bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1223: Accurate inference of genetic ancestry from cancer-derived data
Authors: Belleau Pascal , Deschênes Astrid , Tuveson David A. , Krasnitz Alexander
Published: June 2022

Introduction: For multiple cancer types, epidemiological data exhibit strong correlations between, on the one hand, the incidence of the disease, its severity when diagnosed, and its clinical outcome, and, on the other hand, the ancestral background of the patient. Recent studies point to genetic and phenotypic differences between tumors in patient populations with differing genetic ancestries, and to the need for more data collection to power research in this area. We sought to facilitate such analysis by developing computational tools for genetic ancestry inference from cancer-derived molecular data, without the need for the patient’s cancer-free genotype or self-declared race or ethnicity. The ability to perform such inference accurately would unlock vast amounts of such data for ancestry-oriented studies of cancer from two major sources. One is the body of data stored by similar massive digital repositories. The other is the body of archival tumor tissues from which molecular data may be generated.

Methods: We developed methods for genetic ancestry inference from cancer-derived whole exomes, transcriptomes and targeted gene panels, in the absence of matching cancer-free genomic data. These are adaptive, endowed with the ability to optimize their performance for each input cancer-derived molecular profile. As a result, these inference methods perform consistently, and with quantifiable accuracy, across a range of profiling depths and qualities, and mitigate cancer-related damage to the genome, such as somatic copy-number variation.

Results: We examined the performance of these tools with molecular data from three cancer types: pancreatic and ovarian cancers as representative of epithelial tumor types, and acute myeloid leukemia as an example of hematopoietic malignancy. Three molecular data types were considered: whole-exome sequences, exome sequences targeting a panel of cancer-related genes and RNA sequences. The inference accuracy was found to be consistently above 97% across the three cancers and the three data types.

Conclusion: Our study demonstrates the feasibility of accurate inference of genetic ancestry from cancer-derived data, with no need for matching cancer-free genotypes. Computational tools for this purpose will be made available as open-source software.

Citation Format: Pascal Belleau, Astrid Deschênes, David A. Tuveson, Alexander Krasnitz. Accurate inference of genetic ancestry from cancer-derived data [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 1223.
Abstract 1226: A genome wide pharmacological perturbation platform
Authors: Correia Cristina , Ung Choong Yong , Zhang Cheng , Kaufmann Scott H. , Li Hu
Published: June 2022

Background: Large scale genetic perturbations screens have been instrumental for many key discoveries. In the last decade sequence specific genetic perturbations created RNAi and CRISPR (PMID: 25446316). Recently, specific genome targeting approaches using single guide RNAs (sgRNAs) for the CRISPR-Cas9 system have been repurposed to induce sequence-specific repression or activation of gene expression at a genome-wide scale. Different perturbation approaches have different effects and encompass a trade-off between specificity and duration in establishing a measurable perturbation which can result in different biological readouts. However, bench-side strategies are time consuming and expensive. The method described here aims to build a systems-based computational platform to assess pharmacological and biological perturbations at genome-wide network level.Methods: Our network tool NetDecoder (Nucleic Acids Res. 2016; 44(10):e100) that models genome-wide information flow will be used to build context specific protein-protein interaction networks. These networks will be subsequently subjected to in silico perturbations and their impacts on network properties will be assessed. AVANA and Demeter datasets will be used during method validation procedures. Results: Using publicly available expression datasets we generated context specific Breast Cancer networks and individually manipulated nodes and edges of context-specific networks associated with pharmacological FDA approved targets. Our perturbation networks captured cellular rewiring by engulfing new gene players, alterations of the network size and its connectivity. Conclusions: Our in-silico genome wide perturbation platform provides a flexible approach to fine-tune single and tandem manipulations, allowing for inexpensively monitoring a range of pharmacological interventions using large omics data.

Citation Format: Cristina Correia, Choong Yong Ung, Cheng Zhang, Scott H. Kaufmann, Hu Li. A genome wide pharmacological perturbation platform [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 1226.
Abstract 131: The role of p66ShcA in the melanoma oncogenesis process
Authors: Canedo Eduardo Cepeda , Rincon Sonia Del , Siegel Peter , Witcher Michael , Ursini-Siegel Josie
Published: June 2022

Background: The identification of crucial driver mutations that affect the MAPK signaling pathway (BRAF, NRAS, NF1) in melanomas, has allowed for the development of targeted therapies and shed light on the process of melanocyte transformation. Gain of function mutations in proteins within the Ras/MAPK pathway in melanocytes leads to the formation of benign neoplasms, or nevi. Multiple mechanisms prevent the transformation of nevi into invasive cancer, namely, oncogene-induced senescence (OIS) and immunosurveillance. Of interest to the melanomagenesis process is the p66ShcA redox protein. We have found that compared to other solid cancers, the majority of primary human melanomas, patient-derived xenografts and melanoma cell lines strongly overexpress p66ShcA, an adaptor protein that induces production of reactive oxygen species (ROS) in response to stress stimuli. Indeed, UV light, increases p66ShcA levels and p66ShcA-induced oxidative stress, which is relevant to this disease as sun exposure contributes significantly to melanoma development. We hypothesized that p66ShcA may play a crucial role in the early steps of melanocyte transformation, possibly by contributing to the overriding of OIS and immune surveillance.

