bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3641: Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma
Authors: Murimwa Gilbert Z. , Rossi Francesca , Fleming Henry K. , Yazgan Zeynep , Alzhanova Dina , Huang Huocong , Brekken Rolf A.
Published: April 2023

Up to 50% of pancreatic ductal adenocarcinomas (PDA) harbor mutations in canonical TGFß pathway genes such as SMAD4 and TGFBR2. SMAD4 mutations have been shown to drive resistance to chemotherapy, metastasis, and margin positivity following surgical resection. To characterize the unique tumor microenvironment of SMAD4 mutant PDA, we developed isogenic, orthotopic mouse models using CRISPR mediated knockout of Smad4 in a cell line derived from KPfC (KrasLSL-G12D;Trp53fl/fl;Pdx1Cre) mice, a genetically engineered mouse model of pancreatic cancer. To characterize the alteration of the secretome caused by the loss of Smad4, we performed proteomic analysis with conditioned media collected from Smad4 wildtype and mutant cancer cell clones. Interestingly, the loss of Smad4 resulted in the production of neuroendocrine related factors. In addition, gene ontology analysis with the secretome suggested that these factors were associated with neuronal processes. These data indicate that loss of Smad4 in PDA cancer cells may lead to a neuroendocrine signature change. In particular, we found one of the most up-regulated secreted factors in the mutant cells was SPP1, which has been shown to promote tumor progression by affecting cancer cell stemness and macrophage polarization. To validate the proteomic results, we established orthotopic models with Smad4 wildtype and mutant clones. We found that loss of Smad4 resulted in larger tumor growth. Moreover, immunohistochemistry showed a significant up-regulation of SPP1 in the cancer cells of Smad4 mutant tumors. To further profile the alteration of immune landscape, we stained for markers of myeloid cells and lymphocytes. We found that Smad4 mutant tumors had an immunosuppressive microenvironment characterized by increased M2 macrophage, neutrophil and MDSC infiltration. Altogether, our study suggests that loss of SMAD4 in PDA may induce a neuroendocrine signature with increased SPP1 secretion in cancer cells. The production of this unique cancer cell secretome may result in a microenvironment marked by immunosuppressive myeloid cells.

Citation Format: Gilbert Z. Murimwa, Francesca Rossi, Henry K. Fleming, Zeynep Yazgan, Dina Alzhanova, Huocong Huang, Rolf A. Brekken. Loss of Smad4 induces SPP1 secretion and immunosuppressive myeloid cell formation in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3641.
Abstract 3650: STAT1 suppresses KRAS-driven lung adenocarcinoma depending on the tumor microenvironment
Authors: Trenk Christoph , Sagmeister Rebecca , Horvath Jaqueline , Homolya Monika , Luca Andreea Corina , Eferl Robert , Moll Herwig , Casanova Emilio
Published: April 2023

Lung cancer is responsible for the majority of cancer-related deaths worldwide. Within this severe disease lung adenocarcinoma (LUAD) belongs to the most common form of lung cancer. LUAD is highly correlated with KRAS mutations. Although, the first KRAS inhibitors entered into clinics recently, therapy resistance arises. Since KRAS-mutant LUAD is an inflammation driven disease, we focus on the JAK-STAT pathway as an alternative target. Preliminary data suggests that human KRAS-mutant LUAD is correlated to enriched JAK-STAT signaling, as well as STAT1 upregulation. Accordingly, we aimed to explore the tumorigenic functions of STAT1 in this form of lung cancer. Genetically engineered C57BL6/N mice which develop autochthonous KRAS-driven and P53-deficient LUAD proficient (KP) and deficient for STAT1 (KPS) were used. In a second mouse model expression of ovalbumin was induced in tumor cells, mimicking a neoantigen to increase immune infiltration in the presence (KPO) or absence of STAT1 (KPOS). Kaplan-Meyer analysis was performed to assess the overall survival. Tumor burden, as well as immune infiltration was analyzed in lungs 6 and 10 weeks after tumor induction via H&E, IHC and IF staining. Furthermore, the lung fluid was collected by bronchoalveolar lavage and used to profile inflammatory cytokines. Deletion of STAT1 reduced the survival in KRAS-driven LUAD mice only when ovalbumin was expressed. Tumor burden and tumor grades were increased in tumors lacking STAT1 at 6 and 10 weeks after tumor initiation. Surprisingly, although tumors of KPOS mice showed a decreased infiltration by T cells and CD4 T cells, CD8 T cell infiltration did not change, suggesting a tumor suppressive function of STAT1 via T cell exhaustion. Moreover, the macrophage attracting cytokine CCL9 secretion was upregulated and the number of tumor infiltrating, immunosuppressive macrophages was increased in KPOS mice. This data implicates a tumor suppressive function of STAT1 by secretion of cytokines that can recruit suppressive myeloid immune cells to the TME leading to CD4 T cell exclusion and CD8 T cell exhaustion. Further studies are needed on the changes in the immune infiltrating cells to explain the mechanism behind these intriguing findings.

