The mechanisms of CAR-T cell mediated anti-tumor immunity and toxicity remain poorly characterized due to few studies examining the intact tumor microenvironment (TME) following CAR T-cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for patients with large B-cell lymphoma. We devised multiplex immunostaining and in-situ hybridization assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B-cell lymphoma following axicabtagene ciloleucel infusion. We found a majority of intratumoral CAR T cells expressed markers of T-cell activation but, unexpectedly, comprised ≤ 5% of all T cells within the TME five days or more after therapy. T cells lacking CAR (non-CAR T cells) were also activated within the TME after axicabtagene ciloleucel infusion, being positive for Ki-67, interferon-γ, granzyme B and/or PD-1, and highest in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and highest in biopsies with CAR T cells. These data indicate that intratumoral CAR T cells are associated with generalized immune cell activation within the TME with both beneficial and pathological effects.
Pei-Hsuan Chen, Mikel Lipschitz, Jason L. Weirather, Caron Jacobson, Philippe Armand, Kyle Wright, F. Stephen Hodi, Zachary J. Roberts, Stuart A. Sievers, John Rossi, Adrian Bot, William Y. Go, Scott J. Rodig
Immunotherapies that modulate T cell function have been firmly established as a pillar of cancer therapy, whereas the potential for B cells in the antitumor immune response is less established. B cell–activating factor (BAFF) is a B cell–activating cytokine belonging to the TNF ligand family that has been associated with autoimmunity, but little is known about its effects on cancer immunity. We find that BAFF upregulates multiple B cell costimulatory molecules; augments IL-12a expression, consistent with Be-1 lineage commitment; and enhances B cell antigen-presentation to CD4+ Th cells in vitro. In a syngeneic mouse model of melanoma, BAFF upregulates B cell CD40 and PD-L1 expression; it also modulates T cell function through increased T cell activation and TH1 polarization, enhanced expression of the proinflammatory leukocyte trafficking chemokine CCR6, and promotion of a memory phenotype, leading to enhanced antitumor immunity. Similarly, adjuvant BAFF promotes a memory phenotype of T cells in vaccine-draining lymph nodes and augments the antitumor efficacy of whole cell vaccines. BAFF also has distinct immunoregulatory functions, promoting the expansion of CD4+Foxp3+ Tregs in the spleen and tumor microenvironment (TME). Human melanoma data from The Cancer Genome Atlas (TCGA) demonstrate that BAFF expression is positively associated with overall survival and a TH1/IFN-γ gene signature. These data support a potential role for BAFF signaling as a cancer immunotherapy.
Mark Yarchoan, Won Jin Ho, Aditya Mohan, Yajas Shah, Teena Vithayathil, James Leatherman, Lauren Dennison, Neeha Zaidi, Sudipto Ganguly, Skylar Woolman, Kayla Cruz, Todd D. Armstrong, Elizabeth M. Jaffee
Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about its function in pancreatic ductal adenocarcinoma (PDAC). In this study, we performed in silico analysis of WASL expression in PDAC patients and found a correlation between low WASL expression and prolonged survival. To clarify the role of Wasl in pancreatic carcinogenesis, we used 2 oncogenic Kras–based PDAC mouse models with pancreas-specific Wasl deletion. In line with human data, both mouse models had an increased survival benefit due to either impaired tumor development in the presence of the tumor suppressor Trp53 or the delayed tumor progression and senescent phenotype upon genetic ablation of Trp53. Mechanistically, loss of Wasl resulted in cell-autonomous senescence through displacement of the N-WASP binding partners WASP-interacting protein (WIP) and p120ctn; vesicular accumulation of GSK3β, as well as YAP1 and phosphorylated β-catenin, which are components of the destruction complex; and upregulation of Cdkn1a(p21), a master regulator of senescence. Our findings, thus, indicate that Wasl functions in an oncogenic manner in PDAC by promoting the deregulation of the p120-catenin/β-catenin/p21 pathway. Therefore, strategies to reduce N-WASP activity might improve the survival outcomes of PDAC patients.
