bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6100: Chemotherapy reduces TGF-beta signaling triggered by blockade of the PD-1/PD-L1 axis
Authors: Iida Masafumi , Horn Lucas A , Qin Haiyan , Fousek Kristen , Schlom Jeffrey , Palena Claudia
Published: June 2022

Combination of immune checkpoint blockade (ICB) with chemotherapy is becoming more commonly used for the treatment of various cancer types, including advanced non-small cell lung cancer, and triple negative breast cancer (TNBC) in patients who have PD-L1 positive tumors, among others. Although chemotherapy-ICB modalities have been shown to improve clinical responses over those observed with ICB alone, long-term disease control is achieved only in a small subset of patients. To provide a rational basis for combination therapy, and to potentially develop more effective approaches, in this study we investigated the impact of chemotherapy on the tumor microenvironment (TME) in the context of PD-1/PD-L1 axis blockade. ICB refractory murine models were utilized, including the TNBC 4T1 model and the Lewis Lung Carcinoma (LLC) model. Following treatments with anti-PD-L1, docetaxel, or the combination anti-PD-L1 plus docetaxel, tumors were collected 4 days after the last treatment to evaluate early changes in the TME. Analyses included the characterization of tumor immune infiltrates via flow cytometry, RNA expression profiling via RT-PCR, RNA in situ hybridization, and immunohistochemistry for detection of immune infiltrates, cancer-associated fibroblasts, and phospho-Smad2 signaling. RNA analysis of 4T1 and LLC tumors revealed a significant upregulation of several transforming growth factor-beta (TGF-beta) downstream target genes in the anti-PD-L1 monotherapy group. This increase was associated with enhanced expression of TGF-beta in the TME, as determined by RNA in situ hybridization, together with enhanced TGF-beta signaling denoted by increased expression of phospho-Smad2. Tumors treated with anti-PD-L1 monotherapy also exhibited increased numbers of alpha-smooth muscle actin positive fibroblasts in the TME. The effects observed with anti-PD-L1 as a monotherapy were not observed in tumors treated with docetaxel monotherapy or in those treated with the combination docetaxel plus anti-PD-L1 therapy, suggesting that upregulation of immunosuppressive TGF-beta signaling in the TME could be prevented by the addition of chemotherapy. Ongoing and future studies are aimed at understanding the mechanisms involved in the upregulation of TGF-beta signaling in the TME in response to anti-PD-L1 therapy in non-responsive tumors, the effect of various chemotherapies in this phenomenon, and the study of additional tumor models for optimization of chemotherapy-ICB modalities.

Citation Format: Masafumi Iida, Lucas A Horn, Haiyan Qin, Kristen Fousek, Jeffrey Schlom, Claudia Palena. Chemotherapy reduces TGF-beta signaling triggered by blockade of the PD-1/PD-L1 axis [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 6100.
Abstract 6102: Single-cell RNA sequencing of lung adenocarcinoma patients reveals age-dependent tumour microenvironment alterations that facilitate response to immunotherapy
Authors: Zhu Xiaoqiang , Wang Hai , Pan Xuefeng , Hong Jie , Wong Jason Wing Hon , Chen Haoyan , Zhang Youwei
Published: June 2022

Background: Immunotherapy has revolutionized the treatment of lung adenocarcinoma (LUAD). While the increase in cancer incidence with age and age-associated changes in immune function have been well characterized, age-related differences in the intratumoral immune populations and how this affects immunotherapy response remain unknown.

Methods: We collected clinical data from 3,544 samples treated with immune checkpoint blockade (ICB) at the pan-cancer level. Single-cell RNA sequencing data from 19 LUAD samples were integrated and analyzed. Cell types were identified based on canonical marker gene expression.

Results: Aged individuals responded more efficiently to ICB treatment at the pan-cancer level. By focusing on LUAD, we showed that patients aged above 46 responded better and had longer progression-free survival than younger counterparts. Further, we presented a 58,031-cell catalog of age-associated single-cell transcriptomes from LUAD patients. Our analysis revealed the accumulation of immune response and chronic inflammation with increasing age. Aged tissues (> 60 years) displayed dominant exhausted natural killer (NK)/T cells and T regulatory cells and were enriched with two subtypes of tumour-associated macrophages marked by SPP1 and CX3CR1 expression, respectively. Furthermore, the ageing dynamics of stroma cells created a pro-tumor microenvironment reflected by increased angiogenesis and collagen formation.

Conclusions: These results revealed an increasing immune-suppressive tumour microenvironment with age, supporting an observation of improved ICB response in older LUAD patients. Our extensive single-cell analysis enhances the understanding of age-dependent molecular and cellular dynamics in LUAD. Our results highlight the importance of considering age as a factor for ICB response and provide a rich resource for developing therapeutic targets in LUAD-microenvironment interactions.

