graham P Heery, Anthony Eglezos, Cameron N Johnstone, Jeremy R A Paull. An SN38 dendrimer nanoparticle, DEP irinotecan (SN38-SPL9111), demonstrates efficacy in mouse models of gastrointestinal cancer and augments anti-tumor effects of immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C167.

BackgroundEpstein-Barr virus (EBV)-associated gastric cancer (GC) (EBVaGC) is a distinct molecular subtype of GC with a favorable prognosis. However, the exact effects and potential mechanisms of EBV infection on immune...

Although radiotherapy (RT) and immune checkpoint blockade (ICB) are effective for treatment of triple-negative breast cancer (TNBC), significant portion of patients do not experience a clinical benefit, partly due to activation of immunosuppressive cells. Capivasertib, an AKT inhibitor, is approved for use in advanced luminal breast cancer, but its immunomodulatory role in breast cancer remains still unclear. Given the ability of PI3K/AKT/mTOR inhibition to regulate immunosuppressive myeloid cells, we hypothesized that capivasertib could enhance the efficacy of RT and ICB in TNBC. In this study, we investigated the antitumor effects and phenotypes of immune cells in 4T1 syngeneic mouse TNBC model treated with RT, dual ICB (PD-L1 and CTLA-4 blockade), and capivasertib. A dual-tumor model was used to examine abscopal effects, with one tumor receiving RT and the other remaining unirradiated. We found that the quadruple therapy (i.e. RT + anti-PD-L1 + anti-CTLA-4 + capivasertib) resulted in greatest growth delay of both irradiated (primary) and unirradiated (secondary) tumors, along with the lowest number of metastatic lung nodules. Notably, compared to the RT + anti-PD-L1 + anti-CTLA-4 treatment, the quadruple therapy significantly reduced tumor-infiltrating macrophages, with an increase in inflammatory M1 macrophages and a decrease in immunosuppressive M2 macrophages. Moreover, CD86+ mature dendritic cells within tumor microenvironment were increased by addition of capivasertib to RT + anti-PD-L1 + anti-CTLA-4 treatment. These changes were not observed within the spleen and tumor-draining lymph node. Proportion and phenotypes of myeloid-derived suppressor cells (MDSCs) and T cells in tumor were not significantly changed by the quadruple therapy. These findings suggest that capivasertib, when combined with RT and ICB, led to less immunosuppressive tumor microenvironment by modulating tumor-infiltrating myeloid cells. Taken together, this combination strategy could be a viable approach to overcome therapeutic resistance of ICB in TNBC patients. (Work supported by grants from KHIDI & AstraZeneca, and the National Research Foundation of Korea #2023R1A2C3003782) Citation Format: Nawon Park, Seung Hyuck Jeon, Yoomin Kim, Seongmin Kim, In Ah Kim. AKT inhibition enhances antitumor efficacy of radiation and immune checkpoint blockade in triple-negative breast cancer by modulating tumor-infiltrating myeloid cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2167.

e14596 Background: Immune-related adverse events (irAEs) are major hurdle to immune checkpoint blockade (ICB). Corticosteroid (CS) is effective in controlling the majority of irAEs. Although recent studies suggested CS use does not jeopardize the anti-tumor efficacy of ICB, prophylactic use of CS remains prohibited with the concerns of attenuating efficacy of ICB. This study aimed to investigate the effects of CS premedication on the efficacy of ICB in murine hepatocellular carcinoma models. Methods: Anti-mCTLA-4 (9D9C, BMS) and anti-mPD-1 antibodies (4H2, ONO) were intraperitoneally (ip) administered to tumor-bearing mice (subcutaneous Hepa 1-6 model and orthotopic BNL 1MEA.7R.1 model) with or without dexamethasone (DEXA) premedication (10 and 200 μg, equivalent to minimal anti-inflammatory dosage and pulse therapy in human, respectively). Efficacy of ICB was evaluated as tumor shrinkage. Tumor-infiltrating lymphocytes (TILs) were isolated for single cell RNA-sequencing and effector function analysis through flow cytometry. Results: In the subcutaneous model, all tumors treated with ICB alone (N=7) or ICB plus DEXA 10 μg (N=5) completely regressed, but 1 out of 7 tumors treated with ICB plus DEXA 200 μg escaped. However, the tumor growth was not significantly different between groups ( P-value >0.05, multiple Mann-Whitney test). In the orthotopic model (N=5/group), the mean (± standard error) tumor weights on day 21 after tumor implantation for isotype control, ICB, ICB plus DEXA 10 μg and ICB plus DEXA 200 μg were 2.45 (± 0.54), 0.73 (± 0.18), 0.98 (± 0.65), and 0.69 (± 0.17) grams, respectively ( P-value >0.05, comparing ICB plus DEXA 10 or 200 μg with ICB). At transcriptomic level, premedication with either dosage of DEXA significantly reduced the percentage of effector memory cells and increased the percentage of exhausted effector cells in the CD8 TIL population, which appeared to counterbalance the effects of ICB. However, at protein level, premedication of DEXA 10 or 200 μg did not reduce the interferon-γ or granzyme B production of CD8 TILs in both models (Table). Conclusions: CS premedication did not attenuate the efficacy of ICB. Our study provides the scientific basis to evaluate the potential of prophylactic CS in preventing ICB-induced irAEs in clinical studies.[Table: see text]

