This study suggests a modified model of TNFR1-induced complex I-mediated NFκB signaling. Evaluation of a panel of five tumor cell lines (HCT116-PIK3CAmut, SK-MEL-23, HeLa-RIPK3, HT29, D10) with TRAF2 knockout revealed in two cell lines (HT29, HeLa-RIPK3) a sensitizing effect for death receptor-induced necroptosis and in one cell line (D10) a mild sensitization for TNFR1-induced apoptosis. TRAF2 deficiency inhibited death receptor-induced classical NFκB-mediated production of IL-8 only in a subset of cell lines and only partly. TRAF5, furthermore, failed to improve DR-induced NFκB signaling in HCT116-PIK3CAmut and HCT116-PIK3CAmut-TRAF2KO cells. These findings argue for a non-obligatory role of TRAF2 in death receptor-induced classical NFκB signaling. Similar as in TRAF2-deficient cells, TNF- and CD95L-induced NFκB signaling was found to be only poorly affected in RIPK1KO cells and in cells treated with the RIPK1-specific PROTAC LD4172. Intriguingly, however, death receptor-induced NFκB signaling was completely inhibited in HCT116-PIK3CAmut cells double deficient for TRAF2 and RIPK1 and in TRAF2-deficient cells treated with LD4172. Moreover, with exception of recruitment of TRADD, acting upstream to TRAF2 and parallel to RIPK1, TNFR1 signaling complex formation was abrogated in TRAF2-RIPK1 DKO cells. Based on our findings, two distinguishable types of TNFR1-interacting complexes promote TNF-induced NFκB signaling: First, a TRADD-TRAF2/cIAP utilizing complex Ia which becomes evident in RIPK1-deficient cells. Second, a non-modified RIPK1 utilizing complex Ib which acts in TRADD- or TRAF2-deficient cells. Complex Ia and Ib may furthermore interact and cooperate to ubiquitinate RIPK1 resulting in a modified complex Ia/b preventing complex Ia and Ib to convert to the established TNFR1-induced cytotoxic complexes IIa and IIb.

Extended data on Figure 5: Low expression of ELOB/C is associated with phenotype switching and EMT.

Synthetic lethal interactions (SLIs) based on genomic alterations in cancer have been therapeutically explored. We investigated the SLI space as a function of differential RNA expression in cancer and normal tissue. Computational analyses of functional genomic and gene expression resources uncovered a cancer-specific SLI between the paralogs cytidine diphosphate diacylglycerol synthase 1 (CDS1) and CDS2. The essentiality of CDS2 for cell survival is observed for mesenchymal-like cancers, which have low or absent CDS1 expression and account for roughly half of all cancers. Mechanistically, the CDS1-2 SLI is accompanied by disruption of lipid homeostasis, including accumulation of cholesterol esters and triglycerides, and apoptosis. Genome-wide CRISPR-Cas9 knockout screens in CDS1-negative cancer cells identify no common escape mechanism of death caused by CDS2 ablation, indicating the robustness of the SLI. Synthetic lethality is driven by CDS2 dosage and depends on catalytic activity. Thus, CDS2 may serve as a pharmacologically tractable target in mesenchymal-like cancers.

Extended data on Figure 4: Elongin B/C interact with AXL and loss of Elongin B/C stabilizes AXL protein levels.

