bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1097: First-line therapy based on kinase inhibitors and antibodies prevents resistance in EGFR-mutated lung cancer
Authors: Marrocco Ilaria , Haga Yuya , Yarden Yosef
Published: June 2022

Introduction: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are commonly used to treat patients with EGFR+ non-small cell lung cancer (NSCLC). Unfortunately, despite the initial response, the long-term efficacy of the five clinically approved TKIs (i.e., gefitinib, erlotinib, afatinib, dacomitinib, osimertinib) is limited by the onset of resistance. Several mechanisms of resistance have been described, including the appearance of new EGFR mutations (e.g., T790M, C797S), activation of bypass pathways (e.g., AXL, HER2, MET and IGF1R) and phenotypic alterations (e.g., epithelial to mesenchymal transition, EMT). Additionally, in vitro treatment using EGFR TKIs leads to the establishment of drug tolerant persisters (DTPs), a cell population that might be responsible for the onset of resistance. In order to prevent resistance, the data we present herein propose to simultaneously target EGFR and other survival receptors (e.g., HER2 or HER3) by combining TKIs and specific monoclonal antibodies.

Materials and methods: Cultures of NSCLC cell lines, patient-derived and cell line xenografts were employed to test the efficacy of the drug combinations. In parallel, to better understand resistance to EGFR-TKIs we established NSCLC dacomitinib resistant (DR) cell lines that were used in vivo and in vitro.

Results: DR cells showed upregulation of AXL and IGF1R, features shared with the persister cells. We also demonstrate that combining TKIs and monoclonal antibodies successfully prevented TKI acquired resistance, by downregulating receptor kinases previously implicated in resistance to TKIs (e.g., MET and AXL), as well as by blocking pathways essential for mitosis (e.g., FOXM1).

Conclusions: Combining kinase inhibitors and antibodies in first line settings effectively avoids TKI resistance in animal models of lung cancer and might represent a long-term solution for EGFR positive NSCLC patients.

Citation Format: Ilaria Marrocco, Yuya Haga, Yosef Yarden. First-line therapy based on kinase inhibitors and antibodies prevents resistance in EGFR-mutated 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 1097.
Abstract 1107: Notch activation drives resistance to Kras(G12C) inhibition in lung adenocarcinoma
Authors: Hwang Elizabeth E. , Lee Alex , Leung Stanley , Breese Marcus , Sweet-Cordero Alejandro
Published: June 2022

Approximately one-third of lung adenocarcinoma (LUAD) tumors contain driver mutations in the Kras GTPase, most commonly occurring as Kras(G12C) activating mutations. Despite the promising development of covalent G12C small molecule inhibitors, early-phase clinical trial data indicate most patients will likely demonstrate disease progression through either intrinsic or acquired resistance. Thus, there is an urgent need to identify such resistance mechanisms to targeting Kras. Our lab previously discovered a unique requirement for Notch3 in tumor-propagating cells from the KrasLSL-G12D p53fl/fl LUAD mouse model as well as in patient-derived xenografts. Here we report activated Notch3 as a novel driver of resistance to Kras(G12C) inhibition in LUAD cell lines.

Aiming to study the Notch3 transcriptional signature in LUAD, we developed an inducible model of Notch3 intracellular domain (NICD) expression in Kras-mutant LUAD cell lines. Our data demonstrate that activated NICD modulates downstream RTK signaling pathways to suppress both cell cycle arrest and apoptosis in response to G12C inhibitor treatment. We subsequently performed RNA-sequencing to identify Notch-dependent genes driving resistance and in parallel, used Cut&Run technology to interrogate the global chromatin binding profiles of Notch3 and associated histone marks in NICD-expressing cell lines. We integrate our transcriptomic and epigenomic data to reveal that NICD acts as a key regulator of Rho family genes, which in turn support cell survival upon Kras inhibition. We additionally find enrichment of cell division and morphological signatures in inhibitor treated NICD cell lines and we elucidate the mechanism of direct Notch target genes in mediating the apoptotic response. Finally, we conduct a focused in vitro CRISPRi screen in the H358 LUAD cell line to identify the subset of Notch target genes required for NICD-induced resistance. Our results define a Notch3 transcriptional landscape in LUAD and demonstrate a role for Notch signaling in promoting resistance to small molecule Kras inhibitors.

Citation Format: Elizabeth E. Hwang, Alex Lee, Stanley Leung, Marcus Breese, Alejandro Sweet-Cordero. Notch activation drives resistance to Kras(G12C) inhibition in lung adenocarcinoma [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 1107.
Abstract 1132: A systems biology approach combining ProLiFiler and Cancer Data Miner for an enhanced preclinical characterization of the WEE-1 inhibitor Adavosertib
Authors: Vuaroqueaux Vincent , Feger Daniel , Peille Anne-Lise , Siedentopf Oliver , Panzade Sadhana , Ulrich Sarah , Dempe Sebastian , Fiebig Heinz-Herbert , Ehlert Jan Erik
Published: June 2022

