bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2001: Autocrine HGF maturation as a targetable regulatory step in cetuximab resistance in colorectal cancer
Authors: Jones Vivian Truong , Graves-Deal Ramona , Cao Zheng , Bogatcheva Galina , Ramirez Marisol A. , Harmych Sarah J. , Higginbotham James N. , Wahoski Claudia C. , Joshi Neeraj , Roland Joseph T. , Ayers Gregory D. , Liu Qi , Coffey Robert J. , Janetka James W. , Singh Bhuminder
Published: March 2024

Although amplifications and mutations in receptor tyrosine kinases (RTKs) act as bona fide oncogenes, in most cancers, RTKs maintain moderate expression and remain wild-type. Consequently, cognate ligands control many facets of tumorigenesis, including resistance to anti-RTK therapies. Herein, we show that the ligands for the RTKs MET and RON, HGF and HGFL, respectively, are synthesized as inactive precursors that are activated by cellular proteases. Our newly generated HGF/HGFL protease inhibitors could overcome both de novo and acquired cetuximab resistance in colorectal cancer (CRC). Conversely, HGF overexpression was necessary and sufficient to induce cetuximab resistance and loss of polarity. Moreover, HGF-induced cetuximab resistance could be overcome by the downstream MET inhibitor, crizotinib, and upstream protease inhibitors. Additionally, HAI-1, an endogenous inhibitor of HGF proteases, (i) was downregulated in CRC, (ii) exhibited increased genomic methylation that correlated with poor prognosis, (iii) HAI-1 expression correlated with cetuximab response in a panel of cancer cell lines, and (iv) exogenous addition of recombinant HAI-1 overcame cetuximab resistance in CC-HGF cells. Thus, we describe a targetable, autocrine HAI-1/Protease/HGF/MET axis in cetuximab resistance in CRC.

Citation Format: Vivian Truong Jones, Ramona Graves-Deal, Zheng Cao, Galina Bogatcheva, Marisol A. Ramirez, Sarah J. Harmych, James N. Higginbotham, Claudia C. Wahoski, Neeraj Joshi, Joseph T. Roland, Gregory D. Ayers, Qi Liu, Robert J. Coffey, James W. Janetka, Bhuminder Singh. Autocrine HGF maturation as a targetable regulatory step in cetuximab resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2001.
Abstract 2003: Chromatin based targeting of a master regulatory complex in estrogen receptor epithelial ovarian cancer
Authors: Jaiswal Sushil K. , Fedkenheuer Kevin , Khamar Ronak , Tan Hua , Gotea Valer , Elkahloun Abdel , Annunziata Christina , Elnitski Laura
Published: March 2024

Ovarian cancer remains a significant contributor to cancer-related mortality in the United States, with low survival rates over the past decades. Hormonal therapies targeting estrogen receptors have shown limited efficacy. To explore mechanism of the inefficacy, our approach involved investigating ESR1 degradation within the higher order chromatin structure formed by Megacomplex. We identified a 1.3MDa Megacomplex containing ESR1, FOXA1 and PITX1, with colocalization at 2,440 genomic positions within the PEO4 cell line. Unlike cytoplasmic ESR1, the nuclear form was degraded minimally by ICI182780 (ICI), moreover, ESR1 incorporated into the Megacomplex macromolecule was least degraded. We hypothesized that this resistance might be due to sequestration within the Megacomplex, thereby preventing ICI from effectively targeting ESR1. Addition of a small molecule inhibitor of active chromatin, JQ1, in combination with ICI effectively enhanced the degradation of ESR1 within Megacomplex. The combination stopped nuclear occupancy of ESR1 and also degraded the cytoplasmic protein, thus breaking the ESR1 signaling circuit responsible for tumor cell proliferation. Pathway enrichment results consistently identified similar gene sets obtained independently for RNA-seq analysis after ESR1 modulation, and ChIP-seq analysis of Megacomplex bound genes. Likewise, proteins enriched within the Megacomplex macromolecule, as revealed from mass-spec analysis, returned similar pathway enrichments, directly implicating Megacomplex in ESR1 ovarian cancer gene regulation. We conclude, ESR1 in Megacomplex is resistant to ICI and sensitized by combining ICI with JQ1. This combined treatment strategy showed inhibition of cell proliferation and viability by uncovering ESR1 within the Megacomplex, thus elucidating a novel drug resistance mechanism.

Citation Format: Sushil K. Jaiswal, Kevin Fedkenheuer, Ronak Khamar, Hua Tan, Valer Gotea, Abdel Elkahloun, Christina Annunziata, Laura Elnitski. Chromatin based targeting of a master regulatory complex in estrogen receptor epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2003.
Abstract 2045: Novel TFRC humanized mouse model for drug delivery to the brain
Authors: Sonego Fabiane , Pappalardo Angela , Martin Gaëlle , Ozgür Burak , Ravn Peter , Cherifi Yacine , Sotty Florence , Isnard-Petit Patricia , Thiam Kader
Published: March 2024

