Copy Number Amplification Research Articles

Functional regulations between genetic alteration-driven genes and drug target genes acting as prognostic biomarkers in breast cancer

Differences in genetic molecular features including mutation, copy number alterations and DNA methylation, can explain interindividual variability in response to anti-cancer drugs in cancer patients. However, identifying genetic alteration-driven genes and characterizing their functional mechanisms in different cancer types are still major challenges for cancer studies. Here, we systematically identified functional regulations between genetic alteration-driven genes and drug target genes and their potential prognostic roles in breast cancer. We identified two mutation and copy number-driven gene pairs (PARP1-ACSL1 and PARP1-SRD5A3), three DNA methylation-driven gene pairs (PRLR-CDKN1C, PRLR-PODXL2 and PRLR-SRD5A3), six gene pairs between mutation-driven genes and drug target genes (SLC19A1-SLC47A2, SLC19A1-SRD5A3, AKR1C3-SLC19A1, ABCB1-SRD5A3, NR3C2-SRD5A3 and AKR1C3-SRD5A3), and four copy number-driven gene pairs (ADIPOR2-SRD5A3, CASP12-SRD5A3, SLC39A11-SRD5A3 and GALNT2-SRD5A3) that all served as prognostic biomarkers of breast cancer. In particular, RARP1 was found to be upregulated by simultaneous copy number amplification and gene mutation. Copy number deletion and downregulated expression of ACSL1 and upregulation of SRD5A3 both were observed in breast cancers. Moreover, copy number deletion of ACSL1 was associated with increased resistance to PARP inhibitors. PARP1-ACSL1 pair significantly correlated with poor overall survival in breast cancer owing to the suppression of the MAPK, mTOR and NF-kB signaling pathways, which induces apoptosis, autophagy and prevents inflammatory processes. Loss of SRD5A3 expression was also associated with increased sensitivity to PARP inhibitors. The PARP1-SRD5A3 pair significantly correlated with poor overall survival in breast cancer through regulating androgen receptors to induce cell proliferation. These results demonstrate that genetic alteration-driven gene pairs might serve as potential biomarkers for the prognosis of breast cancer and facilitate the identification of combination therapeutic targets for breast cancers.

Extra Spindle Pole Bodies-Like 1 Serves as a Prognostic Biomarker and Promotes Lung Adenocarcinoma Metastasis

Extra spindle pole bodies-like 1 (ESPL1), a cysteine endopeptidase, plays a vital role in chromosome inheritance. However, the association of ESPL1 with prognosis and immune infiltration in lung adenocarcinoma (LUAD) has not yet been explored. Here, we analyzed the expression level, prognostic values, diagnostic value, and immune infiltration level in LUAD using various databases. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) assays were used to detect the expression of ESPL1 in LUAD tissues and cell lines. In this study, we found that ESPL1 was upregulated in LUAD and a higher expression of ESPL1 was correlated with unfavorable prognosis in LUAD. Meanwhile, Cox hazard regression analysis results suggested that ESPL1 may be an independent prognostic factor for LUAD. Moreover, we demonstrated that ESPL1 expression was significantly correlated with immune infiltration of Th2 and dendritic cells in LUAD. We also confirmed that DNA copy number amplification and DNA hypo-methylation were positively correlated with ESPL1 expression in LUAD. Additionally, DNA copy number amplification was significantly associated with adverse clinical outcomes in LUAD. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) confirmed that ESPL1 was mainly involved in the DNA replication and glycolysis signaling pathway. Finally, we revealed that ESPL1 was highly expressed in LUAD tissues and cell lines. Knockdown of ESPL1 significantly inhibited cell migration and the invasion abilities of LUAD. Our study comprehensively confirmed that ESPL1 expression may serve as a novel prognostic biomarker for both the clinical outcome and immune cell infiltration in LUAD.

MPZL1 upregulation promotes tumor metastasis and correlates with unfavorable prognosis in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), accounting for 85% of all lung cancer, is one of the leading causes of cancer-related death worldwide. Previously, we demonstrated that MPZL1 gene amplification promotes liver cancer metastasis through activating Src/Cortactin pathway. However, the clinical relevance and biological roles of the MPZL1 gene in lung cancer are still unknown. Here, we found that MPZL1 expression upregulates in human NSCLC, which is partly due to the copy number amplification of this gene. Next, we observed that high MPZL1 expression correlates with unfavorable prognosis of NSCLC patients. We further demonstrated that ectopic MPZL1 overexpression promotes in vitro migratory but not proliferation and colony formation abilities of both H1299 and H460 cells. Consistently, we found that MPZL1 knockdown impairs the migratory abilities of A549 and H1775 cells. Moreover, we found that MPZL1 knockdown inhibits in vivo metastatic but not tumor growth abilities of the A549 cells. Additionally, a total of 297 differentially expressed genes (DEGs) were identified by RNA sequencing in A549 cells upon MPZL1 knockdown. By integrative analysis of DEGs regulated by MPZL1 in A549 cells and human NSCLC tissues, we revealed that COL11A1 is the potential effector gene that positively regulated by MPZL1 and correlates with poor prognosis of NSCLC patients. In conclusion, our work indicates that one of the mechanisms by which MPZL1 promotes NSCLC metastasis is through upregulating the COL11A1, and MPZL1 can be used as a biomarker to predict the prognosis of NSCLC patients.

Gene amplification-driven RNA methyltransferase KIAA1429 promotes tumorigenesis by regulating BTG2 via m6A-YTHDF2-dependent in lung adenocarcinoma.