Ana Hidalgo-Sastre, Judit Desztics, Zahra Dantes, Katharina Schulte, Hilal Kabadayi Ensarioglu, Blessing Bassey-Archibong, Rupert Öllinger, Thomas Engleiter, Lyndsay Rayner, Henrik Einwächter, Juliet M. Daniel, Ali Sameer Abdulghani Altaee, Katia Steiger, Marina Lesina, Roland Rad, Maximilian Reichert, Guido von Figura, Jens T. Siveke, Roland M. Schmid, Clara Lubeseder-Martellato
Colitis is associated with the development of colorectal cancer (CRC) by largely undefined mechanisms that are critical for understanding the link between inflammation and cancer. Intestinal stem cells (ISCs) marked by LGR5 expression are of importance in both the inflammatory response to colitis and progression to colitis-associated colon cancer (CACC). Here, we report in human MUC1-transgenic mouse models of CACC that targeting the MUC1-C oncogenic protein, which is upregulated in inflammation, suppresses the (i) Lgr5+ ISC population, (ii) induction of Myc and core pluripotency stem cell factors, and (iii) severity and progression of colitis to dysplasia and cancer. By extension to human colon cancer cells, we demonstrate that MUC1-C drives MYC, forms a complex with MYC on the LGR5 promoter and activates LGR5 expression. We also show in CRC cells that MUC1-C induces the cancer stem cell (CSC) markers (BMI1, ALDH1, FOXA1, LIN28B) and the OCT4, SOX2 and NANOG pluripotency factors. Consistent with conferring the CSC state, targeting MUC1-C suppresses the capacity of CRC cells to promote wound healing, invasion, self-renewal and tumorigenicity. In analysis of human tissues, MUC1 expression associates with activation of inflammatory pathways, development of colitis and aggressiveness of CRCs. These results collectively indicate that MUC1-C is of importance for integrating stemness and pluripotency in colitis and CRC. Of clinical relevance, the findings further indicate that MUC1-C represents a previously unrecognized target that is druggable for treating progression of colitis and CRC.
Wei Li, Ning Zhang, Caining Jin, Mark D. Long, Hasan Rajabi, Yota Yasumizu, Atsushi Fushimi, Nami Yamashita, Masayuki Hagiwara, Rongbin Zheng, Jin Wang, Ling Kui, Harpal Singh, Surender Kharbanda, Qiang Hu, Song Liu, Donald W. Kufe
Metastatic colorectal cancer (CRC) is poorly immunogenic, with limited neoantigens that can be targeted by cancer vaccine. Previous approaches to upregulate neoantigen have had limited success. In this study, we investigated the role of a DNA methyltransferase inhibitor (DNMTi), 5-aza-2′-deoxycytidine (DAC), in inducing cancer testis antigen (CTA) expression and evaluated the antitumor efficacy of a combinatorial approach with an epigenetically regulated cancer vaccine EpiGVAX and DAC. A murine model of metastatic CRC treated with combination therapy with an irradiated whole-cell CRC vaccine (GVAX) and DAC was used to assess the antitumor efficacy. DAC significantly induced expression of CTAs in CRC, including a new CTA Tra-P1A with a known neoepitope, P1A. Epigenetically modified EpiGVAX with DAC improved survival outcomes of GVAX. Using the epigenetically regulated antigen Tra-P1A as an example, our study suggests that the improved efficacy of EpiGVAX with DAC may due in part to the enhanced antigen-specific antitumor immune responses. This study shows that epigenetic therapy with DNMTi can not only induce new CTA expression but may also sensitize tumor cells for immunotherapy. Neoantigen-based EpiGVAX combined with DAC can improve the antitumor efficacy of GVAX by inducing antigen-specific antitumor T cell responses to epigenetically regulated proteins.