Gliomas are the most common neoplasm in the central nervous system (CNS). Although immunotherapy has shown efficacy in several tumor types, no immunotherapies are approved by the US Food and Drug Administration for treatment of gliomas. One of the biggest challenges in using immunotherapies in the CNS is the existence of the blood-brain barrier (BBB), which limits drug delivery and the subsequent therapeutic drug efficacy. In addition to biodistribution, assessment of PK in preclinical models do not reflect the clinical findings. Conjugation of therapeutic agents with molecules binding to TRFC expressed along the BBB are being used as one of the alternative approaches to deliver drugs into the CNS. Herein, we describe a novel TFRC humanized mouse model (hTFRC), developed to enable assessment of compounds targeting human TFRC and aiming to deliver drug through the BBB. hTFRC model was developed by knock-in at the mouse endogenous locus to enable a physiological expression of TFRC. Splenocytes from hTFRC mice stimulated with labelled pHrodo Red human transferrin show internalization of human transferrin, suggesting that humanization of the transferrin receptor does not change the endocytosis process. In vivo, treatment of hTFRC mice with anti-BACE1/anti-TFRC antibodies enables the transfer of anti-BACE1 to the brain, while anti-BACE1 antibody is not. As a consequence of the drug delivery, mice treated with anti-BACE1/anti-TFRC show a marked reduction in Amyloid-β 1-40 levels in the brain. Altogether, the data suggest that hTFRC is expressed in the BBB and enables the efficient shuttling of therapeutic antibodies to the brain. While assessment of TFRC-targeting antibodies in a glioma model remains to be performed, the hTFRC model is a promising tool to assess delivery and efficacy of immunotherapies in gliomas. Moreover, the hTFRC model is also being intercrossed with the well-established hSA/FcRn humanized mouse model (Viuff et al., 2016), to enable a more translatable PK and biodistribution of compounds targeting TFRC.

Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Burak Ozgür, Peter Ravn, Yacine Cherifi, Florence Sotty, Patricia Isnard-Petit, Kader Thiam. Novel TFRC humanized mouse model for drug delivery to the brain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2045.
Abstract 2060: Automation of 3D cancer spheroid assay for compound screening
Authors: Sirenko Oksana , Lim Angeline , Michlmayr Astrid , Tong Zhisong , Keidel Emilie , Spira Felix
Published: March 2024

Finding efficient drug combinations to treat cancer patients is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient testing drug efficacy to discover new therapeutic targets. 3D cancer models are highly valuable tools for cancer research and drug development. However, the complexity of performing 3D assays remains a hurdle for adopting these methods for compound screening. To alleviate the bottlenecks that come with labor-intensive manual protocols we developed cell culture automation methods using CellXpress.ai instrument. The instrument enables automation of 2D or 3D culture for prolonged complex workflows. The CellXpress.ai provides automated plating, passaging, media exchanges, monitoring organoids, alone with compound treatment and the end point assays with high content image analysis. In the present study we describe full automation of imaging, analysis, and cell culture process which enables scaling up complex 3D cell-based assays and compound screening. As an example of 3D protocol, we automated culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell line in U-shape low attachment plates. HCT116 cells were expanded in 2D, then spheroids were formed after automated dispensing of cell suspension into U-shape 96 or 384 plates. After 48h spheroids were treated with 12 anti-cancer compounds, at multiple concentrations for 3-5 days, then stained and imaged. Cell plating, compound additions, media exchange, and staining were performed automatically by CellXpressAI instrument. During the culture spheroids were monitored using transmitted light, with immediate analysis of phenotypic changes, including inhibition of growth or spheroid disintegration. For endpoint assay spheroids were stained with combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD, then imaged, and analyzed for multiple read-outs including spheroid size, marker intensities, and live-dead cell scoring. We observed concentration-dependent inhibition of spheroid growth and cell death in response to anti-cancer compounds and evaluated effective compound concentrations for cytotoxic or cytostatic effects. Cell culture process automation for 3D assays, powered by imaging and image analysis has a potential to bring 3D biology into another level, allowing to increase throughput and reproducibility, and enabling variety of high throughput drug discovery and precision medicine applications.

Citation Format: Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Zhisong Tong, Emilie Keidel, Felix Spira. Automation of 3D cancer spheroid assay for compound screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2060.
Abstract 2066: Measuring the activity of SMUG1 DNA glycosylase with a novel sSTRIDE-SMUG1 assay
Authors: Stepien Karolina , Sierpowski Franek , Prucsi Zsombor , Koman Szymon , Jarosz Monika , Waligorska Agnieszka , Kordon-Kiszala Magdalena , Solarczyk Kamil
Published: March 2024