BackgroundEpigenetic alterations have been shown to contribute immensely to human carcinogenesis. Dynamic and reversible N6‐methyladenosine (m6A) RNA modification regulates gene expression and cell fate. However, the reasons for activation of KIAA1429 (also known as VIRMA, an RNA methyltransferase) and its underlying mechanism in lung adenocarcinoma (LUAD) remain largely unexplored. In this study, we aimed to clarify the oncogenic role of KIAA1429 in the tumorigenesis of LUAD.MethodsWhole‐genome sequencing and transcriptome sequencing of LUAD data were used to analyze the gene amplification of RNA methyltransferase. The in vitro and in vivo functions of KIAA1429 were investigated. Transcriptome sequencing, methylated RNA immunoprecipitation sequencing (MeRIP‐seq), m6A dot blot assays and RNA immunoprecipitation (RIP) were performed to confirm the modified gene mediated by KIAA1429. RNA stability assays were used to detect the half‐life of the target gene.ResultsCopy number amplification drove higher expression of KIAA1429 in LUAD, which was correlated with poor overall survival. Manipulating the expression of KIAA1429 could regulate the proliferation and metastasis of LUAD. Mechanistically, the target genes of KIAA1429‐mediated m6A modification were confirmed by transcriptome sequencing and MeRIP‐seq assays. We also revealed that KIAA1429 could regulate BTG2 expression in an m6A‐dependent manner. Knockdown of KIAA1429 significantly decreased the m6A levels of BTG2 mRNA, leading to enhanced YTH m6A RNA binding protein 2 (YTHDF2, the m6A “reader”)‐dependent BTG2 mRNA stability and promoted the expression of BTG2; thus, participating in the tumorigenesis of LUAD.ConclusionsOur data revealed the activation mechanism and important role of KIAA1429 in LUAD tumorigenesis, which may provide a novel view on the targeted molecular therapy of LUAD.

PRKCI Mediates Radiosensitivity via the Hedgehog/GLI1 Pathway in Cervical Cancer.

ObjectiveInsensitivity to radiotherapy accounts for the majority of therapeutic failures in cervical cancer (CC) patients who undergo radical radiotherapy. We aimed to elucidate the molecular mechanisms underlying radiosensitivity to identify methods to improve the overall 5-year survival rate. The atypical protein kinase C iota (aPKCι) gene PRKCI exhibits tumor-specific copy number amplification (CNA) in CC. We investigated how PRKCI decreases radiosensitivity in CC and assessed the interplay between PRKCI and the Hedgehog (Hh)/GLI1 pathway in the present research.MethodsThe biological functions of PRKCI in CC radiosensitivity were explored through immunohistochemistry, colony formation, Cell Counting Kit-8 (CCK-8), cell cycle, apoptosis assays, and xenograft models. qRT-PCR, Western blotting analysis, and immunofluorescence assays were utilized to evaluate the interplay between PRKCI and the Hh/GLI1 pathway and its mechanism in PRKCI-decreased radiosensitivity in CC. Furthermore, the effect of auranofin (AF), a selective inhibitor of PKCι, on CC cells was explored through biochemical assays in vitro and in vivo.ResultsWe found that high PRKCI expression was responsible for decreased survival in CC. PRKCI was intimately associated with radiation-triggered alterations in proliferation, the cell cycle, apoptosis, and xenograft growth. The Hh/GLI1 pathway was activated when PRKCI expression was altered. PRKCI functions downstream of the Hh/GLI1 pathway to phosphorylate and activate the transcription factor GLI1. AF acts as a radiosensitizer and showed biological effects in vitro and in vivo.ConclusionsPRKCI is a therapeutic target for regulating radiosensitivity in CC. This molecule regulates radiosensitivity by modulating GLI1 relocalization and phosphorylation in CC via the Hh/GLI1 pathway.

Abstract 1103: MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC

Abstract ROS1 TKIs entrectinib and crizotinib have significantly improved outcomes for ROS1+ lung adenocarcinoma patients. However, drug resistance inevitably develops, leading to disease progression. Approximately 1/3 of patients resistant to ROS1 TKIs demonstrate ROS1 kinase domain (KD) mutations (Dziadziuszko et al. ESMO 2019), but the mechanism of resistance in most patients is unknown or poorly characterized. To model and characterize acquired resistance to ROS1 TKIs, we used patient-derived cell line CUTO28 (TPM3-ROS1) to generate an entrectinib-resistant derivative (CUTO28-ER) in vitro. We utilized several techniques to probe the mechanisms driving resistance: DNA and RNA sequencing (seq), fluorescence in-situ hybridization (FISH), cell proliferation assays, and western blotting. DNA seq of the ROS1 KD in CUTO28-ER failed to reveal mutations. CUTO28-ER cells displayed sensitivity to MET-selective TKIs in proliferation assays, and MAPK and AKT pathways were inhibited only with MET-selective TKI. RNA seq and western blot showed MET overexpression, and interphase FISH confirmed MET amplification compared to parental cells (MET:CEP7 ratio 4.2 vs 1.0). We substantiated in vitro findings in patient tissue, utilizing tumor samples at 2 different points of a single CD74-ROS1 NSCLC patient’s tumor progression. The first tumor sample did not display MET amplification while on ROS1 TKI (MET:CEP7 ratio 0.9, 3.4 copies of MET). However, the second tumor sample collected 5 months later displayed a MET:CEP7 ratio of 2.5 (9.5 copies of MET), indicating progression on ROS1 TKI was likely MET-driven. To determine prevalence of MET amplification in entrectinib-resistant ROS1+ NSCLC, we analyzed circulating tumor DNA (ctDNA) by FoundationOne Liquid CDx from patients with ROS1+ NSCLC in the STARTRK-2 entrectinib trial. Of 105 ROS1+ NSCLC patients with ctDNA analysis both at enrollment and progression, 2 (1.9%) displayed copy number amplification (CNA) of MET. Of these, 1 patient had no detectible CNA at study baseline but MET CNA gain by day 166 of entrectinib therapy and the other had detectable MET CNA gains both at baseline and at progression only 28 days later. Both received 3 lines of therapy prior to entrectinib, none of which targeted ROS1 or MET. In conclusion, we demonstrated MET gene amplification as a potential mechanism of resistance to ROS1 TKI entrectinib. The prevalence of MET amplification at resistance was 2 of 105, but may be greater than was detected in STARTRK-2 due to the sensitivity of ctDNA assays and challenges of measuring CNA in ctDNA. Utilization of the ROS1/MET TKI crizotinib or combination of entrectinib with capmatinib should be explored in patients with ROS1+ NSCLC that display MET CNA to overcome MET-driven resistance following entrectinib. Citation Format: Logan C. Tyler, Anh T. Le, Hala Nijmeh, Liming Bao, Timothy R. Wilson, Brian Simmons, David Chen, Robert C. Doebele. MET gene amplification is a mechanism of resistance to entrectinib in ROS1+ NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1103.