BackgroundProgrammed death-ligand 1 (PD-L1) positivity is associated with a favorable response to immune checkpoint blockade (ICB) in urothelial bladder cancer (BLCA). However, the efficacy of ICB in BLCA exhibits considerable heterogeneity, leading to the need for complementary predictive biomarkers. Recent studies suggest that a high degree of plasma cell infiltration is correlated with improved benefit from ICB, but a specific plasma cell marker in BLCA has not been identified. The aim of this study was to evaluate tumor necrosis factor receptor superfamily member 17 (TNFRSF17) as a plasma cell-specific marker in BLCA and test its utility, combined with PD-L1, for patient stratification receiving ICB therapy.MethodsTranscriptomic and clinical data from publicly available cohorts were analyzed. Plasma cell-associated markers were identified based on expression specificity and correlation analyses. The clinical relevance of TNFRSF17, alone and in combination with CD274, was evaluated by comparisons of survival and the response rate. Associations with immunotherapy-related features were examined using established surrogate measures, including the immunophenoscore. In silico deconvolution analyses were performed to characterize the immunogenic tumor microenvironment by comparing distinct immune infiltration patterns and differential gene expression pathways between the subgroups.ResultsPlasma cell infiltration correlated with favorable survival in BLCA patients. Higher expression of TNFRSF17, a plasma cell-specific marker (R = 0.73 ± 0.15; z score = 1.88 ± 0.41), correlated with increased immunophenoscores, more favorable overall survival outcomes (HR = 0.59) and increased responsiveness to ICB therapy. Tumors with concurrent high TNFRSF17 and CD274 expression exhibited the most favorable survival outcomes (HR = 0.38) and demonstrated an immune-inflamed transcriptional profile, including enrichment of antigen presentation and immune signaling pathways.ConclusionsTNFRSF17 serves as a potential marker to characterize an immune-distinct and prognostically favorable subgroup within CD274High tumors, and to refine stratification for ICB.

Although immunotherapy has shown promise in improving outcomes for bladder cancer (BCa) patients, treatment responses remain highly variable. A comparative examination of the tumor microenvironment (TME) between responders and non-responders may reveal key resistance mechanisms and identify potential therapeutic targets. We integrated spatial transcriptomics, single-cell RNA sequencing, and multiplexed immunofluorescence to characterize spatial structures within the TME that influence response to anti-PD-1 therapy in BCa patients. In non-responders, we observed an accumulation of stem-like malignant epithelial cells with high MYBL2 expression near the tumor boundary. Furthermore, we identified a spatial triad structure-composed of SPP1+ tumor-associated macrophages (TAMs), POSTN+ cancer-associated fibroblasts (CAFs), and endothelial cells-located at the tumor periphery. This structure was associated with T-cell exclusion and reduced efficacy of immune checkpoint blockade. In a preclinical model, inhibiting SPP1 enhanced the response to anti-PD-1 therapy, resulting in reduced CAF infiltration and increased recruitment of cytotoxic T cells. Our study reveals a triad cellular structure mediated by SPP1+ TAMs, POSTN+ CAFs, and endothelial cells that contribute to immunotherapy resistance in BCa. Targeting this structure, particularly through SPP1 blockade, represents a promising strategy to augment the efficacy of immune checkpoint inhibitors.

Molecular Pathways Responsible for Adaptive Resistance to Single-Checkpoint Inhibition May Reveal Biomarkers and Immunotherapy Targets in Multiple Myeloma Patients

Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising and immunotherapies have benefits over traditional chemotherapeutics, a sizable percentage of patients are non-responders to all types of immunotherapy as a treatment option. These differences in sensitivity can be either innate or acquired. Yet, there has been limited 3D in vitro models to assess tumor immune-reactivity. These systems are ideal for isolating specific molecular mechanisms that dictate cell behavior and interactions. Our goal was to create an organoid model containing cancer cells paired with cytotoxic T-cells to model immune checkpoint blockade (ICB) efficacy. This model could then be used to examine novel microbiome-ICB interactions shown by recent research to alter therapeutic response levels in patients. Methods: We created tumor organoids consisting of matched tumor and immune cells, embedded in extracellular matrix (ECM)-like hydrogels. Organoids were treated with therapeutic equivalent doses of anti-PD-1 and anti-CTLA-4 or single dose of anti-CD-47. The organoids were also exposed to physiologic concentrations of metabolites 3-indolepropionic acid derived from the bacterial species Clostridium sporogenes, hippurate derived from Clostridiales, Faecalbacterium prausnitzii, and Eubacterium, pyocyanin derived from Pseudomonas aeruginosa, butyrate derived from Faecalbacterium prausnitzii, and inosine derived from Bifidobacterium pseudolongum. Each of these bacterial species and the associated metabolite represent a likely effector of host immune function described in literature and therefore a potential effector of ICB response. Organoids were analyzed with cell viability assays, flow cytometry, RT-qPCR, and immunohistochemistry staining to determine the effects of the metabolites on ICB response. Results and Discussion: We showed that ICB therapy stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing. ICB treated samples resulted significant loss of viability with corroborating readings from the other methods of characterization. RT-qPCR and flow cytometry demonstrated the cellular changes due to bacterial metabolite co-administration. These results include increased expression of CD-8 T-cell co-receptor, increased cytokine production, and increased effector T-cell viability. Conclusion: We have created an ex-vivo tumor immune-reactive organoid model for studying immunotherapy. We are working to elucidate the effects of microenvironment factors, such as microbiome metabolites, and observe their impacts on immunotherapy efficacy to better understand what conditions are conducive or detrimental to successful ICB treatment. This abstract is also being presented as Poster P002. Citation Format: Ethan Shelkey, Yong Lu, David Soto-Pantoja, Shay Soker. Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr PR03.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

Antibiotic use can result in reduced efficacy of immune checkpoint blockade (ICB), presumably because of dysbiosis of the intestinal microbiome. We sought to determine the precise temporal relation between antibiotic therapy and its possible effects on ICB efficacy. We also investigated the histologic changes in the tumor microenvironment secondary to antibiotics use. This was a single institution retrospective study that evaluated the impact of antibiotics on outcomes of patients with advanced or metastatic malignancy who were treated with ICB. Use of antibiotics among patients treated with ICB was assessed during a 12-week period before and after initiation of ICB. The primary outcome was response to ICB. Histologic changes in the tumor microenvironment following antibiotics use were also examined. Between January 1, 2011 and December 31, 2018, 414 patients were identified who received ICB, and 207 patients (50%) received antibiotics within 12 weeks (before/after) of initiation of ICB. In univariate analysis, antibiotic use following initiation of ICB was associated with a significantly reduced response (odds ratio [OR]: 0.33, 95% confidence interval [CI]: 0.2-0.52, P<0.001). There was no significant negative impact on response to immunotherapy when antibiotics were used before ICB initiation (OR: 0.87, 95% CI: 0.55-1.34, P=0.52). The maximal negative impact of antibiotics occurred in the first 6 weeks after initiating ICB, and was independently associated with significantly reduced likelihood of response to immunotherapy in multivariable analysis (OR: 0.48, 95% CI: 0.29-0.8, P=0.01). This study demonstrates that the use of antibiotics during ICB significantly negatively impacts the efficacy of immunotherapy. The maximal negative impact occurs if the antibiotics are used in the first 6 weeks after initiating ICB.

BackgroundCold tumor is one of the most refractory solid tumors and experience dismal response to immune checkpoint blockade (ICBs). Most therapeutic vaccines provided prerequisite for ICBs by priming tumor specific...

