bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2278: CTLA-4 targeted engineered toxin bodies designed to deplete regulatory T cells (Tregs)
Authors: Iberg Aimee , Acquaye-Seedah Edith , Rabia Lilia A. , Robinson Garrett L. , Ramos Hilario J. , Dekker Joseph D. , Zhao Jay , Willert Erin K.
Published: September 2020

Tumor resident regulatory T cells (Tregs) are important mediators of an immunosuppressive tumor microenvironment (TME) promoting tumor immune evasion. The presence of Tregs, and a higher ratio of Tregs to effector T cells in the TME, are associated with poor prognosis. The depletion of Tregs in the TME is expected to re-expose the tumor to the immune system to allow for tumor control. CLTA-4 is expressed on the cell surface of Tregs and is a clinically validated target. Better responses to CTLA-4 monoclonal antibody (mAb) treatment are correlated with stronger ADCC-mediated Treg depletion in preclinical models, and patient FcγR polymorphism has been reported to correlate with response to CTLA-4 mAb therapy. Towards improving on current therapies, many efforts to increase effective depletion of Tregs (such as by Fc effector modification) are being pursued. Engineered toxin bodies (ETBs) are comprised of a proprietarily engineered form of Shiga-like Toxin A subunit (SLT-A) genetically fused to antibody-like binding domains. ETBs work through novel mechanisms of action and are capable of forcing internalization, self-routing through intracellular compartments to the cytosol, and inducing potent cell-kill via the enzymatic and permanent inactivation of ribosomes. Targeted ETBs are being developed to specifically target and destroy CTLA-4 expressing cells. In vitro, cells expressing CTLA-4 are effectively and specifically killed by targeted ETBs. This direct cell kill activity of the ETB has the potential to deplete Tregs in the TME without a requirement for ADCC mechanisms. ETBs have been designed to bind various CTLA-4 epitopes, and to comprise of different targeting domains formats, including monomeric and diabody single chain variable fragments (scFvs) as well as single domain and biparatopic antibody fragments. The development of a CTLA-4 targeted ETB is ongoing. The entry of a protein with direct cell kill properties into the therapeutic space represents a differentiated mechanism of action to deplete Tregs for ultimate re-invigoration of the anti-tumor immune response, and has the potential to provide benefit to patients, including in the relapsed or refractory setting.

Citation Format: Aimee Iberg, Edith Acquaye-Seedah, Lilia A. Rabia, Garrett L. Robinson, Hilario J. Ramos, Joseph D. Dekker, Jay Zhao, Erin K. Willert. CTLA-4 targeted engineered toxin bodies designed to deplete regulatory T cells (Tregs) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2278.
Abstract 2352: Sexual morbidity and gender disparities among young-onset colorectal cancer patients and survivors
Authors: Springer Tamara , Newcomer Kimberly , Peterson Danielle , Yarden Ronit
Published: September 2020

Rectal cancer incidence is rising dramatically among people younger than 50 years old. The risk has doubled four times for individuals born in the 1990s relative to those born in the 1970s. Previously, we and others showed that young-onset patients are more likely to be diagnosed at an advanced stage of colorectal cancer when compared to patients of all ages, which would render aggressive treatments. There is limited data and public discussion on the long-term effects of colorectal cancer treatments on intimacy and sexual morbidity and their long-term impact on the quality of life especially among young cancer survivors who were developing their own social and financial responsibilities and establishing their life-long goals. To explore the unique challenges and unmet needs of the young-adult patient population, a cross-sectional study was conducted. Colorectal cancer patients and survivors (N=885) diagnosed between the ages of 20 to 50 years old (median age 42 ± 7.0) completed an online questionnaire that was based on established instruments including EORTC-QOL-30, EORTC-CR-29, and EORTC-SHC-22. Patients and survivors diagnosed at stage III reported worse outcomes. Patients' comprehension of radiation sexual-side effects was assessed based on self-reported discussions with their physicians. Prior to radiation, providers were significantly less likely to discuss the many sexual side-effects and long-term consequences of radiation and surgery with women than with men. Providers were also less likely to discuss fertility preservation, a notable issue with a strong impact on young cancer patients, with women than men prior to and during treatments, regardless of their parental status. While the majority of patients reported having sexual-dysfunction after treatment, women more frequently classified different aspects of their sexual dysfunction as being 'severe.' and expressed worse body image than men. Women were more likely to experience pain while more men reported reduction in sex drive. Additionally, a strong correlation was noted between sexual dysfunction and emotional exhaustion in all young-onset patients and survivors. Together, our survey demonstrates inadequate communications between patients and providers about the irreversible sexual side-effects of colorectal cancer treatments. Younger patients and survivors are facing unique long-term challenges and require further information about fertility preservation options and emotional support regarding their sexuality post-treatment.