Adavosertib (AZD1775, MK-1775), a clinical stage inhibitor of the tyrosine kinase WEE1, was investigated in a cell proliferation and survival assay with 140 human cancer cell lines (CLs) representing all major tumor types (ProLiFiler platform of Reaction Biology) followed by mechanism of action (MoA) and biomarker analyses using 4HF Biotec’s Cancer Data Miner in silico platform. Adavosertib exhibited a broad anti-cancer activity across all hematological and solid tumor types with IC50 values ranging from 0.06 to 10 µM (median: 0.38 µM), matching the consistently high expression of the WEE1 gene. Among 900 reference compounds, the activity profile of adavosertib correlated best with the profiles of compounds targeting the replication stress response including other WEE1 inhibitors but also inhibitors of checkpoint kinase 1 and 2 (CHK1/2) or ataxia telangiectasia-mutated (ATM). Significant correlations were also seen with compounds blocking mitosis, DNA replication and DNA repair. Interestingly, we observed a subset of cell lines that were resistant to both DNA synthesis and PARP inhibitors but were sensitive to WEE1 inhibition. Moreover, by using multiple datasets of WEE1 inhibitors connected to the molecular annotations of CLs for a data driven biomarker screening, we identified MYC mutations as a predictive marker of sensitivity and PIK3CA or ERBB2 gene amplifications as predictors of resistance. Transcriptome analysis identified up to 900 genes for which higher expression in CLs was associated with CL sensitivity to the compound. Preliminary pathway analysis indicated that these genes are well represented among nuclear factor and Myc-regulated genes. In conclusion, our studies demonstrate broad anticancer activity of adavosertib and confirm its proposed MoA. The biomarkers we identified will facilitate the selection of pre-clinical in vivo tumor models and, if confirmed, even patient selection for clinical trials. The combined use of the ProLiFiler and Cancer Data Miner Platforms has the potential to accelerate and de-risk the development of anti-cancer agents.

Citation Format: Vincent Vuaroqueaux, Daniel Feger, Anne-Lise Peille, Oliver Siedentopf, Sadhana Panzade, Sarah Ulrich, Sebastian Dempe, Heinz-Herbert Fiebig, Jan Erik Ehlert. A systems biology approach combining ProLiFiler and Cancer Data Miner for an enhanced preclinical characterization of the WEE-1 inhibitor Adavosertib [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 1132.
Abstract 1133: Novobiocin-mediated polymerase theta inhibition induces cGAS/STING pathway activation and T-cell infiltration in BRCA-associated cancers
Authors: Patterson-Fortin Jeffrey , Jadhav Heta , Phan Tin , D'Andrea Alan , Shapiro Geoffrey I.
Published: June 2022

Recently developed inhibitors of polymerase theta have demonstrated synthetic lethality in pre-clinical tumor models of BRCA-associated cancers. Given that other DNA damage repair inhibitor therapies, including PARP inhibitors, induce changes in the tumor immune microenvironment, we asked whether polymerase theta inhibition would also alter the tumor microenvironment in BRCA-associated cancers. We have demonstrated that novobiocin, a first-in-class inhibitor of the polymerase theta ATPase domain, induces both innate and adaptive immune responses through activation of the cGAS/STING pathway in BRCA-deficient tumor cells. Novobiocin-mediated polymerase theta inhibition generates micronuclei, activating the cGAS/STING pathway in both BRCA1-deficient MDA-MB-436 triple-negative breast cancer and BRCA2-deficient CAPAN1 pancreatic cancer models in vitro. Activation of the STING pathway upregulated the expression of interferon response genes, which was abrogated upon siRNA-mediated STING depletion. As an adaptive response, novobiocin also increased expression of PD-L1 in BRCA-deficient tumor cells. These effects were not observed in isogenic cell lines in which wild-type BRCA expression was corrected by add-back. Using a genetically engineered syngeneic mouse model of BRCA1-deficient breast cancer, we confirmed novobiocin-mediated activation of the cGAS/STING pathway in vivo, resulting in the induction of pro-inflammatory signaling and ultimately robust CD8+ T-cell infiltration and activation. Taken together, our findings demonstrate that novobiocin-mediated polymerase theta inhibition induces local and systemic immune responses in a cGAS/STING-dependent manner and provide a rationale for combining polymerase theta inhibition with immunotherapies for the treatment of BRCA-associated cancers.

Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Tin Phan, Alan D'Andrea, Geoffrey I. Shapiro. Novobiocin-mediated polymerase theta inhibition induces cGAS/STING pathway activation and T-cell infiltration in BRCA-associated cancers [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 1133.
Abstract 1168: From clinical mutational data to a novel lung cancer mouse model -NF1inactivation cooperates withRASA1inactivation to drive non-small cell lung cancer (NSCLC)
Authors: Song Lin , Rodriguez Thaidy Y. , Olshen Adam , Collisson Eric
Published: June 2022

11% of non-small cell lung cancer (NSCLC) patients harbor NF1 mutations. The enrichment of NF1 mutations in lung adenocarcinoma (LUAD) cases that otherwise lack activated oncogene mutations indicates that NF1 loss may play a necessary role in the activation of the RAS pathway. However, conditional, biallelic loss of Nf1 in the mouse lung is insufficient for LUAD initiation over 2 years, indicating additional genetic or epigenetic events cooperate with Nf1 loss in lung tumorigenesis. To better understand NF1 mutant lung cancers, we analyzed a lung cancer dataset with 47227 patients’ genomic mutation data from Foundation Medicine (FMI) and found that NF1 mutations (5270 patients) are associated with a higher mutational burden compared to NF1 wild type lung cancer patients in the lung cancer patients’ clinical database. We then analyzed the AACR GENIE non-small cell lung cancer datasets for genes mutated concurrently with NF1 mutation. We found that NF1 mutation statistically significantly cooccurs with RASA1 (p <0.001 for concurrency) and with TP53 mutations in the non-small lung cancer dataset. NF1-mutated samples lack other key driver oncogene mutations such as EGFR (p <0.001 for mutual exclusivity). To functionally validate our findings of genetic cooperativity, we conditionally deleted the two key GAPs: Nf1 and Rasa1 (with Trp53) in vivo. Deletion of both Nf1 and Rasa1 but neither the Nf1 or Rasa1 deletion alone lead to LUAD when combined with Trp53 deletion. Our new mouse model provides a unique tool for our ongoing effort to understand the mechanism of Nf1 & Rasa1 tumor initiation in the lung and will aid preclinical investigations of targeted therapies.