On average, spontaneous deamination of cytosine to aberrant base uracil happens 70-200 times per human genome per day. These mutagenic U/G mispairs are, however, corrected by error-free base repair (BER) initiated by uracil-DNA glycosylases. SMUG1 (single-strand selective monofunctional uracil DNA glycosylase), as one of the major glycosylases, removes uracil and oxidized pyrimidines from DNA. In spite of the name, SMUG1 removes uracil also from double-stranded DNA. Unrepaired by SMUG1 U/G mispairs are mutagenic, and alterations in SMUG1 expression levels have been linked to cancer development. Low SMUG1 transcripts are associated with poor prognosis in breast cancer and SMUG1 loss has been shown to cause PARPi resistance in HR-deficient background. Hence, tools describing SMUG1 activities in DNA damage response may provide new approaches for cancer cell treatment. We report here the development, optimization and validation of an assay measuring the activity of SMUG1. The new assay is based on the STRIDE platform technology, which enables direct and sensitive detection of single- or double-strand DNA breaks in situ, in fixed cells. sSTRIDE-SMUG1 assay detects DNA nicks localized in close proximity to the SMUG1 protein and can thus be utilized as a direct reporter of its activity. The experiments were performed in HAP1 cells. First, in untreated cells we have shown that the sSTRIDE-SMUG1 signals constitute ca. 1-2% of total single-strand DNA breaks foci detected by the classic sSTRIDE assay variant, while the negative controls have shown that the number of false-positive foci does not exceed 15% of the total number of signals. As expected, treatment of cells with a cytotoxic nucleotide hydroxymethyl-deoxyuridine (hmdU) resulted in an increase in the number of detected sSTRIDE-SMUG1 signals. To verify the specificity of the assay, SMUG1 was knocked-down in HAP1 wild-type cells using siRNA and then the level of sSTRIDE-SMUG1 signals was measured in both untreated and hmdU-treated cells. In all tested conditions, SMUG1 expression silencing has resulted in a decrease in sSTRIDE-SMUG1 foci. We believe that the sSTRIDE-SMUG1 assay can prove to be a very useful solution to study DNA repair mechanisms involving aberrant base excision. Additionally, sSTRIDE-SMUG1 measurements may be of the utmost importance in characterization of agents inducing synthetic lethality or trapping BER intermediates.

Citation Format: Karolina Stepien, Franek Sierpowski, Zsombor Prucsi, Szymon Koman, Monika Jarosz, Agnieszka Waligorska, Magdalena Kordon-Kiszala, Kamil Solarczyk. Measuring the activity of SMUG1 DNA glycosylase with a novel sSTRIDE-SMUG1 assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2066.
Abstract 2119: Translational research to facilitate development of novel therapeutic combinations of letrozole for the treatment of glioblastoma
Authors: Karve Aniruddha Sunil , Gabani Bhavesh Babulal , Gadgil Sidharth Nitin , Kulkarni Shravani Parag , Gudelsky Gary A. , Desai Pankaj B.
Published: March 2024

Letrozole (LTZ), a third-generation aromatase inhibitor, appears to be a promising novel therapeutic for the treatment of glioblastoma (GBM). It synergistically enhances the efficacy of the DNA alkylating agent temozolomide, one of the few approved chemotherapeutics for GBM, against patient-derived GBM cells. RNA-seq analysis of tumor from recurrent GBM patients treated with LTZ prior to surgery in a “window of opportunity” phase 0/1 dose escalation clinical trial revealed that LTZ downregulates genes encoding for DNA-damage repair proteins (BRCA2) in a dose dependent manner. Our central hypothesis for this project is that combination of LTZ with targeted therapeutic agents that promote DNA damage results in synergistic effects against GBM. The objective of this study is to evaluate the effects of poly-ADP ribose polymerase (PARP) inhibitors in combination with LTZ against patient-derived GBM cells. Our secondary aim for this project is to delineate the blood-brain barrier (BBB) permeability of PARP inhibitors. Employing patient-derived G43, G75 and JHH-136 GBM lines we assessed the influence of PARP inhibitors (olaparib, pamiparib, veliparib and niraparib) in combination with LTZ on cell viability and neurosphere growth. We then utilized male CD-IGS rats (n=6; body wt., 250g) to assess plasma and brain extracellular fluid (ECF) pharmacokinetics (PK) of PARP inhibitors using cerebral microdialysis. The IC50 values for PARP inhibitors in these assays ranged from 0.06 to 11.31 µM. Treatment in combination with LTZ (0.05 µM), a non-cytotoxic concentration of LTZ in these lines, significantly decreased the IC50 values for PARP inhibitors (0.012 to 0.51 µM). Combination Index analysis indicated that this combination was strongly synergistic. Furthermore, the plasma and brain ECF PK analyses suggested that PARP inhibitors such as pamiparib and niraparib exhibit facile transport across the BBB barrier as reflected in Kp,uu,brain (ratio of AUCecf/AUCplasma, unbound) values of 0.98 and 0.45, respectively. Overall, these results provide a strong foundation for pursuing further development of combination therapy of GBM with LTZ and PARP inhibitors. A careful assessment of the clinical efficacy and safety profile of these agents will further guide shortlisting combination of LTZ for pre-clinical and clinical development.

Citation Format: Aniruddha Sunil Karve, Bhavesh Babulal Gabani, Sidharth Nitin Gadgil, Shravani Parag Kulkarni, Gary A. Gudelsky, Pankaj B. Desai. Translational research to facilitate development of novel therapeutic combinations of letrozole for the treatment of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2119.
Abstract 212: Repression of NKX2-1 promote epithelial-mesenchymal transition and tumor neutrophil recruitment in lung cancer progression
Authors: Chiou Shih-Hwa , Tsai Ping-Hsing , Wang Mong-Lien
Published: March 2024

Background: Malignant transformation is associated with tumor aggressiveness and poor patient outcome. This cellular alteration is due to either activation of oncogenes or the silencing of tumor suppressor genes. NKX2-1 was identified as a lineage-specific gene downregulated in poorly differentiated lung adenocarcinoma (LUAD). However, the molecular mechanism and functional role of infiltrated immune cells in NKX2-1-negative tumors are not clearly understood.

