Immune checkpoint inhibitors1 result in impressive clinical responses2–5 but optimal results will require combination with each other6 and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells (Tregs) to increase the CD8 T cell to Treg (CD8/Treg) ratio. RT enhances the diversity of the T cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while RT shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligo-clonal T cell expansion. Similar to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumors to escape anti-CTLA4-based therapy, and the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms.

SUMMARY Therapeutic blocking of the PD1 pathway results in significant tumor responses but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB), and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways, and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.

SUMMARY Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely non-coding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent anti-viral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine anti-viral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of anti-viral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate cross-talk with BrCa cells by utilizing exosomes to instigate anti-viral signaling. This expands BrCa subpopulations adept at resisting therapy and re-initiating tumor growth.

Immunotherapy in pancreatic ductal adenocarcinoma (PDA) remains a difficult clinical problem despite success in other disease types with immune checkpoint blockade (ICB) and chimeric antigen receptor T cell therapy. Mechanisms driving immunosuppression and poor T cell infiltration in PDA are incompletely understood. Here we use genetically engineered mouse models of PDA that recapitulate hallmarks of human disease to demonstrate that CD40 pathway activation is required for clinical response to radiotherapy (RT) and ICB with αCTLA-4 and αPD-1. The combination of an agonist αCD40 antibody, RT, and dual ICB eradicated irradiated and unirradiated (i.e. abscopal) tumors, generating long-term immunity. Response required T cells and also short-lived myeloid cells and was dependent on the long non-coding RNA myeloid regulator Morrbid. Using unbiased random forest machine learning, we built unique, contextual signatures for each therapeutic component, revealing that (i) RT triggers an early proinflammatory stimulus, ablating existing intratumoral T cells and upregulating MHC class I and CD86 on antigen presenting cells, (ii) αCD40 causes a systemic and intratumoral reorganization of the myeloid compartment, and (iii) ICB increases intratumoral T cell infiltration and improves the CD8 T cell:regulatory T cell ratio. Thus, αCD40 and RT non-redundantly augment anti-tumor immunity in PDA, which is otherwise refractory to ICB, providing a clear rationale for clinical evaluation.

Background & Aims IκB kinase-β (IKKβ) mediates activation of the nuclear factor-κB (NFκB), which regulates immune and inflammatory responses. Although NFκB is activated in cells from patients with inflammatory diseases or cancer, little is known about its roles in development and progression of esophageal diseases. We investigated whether mice that express an activated form of IKKβ in the esophageal epithelia develop esophageal disorders. Methods We generated ED-L2-Cre/Rosa26-IKK2caSFL mice, in which the ED-L2 promoter activates expression of Cre in the esophageal epithelia, leading to expression of a constitutively active form of IKKβ (IKKβca) in epithelial cells but not inflammatory cells or the surrounding stroma (IKKβca mice). Mice lacking the Cre transgene served as controls. Some mice were given intraperitoneal injections of neutralizing antibodies against granulocyte macrophage colony-stimulating factor (GMCSF) or tumor necrosis factor (TNF), or immunoglobulin G1 (control), starting at 1 month of age. Epithelial tissues were collected and analyzed by immunofluorescence, immunohistochemical, and quantitative real-time PCR assays. Transgenes were overexpressed from retroviral vectors in primary human keratinocytes. Results IKKβca mice developed esophagitis and had increased numbers of blood vessels in the esophageal stroma, compared with controls. Esophageal tissues from IKKβca mice had increased levels of GMCSF. Expression of IKKβca in primary human esophageal keratinocytes led to 11-fold overexpression of GMCSF and 200-fold overexpression of TNF. Incubation of human umbilical vein endothelial cells with conditioned media from these keratinocytes increased endothelial cell migration by 42% and promoted formation of capillary tubes; these effects were blocked by a neutralizing antibody against GMCSF. Injections of anti-GMCSF reduced angiogenesis and numbers of CD31+ blood vessels in esophageal tissues of IKKβca mice but did not alter the esophageal vasculature of control mice and did not alter recruitment of intraepithelial leukocytes to esophageal tissues of IKKβca mice. Injections of anti-TNF prevented development of esophagitis in IKKβca mice. Conclusions Constitutive activation of IKKβ in the esophageal epithelia of mice leads to inflammation and angiogenesis, mediated by TNF and GMCSF, respectively.

