Transcriptomic analysis of cutaneous squamous cell carcinoma reveals a multigene prognostic signature associated with metastasis.

BACKGROUND: Metastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumors at high risk of metastasis would have a significant impact on management. OBJECTIVE: To develop a robust and validated gene expression profile signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach. METHODS: Archival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 nonmetastasizing and 86 metastasizing) were collected retrospectively from four centers. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets. RESULTS: A 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk. LIMITATIONS: This was a retrospective 4-center study and larger prospective multicenter studies are now required. CONCLUSION: The 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC.

Preclinical Evaluation of AZ12601011 and AZ12799734, Inhibitors of Transforming Growth Factor ß Superfamily Type 1 Receptors.

The transforming growth factor ß (TGFß) superfamily includes TGFß, activins, inhibins, and bone morphogenetic proteins (BMPs). These extracellular ligands have essential roles in normal tissue homeostasis by coordinately regulating cell proliferation, differentiation, and migration. Aberrant signaling of superfamily members, however, is associated with fibrosis as well as tumorigenesis, cancer progression, metastasis, and drug-resistance mechanisms in a variety of cancer subtypes. Given their involvement in human disease, the identification of novel selective inhibitors of TGFß superfamily receptors is an attractive therapeutic approach. Seven mammalian type 1 receptors have been identified that have context-specific roles depending on the ligand and the complex formation with the type 2 receptor. Here, we characterize the biologic effects of two transforming growth factor ß receptor 1 (TGFBR1) kinase inhibitors designed to target TGFß signaling. AZ12601011 [2-(2-pyridinyl)-4-(1H-pyrrolo[3,2-c]pyridin-1-yl)-6,7-dihydro-5H-cyclopenta[d]pyrimidine]; structure previously undisclosed] and AZ12799734 [4-({4-[(2,6-dimethyl-3-pyridinyl)oxy]-2-pyridinyl}amino)benzenesulfonamide] (IC50 = 18 and 47 nM, respectively) were more effective inhibitors of TGFß-induced reporter activity than SB-431542 [4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide] (IC50 = 84 nM) and LY2157299 [4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide monohydrate]] (galunisertib) (IC50 = 380 nM). AZ12601011 inhibited phosphorylation of SMAD2 via the type 1 receptors activin A receptor type 1B (ALK4), TGFBR1, and activin A receptor type 1C (ALK7). AZ12799734, however, is a pan TGF/BMP inhibitor, inhibiting receptor-mediated phosphorylation of SMAD1 by activin A receptor type 1L, bone morphogenetic protein receptor type 1A, and bone morphogenetic protein receptor type 1B and phosphorylation of SMAD2 by ALK4, TGFBR1, and ALK7. AZ12601011 was highly effective at inhibiting basal and TGFß-induced migration of HaCaT keratinocytes and, furthermore, inhibited tumor growth and metastasis to the lungs in a 4T1 syngeneic orthotopic mammary tumor model. These inhibitors provide new reagents for investigating in vitro and in vivo pathogenic processes and the contribution of TGFß- and BMP-regulated signaling pathways to disease states.

A Unique Panel of Patient-Derived Cutaneous Squamous Cell Carcinoma Cell Lines Provides a Preclinical Pathway for Therapeutic Testing.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. METHODS: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. RESULTS: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. CONCLUSIONS: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.

The Promise of Genomics and the Development of Targeted Therapies for Cutaneous Squamous Cell Carcinoma.

Targeted therapies for melanoma and basal cell carcinoma have evolved from deciphering the molecular mechanisms involved in their tumorigenesis. Mutations in BRAF have led to clinical use of BRAF-inhibitors in advanced melanoma, and mutations in Hedgehog signaling to smoothened inhibitors in basal cell carcinoma. The development of tumor resistance to these treatments is leading to many new drug development initiatives and the exploration of multiple signalling pathways. Cutaneous squamous cell carcinoma continues to rise steeply in incidence with very limited therapeutic options for locally advanced or metastatic disease. New genetic technologies find significant levels of mutation in Notch gene family as well as other already recognized gene mutations, such as TP53. The mutational burden in cutaneous squamous cell carcinoma is massive, challenging the identification of driver genes and inhibiting translation from genomics to the clinic. Clinical experience with targeted therapies, such as epidermal growth factor receptor inhibitors, or immune modulatory drugs suggests that these agents may be of benefit to patients, while a more complete understanding of the mechanisms behind squamous cell carcinogenesis awaits further progress.