Citation Format: Lin Song, Thaidy Y. Rodriguez, Adam Olshen, Eric Collisson. From clinical mutational data to a novel lung cancer mouse model -NF1inactivation cooperates withRASA1inactivation to drive non-small cell lung cancer (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 1168.
Abstract 1200: COSMIC cancer mutation census: Classifying somatic coding variants by their potential to drive cancer
Authors: Sondka Zbyslaw , Harsha Bhavana , Pedro Helder , Dhir Nidhi Bindal , Hathaway Charlie , Nair Sumodh , Sondheimer Doron , Forbes Simon A.
Published: June 2022

Somatic mutations accumulate in cells throughout their life. Most of them do not bring any negative effect. However, certain mutations change protein behaviour, structure, or level of expression. More importantly, some mutations are known to initiate and drive oncogenic transformation. These mutations often make good therapeutic targets but recognising this small subset in a cancer sample is a major challenge. The average cancer cell carries a life-long baggage of somatic mutations, and the mutational process is sped up in these cells through genomic instability (one of the hallmarks of cancer). As a result, there are hundreds of thousands of variants of unknown significance identified through sequencing of cancer DNA. COSMIC Cancer Mutation Census (CMC) answers this challenge by identifying coding mutations with a potential to drive cancer. This is achieved by combining manually curated information regarding cancer genes and genetic variants with data on variant frequencies in cancer and non-cancer populations, and algorithmic evaluation of variant significance. It applies a simple and transparent set of rules to the whole set of coding mutations in COSMIC to identify variants with the highest potential of clinical relevance. In current version (v95, November 2021) the CMC describes 4.7 million somatic variants and segregates them into four tiers. Tier 1 is the highest confidence set. This set includes 1558 mutations that are found in Cancer Gene Census genes and are also described as pathogenic in cancer by ClinVar. Tiers 2 and 3 contain variants with less extensive evidence of involvement in carcinogenesis. The dN/dS algorithm is used to include variants that are under positive selection in cancer cells. Finally, mutations without evidence for driving cancer are classified as Tier 4. In addition to this classification, CMC integrates and presents the information used to prioritise variants, including their frequencies in various cancer types (COSMIC), germline frequencies (gnomAD), ClinVar annotations, dN/dS analysis results, and nucleotide and amino acid conservation. Data can be accessed and scrutinised through a dedicated website at https://cancer.sanger.ac.uk/cmc.

Citation Format: Zbyslaw Sondka, Bhavana Harsha, Helder Pedro, Nidhi Bindal Dhir, Charlie Hathaway, Sumodh Nair, Doron Sondheimer, Simon A. Forbes. COSMIC cancer mutation census: Classifying somatic coding variants by their potential to drive 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 1200.
Abstract 1210: Resolving clonal substructure from single cell genomic data in primary and metastatic tumors using CopyKit
Authors: Minussi Darlan Conterno , Wang Junke , Schalck Aislyn , Yan Yun , Wu Hua-Jun , Peng Cheng , Hu Min , Sei Emi , Edgerton Mary , Chen Hui , Contreras Alejandro , Hui David , Damodaran Senthil , Kopetz Scott , Lim Bora , Navin Nicholas
Published: June 2022

Advances in single cell copy number sequencing technologies have enabled the generation of data on hundreds to thousands of cells in parallel. Despite the rapid development in these technologies, there is a significant bottleneck for the analysis of the resulting large-scale datasets. Here we present CopyKit, a comprehensive and user-friendly toolkit for the analysis of single cell copy number data. CopyKit provides a suite of tools for pre-processing, QC, copy number inference, and subclone clustering to delineate the clonal diversity of tumors. We performed single cell copy number sequencing of 2977 cells from two breast tumor liver metastasis. CopyKit identified 4 and 12 subclonal populations, including amplification of PDGFRA, KRAS, and MYC as well as losses of PTEN, FOXO1, RB1, and BRCA1, many of which were spatially segregated in the tumor mass of the two tumors. Additionally, we applied CopyKit to study metastatic dissemination from a primary ER+ breast tumor with two matched metastatic sites and two colorectal carcinomas with matched liver metastatic tissues. This analysis demonstrated that the breast metastatic tumors selected for subclones that were lacking ERBB2 amplification in the primary site. Furthermore, the liver metastasis had deletions of chromosomes 18, 19, and 20p and a focal gain of FGFR1, while the pleural effusion acquired two additional focal gains on chromosome 8, including MYC. The colorectal metastatic tumors diverged from the primary tumor with copy number events affecting important cancer genes such as gain of SOX4, MYC, CDK8 and deletions of FHIT, CHEK1, and CHEK2. Collectively, this study shows that CopyKit provides a comprehensive set of tools for resolving clonal substructure from single cell copy number data for diverse applications in cancer biology.