Methods: Clinical relevance of NKX2-1 expression and disease status were evaluated by immunohistochemical analysis of LUAD tissue arrays and the overall survival analysis of TCGA databases. In vitro and in vivo experiments were applied to confirm the role and mechanisms of NKX2-1 actions in transformed cells. The secretion of chemokines was evaluated by using chemokine array. Flow cytometry and single-cell RNA sequencing analysis of mouse tumor tissue were used to demonstrate the infiltration of neutrophils in mouse models.

Results: Low expression of NKX2-1 was observed in high-grade LUAD tumors. LUAD patients who express a pattern of NKX2-1high/TGF-βlow had better survival outcomes than those with NKX2-1low/TGF-βhigh. Mechanistically, TGF-β induced the downregulation of NKX2-1 which led to enhanced epithelial-mesenchymal transition (EMT), cell motility, and CXC chemokine expression. The high release of these chemoattractants promoted the recruitment of neutrophils. Meanwhile, NKX2-1-dependent suppression of CXC chemokines gene expression may be via repressive histone methylation at the promoter region of CXC chemokines in lung cancer cells. In the in vivo syngeneic LUAD models, exacerbated neutrophil infiltration was noted. Single-cell RNA sequencing and flow cytometry both revealed that neutrophils were recruited via CXCLs/CXCR2 activation in LUAD tumors with NKX2-1 knockdown.

Conclusion: Our data showed that NKX2-1 represses tumor growth, metastasis, and neutrophil recruitment via the CXCLs/CXCR2-dependent mechanisms. Notably, targeting CXCR2 in NKX2-1-negative tumors may improve LUAD patient outcomes.

Citation Format: Shih-Hwa Chiou, Ping-Hsing Tsai, Mong-Lien Wang. Repression of NKX2-1 promote epithelial-mesenchymal transition and tumor neutrophil recruitment in lung cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 212.
Abstract 2134: Disaggregating breast cancer mortality trends in Asian American women from 2005-2020
Authors: Marcotte Mia , Chauhan Yashi , Penmetcha Vedha , Thompson Caroline , Thompson Candice , Srinivasan Malathi , Kim Gloria , Qi Xinran , Huang Robert , Bacong Adrian
Published: March 2024

Background: Breast cancer (BC) is the second leading cause of death among women and has disproportionately higher rates of mortality in certain racial groups, including Asian Americans (AA). While differences in BC mortality between AA and other racial groups have been identified, few studies have disaggregated nationwide AA mortality data.

Methods: Using National Vital Statistics System mortality data, BC-related deaths were analyzed among AA groups (Asian Indian, Chinese, Filipina, Japanese, Korean, Vietnamese), Native Hawiian and Pacific Islanders (NHPIs), and non-Hispanic Whites (NHWs) from 2005-2020. We calculated cancer proportional mortality ratios (PMRs), age-adjusted mortality rates (AAMRs), standard mortality ratios, and used JoinPoint regression for average annual percentage change (AAPC).

Results: Among 13677 AA, 522 NHPI, 501282 NHW descendants, 14.3% cancer deaths were due to BC. PMRs increased for all racial groups except Filipina. AAMRs significantly decreased in NHWs (AAPC -1.61; CI -1.70 to -1.48) while significantly increasing for aggregate AAs (AAPC 0.45; CI 0.08 to 0.94). Among AAs, Filipinas had the highest AAMR. AAMRs significantly increased for Asian Indians (AAPC 2.05; CI 1.20 to 3.46), Filipinas (AAPC 0.61; CI 0.15 to 1.46), Koreans (AAPC 2.72; CI 1.57 to 4.65) and Vietnamese women (AAPC 3.63; CI 2.26 to 6.20), but significantly decreased for Japanese (AAPC -1.41; CI -2.22 to -0.53).

Conclusions: We observed heterogenous and converging BC mortality trends across AA groups. These trends elucidate which populations within the broader AA group have disproportionately higher death rates and therefore should be targeted for BC prevention protocols (e.g., Asian Indian and Filipina). Identifying such trends can also help elucidate disparities in cancer care and the overall social determinants that affect women with BC. Understanding disaggregated trends can inform more inclusive screening practices and culturally tailored interventions and treatments to reduce BC deaths in vulnerable populations.

Citation Format: Mia Marcotte, Yashi Chauhan, Vedha Penmetcha, Caroline Thompson, Candice Thompson, Malathi Srinivasan, Gloria Kim, Xinran Qi, Robert Huang, Adrian Bacong. Disaggregating breast cancer mortality trends in Asian American women from 2005-2020 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2134.
Abstract 2156: Exploring barriers to colorectal cancer screening among adult male immigrants in Salt Lake City
Authors: Onigbogi Olanrewaju Olusola , Kah Kebba , Okuyemi Kolawole
Published: March 2024

Background: The United States (U.S) foreign-born population aged 65+ increased substantially between 1990 and 2010 with migrants from Africa and the Middle East being a part of these new refugee admissions. According to 2010 data, socioeconomic disparities and reduced access to health care is common in this population with 16 percent of foreign-born elderly living below the poverty threshold in comparison with 8 percent of U.S.-born elderly. Colorectal Cancer (CRC) is the second leading cause of cancer related deaths in the U.S despite being easily preventable and treatable. Studies among male African Americans reveal a CRC incidence >20% higher than what obtains among whites. Since, genetic factors are believed to contribute as much as 35% to the overall risk of CRC, male adult immigrants aged 45 years and above from Africa and the Middle East may be at increased risk of developing the disease. Determining CRC screening uptake in this population will be critical in planning interventions which may ultimately reduce disparities in CRC-related morbidity and mortality in the future.

Methods: We conducted a mixed methods study comprising administration of questionnaires and organizing four Focus Group Discussion (FGD) sessions among adult males in Salt Lake City who identified as refugees from Africa or the Middle East. The sessions were audio-taped, transcribed and coded using the Dedoose software.

Results: We had thirty-one participants who were between 45 and 64 years of age. Twenty-five of them (83%) reported awareness of the existence of CRC screening tests. Ten participants (33%) had undergone a CRC test in the past. However, only nine participants (30%) indicated that they will be willing to participate (WTP) in CRC screening if offered for free. Increased WTP was associated with younger age (OR=1.45, 95% CI: 1.23-1.81), length of stay in the United States (OR=1.41, 95% CI: 1.13-1.78) and higher education (OR= 1.26, 95% CI 1.13-1.37). Three major themes identified in the FGDs include: (1) Mistrust - distrust of the US health system (2) Fear - fear of cancer and nature of test and (3) Fatalism - the belief that screening cannot lead to cancer cure.

Conclusion: The low level of WTP in CRC screening tests among the respondents may be associated with past experiences. There may be a need to consider the correlates of increased WTP and explore culture-specific CRC prevention measures in planning interventions designed to improve screening uptake in this population.

Citation Format: Olanrewaju Olusola Onigbogi, Kebba Kah, Kolawole Okuyemi. Exploring barriers to colorectal cancer screening among adult male immigrants in Salt Lake City [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2156.
Abstract 224: Combining functional precision medicine and radiotherapy with bio-printed organoids in head and neck squamous cell carcinoma
Authors: Lin Luda , Bommakanti Krishna K. , Sannajust Andreanne V. , Alhiyari Yazeed , Mo Brandon , Evans Lauran , Tebon Peyton , St. John Maie , Soragni Alice
Published: March 2024

The study aims to establish a high throughput ex vivo organoid-based drug screening platform for head and neck squamous cell carcinoma (HNSCC) to investigate interactions between radiotherapy and different chemotherapies or targeted therapy agents and ultimately provide functional precision medicine therapeutic suggestions. HNSCC is the sixth most common cancer worldwide. Both HPV+ and HPV- cancers can be treated with surgery, radiotherapy, chemotherapy, or a combination of these. Cetuximab is currently the only FDA-approved targeted agent for HNSCC. Few clinical trials to date have shown an improved clinical outcome when combining targeted agents with radiotherapy, thus there is a need to identify new therapeutic combinations to improve outcomes.

In order to characterize HNSCC tumor behavior and sensitivity to therapy, we have leveraged our high throughput 3D tumor organoid screening platform. Our approach entails seeding tumor cells in extracellular matrix around the perimeter of tissue culture wells in ring-like structures, which we have successfully deployed in other cancer types (Phan et al, 2019; Al Shihabi et al, 2022; Nguyen et al, 2022; Al Shihabi et al 2023). To optimize a chemoradiation platform, we used the HN30, HN31, and SCC154 cell lines representing HPV- primary, HPV- metastatic, and HPV+ head and neck tumors. The cell-line-derived 3D models are exposed to both radiotherapy and various chemotherapy or targeted therapy drugs. We measured cell responses by ATP-release assays and by applying our machine learning-based brightfield image analysis pipeline (Al Shihabi et al, 2022).

Our results showed that the HPV+ SCC154 was the most sensitive line to radiotherapy while HPV- metastatic HN31 was the most resistant. HN31 was resistant to most drug treatments except for gemcitabine, docetaxel, and mitomycin c, while HN30 and SCC154 were sensitive to a broader set of agents. We have investigated combinatorial efficacy of radiation and chemotherapy as well as targeted agents and extended this platform to HNSCC patient-derived tumor organoids. Our proof-of-concept study highlights how our organoid-based drug screening platform can be used to test chemoradiation approaches that are more effective and personalized.

Citation Format: Luda Lin, Krishna K. Bommakanti, Andreanne V. Sannajust, Yazeed Alhiyari, Brandon Mo, Lauran Evans, Peyton Tebon, Maie St. John, Alice Soragni. Combining functional precision medicine and radiotherapy with bio-printed organoids in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 224.
Abstract 2262: Genomic profiling of leukemic cell lines, responders vs. non-responders to Narazaciclib (HX301 or ON123300), a novel kinase inhibitor with activity against CSF1R and FLT3
Authors: Xue Jia , Yang Tao , Ke Hang , Guos Sheng , Li Henry Qixiang
Published: March 2024

