Immunotherapy has revolutionized cancer treatment, yet its efficacy in hepatocellular carcinoma (HCC) remains limited and the mechanisms of resistance are poorly defined. Using in vivo CRISPR-Cas9 screens, we identify serine/threonine kinase 40 (STK40) as a previously unrecognized regulator of immune evasion. Stk40 ablation synergizes with PD-1 blockade to induce tumor regression. Hepatocyte-specific Stk40 deletion abolishes tumorigenesis in hydrodynamic plasmid-driven HCC models. Mechanistically, STK40 scaffolds the COP1 ubiquitin ligase to promote interferon gamma receptor 1 (IFNGR1) degradation. Genetic depletion of Stk40 stabilizes IFNGR1, restoring tumor cell sensitivity to T cell cytotoxicity. Concurrently, Stk40 loss triggers autonomous GM-CSF secretion, enhancing the infiltration and activation of conventional type 1 dendritic cells, which promotes antigen cross-presentation and CD8+ T cell activation. Pharmacological inhibition of STK40 using LNP-siRNA, combined with PD-1 blockade, elicits potent anti-tumor responses across multiple cancer types. These findings establish STK40 as a dual-action therapeutic target to overcome resistance to anti-tumor immunity.

Supplementary table 6: Stress-focused drug screens AUC differences in SW-480 cells Area Under the Curve (AUC) from each compound of the stress-focused drug screen in the presence or absence of LB-100. Compounds are ranked by the difference in the AUC between LB-100-treated and untreated samples.

Figure S1: LB-100 engages stress-related, inflammatory response, and mitogenic signaling transcriptional programs in CRC cells (A) Dose-response assays show the effect of LB-100 in 7 CRC models. Cell viability was estimated by resazurin fluorescence after 5 days in the presence of the drug or DMSO control. The normalized values are plotted. (B) The heat map shows all “Hallmarks” and “KEGG” molecular signatures significantly enriched by LB-100 (4 µM) in both HT-29 and SW-480 cells in at least one of the addressed time points. Asterisks indicate significance level (* p-value <0.05, ** p-value <0.01, *** p-value <0.001)

Figure S10: Acquired resistance to the combination of LB-100 and Adavosertib suppress anchorage-independent and tumor growth in CRC models (A) Endpoint proliferation of HT-29 and SW-480 parental and resistant cells growing attached or in anchorage-independent conditions. Cells were plated in the same density on regular or cell-repellent culture plates in the absence of drugs and grown for 5-6 days. Proliferation was addressed by CellTiter-Glo 3D® and is expressed as fold change over T0. (B) Growth curves of the individual tumors from SW-480 parental and resistant cells measured 3 times per week. The dashed line indicates the 1500 mm3 ethical sacrifice.

Senescence contributes to aging and cancer-treatment-related decline, and is increasingly viewed as a therapeutic target. In this issue, Rajesh et al. and Wang et al. identify cyclin D–CDK4–CDK6 (CDK4/6) signaling as a regulator of the inflammatory senescence secretome, which suggests that approved CDK4/6 inhibitors may be repurposed as senomorphic therapies.

Figure S6: LB-100 and adavosertib induce histological response in orthotopic CRC PDXs (A) Representative Hematoxylin & Eosin (H&E) stainings at endpoint from PDOX2 and PDOX3 treated as indicated. Original magnification middle images: 15x, scale bar 1000 µm; right images: 200x. S indicates stroma, and the arrows point to the tumor-cell component. (B) Mice body weight variation of the 3 CRC PDXs across the experiments.

Figure S8: Acquired resistance to the combination of LB-100 and adavosertib suppressed malignant traits in CRC models (A) IncuCyte-based proliferation assays from HT-29 and SW-480 parental and resistant cells in the absence or presence of the combination (LB-100 4 µM + adavosertib 400 nM). (B) Chromosome counting and representative chromosome spreads from HT-29 and SW-480 parental and resistant cells. Nocodazole was added for 3h to block cells in mitosis. Cells were harvested by mitotic shake-off for spreading. Over 40 (HT-29 and HT-29-R) or 50 (SW-480 and SW-480-R) spreads were counted per cell line. Asterisks indicate significance level (**** p-value <0.0001) by two-tailed Mann-Whitney test. (C) Heatmaps showing the marker genes of each cluster from the scRNAseq analyses of HT-29 and SW-480 cells

Supplementary table 5: Stress-focused drug screens AUC differences in HT-29 cells Area Under the Curve (AUC) from each compound of the stress-focused drug screen in the presence or absence of LB-100. Compounds are ranked by the difference in the AUC between LB-100-treated and untreated samples.

Ever since immune checkpoint blockade showed activity in the treatment of cancer, the search has been on for combination regimens that make this therapy more effective. In this issue, Blagg and colleagues describe an unorthodox approach to increasing the effectiveness of cancer immunotherapy. See related article by Blagg et al., p. 1649.