Citation Format: Xiaoqiang Zhu, Hai Wang, Xuefeng Pan, Jie Hong, Jason Wing Hon Wong, Haoyan Chen, Youwei Zhang. Single-cell RNA sequencing of lung adenocarcinoma patients reveals age-dependent tumour microenvironment alterations that facilitate response to immunotherapy [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 6102.
Abstract 6191: A novel anti-IL33 antibody that neutralized Th1-type and Th2-type pro-inflammation responses induced by IL-33/ST2 axis pathway
Authors: Zhang Xuesai , Li Qingrou , Liu Cui , Zhao Le , Wang Lujie , Deng Lan , Xu Wei , song Hongli , Zhu Zhenping , Huang Haomin
Published: June 2022

IL-33, the ligand for ST2, is a member of IL-1 family. IL-33 was described to activate many immune cell types involved in type-2 immunity and allergic inflammation. Here we describe a novel anti-IL33 humanized monoclonal antibody, SSGJ-621, which bound IL-33 with high affinity and blocked the interaction between IL-33 and ST2. Our study has shown that SSGJ-621 was capable of effectively neutralizing IL-33-induced IL-6 secretion from HUVECs and IL-33-dependent IFNγ production in PBMCs and NK cells, suggesting a role that IL-33 might be involved in Th1-type of immunity. In addition, SSGJ-621 also exhibited potent inhibitory effects on IL-33 induced IL-5 and IL-13 production in KU812 cells. In vivo study demonstrated that SSGJ-621 can inhibit the activation of splenocytes in spleen and the production of eosinophils and IL-5 in peripheral blood. Thereby, our results highlight the dual functions of IL-33 in promoting Th1 and Th2 immune responses and SSGJ-621 has potentials to treat not only chronic inflammatory diseases induced by IL-33, but also cancers by favoring Th1 antitumor immunity in the tumor microenvironment.

Citation Format: Xuesai Zhang, Qingrou Li, Cui Liu, Le Zhao, Lujie Wang, Lan Deng, Wei Xu, Hongli song, Zhenping Zhu, Haomin Huang. A novel anti-IL33 antibody that neutralized Th1-type and Th2-type pro-inflammation responses induced by IL-33/ST2 axis pathway [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 6191.
Abstract 6197: The role of the integrated stress response in pancreatic cancer adaptation to tumor microenvironmental nutrient stress
Authors: Cai Chufan
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, with a five-year survival rate ~10%1. PDAC is characterized by an altered/abnormal nutrient tumormicroenvironment (TME)2, and cancer cells must adapt metabolically, which exposes potentialtherapeutic targetable vulnerabilities. Therefore, we seek to identify metabolic pathways thatallow PDAC adaptation to such abnormal TME. The integrated stress response (ISR) is anintracellular stress response pathway that senses diverse stressors, and subsequently initiatesdownstream stress adaptations3. We find both murine and human PDAC have elevatedexpression of ATF4, the most well-characterized ISR effector, compared to paired healthypancreas by immunohistochemistry staining. Further, I find the ISR is activated and critical forPDAC survival/proliferation upon physiological PDAC TME nutrient stress. What is triggering theISR activation in PDAC? Through a variety of studies, I determined that it was the amino acidstress, specifically the depletion of arginine and asparagine, that activated the ISR upstreamkinase GCN2, which is responsible for ATF4 upregulation upon PDAC TME nutritional stress.Through CRISPR-based competition assays, I determined that ATF4 activity was critical forPDAC cells to cope with the depletion of arginine and asparagine, and ATF4 activity wasdispensable when these two amino acids were supplied even though cultured in PDACphysiological TME conditions. Currently, I am seeking to validate these findings in murine PDACmodels, and to determine if ATF4 or GCN2 could be relevant therapeutic targets, using an invivo tumor competition assay that I am developing. Altogether, I find the ISR is active in PDACboth ex vivo and in vivo, and suggest that the ISR is critical for PDAC survival/proliferation byallowing adaptation to abnormal PDAC amino acid TME. This work suggests that the ISR is anovel therapeutic target for PDAC treatment, which ultimately can benefit PDAC patients.

References: 1. Cancer Facts & Figures 2021. https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2021.html. 2. Sullivan, M. R. et al. Quantification of microenvironmental metabolites in murine cancersreveals determinants of tumor nutrient availability. Elife 8, (2019).3. Pakos-Zebrucka, K. et al. The integrated stress response. EMBO Rep. 17, 1374-1395(2016).

Citation Format: Chufan Cai. The role of the integrated stress response in pancreatic cancer adaptation to tumor microenvironmental nutrient stress [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 6197.
Abstract 6209: TumOC: A tumor organoid-on-chip platform for real-time cell vitality measurements
Authors: Flechner Marie , Loskutov Juergen , Pfohl Ulrike , Osman Katja , Regenbrecht Christian R. , Uhlig Katja , Wedeken Lena
Published: June 2022