Citation Format: Tamara Springer, Kimberly Newcomer, Danielle Peterson, Ronit Yarden. Sexual morbidity and gender disparities among young-onset colorectal cancer patients and survivors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2352.
Abstract 238: Targeting glutaminolysis potentiates the efficacy of chemotherapy in RAS-driven pancreatic cancers
Authors: Mukhopadhyay Suman , Goswami Debanjan , Nissley Dwight V. , McCormick Frank
Published: September 2020

Pancreatic cancer is an extremely aggressive and deadly malignancy. Despite a detailed understanding of pancreatic ductal adenocarcinoma (PDAC)'s biology, the outcome of the current therapeutic regimen remains grim. Chemotherapeutic treatment is one of the first-line systemic treatments for PDAC. However, creating resistance against chemotherapy remains a major challenge. Oncogenic K-Ras mutation is one of the most common events responsible for the initiation and progression of PDAC. K-Ras mutation also promotes the activation of a transcription factor called NRF2 that is crucial for tumor development and pancreatic cancer proliferation. Our study detects differential basal levels of NRF2 in a panel of PDAC cell lines. We hypothesized that this phenomenon might reflect a greater intrinsic capacity of PDAC cells to become resistant to chemotherapy. We then demonstrated that up-regulation of NRF2 creates resistance against chemotherapy in PDAC cells. Consequently, NRF2 activation rewires metabolic pathway networks. Importantly, our metabolomics analysis revealed that, upon NRF2 activation, several metabolic pathways are reprogrammed. Recent advances in tumor metabolism signify the critical role of glutamine as a key regulator of cancer therapy. Moreover, we observed that inhibiting glutamine metabolism enhances the efficacy of chemotherapeutic drugs in PDAC cells. Taken together, our finding holds promise as a novel combination therapy for patients with pancreatic cancer marked by developed resistance against chemotherapy. Finally, this treatment strategy further establishes the potential for exploiting metabolic changes in RAS-driven cancer cells that confer novel therapeutic opportunities.

Citation Format: Suman Mukhopadhyay, Debanjan Goswami, Dwight V. Nissley, Frank McCormick. Targeting glutaminolysis potentiates the efficacy of chemotherapy in RAS-driven pancreatic cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 238.
Abstract 2483: NELFE modulates chromatin accessibility to amplify the transcription of MYC-associated genes in hepatocellular carcinoma
Authors: Zhang Kai , Dang Hien , Barry Anna
Published: September 2020

Global transcriptomic alterations are often observed in cancers, including hepatocellular carcinoma (HCC). How these large changes are modulated remain elusive. We have previously demonstrated that the overexpression of Negative elongation factor E (NELFE) contributes to the transcriptomic imbalance in HCC through MYC signaling, which regulates more than 15% of the genome. However, how NELFE modulates MYC to enhance the transcription of pro-oncogenic mRNAs is unknown. It is known that the status of the chromatin is essential for gene expression. Previous studies have shown that NELF-mediated stalling of Pol II can enhance gene expression by blocking promoter-proximal nucleosome assembly in Drosophila. Here, we propose that NELFE modulates the status of the chromatin to regulate gene expression to promote hepatocarcinogenesis. Accordingly, the overexpression of NELFE and MYC in the h-TERT immortalized hepatocyte cells, HHT4, significantly enhanced cancer associated phontypes, including proliferation and colony and spheroid formation. NELFE and MYC ChIP-PCR demonstrated that both NELFE and MYC co-occupy the promoter regions of two proliferative genes, cyclin E2 and PA2G4. Furthermore, the signals of MYC and H3K27ac, a histone marker associated with open and active promoter and enhancer elements, were decreased in these regions by NELFE-depletion. These results provide a possible mechanism whereby NELFE plays an essential role in modulating the status of the chromatin to regulate MYC-induced gene expression. Current studies include the use of CRISPR/Cas9 tools to generate NELFE mutants and ATAC-seq and ChIP-seq (MYC and NELFE) to explore NELFE's role in chromatin modulation genome-wide. These assays will allow us to determine the dynamic function and pro-oncogenic roles of NELFE and their effect on the MYC targets, including cyclin E2 and PA2G4 transcripts. Key words: NELFE, chromatin, transcription, HCC, liver, MYC.

Citation Format: Kai Zhang, Hien Dang, Anna Barry. NELFE modulates chromatin accessibility to amplify the transcription of MYC-associated genes in hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2483.
Abstract 2487: Mutation of the STK11 gene predicts recurrence of breast cancer
Authors: Wang Chih-Yang , Chang Yung-Chieh , Kuo Yao-Lung , Lee Kuo-Ting , Chen Pai-Sheng , Cheung Chun Hei Antonio , Shen Che-Hung , Chang Chih-Peng , Lai Ming-Derg , Shen Meng-Ru , Hsu Hui-Ping
Published: September 2020

Introduction: Breast cancer is the most common cancer in women, and some patients develop recurrence after standard therapy. Effective predictors are urgently needed to detect recurrence earlier.

Methods: We conducted a comprehensive study via an experimental and bioinformatics approach to detect mutated genes in breast cancer. Twenty-seven breast cancer patients who developed recurrence within 24 months postoperatively and 22 control cancer patients without recurrence were enrolled from National Cheng Kung University Hospital in Taiwan. Targeted deep sequencing was performed to assess the mutations among individuals with breast cancer using a panel of 143 cancer-associated genes. Bioinformatics and public databases were used to predict the protein functions of the mutated genes.

Results: Mutations were identified in 49 breast cancer specimens, and the most frequently mutated genes were BRCA2, TP53, APC, ATM, BRCA1, NOTCH1, TET2, NF1, TSC2, PIK3CA, TSC1, PTEN, MSH2, PTCH1, PIK3R1, STK11, RB1, BAP1, CDH1 and FBXW7. Mutation of these genes was correlated with protein phosphorylation and autophosphorylation. Among these highly mutated genes, mutations of STK11 were associated with poor prognosis and increased recurrence of breast cancer. Knockdown of STK11 in triple negative breast cancer cell lines increases transcription of cytokines and modulates immune response.

Conclusion: Our findings suggest that mutation of STK11 is correlated with early recurrence of breast cancer patients and it will become a powerful prognostic marker for recurrence of breast cancer. Suppression of STK11 signaling by gene mutation may contribute to immune escape.

