Robert T. Manguso

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR–Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity

It is unclear whether PD-L1 on tumor cells is sufficient for tumor immune evasion or simply correlates with an inflamed tumor microenvironment. We used three mouse tumor models sensitive to PD-1 blockade to evaluate the significance of PD-L1 on tumor versus nontumor cells. PD-L1 on nontumor cells is critical for inhibiting antitumor immunity in B16 melanoma and a genetically engineered melanoma. In contrast, PD-L1 on MC38 colorectal adenocarcinoma cells is sufficient to suppress antitumor immunity, as deletion of PD-L1 on highly immunogenic MC38 tumor cells allows effective antitumor immunity. MC38-derived PD-L1 potently inhibited CD8+ T cell cytotoxicity. Wild-type MC38 cells outcompeted PD-L1–deleted MC38 cells in vivo, demonstrating tumor PD-L1 confers a selective advantage. Thus, both tumor- and host-derived PD-L1 can play critical roles in immunosuppression. Differences in tumor immunogenicity appear to underlie their relative importance. Our findings establish reduced cytotoxicity as a key mechanism by which tumor PD-L1 suppresses antitumor immunity and demonstrate that tumor PD-L1 is not just a marker of suppressed antitumor immunity.

Abstract A16: Defining molecular mechanisms of resistance to tumor immunity

Recent success of immune checkpoint blockade solidifies the importance of the immune system in the defense against cancer. The clinical impact of the immune response is, however, very heterogeneous, with some patients achieving dramatic responses while others fail to respond. Known genomic correlates of response to immunotherapy are not perfectly predictive of clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. I hypothesize somatic acquired mutations of individual tumors may account for heterogeneity in the spontaneous response to tumors and response to immunotherapy. I have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to discover new mechanisms of immune resistance, define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapeutic strategies. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations from the Pan Cancer analysis within The Cancer Genome Atlas (TCGA). These cell lines form tumors when implanted subcutaneously in immunocompetent animals. Tumor-bearing animals were then subjected to immunotherapy with either therapeutic vaccination or checkpoint blockade with anti-PD-1. Barcode relative representation was measured by next generation sequencing at the time of tumor implantation and at the time of tumor harvest post-immunotherapy. Barcoded mutant ORFs that confer immune resistance increased in representation under immune pressure in comparison to untreated or immunodeficient animals. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A>G, consistently increased in representation in both B16 and MC38 immunotherapy-treated tumors. This mutation encodes a constitutively active mutant catalytic domain of PI3K, PIK3CA H1047R. MC38 tumors homogenously expressing PIK3CA H1047R and implanted into wild type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene or non-scoring mutation in PI3K regressed after treatment with anti-PD-1. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry of tumor infiltrating lymphocytes. I conclude that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion. As such, activation of PI3K may be useful as a predictor of resistance to immunotherapy. Importantly, these findings also provide a rationale for therapeutic combination trials of immune checkpoint blockade and PI3K inhibition. Citation Format: Natalie B. Collins, Robert Manguso, Hans Pope, W. Nicholas Haining. Defining molecular mechanisms of resistance to tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A16.

Abstract 417: Defining molecular mechanisms of resistance to glioblastoma immunity using a novel CRISPR/Cas9in vivoloss-of-function screening platform