Methods: Melanoma initiation and progression will be examined in a known transgenic mouse model (Tyr::CRE/brafCA/ptenlox/lox) either in the presence of endogenous levels of p66ShcA or in an inducible p66ShcA over-expression setting. The necessity of p66ShcA-high expression for melanocyte BRAFV600E-transformation will be tested in immortalized melanocytes. Lastly, to explore the potential selection advantage conferred by p66ShcA, its expression will be silenced in a panel of known melanoma cell lines. Clonogenic assays as well as mouse xenografts will be performed.

Results: TCGA melanoma datasets, immunoblot analysis of human melanoma cell lines (n>10), and a collection of metastatic patient-derived xenografts (PDX) (n=6) indicate uniformly elevated p66ShcA levels compared to other cancer types. Knock-down of p66ShcA in multiple melanoma cell lines reduces their clonogenic potential. This reduction in colony formation is independent of driver mutation (e.g., BRAF, NRAS, NF1). Suggesting that p66ShcA may sustain melanoma proliferation. Furthermore, xenografts of the murine YUMM1.7 cell line (braf mutant, cdkn2a null and pten null) in immune-deficient and immune-competent mice indicate that loss of p66ShcA (YUMM1.7 p66ShcA-KO) delays tumor formation, specifically in mice with an intact immune system.

Citation Format: Eduardo Cepeda Canedo, Sonia Del Rincon, Peter Siegel, Michael Witcher, Josie Ursini-Siegel. The role of p66ShcA in the melanoma oncogenesis process [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 131.
Abstract 1319: Complement activation in pancreatic ductal adenocarcinoma
Authors: Bell Brett I. , Pandey Sanjay , Asp Patrik , Guha Chandan
Published: June 2022

The complement system is an innate immune cascade which recognizes and clears microbes and damaged host cells. Complement activation has been linked to the promotion of tumor growth and metastasis across various anatomical sites with context-dependent effects. We therefore aim to understand the role for complement activation in pancreatic cancer. We have generated a tamoxifen-inducible autochthonous mouse model of PDAC which recapitulates genetic mutations found in humans using LSL-KrasG12D/+; Tgfbr2fl/fl; Ptf1a-CreER (KTC) mice. Using these mice, we assessed for complement activation in pancreatic tumors by western blot and confocal immunofluorescence. Western blotting for neoepitopes generated upon cleavage of Complement Component 3 (C3) demonstrated significantly increased complement activation in KTC tumors when compared to normal pancreas from at least 4-7 months post-induction. Using confocal immunofluorescence, we observed deposition of complement fragments in tumor stroma and on ductal carcinoma cells identified by co-staining with cytokeratin 19. We therefore hypothesized complement blockade would reduce PDAC tumor growth and sought to test this using C3 knockout (C3 KO) mice which lack all downstream complement function due to the central role of C3. We assessed flank tumor growth using two Kras; Tp53 (KPC) mutant-derived tumor cell lines in wild type (WT) C57BL/6J and C3 KO mice yet did not find a biologically significant reduction in flank tumor growth between WT and C3 KO. To validate the knockout, we assessed C3 activation by western blot but surprisingly found increased complement activation in C3 KO tumors. RT-qPCR demonstrated local transcription of C3 mRNA in both WT and C3 KO tumors as well as transcription of C3 mRNA in KPC cells in culture. This demonstrates that KPC cells are capable of autocrine production of C3. Using immunocytochemistry and RT-qPCR we identified expression of the C3a receptor in KPC cells which we hypothesized could serve as a sensor for feedback to control local C3 production. We applied 10 nM recombinant C3a to KPCY cells in vitro and found a 20% reduction in C3 mRNA transcription 1-hour post-treatment. Pre-treatment with 500 nM C3aR Antagonist (SB290157) ablated this effect. In summary, complement activation is significantly increased in PDAC and appears to be activated in part by tumor cells themselves. Autocrine production of C3 by tumor cells is sufficient to produce complement activation in the absence of systemic C3. Additional experiments are warranted to determine the role of complement in PDAC using C3 mice crossed with KTC mice to assess tumorigenesis in the total absence of C3.

Citation Format: Brett I. Bell, Sanjay Pandey, Patrik Asp, Chandan Guha. Complement activation in pancreatic ductal adenocarcinoma [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 1319.
Abstract 1357: T cell memory and the critical effectors of successful anticancer immune response
Authors: Daniels Andrew , Damsky William , McGeary Meaghan , Iwasaki Akiko , Bosenberg Marcus
Published: June 2022

Despite the initial clinical success of immunotherapies in the treatment of melanoma, most patients fail to achieve long-term responses. While the majority of patients initially respond to these treatments, most go on to develop secondary resistance. The mechanisms underlying this failure of long-term response and successful responses is poorly understood.

To better understand the factors contributing to successful long-term anti-cancer immune responses, we utilized the YUMMER1.7 (YR1.7) melanoma line to develop a novel model for the evaluation of immunologic memory. We found that mice engrafted with a YR1.7 tumor can be cured by resection, and that subsequent challenge of these mice with the same tumor, even at 20x the cell number, led to expeditious rejection. Using depleting antibodies, we found that memory response were dependent on CD8 and CD4 T cells, and that CD4 depletion alone does not compromise initial responses, but leads to eventual failure of memory. We found no effect of natural killer cell (NK) depletion.