Citation Format: Chih-Yang Wang, Yung-Chieh Chang, Yao-Lung Kuo, Kuo-Ting Lee, Pai-Sheng Chen, Chun Hei Antonio Cheung, Che-Hung Shen, Chih-Peng Chang, Ming-Derg Lai, Meng-Ru Shen, Hui-Ping Hsu. Mutation of the STK11 gene predicts recurrence of breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2487.
Abstract 2502: Genetic and functional heterogeneity of DCIS as predictors of invasive cancer
Authors: Fortunato Angelo , Mallo Diego , King Lorraine , Hardman Timothy , Hall Allison , Marks Jeffrey R. , Hwang Shelley Shelley , Maley Carlo C.
Published: September 2020

Genetic diversity both between and within individual tumors constitutes a challenge to personalized cancer medicine. Intra-tumor heterogeneity provides the genetic fuel for natural selection in clonal evolution and cancer progression. Tumors with high levels of genetic heterogeneity are hypothesized to be more likely to demonstrate aggressive behavior and progress to invasion and metastasis.

We analyzed the mutational loads from separate areas of pure DCIS and compared this to genetic heterogeneity in DCIS lesions found adjacent to invasive and metastatic cancer. Two spatially distinct areas of DCIS from each case were macro-dissected and the DNA extracted from FFPE samples. To analyze the data, we developed new bioinformatics methods that allowed analysis of small amounts of degraded DNA extracted from FFPE samples across multiple regions. Our bioinformatics pipeline was optimized on a series of 28 independent technical replicates of the same DNA sample sequenced twice, as training tools to find the best filtering parameters.

Whole exome sequencing was performed on each of the two geospatially separated samples for each case. Minimum coverage for inclusion in this study was 40X over at least 50% of the exome. We used the ratio of private mutations (only in 1 area) to public (found in both areas) mutations as a measure of intra-tumor heterogeneity.

We present an approach to measure clonal heterogeneity using a bulk sequencing strategy applied to geospatially distinct foci of DCIS. We found statistically significant difference between DCIS adjacent to invasive disease and metastatic patients' genetic divergence (t-test, p=0.013). Our findings suggest that genetic and functional heterogeneity may play an important evolutionary role as a driver for invasive progression.

Citation Format: Angelo Fortunato, Diego Mallo, Lorraine King, Timothy Hardman, Allison Hall, Jeffrey R. Marks, Shelley Shelley Hwang, Carlo C. Maley. Genetic and functional heterogeneity of DCIS as predictors of invasive cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2502.
Abstract 2531: The mechanism of HPV oncogenes in the regulation of upstream microRNA pathway in cervical cancer
Authors: Song Yi , Deng Yan , Tang Tao , Wang Chi Chiu
Published: September 2020

High risk Human Papillomavirus (HPVs) is a very important etiological agent in cervical cancer. HPV types have been subdivided into low-risk types and high-risk types, which are frequently associated with invasive cervical cancer. HPV16 and HPV18 count for 70% of cervical cancer. The E6, E7 genes in the early region of HPV genome are proved to have a significant impact on progression of cervical cancer. miRNA is a kind of short non-coding RNA which regulates the gene expression on post-transcriptional and translational level, and it has been proved to have a significant impact on tumor development. Our group previously investigated the correlation of miRNAs and HPV oncogenes, and identified several differentially expressed miRNAs in cervical cancer cell lines and clinical specimens. The expression level of miR-145, miR-195 and miR-142-3p were down-regulated in cervical cancer patients. However, whether the HPV oncogenes regulate miRNAs is still unknown. In order to determine the relationship between HPV oncogenes and miRNAs. We did several experiments. We found that up/down-regulate HPV 16/18 E6 and E7 caused an decrease/increase of BRCA1, miR-145 and miR-195, and BRCA1 could increase the expression of miR-145 and miR-195 in different cell lines transfected with HPV 16/18 E6 and E7 by using qRT-PCR and Western-blot. We also found the binding activity of E2F1 and MYC to the promoter region of BRCA1, which validated by ChIP-PCR and Luciferase assay. These pathways were also validated in tumor xenograft nude mice. We also tried to find the target genes of miR-145 and miR-195, and investigated how they affect tumorigenesis in cervical cancer. These findings provided a solid foundation that HPV 16/18 E6 and E7 regulated miR-145 and miR-195 via BRCA1 in cervical cancer. It can be an important pathway for the pathogenesis of cervical cancer induced by high risk HPV. These findings could provide new ways in diagnosis and treatment for cervical cancer. They also give a new insight to investigate molecular pathways for HPV-associated cancers. We believe that further investigation will be done for the mechanisms in the future.

