Pub Date : 2024-09-19DOI: 10.1101/2024.09.18.613595
Aya Ludin, Georgia L. Stirtz, Asaf Tal, Ajit J. Nirmal, Naomi Besson, Stephanie M. Jones, Kathleen L. Pfaff, Michael Manos, Sophia Liu, Irving Barrera, Qiyu Gong, Cecilia Pessoa Rodrigues, Aditi Sahu, Elizabeth Jerison, Joao V. Alessi, Biagio Ricciuti, Douglas S. Richardson, Jodi D. Weiss, Hadley M. Moreau, Meredith E. Stanhope, Alexander B. Afeyan, James Sefton, Wyatt D. McCall, Emily Formato, Song Yang, Yi Zhou, David P. Hoytema van Konijnenburg, Hannah L. Cole, Miguel Cordova, Liang Deng, Milind Rajadhyaksha, Stephen R. Quake, Mark M. Awad, Fei Chen, Peter K. Sorger, F. Stephen Hodi, Scott J. Rodig, George F. Murphy, Leonard I. Zon
Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8+ T cells in the tumor. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8+ T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response.
尽管肿瘤处于免疫抑制环境中，免疫疗法仍能导致癌症的根除。在这里，我们利用对斑马鱼内源性黑色素瘤的长期体内成像和高分辨率空间转录组学，以及对人类黑色素瘤的多重成像，确定了在免疫治疗过程中促进免疫反应的区域。我们在斑马鱼和人类黑色素瘤的边缘发现了火山口状的凹陷，其中富含β-2微球蛋白（B2M）和抗原识别分子。陨石坑是肿瘤中 CD8+ T 细胞密度最高的地方。在斑马鱼体内，CD8+ T 细胞与嵴内的黑色素瘤细胞形成了长时间的相互作用，这是抗原识别的特征。经过免疫刺激治疗后，陨石坑扩大，成为活化的 CD8+ T 细胞聚集和肿瘤杀伤的主要部位，这种杀伤依赖于 B2M。在人体中，陨石坑可预测对 ICB 治疗的免疫反应，其反应优于高 T 细胞浸润。这标志着陨石坑可能成为免疫疗法成功与否的新诊断工具，并成为增强 ICB 反应的目标。
{"title":"Craters on the melanoma surface facilitate tumor-immune interactions and demonstrate pathologic response to checkpoint blockade in humans","authors":"Aya Ludin, Georgia L. Stirtz, Asaf Tal, Ajit J. Nirmal, Naomi Besson, Stephanie M. Jones, Kathleen L. Pfaff, Michael Manos, Sophia Liu, Irving Barrera, Qiyu Gong, Cecilia Pessoa Rodrigues, Aditi Sahu, Elizabeth Jerison, Joao V. Alessi, Biagio Ricciuti, Douglas S. Richardson, Jodi D. Weiss, Hadley M. Moreau, Meredith E. Stanhope, Alexander B. Afeyan, James Sefton, Wyatt D. McCall, Emily Formato, Song Yang, Yi Zhou, David P. Hoytema van Konijnenburg, Hannah L. Cole, Miguel Cordova, Liang Deng, Milind Rajadhyaksha, Stephen R. Quake, Mark M. Awad, Fei Chen, Peter K. Sorger, F. Stephen Hodi, Scott J. Rodig, George F. Murphy, Leonard I. Zon","doi":"10.1101/2024.09.18.613595","DOIUrl":"https://doi.org/10.1101/2024.09.18.613595","url":null,"abstract":"Immunotherapy leads to cancer eradication despite the tumor's immunosuppressive environment. Here, we used extended long-term in-vivo imaging and high-resolution spatial transcriptomics of endogenous melanoma in zebrafish, and multiplex imaging of human melanoma, to identify domains that facilitate immune response during immunotherapy. We identified crater-shaped pockets at the margins of zebrafish and human melanoma, rich with beta-2 microglobulin (B2M) and antigen recognition molecules. The craters harbor the highest density of CD8+ T cells in the tumor. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within craters, characteristic of antigen recognition. Following immunostimulatory treatment, the craters enlarged and became the major site of activated CD8+ T cell accumulation and tumor killing that was B2M dependent. In humans, craters predicted immune response to ICB therapy, showing response better than high T cell infiltration. This marks craters as potential new diagnostic tool for immunotherapy success and targets to enhance ICB response.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249844","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-19DOI: 10.1101/2024.09.18.613673
Xueqing Peng, Peiluan Li, Luonan Chen
There generally exists a critical state or tipping point from a stable state to another in dynamic biological processes, beyond which a significant qualitative transition occurs. Identifying this tipping point and its driving network is essential to prevent or delay catastrophic consequences. However, most traditional approaches based on undirected networks still suffer from the problem of the robustness and effectiveness when applied to high-dimensional small sample data, especially for single-cell data. To address this challenge, we developed a directed-network flow entropy (DNFE) method which can transform measured omics data into a directed network. This method is applicable to both single-cell RNA-sequencing (scRNA-seq) and bulk data. By applying this method to five real datasets, including three single-cell datasets and two bulk tumor datasets, the method can not only successfully detect the critical states as well as their dynamic network biomarkers, but also help explore regulatory relationships between genes. Numerical simulation indicates that the DNFE method is robust and superior to existing methods. Furthermore, DNFE has predicted active transcription factors (TFs), and further identified 'dark genes', which are usually overlooked by traditional methods.