Victoria M. Kim, Xingyi Pan, Kevin C. Soares, Nilofer S. Azad, Nita Ahuja, Christopher J. Gamper, Alex B. Blair, Stephen Muth, Ding Ding, Brian H. Ladle, Lei Zheng
Tumor-Associated Macrophages (TAMs) contribute to the maintenance of a strong immunosuppressive environment, supporting tumor progression and resistance to treatment. To date, the mechanisms that drive acquisition of these immunosuppressive features are still poorly defined. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme that catabolizes free heme. It displays important cytoprotective, anti-inflammatory and antioxidant properties. A growing body of evidence suggests that HO-1 may also promote tumor development. Herein, we show that HO-1 is highly expressed in monocytic cells in the tumor microenvironment (TME) once they differentiate into TAMs. Deletion of HO-1 in the myeloid compartment enhances the beneficial effects of a therapeutic antitumor vaccine by restoring CD8 T-cell proliferation and cytotoxicity. We further show that induction of HO-1 plays a major role on monocyte education by tumor cells by modulating their transcriptional and epigenetic programs. These results identify HO-1 as a valuable therapeutic target to reprogram the TME and synergize with current cancer therapies to facilitate antitumoral response.
Emmanuelle Alaluf, Benoît Vokaer, Aurélie Detavernier, Abdulkader Azouz, Marion Splittgerber, Alice Carrette, Louis Boon, Frédérick Libert, Miguel P. Soares, Alain Le Moine, Stanislas Goriely
BACKGROUND. The reshaping of the immune landscape by nivolumab (NIVO) and ipilimumab (IPI) and its relation to patient outcomes is not well-described. METHODS. We used high-parameter flow cytometry and a novel computational platform, CytoBrute, to define immunophenotypes of up to 15 markers to assess peripheral blood samples from metastatic melanoma patients receiving sequential NIVO>IPI or IPI>NIVO (CheckMate-064). RESULTS. The two treatments were associated with distinct immunophenotypic changes and had differing profiles associated with response. Only two immunophenotypes were shared but had opposing relationships to response/survival. To understand the impact of sequential treatment on response/survival, phenotypes that changed after the initial treatment and differentiated response in the other cohort were identified. Immunophenotypic changes occurring post-NIVO were predominately associated with response to IPI>NIVO, but changes occurring post-IPI were predominately associated with progression after NIVO>IPI. Among these changes, CD4+CD38+CD39+CD127–GARP– T-cell subsets were increased after IPI treatment and were negatively associated with response/survival for the NIVO>IPI cohort. CONCLUSION. Collectively, these data suggest that the impact of IPI and NIVO on the immunophenotypic landscape of patients is distinct and that the impact of IPI may be associated with resistance to subsequent NIVO therapy, consistent with poor outcomes in the IPI>NIVO cohort of Checkmate-064.
David M. Woods, Andressa S. Laino, Aidan F. Winters, Jason M. Alexandre, Daniel Freeman, Vinay Rao, Santi S. Adavani, Jeffrey S. Weber, Pratip K. Chattopadhyay
BACKGROUND. Epidemiologic studies suggest that metformin has antitumor effects. Laboratory studies indicate metformin impacts cancer stem-like cells (CSCs). As part of a phase II trial, we evaluated the impact of metformin on CSC number, and carcinoma associated mesenchymal stem cells (CA-MSC), and clinical outcomes in non-diabetic patients with advanced stage epithelial ovarian cancer (EOC). METHODS. Thirty-eight patients with confirmed stage IIC(n = 1)/III(n = 25)/IV(n = 12) EOC were treated with either (i) neoadjuvant metformin, debulking surgery and adjuvant chemotherapy + metformin, or (ii) neoadjuvant chemotherapy and metformin, interval debulking surgery, and adjuvant chemotherapy + metformin. Metformin treated tumors, compared to historical controls, were evaluated for CSC number and chemotherapy response. Primary endpoints were (i) a greater than 2-fold reduction in ALDH+CD133+ CSC and (ii) a relapse free survival at 18 months of greater than 50%. RESULTS. Metformin was well-tolerated. Median progression-free survival was 18.0 months (95% CI 14.0–21.6) with relapse-free survival at 18 months of 59.3% (95% CI 38.6–70.5). Median overall survival was 57.9 months (95% CI 28.0 – Not Estimable). Tumors treated with metformin had a 2.4-fold decrease in ALDH+/CD133+ CSC and increased sensitivity to cisplatin ex vivo. Furthermore, metformin altered the methylation signature in CA-MSC which prevented CA-MSC driven chemoresistance in vitro. CONCLUSIONS. Translational studies confirm an impact of metformin on EOC CSC and suggest epigenetic change in the tumor stroma may drive the platinum sensitivity ex vivo. Consistent with this, metformin therapy was associated with better than expected overall survival, supporting the use of metformin in phase-III studies.
