Checkpoint Blockade In Breast Cancer Research Articles

Tumor-intrinsic CDC42BPB confers resistance to anti-PD-1 immune checkpoint blockade in breast cancer

Immune checkpoint blockade has been used to treat breast cancer, but the clinical responses remain relatively poor. We have used the CRISPR-Cas9 kinome knockout library consisting of 763 kinase genes to identify tumor-intrinsic kinases conferring resistance to anti-PD-1 immune checkpoint blockade. We have identified the CDC42BPB kinase as a potential target to overcome the resistance to anti-PD-1 immune checkpoint blockade immunotherapy. We found that CDC42BPB is highly expressed in breast cancer patients who are non-responsive to immunotherapy. Furthermore, a small-molecule pharmacological inhibitor, BDP5290, which targets CDC42BPB, synergized with anti-PD-1 and enhanced tumor cell killing by promoting Tcell proliferation in both invitro and invivo assays. Moreover, anti-PD-1-resistant breast cancer cells showed higher expression of CDC42BPB, and its inhibition rendered the resistant cells more susceptible to Tcell killing in the presence of anti-PD-1. We also found that CDC42BPB phosphorylated AURKA, which in turn upregulated PD-L1 through cMYC. Our results have revealed a robust link between tumor-intrinsic kinase and immunotherapy resistance and have provided a rationale for a unique combination therapy of CDC42BPB inhibition and anti-PD-1 immunotherapy for breast cancer.

Abstract 7514: Tumor intrinsic CDC42BPB confers resistance to anti-PD-1 immune checkpoint blockade in breast cancer

Abstract Immune checkpoint blockade has been used to treat breast cancer, but the clinical responses remain relatively poor, especially for the triple-negative subtype. The weak responsiveness is attributed to tumor intrinsic as well as tumor extrinsic factors, suggesting a need for novel approaches. We have used the CRISPR Cas9 kinome knockout library consisting of 763 kinase genes to identify tumor intrinsic kinase conferring resistance to anti-PD-1 immune checkpoint blockade and identified the CDC42BPB kinase as a potential target to overcome the resistance to anti-PD-1 immunotherapy. We found that CDC42BPB is highly expressed in breast cancer patients who are non-responsive to immunotherapy. Importantly, a small molecule pharmacological inhibitor, BDP5290, which targets CDC42BPB synergized with anti-PD-1 and enhanced tumor cell killing by promoting T-cell proliferation in both in vitro and in vivo assays. Moreover, anti-PD-1 resistant breast cancer cells showed higher expression of CDC42BPB, and its inhibition rendered the resistant cells more susceptible to T cell killing in the presence of anti-PD-1. We also found that CDC42BPB phosphorylated AURKA which in turn upregulated PD-L1 through cMYC. Our results have revealed a robust link between tumor intrinsic kinase and immunotherapy resistance and provided a rationale for a novel combination therapy of CDC42BPB inhibition and anti-PD-1 immunotherapy for breast cancer. Citation Format: Ravindra Pramod Deshpande, Kerui Wu, Shih-Ying Wu, Abhishek Tyagi, John Hunting, Jimmy Ruiz, Wencheng Li, Kounosuke Watabe. Tumor intrinsic CDC42BPB confers resistance to anti-PD-1 immune checkpoint blockade in 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 7514.

IL-5-producing CD4+ Tcells and eosinophils cooperate to enhance response to immune checkpoint blockade in breast cancer.