Abstract 6376: Effect of MET tyrosine kinase inhibitors on MET amplified gastro-esophageal cancer cells

Abstract Background: Many studies have suggested that MET protein overexpression or MET amplification plays a critical role in the progression of gastro-esophageal cancer (GC) and negatively affects survival in patients with GC. However, the criteria used to define overexpression of MET protein have differed among many studies, and in contrast to the initial phase II studies, the phase III trials failed to show any clinical benefit from anti-MET therapies in GCs, even in patients with MET-positive disease. Several studies have used consistent criteria to define MET amplification on FISH, and recent MET inhibitor trials for MET amplified cancers have been successful. Furthermore, hyperactivation of MET pathway are known to reduce the growth-inhibitory effect of trastuzumab in HER2 positive GC. Currently, there is no FDA approved MET inhibitors for GC. Capmatinib and savolitinib, which are selective MET inhibitors, has been evaluated for various MET positive cancers. The aim of this study was to test these MET inhibitors on MET amplified gastro-esophageal cancer (GC) cells. Material and Methods: Among a total of 37 GC cells in our lab, the following cells were positive for MET by copy number amplification (CNV > 10): SNU-620, ESO51, MKN-45, SNU-5 and OE-33. We assessed cytotoxic response of these GC cells to capmatinib or savolitinib alone by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay and anchorage independent growth in soft agar. Western blots were performed to assess the cellular activity of capmatinib or savolitinib in MET signal pathway. Xenograft studies were performed to evaluate in vivo therapeutic efficacy of savolitinib with MKN-45 cells. Results: Savolitinib and capmatinib showed anti-proliferative activity against MET amplified GC cell lines in dose-dependent manner. There was no inhibitory effect on GC cells without MET amplification, such as SNU-216 and SNU-1750. Savolitinib inhibited phosphorylation of MET and down-stream signaling pathway, such as AKT or ERK, in MET amplified GC cells. We observed that treatment with savolitinib induced a significant decrease in the number of colonies formed in soft agar. In addition, Savolitinib exhibited dose-responsive anti-tumor efficacy in xenograft model with MKN-45 GC cells. Furthermore, the combination of Trastuzumab and capmatinib exhibited enhanced inhibition of AKT and ERK activation in OE-33 cells, which is positive for both HER2 and MET, and resistant to trastuzumab alone. Conclusion: Collectively, these data support that targeting MET with Savolitinib and capmatinib efficiently suppresses the growth of MET amplified GC cells. Moreover, MET inhibitor could synergize with trastuzumab to induce the therapeutic effect on the HER2 and MET amplified GC cells. Further studies are warranted to test this concept in clinical setting. Citation Format: Jin-Soo Kim, Sungyoul Hong, Mi Young Kim. Effect of MET tyrosine kinase inhibitors on MET amplified gastro-esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6376.

Abstract 5790: Genomic landscape of circulating tumor DNA alterations in patients with paraganglioma and pheochromocytoma

Abstract Background: Paragangliomas (PGLs) and Pheochromocytomas (PCCs) are rare neuroendocrine tumors (NETs). PGLs arise from chromaffin cells in the ganglia of the autonomic nervous system, while PCCs arise from chromaffin cells in the adrenal medulla. The genomic landscapes of PGLs and PCCs have not been well studied. Thus, the aim of this study is to report differences of mutational occurrences and confirm the feasibility of next generation sequencing (NGS) testing circulating tumor DNA (ctDNA) in patients with PGL and PCC. Patients and Methods: Molecular alterations in 46 plasma samples were evaluated using a commercially available ctDNA assay (Guardant360® or Guardant360® CDx) across multiple institutions from 2016-2021. The tests detect single nucleotide variants and indels in 54-83 genes with copy number amplifications and fusions in selected genes. Results: Of the 24 PGL patients, there was a median age of 55 (range:28-78) years and 14 (58%) patients were male. Of the 22 PCC patients, there was a median age of 56 (range:28-86) years and 12 (54.5%) patients were male. Genetic alterations were identified in 16 (67%) PGL and 17 (77%) PCC patients. Of the 16 PGL samples with alterations, TP53 associated genes were most commonly altered (44%), followed by ATM (25%), FGFR2 (19%), APC (13%), BRAF (13%), BRCA1 (13%), CCND2 (13%), FGFR3 (13%), IDH2 (13%), KRAS (13%), PDGFRA (13%), RB1 (13%), TERT(13%), ALK (6%), ARID1A (6%), BRCA2 (6%), CCND1 (6%), CDK6 (6%), CDK12 (6%), EGFR (6%), FGFR1 (6%), KIT (6%), MET (6%), NF1 (6%), NRAS (6%), PIK3CA (6%), PTEN (6%), and ROS1 (6%). Of the 17 PCC samples with alterations, TP53 was most commonly altered (41%), followed by ATM (35%), NF1 (24%), FGFR1 (18%), APC (13%), EGFR (12%), MET (12%), MYC (12%), NOTCH1 (12%), PDGFRA (12%), TSC1 (12%), AR (6%), ARID1A(13%), BRAF (6%), BRCA1 (6%), BRCA2 (6%), CCND1 (6%), CDK6 (6%), CHEK2 (6%), ERBB2 (6%), EZH2 (6%), FGFR2 (6%), IDH2 (6%), KIT (6%), KRAS (6%), NRAS (6%), NTRK1 (6%), NTRK2 (6%), and VHL (6%). 21% of PGL and 41% of PCC patients reported alterations associated with therapies approved in other indications including genes in the MAPK pathway and the homologous recombination repair pathway. Conclusions: Liquid biopsy is a non-invasive method that can provide personalized treatment options for patients. In this study, we found that evaluation of ctDNA was feasible among individuals with advanced PGL and PCC. We report a high rate of homologous recombinant deficiencies that are in need of evaluation in future Percentage refers to frequency of patients with an alteration detected. Citation Format: Lana Khalil, Jill Tsai, Leylah Drusbosky, Olatunji Alese, Maria Diab, Mehmet Akce, Christina Wu, Olumide Babjide Gbolahan, Bassel El-Rayes, Walid Shaib. Genomic landscape of circulating tumor DNA alterations in patients with paraganglioma and pheochromocytoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5790.