Citation Format: Christoph Trenk, Rebecca Sagmeister, Jaqueline Horvath, Monika Homolya, Andreea Corina Luca, Robert Eferl, Herwig Moll, Emilio Casanova. STAT1 suppresses KRAS-driven lung adenocarcinoma depending on the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3650.
Abstract 3673: Plk1 phosphorylation of PHGDH to regulate serine metabolism
Authors: Rao Xiongjian , Allison Derek B. , Flight Robert M. , Wu Yuanyuan , Scott Timothy , He Daheng , Wang RuiXing , Li Zhiguo , Li Chaohao , Kong Yifan , Mao Fengyi , Wang Chi , Higashi Richard M. , Lane Andrew N. , Moseley Hunter NB , Liu Xiaoqi
Published: April 2023

Polo-like kinase 1 (Plk1) has been reported to be highly expressed in most tumors, especially in advanced tumors, while how Plk1 elevation benefits tumor growth remains elusive. Metabolic reprogramming is one of the hallmarks of cancer, but how the tumors fully take advantage of deregulated metabolism is an enigma. Here, we found that Plk1 could divert serine metabolism from de novo synthesis to exogenous uptake by regulating PHGDH (phosphoglycerate dehydrogenase), the first rate-limiting enzyme of de novo serine biosynthesis. We show that PHGDH is phosphorylated by Plk1 at a cluster site (S512, S513, S517) and that Plk1-associated phosphorylation of PHGDH results in its protein degradation, thus reduced de novo serine biosynthesis. As a compensatory response, cells with an elevated level of Plk1 significantly increase the uptake of serine to produce more sphingosines, which are pivotal metabolites for the growth of cancer cells. Our finding may provide guidance on how to target de novo biosynthesis of serine, serine uptake or sphingosine metabolism to treat advanced prostate cancer.

Citation Format: Xiongjian Rao, Derek B. Allison, Robert M. Flight, Yuanyuan Wu, Timothy Scott, Daheng He, RuiXing Wang, Zhiguo Li, Chaohao Li, Yifan Kong, Fengyi Mao, Chi Wang, Richard M. Higashi, Andrew N. Lane, Hunter NB Moseley, Xiaoqi Liu. Plk1 phosphorylation of PHGDH to regulate serine metabolism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3673.
Abstract 3680: Multiomics analysis of triple negative breast cancer identifies potential metabolic vulnerability for overcoming the resistance of neoadjuvant therapy
Authors: Ren Zuen , Kurmi Kiran , Morris Robert , Joshi Shakchhi , Zaniewski Eric , Kreuzer Johannes , Gioia Gabrielle Elena , Haas Wilhelm , Haigis Marcia C. , Ellisen Leif W.
Published: April 2023

Triple-negative breast cancer (TNBC) is a heterogeneous disease that remains currently medically incurable. We comprehensively analyzed clinical, proteomic, and metabolomic data of a cohort of 12 highly selected primary TNBCs. Among them, 7 patients after neoadjuvant chemotherapy had residual disease and micro- or macro-metastases that were non-pathologically complete response (non-pCR); 5 patients had pathologically complete response (pCR). Interestingly, both proteomic and metabolic profiling results revealed dramatically different molecular signatures in non-pCR relative to pCR. Non-pCR were enriched in metabolites involving aspartic acid, fumarate, nicotinamide, and adenosine that are critical intermediates for nitrogen recycling and tricarboxylic acid (TCA) cycle, which are essential fuels for tumor biomass. Moreover, non-pCR were enriched in urea cycle, oxidative phosphorylation, glycolysis, sirtuin signaling, nucleotide De Novo biosynthesis, which are resistance-associated. Putative therapeutic targets were identified in non-pCR patients. Collectively, our multiomics analysis demonstrated the heterogeneity of TNBCs at multi-levels and enabled the development of personalized therapies targeting unique tumor metabolic profiles.

Citation Format: Zuen Ren, Kiran Kurmi, Robert Morris, Shakchhi Joshi, Eric Zaniewski, Johannes Kreuzer, Gabrielle Elena Gioia, Wilhelm Haas, Marcia C. Haigis, Leif W. Ellisen. Multiomics analysis of triple negative breast cancer identifies potential metabolic vulnerability for overcoming the resistance of neoadjuvant therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3680.
Abstract 3686: Blocking fatty acid oxidation suggests a potential new therapeutic approach for pancreatic cancer
Authors: Woo Sang M. , Han Sung-Sik , Lee Ho , Kim Soo-Youl
Published: April 2023

Background: Glycolysis is known as the main pathway for ATP production in cancer cells. However, in pancreatic ductal adenocarcinoma (PDAC) cells, glucose deprivation for 24 h did not reduce ATP levels, whereas it suppressed lactate production. We found that ATP production in PDAC cells critically depended on fatty acid oxidation (FAO) while normal cells showed no dependency on fatty acid. Therefore, FAO inhibition significantly decrease oxygen consumption rate as well as ATP production only in cancer cells.

Methods: To test whether cancer depends on fatty acid for energy metabolism, high- or low-fat diet was tested in spontaneous PDAC cancer model (KPC mouse: Kras(G12D)/p53(R172H)/Pdx1-Cre model). To test whether FAO is absolute requirement for ATP production in cancer, overall survival (OS) was monitored in KPC mice by crossbreeding with FAO gene knock out mice.