Despite advances in targeted anti-cancer therapy in EGFR-mutant Non-Small Cell Lung Cancer (NSCLC), resistance is still almost inevitable. Identifying combination strategies that can treat drug-resistant tumors effectively is an urgent clinical need. Importantly, accurate mechanistic understanding of these combinations is essential for their optimal clinical development. Here, we aimed to identify stress-related vulnerabilities associated with resistance to EGFR inhibition in NSCLC cells. Using a stress-focused compound library, we screened gefitinib-resistant PC9 cells and identified barasertib, an Aurora kinase B inhibitor, as a vulnerability of drug-resistant cells. Surprisingly, this vulnerability was strictly dependent on the presence of EGFR inhibitor gefitinib, but independent of EGFR signaling inhibition. Mechanistic investigation, including a genome-wide CRISPR screen, revealed that the synergistic interaction between gefitinib and barasertib is mediated by ABCG2 (ATP Binding Cassette Subfamily G Member 2), a critical drug efflux pump. Our data indicate that gefitinib reduces barasertib efflux, thereby increasing its intracellular accumulation and target engagement at lower, non-toxic concentrations. This combination strategy was validated in multiple EGFR inhibitor-resistant models in vitro and in vivo using patient-derived xenograft models. Furthermore, we demonstrate that this concept is generalizable. By performing a large-scale drug repurposing screen, we identified multiple clinically approved drugs with diverse targets that act as ABCG2 inhibitors and sensitize cancer cells to barasertib. We conclude that the ability of a drug to act as an ABC transporter inhibitor – beyond its original target inhibition – represents an underappreciated mechanism with potential for increasing the efficacy of targeted anti-cancer agents. This approach provides a rational, highly translatable paradigm for designing drug combinations that can reduce effective dosage and thereby potentially revive drugs previously discontinued due to toxicity.

While many antagonistic antibodies are in routine clinical use, only a single agonistic antibody has received regulatory approval to date. While antibodies that activate Death Receptor 5 (DR5) were thought to have utility in the treatment of cancer by enhancing extrinsic apoptosis signaling, to date all clinical studies with these DR5 agonists have failed to deliver significant clinical benefit. A notable example of this is the DR5 agonistic antibody conatumumab. Here, we provide two potential avenues to improve the activity of DR5 agonists. First, we show that a dimeric IgA version (dIgA2) of the conatumumab antibody has a higher toxicity to cancer cells and a shorter half-life in vivo compared to the original IgG version of the antibody. Moreover, we conducted a genome-wide CRISPR screen to identify genes for which inactivation enhances the sensitivity of cancer cells to the dIgA2 DR5 antibody. We found that inhibition of mitochondrial protein translation synergizes with DR5 agonists. Consequently, antibiotics that inhibit mitochondrial protein translation also synergize with DR5 agonists. Finally, we show that these antibiotics activate the Integrated Stress Response (ISR) and upregulate DR5 through the EIF2a-ATF4 axis, which sensitizes cancer cells to DR5 activation. These data suggest a potential combination strategy for the effective use of DR5 agonistic antibodies.

PURPOSE:To determine the safety and efficacy of taselisib, a selective PI3K inhibitor, in combination with tamoxifen. PATIENTS AND METHODS:POSEIDON is a phase II, randomized, placebo-controlled trial conducted from June 2016 to March 2020. Eligible patients were refractory upon prior endocrine therapy. Prior treatment with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors and everolimus was allowed. Patients were randomized (1:1) to receive either taselisib (4 mg) + tamoxifen (20 mg) or placebo + tamoxifen. The primary endpoint of the trial was investigator-assessed progression-free survival (PFS) in the intention-to-treat (ITT) population (two-sided α 0.2, 90% power). Exploratory biomarker analysis with regards to prognosis and treatment resistance was conducted in circulating tumor (ct)DNA. RESULTS:POSEIDON met its primary endpoint, in which patients treated with taselisib + tamoxifen had improved PFS compared with patients treated with placebo + tamoxifen in the ITT population (median PFS 4.8 months vs. 3.2 months; stratified hazard ratio 0.69; 80% confidence interval, 0.49-0.98, P = 0.17). However, toxicity of taselisib was significant, with diarrhea (40% any grade) as the most common adverse event. Exploratory analyses indicated that high tumor fraction (TF) determined in ctDNA at baseline is associated with worse PFS and overall survival (P < 0.0001). CONCLUSIONS:Our findings suggest efficacy of PI3K inhibition + tamoxifen beyond second-line treatment and after prior targeted therapies, including CDK4/6 inhibition in metastatic HR+/HER2- breast cancer, although the magnitude of benefit did not outweigh the tolerability of this combination. Exploratory biomarker analysis indicates that TF determined in ctDNA differentiates patients based on prognosis and may help optimize patient selection for targeted treatment strategies.

Supplementary table 4: The composition of the stress-focused drug library Compounds comprising the stress-focused drug library with their respective targets.