We performed single cell RNA sequencing on primary and rechallenge tumors from the same mouse, with the same tumor line. We found a phenotype of clonal replacement, in which the effective T cell clones seen in the memory response were non-overlapping with those found in the primary tumor. This implies an extra-tumoral location for these clones in the primary setting or the potential for additional priming of novel T cell clones upon re-challenge.

Together, these findings suggest a novel insights on the manner and location of T cells needed for anti-cancer memory responses.

Citation Format: Andrew Daniels, William Damsky, Meaghan McGeary, Akiko Iwasaki, Marcus Bosenberg. T cell memory and the critical effectors of successful anticancer immune response [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 1357.
Abstract 1364: Spatially resolved T-cell receptor profiling elucidates relationships between TCR diversity, immune infiltration, and cancer-associated pathways
Authors: Kriner Michelle A. , van Raay Katrina , Reeves Jason W. , Fuhrman Kit A. , Klock Andrew , Kutchma Alecksandr , Piazza Erin , Beechem Joseph
Published: June 2022

As T-cells mature, genes encoding T-cell receptor (TCR) segments are somatically recombined to generate a diverse repertoire of receptors specific to unique antigens. The resultant TCR diversity, and subsequent clonal expansion events, are critical in understanding the adaptive immune response to pathogens and cancers. While many methods have been developed to determine specific clonotypes and overall TCR diversity present in various tissues, these methods to date have failed to capture spatial orientation and arrangement of T-cells engaging with their microenvironment. Tracking T-cell migration and infiltration into the tumor has thus been limited to post-hoc low-plex profiling methods. To better understand T-cell localization during tumor development, we have developed a TCR profiling panel for the GeoMx® Digital Spatial Profiler that can be combined with the GeoMx Cancer Transcriptome Atlas (CTA) or Human Whole Transcriptome Atlas (WTA). This novel spatial assay enables simultaneous quantification of all functional TCR constant, variable and joining segments in situ along with transcriptome-wide gene expression profiling in spatially defined regions of a tissue. We validated the performance of the TCR probe pool in inflamed tonsil and cell pellet arrays, demonstrating comparable sensitivity and specificity relative to orthogonal methods. We next used the GeoMx TCR spike-in panel to characterize intra- and inter-patient TCR heterogeneity in a cohort of 68 T-cell lymphomas to track clonal interactions between the malignant and non-malignant immune microenvironment. We demonstrate the ability to link the spatial context of TCR segment expression to activation of malignant signaling cascades as well as non-cancerous T-cell response to the presence of other immune cells and cancer-associated signaling. Together, the combination of our TCR spike-in panel with the CTA or WTA illuminates spatial distribution of T-cell clones paving the way for studies to comprehensively link TCR clonality changes to the tumor microenvironment.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Citation Format: Michelle A. Kriner, Katrina van Raay, Jason W. Reeves, Kit A. Fuhrman, Andrew Klock, Alecksandr Kutchma, Erin Piazza, Joseph Beechem. Spatially resolved T-cell receptor profiling elucidates relationships between TCR diversity, immune infiltration, and cancer-associated pathways [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 1364.
Abstract 1380: Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance
Authors: McGeary Meaghan K. , Damsky William , Daniels Drew , Micevic Goran , Song Eric , Lou Hua Jane , Calderwood Clotilde , Paradkar Sateja , Iwasaki Akiko , Calderwood David , Turk Benjamin , Bosenberg Marcus
Published: June 2022

While immunotherapies have been revolutionary for the treatment of many cancers including melanoma, most patients do not respond, or develop resistance to these therapies. Several epigenetic modulators have been identified as potential targets for augmenting anti-tumor immunity, however the full breadth of their role is not well-understood.

To identify modulators of anti-tumor immunity in melanoma, we performed a whole-genome CRISPR screen using the YUMMER-G model and identified members of the HUSH complex and its associated methyltransferase, Setdb1, as drop-out hits. To validate these findings, we used CRISPR-Cas9 to knockout Setdb1 individually and observed that Setdb1-/- tumors are cleared by their host mice in a CD8+ T-cell dependent manner.

To determine the source of immunogenicity in Setdb1-/- cells, we performed RNA-sequencing, cytokine profiling, and evaluated antigen presentation by flow cytometry. These assays revealed increased MHC-I levels, elevated secretion of Ifn-, and induction of an interferon-stimulated gene (ISG) expression program, consistent with the observed immunogenicity. We further determined that treating Setdb1-/- cells with IFNAR-blocking antibodies restores MHC-I levels to wild-type levels, suggestive of a tumor-intrinsic interferon loop.

We hypothesized that type-I interferon signaling is generated through sensing of expressed endogenous retroviral elements (ERVs), and consistently observe upregulation of ERVs in Setdb1-/- cells. Previous studies have described the potential for ERV-derived antigens to act as substrates for anti-tumor activity by CD8+ T cells. We identified T cells in the tumor microenvironment of Setdb1-/- tumors that bind tetramer carrying the P15E peptide derived from ERV envelope.

Together, these findings suggest that tumors lacking expression of Setdb1-/- upregulate ERV transcripts, driving expression of type-I interferons which generate an inflamed tumor microenvironment and potentiate efficient tumor clearance of Setdb1-/- tumors. Overall, this study emphasizes a critical role for regulators of ERV expression and type-I interferon expression, and suggests their potential as therapeutic targets for augmenting anti-cancer immune responses.

Citation Format: Meaghan K. McGeary, William Damsky, Drew Daniels, Goran Micevic, Eric Song, Hua Jane Lou, Clotilde Calderwood, Sateja Paradkar, Akiko Iwasaki, David Calderwood, Benjamin Turk, Marcus Bosenberg. Setdb1-loss reactivates ERV expression and interferon signaling to induce immune-mediated melanoma clearance [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 1380.
Abstract 1436: ABCF1 and ABCF2 genetic variants in association with hepatocellular carcinoma (HCC) risk
Authors: Yeung Philip Chun , Ng Kelvin Kwok Chai , Cheung Tan To , Chong Charing Ching-Ning , Lai Paul Bo San , Cheung Siu Tim
Published: June 2022

Background/Aims: ATP-binding cassette (ABC) transporters have been shown to regulate tumor initiating cells in various cancer types and specific genotype associated with cancer risk. ABCF1 was on priority among the ABC transporters because the gene located at 6p21, the chromosome region associated with a number of HCC risk genetic loci including the HLA genes. Its family member ABCF2 was also examined as their expression levels were highly correlated and associated with HCC recurrence.

Subjects and Methods: A total of 300 HCC and 300 healthy blood samples (99.3% and 94.7% Chinese respectively) were prospectively collected with informed consent. All patients had been diagnosed with primary HCC and underwent partial hepatectomy. Clinical information including sex, age, tumor stage and survival outcomes were collected prospectively. Genomic DNA were extracted from blood samples and SNPs were examined by TaqMan genotyping assay, Sequenom MassARRAY platform and direct sequencing.

Results: Sixteen SNPs and three INDELs were examined in ABCF1 and ABCF2 in the germline DNA of 300 HCCs and 300 healthy individuals. Among the 19 loci investigated, ABCF1 rs1264440 (916-99A>G) and rs4148252 (*1delA), ABCF2 rs3823589 (-43+219C>A) and rs75100208 (368-94C>A) were significantly associated with HCC risk (OR: 1.424, 95%CI: 1.03-1.97, P=0.032; OR: 1.446, 95%CI: 1.04-2.00, P=0.026; OR: 1.635, 95%CI: 1.09-2.46, P=0.018; OR: 1.479, 95%CI: 1.09-2.18, P=0.049, respectively). Patients with any two of the four variant genotype, compared with those with all wild type, revealed elevated HCC risks (OR: 1.657, 95%CI: 1.16-2.37, P=0.006). Importantly, patients with all four variant genotypes demonstrated augmented HCC risk (OR: 2.295, 95%CI: 1.27-4.15, P=0.006).

Conclusions: ABCF1 genetic variants rs1264440, rs4148252 and ABCF2 genetic variants rs3823589, rs75100208 were associated with significantly increased HCC risk. Augmented cancer risk was observed among patients possessed all four variant genotypes in ABCF1 and ABCF2. Further mechanistic studies are warranted to comprehend the underlying mechanisms.

Citation Format: Philip Chun Yeung, Kelvin Kwok Chai Ng, Tan To Cheung, Charing Ching-Ning Chong, Paul Bo San Lai, Siu Tim Cheung. ABCF1 and ABCF2 genetic variants in association with hepatocellular carcinoma (HCC) risk [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 1436.
Abstract 1469: 14-3-3 binding during G1 stabilizes and sequesters PTOV1 to the cytoplasm
Authors: Pennington Katie L. , McEwan Colten M. , Woods James , Muir Colin M. , Sahankumari A G Pramoda , Eastmond Riley , Balasooriya Eranga R. , Egbert Christina M. , McCormack Katherine K. , Andersen Joshua L.
Published: June 2022

Cells must respond to changing environments by modulating signaling and gene expression patterns to maintain homeostasis, health, and viability. Inappropriate responses lead to disease processes, such as cancer. Despite improvements in targeted treatment options, cancer remains the second leading cause of death in the United States, illustrating the need to develop new highly effective targeted therapies. 14-3-3 interacts with a network of proteins to support signaling pathways promoting oncogenesis, metastasis, growth, cellular survival, and chemoresistance in a variety of cancers, including lung, prostate, and breast. We recently identified prostate tumor overexpressed 1 (PTOV1) as a novel 14-3-3 interacting protein promoting tumorigenic gene expression patterns. We showed that 14-3-3 binding sequesters PTOV1 in the cytoplasm and protects PTOV1 from proteasomal degradation. Using double thymidine block synchronization, we now demonstrate that both PTOV1 phosphorylation and total PTOV1 levels peak in G1, prior to PTOV1 translocating into the nucleus during S phase. These data support our model that 14-3-3-PTOV1 binding maintains PTOV1 in the cytoplasm during G1, where it is stabilized to carry out translational regulation. As the cell enters S phase, loss of phosphorylation and the accompanying loss of 14-3-3 binding allows PTOV1 to translocate into the nucleus, carry out relevant nuclear functions, and then be degraded via the proteasome.