Colorectal carcinomas (CRC) are one of the most common malignant tumors. Due to its heterogeneity and high probability to metastasize, the disease is often incurable and novel, improved therapies are needed. Enhanced, physiologically relevant in-vitro tumor models significantly increase the success rate in new drug development, reduce the number of animal experiments and result finally in better therapies for cancer patients. Furthermore, they have the potential to offer new approaches to personalized oncology. In recent years, 3D cell cultures were established as relevant preclinical cancer models, suitable for high-throughput-screening. However, their capacity to model and/or measure physiological processes can still be enhanced. In this project, we developed a tumor organoid-on-chip platform (TumOC) by combining most recent technological advances: We integrated patient-derived 3D cell cultures (organoids) from colorectal carcinomas and microsensor particles measuring oxygen concentration and therefore allowing assessment of cell vitality in real time in a microfluidic system. The resulting TumOC platform allows for defined exposition of cytostatic drugs including dynamic treatments. Measurements of cell vitality in real time enables analysis of not only the final effect of a drug treatment, but also the kinetics of drug response. At the same time, utilizing organoids allows for recapitulating tumor architecture and heterogeneity. Here we report the first proof of principle results generated with this system. We measured the cell vitality in real time over 72 hours during treatment with classic chemotherapeutics or targeted cancer drugs and compared the results to end-point measurements on the same organoids in a static system. In conclusion, our TumOC platform with its ability to recapitulate tumor heterogeneity in combination with dynamic treatments and real-time cell vitality assessment is an in-vitro tumor model closely recapitulating the physiological situation in a patient. TumOC provides an impressive opportunity to test and, consequently, predict the effectiveness of anti-cancer therapies. Therefore, this system is of interest not only for pre-clinical drug development but also for personal oncology.

Citation Format: Marie Flechner, Juergen Loskutov, Ulrike Pfohl, Katja Osman, Christian R. Regenbrecht, Katja Uhlig, Lena Wedeken. TumOC: A tumor organoid-on-chip platform for real-time cell vitality measurements [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 6209.
Abstract 6215: The immune cell atlas of human neuroblastoma
Authors: Verhoeven Bronte Manouk , Mei Shenglin , Olsen Thale K. , Gustafsson Karin K. , Valind Anders , Lindström Axel , Nord David G. , Fard Shahrzad S. , Hagerling Catharina , Kharchenko Peter V. , Kogner Per , Johnsen John I. , Baryawno Ninib
Published: June 2022

Our current knowledge of the different immune cells in neuroblastoma is based on in vitro and in vivo studies mainly focusing on a single cell type. Different studies have conveyed conflicting results and despite the use of anti-GD2 immunotherapy in the clinic, a comprehensive immune cell overview at the single-cell level is still missing and understanding the complete composition of immune cells neuroblastoma will be crucial for the development of novel immunotherapeutics against the disease. In this study, we performed single-cell RNA-sequencing on human neuroblastoma coupled with multiplex immunohistochemistry and survival analysis to provide a complete cellular and molecular immune cell landscape of human neuroblastoma. Further, we contrasted the neuroblastoma data with single-cell RNA-sequencing data from normal fetal adrenal gland to characterize cell-state changes from normal tissue to neuroblastoma. Our analysis revealed 27 immune cell subtypes including distinct subpopulations of myeloid, NK, B and T cells that were associated with a survival benefit. Multiple subtypes of B and NK cells, not seen in neuroblastoma before, were identified. We propose the presence of tertiary lymphoid structures and showed that Active NK cells correlated with improved survival. In contrast to adult cancers, we detected no difference in cytotoxicity and exhaustion score for cytotoxic T cells, nor Treg activity. However, we demonstrated an increase in inflammatory monocyte signature score from normal to tumor derived myeloid cells. Finally, we performed receptor-ligand interaction analysis between tumor, stroma and immune cells, where we highlight several interactions that we suggest for future studies of how to exploit immune cells as a therapeutic option in neuroblastoma. Our findings significantly broaden the understanding of the immune composition in neuroblastoma and provides a resource for the development of novel immunotherapeutics.

Citation Format: Bronte Manouk Verhoeven, Shenglin Mei, Thale K. Olsen, Karin K. Gustafsson, Anders Valind, Axel Lindström, David G. Nord, Shahrzad S. Fard, Catharina Hagerling, Peter V. Kharchenko, Per Kogner, John I. Johnsen, Ninib Baryawno. The immune cell atlas of human neuroblastoma [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 6215.
Abstract 6242: Association of prostate cancer risk variants with gene expression in prostate stroma
Authors: Ma Chaoran , Zhou Yinglu , Plym Anna , Fanelli Giuseppe Nicolò , Loda Massimo , Mucci Lorelei , Tyekucheva Svitlana , Penney Kathryn
Published: June 2022

Background: Prostate cancer genome-wide association studies have identified >260 germline risk variants which provide strong risk prediction for prostate cancer. Functional interpretation and biological mechanisms of these risk variants are not always clear. Many of the prostate cancer risk variants are expression quantitative trait loci (eQTLs), but studies have mostly been performed in tumor or epithelial-enriched normal prostate tissue. Given emerging evidence supporting that stroma contributes to the carcinogenic process, we examined the associations of prostate cancer risk variants with the expression of genes in prostate stroma.

Methods: We generated mRNA expression data for 13,577 genes with the Illumina TruSeq RNA Exome library preparation kit from stromal samples in specimens from participants of the Health Professionals Follow-up Study and Physicians’ Health Study cohorts. Specifically, we profiled 172 stromal samples adjacent to tumor and 86 paired stroma distant from tumor. We included 264 prostate cancer risk variants genotyped data and imputed using a common reference panel (1,000 Genomes Phase I release). We performed cis- (≤1 Mb from the SNP (single nucleotide polymorphism) to the gene) and trans- eQTL analyses using a linear regression model in the R package Matrix eQTL.

Results: Thirty-four SNPs were significantly (P < 0.01) associated with cis-expression of 41 nearby genes in prostate stroma adjacent to tumor, and 41 SNPs with 52 nearby genes for stroma distant from tumor. Genome-wide, 86 SNPs were significantly (FDR < 0.10) associated with the expression of 126 genes in stroma adjacent to tumor, and 64 SNPs with 291 genes for stroma distant from tumor.