Citation Format: Darlan Conterno Minussi, Junke Wang, Aislyn Schalck, Yun Yan, Hua-Jun Wu, Cheng Peng, Min Hu, Emi Sei, Mary Edgerton, Hui Chen, Alejandro Contreras, David Hui, Senthil Damodaran, Scott Kopetz, Bora Lim, Nicholas Navin. Resolving clonal substructure from single cell genomic data in primary and metastatic tumors using CopyKit [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 1210.
Abstract 124: PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic cancer
Authors: Malbeteau Lucie , Jain Pallavi , Harding Shane , Wouters Bradley , Koritzinsky Marianne
Published: June 2022

The prognosis of pancreatic ductal carcinoma (PDAC) remains dismal, with an overall 5-year survival rate of 7%. Immunotherapy (IT) has provided new hope for other hard-to-treat cancers, but PDAC has shown striking resistance to IT by orchestrating mechanisms of immune escape. This spurs an interest to identify novel strategies to improve PDAC sensitivity to IT. We recently unveiled that perturbating redox homeostasis by targeting the antioxidant protein peroxiredoxin 4 (PRDX4) render pancreatic cancer cells highly vulnerable. PRDX4 supports redox homeostasis by metabolizing H2O2 in the endoplasmic reticulum (ER), and its loss leads to oxidative stress and toxicity. Interestingly, cell death induced by PRDX4 depletion is accompanied by DNA damage and accumulation of cytosolic DNA, a potent primer for immune therapy. In line with that, PRDX4 knockdown results in the transcriptional upregulation of inflammatory genes, including type I interferons (IFNs), primary cytokines and interferon-stimulated genes (ISGs). Importantly, our data indicate that this induction is dependent on the critical regulator of the innate immune responses, STING, as treatment with a STING inhibitor, or a STING knockdown, substantially blunts their upregulation. It is known that following DNA damage detection, the ER-localized STING translocates to the Golgi, where it associates with the kinase TBK1 and mediates the induction of inflammatory target genes, depending on IRF3 and NF-κB transcription factors. We observe an activation of downstream STING effectors upon PRDX4 loss, and co-depletion with NFκB significantly rescues the upregulation of those genes. As such, we propose that PRDX4 links ER-specific redox vulnerability to innate immunity signaling in PDAC. Since PRDX4 loss causes increased DNA damage and inflammatory signaling, our preliminary data suggest that PRDX4 could be a novel therapeutic target that may act synergistically with immune checkpoint inhibitors.

Citation Format: Lucie Malbeteau, Pallavi Jain, Shane Harding, Bradley Wouters, Marianne Koritzinsky. PRDX4 links ER-specific redox vulnerability to innate immunity in pancreatic 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 124.
Abstract 126: Defining how oxidative stress drives the evolution of aggressive breast cancers
Authors: Mirabelli Caitlynn N. , La Selva Rachel , Heath John , Hébert Steven , Steinberger Jutta , Jiang Jialin , Kleinman Claudia , Huang Sidong , Ursini-Siegel Josie
Published: June 2022

Breast cancer is the most commonly diagnosed malignancy in women worldwide. It is a molecularly heterogeneous disease, which poses difficulties in the treatment of more advanced cancers. Despite this fact, the mechanisms contributing to the emergence of aggressive breast cancers remains poorly understood. We focus on reactive oxygen species (ROS), which are induced by a variety of stimuli in breast cancer cells. Moderately elevated ROS levels are tumor-promoting and facilitate the emergence of more aggressive tumours. Our laboratory has developed a unique model of HER2+ breast cancer that evolved to acquire more aggressive properties under conditions of chronic oxidative stress. We identified 20 transcription factors/co-regulators that were specifically overexpressed in these aggressive breast cancers, compared to their parental counterparts. We hypothesize that these transcription factors are central to the ability of breast tumors to adapt to chronic oxidative stress and acquire more aggressive properties. To test this, I have performed an in vivo functional shRNA screen to identify those transcriptional regulators that drive an aggressive phenotype. This will be followed by functional validation studies to interrogate whether and how these unique transcription factors overexpressed in aggressive breast cancers impact tumour growth, metastasis, and drug resistance. Mechanistically, we will explore whether and how these transcriptional responses mediate adaptive responses to tumor microenvironmental stressors, including altered metabolism, hypoxia, and tumor immune responses. This research will broaden our understanding of how breast cancers adapt to oxidative stress responses and represents a necessary first step in the development of novel therapeutics against these invulnerable malignancies.

Citation Format: Caitlynn N. Mirabelli, Rachel La Selva, John Heath, Steven Hébert, Jutta Steinberger, Jialin Jiang, Claudia Kleinman, Sidong Huang, Josie Ursini-Siegel. Defining how oxidative stress drives the evolution of aggressive breast cancers [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 126.
Abstract 127: Pre-exposure to stress-inducing agents increase the anticancer efficacy of focused ultrasound against aggressive prostate cancer cells
Authors: Murad Hakm Y. , Chandra Partha K. , Kelly Charles A. , Khurana Namrata , Yu Heng , Bortz Emma P. , Hong Shirley N. , Mondal Debasis , Khismatullin Damir B.
Published: June 2022

Despite the initial success in treatment of localized prostate cancer (PCa) using surgery, radiation or hormonal therapy, recurrence of aggressive tumors dictates morbidity and mortality. Focused ultrasound (FUS) is being tested as a targeted noninvasive approach to eliminate the localized PCa foci, and strategies to enhance the anticancer potential of FUS have a high translational value. Since aggressive cancer cells utilize oxidative stress (Ox-stress) and endoplasmic reticulum stress (ER-stress) pathways for their survival and recurrence, we hypothesized that pre-treatment with drugs that disrupt stress-signaling pathways in tumor cells may increase FUS efficacy. Using four different PCa cell lines, i.e. LNCaP, C4-2B, 22Rv1 and DU145, we tested the in vitro effects of FUS, alone and in combination with two clinically-tested drugs that increase Ox-stress (i.e. CDDO-me) or ER-stress (i.e. Nelfinavir). As compared to standalone FUS, significant (p<0.05) suppressions in both survival and recurrence of PCa cells were observed following pre-sensitization with low dose CDDO-me (100 nM) and/or nelfinavir (2 µM). In drug pre-sensitized cells, significant anticancer effects were evident at a FUS intensity of as low as 0.7 kW/cm2. This combined mechanochemical disruption (MCD) approach decreased cell proliferation, migration and clonogenic ability, and increased apoptosis/necrosis and reactive oxygen species (ROS) production. Furthermore, although activated in cells that survived standalone FUS, pre-sensitization with CDDO-me and/or nelfinavir suppressed both total and activated (phosphorylated) NF-κB and Akt protein levels in surviving cells. Thus, a combined MCD therapy may be an effective noninvasive approach towards targeted elimination of aggressive PCa cells.

Citation Format: Hakm Y. Murad, Partha K. Chandra, Charles A. Kelly, Namrata Khurana, Heng Yu, Emma P. Bortz, Shirley N. Hong, Debasis Mondal, Damir B. Khismatullin. Pre-exposure to stress-inducing agents increase the anticancer efficacy of focused ultrasound against aggressive prostate 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 127.
Abstract 1283: Single intra-tumoral injection of gaseous nitric oxide induces an adaptive immune response in a mouse CT-26 solid tumor model
Authors: Confino Hila , Goldshtein Matan , Levi Yakir , Malka Lidor , Avniel Amir , Lisi Steve , Wolf Ido
Published: June 2022

Background: Metastases are responsible for a significant portion of the morbidity and mortality in cancer patients. In situ destruction of tumor mass has been reported to provide the immune system with an antigen source for the induction of antitumor immunity, which can then attack distant metastases. Nitric oxide (NO) acts as a signaling molecule in multiple disease states, including cancer. NO has been shown to activate innate and adaptive responses of the immune system against tumors. In a previous Beyond Air study, 89% of CT26 tumor bearing mice treated with 50,000 parts per million (ppm) gaseous NO (gNO), rejected a secondary tumor induction. Here, we assessed the effects of a single intra-tumoral injection of gNO at concentrations of 20,000-50,000 ppm on immune cell recruitment in the tumor, spleen, and blood.

Methods: Mouse colon carcinoma (CT26) tumor bearing Balb/c mice underwent a single intra-tumoral treatment with 20,000 (n=3) or 50,000 (n=4) ppm gNO for 5 minutes. Mice treated with N2 (n=3), under the same conditions, served as a control. Tumors were removed 14 days post treatment and were histologically analyzed to test for immune cell penetration, inflammation, and necrosis. Immune cells from the spleen and blood were harvested 21 days post treatment and assessed via flow cytometry.

Results: 1. Treatment with 50,000 ppm gNO resulted in higher levels of inflammation, lymphocytes and T-cell infiltration, and necrosis within the tumor compared to both N2 and 20,000 ppm NO, as viewed histologically. 2. Flow cytometry analysis of immune cells from the spleen revealed a 1.3-fold increase of both B cells and IFN-γ expressing T cells with 50,000 NO treatment, and a 1.3-fold increase of B cells and 1.1-fold increase of IFN-γ expressing T cells with 20,000 NO treatment, compared to N2. 3. A 29% reduction of myeloid-derived suppressor cells (MDSCs) was detected in spleens of mice treated with 50,000 ppm gNO, and 40% for 20,000 ppm, compared to mice treated with N2.

Conclusion: Intra-tumoral injection of ultra-high doses of NO at 20,000 and 50,000 ppm gNO led to an increased recruitment and concentration of T and B cells. Additionally, 50,000 ppm NO led to an increase of lymphocyte infiltration to the tumor. Taken together, the data suggest that the increase in lymphocyte infiltration, B and T cell population, and the reduction of MDSCs, are indicative of an adaptive immune response that results in rejection of secondary tumors in gNO-treated mice.

Citation Format: Hila Confino, Matan Goldshtein, Yakir Levi, Lidor Malka, Amir Avniel, Steve Lisi, Ido Wolf. Single intra-tumoral injection of gaseous nitric oxide induces an adaptive immune response in a mouse CT-26 solid tumor model [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 1283.
Abstract 1285: Chemotherapy induces PD-L1 overexpression in CRC through NF-κBp65
Authors: Fan Fan , Ghosh Susmita , Ellis Lee , Bhattacharya Rajat
Published: June 2022

Background: The programmed cell death ligand (PD-L1) is an immune-inhibitory molecule that suppresses the activation of T cells, leading to the survival and growth of tumors. Overexpression of PD-L1 on cancer cells is associated with poor clinical outcomes in many types of cancers. PD-L1 upregulation contributes to chemoresistance in several types of cancer, but little is known about the mechanisms for alterations in PD-L1 expression associated with chemotherapy, especially in patients with CRC. Therefore, a better understanding of the mechanisms underlying changes in PD-L1 levels in response to standard of care chemotherapeutics in CRC is important. In this study, we examined the effects of thechemotherapeutics that are approved for the treatment of patients with CRC (oxaliplatin, 5FU, and irinotecan whose metabolite is SN38) in inducing PD-L1 expression and determining the molecular pathwaysinvolved in PD-L1 regulation.