Immune checkpoint blockade (ICB) has heralded a new era in cancer therapy. Research into the mechanisms underlying response to ICB has predominantly focused on Tcells; however, effective immune responses require tightly regulated crosstalk between innate and adaptive immune cells. Here, we combine unbiased analysis of blood and tumors from metastatic breast cancer patients treated with ICB with mechanistic studies in mouse models of breast cancer. We observe an increase in systemic and intratumoral eosinophils in patients and mice responding to ICB treatment. Mechanistically, ICB increased IL-5 production by CD4+ Tcells, stimulating elevated eosinophil production from the bone marrow, leading to systemic eosinophil expansion. Additional induction of IL-33 by ICB-cisplatin combination or recombinant IL-33 promotes intratumoral eosinophil infiltration and eosinophil-dependent CD8+ Tcell activation to enhance ICB response. This work demonstrates the critical role of eosinophils in ICB response and provides proof-of-principle for eosinophil engagement to enhance ICB efficacy.

T cell-eosinophil crosstalk-A new road for effective immune checkpoint blockade in breast cancer?

Immune checkpoint blockade (ICB) has revolutionized the landscape of cancer treatment. Nevertheless, most cancer patients still do not respond to ICB. In this issue of Cancer Cell, Blomberg etal. illustrate a critical cooperation between Tcells and eosinophils, which jointly enhance effectiveness of ICB in breast cancer.

Abstract P2-08-23: Early intratumoral changes after a single dose of anti-PD-1 treatment in patients with early breast cancer (BC)

Abstract Intro: Despite the growing success of immune checkpoint blockade in BC, the underlying mechanisms of response or resistance to these treatments is still poorly understood. We recently conducted a window-of-opportunity study (BioKey) including 54 patients with early BC (NCT03197389) in which patients were treated with one dose of pembrolizumab 10 ± 4 days before surgery. A tumor biopsy was taken before and after the administration of pembrolizumab. Using single-cell transcriptome profiling, we identified patients with clonotype expansion of PD-1-expressing T-cells after treatment with pembrolizumab (Bassez A, Vos H et al, Nature Medicine 2021). Expansion mainly involved CD8+ T - and CD4+ T cells. We designated these as patients with clonotype expansion or ‘expanders’ (E), whereas patients lacking clonotype expansion were considered ‘non-expanders’ (NE). In this study, we aimed at investigating the association between stromal tumor infiltrating lymphocytes (sTIL) and various immune markers using immunohistochemistry with the E phenotype. Methods: The BioKey trial has 2 cohorts. The first one included 39 treatment-naive patients who were scheduled for upfront surgery. The second cohort included 15 patients with an estimated residual tumor size of at least 1cm on imaging after neo-adjuvant chemotherapy. All molecular subtypes were represented: triple-negative (TN) BC (n= 18, 8 E and 10 NE), HER2-positive BC (n= 5, 1 E and 4 NE) and hormone receptor (HR)-positive/HER2-negative BC (n= 17, 3 E and 14 NE). 7 core biopsies and 5 resection slides were not contributive and were excluded. The expander status of 14 patients was unknown. sTIL were evaluated and immunohistochemical stains (CD3, CD4, CD8, CD68, CD73, FoxP3, Ki67, PD-1 and PD-L1) were performed. Associations between expander status and biomarkers were assessed using firth logistic regressions, adjusting per cohort. Biomarkers were considered as continuous variables and Odds Ratio computed per one unit increase on a non-transformed scale. Given the low number of E in cohort B, subgroup analyses were performed only in cohort A. Results: We observed that 1) higher levels of sTIL, PD-1+ sTIL and FOXP3+ cells in the pre-treatment biopsies, 2) higher levels of sTIL, PD1+ sTIL, PD-L1+, CD3+, CD4+, CD8+, CD68+, FOXP3+ cells and Ki67 in the post-treatment surgical specimen, as well as, 3) higher delta values (surgical specimen - biopsy) of sTIL, CD3+, CD4+, CD8+, CD68+ and Ki67 are associated with the E phenotype (Table 1). With regard to the subgroup analyses, we observed that higher levels of PD-1+ sTIL and CD68+ cells at diagnosis and surgery respectively, as well as higher delta values for CD68+, Ki67 and sTIL were associated with the E phenotype in HR+/HER2- BC, while high levels of FOXP3+ cells and PD-1+ sTILs in the biopsies, high levels of sTIL, CD3+, CD4+, CD8+, CD68+, FOXP3+, PD-1+ sTILs, PD-L1+ and Ki67 as well as higher delta values for PD-L1+ in the resection specimen were associated with the E phenotype in TNBC. Conclusion: We were able to detect associations between various cell populations of the tumor immune microenvironment evaluated before and after a single dose of pembrolizumab and the T-cell expansion phenotype previously defined at the single-cell level. It highlights the possible clinical utility of a biopsy taken early during treatment. Future neo-adjuvant studies will allow the validation of our findings and investigate the association between T-cell expansion and clinical outcomes. Table 1.Association between the various biomarkers and the expanding T-cell phenotypeVariableOdds RatioLower 95% CIUpper 95% CIp-valuePre-treatment. Core biopsyCD31.071.001.160.061CD41.060.981.170.156CD81.130.951.380.161CD681.050.901.230.494CD73.tumor1.070.821.370.609CD73.on sTIL1.010.961.060.713FoxP32.251.394.030.001Ki671.021.001.050.107sTIL1.061.021.140.005PD-1 on sTIL1.731.212.830.001PD-L11.041.011.070.002Post-treatment. Resection specimenCD31.091.031.190.001CD41.151.051.29<0.001CD81.581.192.34<0.001CD681.471.152.15<0.001CD73.tumor1.090.771.520.608CD73.on sTIL1.030.991.080.110FoxP32.051.313.82<0.001Ki671.041.011.070.004sTIL1.081.031.15<0.001PD-1 on sTIL1.641.152.650.004PD-L11.051.021.110.002Delta (resection-biopsy)CD31.071.011.250.029CD41.091.011.280.030CD81.211.021.690.025CD681.221.051.580.007CD73.tumor0.970.731.290.794CD73.on sTIL1.040.991.120.132FoxP31.220.891.810.219Ki671.051.001.130.031sTIL1.071.001.160.046PD-1 on sTIL0.960.701.330.765PD-L11.000.961.040.941 Citation Format: Hanne Vos, Kathleen Lambein, François Richard, Ines Nevelsteen, Ayse Bassez, Diether Lambrechts, Kevin Punie, Hans Wildiers, Giuseppe Floris, Christine Desmedt, Ann Smeets. Early intratumoral changes after a single dose of anti-PD-1 treatment in patients with early breast cancer (BC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-23.

