Search

Table 1 compound list

We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in an H-2b–restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell–mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell–mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.

Table 3 SCCHN clinical info

Suppl. Fig. 1-6

Figure S1. Characterization of cells and CDK4/6 inhibitors; Figure S2. CDK6 phosphorylates serine residues of the regulatory domain of NFAT4 (NFATc3); Figure S3. Analysis of lung tumor immune infiltrates after CDK4/6 inhibition from KrasG12D (Kras), KrasG12DLkb1 (KL) or KrasG12DTrp53fl/fl (KP) mice; Figure S4. T cell proliferation and cytokine/chemokine profiling of KrasG12DTrp53fl/fl GEMM mice; Figure S5. Tumor antigen experienced T cells are more sensitive to CDK4/6 inhibition; Figure S6. Short-term CDK4/6 inhibition alters the cell cycle status of tumor infiltrating T cells; Figure S7. CDK4/6 inhibition induces changes in the expression of activation and suppression marker genes in tumor-infiltrating T cells; Figure S8. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody elicits anti-tumor immunity; Figure S9. Combination treatment of CDK4/6 inhibitor and anti-PD-1 antibody on established tumor; Figure S10. Effect of TCR stimulation and CDK4/6 inhibition on phosphorylation of NFkB; Supplementary Table S1; Supplmentary Table S2; Supplementary Table S3: Selected genes reported to be regulated by NFAT

Table 2 sgRNA genes and sequences

Purpose:Surgery often represents the best chance for disease control in locoregionally recurrent squamous cell carcinoma of the head and neck (SCCHN). We investigated dual immune-checkpoint inhibition [anti–PD-1, nivolumab (N), and anti-KIR, lirilumab (L)] before and after salvage surgery to improve disease-free survival (DFS).Patients and Methods:In this phase II study, patients received N (240 mg) + L (240 mg) 7 to 21 days before surgery, followed by six cycles of adjuvant N + L. Primary endpoint was 1-year DFS; secondary endpoints were safety, pre-op radiologic response, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 scoring, and immunoprofiling.Results:Among 28 patients, the median age was 66, 86% were smokers; primary site: 9 oral cavity, 9 oropharynx, and 10 larynx/hypopharynx; 96% had prior radiation. There were no delays to surgery. Grade 3+ adverse events: 11%. At the time of surgery, 96% had stable disease radiologically, one had progression. Pathologic response to N + L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤ 10%) and 8/28 (29%) partial (TV ≤ 50%). PD-L1 combined positive score (CPS) at surgery was similar regardless of pathologic response (P = 0.71). Thirteen (46%) recurred (loco-regional = 10, distant = 3). Five of 28 (18%) had positive margins, 4 later recurred. At median follow-up of 22.8 months, 1-year DFS was 55.2% (95% CI, 34.8–71.7) and 1-year OS was 85.7% (95% CI, 66.3–94.4). Two-year DFS and OS were 64% and 80% among pathologic responders.Conclusions:(Neo)adjuvant N + L was well tolerated, with a 43% pathologic response rate. We observed favorable DFS and excellent 2-year OS among high-risk, previously treated patients exhibiting a pathologic response. Further evaluation of this strategy is warranted.

Supplementary Data from Neoadjuvant and Adjuvant Nivolumab and Lirilumab in Patients with Recurrent, Resectable Squamous Cell Carcinoma of the Head and Neck

Purpose: Surgery often represents the best chance for disease control in locoregionally recurrent squamous cell carcinoma of the head and neck (SCCHN). We investigated dual immune-checkpoint inhibition [anti–PD-1, nivolumab (N), and anti-KIR, lirilumab (L)] before and after salvage surgery to improve disease-free survival (DFS). Patients and Methods: In this phase II study, patients received N (240 mg) + L (240 mg) 7 to 21 days before surgery, followed by six cycles of adjuvant N + L. Primary endpoint was 1-year DFS; secondary endpoints were safety, pre-op radiologic response, and overall survival (OS). Correlatives included tumor sequencing, PD-L1 scoring, and immunoprofiling. Results: Among 28 patients, the median age was 66, 86% were smokers; primary site: 9 oral cavity, 9 oropharynx, and 10 larynx/hypopharynx; 96% had prior radiation. There were no delays to surgery. Grade 3+ adverse events: 11%. At the time of surgery, 96% had stable disease radiologically, one had progression. Pathologic response to N + L was observed in 43% (12/28): 4/28 (14%) major (tumor viability, TV ≤ 10%) and 8/28 (29%) partial (TV ≤ 50%). PD-L1 combined positive score (CPS) at surgery was similar regardless of pathologic response (P = 0.71). Thirteen (46%) recurred (loco-regional = 10, distant = 3). Five of 28 (18%) had positive margins, 4 later recurred. At median follow-up of 22.8 months, 1-year DFS was 55.2% (95% CI, 34.8–71.7) and 1-year OS was 85.7% (95% CI, 66.3–94.4). Two-year DFS and OS were 64% and 80% among pathologic responders. Conclusions: (Neo)adjuvant N + L was well tolerated, with a 43% pathologic response rate. We observed favorable DFS and excellent 2-year OS among high-risk, previously treated patients exhibiting a pathologic response. Further evaluation of this strategy is warranted.