Citation Format: Katie L. Pennington, Colten M. McEwan, James Woods, Colin M. Muir, A G Pramoda Sahankumari, Riley Eastmond, Eranga R. Balasooriya, Christina M. Egbert, Katherine K. McCormack, Joshua L. Andersen. 14-3-3 binding during G1 stabilizes and sequesters PTOV1 to the cytoplasm [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 1469.
Abstract 148: New conformational details help in clarifying Raf activation by dimerization
Authors: Zhang Mingzhen , Maloney Ryan , Jang Hyunbum , Nussinov Ruth
Published: June 2022

Raf is a family of serine/threonine kinases in the mitogen-activated protein kinase (MAPK) pathway for cell proliferation. The full activation of Raf requires the dimerization of the kinase domain. The dimer interface of Raf has been regarded as an important therapeutic target against cancer. The next generation paradox breakers target the Raf dimerization. Despite the clinical importance, it remains unclear that how the transverse side-to-side dimerization promotes the OFF-to-ON transition of Raf’s kinase domain, and how the activated ON-state kinase domain can be stabilized in the dimer for Raf signaling. In this work, we uncover an atomic-level mechanism of Raf activation through dimerization, addressing these questions. The mechanism shows that the replacement of the intramolecular π-π stacking by the intermolecular π-π stacking at the dimer interface promotes the Raf activation. It releases the structural constraint of the αC-helix, inducing a series of conformational changes for the OFF-to-ON transition, such as the disruption of the inhibitory hydrophobic interactions and the approach of phosphorylation sites in A-loop to HRD motif for cis-autophosphorylation. Once activated, the ON-state kinase domain can be stabilized by the N-terminal basic (NtB) motif in the dimer for Raf signaling. This work provides the high-resolution structural insights into the Raf activation, suggesting a new structural strategy for drug discovery against Raf dimerization. Funded by Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261201500003I.

Citation Format: Mingzhen Zhang, Ryan Maloney, Hyunbum Jang, Ruth Nussinov. New conformational details help in clarifying Raf activation by dimerization [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 148.
Abstract 1485: Identification and in vitro validation of miRNA biomarker candidates in head and neck squamous cell carcinoma using a network biology approach
Authors: Shah Laiba , Shah Maryam , Salam Abdus , Salim Khudeja , Khan Faisal F.
Published: June 2022

Micro-RNAs are small non-coding RNAs that are able to modulate the gene expression. Aberrant expression of micro-RNAs leads to different diseased conditions such as cancer. Head and Neck Squamous Cell Carcinoma (HNSCC) is the second most prevalent cancer in Pakistan and contributes to 9.1% of mortality annually due to late stage diagnosis and ineffective treatment. This research attempts to identify novel miRNAs candidates for their potential role as diagnostic, prognostics or therapeutic biomarkers in HNSCC patient-derived primary cells in the Pakistani population. miRNA species and their gene targets reported in HNSCC were identified using the miRbase, miRNet, and miRCancer databases, and a total of 69 miRNAs with differential expression levels in HNSCC were selected to seed a miRNA-Gene interaction network that was created using BioGrid and STRING data. Hub genes that were targeted by over-expressed miRNAs were predicted using miRTarbase. GO and KEGG analysis was carried out for the 181 interacting genes including TP53, SMAD4, PTEN, NOTCH2, and STAT3. Enrichment analysis demonstrated the increased involvement of these miRNAs in Cancer Pathways such as Hippo signaling Pathways and FoxO signaling pathways. We prioritized hub genes that were targeted by the highest number of tumor suppressor genes, and identified five miRNA species, including miR-21-5p, miR-16-5p, miR30a-5p, miR-93-5p, and miR106b-5p, as the best biomarker candidates for experimental validation. To validate the in silico findings, the top five microRNA candidates are being screened for expression in a library of local patient-derived primary cell lines from Pakistani HNSCC patients.

Citation Format: Laiba Shah, Maryam Shah, Abdus Salam, Khudeja Salim, Faisal F. Khan. Identification and in vitro validation of miRNA biomarker candidates in head and neck squamous cell carcinoma using a network biology approach [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 1485.
Abstract 1519: ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression
Authors: Lavudi Kousalya , Banerjee Ananya , Cai Shurui , Li Na , Bai Xuetao , Qi-En Wang
Published: June 2022