Immunogenic cell death (ICD) converts the death of a tumor cell into an event sensed by the immune system. Recent studies show that distinct ICD modalities, including immunogenic apoptosis, pyroptosis, necroptosis, and hybrid forms such as PANoptosis, release defined sets of danger signals and cytokines that reshape the immune composition of the tumor microenvironment. In this review, we examine how ICD activates antitumor immunity and which immune cell subsets drive these responses. We also discuss how the benefits of ICD rely on its acute and transient nature, whereas prolonged or chronic exposure to the same inflammatory cues can ultimately dampen immune activation and promote oncogenesis. Finally, we outline the role of ICD and its clinical relevance in combination with immunotherapies.

Supplementary table 1: Cancer cell lines with oncogenic drivers The mutational status of the cell lines was compiled from the ATCC, Catalogue of Somatic Mutations in Cancer (COSMIC) and Cell Model Passport, Wellcome Trust Sanger Institute, and Depmap portal databases.

Figure S1: LB-100 engages stress-related, inflammatory response, and mitogenic signaling transcriptional programs in CRC cells (A) Dose-response assays show the effect of LB-100 in 7 CRC models. Cell viability was estimated by resazurin fluorescence after 5 days in the presence of the drug or DMSO control. The normalized values are plotted. (B) The heat map shows all “Hallmarks” and “KEGG” molecular signatures significantly enriched by LB-100 (4 µM) in both HT-29 and SW-480 cells in at least one of the addressed time points. Asterisks indicate significance level (* p-value <0.05, ** p-value <0.01, *** p-value <0.001)

Pancreatic ductal adenocarcinoma (PDAC) is often driven by KRAS mutations, but inhibitors targeting the most frequent KRAS substitutions in PDAC are not yet approved in the clinic. We previously discovered that KRAS-mutant PDAC is sensitive to the combination of SHP2 and ERK inhibitors, recently investigated in the Phase I/Ib clinical trial NCT04916236. Lately, RAS(ON) multi-selective inhibitors have entered clinical development, representing a promise for mono or combination therapies in PDAC. However, resistance may arise even for combination therapies. Here, we aimed at anticipating mechanisms of resistance to SHP2 plus ERK or RAS(ON) multi-selective inhibitors. We performed a genome-wide CRISPR-KO screening, followed by four follow-up focused screenings, leading to the identification of resistance mediators, which were further validated through functional genetic and pharmacological experiments, both in vitro and in vivo. Through unbiased CRISPR-based screenings, we identified mTOR and JUN hyperactivation as interconnected mechanisms that overcome MAPK suppression. Further investigation pointed at JUN as the most downstream resistance mediator, and indirect therapeutic target, using MAP2K4 inhibitors. Alterations in the PI3K/AKT/mTOR and JUN pathways can induce resistance to multiple combinations of MAPK pathway inhibitors, and may serve as biomarkers for sensitivity/resistance in clinical trials exploring such combinations in KRAS-mutant PDAC.

Figure S5: Combined toxicity of LB-100 and Adavosertib in PDAC and CCA models (A) and (B) Dose-response assays show the effect of LB-100 or Adavosertib in 4 PDAC and 4 CCA models, respectively. Cell viability was estimated by resazurin fluorescence after 5 days in the presence of the drug or DMSO control. The normalized values are plotted. (C) and (D) Long-term viability assays show 4 PDAC and 4 CCA models, respectively, treated with LB-100 or Adavosertib at the indicated concentrations. Treatments were refreshed every 2-3 days, and the cells were grown for 10-14 days before fixing, staining, and imaging. (E) and (F) IncuCyte-based proliferation assays from 4 PDAC and 4 CCA models, respectively, in the absence or presence of LB-100, Adavosertib, or the combination at the indicated concentrations.

Figure S3: High N-Myc levels sensitize neuroblastoma cells to LB-100, and PP2A knockdown sensitizes CRC cells to WEE1 inhibition (A) Western blots comparing N-Myc levels in isogenic neuroblastoma models. GAPDH was used as a loading control. (B) long-term viability assays compares the toxicity of LB-100 across these neuroblastoma models. LB-100 was refreshed every 2-3 days, and the cells were grown for 10 days before fixing, staining, and imaging. (C) Western blots show the knockdown of PPP2R1A in HT-29 and SW-480 cells. GAPDH was used as a loading control. (D) Dose-response assays show the effect of Adavosertib after PPP2R1A knockdown compared to a control shRNA in HT-29 and SW-480 cells. Cell viability was estimated by resazurin fluorescence after 5 days in the presence of the drug or DMSO control. The normalized values are plotted. (E) The heat map shows all “Hallmarks” and “KEGG” molecular signatures significantly enriched by LB-100 (4 µM), calyculin A (5 nM), or okadaic acid (10 nM) in HT-29 and SW-480 cells after 8 hours. Asterisks indicate significance level (* p-value <0.05, ** p-value <0.01, *** p-value <0.001).