Methods: PD-L1 expression in CRC cell lines was determined in vitro using western blotting and FACS analysis. Changes in PD-L1 transcript levels were measured using real time PCR. RPPA analyses were performed to identify proteins/pathways affected by chemotherapeutics. Pharmacological inhibitors and siRNAs were used to target specific proteins in determining their role in PD-L1 expression.

Results: We observed that oxaliplatin, 5FU, and SN38, resulted in induction of PD-L1 to various degrees. Interestingly this induction was observed in KRAS or BRAF mutated CRC cells but not in CRC cells with wild-type KRAS and BRAF. Increased levels of PD-L1 were due to increased transcription of PD-L1 resulting from exposure to chemotherapeutics. Chemotherapeutics lead to the activation of the DNA damage signaling mediator Chk1 and the downstream transcription activator NF-κBp65. Inhibition of Chk1 or NF-κBp65 resulted in abrogated PD-L1 induction in response to chemotherapeutic treatment.

Conclusion: PD-L1 can be induced following treatment with chemotherapy in CRC cells harboring mutations in BRAF or KRAS. Our findings provide a rationale for studies combining inhibitors of NF-κBp65 with standard of care chemotherapeutics in CRC to block PD-L1 expression and enhance the efficacy of chemotherapy damaged-tumor cell clearance by innate immunity that, in turn, should improve patient outcomes.

Citation Format: Fan Fan, Susmita Ghosh, Lee Ellis, Rajat Bhattacharya. Chemotherapy induces PD-L1 overexpression in CRC through NF-κBp65 [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 1285.
Abstract 1299: Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent cells
Authors: Chaturvedi Pallavi , George Varghese , Liu Bai , Shrestha Niraj , Wang Meng , Dee Micheal , Zhu Xiaoyun , Egan Jack , Jiao Jin-an , Spanoudis Catherine , Xing Jilan , Gallo Victor , Echeverri Christian , You Lijing , Kong Lin , Muniz Gabriela , Rhode Peter , Wong Hing
Published: June 2022

Advances in immunostimulatory and anti-immunosuppressive therapeutics have revolutionized cancer treatment. Here, we report a novel heterodimeric bifunctional fusion molecule, HCW9218, constructed using our soluble tissue factor (TF)-based scaffold technology comprising extracellular domains of the human transforming growth factor-β (TGF-β) receptor II and a human interleukin (IL)-15/IL-15 receptor α complex. This fusion complex exhibited TGF-β neutralizing activity in vitro and sequestered plasma TGF-β in vivo. Subcutaneous administration of HCW9218 was well tolerated in mice, with a half-life sufficient to provide long lasting biological activity. HCW9218 enhanced metabolic and cytotoxic activities of immune cells (CD8+ T cells, NK cells) and reduced tumor progression in models of chemotherapy induced senescence (TIS, docetaxel for B16F10 tumors and gemcitabine plus nab-paclitaxel for SW1990 tumor models) and non-TIS in vivo. Mechanistically, HCW9218 treatment not only affected the immunosuppressive tumor microenvironment but also enhanced CD8+ T cells and NK cells infiltration and cytotoxicity in the tumors to eliminate TIS cancer cells. Immune-depletion studies showed that HCW9218-activated NK cells played a pivotal role in TIS cancer cell removal. HCW9218 treatment following docetaxel chemotherapy further enhanced efficacy of tumor antigen-specific and anti-PDL-1 antibodies in B16F10 tumor-bearing mice. We also show that HCW9218 treatment lowered senescence associated secretory phenotype (SASP) factors in off-target tissues in chemotherapy treated tumor-bearing mice. Thus, HCW9218 may serve as a novel therapeutic (monotherapy or in combination with chemotherapy) to simultaneously provide immunostimulation and lessen immunosuppression associated with tumors. HCW9218 has been cleared by the U.S. Food and Drug Administration to proceed for evaluation in a first-inhuman Phase 1b clinical trial in patients with advanced pancreatic cancer.

Citation Format: Pallavi Chaturvedi, Varghese George, Bai Liu, Niraj Shrestha, Meng Wang, Micheal Dee, Xiaoyun Zhu, Jack Egan, Jin-an Jiao, Catherine Spanoudis, Jilan Xing, Victor Gallo, Christian Echeverri, Lijing You, Lin Kong, Gabriela Muniz, Peter Rhode, Hing Wong. Immunotherapeutic hcw9218 enhances anti-tumor activity of chemotherapy by facilitating immune-mediated elimination of therapy induced senescent 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 1299.
Abstract 13: Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro
Authors: Davidson Brad A. , Miranda Adam X. , Croessmann Sarah , Park Ben Ho
Published: June 2022