Results: First, the effects of calorie balanced high- or low-fat diets were tested to determine whether cancer growth is modulated by fatty acid instead of calories. A low-fat diet caused a 70 % decrease in pancreatic preneoplastic lesions accompanying decrease of body weight 20% compared with the control, whereas high-fat diet caused a 2-fold increase in preneoplastic lesions accompanied with 25% increase of body mass index in Kras(G12D)/p53(R172H)/Pdx1-Cre model (KPC model; spontaneous PDAC model). Second, the effect of FAO gene knock down was tested to determine whether OS is increased in KPC model. The crossbreeding of FAO gene knockout (+/-) and KPC mice resulted in 2-fold increase of OS.

Conclusions: The two results suggest that controlling obesity by diet as well as targeting FAO can enhance anti-cancer effect of cancer therapeutics including conventional anti-cancer drug or immune-oncology drug.

Clinical Trial Identification: IND for PDAC therapy with drugs targeting FAO will be submitted in 2022.

Legal Entity Responsible for the Study: This study is legally under review by IRB in National Cancer Center, Korea, and sponsored by National Research Foundation of Korea (NRF) and NCC-Bio Co.

Funding: Funding source: Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2019M3A9G1104345)

Citation Format: Sang M. Woo, Sung-Sik Han, Ho Lee, Soo-Youl Kim. Blocking fatty acid oxidation suggests a potential new therapeutic approach for pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3686.
Abstract 3694: Metabolic response in human cervical cancer cells to modifications in bioavailable ATP
Authors: Jayaswal Parth K. , Muhammad Taha , Brown Ashley , Norris Jeffrey , Korch Shaleen B.
Published: April 2023

Introduction: Cancer cells utilize a markedly reduced oxidative phosphorylation (OXPHOS) when experiencing conditions of high oxygen stress (Warburg Effect/aerobic glycolysis), a phenomenon first studied by Otto Warburg. While there have been many discoveries associated with metabolic differences across cancer cells, one of the most common alterations is the primary means of generating ATP. The importance of deciphering these cellular decisions will enable an understanding of the multifaceted role ATP has in cancer proliferation, senescence, and chemotherapeutic resistance. The use of synthetic protein DX, a man-made ATP chelator with high specificity and affinity, is a proposed method of investigating the metabolic response in cancer cells. Hypothesis: Following DX expression, HeLa cells respond to energy stress (reduced intracellular ATP) via metabolic adaptations, specifically inducing the spare respiratory capacity.

Methods: The impact of DX on cell viability was determined using a tetrazolium-based colorimetric cell viability assay and a caspase 3/7 assay. To correlate phenotypic/viability change with DX activity, bioavailable ATP levels were measured at specific time points following DX expression. Additionally, the relative contribution of glycolysis and OXPHOS to the total ATP production rate was measured using the label-free XF Real Time ATP Rate Assay and XF Cell Mito Stress Test (XFe96 Seahorse, Agilent Technologies) over time post DX expression. To determine the impact of ATP stress on mitochondrial and nuclear content, both were quantified following DX-expression.

Results: In a time- and dose- dependent manner, DX negatively impacted cell growth and induced cell death via apoptosis, at a time concomitant with a decrease in bioavailable ATP. In response to DX over time, the total ATP production rates in HeLa cells significantly decreased. Importantly, this reduction in the rate of ATP production was associated with a significant downregulation in both OXPHOS and glycolysis.

Conclusion: Advances in synthetic biology have allowed for the intentional design of artificial proteins that maintain function in vivo. These proteins can be developed into powerful investigative tools to study biological questions, such as cellular decisions involved in cancer cell metabolism. In response to DX-potentiated ATP stress, the significant reduction in both glycolysis and oxidative phosphorylation suggests metabolic downregulation is required to maintain HeLa cell viability.

Citation Format: Parth K. Jayaswal, Taha Muhammad, Ashley Brown, Jeffrey Norris, Shaleen B. Korch. Metabolic response in human cervical cancer cells to modifications in bioavailable ATP. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3694.
Abstract 3707: UXS1 is a synthetic lethal target in cancer
Authors: Cicchini Michelle Patricia , Wu Shuzhen , Peng Yang , Rao Yanhua
Published: April 2023

Studies presented here were conducted to validate UXS1 as a synthetic lethal target in UGDHhigh expressing cancer. In vitro and in vivo knockout studies were used to investigate the role of UXS1 in UGDH-high cancers. UXS1 has been validated as a synthetic lethal target in cancer cell lines highly expressing UGDH in vitro by CRISPR-mediated knockout of UXS1 in a panel of lung cancer cell lines. In addition to UGDH expression predicting UXS1 dependency, we have shown UGDH overexpression functional drives UXS1 dependency, such that knockout of UGDH rescues cellular growth defects seen with UXS1-knockout alone. Furthermore, catalytic activity of UXS1 was shown to be required, as wildtype but not catalytic deficient UXS1 was able to rescue cellular growth defects seen with endogenous UXS1 knockout in UGDH-high cancer cell lines. Together this data suggests that dysregulation of some metabolite(s) is driven by high expression of UGDH when knockout of UXS1 prevents normal pathway flux of UDP-Glucuronic Acid (UDP-GA) to be converted to UDP-Xylose (UDP-X), leading to the synthetic lethal effect observed as impaired cellular growth. To validate UXS1 as a target in vivo, an inducible genetic knockout system to knockout UXS1 showed impaired tumor growth, but no tumor regression. Sanger sequencing of UXS1 knockout efficiency at different time course suggested a slow and incomplete dropout of the UXS1-KO cells in vivo. Although in vitro efforts have shown UXS1 to a synthetic lethal target, the failure to induce a robust in vivo effect of UXS1 knockout have casted doubts on the promise of UXS1 being an effective target for the proposed indication.

