A high-throughput RNAi screening identifies olfactory receptor signaling as a novel immune checkpoint in solid tumors

Immune checkpoint blockade has revolutionized immunotherapy against cancer with tremendous clinical benefits for patients. Despite these achievements, tumors utilize a plethora of suppressive mechanisms to evade immune destruction which are yet to be understood and matched by today’s immunotherapy. Our group developed a high-throughput RNAi screening to unravel the arsenal of immune checkpoints of cancer. We screened a siRNA library (around 2880 kinases and surface-associated genes) with patient-derived tumor cells and HLA-matched tumor-infiltrating lymphocytes (TILs). The library was reverse-transfected into M579-A2 melanoma cells and these were co-cultured with MART1- and gp100-specific TILs to determine TIL-mediated lysis. We identified 75 genes in tumor cells that impaired TIL-mediated cytotoxicity. Interestingly, we found that several genes and their associated pathways were found in pancreatic adenocarcinoma and multiple myeloma as well. This suggests that different cancer entities might share inhibitory modes of action. In order to distinguish between genes altering tumor susceptibility towards TIL-mediated killing and those impairing TIL activity, we established a secondary screening assaying multiple T cell activation marker, including effector cytokines. The olfactory receptor OR10H1 was one of the strongest candidates from our primary screening as its knockdown increased TIL-mediated killing in melanoma, PDAC and colorectal carcinoma. Furthermore, TILs were activated stronger after interaction with OR10H1-deficient cells as sensed by the increased secretion of type 1-associated cytokines and a reduced T cell apoptosis. We confirmed the role of OR10H1 as an immune checkpoint in vivo using a xenograft mouse model in combination with adoptive T cell transfer. We performed mode of action analyses in order to understand how OR10H1 affects T cell activity. These analyses revealed that tumor-associated OR10H1 controls cAMP-dependent signaling inside T cells. Inside TILs, cAMP activates protein kinase alpha (PKA) and PKA in turn activates C-terminal Src kinase (Csk. Csk phosphorylates an inhibitory tyrosine residue of Lck impairing its activity and shutting down TCR-associated signaling. Furthermore, PKA activates CREB and thus induces an anergy-associated gene expression profile in TILs. Our data suggest that OR10H1 alters the balance between the inhibitory (GαI) and the stimulatory/olfactory G-Protein alpha (GαS/Olf) inside tumor cells depending on the encounter of TILs. This results into increased production of cAMP in tumor cells and its subsequent transport into T cells. In summary, we established a discovery platform aiding the search for immune checkpoints in cancer. We identified OR10H1 and its associated olfactory receptor signaling as a novel pathway inhibiting TIL responses by inducing cAMP-dependent Lck inhibition

Common clonal origin of conventional T cells and induced regulatory T cells in breast cancer patients

Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation

Acid-Mediated Prevention of Aspartimide Formation in Solid Phase Peptide Synthesis

Aspartimide formation is one of the major obstacles that impedes the solid phase synthesis of large peptides and proteins. Until now, no cost-effective strategy to suppress this side reaction has been developed. Here it is demonstrated that addition of small amounts of organic acids to the standard Fmoc cleavage agent piperidine efficiently prevents formation of aspartimide side products. This effect is shown to be virtually independent of the acid strength

CAMK1D Triggers Immune Resistance of Human Tumor Cells Refractory to Anti–PD-L1 Treatment

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 CTLs in anti-PD-L1 treatment-refractory human tumors. Using PD-L1-positive multiple myeloma cells cocultured 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 coexpressed 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 pharmacologic inhibition of CAMK1D, on the other hand, restored the sensitivity toward Fas-ligand treatment in multiple myeloma and uveal melanoma cells in vitro. Thus, rapid inhibition of the terminal apoptotic cascade byCAMK1D-expressed in anti-PD-L1-refractory tumors via T-cell recognition may have contributed to tumor immune resistance

Salt-inducible kinase 3 protects tumor cells from cytotoxic T-cell attack by promoting TNF-induced NF-κB activation

Background Cancer 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. Methods To 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. Results The 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. Conclusion Our data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible