bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1977: Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry
Authors: Saederup Noah , Yao Huihui , Cohen Michael , Fung Leslie
Published: June 2022

Mass cytometry, empowered by CyTOF® technology, utilizes monoisotopic metal-tagged antibodies and a high-sensitivity mass cytometer to allow high-dimensional single-cell analysis in complex biological samples. The 30-marker panel Maxpar® Direct™ Immune Profiling Assay™ (Cat. No. 201325) for suspension mass cytometry provides an unprecedented sample-to-answer solution for detecting and analyzing 30 surface markers in a single experiment. With 18 open mass channels for additional biological markers, the Maxpar Direct Assay facilitates panel expansion and enables flexibility for higher multiplexity and applications. Among the potential complementary applications with the Maxpar Direct Assay, intracellular cytokine staining (ICS) is of particular interest as it may be used to assess infiltrating immune cell phenotypes in the tumor microenvironment. However, for the purpose of assessing cell viability in this workflow, the effectiveness of the Cell-ID™ Intercalator-Rh (Cat. No. 201103) that is included in the Maxpar Direct Assay is in question, since cell permeabilization during ICS can potentially damage the DNA-intercalator bondIn this study, we investigated the compatibility of the Cell-ID Intercalator-Rh (103Rh) with intracellular staining. To do this, we stained either human peripheral blood mononuclear cell (PBMC) or whole blood samples (FLDM-400287) with the Maxpar Direct Assay followed by intracellular staining for the detection of expressed cytokines. The intercalator was evaluated for its ability to discriminate live and dead cells when the sample undergoes surface antibody staining. The monoisotopic Cell-ID Cisplatin-194Pt (Cat. No. 201194) was used as the control to provide benchmark measurement of cell viability of the samples. For both sample types, known percentages of heat-killed PBMC were spiked into the samples in order to evaluate the influence of dead cells. We demonstrate that 103Rh provides equivalent functionality as a cell viability indicator during intracellular staining for cytoplasmic proteins compared to Cell-ID Cisplatin-194Pt. This work was designed to support the use of the Maxpar Direct Immune Profiling Assay in combination with additional intracellular markers. Overall, these findings expand the applicability of Cell-ID Intercalator-Rh (103Rh) to processes that involve cytoplasmic staining. For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Noah Saederup, Huihui Yao, Michael Cohen, Leslie Fung. Extending the capabilities of a high-parameter immunophenotyping assay with cytoplasmic staining applications for mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1977.
Abstract 2006: Preclinical evaluation of dual AURKA (LY3295668) and BCL2 (venetoclax) inhibition in neuroblastoma (NB)
Authors: Groff David , Casey Colleen , krytska Kateryna , Tsang Matthew , Acholla Christina , Maris John M. , Mosse Yael P.
Published: June 2022

Introduction: Deregulated expression of the MYCN oncogene is associated with the development of many neural-derived pediatric cancers including NB, medulloblastoma and atypical teratoid rhabdoid tumors. Amplification of the MYCN oncogene is present in over 40% of high-risk NBs that remain lethal in most patients, thus developing better therapies for these patients is essential. While direct therapeutic targeting of MYCN protein remains challenging, we previously demonstrated synergistic activity of dual targeting of BCL-2 (venetoclax) and AURKA (MLN8237) in MYCN-amplified NB (Ham Cancer Cell, 2016). With MLN8237 no longer being developed due to toxicities related to AURKB inhibition, testing of a highly selective AURKA inhibitor, LY3295668, is warranted.

Methods: LY3295668 (Chemie Tek) and venetoclax (Selleck Chem) were tested alone and in combination in six NB PDX models using 2-3 mice/arm. Five Patient Derived Xenograft (PDX) models tested (COG-N-424x, COG-N-453x, NB-1643, Obelix-Rx and Obelix-2x) were MYCN-amplified and one xenograft model (SK-N-AS) was MYCN-non-amplified. First, a comprehensive toxicity study, testing LY3295668 at 30mg/kg with venetoclax at 100mg/kg, 75mg/kg, 50mg/kg, and 25mkg/kg, was performed. Based on tolerability of combination, an optimal dose of 30mg/kg for LY3295668 and 50mg/kg for venetoclax was selected and administered once daily, seven days a week, via oral gavage for 60 days. These were the maximum tolerated doses when tested in combination, that did not result in weight loss greater than 15% or other overt signs of toxicity.

Results: LY3295668 (30mg/kg) and venetoclax (50mg/kg) were well tolerated as monotherapy, and when administered in combination showed a maximum weight loss of 13%. Single agent therapy resulted in minimal if any growth delay. Four of five MYCN-amplified PDX models demonstrated maintained complete responses and one showed significant tumor growth delay. No effect was observed in the MYCN-non-amplified model with single agent or combination treatment compared to vehicle.

Conclusions: Dual targeting of AURKA and BCL2 with LY3295668 and venetoclax is tolerable and demonstrates compelling anti-tumor activity in MYCN-amplified NB PDX models, validating our earlier findings (Ham et al). These preclinical results provisionally support clinical development of this combination for NB patients with MYCN amplification. Assessment of additional PDX models is currently ongoing and will be reported.