Conclusions: This is the first study to comprehensively examine associations between prostate cancer risk variants and gene expression in prostate stroma. We observed some previous reported associations but also novel associations which may suggest genes that contribute to prostate cancer risk within the stromal tissue. Identifications of eQTLs within prostate stroma may suggest potential mechanisms in specific cell types for their effect on disease.

Citation Format: Chaoran Ma, Yinglu Zhou, Anna Plym, Giuseppe Nicolò Fanelli, Massimo Loda, Lorelei Mucci, Svitlana Tyekucheva, Kathryn Penney. Association of prostate cancer risk variants with gene expression in prostate stroma [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 6242.
Abstract 6249: PERK arm of UPR selectively regulates ferroptosis in colon cancer cells by modulating the expression of system xc
- (SLC7A11)
Authors: Saini Krishan K. , Chaturvedi Priyank , Verma Ayushi , Nengroo Mushtaq A. , Sinha Abhipsa , Singh Akhilesh , Meena Sanjeev , Khan Muqtada A. , Singh Manish P. , Datta Dipak
Published: June 2022

Ferroptosis, a genetically and biochemically distinct form of programmed cell death, is characterized by iron-dependent accumulation of lipid peroxides. Ferroptosis is induced in cancer cells by inhibition of lipid peroxide quencher GPx4. System xc - imports cystine into cytosol for the biosynthesis of glutathione. Tumor cells that are resistant to chemotherapeutic drugs called ‘drug tolerant’ or ‘Persister’ cells which have distinct vulnerability towards iron mediated cell death or ferroptosis. Unfolded Protein Response (UPR) plays critical role for cancer cells to become drug tolerant. Tweaking the balance of UPR to make drug tolerant cells susceptible to Ferroptotic cell death could be an attractive therapeutic strategy. To decipher the emerging contribution of ER-stress in ferroptosis, we investigated the status of UPR following treatment of potent ferroptosis inducer RSL3 (Ras Selective Lethal) in colon cancer cells. We observed an overall up-regulation of UPR activators (ATF6, IRE1α and PERK) and their downstream effectors along with a marked overexpression of cystine-glutamate transporter (System xc -). To further delineate the contribution of particular UPR arm in modulating System xc - expression and subsequent ferroptosis, we made stable knock down cells of each UPR arm and discovered that PERK is selective and critical in inducing ferroptosis in colon cancer cells. Loss of PERK function not only promotes ferroptosis via increasing lipid peroxidation but also limits in vivo tumor growth in colon xenograft model. Further, we find that low PERK expression is associated with higher patient survival as per TCGA COLON CANCER (COAD) database. Overall, our experimental data indicate that PERK is a negative regulator of ferroptosis and genetic silencing of PERK sensitizes colon cancer cells to selective Ferroptotic cell death. Therefore, small molecule PERK inhibitors hold huge promise as novel therapeutics that can sensitize apoptosis resistant cancer cells towards Ferroptotic cell death.

Citation Format: Krishan K. Saini, Priyank Chaturvedi, Ayushi Verma, Mushtaq A. Nengroo, Abhipsa Sinha, Akhilesh Singh, Sanjeev Meena, Muqtada A. Khan, Manish P. Singh, Dipak Datta. PERK arm of UPR selectively regulates ferroptosis in colon cancer cells by modulating the expression of system xc - (SLC7A11) [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 6249.
Abstract 6329: Using low-resolution, low-cost histopathology images to predict esophageal squamous cell carcinoma via deep learning
Authors: Alam Mansoor , Amin Ibrar , Sajjad Ayesha , Zamir Hina , Zaheer Saad , Khattak Maria T. , Muhammad Iqbal , Khan Faisal F.
Published: June 2022

Like many cancers, esophageal squamous cell carcinoma, has a substantially better survival rate if detected in early stages, making early detection a critical element in successful treatment. On the other hand, deep learning tools that employ high-resolution whole slide images and therefore substantially greater compute power and are not easily accessible in low-resource settings. In this study we train and test deep learning models using low-resolution and low-cost digital histopathology images acquired using a 5-megapixel Leica ICC50 camera mounted on a Leica DM500 binocular microscope, at two different magnification levels, Under IRB approvals, we acquired a total of 64 Hematoxylin and Eosin (H&E) stained tissue slides of biopsies taken from patient presenting at Rehman Medical Institute from 2016 to 2020 that were anonymized. A total of 2370 images were captured at two different magnification levels, i.e. 10x and 40x, and were labelled with four classes by at least one expert pathologist (677 well differentiated, 1066 moderately differentiated, 152 poor differentiated and 475 normal) to develop the ESCC image dataset. Preprocessing was performed to clean the dataset, generate patches and resize the images. Deep learning models were trained on the ESCC image dataset for feature extractions. This was followed by machine learning classifier for the diagnosis of ESCC. Deep learning models that were used in this study are Resnet50, VGG16, VGG19, and Inception_Resnet_V2 with Logistic Regression (LR) used as classifier. An ensemble model which stacks up the Inception and Resnet. This model preformed with the highest accuracy at 96% with Precision, Recall, and f1 score values at 96%, 96%, and 96%, respectively. This study reports 1) a local ESCC image repository for research purposes, 2) an ensemble ML/DL model that predicts ESCC with 96% accuracy and 3) also a computer-aided diagnosis (CAD) system for ESCC with a user-interface which will assist histopathologists in diagnosing ESCC with higher speed, efficiency and accuracy.