Citation Format: Michelle Patricia Cicchini, Shuzhen Wu, Yang Peng, Yanhua Rao. UXS1 is a synthetic lethal target in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3707.
Abstract 3709: Hyperglycemia induces metabolic reprogramming and promotes epithelial-mesenchymal transitions in pancreatic cancer: an in vitro and in vivo experiment
Authors: Liu Zhao , Hayashi Hiromitsu , Matsumura Kazuki , Ogata Yoko , Sato Hiroki , Shiraishi Yuta , Uemura Norio , Miyata Tatsunori , Higashi Takaaki , Nakagawa Shigeki , Mima Kosuke , Imai Katsunori , Baba Hideo
Published: April 2023

Background: Pancreatic cancer is a serious life-threatening disease due to its highly invasive and metastatic potential. Hyperglycemia is a well-known initial symptom in patients with pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming in cancer, described as the Warburg effect, can induce epithelial-mesenchymal transition (EMT). In this study, we elucidate the biological and clinical impact of hyperglycemia in PDAC progression.

Methods: Biological impact of hyperglycemia on malignant behavior in PDAC was examined by in vitro and in vivoexperiment.

Results: Hyperglycemia promoted EMT by inducing metabolic reprogramming into a glycolytic phenotype via yes-associated protein (YAP)/PDZ-binding motif (TAZ) overexpression, accompanied by GLUT1 overexpression and enhanced phosphorylation Akt in PDAC. In addition, hyperglycemia enhanced chemoresistance by upregulating ABCB1 expression, and triggered PDAC switch into pure-basal-like subtype with activated Hedgehog pathway (GLI1 high, GATA6 low expression) through YAP/TAZ overexpression. PDAC is characterized by abundant stroma that harbors tumor-promoting properties and chemoresistance. Hyperglycemia promotes production of collagen fiber-related proteins (fibronectin, fibroblast activation protein, COL1A1, and COL11A1) by stimulating YAP/TAZ expression in cancer-associated fibroblasts (CAFs). Knockdown of YAP and/or TAZ or treatment with YAP/TAZ inhibitor (K975) abolished EMT, chemoresistance, and a favorable tumor microenvironment even under hyperglycemic conditions in vitro and in vivo. Indeed, YAP/TAZ overexpression was significantly associated with worse prognostic outcomes in human PDAC patients.

Conclusion: Hyperglycemia induce metabolic reprogramming into a glycolytic phenotype and promote EMT via the YAP/TAZ-Hedgehog signaling axis in PDAC. Hyperglycemia as an initial symptom may be the cause of highly invasive and metastatic potential by inducing YAP/TAZ overexpression, while YAP/TAZ could be a novel therapeutic target in PDAC patients.

Citation Format: Zhao Liu, Hiromitsu Hayashi, Kazuki Matsumura, Yoko Ogata, Hiroki Sato, Yuta Shiraishi, Norio Uemura, Tatsunori Miyata, Takaaki Higashi, Shigeki Nakagawa, Kosuke Mima, Katsunori Imai, Hideo Baba. Hyperglycemia induces metabolic reprogramming and promotes epithelial-mesenchymal transitions in pancreatic cancer: an in vitro and in vivo experiment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3709.
Abstract 3735: N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation
Authors: Yang William H. , Yang Wei-Hsiung
Published: April 2023

Cancer is one of the most dreaded diseases of the 21st century for humans. Currently, the human cancer mortality rate is approximately 11-25%. However, for some animals, such as elephants, the cancer mortality rate is lower than 5%. p53 is a significant cellular regulator in human cancers, and its important role in response to DNA damage has been highlighted by the discovery of p53 retrogene found in elephants (19 copies) but not humans (0 copies). Therefore, the main question is whether (1) more copies of p53 are better at combating cancer development or (2) is elephant p53 is better than human p53 in p53-mediated function? Herein, we demonstrate for the first time that N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation. First, we created elephant-to-human p53 variants (such as T387R, L383P, LSTEL, etc.) and truncations at the N-terminus and C-terminus of human p53. Secondly, R175H and R273H p53 mutants (conformational and contact mutants, respectively) were used as negative control. Using a p53 (14X) response element LUC and several natural p53 downstream gene promoters, we demonstrated that several N-terminal and C-terminal p53 variants and truncations increases p53 transactivation compared to WT p53. Moreover, we found that deletion of the region between aa374 and aa382 is integral for p53 transactivation. Taken together, our preliminary results demonstrate that several N-terminal and C-terminal elephant-to-human p53 variants enhance p53 transactivation and possible stability. This data is the first step to support the notion that elephant p53 is better than human p53 in p53-mediated function.