Abstract CD8+ T cell-tumor cell proximity correlates with immunotherapy response. However, it is unknown whether these cells can be captured as functional clusters from clinical samples. In defined co-cultures, tumor antigen-recognizing T cells outcompeted unmatched T cells in forming heterotypic clusters with tumor cells, showing >10-fold enrichment when comprising only 1% of the total T cell population, which was seen in across cancer cell lines. This prompted us to investigate whether this feature could be used to isolate tumor-reactive CD8+ T cells directly from cancer specimens. By regular and imaging flow cytometry, we could identify heterotypic clusters from 21/21 human melanoma metastases, comprising CD8+ T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs). These results prompted us to investigate whether these heterotypic T cell clusters could be used to better understand biology and whether they hold therapeutic potential. Regarding biology, we observed that CD8+ T cells from clusters isolated from clinical samples were significantly enriched for tumor-reactive and exhausted gene signatures. Integration with T cell receptor (TCR)-sequencing revealed increased clonality of clustered T cells, indicative of expansion. Whereas single CD8+ T cells showed enrichment of viral antigen-specific signatures, T cells from clusters were enriched for tumor antigen-reactivity signatures. In-depth single cell RNA analyses revealed conjugation of these T cells to tumor cells and different APC types. These were not random events, as both T cell-associated APCs and tumor cells were enriched for specific cell states and associated with differential cell-cell communication. CD8+ T cells carrying identical TCRs showed more exhaustion and co-inhibition when conjugated to APCs than to tumor cells. Regarding therapeutic opportunities, we found that upon rapid expansion (REP), CD8+ TIL from clusters exerted on average 9-fold increased melanoma-killing activity in ex vivo assays, accompanied by enhanced production of cytokines. Upon ACT, T cells from clusters showed superior in vivo killing of matched patient-derived xenografts (PDX), which was associated with more infiltration and activation than for ACT of single T cells. Together, these results demonstrate that tumor-reactive CD8+ T cells are enriched in functional clusters with tumor cells and/or APCs, and that they can be isolated and expanded from clinical samples. Being often excluded during sorting for single-cell studies, these distinct heterotypic CD8+ T cell clusters serve as a valuable source amenable to deciphering functional tumor-immune cell interactions, while they may also be therapeutically explored. Citation Format: Daniel S Peeper, Sofia Ibáñez-Molero, Johanna Veldman, Juan Simón-Nieto, Joleen Traets, Austin George, Kelly Hoefakker, Anita Karomi, Jonas Nilsson, John Haanen, Winan van Houdt. Heterotypic CD8 T cell clusters isolated from clinical samples are distinct and enriched for antitumor activity [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr PR007.

The efficacy of immunotherapy fundamentally depends on effective T cell-tumor cell interactions, yet the molecular determinants orchestrating this engagement are not fully understood. Here, we systematically mapped and perturbed key regulators of the T cell-tumor cell interactome using innovative cell-cell interaction assays. We performed genome-wide CRISPR screens to identify genes critically required for physical and functional interactions between T cells and tumor cells. Using TurboID proximity labeling and T cell-tumor cell conjugates, we identified the complex N-glycan biosynthesis pathway as a crucial regulator of T cell-tumor cell interactions. Recognizing the role of N-glycans in protein trafficking and surface retention, we subsequently performed cell surface proteomics and TurboID-based interactome profiling on complex N-glycan-deficient T cells, revealing increased expression of cell adhesion and co-stimulatory molecules. T cells with perturbed N-glycans show a shift towards an effector memory phenotype with elevated granzyme and integrin expression. In vivo, both genetic and pharmacological interventions that decrease the number of complex N-glycans on the surface of T cells enhanced intra-tumoral infiltration. Our findings provide a comprehensive functional atlas of molecules modulating T cell-tumor cell interactions and position the complex N-glycan biosynthesis pathway as a potential therapeutic target to strengthen T cell-mediated cytotoxicity in cancer immunotherapy. Adriaan F. de Groot, Zowi R. Huinen, Juan Simon Nieto, Daniel S. Peeper. Genome-wide CRISPR screens for genes inhibiting T cell-tumor cell interactions identify complex N-glycans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3960.

Supplementary Data file, Figure S1-S9, Table S1

Extended data on Figure 2: Elongin BC complex members serve as keyAXL regulators.

Modulation of AXL neither affects cell proliferation nor sensitivity to MEK inhibition.

Extended data on Figure 3: Elongin B/C regulate AXL independently of hypoxia.

Extended data on Figure 6: Low expression of ELOB/C is associated with dedifferentiation in human melanomas.

Extended data on Figure 4: Elongin B/C interact with AXL and loss of Elongin B/C stabilizes AXL protein levels.

