Although functionally competent cytotoxic, T cells are frequently observed in malignant diseases, they possess little ability to react against tumor cells. This phenomenon is particularly apparent in multiple myeloma. We here demonstrate that cytotoxic T cells reacted against myeloma antigens when presented by autologous dendritic cells, but not by myeloma cells. We further show by gene expression profiling and flow cytometry that, similar to many other malignant tumors, freshly isolated myeloma cells expressed several carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) at varying proportions. Binding and crosslinking of CEACAM-6 by cytotoxic T cells inhibited their activation and resulted in T-cell unresponsiveness. Blocking of CEACAM-6 on the surface of myeloma cells by specific monoclonal antibodies or CEACAM-6 gene knock down by short interfering RNA restored T-cell reactivity against malignant plasma cells. These findings suggest that CEACAM-6 plays an important role in the regulation of CD8+ T-cell responses against multiple myeloma; therefore, therapeutic targeting of CEACAM-6 may be a promising strategy to improve myeloma immunotherapy.

The success of T cell-based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune-suppressive ligands have been identified. We here describe a rapid high-throughput siRNA-based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL-mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune-regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T-helper-1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor-specific T cells in vivo. Taken together, this method allows a rapid and comprehensive determination of immune-modulatory genes in human tumors which, as an entity, represent the ‘immune modulatome’ of cancer.

The success of cancer immunotherapy is limited by resistance to immune-checkpoint blockade. We therefore conducted a genetic screen to identify genes that mediated resistance against cytotoxic T lymphocytes (CTL) in anti-PD-L1 treatment refractory human tumors. Using PD-L1 positive multiple myeloma cells co-cultured with tumor-reactive bone marrow-infiltrating CTL as a model, we identified calcium/calmodulin-dependent protein kinase 1D (CAMK1D) as a key modulator of tumor intrinsic immune resistance. CAMK1D was co-expressed with PD-L1 in anti-PD-L1/PD-1 treatment refractory cancer types and correlated with poor prognosis in these tumors. CAMK1D was activated by CTL through Fas-receptor stimulation, which led to CAMK1D binding to and phosphorylating caspase-3,-6 and-7, inhibiting their activation and function. Consistently, CAMK1D mediated immune resistance of murine colorectal cancer cells in vivo. The pharmacological inhibition of CAMK1D on the other hand, restored the sensitivity towards Fas-ligand treatment in multiple myeloma and uveal melanoma cells in vitro. Thus, rapid inhibition of the terminal apoptotic cascade by CAMK1D expressed in anti-PD-L1 refractory tumors via T cell recognition may have contributed to tumor immune resistance.

BackgroundCancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies.MethodsTo identify novel genes that protect tumor cells from effective TC-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with tumor-infiltrating lymphocytes and antigen-specific TCs.ResultsThe screening revealed 108 potential genes that protected tumor cells from TC attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibitions of SIK3 in tumor cells dramatically increased TC-mediated cytotoxicity in several in vitro coculture models, using different sources of tumor and TCs. Consistently, adoptive TC transfer of TILs led to tumor growth inhibition of SIK3-depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to tumor necrosis factor (TNF) secreted by tumor-activated TCs. SIK3 promoted nuclear factor kappa B (NF-κB) nuclear translocation and inhibited caspase-8 and caspase-9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of prosurvival genes under the TNF–NF-κB axis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-κB upstream regulators inhibitory-κB kinase and NF-kappa-B inhibitor alpha on the one side, and to inhibition of histone deacetylase 4 on the other side, thus sustaining NF-κB activation and nuclear stabilization. A SIK3-dependent gene signature of TNF-mediated NF-κB activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic TC activity and poor prognosis.ConclusionOur data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible.

Cytotoxic T lymphocytes (CTLs) form an indispensable component of body's defense against cancer and therefore lie at the heart of the cancer immunotherapy program. However, evasion and suppression of immune surveillance by tumor cells remains a reality and a major roadblock for the clinical success of cancer immunotherapy. In this study, we established a novel RNAi silencing screen based on a siRNA library, which contained multiple siRNA sequences to individually target more than 500 G-protein coupled receptors (GPCRs) in MCF7 breast cancer cells, to identify those tumor-associated receptors that promote tumor resistance to immunological destruction by CTLs. Briefly, luciferase-tagged MCF7 cells were reverse transfected with the siRNA library, followed by co-culture with the reactive CTLs. Effect of individual gene knockdown on CTL-mediated tumor lysis was determined based on the luciferase activity of the remaining live cells in each well. Gene knockdowns that affected cell viability by themselves were excluded from the hit analysis. Z-score based normalization resulted in a hit list comprising of 79 candidates that negatively regulate CTL activity and 27 candidates that positively modulate the T cell response. Using antigen-specific T cell clones in secondary assays, we have validated two of such high scoring hits, namely NiS-6 and NiS-1, that negatively regulate CTL response against tumor. siRNA-mediated knockdown of NiS-6 increases the susceptibility of both MCF7 and MDA-MBD-231 breast cancer cells to immunological destruction by survivin-specific T cells, as measured in standard chromium-release kill assays. Blocking of NiS-6 in MCF7s via siRNA also increases the interferon-gamma and granzyme B secretion by survivin-TCs in ELISpot readouts. Moreover, elevated levels of IL-2 and TNF-alpha, along with significantly diminished levels of immunosuppressive cytokine IL-10, were observed in the supernatant of the MCF7 and T cell coculture upon NiS-6 knockdown. Preliminary knockdown studies with NiS-1 receptor in tumor cells demonstrated that NiS-1 might be similarly involved in de-sensitizing tumor cells to CTL-mediated lysis. Here we report a systematic and high throughput screening strategy for the identification of novel immune modulators associated with breast cancer. Based on our findings, we suggest a new and previously undefined role for the tumor-associated G-protein coupled receptors- NiS-6 and NiS-1 – in downregulating T cell responses. These surface receptors are therefore an attractive therapeutic target for the treatment of breast cancer in either a monotherpay setting or in conjunction with adoptive immunotherapy, so as to override tumor's acquired resistance to immunological destruction. Citation Format: Nisit Khandelwal, Marco Breinig, Tobias Speck, Heinke Conrad, Ludmila Umansky, Michael Boutros, Philipp Beckhove. A high-throughput RNAi screen unravels new immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A97.

SIK3 is an intracellular serine/threonine kinase belonging to the AMPK superfamily. We recently discovered a novel role of SIK3 in conferring TNF resistance to tumor cells. Resistance to TNF is an emerging mode of immune evasion in multiple solid tumors. In fact, while treating a broad panel of tumor cell lines with TNF we observed that almost 70% were either resistant or even proliferative in response to TNF. SIK3 knockout using CRISPR re-sensitized human PANC-1 and murine MC38 tumor cells to TNF-mediated death. Intratumoral SIK3 induces TNF resistance by retaining HDAC4 in the cytoplasm thus keeping the chromatin open and potentiating the TNF-driven pro-tumorigenic activity of NF-κB. To translate these findings into the clinic, we have developed a potent (low nM range) inhibitor of SIK3, OMX-0370. Firstly, OMX-0370 exhibits dose-dependent inhibition of HDAC4 phosphorylation and nuclear NF-κB activity in response to TNF, thereby effectively neutralizing the SIK3-HDAC4-NF-κB axis. Consequently, treatment of human as well as murine tumor cells with OMX-0370 induces significant TNF-mediated apoptosis while sparing non-TNF treated cells. In mouse DMPK studies, OMX-0370 was found to be orally bioavailable with a favorable pharmacokinetic profile and was well tolerated at 100 mg/kg twice daily dosing in wild type C57BL/6 mice. Having shown a potent and favorable biochemical, functional as well as pharmacokinetic profile of the molecule, we next investigated the ability of OMX-0370 to inhibit the growth of established tumors in multiple syngeneic tumor models, including MC38, EMT6 and RENCA. Notably, OMX-0370 showed significant tumor growth inhibition as a single agent, which was found to be even superior to anti-PD-1 antibody treatment in RENCA and EMT-6 models. Immune profiling showed enhanced activation of intratumoral T cells, improved ratio of CTLs to Tregs and depletion of tumor-associated M2 macrophages, while no effect on peripheral leukocyte counts was observed. To monitor target engagement and pharmacodynamics of OMX-0370 in vivo, we developed a reporter MC38 cell line expressing luciferase under a NF-κB promotor. Using this reporter cell line, we could show that OMX-0370 inhibits TNF-induced NF-κB activation in a dose-dependent manner. Encouraged by the strong single-agent activity of OMX-0370 in solid tumor models, we have developed follow-on variants that exhibit higher potency as well as improved exposure in vivo. In summary, we here report that OMX-0370, a first-in-class inhibitor of SIK3 kinase, is an active immunotherapeutic drug in vivo which effectively abolishes the TNF-driven NF-κB activity in tumors and re-sensitize them to TNF-induced apoptosis. OMX-0370 and its follow-on improved variants address the high unmet medical need of effective immunotherapeutic agents that neutralize clinically relevant and key orthogonal immune evasion axes in solid tumors as a monotherapy regimen. Citation Format: Tillmann Michels, Stefan Bissinger, Peter Sennhenn, Hannes Loferer, Catarina Martins Freire, Olivia Reidell, Sebastian Meier-Ewert, Apollon Papadimitriou, Philipp Beckhove, Nisit Khandelwal. A first-in-class SIK3 inhibitor, OMX-0370, effectively inhibits tumor growth in syngeneic tumor models, as single agent, by abolishing tumor resistance to immune-derived TNF [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6698.

<p>Supplementary Table 2</p>

<p>Supplementary Figures 1-7</p>