We developed a screening assay in which luciferized ID8 expressing OVA was cocultured with transgenic CD8+ T cells specifically recognizing the model antigen in an H-2b–restricted manner. The assay was screened with a small-molecule library to identify compounds that inhibit or enhance T cell–mediated killing of tumor cells. Erlotinib, an EGFR inhibitor, was the top compound that enhanced T-cell killing of tumor cells. Subsequent experiments with erlotinib and additional EGFR inhibitors validated the screen results. EGFR inhibitors increased both basal and IFNγ-induced MHC class-I presentation, which enhanced recognition and lysis of tumor cell targets by CD8+ cytotoxic T lymphocytes. The ID8 cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen, utilizing the same target tumor cell/T-cell assay, identified single-guide (sg)RNAs targeting EGFR that sensitized tumor cells to T cell–mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in a syngeneic model, further validating EGFR inhibitors as immunomodulatory agents that enhance checkpoint blockade. This assay can be screened in high-throughput with small-molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds and target genes, respectively, that enhance or inhibit T-cell recognition and killing of tumor cells. Retrospective analyses of squamous-cell head and neck cancer (SCCHN) patients treated with the combination of afatinib and pembrolizumab demonstrated a rate of clinical activity exceeding that of each single agent. Prospective clinical trials evaluating the combination of an EGFR inhibitor and PD-1 blockade should be conducted.

Programmed cell death protein 1 (PD-1) inhibitors have efficacy in treating squamous cell carcinoma of the head and neck (SCCHN), but objective response rates are low. PD-1 ligand (PD-L1) expression alone is not considered a robust predictor of response and additional biomarkers are needed. This 3-year observational cohort followed 126 SCCHN patients treated with anti–PD-1/L1 therapy. Prior to treatment, 81 (64%) had targeted massively parallel tumor sequencing. Of these, 42 (52%) underwent fluorescence-activated cell sorting and PD-L1 immunohistochemistry for tumor immunoprofiling. Six (5%) complete responses (CRs) and 11 (9%) partial responses (PRs) were observed. Those treated with prior chemotherapy (98, 78%) versus only surgery and/or radiation had longer overall survival (OS) (10 vs. 3 months, P = 0.02). Smokers had a higher total mutational burden (TMB) (P = 0.01). Virus-positive patients had a lower TMB (P < 0.01) and improved OS (P = 0.02). Among virus-negative responders, NOTCH1 and SMARCA4 were more frequently mutated and frameshift events in tumor suppressor genes occurred more frequently (P = 0.03). Higher TMB and CD8+ T cell infiltrates predicted anti–PD-1/L1 benefit (P < 0.01, P < 0.01, respectively) among virus-negative tumors. TIM-3/LAG-3 coexpression with PD-1 was higher on T cells among nonresponders (P = 0.03 and 0.02, respectively). Somatic frameshift events in tumor suppressor genes and higher TMB among virus-negative SCCHN tumors predict anti–PD-1/L1 response.

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies. Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216–33. ©2017 AACR. See related commentary by Balko and Sosman, p. 143. See related article by Jenkins et al., p. 196. This article is highlighted in the In This Issue feature, p. 127

Objectives Immune checkpoint inhibitors have demonstrated clinical benefit in recurrent, metastatic (R/M) squamous cell carcinoma of the head and neck (SSCHN), but lacking are biomarkers that predict response. We sought to define an inflamed tumor immunophenotype in this R/M SCCHN population and correlate immune metrics with clinical parameters and survival. Methods Tumor samples were prospectively acquired from 34 patients to perform multiparametric flow cytometry and multidimensional clustering analysis integrated with next-generation sequencing data, clinical parameters and outcomes. Results We identified an inflamed subgroup of tumors with prominent CD8+ T cell infiltrates and high PD-1/TIM3 co-expression independent of clinical variables, with improved survival compared with a non-inflamed subgroup (median overall survival 84.0 vs. 13.0 months, p = 0.004). The non-inflamed subgroup demonstrated low CD8+ T cells, low PD-1/TIM3 co-expression, and higher T regs . Overall non-synonymous mutational burden did not correlate with response to PD-1 blockade in a subset of patients. Conclusion R/M SCCHN patients with an inflamed tumor immunophenotype demonstrate improved survival. Further prospective studies are needed to validate these findings and explore the use of immunophenotype to guide patient selection for immunotherapeutic approaches.