Abstract 6058: The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme

Abstract Purpose: Glioblastoma multiforme (GBM) remains a fatal disease despite aggressive treatment approaches. Post-surgical radiation therapy (RT) is the only treatment that significantly improves survival of GBM patients but recurrence is inevitable. Elucidating the mechanisms of resistance to RT could transform GBM patient outcomes. The purpose of this study was to elucidate the effect of radiation on the serine synthesis pathway (SSP) and the role of SSP in boosting antioxidant defenses in irradiated GBM cells and promoting radioresistance. Methods: We compared copy number alterations (CNA) and amplification of SSP enzymes (PHGDH, PSAT1, PSPH, SHMT1/2) in low-grade gliomas (LGG, n = 511) and GBM (n = 575) via TCGA pan-cancer analysis. In vitro HPLC/MS-based metabolomics assays were used to study the changes in overall metabolite levels induced by radiation in patient-derived GBM specimens. Targeted metabolomics with 13C-labeled glucose was used for tracking the fate of glucose towards SSP intermediates and antioxidant species. Gene expression levels after radiation were measured via molecular biology approaches. De novo SSP was targeted via pharmacological inhibition of PHGDH. Modified clonogenic, sphere forming assays (SFA) on gliomasphere cultures were used to determine radiosensitivity. Dependence on extracellular serine/glycine was determined via serine/glycine depletion experiments. Results: The TCGA analysis revealed increased CNAs in all the de novo SSP enzymes in GBM tumors relative to the LGGs. 82% of GBM tumors had CNAs or amplification of the PSPH gene, which codes for the ultimate enzyme in serine synthesis. In vitro metabolomics assays revealed that radiation increases the levels of serine, glycine and cystine in gliomaspheres, and this is accompanied by elevated levels of reduced glutathione (GSH). Targeted glucose metabolomics revealed radiation-induced upregulation of the de novoSSP, with a concomitant increase in gene expression of SSP enzymes. Downstream of the SSP, metabolomics data show that irradiated GBM cells elevate nucleotide and precursor levels, likely to support DNA damage repair mechanisms after radiation. The inhibition of PHGDH, the first rate-limiting enzyme in de novo SSP, significantly reduced GBM cell survival in vitro. Extracellular serine/glycine appeared to also be important contributors to radiation survival. Gene expression of SLC1A4, an amino acid transporter, was upregulated by radiation, while depletion of extracellular serine/glycine led to significant decreased survival in in vitro SFAs. Conclusion: Here we provide evidence that the de novo serine synthesis pathway is an important contributor to the metabolic reprogramming of GBM cells after radiation that promotes radiation resistance. These data call for further investigations to evaluate the radiosensitizing potential of targeting de novo SSP in GBM. Citation Format: Justine Bailleul, Yangjingyi Ruan, Erina Vlashi. The serine synthesis pathway contributes to the radiation-induced metabolic plasticity in glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6058.

Abstract 45: Screening somatic genomic alteration for rare adult cancers: the SPECTA-ARCAGEN project of the EORTC/EURACAN

Abstract Introduction: Rare cancers are defined as having an incidence lower than 6 per 100,000 new cases per year. Despite advances in treatments and personalised medicine for cancer, rare cancers are often left behind due to limited knowledge regarding molecular characterization, heterogeneity of the population and poor access to screening platform or interventional trials.To bridge this gap, EORTC, in collaboration with EURACAN, has developed a project within the EORTC-SPECTA platform to assess the molecular landscape of rare cancers in Europe and the actionability of the different findings. Material and Methods: The pan-European Arcagen project is prospectively analyzing tumor material or blood of patient with rare cancers, using Foundation Medicine NGS panels, while collecting baseline and follow-up clinical data. Results: At the cut-off date of 10/10/2021, 629 patients were evaluable (baseline clinical data and successful molecular profiling performed by a Foundation Medicine test), molecular alterations were found in 620 patients, theoretically actionable ones in 421 patients. Importantly, approved treatment in the right tumor type was proposed in 58 patients (9.2%). Longer follow-up is needed to assess treatment adaptation and response. Most analyses were performed based on FFPE material (451 out of 629), but in case of low sample quality, low content of tumor cells, or old sample, rescue analysis was performed on liquid biopsy (177 out of 629). The main reason for switching to liquid biopsy was due to low FFPE tissue quality (90 out of 178). This rescue pathway was used on average in 28.1% of cases, ranging from 14.7% in salivary gland cancers to 48% for Cancer of Unknown Primary. Comparing per tumor type the molecular profile obtained either with FFPE or blood analysis, we noticed that SNVs are nicely detected in both compartments. However, copy number loss and amplifications are mainly detected when FFPE material is used. This is especially true for copy number loss of CDKN2A/B (cholangiocarcinoma, mesothelioma and salivary gland), or MTAP (mesothelioma), and MYC amplification (brain and rare skin cancer).As expected according to other datasets, some clonal hematopoiesis of indeterminate potential (CHIP) was observed on liquid biopsy, with DNMT3A being in the top ten mutations detected in the blood of patients with salivary gland tumors, endocrine tumors, Merkel cell carcinoma and uveal melanoma. Conclusion: This project highlights the clear need for more collaboration, research and clinical trials on rare cancers, to improve the treatment accessibility for targeted therapy for these patients. The use of liquid biopsy, despite its limitations, is an ideal candidate to gain understanding of a patient tumor molecular profile, while being non-invasive and easily performed longitudinally. Citation Format: Marie Morfouace, Aleksandra Stevovic, Julio Oliveira, Nicolas Penel, Julien Péron, Paolo G. Casali, Michael J. SECKL, Jourik A. GIETEMA, Wouter de HERDER, Lucjan WYRWICZ, Christelle de la FOUCHARDIERE, Lisa LICITRA, Nicolas GIRARD, Sophie PIPERNO-NEUMANN, Ellen KAPITEIJN, Ahmed IDBAIH, Jean-Yves Blay. Screening somatic genomic alteration for rare adult cancers: the SPECTA-ARCAGEN project of the EORTC/EURACAN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 45.