Acute myeloid leukemia (AML) is an aggressive myeloid leukemia with global annual mortality ~150,000. While standard of care (SOC) of the 1st -line treatment being mainly induction chemotherapy, available targeted therapies cover rather limited patients carrying specific driver mutations (IDH mutations and FLT3-ITR). We previously reported a novel kinase inhibitor, Narazaciclib with strong anti-CSF1R activity (IC50 0.7nM) as well as anti-FLT3 (IC50 7nM), and also thus anti-AML activity for certain AML experimental models, as shown in vitro and in vivo (AACR Annual Meeeting, 2023). In the present study, we assessed the impact of HX301 on the in vitro proliferations of a panel of leukemic cell lines (n = 54), including a panel of AML cell lines (n = 33), that have been parallelly genomic-profiled by whole-exome sequencing and whole-transcriptome sequencing. We then set out to analyze the proliferation data and the genomic data, in attempt to elucidate genes and pathways that may be associated with the treatment responses. The biomarker analysis has been performed on gene expression, mutation and copy number variation levels, and the data been presented using common methodologies, including principal component analysis, differential expression analysis, correlation analysis, Gene Ontology enrichment analysis, gene set variation analysis, somatic gene mutation analysis, and copy number variation analysis, etc. Some of the observations have been made: 1) differential expression analysis on all leukemic cell lines shows that 254 genes have been positively associated with the responders; while expression of 191 genes are negatively correlated with AUC, i.e. positively associated with response, as revealed by Spearman correlation analysis; 2) Gene Ontology enrichment analysis revealed these response related genes are mostly involved in myeloid leukocyte activation and defense response; 3) all FLT3-ITD AML lines are responders, presumably due to the FLT3i activity of HX301; 4) CSF1R expression levels seem not correlated to drug responses; 5) pathway analysis on non FLT3-ITD AML lines reveals that STAT5 activation downstream of FLT3 signaling, IL7 pathway and NFKB pathway are significantly associated with better response. Finally, we are also attempted to use statistics learning to reveal genes, as a potential predictive biomarker, that may be correlated to drug response.

Citation Format: Jia Xue, Tao Yang, Hang Ke, Sheng Guos, Henry Qixiang Li. Genomic profiling of leukemic cell lines, responders vs. non-responders to Narazaciclib (HX301 or ON123300), a novel kinase inhibitor with activity against CSF1R and FLT3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2262.
Abstract 2272: Unlocking the potential of miRNA sequencing for early detection and monitoring of pancreatic cancer progression and recurrence
Authors: Fuller Ryan N. , Morcos Ann , Escalera Desirae , Wall Nathan R.
Published: March 2024

Pancreatic cancer (PC) is an elusive and deadly disease. Many patients with PC are diagnosed with metastatic or locally advanced tumors, limiting surgical resection, and increasing the risk of relapse after treatment. Relapse is recorded to be as high as 75% in the first 2 years after clinical intervention, resulting in a mean 5-year survival of 12% in the US, and as low as 2% worldwide. It is vital to develop diagnostic and monitoring techniques for PC. MicroRNAs (miRNAs) are robustly expressed in patient tumors and provide a rich source of biomarkers for disease detection and status. miRNA are short nucleic acid strands that bind to catalytic proteins to guide the regulation of large RNAs within the cellular space. This system is altered in tumors, leading to abhorrent expression of miRNA. Due to significant advancements in sequencing technology, miRNA-seq has become more accurate and affordable in detecting profile changes. To identify biomarkers for PC progression, we developed from the Gemcitabine-sensitive MIA-PaCa-2 (MP2) cell line the Gemcitabine and FOLFIRINOX resistant MP2-GR cell line. MiRNA-seq was conducted on the parental MP2 and resistant MP2-GR cells to identify those miRNAs that were differentially expressed between the cell lines. These miRNAs were analyzed via pathway analysis and gene ontology to find predicted functions followed by annotations from the miRNA Enrichment Analysis and Annotation tool (miEAA) to identify differentially expressed miRNAs associated with PC progression. This comparison indicated several miRNAs related to PC resistance. This dataset was then compared to The Cancer Genome Atlas (TCGA), which contains data from over 300 PC patients. The miRNA set was used to predict both PC progression after treatment, and recurrence. We are currently refining this data set to increase its predictive sensitivity and widen our patient data set comparison to further elucidate this approach in a clinical setting.

Citation Format: Ryan N. Fuller, Ann Morcos, Desirae Escalera, Nathan R. Wall. Unlocking the potential of miRNA sequencing for early detection and monitoring of pancreatic cancer progression and recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2272.
Abstract 2308: Semi-automated Ki-67 index assessment using top-k hotspot recommendation in Ki-67 IHC stained WSIs
Authors: Yang Hyeon Seok , Hwang Yunseob , Lee Yongeun , Jung Kyungsoo , Sung Minjung , Kwak Tae-Yeong , Kim Sun Woo , Chang Hyeyoon
Published: March 2024

Ki-67 index is commonly used as a breast cancer proliferation marker. However, it is a laborious and time-consuming task for a pathologist to directly measure the Ki-67 index. Recently, automation techniques using machine learning techniques such as deep learning have been proposed. In this study, we propose a top-k hotspot recommendation method to help Ki-67 index assessment based on nuclei detection detected by a deep learning model. The dataset included a total of 32 diaminobenzidine-hematoxylin (DAB-H) Ki-67 IHC whole slide images (WSIs) of breast cancer obtained by core biopsy. Each of these WSIs was evaluated by a pathologist using the interval of the Ki-67 index estimate and the scores of 1+, 2+, 3+, and 4+. Additionally, the search area was manually designated to exclude internal control. We developed a Ki-67 index analysis model based on deep learning and image analysis. Nuclei segmentation used the StarDist model. The detected results were filtered based on nucleus size and nucleus eccentricity. The filtering threshold was determined experimentally. To recommend top-k hotspots, a coarse search is performed to move the circle region horizontally and vertically in specific units, and the top k regions based on Ki-67 index are found among areas containing more than 500 cells. Next, a fine search is performed around the k areas in detail. Finally, the top-k hotspots and each detected positive and negative cells, count of positive cells and negative cells, and predicted Ki-67 index are provided. It was confirmed that the Ki-67 score estimated for the detected top-1 hotspot and the pathologist's Ki-67 score had a correlation of Pearson correlation coefficient=0.8153, R2=0.6648. As k increased, the top-k accuracy was observed to increase from 68.75% when k = 1 and 3 to 75.00% when k = 5, based on the pathologist's score. When a pathologist reviewed 10 cases where the Ki-67 score differed from the prediction, 6 cases were judged to be worth reexamining considering the proposed hotspot and Ki-67 index, and 2 cases included stromal cells in cell detection. In two cases, the WSI was judged to be blurry. Cell analysis and top-k hotspot recommendation using deep learning and image analysis were performed on Ki-67 IHC stained WSIs and compared with the pathologist's Ki-67 score. Through semi-automatic top-k hotspot recommendation, the reliability of diagnosis can be increased as an auxiliary test to Ki-67 index assessment.