Citation Format: Yi Song, Yan Deng, Tao Tang, Chi Chiu Wang. The mechanism of HPV oncogenes in the regulation of upstream microRNA pathway in cervical cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2531.
Abstract 2540: Chemoprevention of aerosolized Let-7b in mouse lung cancer model
Authors: Zhang Qi , Pan Jing , Wang Yian , Miller Mark S. , Izzotti Alberto , You Ming
Published: September 2020

The delivery of microRNA (miRNA) via inhalation is a potential strategy for lung cancer prevention in high risk individuals such as current and former smokers. Previous studies have shown that intratracheal or intranasal exposure of genetically engineered mice to viral vectors expressing let-7 miRNA resulted in a reduced lung tumor burden. However, these studies were done in genetically engineered mouse models that developed highly aggressive tumors more relevant for a treatment regimen. In this study, we investigated the efficacy of let-7b in lung cancer prevention. An aerosolized let-7b miRNA formulation was tested in the benzo(a)pyrene (B[a]P) mouse model. The particle size of the let-7b miRNA aerosol has been systematically characterized as particles with sizes between 1.9 to 35 nm (average size of 29.0 nm). Following the aerosol formulation evaluation, the biodistribution of let-7b in the lung by tail vein injection and aerosolized delivery were compared. The distribution of the aerosolized let-7b produced higher levels of the miRNA in the lung compared with systematic delivery. The aerosol delivery of let-7b had no effect on animal body weight nor were there any other signs of toxicity. The development of an aerosolized let-7b formulation inhibited B[a]P - induced lung adenoma volume by 72% in A/J mice. Collectively, these findings show that aerosolized miRNA is a promising approach for lung cancer prevention.

Citation Format: Qi Zhang, Jing Pan, Yian Wang, Mark S. Miller, Alberto Izzotti, Ming You. Chemoprevention of aerosolized Let-7b in mouse lung cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2540.
Abstract 2616: RhoC decreases tight junction stabilization in breast cancer cells, revealing a potential therapeutic target
Authors: Abraham Hannah , Goo Laura , Little Andrew , Yates Joel , Wu Zhifen , Merajver Sofia D.
Published: September 2020

RhoC, a member of the Rho GTPase family, has been shown to have a role specifically in facilitating metastasis of breast cancer cells via influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. As these processes require disruptions in cell-cell junctions, we use wild-type and CRISPR RhoC and RhoA knockout lines of the breast cancer cell lines SUM-149 (inflammatory breast cancer, IBC) and MDA-MB-231 (triple-negative breast cancer, TNBC) in order to assess the influence of RhoC and RhoA on their cell-cell junctions. Protein lysates from these cell lines were blotted for the adherens junction markers E-cadherin and β-catenin, as well as the tight junction markers ZO-1 and Occludin. Cells were also fixed and stained for these junction markers to further assess expression and localization. In order to measure junction stability, we conducted FITC-Dextran assays, wherein cells were grown to confluency on Transwell plates, the apical chambers were incubated with FITC-Dextran solution for 24 hours, and the fluorescence of the basal chambers was subsequently measured. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM-149 IBC and MDA-MB-231 TNBC cells results in increased epithelial morphology, cell-cell connections, and tight junction marker colocalization, as measured via immunofluorescent staining. Furthermore, RhoC knockout cells have increased junctional proteins compared to wild-type cells, as measured by Western blot. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity compared to wild-type cells, by the FITC-Dextran leakage assay. These results suggest that high expression of RhoC in cancer cells destabilizes tight junctions, although loose clusters of cells are observed depending on cell type; taken together, upregulation of RhoC activation may contribute to increased metastatic potential, in part through destabilization of tight junctions. We will report on further invasion functional assays and RNA-seq data on the RhoC knockout cell lines. Taken together, these data support an important role for RhoC in tight junction modulation, thereby advancing the evidence for a potential highly specific new therapeutic target in RhoC-driven metastases control.

Citation Format: Hannah Abraham, Laura Goo, Andrew Little, Joel Yates, Zhifen Wu, Sofia D. Merajver. RhoC decreases tight junction stabilization in breast cancer cells, revealing a potential therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2616.
Abstract 2634: Oral bacteria effects on oral cancer initiation and progression
Authors: Kovac Stefan , Napier Tiara S. , Scoffield Jessica , Amm Hope M.
Published: September 2020

Oral squamous cell carcinoma (OSCC) is the most common site of head and neck squamous cell carcinoma. It is estimated to be diagnosed in 50,000 patients this year with an overall 5-year survival rate of 64%. The tumor microenvironment has been shown to play a role in the progression of many human cancers, including OSCC. Given the complexity of the oral microbiome, we hypothesize that OSCC and oral bacteria interact to affect the tumorigenic properties of OSCC.

Methods: OSCC cells were cultured in the presence of oral pathogenic (Aggregatibacter actinomycetemcomitans (Aa), Enterococcus faecalis (Ef), Fusobacterium nucleatum (Fn)) and non-pathogenic (Streptococcus parasanguinis (Sp)) bacteria. Human oral keratinocytes (HOK) and OSCC cells were treated with oral bacteria and assessed for changes in cell proliferation and gene expression.

Results: HOK treated with Ef demonstrated significantly increased proliferation and increased expression of HES1, MMP11, and CDKN1A(p21). Of the five OSCC cell lines, Ef increased proliferation in the two cell lines that were human papilloma virus positive (HPV+) (2A3, SCC152) and in one of three cell lines that were HPV negative (HPV-, Cal27). Ef increased the expression of HES1, MMP2, CDKN1A, and GLI1 in SCC152 cell and HES1, MMP11, and CDKN1A in 2A3 cells. Ef also increased the invasion of SCC152 cells in a transwell experiment. In HPV- Cal27 cells, Ef increased MMP2 and GLI1 expression. Aa also increased proliferation in 2A3 HPV+ cells. Fn did not increase proliferation of OSCC cells, but it did increase the expression of HES1, CDKN1A, and GLI1 in SCC152 HPV+ cells.

Conclusions: Oral bacteria have direct interactions with human oral keratinocytes and cancer cells. In particular, Ef may be involved in both the initiation and progression stages of oral cancer.