Citation Format: Mansoor Alam, Ibrar Amin, Ayesha Sajjad, Hina Zamir, Saad Zaheer, Maria T. Khattak, Iqbal Muhammad, Faisal F. Khan. Using low-resolution, low-cost histopathology images to predict esophageal squamous cell carcinoma via deep learning [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 6329.
Abstract 634: Mutation agnostic diagnosis of clonal hematopoiesis of indeterminate potential using fluctuating methylation clocks
Authors: Schenck Ryan O. , Jakobsen Niels A. , Turati Virginia , Shibata Darryl , Vyas Paresh , Leedham Simon , Anderson Alexander R.
Published: June 2022

Clonal hematopoiesis (CH), such as clonal hematopoiesis of indeterminant potential (CHIP), is diagnosed based on somatic genomic alterations in the absence of hematologic malignancy. At present, CHIP is diagnosed using peripheral blood where putative driver point mutations and small insertions/deletions whose variant allele frequency is greater or equal to two percent. Generally, the prevalence of CH increases as an individual ages and conveys a risk for progression to a malignancy. Previously, we developed a method using fluctuating CpG (fCpG) sites to serve as a fluctuating methylation clock to uncover stem cell dynamics in glandular tissues and orthogonally validated our method using publicly available datasets of human blood from normal cohorts and malignant cohorts. Here we expand on this work by presenting 38 new patients with distinct VAF groups from 1-2% VAF up to greater than 10% VAF for putative drivers with corresponding DNA methylation profiles using the Illumina EPIC array platform. We identify fCpG from our normal and CHIP cohorts to train and validate a machine learning method that allows us to diagnose CHIP without DNA sequencing. This method allows for the identification of patients who may have CH driven by structural variants such as copy number variants. We use this method to examine a cohort of 656 normal patients without evidence of CHIP based on mutation showing that we identify the presence of CHIP roughly a third of these patients. Clonal hematopoiesis is driven by the underlying hematopoietic stem cells of an unknown quantity, with estimates for stim cell numbers differing by orders of magnitude.Finally, using a mechanistic model of clonal hematopoeisis and fCpGs we examine the temporal ability to diagnose CHIP given single and multiple expanding clones. We illustrate that multiple driver mutations within the same clone leads to an increased detection ability with an increased expansion rate (i.e. fitness) and in cases where drivers are found in separate, competing clones.

Citation Format: Ryan O. Schenck, Niels A. Jakobsen, Virginia Turati, Darryl Shibata, Paresh Vyas, Simon Leedham, Alexander R. Anderson. Mutation agnostic diagnosis of clonal hematopoiesis of indeterminate potential using fluctuating methylation clocks [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 634.
Abstract 6378: The cross-talk between FTH1 and PYCR1 drives pancreatic cancer progression
Authors: Park Ji Min , Chiu Ching-Feng
Published: June 2022

This study is designed to explore the effect of FTH1 on PDAC human pancreatic cancer cells, proline metabolism, and their crosstalk. Considering the high prevalence of pancreatic cancer with an absence of efficient therapy against PDAC, the present study may provide experimental evidence in support of the novel role of FTH1 in proline metabolism and suggest FTH1 as a potential target for PDAC patients. We further focus on the underlying molecular mechanism of how FTH1 cross-talks with PYCR1 and leads KRAS-mutated PDAC cell growth, investigating whether miRNAs regulation involves in FTH1 mediating PYCR1 expression. Taken together, our study provides a novel function of FTH1 and its cross-talk with PYCR1 may be a novel target for pancreatic cancer research and thus these findings will help us to find metabolite-based therapeutic strategies to improve KRAS-mutated PDAC.

Citation Format: Ji Min Park, Ching-Feng Chiu. The cross-talk between FTH1 and PYCR1 drives pancreatic cancer progression [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 6378.
Abstract 6401: PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state
Authors: WATANABE RYUTA , MAEKAWA MASASHI , KIYOI TAKASHI , KURATA MIE , MIURA NORIYOSHI , KIKUGAWA TADAHIKO , HIGASHIYAMA SHIGEKI , SAIKA TAKASHI
Published: June 2022

Introduction & objectives: Prostate-specific membrane antigen (PSMA) is highly expressed in poorly differentiated, metastatic, and castration-resistant prostate cancers. Recently, 68Ga-PSMA Positron Emission Tomography/Computed Tomography (PET/CT) has been successfully developed as an effective diagnostic tool for prostate cancer. However, the pathophysiological functions of PSMA in prostate tumors remain unclear.