Citation Format: William H. Yang, Wei-Hsiung Yang. N-terminal and C-terminal elephant-to-human p53 variants and truncations alter p53 transactivation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3735.
Abstract 3745: GRAS1 long non-coding RNA binds and stabilizes NF-kB activating protein to protect lung cancer cells from DNA damage
Authors: Su Tong , Kong Bobby , Zhu Jonathan , McHugh Colleen A.
Published: April 2023

Long non-coding RNAs (lncRNAs) control numerous biological processes, including epigenetic regulation, gene expression, proliferation and metabolism. However, the detailed biological mechanisms by which lncRNAs regulate cellular processes are still poorly understood. We identified a set of growth regulator non-coding RNAs based on data mining of CRISPR/Cas9 screens and high-throughput sequencing studies from patient normal and tumor cells. We hypothesized that growth regulator lncRNAs play a role in cancer development by recruiting effector proteins to regulate gene expression. In our screen, the Growth Regulator Antisense 1 RNA (GRAS1) was identified as a key factor controlling growth in non-small cell lung cancer cells. Loss of GRAS1 resulted in altered expression of genes involved in DNA repair, cell cycle progression, and p53 and NF-ΚB signaling pathways. Extensive DNA damage occurred within 48 hours after GRAS1 knockdown, with a significant increase in the number of γH2AX loci visualized by immunofluorescence assay. We used RNA antisense purification combined with mass spectrometry to identify the NF-ΚB activating protein (NKAP) as the top directly interacting partner of GRAS1 lncRNA. NKAP stability decreased after GRAS1 knockdown, and this phenotype could be reversed with proteasome inhibition. Further, RelA levels were decreased in GRAS1 knockdown cells. We found that GRAS1 lncRNA binds and stabilizes NKAP to enable survival and protect against DNA damage in lung cancer cells.

Citation Format: Tong Su, Bobby Kong, Jonathan Zhu, Colleen A. McHugh. GRAS1 long non-coding RNA binds and stabilizes NF-kB activating protein to protect lung cancer cells from DNA damage. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3745.
Abstract 376: DKK1 inhibition can overcome sorafenib resistance in hepatocellular carcinoma cells through beta-catenin suppression
Authors: Li Lin , Hsu Chia-Lang , Lin Liang-In , Lee Bin-Shyun , Ou Ping-Yun , Cheng Ann-Lii , Hsu Chiun , Ou Da-Liang
Published: April 2023

As part of our previous studies on the anti-tumor mechanisms of sorafenib, one of the primary treatments for advanced hepatocellular carcinoma (HCC), we found that suppression of cyclin E1 in HCC cells correlates with sensitivity to sorafenib (Chiun Hsu et al. Clin Cancer Res. 2016:2555). Furthermore, the combination of sorafenib and a cyclin-dependent kinase inhibitor was found to have synergistic anti-tumor effects. Specifically, we found that inhibiting DKK1 (dickkopf1), an essential regulator of Wnt signaling, had a synergistic effect. In sorafenib-sensitive HCC cells, DKK1 mRNA and protein expressions were suppressed following treatment with sorafenib, but not in sorafenib-resistant cells. HCC cells resistant to sorafenib were induced to apoptosis with knockdown of DKK1 expression. The apoptosis-inducing effects of sorafenib were enhanced by DKK1 inhibitors, and beta-catenin suppression was found to be crucial to this effect. In addition, mouse tumors secrete DKK1 into the blood and its presence correlates positively with tumor size and survival during sorafenib treatment. Suppression of DKK1 in hepatocellular carcinoma cells may serve as a pharmacodynamic biomarker for predicting sorafenib or CDK inhibitor efficacy. Sorafenib combined with a DKK1 inhibitor may improve sorafenib's efficacy in hepatocellular carcinoma. (Supported by MOST 106-2314-B-002-229-MY3, MOST 109-2634-F-002-043, MOST 109-2314-B-002-229 -MY3, MOST 110-2634-F-002-044 from Ministry of Science and Technology, Taiwan)

Citation Format: Lin Li, Chia-Lang Hsu, Liang-In Lin, Bin-Shyun Lee, Ping-Yun Ou, Ann-Lii Cheng, Chiun Hsu, Da-Liang Ou. DKK1 inhibition can overcome sorafenib resistance in hepatocellular carcinoma cells through beta-catenin suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 376.
Abstract 383: Trastuzumab deruxtecan resistance is associated with reduced responsiveness to topoisomerase inhibitors (payload resistance) but no reduction in sensitivity to HER2 tyrosine kinase inhibitors
Authors: Nilsson Monique B. , Poteete Alissa , Udagawa Hibiki , Yu Xiaoxing , He Junqin , Le Xiuning , Heymach John
Published: April 2023