Citation Format: David Groff, Colleen Casey, Kateryna krytska, Matthew Tsang, Christina Acholla, John M. Maris, Yael P. Mosse. Preclinical evaluation of dual AURKA (LY3295668) and BCL2 (venetoclax) inhibition in neuroblastoma (NB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2006.
Abstract 2010: Transcriptional antagonism by CDK8 inhibition improves therapeutic efficacy of MEK inhibitors
Authors: Malone Clare F. , Kim Minjee , Alexe Gabriela , Forman Alexandra B. , Robichaud Amanda , Conway Amy Saur , Goodale Amy , Hatcher John M. , Gray Nathanael S. , Piccioni Federica , Stegmaier Kimberly
Published: June 2022

Mutations in the RAS/MAPK pathway are frequent drivers of oncogenesis. Clinically approved MEK inhibitors are effectively used to target RAS-pathway driven cancers in combination with RAF inhibitors in BRAF mutant cancers such as melanoma and non-small cell lung cancer. However, single agent treatment with MEK inhibitors is not typically sufficient for clinical benefit. Therefore, combination therapy approaches will be required for maximal efficacy in most advanced cancers. Here, we sought to identify MEK inhibitor-based combination therapy approaches in the setting of RAS-mutant neuroblastoma. Neuroblastoma is a cancer that arises in the developing peripheral nervous system. Children with low-risk disease frequently benefit from therapy, but 50-60% of children with high-risk disease will ultimately relapse, and relapsed disease is generally incurable. We performed genome-scale functional genomic drug modifier screens and identified mediator kinase module members CCNC and CDK8 as two genes that when lost sensitize RAS-mutant NBL to MEK inhibition. We used small molecule inhibitors of CDK8 and found that combined inhibition of MEK and CDK8 leads to an improved therapeutic response in vitro and in vivo in a xenograft model of neuroblastoma. Using transcriptional profiling, we established that CDK8 loss and MEK inhibition induce opposite transcriptional signatures. When combined, CDK8 loss prevents the compensatory upregulation of the pro-growth gene expression program induced by MEK inhibition, while the downregulation of MAPK signaling is maintained. Finally, we show that this combination is effective in a subset of other RAS-mutant cancers including pancreatic and lung cancers. Together these data suggest that the mediator kinase module is critical for the adaptive pro-survival transcriptional response induced by MEK inhibition in RAS-mutant neuroblastoma and other RAS-driven malignancies, and that the addition of a CDK8 inhibitor may improve clinical response to MEK inhibitors. Our data supports a model where agents with antagonistic transcriptional programs can have synergistic therapeutic effects when combined, which may be a broadly relevant approach in combination therapies.

Citation Format: Clare F. Malone, Minjee Kim, Gabriela Alexe, Alexandra B. Forman, Amanda Robichaud, Amy Saur Conway, Amy Goodale, John M. Hatcher, Nathanael S. Gray, Federica Piccioni, Kimberly Stegmaier. Transcriptional antagonism by CDK8 inhibition improves therapeutic efficacy of MEK inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2010.
Abstract 2012: A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS)
Authors: Guerra Alberto D. , Kennedy Annie , Adams Cynthia , Shraim Rawan , Weiner Amber K. , Chen Chuan , Krytska Kateryna , Larmour Colleen , Matkar Smita , Dimitrov Dimiter , Jelev Dontcho V. , Mossé Yael P.
Published: June 2022

Introduction: ALK is a lineage restricted oncoprotein expressed on the cell surface of NBL as wild-type (WT), mutated, or amplified, and recently shown to be overexpressed in FP RMS. Small molecule inhibitors have shown potent efficacy in NBL with ALK mutations or amplifications, but is limited to a minority of the high-risk population and have demonstrated conferred resistance. We have previously shown the potential of an ADC approach for WT ALK-expressing NBL with a chimeric ALK-directed ADC CDX-0125.

Methods: A highly specific humanized ALK antibody KTN0239 was developed with site-specific conjugation of an amine-azido linker conjugated to pyrrolobenzodiazepine (PBD) dimers or duocarmycin using click chemistry. ALK surface expression in ALK amplified (NB-1), WT non-amplified (IMR-32), mutant (Felix, SH-SY5Y, Kelly, NB1-643), and WT null (SK-N-AS) cell lines were quantified with flow cytometry using the primary KTN0239 antibody. Internalization kinetics of KTN0239 were measured using Incucyte® Fabflour-pH labeling. In vitro ADC IC50 values and viability rescue were assessed with a CellTiter-Glo® assay with ADC concentrations ranging from 10 fM to 10 nM. Western blot was used to elucidate cytotoxic mechanism of the ADC.

Results: ALK surface protein molecule expression was 21,820 in NB-1 cells, 3,708 in Felix, and 1,426 in SK-N-AS cells. KTN0239 was internalized into NB1 cells with a logarithmic trend, with >65% of antibody internalized within 8 hours.

NB-1 cell line viability was rescued to >80% when 2.5 nM of KTN0239 was in competition with the PBD-conjugated ADC at the IC50 concentration. The ADC induced activation of PARP, caspase-3, and γH2AX without attenuation of ALK signaling in NB-1 cells.

Conclusions: ADCs directed to ALK-expressing cancers are a promising therapeutic approach. The efficacy of the ADCs are currently being tested in NBL and FP RMS mouse models, and will be reported.