Citation Format: Stefan Kovac, Tiara S. Napier, Jessica Scoffield, Hope M. Amm. Oral bacteria effects on oral cancer initiation and progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2634.
Abstract 2813: Tumor models driven by EGFR: Optimizing the preclinical profiling of EGFR-targeting agents
Authors: Posch Markus , Klett Hagen , Peille Anne-Lise , Kelter Gerhard , Maier Armin , Schüler Julia , Metz Thomas M.
Published: September 2020

The epidermal growth factor receptor (EGFR, HER1, ERBB1) is a driver of many human cancers. Standard of care treatment for colon, head and neck and non-small cell lung cancer includes drugs targeting EGFR. Numerous molecular alterations activating the oncogenic potential of the EGFR gene have been described including activating point mutations, point mutations causing resistance against EGFR-targeting drugs, activating deletions and truncations as well as overexpression of EGFR and its ligands, occasionally induced by gene amplification. In other situations, EGFR has been identified as a driver in the absence of any obvious molecular alterations of the EGFR gene. The discovery and development of EGFR-targeting agents depends on the availability of relevant tumor models. We provide an overview of our collection of EGFR-driven tumor models, including PDXs along with PDX-derived cell lines and human tumor cell lines. We compare sensitivity profiles of tumor models for EGFR-targeting agents obtained in vivo and in vitro and demonstrate that for EGFR-targeting agents, data obtained in 2D and 3D assays are predictive for the in vivo situation. We propose an optimized strategy for the preclinical profiling of EGFR-targeting anti-cancer agents.

Citation Format: Markus Posch, Hagen Klett, Anne-Lise Peille, Gerhard Kelter, Armin Maier, Julia Schüler, Thomas M. Metz. Tumor models driven by EGFR: Optimizing the preclinical profiling of EGFR-targeting agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2813.
Abstract 2894: XMT-1592, a site-specific Dolasynthen-based NaPi2b-targeted antibody-drug conjugate for the treatment of ovarian cancer and lung adenocarcinoma
Authors: Fessler Shawn , Dirksen Anouk , Collins Scott D. , Xu Ling , Lee Winnie , Wang Jason , Eydelloth Ron , Ter-Ovanesyen Elena , Zurita Jeffrey , Ditty Elizabeth , Nehilla Barrett , Clardy Susan , Clardy Susan , Carter Tyler , Avocetien Kenneth , Nazzaro Mark , Le Nam , Catcott Kalli C. , Uttard Alex , Du Bingfan , Chin Chen-Ni , Mosher Rebecca , Slocum Kelly , Qin Liuliang , Lee David , Toader Dorin , Damelin Marc , Lowinger Timothy B.
Published: September 2020

The Dolasynthen platform incorporates the highly potent anti-mitotic agent auristatin F-HPA (AF-HPA), with its associated DolaLock mechanism of controlled bystander effect, and enables the synthesis of antibody-drug conjugates (ADCs) with precise control of the drug-to-antibody ratio (DAR) and site-specific bioconjugation. XMT-1592 is a novel ADC comprised of an anti-NaPi2b antibody and Dolasynthen, conjugated in a site-specific manner to yield DAR 6.

NaPi2b, also known as SLC34A2, is a transmembrane sodium-phosphate transporter that is broadly expressed on tumor cells in ovarian carcinoma, NSCLC lung adenocarcinoma and other tumor types. Recent studies have shown that NaPi2b expression is enriched in the EGFR and KRAS mutant subtypes of lung adenocarcinoma.

Binding studies showed a specific, high-affinity interaction of XMT-1592 with NaPi2b that was not affected by conjugated Dolasynthen. XMT-1592 elicited potent and specific in vitro cytotoxicity against NaPi2b-expressing ovarian carcinoma cells. XMT-1592 exhibited potent and specific in vivo activity in NaPi2b-expressing tumor xenografts derived from ovarian carcinoma or lung adenocarcinoma. Consistent with the targeted delivery benefits of the ADC approach, XMT-1592 yielded high and sustained concentrations of AF-HPA to tumors but not normal tissues.

To evaluate the benefits of site-specific bioconjugation of Dolasynthen, we conducted in vitro and in vivo comparisons of XMT-1592 to a stochastically conjugated version of the ADC. XMT-1592 had improved in vivo activity, pharmacokinetics, and clinical pathology relative to its stochastic counterpart. Taken together, these results support XMT-1592 as a development candidate for the treatment of NaPi2b-expressing tumors.

Citation Format: Shawn Fessler, Anouk Dirksen, Scott D. Collins, Ling Xu, Winnie Lee, Jason Wang, Ron Eydelloth, Elena Ter-Ovanesyen, Jeffrey Zurita, Elizabeth Ditty, Barrett Nehilla, Susan Clardy, Susan Clardy, Tyler Carter, Kenneth Avocetien, Mark Nazzaro, Nam Le, Kalli C. Catcott, Alex Uttard, Bingfan Du, Chen-Ni Chin, Rebecca Mosher, Kelly Slocum, Liuliang Qin, David Lee, Dorin Toader, Marc Damelin, Timothy B. Lowinger. XMT-1592, a site-specific Dolasynthen-based NaPi2b-targeted antibody-drug conjugate for the treatment of ovarian cancer and lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2894.
Abstract 2934: The role of Globo H in cancer cell survival
Authors: Kuo Tzer-Min , Tsai Yi-Chien , Lee Chin-Chan , Lai Jiann-Shiun
Published: September 2020

BACKGROUND: Globo H (GH), a glycosphingolipid (GSL) carbohydrate antigen, is found on a variety of epithelial tumors and is a promising target for cancer immunotherapy given the marked difference in expression of GH between cancer cells and normal tissues. Globo series antigens (Globo H, SSEA-4) have been implicated in playing important roles in protecting tumor cells from apoptosis, suppression of immune cell activity in the tumor microenvironment, and promoting endothelial cell angiogenesis through shedding the glycosphingolipids to immune cells and endothelial cells, respectively. Recently, apoptosis regulation by protein interaction with Globo-series GSLs in breast cancer cells was reported. This study aims to investigate the GH and anti-GH antibody effects on cancer cell survival.