BRAF inhibitor-resistant melanomas (BRAFiR) acquire (epi)genetic and functional alterations that enable them to evade alternative treatments. Identifying these alterations is critical to advancing treatment strategies. Here, we explored the effect of Chk1 inhibition (Chk1i) on BRAFiR cells, revealing higher sensitivity compared to treatment-naïve cells both in vitro and in vivo. Using FUCCI-labeling and time-lapse microscopy, we show that S phase progression is required for Chk1i-induced cytotoxicity in BRAFiR cells, but not in treatment-naïve cells. Replication stress markers, including reduced BrdU incorporation and increased phospho-RPA and γH2AX, were observed mostly in BRAFiR cells with increased sensitivity to Chk1i. Untreated BRAFiR cells exhibited upregulated DNA replication genes, reduced progressing forks and increased origin firing, suggesting intrinsic replication changes. MAPK pathway reactivation in treatment-naïve cells mimicked BRAFiR traits, increasing sensitivity to Chk1i. These findings indicate that Chk1i exploits elevated replication stress specifically in BRAFiR cells, highlighting its therapeutic potential in overcoming MAPK inhibitor resistance in BRAF600-mutant melanoma.

Extended data on Figure 1: Whole genome-wide CRISPR-Cas9 screenidentifies several negative regulators of AXL.

Resistance to immune cell-mediated cytotoxicity poses a significant challenge in cancer therapy, compromising the efficacy of immunotherapeutic approaches such as immune checkpoint blockade (ICB) treatment. To enhance therapy outcomes, it is crucial to identify interventions that can synergize with ICB therapy to overcome tumor resistance. Therefore, we need to define the cellular mechanisms that sensitize tumors to cytotoxic T cells. CD8 T cells rely on cytokines such as TNF to carry out their cytotoxicity against tumors, and recent findings link select tumor mutations in the TNF pathway to increased T cell killing, in a manner dependent on RIPK1 kinase. Here, we demonstrate that sensitized tumor cells fail to initiate inhibitory RIPK1 phosphorylation at site S25 upon T cell attack, thereby foregoing a pro-survival checkpoint early in TNF signal transduction. Consequently, tumor cells experiencing a loss of TNF-induced RIPK1 S25 phosphorylation exhibit increased RIPK1 activation and fail to recruit non-canonical IKK kinases (TBK1 and IKKε) to the TNFR1 complex. Functional knockouts of TBK1 and IKKε in melanoma cells result in heightened sensitivity not only in CD8 T cell but also in Natural Killer cell attacks. Our findings indicate that preventing TBK1 and IKKε recruitment to the TNF signaling complex, thereby blocking RIPK1 pro-survival phosphorylation and promoting direct RIPK1 activation, is a tractable strategy to increase tumor sensitivity to immune cell killing and has the potential to benefit current immunotherapy interventions.

Summary: Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. However, our understanding of the proteins involved is limited. Here, we present HySic (hybrid quantification of stable isotope labeling by amino acids in cell culture [SILAC]-labeled interacting cells) as a method to quantify protein and phosphorylation dynamics between and within physically interacting cells. Using co-cultured T cells and tumor cells, we directly measure the proteome and phosphoproteome of engaged cells without the need for physical separation. We identify proteins whose abundance or activation status changes upon T cell:tumor cell interaction and validate our method with established signal transduction pathways including interferon γ (IFNγ) and tumor necrosis factor (TNF). Furthermore, we identify the RHO/RAC/PAK1 signaling pathway to be activated upon cell engagement and show that pharmacologic inhibition of PAK1 sensitizes tumor cells to T cell killing. Thus, HySic is a simple method to study rapid protein signaling dynamics in physically interacting cells that is easily extended to other biological systems.