Activating ERBB2 (HER2) mutations occur across multiple malignancies, and although mutational hotpots vary by disease, HER2 mutations most frequently occur in the tyrosine kinase domain. Among lung cancer patients, the most common HER2 mutation is the exon 20 insertion mutations, Y772dupYVMA, which accounts for over 30% of all HER2 mutations in lung cancer. Recent clinical testing of trastuzumab deruxtecan (T-DXd), a HER2 antibody-drug conjugate (ADC), against HER2 mutant non-small cell lung cancer (NSCLC) demonstrated a confirmed objective response of 55%, a median duration of response of 9.3 months, and a median overall survival of 17.8 months, which ultimately led to the recent approval of T-DXd for HER2 mutant NSCLC. Acquired resistance to targeted agents remains a major clinical challenge, and HER2-mutant patients that initially respond to T-DXd will eventually develop progressive disease. Thus, understanding mechanism of T-DXd resistance and identifying targeted agents that remain effective in cells with acquired T-DXd resistance is of great clinical importance. We generated Ba/F3 cells expressing the HER2 YVMA mutation and assessed sensitivity to HER2 targeting agents by Cell Titer Glo assay. HER2 YVMA cells were sensitive to the EGFR/HER2 tyrosine kinase inhibitor (TKI) poziotinib and the HER2 ADC T-DXd. To generate HER2 ADC resistant cells, we cultured Ba/F3 HER2 YVMA cells in T-DXd continuously for 8 weeks at which point cells resumed rapid proliferation. Cell Titer Glo assay confirmed that Ba/F3 HER2 YVMA cells were sensitive to T-DXd with an IC50 of 195 μg/ml, whereas Ba/F3 HER2 YVMA T-DXd-resistant cells had an IC50 of greater than 1000 μg/ml. Notably, T-DXd resistant cells remained highly sensitive to the HER2 TKI poziotinib with an IC50 of 14 nM and 7.5 nM for T-DXd-resistant and parental cells, respectively. Moreover, T-DXd-resistant cells demonstrated reduced sensitivity to the topoisomerase inhibitor topotecan with an IC50 of 200 nM whereas parental cells had an IC50 of 66 nM, suggesting that acquired T-DXd resistance may be mediated by loss of sensitivity to the ADC payload. Collectively, these data demonstrate that HER2 TKIs such as poziotinib may retain anti-tumor cell activity in HER2 mutant tumor cells with acquired resistance to HER2 ADCs.

Citation Format: Monique B. Nilsson, Alissa Poteete, Hibiki Udagawa, Xiaoxing Yu, Junqin He, Xiuning Le, John Heymach. Trastuzumab deruxtecan resistance is associated with reduced responsiveness to topoisomerase inhibitors (payload resistance) but no reduction in sensitivity to HER2 tyrosine kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 383.
Abstract 3831: The molecular composition of a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea that displays anti-cancer activity against a triple negative breast cancer cell line
Authors: Alaouna Mohammed , Penny Clement , Ulll Rodney , Dlamini Zodwa
Published: April 2023

Approximately 20% of all breast cancer cases are Triple negative breast cancer (TNBC), whichis more difficult to treat and has a higher mortality rate than other types of breast cancer. Thereis an active practice of traditional medicine in in South Africa, in the form of infusions madeusing plant material. Previously we demonstrated that water soluble extracts from the leaves ofone of these plants, African garlic (Tulbaghia violacea), is able to induce apoptosis in TNBCcell lines, while also inhibiting the ability of these cells to migrate or penetrate a basementmembrane. In an attempt to isolate the molecular compounds that are present in this extract,water soluble leaf extracts were subjected to Fourier transform infrared spectroscopy (FITR),Gas chromatography–mass spectrometry (GC-MS) and Nuclear magnetic resonance (NMR)spectroscopy. Identified compounds of interest were then used in computational dockingstudies with the anti-apoptotic protein Cox2. The FITR analysis indicated that the extractconsisted of multiple compounds, belonging to a variety of classes. GC-NS and NMRspectroscopy identified 36 and 61 separate compounds respectively. Many of these compoundswere similar in structure to compounds with known anti-cancer properties. Many compoundswith established medical uses were also identified in the extract. Five compounds identified inthe extract, d-Glycero-d-galacto-heptose, H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, and Benzaldehyde and 4-(1-methylethyl)- demonstrated high binding affinity towardsCOX2. With d-Glycero-d-galacto-heptose having a higher docking score than manycommercially available COX2 inhibitors. Based on our findings, the compounds within theextract could be used as the basis for the development of safe and effective drugs for thetreatment of TNBC and other cancers. In order to fulfil this potential, future work shouldexamine the molecular pathways affected by the treatment of TNBC cells with this extract orindividual compounds.

Citation Format: Mohammed Alaouna, Clement Penny, Rodney Ulll, Zodwa Dlamini. The molecular composition of a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea that displays anti-cancer activity against a triple negative breast cancer cell line. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3831.
Abstract 3926: Activating missense mutations in ROS1 receptor harbor oncogenic potential and are sensitive to tyrosine kinase inhibition
Authors: Iyer Sudarshan R. , Nusser Kevin , Jones Kristen , Shinde Pushkar , Beach Catherine Z. , Keddy Clare , Aguero Erin , Force Jeremy , Shinde Ujwal , Davare Monika A.
Published: April 2023

ROS1 encodes the largest receptor tyrosine kinase (RTK) in the human genome. Chromosomal rearrangements of the ROS1 gene generate oncogenic ROS1 fusion proteins that have been successfully targeted with tyrosine kinase inhibitors (TKIs), most notably in lung cancer. To date, ROS1 fusions are the only known biomarkers for therapeutic implementation of ROS1 TKIs. However, unlike known oncogenic mutations in EGFR and ALK RTKs, the functional impact of point mutations within the full-length ROS1 RTK is unknown. Here we queried the AACR Genie database, which consists of pan-cancer clinical genomic sequencing data from thousands of patient samples, for ROS1 aberrations and nominated thirty-four missense mutations residing in the ROS1 tyrosine kinase domain for functional interrogation. Immunoblotting revealed distinct impact of the mutations on kinase function, ranging from complete loss to significant increase in catalytic activity. Notably, Asp and Gly substitutions at the D2113 position within the ROS1 kinase domain dramatically increased autophosphorylation and activation of downstream signaling pathways. ROS1D2113N/G were found to be ROS1 TKI-sensitive transformative oncogenes in independent cell-based models. Molecular dynamic simulations of ROS1D2113N kinase revealed dramatic alterations in the activation loop of the mutant kinase as compared to wildtype. Global proteomics and phosphoproteomics showed that ROS1D2113N upregulates signaling via phosphorylation of known effectors (SHP2 and STAT3) similar to ROS1 fusions, but also upregulates other pathways, including mTORC2, JNK1/2, AP-1 and TGFB1. In vivo, ROS1D2113N drove tumor formation that was inhibited with ROS1 TKIs. Taken together, these data demonstrate for the first time that select point mutations within ROS1 drive tumorigenesis and can be therapeutically targetable with existing FDA-approved ROS1-TKIs.