As immune checkpoint blocking antibodies increasing become foundational therapies for the treatment of cancer, there is a pressing need to identify compounds that synergize with checkpoint blockade as the basis of combinatorial treatment regimens. We have developed a screening assay in which a luciferized tumor cell line expressing a model antigen is co-cultured with a transgenic CD8+ T cell specifically recognizing the model antigen in a H-2b-restricted manner. The target tumor cell/T cell assay was screened with a small molecule library to identify compounds that inhibit or enhance T cell-mediated killing of tumor cells in an antigen-dependent manner. The EGFR inhibitor Erlotinib was the top hit that enhanced T cell killing of tumor cells. Subsequent experiments with Erlotinib and additional EGFR inhibitors validated the screen result. EGFR inhibitors increase both basal and IFN-γ-induced antigen processing and presentation of MHC class-I, which enhanced recognition and lysis by CD8+ cytotoxic T lymphocytes. The tumor cell line was also transduced to constitutively express Cas9, and a pooled CRISPR screen utilizing the same target tumor cell/T cell assay identified sgRNAs targeting EGFR as sensitizing tumor cells to T cell-mediated killing. Combination of PD-1 blockade with EGFR inhibition showed significant synergistic efficacy in the MC38 syngeneic colon cancer model that was superior to PD-1 blockade or EGFR inhibition alone, further validating EGFR inhibitors as immunomodulatory agents that enhance PD-1 checkpoint blockade. This novel target tumor cell/T cell assay can be screened in high-throughput with small molecule libraries and genome-wide CRISPR/Cas9 libraries to identify both compounds AND target genes, respectively, that enhance or inhibit T cell recognition and killing of tumor cells. Citation Format: Patrick H. Lizotte, Troy Luster, Megan E. Cavanaugh, Luke J. Taus, Abha Dhaneshwar, Naomi Mayman, Aaron Yang, Mark Bittinger, Paul Kirschmeier, Nathanael S. Gray, David A. Barbie, Pasi A. Janne. High-throughput immune-oncology screen identifies EGFR inhibitors as potent enhancers of CTL antigen-specific tumor cell killing [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 4935.

8555 Background: Defactinib is an oral Focal Adhesion Kinase (FAK) inhibitor with preclinical activity in MPM. We assessed responses to defactinib treatment prior to planned surgical resection in naive patients with MPM. Methods: Three cohorts of 10 participants each received defactinib 400mg BID for 12, 35 and 21 days. Pre- and post-treatment blood, tumor biopsies and imaging were obtained for biomarker, immune cell and tumor response (modified RECIST, Tumor volume and SUV max) assessment. Toxicity was monitored for 30 days post treatment. Results: Between 12/2013 and 12/2017, 31 participants were registered at our center; 1 withdrew prior to intervention. Among 30 treated, 24 (80%) were male; median age 70 (47-83) years; surgery was EPP 7%, complete pleurectomy decortication (PD) 10%, extended PD 60%, partial PD 10%, unresectable 13%; MPM subtype was epithelioid 67%, biphasic 17%, sarcomatoid 17%. Expected complications of FAK inhibition, diagnostic/staging/operative procedures occurred in 83% (grade 1, 30%; grade 2, 43%; grade 3, 10%). Unexpected adverse events occurred in 77% (grade 1, 63%; grade 2, 20%; grade 3, 17% [wound-infection, prolonged QT interval, and hyperglycemia in 3% each; increased INR in 7%]; grade 5, 7% [due to progressive disease in 3%, intraoperative anaphylactoid reaction unrelated to the drug in 3%]). Objective partial response was observed in 13%, stable disease in 67%, progression in 17%. Tumor volume decreased 3-72% in 47% patients and increased 1-82% in 53%. SUV max decreased 3-69% in 50% and increased 1-61% in 50%. Biological correlates of treatment included target inhibition (75% pFAK reduction); tumor immune microenvironment changes: increased naïve (CD45RA+PD-1+CD69+) CD4 and CD8 T cells, reduced myeloid and Treg immuno-suppressive cells, reduced exhausted T cells (PD-1+CD69+), reduced peripheral MDSCs; and histological subtype change (pleomorphic or biphasic to epithelioid) in 13% of cases. Conclusions: Brief preoperative defactinib exposure was well tolerated, did not alter resectability or mortality compared to prior series, and showed evidence of therapeutic and immunomodulatory effects. Clinical trial information: NCT02004028.