Materials & methods: We examined the protein expression of PSMA in tumor endothelial cells in human prostate tumors by immunohistochemistry. Prostate cancer tissues were resected by robotic surgery in 2019 at Ehime University from patients with prostate cancer. In vitro, we prepared conditioned medium derived from a PSMA-positive human prostate cancer cell line, LNCaP, cultured on collagen I gels. We then examined PSMA expression in human umbilical vascular endothelial cells (HUVECs) cultured with the conditioned medium. We assessed angiogenic activities by treatment of HUVECs with LNCaP-derived conditioned medium using a tube formation assay that mimics angiogenesis. Results: Immunohistochemical positivity of PSMA and CD31, a marker of endothelial cells, and PSMA-expressing tumor endothelial cells were observed in four of 33 prostate cancer patients (12.1%). We also found that the 10,000 × g pellet fraction of the LNCaP-derived conditioned medium containing PSMA-positive membranes, including microvesicles, transformed HUVECs from “PSMA-negative” to “PSMA-positive.” Mass spectrometry revealed that the fraction contained an important growth factor. Furthermore, treating HUVECs with the 10,000 × g pellet fraction of the LNCaP-derived conditioned medium significantly promoted tube formation, mimicking angiogenesis in a PSMA-dependent manner.

Conclusions: Our findings revealed the existence of PSMA-positive tumor endothelial cells in human prostate tumors, which enhances tumor angiogenesis in prostate cancer tissues. The endocytic process of the PSMA-positive microvesicles in normal endothelial cells may be an attractive target to develop novel anti-angiogenic drugs, which could inhibit transformation of normal endothelial cells to tumor endothelial cells (Watanabe et al., The Prostate. 2021. in press).

Citation Format: RYUTA WATANABE, MASASHI MAEKAWA, TAKASHI KIYOI, MIE KURATA, NORIYOSHI MIURA, TADAHIKO KIKUGAWA, SHIGEKI HIGASHIYAMA, TAKASHI SAIKA. PSMA-positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state [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 6401.
Abstract 663: Systemic targeting of therapeutic RNA to cancer via a novel, cell-penetrating and nucleic acid binding, monoclonal antibody
Authors: Quijano Elias , Saucedo Diana Martinez , Khang Minsoo , Liu Yanfeng , Ludwig Dale , Turner Bruce C. , Squinto Stephen , Bindra Ranjit , Saltzman W. Mark , Escobar-Hoyos Luisa , Glazer Peter M.
Published: June 2022

There is intense interest in the development of nucleic acid ligands for immune stimulation of the tumor microenvironment via pattern recognition receptors (PRRs), particularly for the treatment of “cold” tumors. However, delivery of these ligands in a tumor-specific manner to avoid systemic toxicity has been a challenge. Many current efforts rely on direct intra-tumoral injection of RNAs or other stimulatory immune ligands, which is therapeutically sub-optimal, especially for metastatic disease. Here we report studies evaluating the utility of a lupus-derived, cell-penetrating antibody, to deliver nucleic acids to tumors in vivo. This antibody, a modified version of 3E10-D31N, designated GMAB, forms non-covalent complexes with RNAs and can mediate highly specific delivery into tumors via intravenous injection by targeting the nucleoside transporter ENT2, which is highly expressed in tumor cells. Studies with labeled RNAs showed tumor specific delivery and functional expression by imaging, with minimal delivery to healthy tissues. Following these proof-of-concept studies, we investigated whether our antibody could be used to deliver RIG-I agonists to tumors. Using a known agonist of RIG-I, 3p-hpRNA, we demonstrated single agent activity of our GMAB/RNA complexes in multiple tumor models, including a mouse model of melanoma (B16) and an orthotopic model of pancreatic cancer (KPC). Measuring 3p-hpRNA by RT-PCR, there was a 1000X fold difference in RNA uptake in KPC tumor cells relative to CD45+ cells isolated from tumors. In addition, synergy between our GMAB/RNA complexes and anti-PD-1 was additionally observed in mouse models of breast (EMT6) and colon (MC38) cancer. Given the expression of ENT2 along the blood brain barrier (BBB), we demonstrate single agent activity of our GMAB/RNA complexes in an orthotopic mouse model of medulloblastoma, resulting in suppression of tumor growth and spinal metastases. Together, these studies demonstrate that the GMAB antibody can 1) localize to orthotopic and flank tumor models, 2) cross the BBB, and 3) deliver RNA payloads to tumors, providing a novel platform for delivery of immunogenic RNAs, as well as mRNAs and siRNAs, directly to tumors with high specificity following systemic administration.

Citation Format: Elias Quijano, Diana Martinez Saucedo, Minsoo Khang, Yanfeng Liu, Dale Ludwig, Bruce C. Turner, Stephen Squinto, Ranjit Bindra, W. Mark Saltzman, Luisa Escobar-Hoyos, Peter M. Glazer. Systemic targeting of therapeutic RNA to cancer via a novel, cell-penetrating and nucleic acid binding, monoclonal antibody [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 663.
Abstract 71: Integrated-omics analysis identifies the druggable achilles targets to sensitize cancer cells with gefitinib resistance
Authors: Kwon Eun-Ji , Lee Haeseung , Cha Hyuk-Jin
Published: June 2022