{"title":"DNFE: Directed-network flow entropy for detecting the tipping points during biological processes","authors":"Xueqing Peng, Peiluan Li, Luonan Chen","doi":"10.1101/2024.09.18.613673","DOIUrl":"https://doi.org/10.1101/2024.09.18.613673","url":null,"abstract":"There generally exists a critical state or tipping point from a stable state to another in dynamic biological processes, beyond which a significant qualitative transition occurs. Identifying this tipping point and its driving network is essential to prevent or delay catastrophic consequences. However, most traditional approaches based on undirected networks still suffer from the problem of the robustness and effectiveness when applied to high-dimensional small sample data, especially for single-cell data. To address this challenge, we developed a directed-network flow entropy (DNFE) method which can transform measured omics data into a directed network. This method is applicable to both single-cell RNA-sequencing (scRNA-seq) and bulk data. By applying this method to five real datasets, including three single-cell datasets and two bulk tumor datasets, the method can not only successfully detect the critical states as well as their dynamic network biomarkers, but also help explore regulatory relationships between genes. Numerical simulation indicates that the DNFE method is robust and superior to existing methods. Furthermore, DNFE has predicted active transcription factors (TFs), and further identified 'dark genes', which are usually overlooked by traditional methods.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-19DOI: 10.1101/2024.09.13.612820
Marie-Jose Truong, Geoffrey Pawlak, Jean-Pascal Meneboo, Sheherazade Sebda, Marie Fernandes, Martin Figeac, Mohamed Elati, David Tulasne
The MET exon 14 skipping mutation (named METex14del) described in lung cancer leads to prolonged activation of signaling pathways and aberrant cell responses, but the link between HGF signaling and cell responses remains unclear. A putative regulatory network of influential regulators of target genes was constructed from the transcriptomes of lung cancer cell lines. Overlaying this reference network with transcriptomic data from METex14del-expressing cells, stimulated or not by HGF, revealed a major regulatory node consisting mainly of the transcription factors ETS1, FOSL1 and SMAD3. HGF activation of METex14del induced the phosphorylation of these master regulators and the expression of their predicted target genes in a RAS-ERK pathway-dependent manner. Furthermore, most of the transcription factors in the regulatory node are known regulators of epithelial-mesenchymal transition, consistent with their involvement in migration and invasion. New modeling with transcriptomic data from MEK inhibitor-treated METex14del cells validated the key role of RAS-ERK pathway regulators and their target genes in METex14del receptor activation. Thus, we report an original strategy to identify key transcriptional nodes associated with specific signaling pathways that may become novel therapeutic targets.