Aldehyde dehydrogenase (ALDH) is a superfamily of 19 known enzymes that participated in the metabolism of endogenous and exogenous aldehydes. ALDH1A1 is a major subtype contributing to high ALDH activity out of all ALDH isoforms and plays a critical role in the maintenance of cancer stem cells. Our previous studies have shown that ALDH1A1 is able to reduce the sensitivity of BRCA2-/- ovarian cancer cells to PARP inhibitors by enhancing microhomology-mediated end joining (MMEJ). MMEJ uses 5-25 base pair micro homologous sequences to align the broken strands before joining, and this repair pathway requires DNA polymerase θ (Pol θ, encoded by POLQ). Here we show that ALDH1A1 can increase the expression of Pol θ, at both mRNA and protein levels, in ovarian cancer cell lines. ALDH1A1 is reported to catalyze the production of all-trans retinoic acid (ATRA), which can bind to the retinoic acid receptor (RAR), and promote transcription of the downstream target genes that possess retinoic acid response element (RARE) in their promoter regions. Interestingly we identified several RAREs in the promoter region of the POLQ gene. Thus, we hypothesized that ALDH1A1 promotes Pol θ expression via activation of the RAR signaling pathway. We found that ATRA and RAR agonist (CH55) increased but pan RAR antagonist (AGN193109) treatment decreased the expression of POLQ, indicating that the RAR pathway is involved in the transcription of POLQ. We also demonstrated that RARα can bind to the promoter region of Pol Q, activation of RAR signaling by treatment with ATRA or CH55 increased the enrichment of K27 acetylated histone H3 in this promoter region. Moreover, we showed that mutated ALDH1A1 lacking the enzyme activity was unable to enhance POLQ transcription. These data suggest that ALDH1A1 enhances the Pol Q expression by increasing the production of ATRA which further activates the RAR signaling pathway.

Citation Format: Kousalya Lavudi, Ananya Banerjee, Shurui Cai, Na Li, Xuetao Bai, Wang Qi-En. ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression [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 1519.
Abstract 1543: Regulatory role of miR-410 in prostate cancer
Authors: Sreekumar Amritha , Patel Nikhil , Saini Sharanjot
Published: June 2022

Prostate cancer (PCa), a leading cause of male cancer related mortality, is dependent upon androgens acting via androgen receptor (AR) signaling. Androgen targeted therapies are often used as the first line of treatment for prostate cancer that are effective initially. However, PCa eventually progresses to castration-resistant stage that has limited treatment options. Castration-resistant prostate cancer (CRPC) is treated by second generation of AR pathway inhibitors such as Enzalutamide and Abiraterone. Resistance to these agents develop owing to various mechanisms such as evolution to androgen-independent neuroendocrine prostate cancer (NEPC). In this study, we examined the potential role of miR-410 in different stages of prostate cancer. Analyses of miR-410 in PCa clinical samples and patient-derived xenograft (PDX) models showed that miR-410 is frequently upregulated in PCa. Its expression was further increased in CRPC cases with neuroendocrine differentiation (NED). We examined the functional role of miR-410 by stably expressing miR-410 in androgen dependent and androgen independent cell lines. Our data suggests a context dependent role of miR-410 in prostate cancer cell lines. In androgen independent cell line, miR-410 plays an oncogenic role by impacting epithelial to mesenchymal transition (EMT) via directly targeting SNAIL. Also, it was found to increase the expression of neuronal genes such as Synaptophysin and neural transcription factor BRN2. However, in androgen dependent cell line, miR-410 was found to target PTEN. In conclusion, our data suggests that miR-410 play an important regulatory role in prostate cancer with effects on EMT and neuronal differentiation programs.

Citation Format: Amritha Sreekumar, Nikhil Patel, Sharanjot Saini. Regulatory role of miR-410 in prostate 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 1543.
Abstract 1557: Defining the regulatory effects of candidate oncogenic non-coding RNA LINC00473 in fibrolamellar carcinoma
Authors: Ma Rosanna K. , Kanke Matt , Vakili Khashayar , Sethupathy Praveen
Published: June 2022

Fibrolamellar carcinoma (FLC) is a rare form of liver cancer that disproportionately affects adolescents, usually presents at advanced stages with no known risk factors or serum biomarkers, and exhibits high intrinsic drug resistance. The signature genetic event of FLC was identified in 2014 as a ~400kb heterozygous deletion resulting in the DNAJB1-PRKACA (or DP) fusion oncogene. The DP fusion occurs in over 80% of FLC patients and is sufficient for tumorigenesis in mice. Targeting DP pharmacologically has proved challenging; therefore, we performed multiple genome-scale studies to identify and target candidate downstream effectors that drive FLC development and progression. LINC00473, a primate-specific long non-coding RNA (lncRNA), emerged as a top candidate oncogene. Multiple models reveal strong induction of LINC00473 in FLC, including: (1) primary tumor relative to normal liver; (2) FLC patient-derived xenograft tumors (PDX); and (3) a newly developed FLC cell line derived from the PDX. Importantly, LINC00473 is upregulated in response to the fusion oncogene in HEK293 cells that express DP via CRISPR/Cas9-mediated deletion, and is dramatically downregulated in FLC cells upon DP knockdown by three independent siRNAs (collaboration with Alnylam Pharmaceuticals, Inc). Stable in vitro suppression of LINC00473 using two distinct short hairpin RNAs results in RNA depletion and significant reduction of cell proliferation. Concordantly, the stable overexpression of LINC00473 in FLC cells leads to a robust increase in gene expression and elevated cell proliferation. These data suggest that LINC00473 is a key regulator of FLC cell growth and survival. Next, to investigate the regulatory effects of LINC00473, we first determined that the lncRNA is enriched in the nucleus of FLC cells using subcellular fractionation followed by qRT-PCR, suggesting a role in transcriptional regulation. To investigate if LINC00473 acts downstream of the DP fusion to regulate gene expression programs mediating FLC phenotypes, we performed RNA-seq on FLC cells that harbor stable LINC00473 knockdown or overexpression. These data enabled us to define the altered gene expression profiles that result from LINC00473 dysregulation. Overall, this study elucidates the functional role of LINC00473 in the progression of FLC and helps advance the field by providing important biological insights into this disease.