Mean IC50 values (pM) of ADCs with a drug antibody ratio (DAR) of 6 in NBL cell lines ADC: PBD-conjugated Duocarmycin-conjugated NB-1 14 71 IMR-32 12 741 Felix 51 110 NB-1643 6.6 1,196 Kelly 517 34,700 SH-SY5Y 693 3,024 SK-N-AS 533 1,420

Citation Format: Alberto D. Guerra, Annie Kennedy, Cynthia Adams, Rawan Shraim, Amber K. Weiner, Chuan Chen, Kateryna Krytska, Colleen Larmour, Smita Matkar, Dimiter Dimitrov, Dontcho V. Jelev, Yael P. Mossé. A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2012.
Abstract 2018: Therapeutic benefit of dual ALK and FAK inhibition in neuroblastoma
Authors: Chugh Seema , Tien Jean C. , Hon Jennifer , Bawa Pushpinder Singh , Kenum Carson , Li Chi Chiang , Cheng Yunhui , Delekta Andrew D. , Choi Jae Eun , Mannan Rahul , Qiao Yuanyuan , Mody Rajen , Chinnaiyan Arul
Published: June 2022

Neuroblastoma (NB) is the most common extracranial pediatric tumor, and accounts for approximately 12% of cancer-related deaths in children. Though several genomic drivers have been identified, the development of targeted therapy for treating this aggressive pediatric malignancy has yet to be realized. Thus, there exists an urgent need to develop alternative therapeutic strategies for treating aggressive and metastatic NB. One novel approach is to therapeutically target anaplastic lymphoma kinase (ALK) and focal adhesion kinase (FAK), which are multi-functional tyrosine kinases implicated in neural development and play an important role in growth and metastasis of several cancers. Genomic aberrations of ALK such as overexpression, amplifications, and mutations, have been reported in NB. The role of FAK has also been delineated in NB as well as associated with disease metastasis. Using in silico analysis of sequencing data from NB patients procured from Pediatric MI-ONCOSEQ (precision oncology program), we found a cohort of patients that express high ALK and FAK expression. Given the potential roles of ALK and FAK in NB, we evaluated the efficacy of a novel compound ESK440, which is a dual ALK and FAK inhibitor. ESK440 has passed Phase 1 clinical trials in adult patients with solid tumors. It is capable of crossing the blood brain barrier and binds to several mutated forms of ALK, thus making ESK440 an attractive therapeutic candidate for treating NB. A wide panel of NB cancer cell lines were screened with ESK440, and a CellTiter-Glo® Luminescent Cell Viability Assay was used to determine the IC-50s. Our results revealed that NB cells with ALK genomic aberrations (mutations and amplifications) showed low IC-50 values. Further, treatment with ESK440 inhibited ALK and FAK associated signaling pathways. We also observed decreased expression of MYCN. Intriguingly, ESK440 studies in NB xenograft model (NB-1) resulted in tumor regression in treated animals. Altogether, our study has discovered the therapeutic potential of dual ALK and FAK inhibitor in NB, and our findings may provide the foundation for initiating clinical trials targeting this disease in pediatric patients.

Citation Format: Seema Chugh, Jean C. Tien, Jennifer Hon, Pushpinder Singh Bawa, Carson Kenum, Chi Chiang Li, Yunhui Cheng, Andrew D. Delekta, Jae Eun Choi, Rahul Mannan, Yuanyuan Qiao, Rajen Mody, Arul Chinnaiyan. Therapeutic benefit of dual ALK and FAK inhibition in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2018.
Abstract 2117: IFN-γ signaling in myeloid cells regulates pancreatic cancer growth and progression
Authors: Ivleva Elena , Andreeva Natalia , Grivennikov Sergei
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is characterized by low survival, early metastasis, and rapidly emerging resistance to therapy. Interferon gamma (IFN-γ) signaling pathway is one of the key regulators of the tumor microenvironment in numerous cancer settings and its possible key role in myeloid cells is suggested. Here we investigated the IFN-y signaling in myeloid cells during PDAC development, progression, and metastasis using mice with conditional ablation of IFNgR2 in myeloid cells (IFNgR2-Delta-Mye). We performed an orthotopical injection of cells derived from conditional murine pancreatic model (Pdx1Cre-Kras-P53, or “KPC” cells) into IFNgR2-LysMCre (IFNgR2-Delta-Mye) mice. Ablation of IFN-γ signaling in myeloid cells led to a higher tumor burden compared to controls. Subsequent RNA sequence analysis demonstrated that IFN-γR2 deficiency in myeloid cells resulted in increased TGFβ1 gene expression as well as changes in metabolic and growth factor pathways in macrophages isolated from tumor tissue. Immunofluorescent analysis demonstrated that IFN-γR2 deletion in myeloid cells led to changes in cancer cell differentiation, resulting in a loss of characteristic epithelial histological pattern and shifting it towards more aggressive phenotype, possible via TGFβ1 dependent mechanism. Using murine model of liver metastasis, we found that loss of IFN-γR2 in myeloid cells leads to increased metastatic outgrowth of PDAC KPC cells. This effect was partially dependent on the gut microbiota, as shown by “separated and co-housed mice” experiments. Deficiency of IFN-γR2 in myeloid cells resulted in increased recruitment and infiltration of monocytes in normal and tumor/metastatic liver tissues. Our data identifies myeloid cell type specific IFN-γ signaling pathway as an important regulator of pancreatic cancer progression and metastasis, that can be potentially exploited as a novel immunooncology target.