Abstract 3989: DSTYK inhibition sensitizes lung cancer cells to immunotherapy by collapsing cytoprotective autophagy and destroying mitochondrial wellness

Abstract Lung cancer is the leading cause of cancer-related death worldwide. The incidence and mortality associated to lung cancer is a major challenge of the Public Health Policy in advanced societies. Although the great effort has been done in NSCLC therapeutic strategies, there are still many NSCLC patients who cannot benefit either from molecular targeted or immune related therapies. Here, we identified DSTYK, a dual serine/threonine and tyrosine non-receptor protein kinase, as a novel candidate with a crucial role in NSCLC. According to data obtained from the TCGA, genomic alterations (mainly copy number alterations and mutations) of DSTYK are present in 40% of lung cancer patients, from which 4.7% LUAD and 3.4% LUSC correspond to copy number (CN) amplification (more than 3 genomic copies). Besides, DSTYK amplification is found to be a poor prognostic factor in lung cancer patients. Accordingly, high expression of DSTYK predicted both, a lower overall survival (OS) and poorer progression free survival (PFS). We next depleted DSTYK expression in both murine and human NSCLC cell lines. DSTYK abrogation prevented cytoprotective autophagy via mTOR regulation, accumulating LC3 and p-P62. In addition, DSTYK inhibition impaired lysosomal biogenesis and maturation, which contributed to dysfunctional autophagy. Furthermore, DSTYK depleted cells showed increased ROS levels caused by depolarized and damaged mitochondria. All together, these results demonstrate that knockdown of DSTYK promoted a dramatic reduction of mitophagy.As it has been shown that autophagy in cancer cells can limit antitumor immunity, we evaluated the role of immune-mediated tumor cell killing in an altered DSTYK context. We found that inhibition of DSTYK sensitized lung cancer cells to T cell–mediated cytotoxicity, via TNF-ɑ. More importantly, immunocompetent mouse models transplanted with DSTYK-inhibited tumors were sensitized to ɑnti-PD1 treatment.Finally, in a cohort of NSCLC patients that underwent immunotherapy (n=40), DSTYK amplification predicted resistance to the treatment, showing poorer PFS rates. The time to progression for DSTYK amplified patients was on average, around 50% faster than that for non-DSTYK altered patients, diminishing from 232 to 114 months.In conclusion, our results support that DSTYK is a new target and that its inhibition could be the base of new therapies, which could benefit a subgroup of NSCLC patients showing genetic amplification, both at early (adjuvant) or late (single or combined treatment) stages of the disease. Citation Format: Karmele Valencia, Mirari Echepare, Andrea Pasquier, Alvaro Teijeira, Cristina Bertolo, Cristina Sainz, Graziella Bosco, Roman K. Thomas, Jackeline Agorreta, Jose Maria Lopez-Picazo, Joan Frigola, Ramon Amat, Alfonso Calvo, Enriqueta Felip, Luis M. Montuenga. DSTYK inhibition sensitizes lung cancer cells to immunotherapy by collapsing cytoprotective autophagy and destroying mitochondrial wellness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3989.

Abstract 1520: Replication stress and the inability to repair damaged DNA, the potential “Achilles' heel” of ecDNA+ tumor cells

Abstract Replication stress (RS) is a primary source of genomic instability, tumorigenesis, and cancer progression. RS is defined as an uncoupling of the replicative helicase and DNA polymerase, resulting in long stretches of fragile single stranded DNA (ssDNA) that is prone to damage. Excessive RS can result in replication catastrophe and cell death, which could be leveraged as a therapeutic strategy to treat high-RS cancers. While overexpression of certain oncogenes has been implicated as a driver of RS in cancer, the precise cellular conditions that result in RS have been difficult to predict. Here we demonstrate that high copy number amplification of genes on non-native extrachromosomal DNA (ecDNA) is associated with elevated RS in comparison to the same amplification on linear chromosomes. The ecDNA bearing cells display heightened basal levels of phosphorylated RPA, a hallmark of ssDNA, and decreased replication fork velocities. Consistent with this finding, the ecDNA amplified cells display greater sensitivity to inhibitors of deoxyribonucleotide production relative to cells harboring chromosomal gene amplification and to normal cells. Moreover, ecDNA bearing cells demonstrate a dramatic vulnerability to reduction in the conditionally essential amino acid glutamine, the primary nitrogen source for de novo nucleotide biosynthesis. The enhanced sensitivity to glutamine starvation is directly correlated with a reduction in ecDNA in tumor cells, and a corresponding selection for chromosomally integrated gene amplification in the surviving population. These phenotypes are rescued with nucleoside supplementation but not anaplerotic TCA cycle intermediates, further reinforcing a link between ecDNA and RS biology. Collectively, observations made here support a novel mechanism for oncogene-induced RS shaped by ecDNA driven oncogene amplification, revealing a novel molecular strategy to identify high-RS tumors and a therapeutic approach to target this “Achilles heel” of tumor biology. Citation Format: Sudhir Chowdhry, Salvador Garcia, Nam-Phuong Nguyen, Anthony Celeste, Edison Tse, Snezana Milutinovic, Deepti Wilkinson, Christian Hassig, Shailaja Kasibhatla. Replication stress and the inability to repair damaged DNA, the potential “Achilles' heel” of ecDNA+ tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1520.

