Clinical trials with immune-checkpoint blockade antibodies have bolstered the importance of immune therapy as the standard of care for cancer patients, but it has also simultaneously highlighted the heterogeneity in patient responses to such treatment. These heterogeneities can be explained to a certain extent by the lack of tumor-specific expression of the targeted immune checkpoint molecules. But in many cases it can also be conceived that tumors either develop resistance to a targeted immune-checkpoint node by circumventing it or more than one player is involved in a concerted action to subvert the T cell response. In either scenario, we lack a holistic understanding of the putative genes in the tumor genome that could functionally suppress the immune response. To bridge this gap, we employed a high-throughput RNAi-mediated knockdown of upto 2800 genes (~50% associated with surface molecules) in MCF7 (breast), M579-A2 (melanoma) and PANC-1 (pancreatic) tumor cell lines and co-cultured them with either antigen-specific T cell clones or respective patient-derived and tumor-specific infiltrating lymphocytes (TILs) to assess the impact on anti-tumor immunity using a luciferase-based readout. Primary hit-list was further subjected to a secondary screen based on multi-cytokine profiling of the T cells. Our investigation revealed a few salient caveats of tumor-mediated immune suppression. Firstly, we discovered a family of orphan receptors, which were never attributed to the immune system before, to actively suppress the T cells in a manner comparable to the currently defined immune-checkpoint molecules, such as PD-L1. Secondly, the trans-versatility of these novel molecules across the tumor types was highly limited, with only 3-14 common molecules being involved in two or more tumor types. This leads us to our third observation that there exists a complex organ-specific orchestration of peripheral immune tolerance, which needs to be taken into account when devising immune-checkpoint blockade therapies. Amongst the key immunosuppressive candidate genes, CCR9 was validated to directly subvert T cell responses in melanoma, breast and pancreatic cancer in the in vitro and in vivo tumor models. Additionally we have verified TiMi1, an orphan G-protein coupled receptor, to mediate strong immunosuppression in melanoma and pancreatic cancer against the respective TILs. Knockdown of both CCR9 and TiMi1, either via siRNAs or shRNAs, in tumor cells significantly increased Th1 cytokine secretion by TILs along with elevated tumor lysis in vitro and in vivo xenotransplanted mouse models. Moreover, they both induce a highly immunosuppressive genetic signature in the encountering TILs. While TiMi1 appeared to modulate calcium-dependent signaling, CCR9 regulated STAT signaling in T cells leading to an immunosuppressed phenotype. Overall, these candidates represent attractive targets for cancer immunotherapy either through function blocking antibodies or small molecules.
In conclusion, we here report an effective genetic screen strategy in multiple tumor types that has the potential to uncover novel modifiers of anti-tumor immunity. Extensively validated candidates from these screens are attractive targets for cancer immunotherapy that will allow us to further expand our limited arsenal of immune-checkpoint inhibitors, with the overall goal of increasing patient responses to such treatments.
Citation Format: Nisit Khandelwal, Tillmann Michels, Marco Breinig, Antonio Sorrentino, Isabel Poschke, Rienk Offringa, Michal Lotem, Michael Boutros, Philipp Beckhove. Genetic knockdown screens across tumor types unravel a diverse tumor “immune-modulatome” landscape. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A070.
- ©2016 American Association for Cancer Research.