Citation Format: Elena Ivleva, Natalia Andreeva, Sergei Grivennikov. IFN-γ signaling in myeloid cells regulates pancreatic cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2117.
Abstract 2129: Spatial charting of single cell transcriptomes in tissues
Authors: Wei Runmin , He Siyuan , Bai Shanshan , Sei Emi , Hu Min , Thompson Alastair , Chen Ken , Krishnamurthy Savitri , Navin Nicholas
Published: June 2022

Single cell RNA sequencing (scRNA-seq) methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics (ST) assays can profile spatial regions in tissue sections but do not have single cell genomic resolution. To address this issue, computational approaches (e.g., cell2location, RCTD) have been designed to deconvolute ST spots into proportions of different cell types. However, the spatial deconvolution method has the following limitations, 1) it can only infer cell type proportions of each spot and cannot reach higher granular "cell states" mapping; 2) it only predict categorical labels and cannot infer continuous cell information (e.g., lineage trajectories, gene signatures, continuous phenotypes) at a spatial resolution.

Here, we developed a computational approach called CellTrek that combines these two datasets to achieve single cell spatial mapping. Using a machine learning-based metric learning approach, CellTrek learned a cell-spot graph and then transfer spatial coordinates to cells. The CellTrek toolkit also provides two downstream analysis modules, including SColoc for spatial colocalization analysis and SCoexp for spatial co-expression analysis.

We benchmarked CellTrek using a simulation and two in situ datasets. We then applied CellTrek to reconstruct cellular spatial structures in existing datasets from normal mouse brain and kidney tissues. We also performed scRNA-seq and ST experiments on two ductal carcinoma in situ (DCIS) tissues and applied CellTrek to identify subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas.

Our data shows that CellTrek can accurately map single cells in diverse tissue types to resolve their spatial organization into cellular neighborhoods and tissue structures. This method provides a new paradigm that is distinct from ST deconvolution, enabling a more flexible and direct investigation of single cell data with spatial topography.

Citation Format: Runmin Wei, Siyuan He, Shanshan Bai, Emi Sei, Min Hu, Alastair Thompson, Ken Chen, Savitri Krishnamurthy, Nicholas Navin. Spatial charting of single cell transcriptomes in tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2129.
Abstract 2193: Subsets of cancer cells expressing CX3CR1 are endowed with metastasis-initiating properties and resistance to chemotherapy
Authors: Zhang Jieyi , DiNatale Anthony , Kaur Ramanpreet , Chen Qian , Marchioli Michael , Ipe Darin , Castelli Maria , McNair Christopher , Kumar Gaurav , Meucci Olimpia , Fatatis Alessandro
Published: June 2022

Metastasis-initiating cells (MICs) display stem cell-like features, cause metastatic recurrences and defy chemotherapy, which leads to patients’ demise. Here we show that prostate and breast cancer patients harbor contingents of tumor cells with high expression of CX3CR1, OCT4a (POU5F1) and NANOG. Impairing CX3CR1 expression or signaling hampered the formation of tumor spheroids by cell lines from which we isolated small subsets co-expressing CX3CR1 and stemness-related markers, similarly to patients’ tumors. These rare CX3CR1High cells show transcriptomic profiles enriched in pathways that regulate pluripotency and endowed with metastasis-initiating behavior in murine models. Cancer cells lacking these features (CX3CR1Low) were capable of re-acquiring CX3CR1-associated features over time, implying that MICs can continuously emerge from non-stem cancer cells. CX3CR1 expression also conferred resistance to docetaxel, and prolonged treatment with docetaxel selected CX3CR1High phenotypes with de-enriched transcriptomic profiles for apoptotic pathways. These findings nominate CX3CR1 as novel marker of stem-like tumor cells and provide conceptual ground for future development of approaches targeting CX3CR1 signaling and (re)expression as therapeutic means to prevent or contain metastasis initiation.

Citation Format: Jieyi Zhang, Anthony DiNatale, Ramanpreet Kaur, Qian Chen, Michael Marchioli, Darin Ipe, Maria Castelli, Christopher McNair, Gaurav Kumar, Olimpia Meucci, Alessandro Fatatis. Subsets of cancer cells expressing CX3CR1 are endowed with metastasis-initiating properties and resistance to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2193.
Abstract 2215: Cancer awareness, tobacco use and cessation among Malayali tribes, Yelagiri Hills, Tamil Nadu, India
Authors: Francis Delfin Lovelina
Published: June 2022

BACKGROUND: Health is a state of complete wellbeing free from any discomfort and pain. Despite remarkable world-wide progress in the field of diagnostic, curative and preventive medicine, still there are large populations of people living in isolation in natural and unpolluted surroundings far away from civilization, maintaining their traditional values, customs, beliefs and myths. India has the second largest tribal population of the world next to the African countries. The present study was conducted to assess the cancer awareness, tobacco use, and its effect on health among Malayali tribes, Yelagiri Hills, Tamil Nadu, India.