LBA10 Critical role of eosinophils during response to immune checkpoint blockade in breast cancer and other cancer types

LBA10 Critical role of eosinophils during response to immune checkpoint blockade in breast cancer and other cancer types

Prognostic value of immune checkpoint molecules in breast cancer.

Immune checkpoint blockade treatments bring remarkable clinical benefits to fighting several solid malignancies. However, the efficacy of immune checkpoint blockade in breast cancer remains controversial. Several clinical trials of immune checkpoint blockades focused on the effect of CTLA4 and PD1/PDL1 checkpoint inhibitors on breast cancer. Only a small portion of patients benefited from these therapies. Here we systematically investigated the expression of 50 immune checkpoint genes, including ADORA2A, LAG-3, TIM-3, PD1, PDL1, PDL2, CTLA-4, IDO1, B7-H3, B7-H4, CD244, BTLA, TIGIT, CD80, CD86, VISTA, CD28, ICOS, ICOSLG, HVEM, CD160, LIGHT, CD137, CD137L, OX40, CD70, CD27, CD40, CD40LG, LGALS9, GITRL, CEACAM1, CD47, SIRPA, DNAM1, CD155, 2B4, CD48, TMIGD2, HHLA2, BTN2A1, DC-SIGN, BTN2A2, BTN3A1, BTNL3, BTNL9, CD96, TDO, CD200 and CD200R, in different subtypes of breast cancer and assessed their prognostic value. The results showed that the expression patterns of these 50 immune checkpoint genes were distinct in breast cancer. High expression of B7-H3 mRNA was significantly associated with worse overall survival (OS), especially in patients with luminal A and luminal B breast cancer. The mRNA expression levels of TIM-3, ADORA2A, LAG3, CD86, CD80, PD1 and IDO1 had no relationship with OS in breast cancer. High expression levels of CTLA-4 and TIGIT were correlated with favorable prognosis in breast cancer. Interestingly, we observed that B7-H3 expression was negatively correlated with the efficacy of cyclophosphamide (CTX). In summary, our study suggested that B7-H3 has potential prognostic value in breast cancer and is a promising target for immune therapy.