Despite the well-documented benefits of circulating tumor DNA (ctDNA) based liquid biopsy, including screening, prognostication, and treatment monitoring, there remain several concerns with its application. Chief among these concerns are imperfect sensitivity and specificity, particularly pre-diagnosis and after tumor resection, when ctDNA burden is low. Cell death, either through apoptosis or necrosis, has been the most widely accepted mechanism of DNA release. Recent studies have also suggested active release through extracellular vesicles as a paradigm for DNA release. However, this pathway remains highly debated in terms of its contribution, mechanism, and even credibility. To address the deficiencies in ctDNA detection in the clinic and the lack of basic mechanistic knowledge of cf/ctDNA release, we performed an in vitro CRISPR screen on MCF10A cells to identify regulators of DNA release. This normal breast epithelial cell line was chosen because MCF10A cells release DNA into cell culture media with a similar fragmentation pattern to that observed in human blood. After lentiviral infection with the genome-wide Brunello CRISPR-Cas9 knockout library, the population of knockouts identified in the genomic DNA of cells attached to the plates was compared to the population of knockouts found in the DNA released into the cell-depleted culture media. Genes with skewed representation were considered potential regulators of DNA release. Our analysis identified several genes in the TRAIL apoptotic pathway including FADD, as well as several genes encoding RNA-binding proteins including Sam68. Knockout of Sam68 and FADD in MCF10As, MDA-MB-468 breast cancer cells, and NCI-H841 lung cancer cells lead to decreases in DNA release. Previous studies have hypothesized that the fragment size of released DNA is dependent on release mechanism. However, the decreases observed in our knockout cell lines were not limited to a specific fragment size of DNA. Application of the small molecule apoptosis inhibitor Z-VAD-FMK also recapitulated the DNA release phenotype seen in our knockout cell lines. From these results, we conclude that apoptosis is the major source of DNA release from cancer cells, and that fragment size is not the indicator of release mechanism it has been presumed to be. Mechanistic studies of DNA release such as this could lead to the ability to modify the amount of ctDNA in circulation, which would significantly improve accurate detection in the clinic.

Citation Format: Brad A. Davidson, Adam X. Miranda, Sarah Croessmann, Ben Ho Park. Novel CRISPR screening approach identifies regulators of cell-free DNA release in vitro [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 13.
Abstract 1300: Targeted therapies prime lung cancer cells for macrophage-mediated destruction
Authors: Vacarro Kyle , Allen Juliet , Maoz Asaf , Reeves Sarah , Hata Aaron , Weiskopf Kipp
Published: June 2022

The CD47/SIRPa axis is an immune checkpoint that regulates macrophage anti-tumor function. Therapies that block CD47 on cancer cells show promise in clinical trials for solid and hematologic malignancies, particularly when combined with other anticancer agents. However, the best combination strategies for using CD47-blocking therapies remain unknown.

In this study, we developed a novel in vitro screening platform to identify drugs that render cancer cells more vulnerable to macrophage attack. We performed an unbiased screen of 800 FDA-approved drugs using primary human macrophages and PC9 cells, a human EGFR mutant lung cancer cell line. We identified EGFR tyrosine kinase inhibitors (TKIs) as drugs that act on the cancer cells and specifically enhance macrophage-mediated cytotoxicity (>4-fold enhancement, p<0.01). In contrast, conventional chemotherapy drugs either showed no significant enhancement or abrogated macrophage activation. The combination of EGFR TKIs with anti-CD47 antibodies elicited maximal phagocytosis across a range of cell lines and conditions. In long-term co-culture assays with macrophages, the combination of EGFR TKIs and anti-CD47 antibodies eliminated persister cells to prevent TKI resistance. Importantly, these findings extended to lung cancers with other RTK-MAPK pathway mutations, such as ALK-rearranged cancers (treated with lorlatinib, alectinib, or crizotinib) or KRAS G12C mutant cancers (treated with sotorasib or adagrasib). In xenograft and syngeneic mouse models, the combination of targeted therapies with CD47 ablation was able to dramatically reduce tumor burden.

To understand the mechanism of synergy, we generated 8 different lung cancer cell lines resistant to EGFR, ALK, or KRAS inhibitors. The resistant lines significantly upregulated CD47 and concomitantly became more sensitive to macrophage attack in vitro and in vivo. By RNA sequencing, we identified multiple mechanisms contributing to vulnerability, including secretion of the cytokine MIP-3 by the cancer cells and alteration of other immunoregulatory molecules. Overall, we have identified a novel therapeutic strategy to enhance the efficacy of RTK-MAPK pathway inhibitors by combining them with anti-CD47 therapies.

Our findings suggest cross-sensitivity occurs such that lung cancer cells that become resistant to targeted therapies also become more sensitive to macrophage attack. Our findings provide rationale for testing this combination approach in patients with lung cancers bearing driver mutations.

Citation Format: Kyle Vacarro, Juliet Allen, Asaf Maoz, Sarah Reeves, Aaron Hata, Kipp Weiskopf. Targeted therapies prime lung cancer cells for macrophage-mediated destruction [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 1300.
Abstract 1339: Interleukin 4 Producing CD4 T cells initiate a crucial event for lung cancer development
Authors: Jogdand Gajendra , Banerjee Sayantan , Shi Gongping , Brown Jami Willet , Singh Amit , Zhao Yongmei , Shetty Jyoti , Tran Bao , Hu Yinling
Published: June 2022

Lung cancer remains one of the leading causes of mortality of cancer patients. Kinase-dead Ikkα knock-in (KA/KA) mice develop CD4 T cell-derived autoimmune disease. Previously, we reported spontaneous lung squamous cell carcinoma (SCC) associated with increase macrophage and CD4 T cell numbers in KA/KA mice. Depleting macrophages dampens lung SCC development in KA/KA mice, but a lot of CD4 T cells remain in the lungs. The activity of CD4 T cells in lung SCC development is unknown. In this study, we attempted to investigate the role of CD4 T cells in lung SCC development. We performed adoptive CD4 T cell transfer experiment and found that KA/KA CD4 T cell transfer, not WT CD4 T cells, recruited marked macrophages to the lungs and induced lung SCC in KA/KA;Rag1-/- mice. The CD4 T cells isolated from the lungs of KA/KA mice and KA/KA;Rag1-/- mice with KA/KA CD4 T cell transfer expressed elevated PD-1, IL-4, IL-5, Il-13, and Il-21 levels compared to WT mice. We found that Il4 ablation, not Il5 deletion, reduced macrophage recruitment and blocked SCC development in KA/KA;Il4-/- mice as well as unlike KA/KA CD4 T cells, KA/KA;Il4-/- CD4 T cells did not induce spontaneous lung SCC in KA/KA;Rag1-/- mice with KA/KA;Il4-/- CD4 T cell injections. Taken together, the finding highlights a pathogenic role of IL-4-producing CD4 T cells in lung carcinogenesis.