METHODOLOGY: After a study among this population of 660 in 2010, an extended research was carried out in 2018 among 2185 participants. The inhabitants of the 14 villages of the Yelagiri hills, who were willing to participate in the study were included. Data was collected from a cross-sectional survey, using a Survey Proforma, clinical examination and a pre-tested questionnaire which included Demographic data, tobacco habits. A brief tobacco cessation counseling was given to all the users. SPSS version 21 was used for statistical analysis.

RESULTS: Results showed that among 2185 study population, 47% had no formal education. Of those who had the habit of smoking, 29% smoked beedi, 31% smoked cigarette, 39% chewed raw tobacco, 33% chewed Hans and 12% had a combination of smoking and smokeless tobacco usage. Prevalence of oral mucosal lesions 47% and 7% oral cancer in the study population was due to tobacco usage and lack of awareness regarding the deleterious effects of the products used.

CONCLUSION: From the results of this study it may be concluded that the Malayali tribes were characterized by a lack of awareness about oral health, deep rooted dental beliefs, high prevalence of tobacco use and limited access to health services.

Citation Format: Delfin Lovelina Francis. Cancer awareness, tobacco use and cessation among Malayali tribes, Yelagiri Hills, Tamil Nadu, India [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2215.
Abstract 2270: Hyperlipidemia promotes aggressive variant prostate cancer via RNA-binding protein Quaking
Authors: Han Hyun Ho , Sung Jin Sol , Song Dong Wook , Park Cheol Keun , Cho Nam Hoon , Choi Young Deuk , Ko Woo Jin
Published: June 2022

Introduction: Hyperlipidemia has been associated with increased risk of advanced stage and high Gleason grade prostate cancer (PCa), yet the underlying mechanism is not completely understood. Quaking (QKI) is an RNA-binding protein (RBP) that regulate lipid metabolism. For PCa, QKI overexpression has been associated with increased chance of metastatic recurrence.

Method: We performed RNA sequencing of prostate cancer tissues of hyperlipidemia (n = 12) and normolipidemia (n=32). Tissue samples were acquired by MRI-US fusion targeted biopsy. DESeq2 and GSEA was used to identify differently expressed genes and enriched genesets. A web-based RBP motif screening tool TRANSITE was utilized to identify active RBPs in hyperlipidemia-associated PCa.

Results: PCa samples of hyperlipidemia patients exhibited poorer histology than normolipidemia PCA. RNA sequencing found that hyperlipidemia-associated PCa is enriched of Myc-regulated genes as well as interferon or innate immunity-associated genes. Interestingly, hyperlipidemia-associated PCa gene expression signature identified PCa samples devoid of common PCa genetic alterations - TMPRSS2-ERG fusion and PTEN deletion/mutation. RBP motif screening found QKI as one of the top enriched RBP in hyperlipidemia-associated PCa transcriptome. In a separate a consecutive radical prostatectomy series (n=190), we found that QKI-high (immunohistochemistry staining score 2-3) tumors had significantly higher serum cholesterol than QKI-low (score 0-1) tumors. Overexpression and knockdown experiments proved that QKI is responsible of rapid cell proliferation and interferon-related inflammatory gene expressions, In silico analysis predicted QKI overexpressing cancer cells are susceptive to stain-mediated ferroptosis-like cell death. Mechanistically, QKI regulates expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which dictates the sensitivity of QKI overexpressing cells to ferroptosis.

Conclusion: Hyperlipidemia was associated with aggressive pathologic characteristics of PCa without TMPRSS2-ERG fusion or PTEN deletion/mutation, and high tissue expression of QKI, an RNA-binding protein. QKI overexpression in PCa increased sensitivity to ACSL4-mediated ferroptosis by statins. This explains the epidemiological associations between hyperlipidemia and PCa, and will guide molecular subtype-driven drug repurposing studies.

Citation Format: Hyun Ho Han, Jin Sol Sung, Dong Wook Song, Cheol Keun Park, Nam Hoon Cho, Young Deuk Choi, Woo Jin Ko. Hyperlipidemia promotes aggressive variant prostate cancer via RNA-binding protein Quaking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2270.
Abstract 2272: Integration of multi-omics data reveals molecular features of Hispanic hepatocellular carcinoma
Authors: Das Debodipta , Wang Xiaojing , Chiu Yu-Chiao , Bouamar Hakim , Chen Yidong , Zheng Siyuan , Cigarroa Francisco G. , Sun Lu-Zhe
Published: June 2022