Limited eligibility remains one of major drawbacks of target therapeutics as the oncogenic dependency is only determined by mutation status of a target. In order to extend the eligibility of target therapeutics, transcriptome profiles of patients have been examined to better delineate sensitivity of target therapeutics. In this study, we took advantage of multiple large datasets such as cancer cell line transcriptome data (CCLE), drug response data (CTRP), and gene dependency data (Achilles Project) to determine the sensitivity markers of tyrosine kinase inhibitors currently approved by FDA. In particular, EGFR inhibitors (iEGFR: lapatinib, gefitinib and erlotinib) revealed clear sensitivity cluster of cell line regardless of EGFR mutation status. Comparative analysis between sensitive (iEGFR-S) and resistant (iEGFR-R) cell groups revealed that integrated gene signatures such as chemokine, GPCR, and PI3K-Akt signaling pathway, were predicted as putative determinants of iEGFR susceptibility. Through integrated analysis of gene dependency data, among several Achilles’ targets (of which depletion increases vulnerability), predicted in iEGFR-R cells group, druggable Achilles’ targets (available to accompanied drugs) such as CCR6, AURKA and mTOR were identified as putative targets for sensitizing iEGFR-R group to iEGFR. Following biochemical studies validated the clear sensitizing effect of the pharmacological inhibitor for each predicted target, in the isogenic cell models with acquired iEGFR resistance. In addition, prediction and following biochemical validation, we demonstrated that the CCR6 inhibition significantly attenuated resistance of iEGFR by repressing mitochondrial ROS (mt-ROS), of which induction by iEGFR rescued cell death. These data suggest that integrated-omics analysis would be a useful approach to extend eligibility and improve the efficacy of target therapeutics.

Citation Format: Eun-Ji Kwon, Haeseung Lee, Hyuk-Jin Cha. Integrated-omics analysis identifies the druggable achilles targets to sensitize cancer cells with gefitinib resistance [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 71.
Abstract 73: A ubiquitination cascade regulating the integrated stress response and survival in carcinomas
Authors: Cervia Lisa D. , Shibue Tsukasa , Gaeta Benjamin , Borah Ashir A. , Leung Lisa , Li Naomi , Dumont Nancy , Gonzalez Alfredo , Bick Nolan , Kazachkova Mariya , Dempster Joshua M. , Krill-Burger John M. , Piccioni Federica , Udeshi Namrata D. , Olive Meagan E. , Carr Steven A. , Root David E. , McFarland James M. , Vazquez Francisca , Hahn William C.
Published: June 2022

Targeting of mutated oncogenes has led to the identification of new targeted therapies. However, druggable oncogenes do not occur in most cancers. Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 793 cancer cell lines to identify selective co-essentiality modules and found that a ubiquitination ligase complex composed of UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2 and two heterodimeric E3 subunits, respectively, is required for the survival of a subset of epithelial tumors. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization and upregulation of the heme-regulated inhibitor (HRI), a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy.

Citation Format: Lisa D. Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir A. Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua M. Dempster, John M. Krill-Burger, Federica Piccioni, Namrata D. Udeshi, Meagan E. Olive, Steven A. Carr, David E. Root, James M. McFarland, Francisca Vazquez, William C. Hahn. A ubiquitination cascade regulating the integrated stress response and survival in carcinomas [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 73.
Abstract 802: Development of replication protein A (RPA) targeted PROTACs for the treatment of lung and ovarian cancers
Authors: Kelm Jeremy M. , Gaddameedi Jitender Dev , VanderVere-Carozza Pamela S. , Samarbakhsh Amirreza , Vakeesan Nivisa , Kansou Hussein W. , Serafimovski Sara , Pawelczak Katherine S. , Turchi John J. , Gavande Navnath S.
Published: June 2022

Despite remarkable advances within the field of molecular cancer therapeutics over the preceding decades, DNA-damaging agents retain a central role in the pharmacotherapy of neoplastic diseases. The clinical response to DNA-damaging therapies is varied within and across cancer subtypes where a robust DNA damage response (DDR) predicts recalcitrance. Replication protein A (RPA) is the predominant human single-stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and the DNA-damage response (DDR). Inhibition of the RPA−DNA interaction is an approach to cancer drug discovery that holds potential to provide single-agent activity and/or synergy with existing therapeutics. Proteolysis targeting chimera (PROTAC) based drug design presages a paradigm shift within drug discovery as PROTACs begin to carve out a space in the clinical landscape. The PROTAC platform is particularly suited for drugging overexpressed proteins such as RPA because iterative target degradation improves drug:target stoichiometry and precludes RPA’s role in protein scaffolding. Herein, we describe the synthesis and biological evaluation of an RPA targeted PROTAC library conceived by conjugation of the diaryl pyrazole RPA inhibitor TDRL-551 and an E3-ligand via a linker of variable length and composition. Residing within the beyond rule of five (bRo5) chemical space, careful consideration was given to minimize the liability to cell permeability imposed by the high molecular weight of PROTACs. Excitingly, cellular uptake across the compound library was broadly improved relative to the unconjugated warhead TDRL-551. Similarly, RPA engagement by the PROTAC series was widely unhindered and, in many cases, improved relative to the warhead TDRL-551. A subset of the compound library unified by flexible linkers and alkyne attachment of the E3-ligand (typified by GL-3311) display low single-digit micromolar IC50 values in viability assays across H460, A549, and A2780 cell lines.