{"title":"Transcriptional program-based deciphering of the MET exon 14 skipping regulation network","authors":"Marie-Jose Truong, Geoffrey Pawlak, Jean-Pascal Meneboo, Sheherazade Sebda, Marie Fernandes, Martin Figeac, Mohamed Elati, David Tulasne","doi":"10.1101/2024.09.13.612820","DOIUrl":"https://doi.org/10.1101/2024.09.13.612820","url":null,"abstract":"The MET exon 14 skipping mutation (named METex14del) described in lung cancer leads to prolonged activation of signaling pathways and aberrant cell responses, but the link between HGF signaling and cell responses remains unclear. A putative regulatory network of influential regulators of target genes was constructed from the transcriptomes of lung cancer cell lines. Overlaying this reference network with transcriptomic data from METex14del-expressing cells, stimulated or not by HGF, revealed a major regulatory node consisting mainly of the transcription factors ETS1, FOSL1 and SMAD3. HGF activation of METex14del induced the phosphorylation of these master regulators and the expression of their predicted target genes in a RAS-ERK pathway-dependent manner. Furthermore, most of the transcription factors in the regulatory node are known regulators of epithelial-mesenchymal transition, consistent with their involvement in migration and invasion. New modeling with transcriptomic data from MEK inhibitor-treated METex14del cells validated the key role of RAS-ERK pathway regulators and their target genes in METex14del receptor activation. Thus, we report an original strategy to identify key transcriptional nodes associated with specific signaling pathways that may become novel therapeutic targets.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250083","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-18DOI: 10.1101/2024.09.16.613189
Zhuomin Yin, Tao Feng, Qing Xu, Wumin Dai, Maowei Ni, Juan Ni, Hanmei Lou
Purpose: Monitoring of circulating human papillomavirus (HPV) cell-free DNA (cfDNA) is a minimally invasive approach for surveillance in HPV-associated cancers, particularly cervical cancer. The aim of this study was to monitor circulating HPV cfDNA levels in patients with recurrent or metastatic cervical cancer during treatment and follow-up to assess the utility of HPV cfDNA as a tumor marker for disease surveillance and in guiding clinical treatment decisions. Experimental Design: In this prospective pilot observational study, levels of HPV cfDNA in serum samples from 28 patients with recurrent or metastatic HPV+ cervical cancer were measured via digital droplet polymerase chain reaction. Results for HPV cfDNA levels were matched to clinical outcomes and to serum levels of squamous cell carcinoma antigen (SCC-Ag) to assess the clinical potential of HPV cfDNA as a tumor marker. Results: HPV cfDNA was detected in all 28 patients (100%). Notably, median baseline HPV cfDNA levels varied according to the metastatic pattern in individual patients (P=0.019). Specifically, patients with a combined multiple-metastasis pattern had higher median baseline HPV cfDNA levels than patients with a single metastasis (P=0.003). All participants exhibited changes in HPV cfDNA levels over a median monitoring period of 2 months (range 0.3–16.9) before evaluations for treatment response or disease progression. Among 26 patients initially diagnosed with squamous cell cervical cancer, the positivity rate was 100% for HPV cfDNA and 69.2% for SCC-Ag (P=0.004, 95% confidence interval, 0–0.391). Among 20 patients longitudinally monitored for squamous cell cervical cancer, the concordance with changes in disease status was 90% for HPV cfDNA and 50% for SCC-Ag (P=0.014, 95% confidence interval, 0.022–0.621). Conclusions: HPV cfDNA is a promising tumor marker for HPV+ cervical cancer that offers advantages over SCC-Ag. In the context of precision medicine, HPV cfDNA is poised to play an increasingly pivotal role in monitoring treatment efficacy, providing valuable insights into disease progression, and guiding clinical decisions.
{"title":"Monitoring of Cell-free Human Papillomavirus DNA in Metastatic or Recurrent Cervical Cancer: Clinical Significance and Treatment Implications","authors":"Zhuomin Yin, Tao Feng, Qing Xu, Wumin Dai, Maowei Ni, Juan Ni, Hanmei Lou","doi":"10.1101/2024.09.16.613189","DOIUrl":"https://doi.org/10.1101/2024.09.16.613189","url":null,"abstract":"Purpose: Monitoring of circulating human papillomavirus (HPV) cell-free DNA (cfDNA) is a minimally invasive approach for surveillance in HPV-associated cancers, particularly cervical cancer. The aim of this study was to monitor circulating HPV cfDNA levels in patients with recurrent or metastatic cervical cancer during treatment and follow-up to assess the utility of HPV cfDNA as a tumor marker for disease surveillance and in guiding clinical treatment decisions. Experimental Design: In this prospective pilot observational study, levels of HPV cfDNA in serum samples from 28 patients with recurrent or metastatic HPV<sup>+</sup> cervical cancer were measured via digital droplet polymerase chain reaction. Results for HPV cfDNA levels were matched to clinical outcomes and to serum levels of squamous cell carcinoma antigen (SCC-Ag) to