Transforming growth factor-ß directly induces p53-up-regulated modulator of apoptosis (PUMA) during the rapid induction of apoptosis in myc-driven B-cell lymphomas.

c-Myc transformed human Burkitt's lymphoma (BL) cells are highly sensitive to TGF-ß-induced apoptosis. Previously we demonstrated that TGF-ß-mediated cell death in BL cells is regulated via the mitochondrial intrinsic apoptosis pathway, which is dependent on the activation of BAX and/or BAK. TGF-ß directly induces transcription of the BH3-only protein BIK and represses expression of the pro-survival factor BCL-X(L) but has no effect on the direct BAX/BAK "activators" BIM or BID (tBID). Here we show that TGF-ß induces the BH3-only activator PUMA to aid induction of the intrinsic cell death pathway. TGF-ß also induced PUMA in normal germinal center CD77-positive centroblasts isolated from human tonsil tissue. PUMA was a direct TGF-ß target gene in B-cells, and we identify a putative Smad-binding region within the human PUMA promoter that recruits Smad3 and Smad4 in cells in response to TGF-ß signaling. Constitutive activity of the isolated Smad-binding region in luciferase reporter assays was dependent on Smad consensus sequences and was partially dependent on endogenous TGF-ß signaling and Smad4. Knockdown of PUMA in BL cells using lentiviral shRNA resulted in slower kinetics of the TGF-ß-mediated apoptotic response. Analysis of Eµ-Myc cell lines demonstrated that c-myc-driven murine lymphomas are also sensitive to TGF-ß-mediated apoptosis. Moreover, Puma(-/-) Eµ-Myc lines demonstrated significantly delayed kinetics of the apoptotic response when compared with wild type lymphomas. TGF-ß therefore induces a polygenic response in Myc-driven lymphomas involving transcription of PUMA, which is necessary for the rapid induction of cell death.

Involvement of Kindlin-1 in cutaneous squamous cell carcinoma.

Kindler syndrome (KS) is a rare genodermatosis resulting from loss-of-function mutations in FERMT1, the gene that encodes Kindlin-1. KS patients have a high propensity to develop aggressive and metastatic cutaneous squamous cell carcinoma (cSCC). Here we show in non-KS-associated patients that elevation of FERMT1 expression is increased in actinic keratoses compared to normal skin, with a further increase in cSCC supporting a pro-tumorigenic role in this population. In contrast, we show that loss of Kindlin-1 leads to increased SCC tumor growth in vivo and in 3D spheroids, which was associated with the development of a hypoxic tumor environment and increased glycolysis. The metalloproteinase Mmp13 was upregulated in Kindlin-1-depleted tumors, and increased expression of MMP13 was responsible for driving increased invasion of the Kindlin-1-depleted SCC cells. These results provide evidence that Kindlin-1 loss in SCC can promote invasion through the upregulation of MMP13, and offer novel insights into how Kindlin-1 loss leads to the development of a hypoxic environment that is permissive for tumor growth.

Development and external validation of a head and neck cancer risk prediction model.

BACKGROUND: Head and neck cancer (HNC) incidence is on the rise, often diagnosed at late stage and associated with poor prognoses. Risk prediction tools have a potential role in prevention and early detection. METHODS: The IARC-ARCAGE European case-control study was used as the model development dataset. A clinical HNC risk prediction model using behavioral and demographic predictors was developed via multivariable logistic regression analyses. The model was then externally validated in the UK Biobank cohort. Model performance was tested using discrimination and calibration metrics. RESULTS: 1926 HNC cases and 2043 controls were used for the development of the model. The development dataset model including sociodemographic, smoking, and alcohol variables had moderate discrimination, with an area under curve (AUC) value of 0.75 (95% CI, 0.74-0.77); the calibration slope (0.75) and tests were suggestive of good calibration. 384 616 UK Biobank participants (with 1177 HNC cases) were available for external validation of the model. Upon external validation, the model had an AUC of 0.62 (95% CI, 0.61-0.64). CONCLUSION: We developed and externally validated a HNC risk prediction model using the ARCAGE and UK Biobank studies, respectively. This model had moderate performance in the development population and acceptable performance in the validation dataset. Demographics and risk behaviors are strong predictors of HNC, and this model may be a helpful tool in primary dental care settings to promote prevention and determine recall intervals for dental examination. Future addition of HPV serology or genetic factors could further enhance individual risk prediction.

Inhibition of ATR opposes glioblastoma invasion through disruption of cytoskeletal networks and integrin internalization via macropinocytosis.