OVERVIEW: Despite the impact of cancer immunotherapy, expanding its clinical utility requires a rational method for identifying combination therapies and resistance mechanisms. This is pertinent to glioblastoma (GBM), where initial trials show uncertain response rates. Functional genomic screens used to identify new therapies and escape mechanisms were generally conducted in vitro, where interaction with the immune system is absent. Here we present the development of a high-throughput, in vivo, loss-of-function screening platform for GBM immune-escape mechanisms. METHODS: CT2A- and GL261-astrocytoma cells bearing Cas9-endonuclease were engineered to express a library of barcoded single guide RNAs (sgRNAs). These cells form tumors when implanted intracranially in immunocompetent mice. Tumor-bearing mice were treated with vaccination or PD-1 checkpoint blockade. Dropout of sgRNAs targeting putative immune evasion molecules or enrichment of sgRNAs mimicking resistance mechanisms were detected using next-generation sequencing at the time of tumor implantation and harvest post-immunotherapy. RESULTS: We first developed an in vivo, pooled, loss-of-function genetic screen using Cas9/CRISPR genome editing in mouse transplantable tumors subjected to titratable, selective immune-pressure. Screening 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade recovered known immune-evasion molecules PD-L1 and CD47. Novel immunotherapy targets validated individually, identifying essential pathways of immune-evasion. We then sought to recapitulate this approach in the CNS, and showed that 500-1000 genes can be functionally screened under graded immune-pressure. In optimized immune-competent CT2A and GL261 GBM models, tumors derived from cancer stem cell CD133hi-rich neurospheres were sensitive to immunotherapy and more aggressive and infiltrative than tumors derived from adherent tumor cells. A neurosphere based immune-competent model can be scaled up to a whole-genome screen due to a shorter experimental time requirement and improved engraftment allowing for functional screening of >1000 genes. We curated a GBM-specific library based on differential in vitro and in vivo gene expression profiles of CT2A and GL261 cells exposed to graded immune pressure. We are now screening this GBM-specific library in our optimized in vivo pooled loss-of-function genetic screen using immune-pressure titration to identify novel immunotherapy targets in GBM. CONCLUSIONS: This assay provides the first high-throughput method for systematically identifying resistance mechanisms and new candidate targets for immunotherapy in CNS tumors. Our optimized model could be scaled up to whole-genome loss of function screens, serving as an important tool for identification of next-generation and combination immunotherapies. Citation Format: Martha R. Neagu, Robert T. Manguso, Hans Pope, Maria C. Speranza, Gordon J. Freeman, John Doench, Arlene H. Sharpe, William Nicholas Haining. Defining molecular mechanisms of resistance to glioblastoma immunity using a novel CRISPR/Cas9 in vivo loss-of-function screening platform [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 417. doi:10.1158/1538-7445.AM2017-417

Abstract 1019: In vivo CRISPR screening identifies Ptpn2 as a target for cancer immunotherapy

Despite the dramatic clinical success of cancer immunotherapy with PD-1 checkpoint blockade, most patients don’t experience sustained clinical benefit, suggesting that additional therapeutic strategies are needed. Functional genomic screens in cancer cells to discover new therapeutic targets are usually carried out in vitro where interaction with the immune system is absent. Here we report a pooled, loss-of-function genetic screening approach using CRISPR/Cas9 genome editing that is conducted in vivo in mouse transplantable tumors treated with vaccination and PD-1 checkpoint blockade. We tested 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade, and recovered the known immune evasion molecules, PD-L1 and CD47. Loss of function of multiple genes required to sense interferon-y caused resistance to immunotherapy. Deletion of Ptpn2, a pleotropic protein tyrosine phosphatase improved response to immunotherapy. In vivo, Ptpn2 deficient tumors showed increased infiltration of activated CD8+T cells. In vitro, Ptpn2 loss by tumor cells increased antigen presentation to T cells. Biochemical, transcriptional and genetic epistasis experiments demonstrated that loss of function of Ptpn2 sensitizes tumors to immunotherapy by enhancing interferon-y-mediated effects on the tumor cell. Thus, augmenting interferon-y signaling in tumor cells could increase the efficacy of immunotherapy. More generally, in vivo genetic screens in tumor models can identify new immunotherapy targets and rationally prioritize combination therapies. Citation Format: Robert T. Manguso, Hans W. Pope, Margaret D. Zimmer, Flavian D. Brown, Kathleen B. Yates, Brian C. Miller, Natalie B. Collins, Kevin Bi, Martin W. Lafleur, Vikram R. Juneja, Sarah A. Weiss, David E. Fisher, David E. Root, Arlene H. Sharpe, John G. Doench, W Nicholas Haining. In vivo CRISPR screening identifies Ptpn2 as a target for cancer immunotherapy [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 1019. doi:10.1158/1538-7445.AM2017-1019