assess the clinical potential of HPV cfDNA as a tumor marker. Results: HPV cfDNA was detected in all 28 patients (100%). Notably, median baseline HPV cfDNA levels varied according to the metastatic pattern in individual patients (<em>P</em>=0.019). Specifically, patients with a combined multiple-metastasis pattern had higher median baseline HPV cfDNA levels than patients with a single metastasis (<em>P</em>=0.003). All participants exhibited changes in HPV cfDNA levels over a median monitoring period of 2 months (range 0.3–16.9) before evaluations for treatment response or disease progression. Among 26 patients initially diagnosed with squamous cell cervical cancer, the positivity rate was 100% for HPV cfDNA and 69.2% for SCC-Ag (<em>P</em>=0.004, 95% confidence interval, 0–0.391). Among 20 patients longitudinally monitored for squamous cell cervical cancer, the concordance with changes in disease status was 90% for HPV cfDNA and 50% for SCC-Ag (<em>P</em>=0.014, 95% confidence interval, 0.022–0.621). Conclusions: HPV cfDNA is a promising tumor marker for HPV<sup>+</sup> cervical cancer that offers advantages over SCC-Ag. In the context of precision medicine, HPV cfDNA is poised to play an increasingly pivotal role in monitoring treatment efficacy, providing valuable insights into disease progression, and guiding clinical decisions.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249756","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-18DOI: 10.1101/2024.09.16.613213
Amy L Collins, Keara Kirkness, Erik Ramon-Gil, Eleni Tzortzopoulou, Daniel Geh, Ranie Cameron, Saimir Luli, Eman Khurram, Daniel Storey, Hannah Paish, David McDonald, Andrew Filby, Lee A Borthwick, Fiona Oakley, Derek Mann, Jack Leslie
Disease modelling is vital for improving knowledge of disease mechanisms and for development of new therapeutic molecules and strategies. Modelling the intact living tumour microenvironment (TME) is increasingly considered to be vital not only for gaining a better understanding of the biology of cancer but for examining the efficacy of novel oncology drugs. To date, pre-clinical mouse models of cancer have represented the mainstay methodology for studying the evolving TME and for determining the effects of potential therapeutic molecules on tumour evolution and growth. Regarding drug screening, in vivo mouse models are expensive, require the use of large cohorts of mice and involve the administration of drugs with unknown toxicities to animals which often result in adverse effects that can cause animal suffering and the discontinuation of drug investigations. Hepatocellular carcinoma (HCC) is a primary cancer of the liver for which there is an urgent need for improved systemic treatments due to the disease usually being diagnosed at an advanced stage and current treatments having limited efficacy. To provide a practical solution to the screening of drugs for their likely efficacy in HCC we have developed an ex-vivo model in which orthotopic tumours are excised from the liver and subsequently processed to generate precision-cut tumour slices (PCTS) which provide an intact culture model of the HCC-TME. We describe simplified culture conditions that maintain the viability and metabolic activity of live PCTS which maintain the architecture, cellular complexity, drug sensitivity and responsiveness to immunotherapy of the original tumour. Importantly, we show that HCC derived PCTS can be miniaturised to 96-well scale and modified to express soluble luciferase, which in combination enabled non-destructive screening of a library of 26 drugs at two doses using just 5 tumours as the source for PCTS. This screen identified two small molecules, salinomycin and rottlerin, that have potent anti-tumour activities in HCC-PCTS and subsequently validated salinomycin as effective in vivo. In summary, we report a 3Rs (reduction, refinement and replacement) solution for study of HCC biology and for 96-well-scale screening of potential therapeutic agents in the context of an intact, metabolically active TME.
{"title":"Ex-vivo mouse precision cut tumour slices for modelling hepatocellular carcinoma; A 3Rs solution for at-scale drug screening","authors":"Amy L Collins, Keara Kirkness, Erik Ramon-Gil, Eleni Tzortzopoulou, Daniel Geh, Ranie Cameron, Saimir Luli, Eman Khurram, Daniel Storey, Hannah Paish, David McDonald, Andrew Filby, Lee A Borthwick, Fiona Oakley, Derek Mann, Jack Leslie","doi":"10.1101/2024.09.16.613213","DOIUrl":"https://doi.org/10.1101/2024.09.16.613213","url":null,"abstract":"Disease modelling is vital for improving knowledge of disease mechanisms and for development of new therapeutic molecules and strategies. Modelling the intact living tumour microenvironment (TME) is increasingly considered to be vital not only for gaining a better understanding of the biology of cancer but for examining the efficacy of novel oncology drugs. To date, pre-clinical mouse models of cancer have represented the mainstay methodology for studying the evolving TME and for determining the effects of potential therapeutic molecules on tumour evolution and growth. Regarding drug screening, in vivo mouse models are expensive, require the