The resistance of pancreatic ductal adenocarcinoma (PDAC) to immune checkpoint inhibitors (ICIs) is attributed to the immune-quiescent and -suppressive tumor microenvironment (TME). We recently found that CCR2 and CCR5 were induced in PDAC following treatment with anti–PD-1 antibody (αPD-1); thus, we examined PDAC vaccine or radiation therapy (RT) as T cell priming mechanisms together with BMS-687681, a dual antagonist of CCR2 and CCR5 (CCR2/5i), in combination with αPD-1 as new treatment strategies. Using PDAC mouse models, we demonstrated that RT followed by αPD-1 and prolonged treatment with CCR2/5i conferred better antitumor efficacy than other combination treatments tested. The combination of RT + αPD-1 + CCR2/5i enhanced intratumoral effector and memory T cell infiltration but suppressed regulatory T cell, M2-like tumor–associated macrophage, and myeloid-derived suppressive cell infiltration. RNA sequencing showed that CCR2/5i partially inhibited RT-induced TLR2/4 and RAGE signaling, leading to decreased expression of immunosuppressive cytokines including CCL2/CCL5, but increased expression of effector T cell chemokines such as CCL17/CCL22. This study thus supports the clinical development of CCR2/5i in combination with RT and ICIs for PDAC treatment.

Immune checkpoint inhibitors result in impressive clinical responses but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here, we report major tumor regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation (RT) on a phase one clinical trial and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumors, resistance was common. Computational analysis of genome-wide and immune profiles of mice revealed resistance was due to T cell exhaustion driven by adaptive resistance and prolonged interferon-gamma exposure, resulting in STAT1-mediated upregulation of PD-L1 on melanoma cells and tumor macrophages. Accordingly, optimal response in melanoma and other cancer types requires RT, anti-CTLA4, and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T regulatory cells, and macrophage depletion and/or PD-L1 blockade reverses T cell exhaustion. RT promotes the infiltration of intratumoral antigen-specific CD8 T cells and enhances the diversity of the T cell receptor (TCR) repertoire. RT with dual checkpoint blockade shapes the TCR repertoire of the expanded peripheral clones in a manner consistent with antigen-driven selection. Similar to results from mice, patients on our clinical trial with tumors showing high PD-L1 did not respond to RT + anti-CTLA4, demonstrated persistent T cell exhaustion, and rapidly progressed. In contrast, patients with low PD-L1 on melanoma cells or macrophages had markedly improved survival, with the best survival observed among those patients with low PD-L1 on both cell types. Thus, our results suggest that 1) RT can enhance response to anti-CTLA4 when the TCR and/or antigen repertoire are sub-optimal, 2) upregulation of PD-L1 through STAT1-mediated adaptive resistance mechanisms inhibits response to anti-CTLA4-based therapy unless PD-L1/PD-1 is blocked, and 3) the combination of RT, anti-CTLA4, and anti-PD-L1 promotes response and immunity through distinct mechanisms. Finally, although PD-L1 was a dominant resistance mechanism in our models, PD-L1-independent resistance mechanisms were also evident. The next generation of clinical trials based on these findings are underway. Citation Format: Christina Twyman-Saint Victor, Andrew Rech, Joseph Benci, Amit Maity, Ramesh Rengan, Kristen Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela Odorizzi, Ramin Herati, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, Xiaowei Xu, Michael Feldman, Tara Gangadhar, Steve Hahn, John Wherry, Robert Vonderheide, Andy Minn. Mechanisms of tumor response and resistance to radiation and dual checkpoint blockade in mice and patients. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr PR05.

Despite the success of cancer immunotherapy in many disease types, pancreatic ductal adenocarcinoma (PDA) is notably unresponsive to immune checkpoint blockade (ICB) with αPD1 and/or αCTLA4. The mechanism of resistance is poorly understood, but tumor epitopes and the microenvironment, which is immunosuppressive and excludes T cells, are thought to be contributory. To improve response to ICB, we used subcutaneous and orthotopic murine models of PDA to investigate the effect of combination therapy with ICB (CTLA-4 and PD-1 antagonist antibodies), CD40 agonist antibody and radiation therapy (RT). Combination therapy with CD40 agonist antibody, ICB and RT resulted in decrease tumor burden, increase overall survival, and generation of long-term immunity. Response is dependent on T and short-lived myeloid cells, while it is independent of innate activation pathways. Together, these results suggest a dual role for both the innate and adaptive immune response in treating PDA. Citation Format: Hannah Dada, Andrew J. Rech, Christina Twyman-Saint Victor, Andy J. Minn, Robert H. Vonderheide. Radiotherapy and αCD40 non-redundantly augment immunity to checkpoint blockade in refractory pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2985.