Abstract 5762: Genomic correlates of response to immune checkpoint blockade in Epstein-Barr virus-associated pulmonary lymphoepithelioma-like carcinoma

Abstract Introduction: Pulmonary lymphoepithelioma-like carcinoma (PLELC) is a rare and distinct subtype of non-small cell lung cancer (NSCLC). The use of immune checkpoint inhibitors (ICIs) for advanced PLELC and tumor genomic features underlying varied treatment responses remains poorly investigated. Methods: The study involved 33 PLELC patients with clinically annotated outcomes to ICIs. Targeted sequencing covering 520 cancer related genes were performed in 22 patients to identify single nucleotide variations (SNVs), copy number variations (CNVs), tumor mutation burden (TMB) and microsatellite instability. Mutation profiles of PLELC were compared with other NSCLC subtypes including lung adenocarcinoma (LUAD) and squamous cell lung carcinoma (LUSC) as well as EBV (Epstein-Barr virus) positive nasopharyngeal carcinoma (NPC) in public databases. Associations between PLELC mutation profiles and treatment responses to ICIs were analyzed. An external dataset of ICI-treated melanoma was used in further analysis of B2M. Results: The objective response rate (ORR), disease control rate (DCR), durable clinical benefit (DCB) rate, and median PFS was 18.2%, 60.6%, 45.5%, and 4.5 months respectively. Genetic alterations were frequently identified in B2M (9 of 22, 41%), CDKN1B (7 of 22, 32%), TP53 (6 of 22, 27%), SGK1 (5 of 22, 23%), NFKBIA (4 of 22, 18%) and TNFAIP3 (4 of 22, 18%). CNVs were comprehensively presented in PLELC while SVNs were less common. In our study, 8 of 9 tumors with B2M mutations and all tumors with CDKN1B, SGK1, TNFAIP3 mutations harbored copy number amplifications. Mutation profiles of PLELC were largely different from those of LUAD and LUSC but were similar to NPC. The median TMB was 2.99 mutations/Mb. All tumors were microsatellite stable (MSS). Patients with higher TMB showed improved DCR (p=0.029), PFS (p=0.056) and overall survival (p=0.038) than those with lower TMB. Patients with lower baseline plasma EBV DNA level also had longer PFS (p<0.001). The presence of B2M amplifications was significant associated with higher TMB level (p=0.022). The PFS of 7 patients with high TMB and amplified B2M were significantly longer than that of other patients (p=0.046). In the external dataset, higher expression of B2M was associated with better ICI responses for melanoma (p<0.05). Pathways involving anti-tumor immune responses such as NK cell-mediated cytotoxicity, antigen processing and presentation were correlated with high B2M expression while Wnt, TGF-beta and MAPK pathways that define poor response to ICI were correlated with low B2M expression. Conclusion: Our study observed comprehensive CNVs in PLELC patients which is a unique molecular feature compared with other NSCLC subtypes and EBV-positive NPC. Higher TMB level and amplifications of B2M in PLELC patients may be used as potential biomarkers to indicate better treatment responses to ICIs. Citation Format: Li-na He, Xuanye Zhang, Xinze Lv, Yuxi Zhou, Min Li, Ting Hou, Wenjie Sun, Li Zhang, ShaoDong Hong. Genomic correlates of response to immune checkpoint blockade in Epstein-Barr virus-associated pulmonary lymphoepithelioma-like carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5762.

Abstract 2565: CDK9 inhibitor KB-0742 is active in preclinical models of small-cell lung cancer

Abstract Small-cell lung cancer (SCLC) is a highly aggressive malignancy, with a 5-year survival rate below 5%. In contrast to non-small cell lung cancer, in which treatment advancements have shown much success, there has not been significant progress in the development of new therapies for SCLC over the last 40 years. Thus, an urgent need exists to identify new and effective pharmacological interventions for these patients. Recent advancements in the understanding of the pathology of the disease has shown that SCLC tumorigenesis and evolution is governed by increased expression of neuroendocrine-associated and other proto-oncogenic transcription factors such as ASCL1, NEUROD1, and MYC. Thus, targeting transcription may be an effective therapeutic strategy. Cyclin-dependent kinase 9 (CDK9) is a serine-threonine kinase that is involved in transcriptional elongation through the phosphorylation of the RNA polymerase II. Additionally, CDK9 interacts with transcription factors such as MYC to promote activation of target genes. Inhibition of CDK9 results in disrupted transcriptional networks and inhibition of cancer cell proliferation and survival, making CDK9 an attractive target for the treatment of SCLC. We have developed a highly selective and orally bioavailable inhibitor of CDK9, KB-0742. Here we present the evaluation of KB-0742 activity in preclinical models of SCLC. Using the Broad PRISM platform, we evaluated the sensitivity of 24 SCLC cell lines. We observed a correlation between MYC copy number amplification and sensitivity with amplified lines having smaller AUC values than non-amplified lines (Mann-Whitney-Wilcoxon; P>0.05). We observed a similar trend of lower IC50 values with increasing levels of MYC expression in a separate cell-line screen that included 5 SCLC lines (Pearson’s correlation; P=0.71). We then evaluated KB-0742 activity in a panel of 6 patient-derived organoid (PDO) SCLC models with different treatment histories and compared to 4 standard-of-care (SOC) compounds. KB-0742 was more active than the SOC compounds, with maximum inhibition rates of over 90% for 5 of the 6 models. In a separate study of 4 treatment-naïve PDO models, KB-0742 was active in 3 different transcription factor-driven subtypes of SCLC, and the response correlated significantly with c-MYC and MYCL expression. Lastly, we used 4 patient-derived xenograft (PDX) models to evaluate KB-0742 activity in vivo. The tumor growth inhibition (TGI) rate ranged from 54% to 92%, with tumor regressions observed in 2 of the 4 models, and 1 model showed greater TGI with KB-0742 than the SOC. These data support the development of KB-0742 as a potential treatment for SCLC. Patients with relapsed or refractory solid tumors or non-Hodgkin lymphoma are currently being enrolled in a phase 1/2 clinical trial of KB-0742 (NCT04718675) with an expansion arm for SCLC being planned after the recommended phase 2 dose is identified. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. CDK9 inhibitor KB-0742 is active in preclinical models of small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2565.