Citation Format: Rosanna K. Ma, Matt Kanke, Khashayar Vakili, Praveen Sethupathy. Defining the regulatory effects of candidate oncogenic non-coding RNA LINC00473 in fibrolamellar 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 1557.
Abstract 1573: Paradoxical roles of peritumoral myofibroblasts in intrahepatic cholangiocarcinoma growth and metastasis
Authors: Tian Cheng , Li Liyuan , Li Yizhen , Yang Wentao , Fan Li , Brown Anthony , Morrison Michelle , Yang Jun J. , Glazer Evan S. , Zhu Liqin
Published: June 2022

Background and Rationale: Intrahepatic cholangiocarcinoma (iCCA) is an aggressive type of primary liver cancer characterized by its highly desmoplastic stroma. Compared to the ample knowledge on the protumorigenic functions of iCCA intratumoral myofibroblasts (iMFs), little is known about the activation status of MFs in the tumor-surrounding liver (peritumoral myofibroblasts, pMFs) and their potential role in iCCA development.

Results: We examined MF activation in both patient iCCA tumors and an iCCA orthotopic allograft mouse model. We found there was a significant, tumor-induced activation and accumulation of pMFs at the tumor border. Using an in vitro spheroid coculture system, we confirmed that iCCA-liver interaction induced rapid pMF activation at their interface. We found that MFs mixed within tumor spheroids as iMFs had a pro-proliferative effect on iCCA tumor cells similar to the previous reports. However, when the same MFs were seeded around iCCA spheroids as pMFs, they exerted a strong suppressive effect on tumor cells growth. Interestingly, prolonged iCCA-pMF coculture elicited marked tumor cell invasion and dissemination which did not occur in iCCA-iMF coculture. We found vascular cell adhesion protein 1 (Vcam1), a glycoprotein that has been implicated in solid tumor metastasis, was part of the dynamic regulation of tumor cell behaviors by iCCA-pMF interaction. Vcam1 was upregulated in the tumor cells at the early, growth-suppressing phase of iCCA-pMF interaction both in vitro and in vivo, and was then downregulated in tumor cells when they started to disseminate. Systemic blocking of Vcam1 activity in iCCA allograft mouse models using a Vcam1 neutralizing antibody slowed the primary tumor growth but promoted tumor cell dissemination.

Conclusion: Our data suggest that pMFs are beyond simple pro- or anti-tumorigenic in iCCA development. iCCA development induces rapid activation and accumulation of pMFs at the tumor border which suppress tumor growth at early stage but eventually elicit tumor cell dissemination at late stage.

Citation Format: Cheng Tian, Liyuan Li, Yizhen Li, Wentao Yang, Li Fan, Anthony Brown, Michelle Morrison, Jun J. Yang, Evan S. Glazer, Liqin Zhu. Paradoxical roles of peritumoral myofibroblasts in intrahepatic cholangiocarcinoma growth and metastasis [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 1573.
Abstract 1611: Temporal recapitulation of genome instability in an iPSC model of osteosarcoma
Authors: Becklin Kelsie Lynn , Madden Rebecca , Mills Lauren , Thue Nicole , Carroll Nathan , DeFeo Anthony , Moriarity Branden , Webber Beau
Published: June 2022

Osteosarcoma (OS) is characterized by widespread genomic instability, with cellular transformation associated with extensive structural (SV) and copy number variation (CNV). TP53 and RB1 mutations contribute to the initiation and maintenance of genome instability, which serves as a key driver of relapse. Significant effort has been made to model OS genome instability; however, current models have limited capacity to recapitulate genomic instability in real time. To model OS initiation and development, TP53 and RB1 mutations were installed in human iPSC using CRISPR/Cas9, followed by differentiation to mesenchymal (iMSC) and osteoblast (iOB) lineages in vitro. Functional assays of transformation were then conducted at intermediate stages of development for each genotype. Compared to isogenic WT controls, TP53-/- and TP53-/-/RB1-/- iOB exhibited increased proliferation, whereas anchorage independent growth was only observed in TP53-/-/RB1-/- iOB. Overexpression of cMYC and hRAS significantly increased the magnitude of these results in iOB. In contrast to iOB, TP53-/-/RB1-/- iMSC did not form colonies without cMYC and hRAS overexpression. To evaluate this phenomenon in vivo, iMSC and iOB were injected into the calcaneal bone of immunodeficient mice. Consistent with our in vitro observations, TP53-/-/RB1-/- and TP53-/-/RB1-/-+cMYC/hRAS iOB, and TP53-/-/RB1-/-+cMYC/hRAS iMSC gave rise to tumors. Histological evaluation of MSC-derived tumors revealed a largely undifferentiated pleomorphic phenotype, with a progressive increase in complexity in tumors derived from later stage iOB. Global gene expression signatures of xenografted tumors bore a striking similarity to OS patient samples compared to other pediatric tumors. In follow-on experiments, a progressive increase in cytogenetic abnormalities were observed in mutant but not WT iMSC, which became more pronounced in mutant iOB and correlated with a transformed phenotype. We are currently evaluating the temporal kinetics of this process, including through the use of low-coverage whole genome sequencing to elucidate the existence of recurrent patterns. These data highlight the utility of our iPSC approach as a novel model of OS, allowing for the first time the ability to study the evolution of genome instability in real time. We envision that this approach may uncover heretofore undiscovered common patterns underlying the seemingly chaotic nature of OS genomes, providing new opportunities for therapeutic intervention.