Hepatocellular carcinoma (HCC) is the major type of liver cancer and one of the few cancers with a steady increase in incidence and mortality rate over the past several decades in the USA. It disproportionately affects Hispanics in the USA with an incidence and mortality rate about two to three times higher in Hispanics than in non-Hispanic whites. To identify molecular features associated with the Hispanic HCC we have generated genome-wide sequencing (i.e., exome-seq, RNA-seq) data and mass spectrometry-based proteomics data from HCC tumor and matched blood and/or adjacent normal tissues of Hispanic patients based in South Texas. The landscape of somatic mutation in HCC tumors revealed the aflatoxin-induced mutational signature (COSMIC) in some patients indicating an association of aflatoxin mediated toxicity in them. Similar to The Cancer Genome Atlas (TCGA) study, with predominant non-Hispanic HCC tumors, we could identify the most frequent somatic mutations in CTNNB1 and TP53. Other frequently (>10%) mutated genes in our patients appear to be mutated at relatively lower rate in the TCGA patients. Mutation(s) in some of the frequently mutated genes might contribute to the significant enrichment of cellular signaling pathways observed from the Gene Set Enrichment Analysis (GSEA) using paired RNA-seq data. Single sample GSEA (ssGSEA) of our RNA-seq and proteomic data sets revealed two clusters of HCC tumors, denoted as H1 and H2 clusters, with striking differences in various signaling pathways and cellular functions. Patients from the H1 cluster and two sub-clusters within the H2 cluster showed a significant difference in their survival rate based on overall survival information from the TCGA study. The transcriptomic data from Hispanic & non-Hispanic HCC identified activated immune evasion mechanisms in H1 patients. Sorafenib treatment was found to be associated with a lower hazard ratio for overall survival in patients from the H1 cluster compared to the H2 cluster. In sum, we have identified genes more frequently mutated in Hispanic HCC than in non-Hispanic HCC, and cellular and tissue functions significantly enriched only in the Hispanic HCC. We also report a novel classification of HCC tumors with significant differences in various cellular signaling pathways and overall survival. Our findings might be used as biomarkers for the early prediction and better therapeutic management of HCC.

Citation Format: Debodipta Das, Xiaojing Wang, Yu-Chiao Chiu, Hakim Bouamar, Yidong Chen, Siyuan Zheng, Francisco G. Cigarroa, Lu-Zhe Sun. Integration of multi-omics data reveals molecular features of Hispanic hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2272.
Abstract 2306: BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models
Authors: Brown Victoria , Ramsden Phil , House Nealia , Vargas Richard , Guo Jian , Wang Ruduan , Lobbardi Riadh , Chen Maxine , Wilson Douglas , Kim Joseph , Bifulco Neil , Maynard Michelle , Perola Emanuele , Zhang Dean , Wenglowsky Steve , Choi Yoon Jong
Published: June 2022

Background: Cyclin-dependent kinases (CDK) are a class of enzymes that, along with their regulatory cyclin binding partners, drive cell cycle progression. Cell cycle dysregulation is a hallmark of cancer and targeting its genetic drivers can confer therapeutic benefits. Cyclin E1 (CCNE1) gene alterations are common in patients with ovarian cancer and can be found in ~20% of high-grade serous ovarian cancer cases, which tend to be platinum therapy resistant and therefore represent a high medical need. Cyclin E1 is the canonical binding partner of CDK2, which becomes constitutively active when CCNE1 is amplified and overexpressed. Selectively inhibiting CDK2 is an attractive therapeutic option for CCNE1-amplified tumors and may limit off-target CDK-driven toxicities. Here we report preclinical validation studies leading to the development of an orally available CDK2 inhibitor, BLU-222, for the treatment of ovarian cancer harboring a CCNE1 amplification.

Methods: BLU-222 selectivity was measured by enzyme assays, cellular target engagement assays (NanoBRET), and proliferation assays in a panel of ovarian cancer cell lines. In vitro cellular potency was assessed by phospho-Rb levels. In vivo antitumor activity of BLU-222 as a single agent or in combination with carboplatin was measured in an OVCAR-3 cell line-derived xenograft (CDX) tumor model harboring a CCNE1 amplification.

Results: BLU-222 demonstrated selectivity, both biochemically and in cells, with low nanomolar potency for CDK2 vs other CDK family members (CDK1, -4, -6, -7, and -9). In a panel of ovarian cancer cell lines, those with CCNE1 amplifications were highly sensitive to BLU-222. Consistent with the in vitro profiling, BLU-222 exhibited significant antitumor activity in the OVCAR-3 CDX model. While administration of single-agent carboplatin led to stasis in vivo, the combination of BLU-222 + carboplatin induced durable tumor regression even after treatment cessation.

Conclusions: These data provide a strong rationale for advancing BLU-222 towards clinical development in patients with CCNE1-amplified ovarian cancer.

Citation Format: Victoria Brown, Phil Ramsden, Nealia House, Richard Vargas, Jian Guo, Ruduan Wang, Riadh Lobbardi, Maxine Chen, Douglas Wilson, Joseph Kim, Neil Bifulco, Michelle Maynard, Emanuele Perola, Dean Zhang, Steve Wenglowsky, Yoon Jong Choi. BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2306.
Abstract 2323: A bioenergetic functional screening assay workflow to identify, validate, and characterize anti-cancer compounds
Authors: Kam Yoonseok , Winer Lisa , Romero Natalia
Published: June 2022