In silico simulation of a clinical trial with anti-CTLA-4 and anti-PD-L1 immunotherapies in metastatic breast cancer using a systems pharmacology model.

The low response rate of immune checkpoint blockade in breast cancer has highlighted the need for predictive biomarkers to identify responders. While a number of clinical trials are ongoing, testing all possible combinations is not feasible. In this study, a quantitative systems pharmacology model is built to integrate immune–cancer cell interactions in patients with breast cancer, including central, peripheral, tumour-draining lymph node (TDLN) and tumour compartments. The model can describe the immune suppression and evasion in both TDLN and the tumour microenvironment due to checkpoint expression, and mimic the tumour response to checkpoint blockade therapy. We investigate the relationship between the tumour response to checkpoint blockade therapy and composite tumour burden, PD-L1 expression and antigen intensity, including their individual and combined effects on the immune system, using model-based simulations. The proposed model demonstrates the potential to make predictions of tumour response of individual patients given sufficient clinical measurements, and provides a platform that can be further adapted to other types of immunotherapy and their combination with molecular-targeted therapies. The patient predictions demonstrate how this systems pharmacology model can be used to individualize immunotherapy treatments. When appropriately validated, these approaches may contribute to optimization of breast cancer treatment.

Inhibition of histone lysine-specific demethylase 1 elicits breast tumor immunity and enhances antitumor efficacy of immune checkpoint blockade.

Immunotherapy strategies have been emerging as powerful weapons against cancer. Early clinical trials reveal that overall response to immunotherapy is low in breast cancer patients, suggesting that effective strategies to overcome resistance to immunotherapy are urgently needed. In this study, we investigated whether epigenetic reprograming by modulating histone methylation could enhance effector T lymphocyte trafficking and improve therapeutic efficacy of immune checkpoint blockade in breast cancer with focus on triple negative breast cancer (TNBC) subtype. In silico analysis of TCGA data shows that expression of histone lysine specific demethylase 1 (LSD1) is inversely associated with the levels of cytotoxic T cell attracting chemokines (CCL5, CXCL9, CXCL10) and programmed death-ligand 1 (PD-L1) in clinical TNBC specimens. Tiling chromatin immunoprecipitation study showed that re-expression of chemokines by LSD1 inhibition is associated with increased H3K4me2 levels at proximal promoter regions. Rescue experiments using concurrent treatment with siRNA or inhibitor of chemokine receptors blocked LSD1 inhibitor-enhanced CD8+ T cell migration, indicating a critical role of key T cell chemokines in LSD1-mediated CD8+ lymphocyte trafficking to the tumor microenvironment. In mice bearing TNBC xenograft tumors, anti PD-1 antibody alone failed to elicit obvious therapeutic effect. However, combining LSD1 inhibitors with PD-1 antibody significantly suppressed tumor growth and pulmonary metastasis, which was associated with reduced Ki-67 level and augmented CD8+ T cell infiltration in xenograft tumors. Overall, these results suggest that LSD1 inhibition may be an effective adjuvant treatment with immunotherapy as a novel management strategy for poorly immunogenic breast tumors.