Abstract 6193: TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma of myoblast origin. RMS tumors are unable to terminally differentiate into syncytial muscle despite elevated expression of the muscle master regulators, MyoD and Myf5. Fusion-positive RMS (FP-RMS) and fusion-negative RMS (FN-RMS) have emerged as the two major RMS subtypes. The pathogenesis of FP-RMS is commonly defined by the balanced translocation of chromosomes 2 and 3 resulting in the misexpression of a chimeric PAX3/7-FOXO1 oncoprotein that derails normal differentiation pathways. However, the molecular basis of FN-RMS is poorly understood as single nucleotide variations in genes like KRAS, NRAS, and TP53 have limited recurrence (less than 10% of all patient tumors). In contrast, DNA copy number variations are highly recurrent and the genes they encode represent vulnerabilities in FN-RMS. Strikingly, we discovered that 72% of FN-RMS patient tumors harbor copy number amplifications at loci of TWIST1, TWIST2, or both, resulting in the upregulation of TWIST gene expression. We demonstrated that depletion of TWIST2 using a doxycycline inducible shRNA system caused cell cycle arrest and increased myogenic differentiation in xenograft models of FN-RMS. To interrogate whether TWIST2 can directly regulate the expression of these genes, we performed RNA-seq and ChIP-seq for TWIST2, H3K27Ac, and MYOD in TWIST2 knockdown and control FN-RMS cells. Utilizing this genome-wide data of chromatin binding, we comprehensively demonstrated that TWIST2 competes with MYOD preferentially at a subset of enhancers required for myogenic differentiation, causing repression of these loci. Interestingly, while MYOD binding is gained at myogenic loci in the absence of TWIST2, binding of MYOD is not lost at growth-promoting, oncogenic enhancers despite a decrease in their associated gene expression. This suggests that TWIST2 interacts with a different slew of transcription factors or chromatin regulators to activate gene expression. To interrogate this possibility, we are characterizing the TWIST2 protein interactome in FN-RMS cells using BioID as well as motifs enriched at TWIST2-regulated peaks. By studying TWIST2-associated pathways that affect RMS growth and differentiation, we see an opportunity to unlock novel therapeutic strategies for RMS. Citation Format: Akansha Shah, Lei Guo, Priscilla Jaichander, Kenian Chen, Karla Cano Hernandez, Lin Xu, Rhonda Bassel-Duby, Eric Olson, Ning Liu. TWIST2 promotes tumor progression and represses myogenic differentiation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6193.

Androgen receptor genomic alterations and treatment resistance in metastatic prostate cancer

Genomic alterations to the androgen receptor (AR) are common in metastatic castration-resistant prostate cancer (mCRPC). AR copy number amplifications, ligand-binding domain missense mutations, and intronic structural rearrangements can all drive resistance to approved AR pathway inhibitors and their detection via tissue or liquid biopsy is linked to clinical outcomes. With an increasingly crowded treatment landscape, there is hope that AR genomic alterations can act as prognostic and/or predictive biomarkers to guide patient management. In this review, we evaluate the current evidence for AR genomic alterations as clinical biomarkers in mCRPC, focusing on correlative studies that have used plasma circulating tumor DNAto characterize AR genotype. We highlight data that demonstrates the complexity of AR genotype within individual patients, and suggest that future studies should account for cancer clonal heterogeneity and variable tumor content in liquid biopsy samples. Given the potential for cooccurrence of multiple AR genomic alterations in the same or competing subclones of a patient, it is distinctly challenging to attribute blanket clinical significance to any individual alteration. This challenge is further complicated by the varied treatment exposures in contemporary patients, and the fact that AR genotype continues to evolve in the mCRPC setting across sequential lines of systemic therapy. As treatment access and liquid biopsy technology continues to improve, we posit that real-time measures of AR biology are likely to play a key role in emerging precision oncology strategies for metastatic prostate cancer.

Pan-cancer landscape of CD274 (PD-L1) and PDCD1LG2 (PD-L2) structural variations.

3133 Background: PD-1 receptor and PD-L1 ligand interactions are the target of immunotherapies across multiple tumor types. Established biomarkers that predict response to immunotherapy are microsatellite instability, tumor mutational burden, and PD-L1 immunohistochemistry. Structural variations in CD274 (PD-L1) and PDCD1LG2 (PD-L2) have been observed in cancer, but the comprehensive landscape is unknown. Here we describe the genomic landscape of CD274 and PDCD1LG2 structural variations, their potential impact on the tumor microenvironment, and evidence that patients with these alterations can benefit from immunotherapy. Methods: We analyzed sequencing data from 514 cancer cases with CD274 and PDCD1LG2 structural variations across 25 publications and data sources, including large pan-cancer sources: Foundation Medicine, Inc (FMI), The Cancer Genome Atlas (TCGA), and Oncology Research Information Exchange Network. To evaluate immune signature enrichment, we ran the software ImSig on gene expression data. We curated literature reporting clinical outcomes of patients harboring structural variations in CD274 and PDCD1LG2. Results: From 25 studies and datasets, we curated 514 cancer cases with structural variations in PD-L1 and PD-L2, including 158 duplications, 126 deletions, 97 inversions, 178 translocations, and 96 unclassified structural variations, totaling to 655 events. We observed breakpoint ‘hotspots’ in the 3’-untranslated regions (UTRs) of PD-L1 and PD-L2. Leveraging TCGA data, we observed, in CD274-rearranged tumors, significant upregulation in PD-L1 and PD-L2 expression and signatures for interferon signaling, macrophages, monocytes, T cells, and immune cell proliferation (each p < 0.001, compared to CD274 non-rearranged, copy neutral tumors). Furthermore, retrospective review of 12 studies that included patients with structural variations in CD274 or PDCD1LG2, including duplications, inversions and copy number amplifications, revealed a 73% (52/71) response rate to PD-1 immunotherapy with durable responses. Conclusions: Our evaluation of CD274 and PDCD1LG2 structural variations shows that the 3’-UTR is frequently affected and is associated with increased expression of ligands and immune signatures. Enriched interferon signaling in CD274-rearranged tumors is of particular interest, as interferon exposure is known to drive PD-L1 and PD-L2 expression. Retrospective evidence from curated studies suggests that these genomic alterations could identify candidates for PD-1 or PD-L1 immunotherapy. We expect that these findings will better define CD274 and PDCD1LG2 structural variations in cancer and support our pan-cancer prospective clinical trial to target these alterations.