As bioenergetic metabolism is recognized as a critical activity supporting cell proliferation, differentiation, and even death, many proposed targets for cancer therapy are functionally linked to cellular bioenergetic metabolism. Here, we introduce an assay workflow for screening and analyzing potential anticancer drugs affecting cellular metabolism using extracellular flux analysis. The Agilent Seahorse XF Real-Time ATP rate assay can be used as a starting point to identify therapeutic compounds that induce a perturbation in metabolic phenotype. It provides a quantitative comparison of basal mitochondrial and glycolytic activities using the universal unit of ATP production rate. Through this initial comparative screening, the effect of different compounds on basal energetic state and pathway-specific metabolic perturbations can be identified providing information that cannot be assessed by intracellular ATP level measurements or cytotoxicity assays. A more comprehensive functional analysis can then be applied to validate the target mode of action through pathway-specific analysis tools such as the XF Cell Mito Stress Test and the XF Glycolytic Rate Assay. In this study, we tested the proposed workflow in three different NSCLC cell lines with different genetic backgrounds using a mock panel of known metabolic modulators including thiosemicarbazones, EGFR inhibitors, KRAS inhibitors, a glutaminase inhibitor, a Glut1 inhibitor, a chemotherapy drug, and a radiotherapy sensitizer candidate. The ATP production rate data from lung cancer cell lines exposed for 1 hour or 24 hours to the compounds, successfully identified the effects of metabolic pathway-specific modulation as well as metabolic phenotypic switching. In addition, dose-response studies using cell lines with different genetic backgrounds demonstrated differential susceptibility to the tested compounds. The workflow was then applied using a broader panel of 72 potential mitochondrial toxicants compounds. The assay could identify potent mitochondrial and glycolytic suppressor compounds effective on A549, a lung cancer cell line. A more comprehensive XF analysis then validated the mode of action for the identified metabolic suppressors. These results demonstrate the benefit of using the XF Real-Time ATP rate assay as an initial assay to uncover novel metabolic targets in early drug discovery research, guiding further research toward functional validation with the appropriate comprehensive XF assays.

Citation Format: Yoonseok Kam, Lisa Winer, Natalia Romero. A bioenergetic functional screening assay workflow to identify, validate, and characterize anti-cancer compounds [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2323.
Abstract 2381: Interleukin-4 receptor signaling induces glycolysis in triple negative breast cancer
Authors: Williams Demond , Bindeman Wendy , Hargrove-Wiley Ebony , Fingleton Barbara
Published: June 2022

In the context of cancer biology Th2 cytokines such as IL4 and IL13 are most studied for their effects on immune cells such as macrophages. IL-4 receptor is established to cause polarization of macrophages to a pro-tumor M2 phenotype. While much is known about the signaling effects of IL-4 in immune cells there is relatively little known about its effects in cancer cells that express the receptor. Our lab has established a role of signaling in IL4 receptor expressing cancer cells for promoting metastasis. Many of the effects of IL-4 signaling in immune cells are accompanied by metabolic changes. Thus, we aimed to study the effects of IL-4 signaling on metabolism in breast cancer to better understand how these metabolic changes might induce metastatic phenotypes associated with IL-4 stimulation. IL-4 increased glucose uptake in multiple human TNBC cell lines assessed via NBDG uptake assay. Production of lactate was also significantly increased as assessed by colorimetric assay. We then became interested in possible alterations in metabolism downstream of increased glucose uptake. By Seahorse assay we found that IL-4 significantly increased glycolysis, as well as mitochondrial metabolism when assessed by ATP Rate assay. By metabolic flux using C13 labeled glucose we found that IL-4 increased incorporation of carbon from glucose into lactate while not having a significant effect on TCA cycle intermediates. This led us to question whether IL-4 may affect other arms of metabolism that feed into TCA cycle and mitochondrial metabolism. We looked at how IL4 impacts glutaminolysis and found that IL-4 stimulation leads to increased expression of glutamine transporter ASCT2, as well as increased glutamine uptake. We have begun to examine whether these metabolic changes affect epigenetic regulation. Initial data indicates that IL4 treatment leads to increased histone acetylation in TNBC cell lines.

Citation Format: Demond Williams, Wendy Bindeman, Ebony Hargrove-Wiley, Barbara Fingleton. Interleukin-4 receptor signaling induces glycolysis in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2381.
Abstract 2382: Aberrant glycogen accumulation alters gene expression and promotes lung tumor progression in vivo
Authors: Lamb Jessica Faith , Conroy Lindsey R. , James Alexis N. , Buoncristiani Michael D. , Relich Eric R. , Young Lindsay E. , Branson Christine F. , Sun Ramon C.
Published: June 2022

Lung cancer is one of the most prevalent cancers worldwide, accounting for nearly two million new cases each year. Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer and is the leading cause of cancer related mortalities worldwide. LUAD has an extremely poor five-year survival rate, which is likely due to cases being diagnosed at advanced stages and thus being more difficult to treat the metastatic disease. Identifying the molecular processes that contribute to disease progression is a critical goal in lung cancer research that could lead to predictive biomarkers and novel therapies. Glycogen is the primary form of carbohydrate storage in mammalian cells, and its degradation product, Glucose 6-Phosphate (G6P) feeds directly into glycolysis, making glycogen intimately connected to central carbon metabolism. Recently, aberrant glycogen accumulation in lung tumors has been reported to promote lung cancer progression, driven by largely unknown mechanisms. We recently generated a transgenic KRASG12D/p53-/- LUAD mouse model that also lacks the glycogen phosphatase laforin (LKO). This model develops increased glycogen accumulation in the lungs and displays accelerated tumor growth compared to the KRASG12D/p53-/- control (WT), as measured by Ki67 staining. To identify potential biological processes perturbed by LUAD-glycogen, we performed RNA-seq on lung tumors resected from WT and LKO mice. We identified over 300 genes that are differentially expressed between WT and LKO lung tumors. Specifically, gene set enrichment analysis (GSEA) revealed genes that were upregulated in LKO lung tumors were involved in extracellular matrix remodeling, cell-to-cell communication, and cell proliferation. All of these processes are implicated in tumor growth, which supports our initial observation that LKO mice have increased tumor burden and disease progression. Our findings ultimately highlight several potential mechanisms by which LUAD-glycogen promotes LUAD tumor progression, which can be further investigated to identify novel biomarkers and therapeutic targets.