Citation Format: Sudarshan R. Iyer, Kevin Nusser, Kristen Jones, Pushkar Shinde, Catherine Z. Beach, Clare Keddy, Erin Aguero, Jeremy Force, Ujwal Shinde, Monika A. Davare. Activating missense mutations in ROS1 receptor harbor oncogenic potential and are sensitive to tyrosine kinase inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3926.
Abstract 3932: Targeting cyclin K in pancreatic cancer
Authors: Xiao Yi , Dong Jixin , Chen Yuanhong
Published: April 2023

This study focuses on investigating the role of Cyclin K in pancreatic cancer growth and chemotherapeutic sensitivity, aiming to provide pre-clinical evidence for Cyclin K-targeted therapy in pancreatic cancer. Unlike other well-known cyclins, Cyclin K is largely understudied in cancers. Data extracted from the TCGA database indicated that Cyclin K was overexpressed in pancreatic ductal adenocarcinoma (PDAC) and associated with reduced overall survival. Consistently, western blotting showed that Cyclin K was abundantly expressed in human and mouse PDAC cell lines. By using a Tet-On inducible system, we established Cyclin K-depleted and Cyclin K-overexpressed cell lines to evaluate the function of Cyclin K in pancreatic cancer. The proliferation assay showed that Cyclin K depletion led to retarded PDAC cell proliferation, whereas Cyclin K overexpression boosted cell growth. We further confirmed that Cyclin K was required for pancreatic cancer growth in vivo. In addition, cell cycle analysis showed that Cyclin K deficiency resulted in G1-S arrest, while Cyclin K overexpression promoted G1-S progression. By using an RT2 cell cycler array, we identified CDC20 as an important target of Cyclin K that mediated Cyclin K-induced PDAC cell proliferation. To investigate the influence of Cyclin K on chemo-sensitivity, we treated the PDAC cell lines with two newly synthesized Cyclin K molecular glue degraders (HQ461 and NCT02). We found that these degraders specifically ablated Cyclin K and its cognate kinase CDK12 in a very efficient manner. Importantly, we demonstrated that Cyclin K abrogation, either by Cyclin K degrader or Cyclin K knockdown, rendered PDAC cells more sensitive to GemTaxol (gemcitabine plus Taxol) treatment and PARP inhibitors (olaparib or niraparib), as indicated by increased cleavage of PARP or caspase 3. Together, our current results suggest: 1. Cyclin K promotes cell proliferation and G1-S transition in pancreatic cancer; 2. CDC20 mediates the function of Cyclin K on pancreatic cancer cell proliferation; 3. Cyclin K depletion synergizes with GemTaxol or PARP inhibitor in pancreatic cancer treatment. Our study revealed for the first time that Cyclin K plays an essential role in pancreatic cancer growth and chemo-sensitivity, and targeting Cyclin K may offer a great therapeutic avenue for pancreatic cancer patients.

Citation Format: Yi Xiao, Jixin Dong, Yuanhong Chen. Targeting cyclin K in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3932.
Abstract 4074: Cellular immunotherapy targeting kappa myeloma antigen for the treatment of multiple myeloma
Authors: Li Jessica , Haynes Nicole , Cummins Katherine , Gowrishankar Kavitha , Micklewaithe Kenneth , Hilton Halley , Dunn Rosanne , Oliaro Jane , Harrison Simon
Published: April 2023

Multiple myeloma (MM), the second most common blood cancer, is characterized by the accumulation of malignant plasma cells in the bone marrow. Chimeric Antigen Receptor (CAR)-T cell therapy has recently entered the standard of care for relapsed and refractory MM, following the recent FDA-approval of two CAR-T cell products, ide-cel® and cilta-cel®, which target the B cell maturation antigen (BCMA). However, despite impressive response rates, most patients relapse within 1-3 years. Kappa (κ) myeloma antigen (KMA) is a tumour specific membrane associated protein expressed on malignant plasma cells in patients with kappa light-chain restricted (κ-type) MM. KMA is absent on normal plasma cells and haematopoietic stem cells, making it an attractive and alternative target antigen for CAR-T cell therapy for MM. The monoclonal antibody, KappaMab (MDX-1097), binds to a conformational epitope on KMA, and has been assessed in phase I, IIa and IIb clinical trials in relapse refractory myeloma patients (1). Here, we have engineered a lentiviral vector encoding a second-generation CAR expressing a scFv from MDX-1097, fused to a 4-1BB co-stimulatory domain and CD3 zeta chain. We successfully generated human anti-KMA CAR-T cells with high and stable CAR expression and a predominately memory T cell phenotype. The CAR-T cells selectively killed KMA-expressing tumour lines, secreted interferon-gamma upon target recognition, and demonstrated potent anti-tumour activity in a xenograft model. Anti-KMA CAR-T cell therapy therefore represents a novel and potent treatment ready to enter a phase I clinical trial for patients with myeloma. (1) Spencer et al. Blood Cancer Journal (2019) 9:58