Enhancing the Efficacy of Checkpoint Blockade Through Combination Therapies

Antibodies targeting coinhibitory receptors on T cells (“checkpoint blockade”) have emerged as some of the most promising therapies for a broad range of malignancies, including melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin’s lymphoma, and bladder cancer. These coinhibitory molecules include CTLA-4, PD-1, LAG-3, TIM-3, and others. The anti-CTLA-4 antibody ipilimumab was approved in 2011 and the anti-PD-1 antibodies pembrolizumab and nivolumab were approved in 2014 for patients with advanced melanoma. Single agent checkpoint blockade is associated with 20–40 % objective response rates in advanced melanoma with improved overall survival. The combination of anti-CTLA-4 and anti-PD-1 antibodies leads to an increased durable response rate compared to either antibody alone, supporting the concept that combination therapy may result in increased clinical benefit. An important goal in the field is to combine checkpoint blockade with other immunotherapies and other types of therapy (e.g., radiation, targeted therapy, chemotherapy, surgery) to increase the fraction of patients that have objective and durable responses. Here, we discuss the current understanding of the mechanisms underlying checkpoint blockade and the rationale for combination therapy. We then discuss potential immunotherapeutic and non-immunotherapeutic combination therapies. Finally, we discuss critical issues that need to be addressed in order to develop combination strategies to induce long-term clinical responses in patients with cancer.

Abstract A097: Tumor PDL1 blocks CTL cytotoxicity

The programmed cell death (PD)-1 pathway has become an attractive therapeutic target in multiple cancers. Blocking the interaction of PD-1 with its ligands, PD-L1 and PD-L2, leads to impressive anti-tumor responses and clinical benefit in a subset of patients. However, the precise cellular and molecular mechanisms underlying this efficacy are not well understood. To better understand why PD-1/PD-L1 blockade works in some patients but not in others, it is critical to first understand how the PD-1 pathway inhibits anti-tumor immunity. Previous studies demonstrate that clinical responses to PD-1 immunotherapy positively correlate with tumor PD-L1 expression along with other predictive biomarkers such as pre-existing CD8+ T cell infiltration and mutational/neoantigen burden. This has led to the speculation that PD-L1 on tumor cells may act as a “molecular shield” to protect PD-L1+ tumor cells from T cell lysis. We sought to determine whether PD-L1 on tumor cells contributed to the suppression of T cells or simply correlated with an inflamed tumor microenvironment. Using two mouse tumor models that are sensitive to PD-1 pathway blockade, MC38 colorectal adenocarcinoma (MC38) and BRAF.PTEN melanoma (BP), we demonstrate that PD-1 pathway blockade is more effective in MC38 despite similar PD-L1 expression by both tumors. Furthermore, by comparing growth of each tumor cell line in PD-L1 or PD-1 deficient mice to wild-type (WT) mice, we found that host (non-tumor) PD-L1 plays a role in immunosuppression in BP tumors, while PD-L1 on MC38 tumor cells largely mediates suppression of anti-tumor immunity. Thus, the role of PD-L1 on the tumor is tumor-dependent. To further examine the role of MC38 PD-L1 in suppressing the anti-tumor immune response we generated PD-L1-deficient MC38 tumor cells using the CRISPR/Cas9 system. Deletion of PD-L1 on MC38 tumors cells alone leads to effective anti-tumor immunity in mice, analogous to the efficacious anti-tumor immunity seen with PD-1 pathway blockade or in PD-1 deficient mice. Further, wild-type MC38 cells outcompete PD-L1-deleted MC38 cells in vivo, demonstrating MC38-derived PD-L1 is sufficient to mediate immunosuppression. We also found that PD-L1 on MC38 cells potently inhibits CD8+ T cell cytotoxic molecule production. Taken together, our data indicate that PD-L1 expression on MC38 cells alone is sufficient to directly suppress CD8+ T cell cytotoxicity in the tumor microenvironment. These findings demonstrate a direct causal relationship between tumor-derived PD-L1 and inhibition of T cell function and establish a key mechanism by which PD-L1 on tumor cells can suppress T cells. Thus, PD-L1 on tumor cells functions to suppress anti-tumor immunity and is not just a marker of suppressed anti-tumor immunity. Citation Format: Kathleen A. McGuire, Vikram R. Juneja, Robert T. Manguso, Martin W. LaFleur, Natalie Collins, W. Nicholas Haining, Gordon J. Freeman, Arlene H. Sharpe. Tumor PDL1 blocks CTL cytotoxicity [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 A097.