Citation Format: Jessica Faith Lamb, Lindsey R. Conroy, Alexis N. James, Michael D. Buoncristiani, Eric R. Relich, Lindsay E. Young, Christine F. Branson, Ramon C. Sun. Aberrant glycogen accumulation alters gene expression and promotes lung tumor progression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2382.
Abstract 2385: A prospective investigation of serum bile acids with risk of liver cancer, fatal liver disease, and biliary cancer
Authors: Farhat Zeinab , Freedman Neal D. , Sampson Joshua N. , Falk Roni T. , Koshiol Jill , Weinstein Stephanie J. , Albanes Demetrius , Sinha Rashmi , Loftfield Erikka
Published: June 2022

Abstract Background: Laboratory and emerging epidemiologic data suggest that bile acids (BAs), major regulators of the gut microbiota, play an important role in liver disease and cancer etiology. However, few epidemiologic studies have comprehensively examined associations between BAs and either liver endpoints or biliary cancer.

Methods: In the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, we designed 1:1 matched nested case-control studies of primary liver cancer (n=247 cases), fatal liver disease (n=261 cases), and primary biliary cancer (n=92 cases). Using baseline serum collected ≤30 years prior to diagnosis or death, we measured concentrations of 15 BAs with liquid chromatography tandem mass spectrometry. We used multivariable conditional logistic regression to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CIs) between BAs and each endpoint, accounting for multiple comparisons.

Results: Comparing the highest to the lowest quartile, ten BAs were positively associated with liver cancer risk, including taurocholic (OR =4.70, 95% CI=2.51-8.78; Q-trend<0.0001), taurochenodeoxycholic (OR =4.61, 95% CI=2.38-8.91; Q-trend<0.0001), and glycocholic acid OR =4.11, 95% CI=2.11-7.99; Q-trend<0.0001), and 11 were positively associated with fatal liver disease risk, including taurochenodeoxycholic acid (OR =9.65, 95% CI=4.41-21.14; Q-trend<0.0001), taurocholic OR =7.45, 95% CI=3.70-14.97; Q-trend<0.0001), taurochenodeoxycholic (OR =9.05, 95% CI=4.04-20.32; Q-trend<0.0001), and glycocholic acid OR=6.12, 95% CI=2.84-13.77; Q-trend<0.0001). No significant associations were observed between BAs and biliary cancer though ORs were consistently >1.

Conclusion: Conjugated BAs were strongly associated with increased risk of liver cancer and fatal liver disease, suggesting mechanistic links between the gut microbiota, BA signaling pathways, and liver cancer or death from liver disease.

Citation Format: Zeinab Farhat, Neal D. Freedman, Joshua N. Sampson, Roni T. Falk, Jill Koshiol, Stephanie J. Weinstein, Demetrius Albanes, Rashmi Sinha, Erikka Loftfield. A prospective investigation of serum bile acids with risk of liver cancer, fatal liver disease, and biliary cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2385.
Abstract 2402: SMAD4 represses PDAC metastatic colonization through binding FOSL1 enhancer regions
Authors: Rennhack Jonathan P. , Dai Chao , Aguirre Andrew , Doench John , Root David , Hahn William C.
Published: June 2022

Less than 5% of patients diagnosed with pancreatic ductal adenocarcinomas (PDAC) survive more than 5 years, largely due to therapeutic resistance and metastatic disease. Half of all PDAC patients have been shown to have loss of SMAD4, which has been shown to correlate with metastasis. Here we functionally demonstrate that SMAD4 is a key suppressor of metastatic colonization of PDAC. Using an in vitro and in vivo multi-component RNA-seq analysis on isogenic human PDAC cell lines, we defined SMAD4-dependent gene expression changes. Specifically, we identified genes that were suppressed in the presence of SMAD4. These genes could contribute to tumor metastasis. To test this, we performed a pooled open reading frame (ORF) overexpression lung colonization assay with the identified genes. We found that expression of the transcription factor FOSL1 is sufficient for metastatic colonization. Mechanistically, SMAD4 directly binds and modulates the activity of the FOSL1 enhancer. Taken together, these studies demonstrate a direct role for SMAD4 in regulating metastatic colonization and identify FOSL1 as a direct SMAD4-regulated gene involved in distant site colonization.

Citation Format: Jonathan P. Rennhack, Chao Dai, Andrew Aguirre, John Doench, David Root, William C. Hahn. SMAD4 represses PDAC metastatic colonization through binding FOSL1 enhancer regions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2402.