METHODS: In vivo: After allografting murine B16F10 melanoma subcutaneously, C57BL/6 mice, n=5, were treated with Globo-H ceramide (GHCer) by peritumoral injection and monitored for 26 days. In vitro: GH expression on the surface of tumor cells and colony formative activity in GHCer-treated human cells were measured. Cytokine antibody array and Microwestern protein array analyses were performed in the GHCer-treated A549 cells. Cell viability in high GH–expressing lung cancer cells treated with erlotinib was assessed. Erlotinib resistance and cytokine levels in high GH–expressing tumor cells treated with anti-GH antibody were measured.

RESULTS: In vivo: GHCer-treated mice showed accelerated B16F10-tumor growth rates (3.36-fold; P<0.01, n=5) with less tumor necrosis and higher proliferating cell nuclear antigen expression in the internal tumor tissues. In vitro: In GHCer-treated human lung cancer A549 and breast cancer MDA-MB-231 cells, higher colony formative activity and increased exogenous GH expression on the surface of tumor cells compared to the control group were observed, while cell proliferation was not enhanced. Up-regulated erlotinib-resistant cytokine IL-8 and FGF2 and K-RAS-related signaling were observed in A549 cells with exogenous high GH expression. Exogenously GHCer-added lung cancer cells displayed higher cell viability (37.8% increase) during erlotinib challenge, and an anti-GH antibody treatment decreased the GH effects of erlotinib resistance.

CONCLUSION: GH promotes cancer cell viability in vitro and in vivo, indicating an important role in cancer cell survival during tumor progression. Anti-GH antibody treatment was shown to be able to abolish the GH effects on erlotinib resistance of lung cancer cells in vitro.

Citation Format: Tzer-Min Kuo, Yi-Chien Tsai, Chin-Chan Lee, Jiann-Shiun Lai. The role of Globo H in cancer cell survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2934.
Abstract 2958: ONC206, an imipridone derivative, induces cell death through activation of the integrated stress response in serous endometrial cancer in vitro
Authors: Zhang Yingao , Huang Yu , Yin Yajie , Fan Yali , Tucker Katherine , Staley Allison , Prabhu Varun , Allen Joshua , Zhou Chunxiao , Bae-Jump Victoria
Published: September 2020

Objectives: ONC206 (Oncoceutics) is a selective dopamine receptor D2 (DRD2) antagonist and an analogue of ONC201, an imipiridone currently being investigated in phase II clinical trials for serous endometrial cancer (EC). ONC206 has been shown to inhibit cell proliferation through activation of the integrated stress response (ISR) and apoptosis in brain, breast, and colorectal cancer cell lines. Thus, we investigated the efficacy of ONC206 for serous EC as well as its underlying mechanism of action.

Methods: The serous EC cell lines, ARK1 and SPEC-2, were used. Cellular proliferation was measured using MTT assay. Cleaved caspase-3 and 9, markers of apoptosis, were assessed by ELISA assays. Adhesion and invasion were assessed by laminin and wound healing assays, respectively. Relative In vitro ISR was estimated by ROS and JC-1 assays. Western immunoblotting evaluated effects of ONC206 on proteins central to cellular stress and invasion. All statistical analyses were performed using the software Prism 8 (GraphPad).

Results: ONC206 inhibited cellular proliferation in a dose-dependent manner and was more potent than ONC201 in ARK1 (IC50=0.33uM vs IC50=1.59uM) and SPEC-2 (IC50=0.24uM vs IC50=0.81uM) cell lines. Treatment with ONC206 decreased JC-1 fluorescence (p=0.001-0.01), and increased ROS production (p<0.001), cleaved caspase-3 (p=0.008-0.02), and caspase-9 (p=0.02-0.04) activity in both cell lines. ONC206 increased expression of the stress proteins ATF4, PERK, and BiP. Adhesion (p=0.01-0.02) and wound healing (p<0.001) were significantly impaired after treatment with ONC206 and was accompanied by decreases in proteins Slug, Snail, Vimentin, and VEGF-C. Pretreatment with stress inhibitor n-acetylcysteine (NAC) significantly attenuated the efficacy of ONC206 on ROS production (p=0.001-0.01), wound healing (p<0.001), and proliferation (p=0.001-0.002) in both cell lines.

Conclusions: Our results suggest that ONC206 demonstrates nanomolar potency for the inhibition of proliferation in serous EC cell lines. Specifically, ONC206 utilizes ISR activation as a significant pathway in the propagation of its anti-proliferative and anti-metastatic effects. Thus, ONC206 may be a promising agent in serous EC which has limited treatment options.

Citation Format: Yingao Zhang, Yu Huang, Yajie Yin, Yali Fan, Katherine Tucker, Allison Staley, Varun Prabhu, Joshua Allen, Chunxiao Zhou, Victoria Bae-Jump. ONC206, an imipridone derivative, induces cell death through activation of the integrated stress response in serous endometrial cancer in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2958.
Abstract 2979: The effect of interferon gamma on melanoma progression under MAPK inhibitor therapy
Authors: Chen Kevin , McMillan Rachel , Chatrizeh Mona , Hugo Willy
Published: September 2020

About half of metastatic cutaneous melanoma harbor a mutation in the BRAF protein, causing constitutive activation of the MAPK pathway. Currently, patients with such mutation are treated with MAPK pathway inhibitors (MAPKi) targeting the mutant BRAF and its downstream MEK proteins. However, within a span of 6-12 months, these patients will relapse, and their tumors will acquire resistance to this treatment. In this project, we aim to understand the role of IFN gamma (IFNg) to help us determine if the resistance phenotype differs between cells exposed with MAPKi alone versus cells treated with MAPKi and IFNg. Previous literature has reported on the development of a dedifferentiated resistance phenotype which was characterized by a downregulation of MITF and an upregulation of AXL proteins. However, the patient-derived transcriptomic data comparing paired tumors pre- and post-MAPKi therapy did not show such changes in MITF or AXL levels. We posit that the discrepancy may be because most of the earlier studies on MAPKi resistance were done in vitro or immune-suppressed in vivo models. Melanoma biopsies from patients responding to MAPKi treatment showed a substantial increase in the tumor-infiltrating T cell and other immune cells. One of the most dominant immune factors produced by the T cells is the interferon gamma (IFNg). Intriguingly, our data show that IFNg, in the presence of MAPKi, can suppress the dedifferentiation switch that is induced by MAPKi-alone. Specifically, through serial western blots, we discovered that MAPKi and IFNg reduced the previously reported AXL upregulation and MITF downregulation in four different mutant BRAF melanoma lines. RNA sequencing from the same time points revealed that melanoma treated with MAPKi and IFNg upregulate IFN-stimulated genes that are typically expressed by immune cells; this leads us to believe that some novel transcriptomic programs have been activated by the combination of MAPKi and IFNg. By solely examining one immune factor, IFNg, we discovered one potential explanation for the discrepancy in the prevalence of MAPKi-induced dedifferentiation phenotype in the patient-derived melanoma samples. We are in the midst of performing drug screening experiments on MAPKi and IFNg-resistant melanoma lines in order to discover novel therapeutic vulnerabilities that were not observed with MAPKi-alone. Finally, by taking into account the secreted factors in the tumor microenvironment, our study can provide a more accurate depiction of MAPKi resistance evolution in mutant BRAF melanoma patients.

Citation Format: Kevin Chen, Rachel McMillan, Mona Chatrizeh, Willy Hugo. The effect of interferon gamma on melanoma progression under MAPK inhibitor therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2979.
Abstract 2986: NR2F1 underlies persistence of residual disease in melanoma
Authors: Tiago Manoela , Teh Jessica L. , Purwin Timothy J. , Cai Weijia , Hollingworth Connor , Lopez-Anton Melisa , Aguirre-Ghiso Julio A. , Aplin Andrew E.
Published: September 2020

Despite the clinical success of targeted therapy and checkpoint inhibitors in melanoma, therapeutic responses are transient, followed by relapse that may be driven by a small subpopulation of residual or drug-tolerant cells. Understanding residual disease and metastasis mechanisms can provide clues both for developing improved versions of a drug and for guiding the selection of appropriate drug combinations for melanoma therapy. Here, we found that the well-known marker of tumor dormancy, Nuclear Receptor Subfamily 2 Group F Member 1 (NR2F1), was overexpressed in minimal disease residual cells following CDK4/6 and MEK inhibitors (CDK4/6i+MEKi) treatment in vivo. Furthermore, melanoma cells overexpressing NR2F1 were less sensitive to (CDK4/6i+MEKi) or BRAF and MEK inhibitors (BRAFi+MEKi) treatment in vitro and in vivo models, inhibiting apoptosis. Surprisingly, we did not find any evidence of decreased cell growth in our model. Using a three-dimensional tumor spheroid assay in vitro, we found the NR2F1 expression enhanced melanoma invasion following CDK4/6i+MEKi or BRAFi+MEKi treatments. The use of published RNA Seq data sets that were gathered from the GEO database and Single Cell Seq data sets from PDX melanoma samples showed that high expression of NR2F1 is enriched in the undifferentiated cell state and invasive cells, respectively. Furthermore, BRAF mutant patient sample with an acquired mutation in NRAS Q61R following BRAFi+MEKi+CDKi presented a high expression of NR2F1. Altogether, these findings suggest that NR2F1 may play a role in residual disease persistence besides known features of tumor dormancy, especially important in determining responses to dramatic changes in the environment, such as changes induced by anti-cancer therapy.

Citation Format: Manoela Tiago, Jessica L. Teh, Timothy J. Purwin, Weijia Cai, Connor Hollingworth, Melisa Lopez-Anton, Julio A. Aguirre-Ghiso, Andrew E. Aplin. NR2F1 underlies persistence of residual disease in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2986.
Abstract 3003: MET mediates entrectinib resistance in ROS1 gene fusion positiveNSCLC
Authors: Tyler Logan , Le Anh , Nijmeh Hala , Doebele Robert
Published: September 2020