use of large cohorts of mice and involve the administration of drugs with unknown toxicities to animals which often result in adverse effects that can cause animal suffering and the discontinuation of drug investigations. Hepatocellular carcinoma (HCC) is a primary cancer of the liver for which there is an urgent need for improved systemic treatments due to the disease usually being diagnosed at an advanced stage and current treatments having limited efficacy. To provide a practical solution to the screening of drugs for their likely efficacy in HCC we have developed an ex-vivo model in which orthotopic tumours are excised from the liver and subsequently processed to generate precision-cut tumour slices (PCTS) which provide an intact culture model of the HCC-TME. We describe simplified culture conditions that maintain the viability and metabolic activity of live PCTS which maintain the architecture, cellular complexity, drug sensitivity and responsiveness to immunotherapy of the original tumour. Importantly, we show that HCC derived PCTS can be miniaturised to 96-well scale and modified to express soluble luciferase, which in combination enabled non-destructive screening of a library of 26 drugs at two doses using just 5 tumours as the source for PCTS. This screen identified two small molecules, salinomycin and rottlerin, that have potent anti-tumour activities in HCC-PCTS and subsequently validated salinomycin as effective in vivo. In summary, we report a 3Rs (reduction, refinement and replacement) solution for study of HCC biology and for 96-well-scale screening of potential therapeutic agents in the context of an intact, metabolically active TME.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-18DOI: 10.1101/2024.09.11.612571
Kwang-Soo Kim, Junyi Zhang, Victor Andres Arrieta, Crismita Dmello, Elena Grabis, Karl Habashy, Joseph Duffy, Junfei Zhao, Andrew Gould, Li Chen, Jian Hu, Irina Balyasnikova, Dhan Chand, Dan Levey, Peter Canoll, Wenting Zhao, Peter Sims, Raul Rabadan, Surya Pandey, Bin Zhang, Catalina Lee-Chang, Dieter Henrik Heiland, Adam Mendel Sonabend
Background: Glioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. Method: We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single-cell RNA-sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Results: CRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. Conclusion: Our data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.
{"title":"MAPK/ERK signaling in gliomas modulates interferon responses, T cell recruitment, enhances tumor-microglia crosstalk, and drives immune checkpoint blockade efficacy.","authors":"Kwang-Soo Kim, Junyi Zhang, Victor Andres Arrieta, Crismita Dmello, Elena Grabis, Karl Habashy, Joseph Duffy, Junfei Zhao, Andrew Gould, Li Chen, Jian Hu, Irina Balyasnikova, Dhan Chand, Dan Levey, Peter Canoll, Wenting Zhao, Peter Sims, Raul Rabadan, Surya Pandey, Bin Zhang, Catalina Lee-Chang, Dieter Henrik Heiland, Adam Mendel Sonabend","doi":"10.1101/2024.09.11.612571","DOIUrl":"https://doi.org/10.1101/2024.09.11.612571","url":null,"abstract":"Background: Glioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. Method: We conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single-cell RNA-sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. Results: CRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. Conclusion: Our data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142250085","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Lung adenocarcinoma (LUAD) is a malignant tumor with high morbidity and mortality worldwide, and overall survival rates for LUAD patients remain unimproved. RNA modification is a key process in post-transcriptional gene regulation in epigenetics, with N6-methyladenosine (m6A) being a common RNA modification. The molecular mechanisms of LUAD are unclear, but evidence suggests that m6A RNA methylation plays a significant role. This study aimed to clarify the role of YTHDF1 in LUAD development and pathogenesis. These findings confirmed that YTHDF1, a m6A reader protein, is highly expressed in LUAD tissues and is correlated with tumor differentiation and TNM stage. The results of functional loss experiments in LUAD cell lines revealed that downregulating YTHDF1 inhibits proliferation, migration, and invasion and induces apoptosis, with opposite effects observed upon YTHDF1 upregulation. In vivo, YTHDF1 knockout suppressed LUAD xenograft growth. RNA-seq, MeRIP-seq, RIP-seq, and bioinformatics analyses identified EEF1G as a downstream target of YTHDF1 in LUAD, and high expression of EEF1G was confirmed. The interaction between YTHDF1 and EEF1G was validated through RIP-qPCR, Co-IP and Co-IF assays. The overexpression of EEF1G in LUAD cells partially counteracts the tumor suppression induced by YTHDF1 silencing, and the knockdown of EEF1G has the opposite effect, further confirming the regulatory relationship. In summary, this study describes a novel YTHDF1/EEF1G regulatory pathway in which YTHDF1 promotes LUAD progression by recognizing and binding to the m6A-modified mRNA of EEF1G, accelerating its translation, suggesting that YTHDF1 may be a potential biomarker and therapeutic target.