Citation Format: Jeremy M. Kelm, Jitender Dev Gaddameedi, Pamela S. VanderVere-Carozza, Amirreza Samarbakhsh, Nivisa Vakeesan, Hussein W. Kansou, Sara Serafimovski, Katherine S. Pawelczak, John J. Turchi, Navnath S. Gavande. Development of replication protein A (RPA) targeted PROTACs for the treatment of lung and ovarian 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 802.
Abstract 861: Nuclear translocation of Axl during early breast cancer progression
Authors: Graham Darby R. , Hebert Kelly D. , Newton Emma E. , Gomes Angelica M. , Behbod Fariba , Machado Heather L.
Published: June 2022

Axl is a receptor tyrosine kinase (RTK) that is activated by its ligand Gas6 and regulates proliferation, cell survival, and clearance of apoptotic cells in both normal physiology and a number of disease states. In breast cancer, Axl is overexpressed in Her2+ and triple-negative breast cancers and is associated with resistance to RTK targeted therapies. Recent studies have shown that upon kinase inhibition, the Axl intracellular domain (ICD) can translocate to the nucleus, however, the biological significance and functions for nuclear Axl are poorly understood. Furthermore, a functional role for Axl during ductal carcinoma in situ (DCIS) progression, a requisite step for invasive breast cancer, has not been established. To address whether Axl mediates localized invasion during DCIS progression, Axl was deleted in a DCIS cell line (MCF10A-DCIS.com) using a CRISPR-Cas9 approach. Deletion of Axl shows a significant decrease in progression to invasive cancer in vivo, which is accompanied by decreased proliferation, however microinvasion is unchanged as compared to wildtype. Analysis of patient samples of pure DCIS, DCIS with microinvasion, and DCIS with invasive cancer shows a significant increase in Axl expression in patients with evidence of invasion. Interestingly, there is a significant increase in nuclear Axl in all patient samples with evidence of invasion as compared to normal breast or pure DCIS, suggesting an important role for Axl-ICD in progression to invasive cancer. Soft agar assays using normal cells (MCF10A) shows that while full length Axl (Axl-FL) is sufficient to induce colonies, Axl-ICD-transduced cells do not form colonies, suggesting that Axl-ICD alone does not have oncogenic activity. However, Axl-ICD significantly increases colonies in DCIS cells, suggesting that the ICD is important in the progression of transformed cells. Ongoing studies are aimed at identifying transcriptional targets of Axl-ICD in DCIS cells. These studies will advance our understanding of how Axl mediates breast cancer progression and will have critical implications for ongoing therapeutic strategies aimed at targeting Axl. This work is supported by NIH R01CA212518 (H.L.M.) and Susan G. Komen CCR1637765 (H.L.M.).

Citation Format: Darby R. Graham, Kelly D. Hebert, Emma E. Newton, Angelica M. Gomes, Fariba Behbod, Heather L. Machado. Nuclear translocation of Axl during early breast cancer progression [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 861.
Abstract 904: Neural stem cells of the subventricular zone evolve into tumors around the resection cavity after surgical resection of glioblastoma via the CXCL12/CXCR4 axis
Authors: Li Xue , Kim Hyun Jung , Lee Jeong Ho , Wu Hong-Gyun , Lee Joo Ho
Published: June 2022

Glioblastoma multiforme (GBM) is the most common lethal primary brain tumor. Neural stem cells (NSCs) harboring cancer-driving mutations in the subventricular zone (SVZ) have recently been reported as the cells of origin for human glioblastoma (GBM) through migration. However, the pathological role of residual tumor-initiating NSCs (tNSCs) in the SVZ is undetermined for local recurrent tumor after surgical removal of the primary tumor. After the primary resection of GBM, most recurrences still appear as local tumors around the resection cavity (RC). Accordingly, we hypothesized that, after complete removal of the primary tumor, the tNSCs in SVZ might be the source for tumor construction at RC. Here, we examined the role of tNSCs in the development of local tumors around RC using a mouse model and genetic analysis of human tissues. First, cancer-driving mutations, including Trp53, Pten, and Egfr mutations, were introduced to NSCs in SVZ through in vivo electroporation, and the RC was generated in the remote cortex through stereotaxic resection. We found that the NSCs harboring driver mutations migrated specifically to the RC through the aberrant growth of oligodendrocyte-precursor cells (OPCs). Furthermore, the SVZ-originated cells constituted the GBM around the RC at four weeks post-resection in 64% of mouse models. Second, we performed deep sequencing of longitudinal matched tissues consisting of (i) normal SVZ away from the tumor, (ii) primary tumor, (iii) recurrent tumor, and (iv) blood in the patients with GBM, IDH wildtype. We found that the dominant clones of recurrent tumors genetically arose from the SVZ, not directly from the primary tumor. Third, the CXCR4/CXCL12 axis was upregulated through migration from the SVZ and tumor formation at the RC compared with primary tumors without the RC in mouse models. Blockage of the CXCR4/CXCL12 axis decreased the number of immigrating OPC lineage cells specifically and improved tumor control and survival rates. Taken together, our results reveal that the residual tNCSs harboring cancer-driving mutations in the SVZ can re-construct tumors after surgical resection of GBM. Blocking CXCL12/CXCR4 might be a potential treatment strategy for preventing the main local failures after resection.

Citation Format: Xue Li, Hyun Jung Kim, Jeong Ho Lee, Hong-Gyun Wu, Joo Ho Lee. Neural stem cells of the subventricular zone evolve into tumors around the resection cavity after surgical resection of glioblastoma via the CXCL12/CXCR4 axis [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 904.