Citation Format: Gajendra Jogdand, Sayantan Banerjee, Gongping Shi, Jami Willet Brown, Amit Singh, Yongmei Zhao, Jyoti Shetty, Bao Tran, Yinling Hu. Interleukin 4 Producing CD4 T cells initiate a crucial event for lung cancer development [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 1339.
Abstract 137: Abrogating therapy induced senescence driven bone loss to improve quality of life
Authors: Raut Ganesh Kumar , Yao Zhangting , Cole Tom , Luo Xainmin , Ren Qihao , Stewart Sheila A.
Published: June 2022

Despite the central role chemotherapy plays in prolonging survival, the toxicities associated with its use can negatively impact quality of life and in some cases, be so severe that patients forego further life preserving treatments. Therefore, it is critical that we understand the mechanisms that drive these toxicities and develop approaches to mitigate their severity. Recently we demonstrated that chemotherapy-induced senescence drives bone loss by both limiting mineralization of new bone and increasing bone resorption. To establish whether senescent bone resident cells or systemic responses to chemotherapy drove therapy-induced bone loss, we used a vossicle model in which neonatal vertebral bones (L4 and L5) were transplanted from 4-day old wildtype or INKATTAC pups (allow to selectively kill senescent cells) into wildtype or INKATTAC adult mice. Using this approach, we found that the elimination of senescent cells in donor vossicles protects from chemotherapy-induced bone loss, indicating that senescent bone resident cells are responsible for therapy-induced bone loss. To understand the mechanism(s) that contributed to therapy-induced bone loss, we used scRNA-seq to determine which bone resident cells underwent senescence in response to chemotherapy and how their gene expression was impacted. Using this approach, we found that adipocytes, fibroblast, chondrocytes and osteoblasts underwent senescence as evidenced by expression of p16, p21 and loss of Mki67 and displayed evidence of a senescence associated secretory phenotype (SASP), which we postulate contributes to therapy-induced bone loss. Collectively, our data indicated that chemotherapy causes bone loss via senescence and can be protected by eliminating senescent cells.

Citation Format: Ganesh Kumar Raut, Zhangting Yao, Tom Cole, Xainmin Luo, Qihao Ren, Sheila A. Stewart. Abrogating therapy induced senescence driven bone loss to improve quality of life [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 137.
Abstract 1376: Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions
Authors: Roetman Jessica J. , Erwin Megan M. , Apostolova Minna K. , Philip Mary
Published: June 2022

T cells have the potential to carry out potent anti-tumor responses. Most studies examining T cell-cancer interactions have been directed toward late-stage tumors, but much less is known about how T cells respond to premalignant lesions. Using a clinically relevant liver cancer mouse model (Albumin-Stop-Large T Antigen; AST) in which the SV40 Large T Antigen (TAG) serves as oncogene and tumor neoantigen, we previously demonstrated that TAG-specific CD8 T cells (TCR-TAG) rapidly become dysfunctional (unable to produce cytokines and cytolytic molecules) after entering premalignant liver lesions, although they persist long-term. We hypothesized that peripheral tolerance mechanisms, which prevent autoimmunity against self-antigens, drive T cell dysfunction in premalignant lesions. To test this, we compared CD8 T cell (TCR-GAG) responses to a liver self-antigen (GAG) to TCR-TAG responses to the TAG neoantigen in premalignant hepatocytes. We found that liver-reactive TCR-GAG were unable to produce cytokines, similar to neoantigen-specific TCR-TAG; however, in contrast, they failed to persist. To test whether oncogenic transformation of hepatocytes promoted T cell persistence, we crossed Alb-GAG and AST mice to generate a model in which GAG and TAG was expressed in premalignant hepatocytes (ASTxAlb-GAG). We found that both TCR-GAG and TCR-TAG transferred into ASTxAlb-GAG mice bearing premalignant lesions driven by the TAG oncogene rapidly lost the ability to produce cytokines; however, only TCR-TAG persisted while TCR-GAG quickly disappeared. To understand why the TCR-GAG failed to persist, we assessed proliferation and cell cycling after T cells encountered their cognate antigen. While both TCR-GAG and TCR-TAG were rapidly activated and began proliferating, TCR-GAG exited cell cycle and began decreasing in number after four cell divisions, while TCR-TAG continued to divide and increase in number. Antigen amount and localization may regulate T cell proliferation and persistence. We found much higher levels of TAG-encoding versus GAG-encoding mRNA in the liver, suggesting that higher antigen expression may be required for T cell persistence. T cell responses can be directed against both shared tumor self-antigens and neoantigens. By dissecting the determinants of CD8 T cell function and persistence in response to self and non-self-antigens, these studies could inform strategies to boost anti-tumor CD8 T cell responses in cancer patients.

Citation Format: Jessica J. Roetman, Megan M. Erwin, Minna K. Apostolova, Mary Philip. Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions [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 1376.