ROS1 gene fusions are predominantly found in non-small cell lung cancer (NSCLC), and ROS1 gene fusion-positive (ROS1+) tumors comprise 1-2% of all diagnosed lung adenocarcinomas. Currently, there are two ROS1 tyrosine kinase inhibitors (TKIs) which are US FDA approved, crizotinib and entrectinib, both of which generate tumor response rates in excess of 70% and demonstrate prolonged disease control. However, acquired resistance to TKIs is inevitable, and has been reported in ROS1+ NSCLC tumors or cancer cell models by our group and others and include ROS1 kinase domain mutations (e.g., L1951R, S1986Y/F, F2004V, L2026M, and G2032R), activation of bypass signaling pathways (e.g., EGFR, RAS, or KIT), or phenotypic transformation (such as epithelial to mesenchymal transition). In order to better understand mechanisms of acquired TKI resistance in ROS1 NSCLC, our lab utilized primary, patient-derived cancer cell models harboring these rare oncogenic kinase rearrangements. Herein, using a patient-derived NSCLC cancer cell line (CUTO28) harboring a TPM3-ROS1 fusion, we derived an entrectinib-resistant cell line (CUTO28-ER) through in vitro culture under drug selection. In these CUTO28-ER cells, we identified: (1) MET-mediated bypass signaling as an acquired resistance mechanism to entrectinib, (2) upregulation of MET signaling is accomplished via MET gene amplification, and (3) resistance could be overcome by the dual ROS1/MET inhibitor crizotinib. To our knowledge, this is the first reported case of MET-mediated acquired resistance in a ROS1+ cancer. This cell line model finding was supported by another primary, patient-derived cell line (CUTO38) harboring a CD74-ROS1 fusion; this cell line was derived from a NSCLC patient following disease progression on entrectenib in the clinic. CUTO38 is resistant to entrectinib in vitro and displays elevated MET expression, MET activation with increased phosphorylation, and dependence on MET for cell proliferation and survival. Notably, MET activation in CUTO38 was not generated by gene amplification, MET exon 14 splicing, MET gene fusion, or autocrine HGF expression, suggesting alternate means of MET activation. These findings suggest that interrogation of MET gene amplification or other MET biomarkers should be performed in ROS1+ NSCLC patients who experience disease progression on entrectinib or other ROS1 TKIs that do not inhibit MET, since there are clinically available MET inhibitors, some of which are dual ROS1/MET inhibitors, that may overcome drug resistance in these patients.

Citation Format: Logan Tyler, Anh Le, Hala Nijmeh, Robert Doebele. MET mediates entrectinib resistance in ROS1 gene fusion positiveNSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3003.
Abstract 3016: Tucatinib inhibits CYP3A, CYP2C8 and P-gp-mediated elimination and is impacted by CYP2C8 inhibition in healthy volunteers
Authors: Topletz-Erickson Ariel R. , Lee Anthony , Sun Hao , Mayor JoAl , Walker Luke , Endres Christopher J.
Published: September 2020

Background: Tucatinib is a potent, highly selective HER2 tyrosine kinase inhibitor in development for the treatment of patients with HER2+ metastatic breast cancer. In vitro metabolism studies suggest that drug metabolizing enzymes CYP2C8 and CYP3A play a role in tucatinib metabolism. Tucatinib exhibits competitive inhibition of CYP2C8, CYP2C9, CYP3A, and P-gp, and metabolism-dependent inactivation of CYP3A in vitro. ONT-380-012 was a clinical drug interaction study conducted to evaluate the magnitude of potential enzyme and transporter interactions for tucatinib (both as a victim and perpetrator), and the safety of healthy subjects when administered tucatinib doses at therapeutic levels (300 mg BID).

Methods: Healthy volunteers (n=116) at multiple centers were enrolled in the study. Parts A-C evaluated the effects of a strong CYP2C8 inhibitor (gemfibrozil), a strong CYP3A inhibitor (itraconazole), and a CYP3A/CYP2C8 inducer (rifampin) on single-dose tucatinib (300 mg) PK. Parts D and E assessed the effects of steady-state tucatinib (300 mg BID) on single-dose PK of substrate probes for CYP2C8 (repaglinide), CYP2C9 (tolbutamide), CYP3A (midazolam), and P-gp (digoxin). Plasma samples were collected for PK analysis and drug concentrations were measured using validated LC-MS/MS methods.

Results: A strong CYP3A inhibitor (itraconazole) increased tucatinib AUCinf and Cmax 1.3-fold. A CYP3A/CYP2C8 inducer (rifampin) decreased tucatinib AUCinf and Cmax 48% and 37%, respectively. A strong CYP2C8 inhibitor (gemfibrozil) increased tucatinib AUCinf and Cmax 3.1- and 1.6-fold, respectively.

Tucatinib increased the AUCinf and Cmax of the CYP3A substrate (midazolam) 5.7- and 3.0-fold, respectively, the AUCinf and Cmax of the CYP2C8 substrate (repaglinide) 1.7-fold, and the AUCinf of the P-gp substrate (digoxin) 1.5-fold. Tucatinib had no impact on the PK of the CYP2C9 substrate (tolbutamide). Overall, tucatinib was well tolerated in healthy volunteers when administered 300 mg BID.

Conclusions: Together, these data indicate tucatinib is metabolized primarily by CYP2C8 and to a lesser extent via CYP3A. Tucatinib was found to be a strong inhibitor of CYP3A, a weak inhibitor of CYP2C8 and P-gp, and had no impact on CYP2C9-mediated metabolism in vivo.

Citation Format: Ariel R. Topletz-Erickson, Anthony Lee, Hao Sun, JoAl Mayor, Luke Walker, Christopher J. Endres. Tucatinib inhibits CYP3A, CYP2C8 and P-gp-mediated elimination and is impacted by CYP2C8 inhibition in healthy volunteers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3016.