{"title":"YTHDF1 Facilitates Lung Adenocarcinoma Progression via Promotion of EEF1G Translation in a m6A-Dependent Manner","authors":"Lihong Wang, Qihong Sheng, Xiaoyu Wang, Hongjuan Yue, Qian Wang, Mei Zhang, Junling Ma, Ling Wu, Jiaojiao Zhang, Zishuo Cheng, Weifang Yu, Ting Liu, Jia Wang","doi":"10.1101/2024.09.13.612607","DOIUrl":"https://doi.org/10.1101/2024.09.13.612607","url":null,"abstract":"Lung adenocarcinoma (LUAD) is a malignant tumor with high morbidity and mortality worldwide, and overall survival rates for LUAD patients remain unimproved. RNA modification is a key process in post-transcriptional gene regulation in epigenetics, with N6-methyladenosine (m6A) being a common RNA modification. The molecular mechanisms of LUAD are unclear, but evidence suggests that m6A RNA methylation plays a significant role. This study aimed to clarify the role of YTHDF1 in LUAD development and pathogenesis. These findings confirmed that YTHDF1, a m6A reader protein, is highly expressed in LUAD tissues and is correlated with tumor differentiation and TNM stage. The results of functional loss experiments in LUAD cell lines revealed that downregulating YTHDF1 inhibits proliferation, migration, and invasion and induces apoptosis, with opposite effects observed upon YTHDF1 upregulation. In vivo, YTHDF1 knockout suppressed LUAD xenograft growth. RNA-seq, MeRIP-seq, RIP-seq, and bioinformatics analyses identified EEF1G as a downstream target of YTHDF1 in LUAD, and high expression of EEF1G was confirmed. The interaction between YTHDF1 and EEF1G was validated through RIP-qPCR, Co-IP and Co-IF assays. The overexpression of EEF1G in LUAD cells partially counteracts the tumor suppression induced by YTHDF1 silencing, and the knockdown of EEF1G has the opposite effect, further confirming the regulatory relationship. In summary, this study describes a novel YTHDF1/EEF1G regulatory pathway in which YTHDF1 promotes LUAD progression by recognizing and binding to the m6A-modified mRNA of EEF1G, accelerating its translation, suggesting that YTHDF1 may be a potential biomarker and therapeutic target.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-18DOI: 10.1101/2024.09.17.612743
Anthony M. Boutelle, Aicha R. Mabene, David Yao, Haiqing Xu, Mengxiong Wang, Yuning J. Tang, Steven S. Lopez, Sauradeep Sinha, Janos Demeter, Ran Cheng, Brooks A. Benard, Liz J. Valente, Alexandros P. Drainas, Martin D. Fischer, Ravindra Majeti, Dmitri Petrov, Peter K Jackson, Fan Yang, Monte M Winslow, Michael C Bassik, Laura D. Attardi
TP53, the most frequently mutated gene in human cancer, encodes a transcriptional activator that induces myriad downstream target genes. Despite the importance of p53 in tumor suppression, the specific p53 target genes important for tumor suppression remain unclear. Recent studies have identified the p53-inducible gene Zmat3 as a critical effector of tumor suppression, but many questions remain regarding its p53-dependence, activity across contexts, and mechanism of tumor suppression alone and in cooperation with other p53-inducible genes. To address these questions, we used Tuba-seqUltra somatic genome editing and tumor barcoding in a mouse lung adenocarcinoma model, combinatorial in vivo CRISPR/Cas9 screens, meta-analyses of gene expression and Cancer Dependency Map data, and integrative RNA-sequencing and shotgun proteomic analyses. We established Zmat3 as a core component of p53-mediated tumor suppression and identified Cdkn1a as the most potent cooperating p53-induced gene in tumor suppression. We discovered that ZMAT3/CDKN1A serve as near-universal effectors of p53-mediated tumor suppression that regulate cell division, migration, and extracellular matrix organization. Accordingly, combined Zmat3-Cdkn1a inactivation dramatically enhanced cell proliferation and migration compared to controls, akin to p53 inactivation. Together, our findings place ZMAT3 and CDKN1A as hubs of a p53-induced gene program that opposes tumorigenesis across various cellular and genetic contexts.