Abstract Synthetic lethal interactions (SLIs) can provide a therapeutic index, as illustrated by PARP inhibition of BRCA-deficient cancers1–4. Whereas additional SLIs based on genomic alterations in cancer have been identified5–19, we set out to explore the SLI space as a function of differential RNA expression profiles in cancer and normal tissue. By unbiased computational analyses of publicly available functional genomic and gene expression resources we uncovered a cancer-specific SLI between the paralogs cytidine diphosphate synthase 1 (CDS1) and CDS2. The essentiality of CDS2 for cell survival is observed for mesenchymal-like cancers, which express low levels of CDS1. We confirm the CDS1-2 SLI in a panel of cultured cancer cell lines and in tumor-bearing mice. Mechanistically, the CDS1-2 SLI is accompanied by disruption of lipid homeostasis including extensive accumulation of cholesterol esters and triglycerides, and induction of apoptotic cell death. Genome-wide CRISPR-Cas9 knockout screens in a panel of CDS1-negative cancer cell lines failed to identify a common escape mechanism of death caused by CDS2 ablation, indicating the robustness of the SLI. Our findings reveal that CDS2 may serve as a pharmacologically tractable target in mesenchymal cancers, meriting therapeutic exploration.

Background An increasing body of evidence suggests that in addition to the type, density, and state of immune cells in the tumor microenvironment (TME), also their proximity to cancer cells influences immunotherapy outcome. For example, favorable responses to immune checkpoint inhibitors in melanoma are associated with higher densities of CD8+ tumor-infiltrating lymphocytes (TIL) within 20 μm distance of melanoma cells. This notion is in line with the understanding that upon specific antigen recognition, cytotoxic T cells physically engage with their target cells through their TCRs followed by immunological synapse formation. Indeed, structural and functional avidity of cytotoxic CD8+ T cells correlates strongly with their activity against cancer cells. Together, these observations point to the importance of direct interactions between cytotoxic T cells and tumor cells in the TME. This led us to investigate whether tumor-specific CD8+ T cells can be isolated from clinical cancer specimens as heterotypic clusters. Materials and Methods We employed a tumor cell-T cell co-culture in vitro model, patient samples and ex vivo assays. To evaluate functional interactions between human T cells and tumor cells, we made use of a system we engineered previously, comprising melanoma cells expressing both HLA-A*02:01 and the MART-1 tumor antigen. They were challenged with CD8+ T cells from PBMCs that were retrovirally transduced with a MART-1-specific TCR. To asses these interactions in patient material, upon surgical removal tissue was cut into small fragments, digested and analyzed by (image-based) flow cytometry. Interacting (cluster) and not-interacting (singlets) T cells were isolated and expanded in vitro. To characterize tumor cell:T cell interactions single cell TCR and RNA sequencing is used, as well as ex vivo co-cultures with autologous tumor cells. Results We found that in defined co-cultures, tumor antigen-recognizing T cells were commonly enriched over non-specific T cells in heterotypic clusters with tumor cells, prompting us to investigate whether such specific clusters could be isolated also from cancer specimens. We observed that from 10/10 human melanoma metastases, we were able to isolate heterotypic clusters, comprising CD8+ T cells interacting with one or more tumor cells and/or antigen-presenting cells (APCs), which was validated by imaging flow cytometry. Upon expansion, CD8+ T cells from tumor cell clusters and APC clusters exerted on average 7.6-fold increased melanoma-killing activity over T cell singlets, which was associated with enhanced cytokine production. CD8+ T cells from clusters were enriched for tumor-reactive and exhausted gene signatures. Integration with T cell receptor (TCR)-sequencing showed increased clonality of clustered T cells, indicative of expansion upon antigen recognition. Conclusions Together, these results demonstrate that tumor-reactive CD8+ T cells are enriched in functional clusters with tumor cells and/or APCs, and that they can be isolated and expanded from clinical samples. Being often excluded in cell sorting procedures, these distinct heterotypic CD8+ T cell clusters serve as a valuable source amenable to deciphering functional tumor-immune cell interactions, while they may also be therapeutically explored. S. Ibáñez Molero: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL. J. Veldman: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL. J. J H Traets: None. A. George: None. K. Hoefakker: None. S. Pack: None. L. Tas: None. P. Alóndiga-Mérida: None. B. van den Broek: None. R. Harkes: None. M. Nieuwland: None. M. van Baalen: None. E. Mul: None. S. Tol: None. J.B.A.G. Haanen: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Amgen, Asher Bio, BioNTech, BMS, MSD, Novartis, Sastra Cell Therapy. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Neogene Tx. F. Consultant/Advisory Board; Modest; BMS, CureVac, GSK, Imcyse, Iovance Bio, Instil Bio, Immunocore, Ipsen, Merck Serono, MSD, Molecular Partners, Novartis, Pfizer, Roche/Genentech, Sanofi, Scenic, Third Rock Ventures, Achilles Tx, BioNTech US, Instil Bio, PokeAcell, T-Knife, Scenic, Neogene Therapeutics. W.J.V. Houdt: None. D.S. Peeper: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Oncode Institute, Dutch Cancer Society KWF. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; P097110NL, Immagene. F. Consultant/Advisory Board; Modest; Immagene.