Citation Format: Hyeon Seok Yang, Yunseob Hwang, Yongeun Lee, Kyungsoo Jung, Minjung Sung, Tae-Yeong Kwak, Sun Woo Kim, Hyeyoon Chang. Semi-automated Ki-67 index assessment using top-k hotspot recommendation in Ki-67 IHC stained WSIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2308.
Abstract 2339: GPNotebook: A toolkit for identifying cancer-related glycosylation alterations in glycoproteomic analysis
Authors: Zhang Hui , Hoang Trung , Hu Yingwei
Published: March 2024

This study is dedicated to the development and application of 'GPNotebook', an innovative Python-based data analysis tool, specifically designed to enhance glycopeptide research in cancer studies by enabling the detailed investigation of glycosylation patterns and their correlation with various clinical cancer phenotypes. In our study, we recognize the critical role of protein glycosylation in biological processes and its relevance in cancer. To address the lack of specialized tools for glycoproteomic analysis, we developed GPNotebook, a comprehensive Python package designed for detailed investigation of intact glycopeptides (IGPs). This tool integrates multiple functionalities, including statistical profiling, differential expression analysis, categorization of glycosylation subtypes, and exploration of the interactions between glycosylation and phosphorylation. It also encompasses survival analysis and assessments of glycosylation enzymes. We applied GPNotebook in a research project focused on 10 cancer types collected from Clinical Proteomic Tumor Analysis Consortium (CPTAC) projects to validate its efficacy and demonstrate its wide-ranging capabilities. Through this application, we were able to analyze and interpret complex glycoproteomic data, revealing potential IGPs as biomarkers for cancer detection and differentiation of cancer subtypes. We identified hundreds of cancer-specific IGPs, with several showing promise as early-stage cancer biomarkers. In addition, our analysis revealed unique glycosylation patterns linked to specific molecular subtypes and immune cell distributions in tumor microenvironments. We also clarified the roles of certain glycosylation enzymes across different cancer types, highlighting their potential significance in cancer progression. GPNotebook has proven to be a critical tool in glycopeptide research. Our findings not only enhance the understanding of glycosylation in cancer but also demonstrate GPNotebook's potential in identifying novel biomarkers and providing insights into cancer subtype differentiation. This study positions GPNotebook as a valuable resource for exploring the complex relationships between protein glycosylation and cancer phenotypes.

Citation Format: Hui Zhang, Trung Hoang, Yingwei Hu. GPNotebook: A toolkit for identifying cancer-related glycosylation alterations in glycoproteomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2339.
Abstract 2341: Morph: A feature extraction toolset for spatial transcriptomics
Authors: da Costa André Luiz N. Targino , Liu Jingxian , Mo Chia-Kuei , Storrs Erik , Southard-Smith Austin N. , Jayasinghe Reyka G. , Wang Julia T. , Iglesia Michael D. , Wendl Michael , Chen Siqi , Houston Andrew , Karpova Alla , Li Yize , Ding Li
Published: March 2024

Accurately defining spatial characteristics of tumors has been a challenge in cancer research. Specifically, there is still a lack of spatial transcriptomic (ST) bioinformatic methods that infer tumor boundaries, a necessity for tumor microenvironment (TME) analyses, that are fully automated and handle non-rectangular grids (like the one found in Visium). Here we introduce Morph, a toolset that not only addresses these limitations, but also accurately extracts tumor regions, layers surrounding them, and distances related to such regions. Morph was tested on a dataset composed of 117 ST slides across 6 different cancer types, including primary and metastatic tumors. More broadly, Morph is a computationally efficient tool based on mathematical morphology that is designed to work without any approximations on ST slides (that can be composed of spots in a hexagonal lattice) of any resolution. The toolset accepts a variety of input types, such as tumor purity and manual annotation, and can perform a diverse set of morphological operations, such as erosion, dilation, opening and closing, using a number of structuring elements with different shapes and sizes, such as a hexagon of side 1. Moreover, Morph runs quickly (seconds per sample). Its main focus is to unveil the aforementioned features of any spatially distinct tumor region (here defined as microregion) for various downstream analyses, such as spatially-varying copy number variations, cell-type distribution, and tumor-microenvironment interaction. Overall, we developed a spatial transcriptomics toolset that is not limited to any specific technology platform, and that can accurately extract ST features facilitating the discovery of spatial transcriptomic and interaction patterns.