Abstract 1634: CDK4/6 inhibition directly enhances an anti-tumor immune response in breast cancer

Abstract The cyclin D:cyclin-dependent kinases 4 and 6 (CDK4/6) axis is one of the most frequently dysregulated pathways in human cancers, and CDK4/6 inhibitors have shown significant activity against a number of solid tumors, including breast cancer. Analogous to clinical experience, we found that the CDK4/6 inhibitor, abemaciclib, caused significant tumor regression in the MMTV-rtTA/tetO-HER2 mouse model of luminal breast cancer. However, as CDK4/6 inhibitors are known to block tumor cell proliferation, but not directly induce tumor cell apoptosis, as we confirmed in our study, the mechanisms by which CDK4/6 inhibition caused tumor regression were not clear. Notably, abemaciclib therapy increased total CD3+ T cells in these tumors, while decreasing the immunosuppressive CD4+ regulatory T cell population. Further investigation revealed that CDK4/6 inhibition directly suppresses regulatory T cells by inhibiting their proliferation without impacting natural regulatory T cell formation in the thymus, the differentiation of inducible regulatory T cells, or regulatory T cell apoptosis. We are currently exploring potential effects of CDK4/6 inhibition on regulatory T cell function and lineage stability. Analysis of CD8+ and CD4+ T cells in abemaciclib-treated tumors revealed a marked reduction in the expression of the inhibitory immune checkpoint receptors PD-1, Tim-3, CTLA-4, and LAG3. Specifically, the fraction of CD8+ T cells expressing known markers of T cell exhaustion (PD-1/Tim-3 double-positivity or high expression of PD-1) was decreased in abemaciclib-treated tumors. Importantly, antibody-mediated depletion of CD8+ T cells established that response of MMTV-rtTA/tetO-HER2 tumors to abemaciclib is dependent on CD8+ T cells. Finally, given the observed immunomodulatory effects of abemaciclib, we sought to determine if abemaciclib treatment would sensitize MMTV-rtTA/tetO-HER2 tumors to immune checkpoint blockade. Pretreatment with abemaciclib followed by combination abemaciclib and α-PDL1 significantly enhanced tumor regression compared to abemaciclib or α-PDL1 alone. Our studies reveal a novel mechanism by which CDK4/6 inhibitors directly elicit an anti-tumor immune response. These results provide strong rationale for further investigations into combining CDK4/6 inhibitors with immune checkpoint blockade in breast cancer. Citation Format: Molly J. DeCristo*, Shom Goel*, April C. Watt, Haley BrinJones, Jaclyn Sceneay, Ben Li, Jessalyn M. Ubellacker, Shaozhen Xie, Susanne Ramm, Hye-Jung Kim, Sandra S. McAllister, Jean J. Zhao. CDK4/6 inhibition directly enhances an anti-tumor immune response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2017-1634

Immune Checkpoint Blockade in Breast Cancer Therapy.

Cancer immunotherapy is emerging as the most promising novel strategy for cancer treatment. Cancer immunotherapy is broadly categorized into three forms: immune checkpoint modulation, adoptive cell transfer, and cancer vaccine. Immune checkpoint blockade is demonstrated as the most clinically effective treatment with low immune-related adverse events (irAE). Blockade of PD-1/PD-L1 and CTLA-4 has achieved remarkable success in treating various types of tumors, which sparks great interests in this therapeutic strategy and expands the role of immune checkpoint blockade in treating tumors including breast cancer. Based on the notable results obtained from clinical trials, the United States' Food and Drug Administration (FDA) has approved multiple CTLA-4 monoclonal antibodies as well as the PD-1/PD-L1 monoclonal antibodies for treatment of different types of tumors. The theories of immunoediting, T-cell exhaustions, and co-stimulatory/co-inhibitory pathways are immunological foundations for immune checkpoint blockade therapy. Breast cancers such as triple negative breast cancer and HER-2 negative breast cancer respond to immune checkpoint blockade therapy due to their high immunogenicity. PD-1/PD-L1 blockade has just received FDA approval as a standard cancer therapy for solid tumors such as breast cancer. Development of immune checkpoint blockade focuses on two directions: one is to identify proper biomarkers of immune checkpoint blockade in breast cancer, and the other is to combine therapies with PD-1/PD-L1 blockade antibodies to achieve optimal clinical outcomes.