{"title":"Integrative multiomic approaches reveal ZMAT3 and p21 as conserved hubs in the p53 tumor suppression network","authors":"Anthony M. Boutelle, Aicha R. Mabene, David Yao, Haiqing Xu, Mengxiong Wang, Yuning J. Tang, Steven S. Lopez, Sauradeep Sinha, Janos Demeter, Ran Cheng, Brooks A. Benard, Liz J. Valente, Alexandros P. Drainas, Martin D. Fischer, Ravindra Majeti, Dmitri Petrov, Peter K Jackson, Fan Yang, Monte M Winslow, Michael C Bassik, Laura D. Attardi","doi":"10.1101/2024.09.17.612743","DOIUrl":"https://doi.org/10.1101/2024.09.17.612743","url":null,"abstract":"<em>TP53</em>, the most frequently mutated gene in human cancer, encodes a transcriptional activator that induces myriad downstream target genes. Despite the importance of p53 in tumor suppression, the specific p53 target genes important for tumor suppression remain unclear. Recent studies have identified the p53-inducible gene <em>Zmat3</em> as a critical effector of tumor suppression, but many questions remain regarding its p53-dependence, activity across contexts, and mechanism of tumor suppression alone and in cooperation with other p53-inducible genes. To address these questions, we used Tuba-seq<sup>Ultra</sup> somatic genome editing and tumor barcoding in a mouse lung adenocarcinoma model, combinatorial <em>in vivo</em> CRISPR/Cas9 screens, meta-analyses of gene expression and Cancer Dependency Map data, and integrative RNA-sequencing and shotgun proteomic analyses. We established <em>Zmat3</em> as a core component of p53-mediated tumor suppression and identified <em>Cdkn1a</em> as the most potent cooperating p53-induced gene in tumor suppression. We discovered that <em>ZMAT3/CDKN1A</em> serve as near-universal effectors of p53-mediated tumor suppression that regulate cell division, migration, and extracellular matrix organization. Accordingly, combined <em>Zmat3-Cdkn1a</em> inactivation dramatically enhanced cell proliferation and migration compared to controls, akin to p53 inactivation. Together, our findings place <em>ZMAT3</em> and <em>CDKN1A</em> as hubs of a p53-induced gene program that opposes tumorigenesis across various cellular and genetic contexts.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-18DOI: 10.1101/2024.09.16.613045
Saireudee Chaturantabut, Sydney Oliver, Dennie T. Frederick, Jiwan Kim, Foxy P Robinson, Alessandro Sinopoli, Tian-Yu Song, Diego J Rodriguez, Liang Chang, Devishi Kesar, Yao He, Meilani Ching, Ruvimbo Dzvurumi, Adel Atari, Yuen-Yi Tseng, Nabeel Bardeesy, William R Sellers
Translocations involving FGFR2 gene fusions are common in cholangiocarcinoma and predict response to FGFR kinase inhibitors. However, the rate and durability of response are limited due to the emergence of resistance, typically involving acquired FGFR2 kinase domain mutations, and to sub-optimal dosing, relating to drug adverse effects. Here, we report the development of biparatopic antibodies targeting the FGFR2 extracellular domain (ECD), as candidate therapeutics. Biparatopic antibodies can overcome drawbacks of standard bivalent monoparatopic antibodies, which often show poor inhibitory or even agonist activity against oncogenic receptors. We show that oncogenic transformation by FGFR2 fusions requires an intact ECD. Moreover, by systematically generating biparatopic antibodies that target distinct epitope pairs along the FGFR2 ECD, we identified antibodies that effectively block signaling and malignant growth driven by FGFR2-fusions. Importantly, these antibodies demonstrate efficacy in vivo, synergy with FGFR inhibitors, and activity against FGFR2 fusions harboring kinase domain mutations. Thus, biparatopic antibodies may serve as new treatment options for patients with FGFR2-altered cholangiocarcinoma.