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer. The discovery that this subtype has the highest levels of tumor-infiltrating immune cells and programmed death ligand 1 (PD-L1) expression in the tumor microenvironment (TME) has propelled the use of anti-PD-1 immune checkpoint blockade (ICB) therapy, in combination with chemotherapy, as a treatment for TNBC patients. This has advantageously impacted TNBC patient outcomes such as survival, yet there remains a need to improve patient response to ICB therapies. The gut microbiome, in particular, increased abundance of Akkermansia muciniphila was associated with ICB therapy response in other cancer types. A. muciniphila can degrade mucins and produce short-chain fatty acid (SCFA) metabolites. Therefore, we investigated the interplay between A. muciniphila, SCFAs, and ICB therapy efficacy in TNBC models. As diet is the main modifier of the gut microbiome, we first investigated diet-gut microbiome interactions on ICB efficacy. Using the EO771 syngeneic murine model of TNBC, female C57BL/6 tumor-bearing mice consuming a low-fat control, or high-fat Western, or Mediterranean diet, were treated with 3 doses of 200µg of IgG or anti-PD-L1 antibodies and tumor progression was monitored. To assess changes in tumoral immune cell populations, end-of-study tumor tissue was used for immunohistochemistry (IHC). To evaluate modulation on the gut microbiome, metagenomic sequencing was performed on DNA isolated from fecal samples collected at the end-of-study, and targeted SCFA metabolomic analysis was performed on plasma collected at the study endpoint. Results show mice consuming a Mediterranean diet treated with anti-PD-L1 had significant increases in the abundance of gut A. muciniphila and plasma SCFAs, as well as significantly reduced primary tumor volume and increased immune cell activity within the TME. To then investigate if direct supplementation of A. muciniphila or SCFAs could enhance ICB efficacy, EO771 tumor-bearing mice were treated with 3 doses of 200µg of IgG, anti-PD-L1 or anti-PD-1 antibodies with a subset of mice stratified to also receive A. muciniphila or exogenoussupplementation of SCFAs. Tumor progression was measured and changes in tumoral immune cell populations were assessed with end-of-study tumor tissue prepared for flow cytometry and IHC. Results show mice supplemented with A. muciniphila and treated with anti-PD-L1 therapy demonstrated response to treatment, with a significant reduction in tumor volume as well as increased levels of immune cell populations in the TME. Mice supplemented with SCFAs and treated with a combination of anti-PD-1 and chemotherapy demonstrated enhanced response to treatment, with a significant reduction in tumor volume. In conclusion, data from our lab suggest that increased levels of gut A. muciniphila and SCFAs drive ICB efficacy in TNBC. Citation Format: Kenysha YJ. Clear, Adam S. Wilson, Elizabeth R. Stirling, Yu-Ting Tsai, Valerie Payne, David R. Soto-Pantoja, Katherine L. Cook. Harnessing the gut microbiome to modulate immune checkpoint blockade response in triple-negative breast cancer [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 1423.

Overcoming acquired adaptive immune resistance to anti-PD-1 therapy is imperative for enhancing the efficacy of immune checkpoint blockade (ICB) in solid tumors. Regulatory T cells (Tregs) play a prominent role in the suppressive tumor microenvironment (TME) and are major contributors to adaptive immune resistance. Tregs limit CD8+ T cell reinvigoration and are a promising target for combination therapy. While the clinical efficacy of anti-CTLA4 may be partially explained by restriction of Tregs, its co-administration with anti-PD1 causes significant toxicity. Thus, safer approaches to limit Treg activity are needed. To elucidate the dynamic changes in immuno-regulatory circuits within the TME during ICB, we performed deep immune profiling of peripheral blood and tumors from patients with advanced melanoma prior to (n=7) and after 1 cycle of anti-PD-1 therapy with pembrolizumab (n=9). Tregs were abundant in the TME and retained their immunosuppressive phenotype and functionality following anti-PD-1. Epigenetic, transcriptomic, and proteomic analysis of Tregs after ICB identified tumor necrosis factor receptor 2 (TNFR2) signaling as a possible driver of CD8+ T cell suppression. TNFR2 was preferentially expressed by Tregs in the TME (mean 18.03 %, SD +/− 10.13 %) relative to CD8+ T cells (mean 0.64 %, SD +/− 0.82 %) and peripheral Tregs (mean 3.16 %, SD +/− 3.21 %), suggesting it might be a safe and effective target for combination therapy. Indeed, dual blockade of TNFR2 and PD-1 led to potent CD8+ T cell expansion in two mouse tumor models, and restored sensitivity to ICB in a resistant murine model of melanoma. Our data suggest that anti-TNFR2 might synergize with current ICB by countering the development of adaptive immune resistance.

Immune checkpoint blockades (ICBs) have limited efficacy against immunologically cold breast cancers. Although radiotherapy (RT) can potentiate antitumor immunity, many patients fail to benefit, partly due to activation of immunosuppressive cells. Given the ability of PI3K inhibition to regulate immunosuppressive repertoires, we hypothesized that inhibition of Akt, a key effector of PI3K, could enhance the efficacy of RT and ICBs. In this study, we investigated antitumor effects and immune cell phenotypes in 4T1 syngeneic triple-negative breast cancer (TNBC) models treated with RT, ICBs, and an Akt inhibitor. RT + αPD-L1 + Akt inhibitor most reduced tumor size and lung metastases. The Akt inhibitor increased the M1/M2 macrophage ratio and the proportion of CD86+ dendritic cells (DCs). Interestingly, RT + αPD-L1 increased M2 macrophages, while adding the Akt inhibitor partially restored M1/M2 balance. When combined with RT + αPD-L1/αCTLA-4, Akt inhibition further elevated the M1/M2 ratio and CD86+ DC frequency and reduced monocytic myeloid-derived suppressor cells (M-MDSCs). These findings suggest that addition of an Akt inhibitor to RT and ICBs leads to a less immunosuppressive tumor microenvironment by modulating myeloid cells. Taken together, Akt inhibition could be a viable strategy to overcome therapeutic resistance of ICBs and RT in breast cancer.