Citation Format: Jessica Li, Nicole Haynes, Katherine Cummins, Kavitha Gowrishankar, Kenneth Micklewaithe, Halley Hilton, Rosanne Dunn, Jane Oliaro, Simon Harrison. Cellular immunotherapy targeting kappa myeloma antigen for the treatment of multiple myeloma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4074.
Abstract 4105: Improvement of CD133-specific chimeric antigen receptor T cells by secreting anti-PD-L1 single-chain variable fragment against cholangiocarcinoma
Authors: Sangsuwannukul Thanich , Supimon Kamonlapat , Chieochansin Thaweesak , Choomee Kornkan , Sujjitjoon Jatuporn , Junking Mutita , Yenchitsomanus Pa-thai
Published: April 2023

Cholangiocarcinoma (CCA) is rare but hard-to-treat solid cancer affecting people worldwide. Cancer stem cells (CSCs) in CCA contribute to tumor heterogeneity and resistance to conventional therapies. Thus, CSCs are notable targets for cancer immunotherapy. CD133 is a potential CSC surface marker overexpressed in various cancers. T cell-based immunotherapy, namely chimeric antigen receptor (CAR) T-cell targeting CD133 has shown potential in eradication of some solid tumors including CCA. Nevertheless, anti-tumor function of CAR T cell could be decreased under immunosuppressive tumor microenvironment (TME). Thus, to improve its function under TME, the next-generation of CD133-specific CAR T-cell is required. We therefore constructed two lentiviral vectors containing: (i) anti-CD133 single-chain variable fragment (scFv), 4-1BB costimulatory molecule, and CD3ζ (133CAR), and (ii) anti-CD133 scFv, 4-1BB, and CD3ζ in tandem with anti-PD-L1 scFv (133CARsL). The lentiviruses were transduced to generate CD133-specific CAR T cells expressing the second-generation (133CART) or anti-CD133 CAR with self-secreting anti-PD-L1 scFv (133CARTsL). The latter was able to simultaneously target CD133 and PD-L1 molecules on CCA cells. CCA cells endogenously expressing CD133 and PD-L1 were cocultured with either 133CART or 133CARTsL. In short-term coculture, the antitumor effects of 133CARTsL were not different from that of 133CART. However, in long-term coculture and after tumor re-challenge, 133CARTsL showed lower expression of programmed cell death protein (PD-1) and better cytotoxic function against high CD133 and PD-L1 CCA cells. The higher potential of 133CARTsL with reduced exhaustion profile would offer great promise for application of CD133-targeting CAR T cells in solid tumors.

Citation Format: Thanich Sangsuwannukul, Kamonlapat Supimon, Thaweesak Chieochansin, Kornkan Choomee, Jatuporn Sujjitjoon, Mutita Junking, Pa-thai Yenchitsomanus. Improvement of CD133-specific chimeric antigen receptor T cells by secreting anti-PD-L1 single-chain variable fragment against cholangiocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4105.
Abstract 4107: Cytotoxic T cells armed with IL-4 receptor-targeting and PD-L1-blocking peptides inhibits lung tumor growth
Authors: Jo Dong Gyun , Rangaswamy Gunassekaran Gowri , Murugan Poongkavithai Vadevoo Sri , Lee Byungheon
Published: April 2023

Tumor homing of adoptively transferred cytotoxic T cells (CTLs) to solid tumors is still inefficient. In addition, CTLs that arrive at tumor tissues meet the immune-suppressive tumor microenvironment. To reduce these limitations in adoptive T-cell therapy against solid tumors, we here exploited CTLs dual-labeled with IL-4 receptor (IL4R)-targeting peptide and programmed cell death-ligand 1 (PD-L1)-blocking peptide. IL4R is commonly upregulated in many solid tumors including lung tumor. PD-L1, an immune checkpoint, is frequently upregulated in tumor cells and contributes to the suppression of CTLs in the tumor microenvironment. CTLs were isolated from the splenocytes of LLC lung tumor-bearing mice and non-genetically labeled with IL4RPep-1, an IL4R-targeting peptide, and PD-L1Pep2, a PD-L1-blocking peptide, using a sulfhydryl-reactive cross linker. Compared to unlabeled CTLs, IL4R-targeted, PD-L1-blocking CTLs showed more robust cytokine secretion and tumor cell lysis during co-culture with LLC tumor cells. The CTLs armed with the dual-acting peptides exerted higher levels of tumor homing and anti-tumor growth activity in LLC lung tumor-bearing mice, which was accompanied by decrease of immune-suppressive cells in the tumor tissues. These results suggest that the IL4R-targeted, PD-L1-blocking CTLs can be a promising strategy in adoptive T-cell therapy against solid tumors.

Key words: Adoptive T-cell therapy, CTLs, IL-4 receptor, lung tumor, PD-L1

Citation Format: Dong Gyun Jo, Gunassekaran Gowri Rangaswamy, Poongkavithai Vadevoo Sri Murugan, Byungheon Lee. Cytotoxic T cells armed with IL-4 receptor-targeting and PD-L1-blocking peptides inhibits lung tumor growth. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4107.