{"title":"Identification of potent biparatopic antibodies targeting FGFR2 fusion driven cholangiocarcinoma.","authors":"Saireudee Chaturantabut, Sydney Oliver, Dennie T. Frederick, Jiwan Kim, Foxy P Robinson, Alessandro Sinopoli, Tian-Yu Song, Diego J Rodriguez, Liang Chang, Devishi Kesar, Yao He, Meilani Ching, Ruvimbo Dzvurumi, Adel Atari, Yuen-Yi Tseng, Nabeel Bardeesy, William R Sellers","doi":"10.1101/2024.09.16.613045","DOIUrl":"https://doi.org/10.1101/2024.09.16.613045","url":null,"abstract":"Translocations involving FGFR2 gene fusions are common in cholangiocarcinoma and predict response to FGFR kinase inhibitors. However, the rate and durability of response are limited due to the emergence of resistance, typically involving acquired FGFR2 kinase domain mutations, and to sub-optimal dosing, relating to drug adverse effects. Here, we report the development of biparatopic antibodies targeting the FGFR2 extracellular domain (ECD), as candidate therapeutics. Biparatopic antibodies can overcome drawbacks of standard bivalent monoparatopic antibodies, which often show poor inhibitory or even agonist activity against oncogenic receptors. We show that oncogenic transformation by FGFR2 fusions requires an intact ECD. Moreover, by systematically generating biparatopic antibodies that target distinct epitope pairs along the FGFR2 ECD, we identified antibodies that effectively block signaling and malignant growth driven by FGFR2-fusions. Importantly, these antibodies demonstrate efficacy in vivo, synergy with FGFR inhibitors, and activity against FGFR2 fusions harboring kinase domain mutations. Thus, biparatopic antibodies may serve as new treatment options for patients with FGFR2-altered cholangiocarcinoma.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249755","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-18DOI: 10.1101/2024.09.17.612958
Sara Sartini, Lexi Omholt, Neda Moatamed, Alice Soragni
High Grade Serous Ovarian Cancer (HG-SOC), the most prevalent and aggressive gyneco-logical malignancy, is marked by ubiquitous loss of functional p53, largely due to point mutations that arise very early in carcinogenesis. These mu-tations often lead to p53 protein misfolding and subsequent aggregation, yet the alterations in in-tracellular p53 dynamics throughout ovarian can-cer progression remain poorly understood. HG-SOC originates from the fallopian tube epithelium, with a well-documented stepwise progression be-ginning with early pre-malignant p53 signatures. These signatures represent largely normal cells that express and accumulate mutant p53, which then transform into benign serous tubal intraepi-thelial lesions (STIL), progress into late pre-malig-nant serous tubal intraepithelial carcinoma (STIC), and ultimately lead to HGSOC. Here, we show that the transition from folded, soluble to aggregated mutant p53 occurs during the malignant transfor-mation of benign precursor lesions into HGSOC. We analyzed fallopian tube tissue collected from ten salpingo-oophorectomy cases and determined the proportion of cells carrying soluble versus mis-folded/mutant p53 through conformation-sensitive staining and quantification. Misfolded p53 protein, prone to aggregation, is present in STICs and HG-SOCs, but notably absent from pre-neoplastic le-sions and surrounding healthy tissue. Overall, our results indicate that aggregation of mutant p53 is a structural defect that distinguishes pre-neoplastic early lesions from late pre-malignant and malig-nant ones, offering a potential treatment window for targeting p53 aggregation and halting ovarian cancer progression.
{"title":"Mutant p53 Misfolding and Aggregation Precedes Transformation into High-Grade Serous Ovarian Carcinoma","authors":"Sara Sartini, Lexi Omholt, Neda Moatamed, Alice Soragni","doi":"10.1101/2024.09.17.612958","DOIUrl":"https://doi.org/10.1101/2024.09.17.612958","url":null,"abstract":"High Grade Serous Ovarian Cancer (HG-SOC), the most prevalent and aggressive gyneco-logical malignancy, is marked by ubiquitous loss of functional p53, largely due to point mutations that arise very early in carcinogenesis. These mu-tations often lead to p53 protein misfolding and subsequent aggregation, yet the alterations in in-tracellular p53 dynamics throughout ovarian can-cer progression remain poorly understood. HG-SOC originates from the fallopian tube epithelium, with a well-documented stepwise progression be-ginning with early pre-malignant p53 signatures. These signatures represent largely normal cells that express and accumulate mutant p53, which then transform into benign serous tubal intraepi-thelial lesions (STIL), progress into late pre-malig-nant serous tubal intraepithelial carcinoma (STIC), and ultimately lead to HGSOC. Here, we show that the transition from folded, soluble to aggregated mutant p53 occurs during the malignant transfor-mation of benign precursor lesions into HGSOC. We analyzed fallopian tube tissue collected from ten salpingo-oophorectomy cases and determined the proportion of cells carrying soluble versus mis-folded/mutant p53 through conformation-sensitive staining and quantification. Misfolded p53 protein, prone to aggregation, is present in STICs and HG-SOCs, but notably absent from pre-neoplastic le-sions and surrounding healthy tissue. Overall, our results indicate that aggregation of mutant p53 is a structural defect that distinguishes pre-neoplastic early lesions from late pre-malignant and malig-nant ones, offering a potential treatment window for targeting p53 aggregation and halting ovarian cancer progression.","PeriodicalId":501233,"journal":{"name":"bioRxiv - Cancer Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142249753","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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