BACKGROUND: Glioblastomas have highly infiltrative growth patterns that contribute to recurrence and poor survival. Despite infiltration being a critical therapeutic target, no clinically useful therapies exist that counter glioblastoma invasion. Here, we report that inhibition of ataxia telangiectasia and Rad 3 related kinase (ATR) reduces invasion of glioblastoma cells through dysregulation of cytoskeletal networks and subsequent integrin trafficking. METHODS: Glioblastoma motility and invasion were assessed in vitro and in vivo in response to ATR inhibition (ATRi) and ATR overexpression using time-lapse microscopy, two orthotopic glioblastoma models, and intravital imaging. Disruption to cytoskeleton networks and endocytic processing were investigated via high-throughput, super-resolution and intravital imaging. RESULTS: High ATR expression was associated with significantly poorer survival in clinical datasets while histological, protein expression, and spatial transcriptomics using glioblastoma tumor specimens revealed higher ATR expression at infiltrative margins. Pharmacological inhibition with two different compounds and RNAi targeting of ATR opposed the invasion of glioblastoma, whereas overexpression of ATR drove migration. Subsequent investigation revealed that cytoskeletal dysregulation reduced macropinocytotic internalization of integrins at growth-cone-like structures, resulting in a tumor microtube retraction defect. The biological relevance and translational potential of these findings were confirmed using two orthotopic in vivo models of glioblastoma and intravital imaging. CONCLUSIONS: We demonstrate a novel role for ATR in determining invasion in glioblastoma cells and propose that pharmacological targeting of ATR could have far-reaching clinical benefits beyond radiosensitization.

Exogenous heparin binds and inhibits bone morphogenetic protein 6 biological activity.

PURPOSE: The purpose of this study was to explore the effect of heparin on bone morphogenetic protein 6 (BMP6) osteogenic activity. METHODS: Western blot analysis was used to confirm the binding of BMP6 to heparin and to observe its effect on BMP6 signaling in C2C12-BRE-Luc myoblasts. Real-time RT-PCR was performed for the expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) in C2C12 myoblasts treated with BMP6 and heparin for 72 hours. Rat ectopic bone formation assay was performed to explore the effect of heparin on BMP6 osteogenic activity. Two weeks following implantation the implants were analysed morphologically and histologically. A mouse osteoporotic model was used to test the ability of BMP6 to improve the bone quality in vivo in the presence of heparin, followed by DEXA and µCT analyses. Blood coagulation was tested in rats previously treated with BMP6. RESULTS: BMP6 specifically bound to heparin and induced Smad1/5/8 phosphorylation which was inhibited by heparin. After 48 and 72 hours of treatment, heparin inhibited BMP6-induced ALP and OC expression in C2C12 cells. Heparin dose dependently inhibited BMP6-induced new bone and cartilage formation in the rat ectopic bone formation assay, while in osteoporotic mice heparin inhibited the BMP6 potential to improve the bone quality as evidenced by decreased bone mineral density and trabecular bone parameters. Interestingly, BMP6 prevented the effect of heparin on the blood coagulation parameters. CONCLUSION: The interaction of BMP6 with heparin might contribute to the heparin-induced osteoporosis and blood coagulation.

Driver gene combinations dictate cutaneous squamous cell carcinoma disease continuum progression.

The molecular basis of disease progression from UV-induced precancerous actinic keratosis (AK) to malignant invasive cutaneous squamous cell carcinoma (cSCC) and potentially lethal metastatic disease remains unclear. DNA sequencing studies have revealed a massive mutational burden but have yet to illuminate mechanisms of disease progression. Here we perform RNAseq transcriptomic profiling of 110 patient samples representing normal sun-exposed skin, AK, primary and metastatic cSCC and reveal a disease continuum from a differentiated to a progenitor-like state. This is accompanied by the orchestrated suppression of master regulators of epidermal differentiation, dynamic modulation of the epidermal differentiation complex, remodelling of the immune landscape and an increase in the preponderance of tumour specific keratinocytes. Comparative systems analysis of human cSCC coupled with the generation of genetically engineered murine models reveal that combinatorial sequential inactivation of the tumour suppressor genes Tgfbr2, Trp53, and Notch1 coupled with activation of Ras signalling progressively drives cSCC progression along a differentiated to progenitor axis. Taken together we provide a comprehensive map of the cSCC disease continuum and reveal potentially actionable events that promote and accompany disease progression.

Risk prediction models for head and neck cancer: A rapid review.