Analysis of molecular characterization: Pancreatic ductal adenocarcinoma with hepatic metastases.

4129 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. Distant metastasis is the leading cause of cancer death. Approximately 50% of PDAC patients are discovered to have distant metastases, mainly in the liver. Chemotherapy is the main recommended treatment for metastatic PDACs, with a median survival time of only 4-6 months and the 5-year survival of 1%. Therefore, deciphering the molecular mechanisms of metastatic PDAC is urgently for exploring effective therapeutic targets. Methods: 93 normal-paired samples from patients with PDAC were analyzed using hybridization capture-based next generation sequencing. Somatic and germline mutations were identified including 500 genes associated with tumor development. Sequencing data were analyzed to call tumor specific single nucleotide variants (SNV), small insertions and deletions (InDels), copy number alterations (CNA) and gene fusions and rearrangements. Results: A total 93 patients with PDAC were analyzed, including hepatic metastases (15/93), lymph node metastases (1/93), colon metastases (1/93), pelvic metastases (1/93), and no distant metastasis (75/93). In PDAC patients without distant metastasis, the most frequent mutated gene was KRAS (72%), followed by TP53 (60%), CDKN2A (21%), SMAD4 (17%), ARID1A (11%), RNF43 (9%), and RBM10 (8%). In PDAC patients with hepatic metastases, we found that the most frequent one was KRAS (100%), followed by TP53 (87%), CDKN2A (40%), SMAD4 (40%), RNF43 (13%), and RBM10 (6%). Furthermore, four PDAC patients occurred KRAS copy number amplification, including 2 cases of hepatic metastases. And a hepatic metastases PDAC patient had ERBB2 and MET copy number amplification. We also found that seven no-metastasis patients carried pathogenic or likely pathogenic germline variants, including BRIP1, PALB2, RECQL, ATM, RAD50, FANCM and BRCA1. Conclusions: High frequency mutation genes in PDAC patients with hepatic metastases were highly consistent with those of no-metastasis patients. KRAS and TP53 gene co-mutations were extensively mutated in hepatic metastases patients. Our results revealed the molecular mechanisms driving PDAC hepatic metastases should be considered in further study.

The landscape of NFE2L2 alteration in Chinese solid tumor patients.

e15126 Background: NFE2L2 encodes a transcription factor which is a member of a small family of basic leucine zipper (bZIP) proteins. NFE2L2 variations were detected in many tumors, such as lung cancer, esophageal cancer, renal cell carcinoma and other cancer types. The landscape of NFE2L2 gene in solid tumors of Chinese population has not been descripted. Methods: 3193 normal-paired samples from patients with solid tumors were analyzed using hybridization capture-based next generation sequencing, and alterations including single base substitution, short and long insertion/deletions, copy number variations, gene fusions and rearrangements. Results: NFE2L2 gene was altered in 2.0% (65 of 3193) of all solid tumors. The frequency of NFE2L2 variations was assessed in multiple cancer types, including lung cancer (33/1109), liver cancer (10/206), colorectal cancer (6/391), stomach cancer (3/227), cervical cancer (3/112), esophageal cancer (3/53), and other cancer types. A total of 54 variations of NFE2L2 were found, mainly in exon 2 (26/54), exon 5 (11/54), exon 3 (4/54) and exon 4 (2/54), no variation in exon 1. NFE2L2 D16G, D21H, R34_S40del, V36_E45del, F37_E45del, R42P, L48P, K53Tfs*2, L62P, E67Q and L76_E78delinsQ in exon 2 have not been reported before. In addition, we identified copy number amplifications of NFE2L2 in four patients, including three lung squamous cell carcinomas and one cervical squamous cell carcinoma patients. Furthermore, in our cohort, NFE2L2 mutation carriers also owned other actionable or driver mutation, the most frequent one was TP53 (71%), followed by PIK3CA (34%), MLL2 (28%), CDKN2A (31%), and ATR (25%). Analysis of co-mutation correlation of NFE2L2 showed that NFE2L2 variation was correlated with ATR, ACVR1B, TERC and Sox2 ( p<0.001). TERC and SOX2 were mainly copy number amplification at chromosome 3q36, and there is higher amplification frequency at 3q36 in squamous cell carcinoma. In TCGA cohort, the patients with NFE2L2 variations and 3q36 amplification had a longer median survival than those group without 3q36 amplification (63.59 vs 32.04 months, p = 0.0459). Conclusions: NFE2L2 deleterious mutations are highly diverse and present at low to moderate frequencies across many cancers. Our results revealed the potential targeted therapeutic strategies and prognostic analysis among NFE2L2 population with solid tumors should be considered in further study.

Characterization of molecular subtypes of melanoma and development of prognostic and immunotherapy-related genetic characteristics by ferroptosis gene analysis.

e21508 Background: The heterogeneity is a major problem in clinical treatment of skin cutaneous melanoma (SKCM). Objective and reproducible classification systems may help decode SKCM heterogeneity. Methods: Based on ferroptosis-related genes, this study used unsupervised clustering to identify two molecular subtypes of SKCM C1 and C2. A ferroptosis-related prognosis risk score (FPRS) model was constructed through analyzing the differences between C1 and C2 subtypes. Results: C1 showed a poor prognosis, with low infiltration degree of immune cells and TIED score were lower and higher homologous recombination defects, fraction altered, number of segments and copy number amplification and deletion. These characteristics of C2 were the opposite of C1. SKCM patients were divided into two risk groups based on FPRS. There were significant differences in the infiltration and proportion of immune cells, expression of immune checkpoint genes, and response to immune checkpoint therapy and sensitivity to chemotherapeutic drugs between high-risk group and low-risk group. Conclusions: In summary, we have identified two subtypes of SKCM with different molecular and immune characteristics based on ferroptosis-related genes, further developed and verified a FPRS model, which can be used as an independent prognostic indicator for SKCM. This study may provide a new insight for the development of personalized management of patients with SKCM.