Abstract B111: HDAC inhibitors modulate immune checkpoint blockade in breast cancer

Abstract Background: Breast cancer is one of the most common diseases, second only to lung cancer as the leading cause of cancer death in women. In particular hormone sensitive metastatic breast cancer (MBC) and triple-negative breast cancer (TNBC) remains a therapeutic challenge as resistance develops in almost all patients. Despite aggressive treatment strategies, survival is poor due to the development of resistance, prompting the need for novel approaches. Here, we characterize the molecular mechanisms underlying epigenetic modifiers and in particular histone deacetylase inhibitors (HDACi) as priming modulators of immunotherapy, with a specific focus on TNBC and hormone resistant breast cancers. HDACi represent a new class of anticancer agents that can reverse hormone therapy resistance, resulting in prolonged anti-tumor responses in patients. In addition to their effects on estrogen receptor (ER) signaling, HDACi can also influence immune cell function, including but not limited to modulation of Foxp3+ regulatory T-cells (Tregs), tumor-infiltrating lymphocyte (TILs) composition, as well as induction of the co-inhibitory receptors PD-L1 and PD-1. Under normal conditions the PD-1/PD-L1 pathway down-regulates cytotoxic T-cell activity to maintain immune homeostasis. Cancer cells exploit this pathway in the tumor microenvironment to suppress cytotoxic T-cell activation, significantly diminishing the anti-tumor immune response. In breast cancer, PD-L1 expression is less frequent and mainly found in TNBC, HER2+, ER- and PR- tumors. Increased PD-L1 expression correlates with increased TILs and these criteria together are indicative of improved response rates in breast cancer patients. The PD-1/PD-L1 pathway represents one of the primary immunosuppressive drivers in multiple types of cancer. Thus, inhibiting PD-1/PD-L1 interactions may prevent T-cell suppression and reactivate immunosurveillance mechanisms necessary for tumor cell eradication. Methods and Results: Evaluation of basal PD-L1 expression in a range of human and mouse breast cancer cell lines by western blotting and real-time PCR identified TNBC and HER2+ cells as the highest expressing cells. Testing different epigenetic modifiers, we found that HDACi were able to up-regulate PD-L1 mRNA and protein in a time-dependent manner up to 72 hours. This was a direct transcriptional effect induced by HDACi and was confirmed even in tamoxifen resistant breast cancer cells, characterized by increased basal expression of PD-L1 as compared to the parental cells. To define the role of epigenetic priming in promoting immune cell activation, we co-cultured tumor cells and human peripheral blood mononuclear cell (PBMCs) and performed comprehensive immunophenotyping by flow cytometry. HDACi were able to up-regulate PD-L1 on tumor cells independent of PBMCs, while exhibiting a selective decrease in the frequency of immunosuppressive Tregs. Conclusion: Our data demonstrate that the combination of HDACi with immune checkpoint inhibitors represents a novel therapeutic anti-tumor strategy and warrants further clinical evaluation for the treatment of TNBC and hormone resistant breast cancer. Citation Format: Manuela Terranova Barberio, Scott Thomas, Niwa H. Ali, Jeenah Park, Michael D. Rosenblum, Alfredo Budillon, Pamela N. Munster. HDAC inhibitors modulate immune checkpoint blockade in breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B111.

CTLA-4 checkpoint blockade in breast cancer, a case in point report

Meeting abstracts A patient concomitantly affected by breast cancer and melanoma is presented in order to contribute to the ongoing debate on breast cancer immunotherapy [[1][1], [2][2]]. In 1996 the patient was operated on for left early breast cancer and treated with adjuvant radiotherapy and 5