Background: Cancer risk assessment models are used to support prevention and early detection. However, few models have been developed for head and neck cancer (HNC). Methods: A rapid review of Embase and MEDLINE identified n = 3045 articles. Following dual screening, n = 14 studies were included. Quality appraisal using the PROBAST (risk of bias) instrument was conducted, and a narrative synthesis was performed to identify the best performing models in terms of risk factors and designs. Results: Six of the 14 models were assessed as "high" quality. Of these, three had high predictive performance achieving area under curve values over 0.8 (0.87-0.89). The common features of these models were their inclusion of predictors carefully tailored to the target population/anatomical subsite and development with external validation. Conclusions: Some existing models do possess the potential to identify and stratify those at risk of HNC but there is scope for improvement.

Epithelial TGFß engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features.

The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.

TGFß signaling networks in ovarian cancer progression and plasticity.

Epithelial ovarian cancer (EOC) is a leading cause of cancer-related death in women. Late-stage diagnosis with significant tumor burden, accompanied by recurrence and chemotherapy resistance, contributes to this poor prognosis. These morbidities are known to be tied to events associated with epithelial-mesenchymal transition (EMT) in cancer. During EMT, localized tumor cells alter their polarity, cell-cell junctions, cell-matrix interactions, acquire motility and invasiveness and an exaggerated potential for metastatic spread. Key triggers for EMT include the Transforming Growth Factor-ß (TGFß) family of growth factors which are actively produced by a wide array of cell types within a specific tumor and metastatic environment. Although TGFß can act as either a tumor suppressor or promoter in cancer, TGFß exhibits its pro-tumorigenic functions at least in part via EMT. TGFß regulates EMT both at the transcriptional and post-transcriptional levels as outlined here. Despite recent advances in TGFß based therapeutics, limited progress has been seen for ovarian cancers that are in much need of new therapeutic strategies. Here, we summarize and discuss several recent insights into the underlying signaling mechanisms of the TGFß isoforms in EMT in the unique metastatic environment of EOCs and the current therapeutic interventions that may be relevant.

Clinicopathological Determinants of Recurrence Risk and Survival in Mucinous Ovarian Carcinoma.

Mucinous ovarian carcinoma (MOC) is a unique form of ovarian cancer. MOC typically presents at early stage but demonstrates intrinsic chemoresistance; treatment of advanced-stage and relapsed disease is therefore challenging. We harness a large retrospective MOC cohort to identify factors associated with recurrence risk and survival. A total of 151 MOC patients were included. The 5 year disease-specific survival (DSS) was 84.5%. Risk of subsequent recurrence after a disease-free period of 2 and 5 years was low (8.3% and 5.6% over the next 10 years). The majority of cases were FIGO stage I (35.6% IA, 43.0% IC). Multivariable analysis identified stage and pathological grade as independently associated with DSS (p < 0.001 and p < 0.001). Grade 1 stage I patients represented the majority of cases (53.0%) and demonstrated exceptional survival (10 year DSS 95.3%); survival was comparable between grade I stage IA and stage IC patients, and between grade I stage IC patients who did and did not receive adjuvant chemotherapy. At 5 years following diagnosis, the proportion of grade 1, 2 and 3 patients remaining disease free was 89.5%, 74.9% and 41.7%; the corresponding proportions for FIGO stage I, II and III/IV patients were 91.1%, 76.7% and 19.8%. Median post-relapse survival was 5.0 months. Most MOC patients present with low-grade early-stage disease and are at low risk of recurrence. New treatment options are urgently needed to improve survival following relapse, which is associated with extremely poor prognosis.

The Genomic Landscape of Actinic Keratosis.

Actinic keratoses (AKs) are lesions of epidermal keratinocyte dysplasia and are precursors for invasive cutaneous squamous cell carcinoma (cSCC). Identifying the specific genomic alterations driving the progression from normal skin to skin with AK to skin with invasive cSCC is challenging because of the massive UVR-induced mutational burden characteristic at all stages of this progression. In this study, we report the largest AK whole-exome sequencing study to date and perform a mutational signature and candidate driver gene analysis on these lesions. We demonstrate in 37 AKs from both immunosuppressed and immunocompetent patients that there are significant similarities between AKs and cSCC in terms of mutational burden, copy number alterations, mutational signatures, and patterns of driver gene mutations. We identify 44 significantly mutated AK driver genes and confirm that these genes are similarly altered in cSCC. We identify azathioprine mutational signature in all AKs from patients exposed to the drug, providing further evidence for its role in keratinocyte carcinogenesis. cSCCs differ from AKs in having higher levels of intrasample heterogeneity. Alterations in signaling pathways also differ, with immune-related signaling and TGFß signaling significantly more mutated in cSCC. Integrating our findings with independent gene expression datasets confirms that dysregulated TGFß signaling may represent an important event in AK‒cSCC progression.