Citation Format: André Luiz N. Targino da Costa, Jingxian Liu, Chia-Kuei Mo, Erik Storrs, Austin N. Southard-Smith, Reyka G. Jayasinghe, Julia T. Wang, Michael D. Iglesia, Michael Wendl, Siqi Chen, Andrew Houston, Alla Karpova, Yize Li, Li Ding. Morph: A feature extraction toolset for spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2341.
Abstract 2348: A global multiscale map of protein assemblies from integration of protein interactions and images
Authors: Schaffer Leah V. , Hu Mengzhou , Huttlin Edward L. , Qian Gege , Pal Abantika , Soni Neelesh , Latham Andrew , Vaites Laura Pontano , Le Trang , Qin Yue , Churas Christopher , Pratt Dexter , Echeverria Ignacia , Sali Andrej , Harper J Wade , Gygi Steven P. , Lundberg Emma , Ideker Trey
Published: March 2024

Cells regulate growth and function through a hierarchical structure of subcellular protein assemblies, in which alterations can result in cellular dysfunction leading to disease such as cancer. Much of this structure remains uncharted, resulting in recent efforts to map subcellular organization at different physical scales. Here, we report a global architectural map of human cancer cell protein assemblies spanning 10−9 to 10−5 nm, based on integration of near-proteome-wide affinity purification mass spectrometry-based protein interactions and immunofluorescent imaging in U-2 OS osteosarcoma cancer cells. The U-2 OS multi-scale integrated cell map places >5000 proteins into 270 distinct subcellular protein assemblies across biological scales, representing approximately half of expressed proteins. There are known organelles and protein complexes recovered in the map, as well as 152 putative assemblies, such as a putative interferon signaling complex consisting of a serine protease and STAT transcription factors. The map also incorporates 128 previously uncharacterized proteins into protein assemblies, such as C18orf21 association with a canonical RNAse complex. Protein subsystems in the U-2 OS map were evaluated for the potential for downstream integrative structural modeling, where available structural data (e.g. crosslinking, Protein Data Bank structures, AlphaFold predictions) are combined in a Bayesian framework to model the physical structures of protein complexes. Using this framework, we created an integrated structural model of new proteins interacting with the Rag-Ragulator complex, which regulates MAPK and mTOR signaling. In summary, the global proteome architecture map provides a resource for cellular biology discovery and the systematic determination of protein functions and structures.

Citation Format: Leah V. Schaffer, Mengzhou Hu, Edward L. Huttlin, Gege Qian, Abantika Pal, Neelesh Soni, Andrew Latham, Laura Pontano Vaites, Trang Le, Yue Qin, Christopher Churas, Dexter Pratt, Ignacia Echeverria, Andrej Sali, J Wade Harper, Steven P. Gygi, Emma Lundberg, Trey Ideker. A global multiscale map of protein assemblies from integration of protein interactions and images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2348.
Abstract 2368: Intratumoral delivery of HER-2-CD3hFc mRNA-LNP inhibits ovarian tumor growth
Authors: Golubovskaya Vita M. , Sienkiewicz John , Sun Jinying , Zhang Shiming , Huang Yanwei , Hu Liang , Zhou Hua , Harto Hizkia , Xu Shirley , Berahovich Robert , Wu Lijun
Published: March 2024

Her-2 is a well-known driver of carcinogenesis that is overexpressed in breast, ovarian and other tumors. Immunotherapy with bispecific antibodies is a promising cancer therapy approach. Her-2 (Herceptin) antibody was used to test bispecific antibody against ovarian cancer cells. First, Her-2-CAR-T cells with Her-2 (4D5) ScFv and 4-1BB costimulatory domain were generated and tested using in vitro killing and IFN-gamma assays, and in vivo efficacy SKOV-3 xenograft NSG mouse model. Her-2-CAR-T cells killed SKBR-3 breast and SKOV-3 ovarian cancer cells and stopped SKOV-3 xenograft tumor growth. This Her-2-ScFv was used to generate bispecific Her-2 ScFv-CD3-ScFv human Fc antibody using in vitro transcribed mRNA. The 5’capped Her-2-CD3-hFc mRNA with 5’UTR, 3’UTR and poly A tail was generated by in vitro transcription using DNA template. The mRNA was embedded into lipid nanoparticles (LNP) using microfluidic PreciGenome Flex S NanoGenerator. Western blotting detected >160 kDa bispecific antibody secreted from A1847 and 293 cells transfected with mRNA-LNPs. The bispecific antibody was binding Her-2-positive A1847 ovarian cancer cell lines and T cells by FACS analysis. The Her-2-CD3-hFc bispecific antibody with T cells effectively killed Her-2-positive target cells in a dose-dependent manner in RTCA assay and produced high level of IFN-gamma. Moreover, intratumoral delivery of Her-2-CD3-hFc mRNA-LNP with intravenous injection of T cells completely blocked A1847 xenograft tumor growth. In conclusion. intratumoral delivery of Her-2-CD3 mRNA-LNP blocks ovarian xenograft tumor growth and provides a solid basis for future pre-clinical and clinical studies.

Citation Format: Vita M. Golubovskaya, John Sienkiewicz, Jinying Sun, Shiming Zhang, Yanwei Huang, Liang Hu, Hua Zhou, Hizkia Harto, Shirley Xu, Robert Berahovich, Lijun Wu. Intratumoral delivery of HER-2-CD3hFc mRNA-LNP inhibits ovarian tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2368.