Abstract Synthetic lethal interactions (SLIs) can provide a therapeutic index, as illustrated by PARP inhibition of BRCA-deficient cancers. This clinical success, and other examples, have spurred efforts to identify additional cancer-associated SLIs. Whereas additional SLIs based on genomic alterations in cancer have been identified, we set out to explore the SLI space as a function of differential RNA expression profiles in cancer and normal tissue, covering all ∼3.4e8 gene pairs. In our bioinformatic pipeline, synthetic lethality is scored by correlating DepMap dependency and expression data (n = 913 cancer cell lines), while cancer specificity is scored by comparing TCGA expression data for 9264 tumors and 741 healthy samples. Cancer specificity is confirmed by comparing also calibrated GTEx data for 17382 healthy tissue samples with calibrated DepMap expression data. With this pipeline we uncovered a frequent cancer-specific SLI between the paralogs cytidine diphosphate diacylglycerol synthase 1 (CDS1) and CDS2 (p<0.001 in all three analyses). Essentiality of CDS2 is observed in one third of cultured cancers (DepMap). Using CRISPR-Cas9 we confirm the CDS1-dependency for CDS2 essentiality in a panel of 12 cultured cancer cell lines, with lethality by CDS2 ablation reaching up to 98% in CDS1-negative cell lines. We also confirm the SLI using admixing experiments in tumor-bearing mice for two cell lines (6 mice per group, up to 95% synthetic lethality, p<0.001). The essentiality of CDS2 for cell survival is observed for mesenchymal-like cancers, which commonly express low levels of CDS1. To biochemically define the effects of CDS2 perturbation in CDS1-negative cancer cells, we performed multi-omic analyses in a panel of CDS1-negative cancer cell lines. The results show that mechanistically, the CDS1-2 SLI is accompanied by disruption of lipid homeostasis, including extensive accumulation of cholesterol esters and triglycerides. Additionally, quantitative western blotting for cleaved caspase-3 showed induction of apoptotic cell death (p<0.001 in 2 cell lines). To challenge the robustness of the SLI, we performed genome-wide CRISPR-Cas9 knockout screens in a panel of four CDS1-negative cancer cell lines. This failed to identify a common escape mechanism of death caused by CDS2 ablation. These findings suggest that no common escape to the combined loss of CDS1 and CDS2 is possible through loss-of-function. In conclusion, by computational, genetic, biochemical and functional analyses we demonstrate that CDS2 may serve as a target in mesenchymal cancers, meriting therapeutic exploration. Citation Format: Tim Arnoldus, Alex van Vliet, Adriaan F.H. de Groot, Niek Blomberg, Onno B. Bleijerveld, Susan E. van Hal-van Veen, Anita E. Grootemaat, Rolf Harkes, Nicole N. van der Wel, Maarten Altelaar, Martin Giera, Daniel S. Peeper. Cytidine diphosphate diacylglycerol synthase 2 is a synthetic lethal target in mesenchymal cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR009.

Abstract This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR009) of the Conference Program/Proceedings. Citation Format: Tim Arnoldus, Alex van Vliet, Adriaan F.H. de Groot, Niek Blomberg, Onno B. Bleijerveld, Susan E. van Hal-van Veen, Anita E. Grootemaat, Rolf Harkes, Nicole N. van der Wel, Maarten Altelaar, Martin Giera, Daniel S. Peeper. Cytidine diphosphate diacylglycerol synthase 2 is a synthetic lethal target in mesenchymal cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B001.