Jason R Brown, Daniel K. Chan, Jessica J Shank, Kent A. Griffith, Huihui Fan, Robert Szulawski, Kun Yang, R. Kevin Reynolds, Carolyn Johnston, Karen McLean, Shitanshu Uppal, J. Rebecca Liu, Lourdes Cabrera, Sarah E Taylor, Brian C. Orr, Francesmary Modugno, Pooja Mehta, Michael Bregenzer, Geeta Mehta, Hui Shen, Lan Coffman, Ronald J. Buckanovich
Recent studies show gut microbiota modulate antitumor immune responses; one proposed mechanism is cross-reactivity between antigens expressed in commensal bacteria and neoepitopes. We found that T cells targeting an epitope called SVYRYYGL (SVY), expressed in the commensal bacterium Bifidobacterium breve (B. breve), cross-react with a model neoantigen, SIYRYYGL (SIY). Mice lacking B. breve had decreased SVY-reactive T cells compared with B. breve–colonized mice, and the T cell response was transferable by SVY immunization or by cohousing mice without Bifidobacterium with ones colonized with Bifidobacterium. Tumors expressing the model SIY neoantigen also grew faster in mice lacking B. breve compared with Bifidobacterium-colonized animals. B. breve colonization also shaped the SVY-reactive TCR repertoire. Finally, SVY-specific T cells recognized SIY-expressing melanomas in vivo and led to decreased tumor growth and extended survival. Our work demonstrates that commensal bacteria can stimulate antitumor immune responses via cross-reactivity and how bacterial antigens affect the T cell landscape.
Catherine A. Bessell, Ariel Isser, Jonathan J. Havel, Sangyun Lee, David R. Bell, John W. Hickey, Worarat Chaisawangwong, Joan Glick Bieler, Raghvendra Srivastava, Fengshen Kuo, Tanaya Purohit, Ruhong Zhou, Timothy A. Chan, Jonathan P. Schneck
The role CD4+ T-cells play in tumor immunity is less well-appreciated than the cytotoxic role of CD8+ T-cells. Despite clear evidence for CD4+ T-cell dependency across multiple immunotherapies, the mechanisms by which CD4+ T-cells infiltrate tumors remain poorly understood. Prior studies by our group have shown in a mouse model of pancreatic cancer that systemic activation of the cell-surface TNF superfamily member CD40 drives T-cell infiltration into tumors and in combination with immune checkpoint blockade, leads to durable tumor regressions and cures that depend on both CD8+ and CD4+ T-cells. Here, we used single-cell transcriptomics to examine the tumor microenvironment following treatment with agonist CD40 antibody with or without immune checkpoint blockade. We show that intratumoral myeloid cells produce the chemokine CCL5 in response to CD40 agonist and that CCL5 mediates an influx of CD4+ T-cells into the tumor microenvironment. Disruption of CCL5 genetically or pharmacologically mitigates the influx of CD4+ but not CD8+ T-cells into tumors and blunts the therapeutic efficacy of immunotherapy. These findings highlight a previously unappreciated role for CCL5 in selectively mediating CD4+ T-cell tumor infiltration in response to effective immunotherapy.
Austin P. Huffman, Jeffrey H. Lin, Samuel I. Kim, Katelyn T. Byrne, Robert H. Vonderheide
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