Oncogenic BRAF, unrestrained by TGFß-receptor signalling, drives right-sided colonic tumorigenesis.

Right-sided (proximal) colorectal cancer (CRC) has a poor prognosis and a distinct mutational profile, characterized by oncogenic BRAF mutations and aberrations in mismatch repair and TGFß signalling. Here, we describe a mouse model of right-sided colon cancer driven by oncogenic BRAF and loss of epithelial TGFß-receptor signalling. The proximal colonic tumours that develop in this model exhibit a foetal-like progenitor phenotype (Ly6a/Sca1+) and, importantly, lack expression of Lgr5 and its associated intestinal stem cell signature. These features are recapitulated in human BRAF-mutant, right-sided CRCs and represent fundamental differences between left- and right-sided disease. Microbial-driven inflammation supports the initiation and progression of these tumours with foetal-like characteristics, consistent with their predilection for the microbe-rich right colon and their antibiotic sensitivity. While MAPK-pathway activating mutations drive this foetal-like signature via ERK-dependent activation of the transcriptional coactivator YAP, the same foetal-like transcriptional programs are also initiated by inflammation in a MAPK-independent manner. Importantly, in both contexts, epithelial TGFß-receptor signalling is instrumental in suppressing the tumorigenic potential of these foetal-like progenitor cells.

Regulation of cell proliferation and apoptosis in neuroblastoma cells by ccp1, a FGF2 downstream gene.

BACKGROUND: Coiled-coil domain containing 115 (Ccdc115) or coiled coil protein-1 (ccp1) was previously identified as a downstream gene of fibroblast growth factor 2 (FGF2) highly expressed in embryonic and adult brain. However, its function has not been characterised to date. Here we hypothesized that ccp1 may be a downstream effecter of FGF2, promoting cell proliferation and protecting from apoptosis. METHODS: Forced ccp1 expression in mouse embryonic fibroblast (MEF) and neuroblastoma SK-N-SH cell line, as well as down-regulation of ccp1 expression by siRNA in NIH3T3, was used to characterize the role of ccp1. RESULTS: Ccp1 over-expression increased cell proliferation, whereas down-regulation of ccp1 expression reduced it. Ccp1 was able to increase cell proliferation in the absence of serum. Furthermore, ccp1 reduced apoptosis upon withdrawal of serum in SK-N-SH. The mitogen-activated protein kinase (MAPK) or ERK Kinase (MEK) inhibitor, U0126, only partially inhibited the ccp1-dependent BrdU incorporation, indicating that other signaling pathway may be involved in ccp1-induced cell proliferation. Induction of Sprouty (SPRY) upon FGF2 treatment was accelerated in ccp1 over-expressing cells. CONCLUSIONS: All together, the results showed that ccp1 regulates cell number by promoting proliferation and suppressing cell death. FGF2 was shown to enhance the effects of ccp1, however, it is likely that other mitogenic factors present in the serum can also enhance the effects. Whether these effects are mediated by FGF2 influencing the ccp1 function or by increasing the ccp1 expression level is still unclear. At least some of the proliferative regulation by ccp1 is mediated by MAPK, however other signaling pathways are likely to be involved.

Multifaceted transforming growth factor-beta (TGFß) signalling in glioblastoma.

Glioblastoma (GBM) is an aggressive and devastating primary brain cancer which responds very poorly to treatment. The average survival time of patients is only 14-15 months from diagnosis so there is a clear and unmet need for the development of novel targeted therapies to improve patient outcomes. The multifunctional cytokine TGFß plays fundamental roles in development, adult tissue homeostasis, tissue wound repair and immune responses. Dysfunction of TGFß signalling has been implicated in both the development and progression of many tumour types including GBM, thereby potentially providing an actionable target for its treatment. This review will examine TGFß signalling mechanisms and their role in the development and progression of GBM. The targeting of TGFß signalling using a variety of approaches including the TGFß binding protein Decorin will be highlighted as attractive therapeutic strategies.

RIPK1-mediated immunogenic cell death promotes anti-tumour immunity against soft-tissue sarcoma.

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti-tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre-clinical application of an in vivo treatment protocol for soft-tissue sarcoma that directly engages RIPK1-mediated immunogenic cell death. We find that RIPK1-mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1-induced cell death in patients with advanced disease at limb extremities.