Citation Format: Kelsie Lynn Becklin, Rebecca Madden, Lauren Mills, Nicole Thue, Nathan Carroll, Anthony DeFeo, Branden Moriarity, Beau Webber. Temporal recapitulation of genome instability in an iPSC model of osteosarcoma [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 1611.
Abstract 1623: AXL-STAT3 targeting of lung tumor microenvironments
Authors: Taverna Josephine Amalia , Hung Chia-Nung , Chen Meizhen , Michalek Joel , Chen Chun-Liang , Kirma Nameer , Chou Chih-Wei , Tan Xi , Wang Chiou-Miin , Zhao Zhang , Nguyen Duy , DeArmond Daniel , Huang Tim
Published: June 2022

Dynamic tumor microenvironments contain a myriad of cell subtypes, each providing a unique functionality in support of malignant growth. To prepare a metastatic niche, a tight network must be present to coordinate the delivery and sharing of co-stimulatory signals among malignant tumor epithelial cells and supportive host cells. AXL and STAT3 cascades are critical components of intracellular signaling networks, as they translate multiple external stimuli into specific cellular responses. Single cell profiling of lung tumors reveals that AXL collaborates with STAT3 to create a unified tumor ecosystem favoring hybrid epithelial-to-mesenchymal transition states of lung epithelial cells, pro-tumorigenic remodeling of fibroblasts and M2 polarization of macrophages. Disruption of AXL-STAT3 circuit with AXL inhibitor and JAK inhibitor not only compromises this co-dependency in A549 lung cancer cells implanted in xenograft mice, but also limits their ability to conscript murine fibroblasts, macrophages and other immune cells that form a symbiotic community for tumor growth. Our preclinical work suggests that combined targeting of AXL-STAT3 circuit could disturb the communication among diverse cell subtypes in the lung tumor microenvironment, thus limiting the metastatic progression. Our co-culture studies reveal that AXL signaling in lung cancer cells and fibroblasts exerts an IL-11 mediated paracrine effect on macrophages to sustain STAT3 signaling for M2 polarization and cancer stemness mimicry. In vivo drug testing in A549 mouse xenograft model demonstrate that AXL-STAT3 inhibition leads to reduction in host cell recruitment and attenuates M2 polarization of macrophages and pro-tumorigenic remodeling of stromal fibroblasts. AXL-STAT3 targeting to dismantle lung tumor microenvironments is a promising therapeutic strategy and may establish new treatment paradigms for patients with lung adenocarcinoma.

Citation Format: Josephine Amalia Taverna, Chia-Nung Hung, Meizhen Chen, Joel Michalek, Chun-Liang Chen, Nameer Kirma, Chih-Wei Chou, Xi Tan, Chiou-Miin Wang, Zhang Zhao, Duy Nguyen, Daniel DeArmond, Tim Huang. AXL-STAT3 targeting of lung tumor microenvironments [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 1623.
Abstract 1636: A new invasive zebrafish model of Ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism in developing tumors
Authors: Vasileva Elena , Warren Mikako , Triche Timothy , Amatruda James
Published: June 2022

Ewing sarcoma (ES) is a malignant bone and soft-tissue tumor with an extremely poor prognosis for patients with metastatic or relapsed disease. ES is characterized by the appearance of a chimeric fusion oncogene, most frequently EWSR1-FLI1 (EF1). In the absence of a representative in vivo model the role of tumor cell/microenvironmental interactions in tumor initiation and progression remains a subject of constant debate. To address these concerns, we developed an innovative Cre-inducible model of ES in zebrafish which allows broad mosaic expression of human EF1 in zebrafish, which causes rapid onset of ES at high penetrance. This invasive model allows study of the behavior of GFP-labeled cancer cells during tumor initiation and progression, in a complex developmental context which is not currently possible in mammals. We found that observed tumors express canonical EF1 target genes and stain for known ES markers including CD99. Proteomic analysis revealed that EF1 expression affects heparan sulfate proteoglycan (HSPG) metabolism and activates HSPG-mediated ERK signaling in EF1 expressing cells, promoting cancer cell survival and proliferation. Proteoglycans are key regulators of signaling of cell surface receptors, playing an essential role in cell-to-cell communication. Targeting heparan sulfate proteoglycans with the specific heparan sulfate antagonist Surfen reduces ERK1/2 signaling and decreases tumorigenicity of ES cells in vitro and in vivo. These results highlight the important role of the extracellular matrix in ES tumor growth and the potential of agents targeting proteoglycan metabolism as novel therapies for this disease.

Citation Format: Elena Vasileva, Mikako Warren, Timothy Triche, James Amatruda. A new invasive zebrafish model of Ewing sarcoma reveals EWSR1-FLI1-driven dysregulation of heparan sulfate proteoglycan metabolism in developing tumors [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 1636.