bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 825: Evasion of apoptosis in MYC dependent T-ALL through epigenetic control
Authors: Dewson Gabrielle , Anchang Benedict , Sears Rosalie , Liefwalker Daniel F.
Published: June 2022

Cancer is a complex landscape of aberrant cell signaling programs, often initiated by oncogenes. The causal oncogene drives the preponderance of accumulated mutations that enable tumorigenesis. Cancers arising this way can be reliant on the initiating oncogene, a term known as oncogene addiction. The proto-oncogene c-MYC is a transcription factor that regulates much of the genome and is deregulated in many cancers. The transgenic Eµ-tTA/Tet-O-MYC mouse model of T-cell acute lymphoblastic leukemia (T-ALL), allows for modulation of c-MYC expression (ON vs OFF). When c-MYC is expressed, T-ALL progression is observed; and when c-MYC expression is revoked, tumor regression occurs. Using cells and microarray data from this model we employed a nested effects model (NEM) that infers hierarchical relationships anchored to master transcription factors that govern critical aspects of cell biology. We identified a critical node governed by a class of histone demethylases influencing transcriptional availability across the genome. KDM5B/JARID1b is known as a transcriptional repressor, and is downregulated in the T-ALL model when c-MYC is overexpressed. Using CRISPR/Cas9 mediated mutagenesis to disrupt KDM5B expression, we observed a potent reduction in cell death when c-MYC expression is abrogated; suggesting KDM5B mediates cell death responses in T-ALL. KDM5B is downregulated in the primary murine model when c-MYC is overexpressed, and upregulated concordantly with the loss of MYC expression. Human lymphoma cell lines that are sensitive to the bromodomain inhibitor JQ1 show downregulation of c-MYC and KDM5B upregulation. Xenografts of KDM5B knockout cells in NOD-SCIDIL-2Rg-/- (NSG) mice show increased tumor progression and burden, and more aggressive time to relapse after c-MYC expression is revoked. Future studies examining the KDM5B/c-MYC axis and its influence on chromatin architecture are ongoing. These studies aim to demonstrate a novel paradigm in understanding how c-MYC promotes tumor development through repression of a tumor suppressive epigenetic landscape regulated by KDM5B and identify therapeutic options for treating c-MYC-induced T-ALL.

Citation Format: Gabrielle Dewson, Benedict Anchang, Rosalie Sears, Daniel F. Liefwalker. Evasion of apoptosis in MYC dependent T-ALL through epigenetic control [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 825.
Abstract 828: Screen for actionable ERBB3 mutations
Authors: Koivu Marika K. , Chakroborty Deepankar , Kurppa Kari J. , Elenius Klaus
Published: June 2022

Activating mutations and copy number variations in ERBB genes have been shown to serve as oncogenic driver mutations and predictive biomarkers for ERBB inhibitor drugs. To address whether mutations in ERBB3 can affect the potential of ERBB3 to promote growth or affect sensitivity to ERBB inhibitors, we set up an unbiased functional screen for ERBB3 missense or nonsense mutations in the context of ERBB2/ERBB3 heterodimers. To this end, the iSCREAM (in vitro screen of activating mutations) pipeline, recently developed in our laboratory, was chosen. This platform exploits randomly mutated cDNA libraries of the gene of interest created with error-prone PCR and allows an unbiased assessment of growth-advantage conferred by thousands of mutations in parallel. To set up the model for screening actionable ERBB3 mutations, interleukin-3 (IL-3)-dependent Ba/F3 cells were engineered to express a homodimerization-incompetent ERBB2 V956R mutant together with ERBB3 constructs. The model was validated to serve as a readout for ERBB3's ability in activating ERBB2 kinase by demonstrating that cells expressing known transforming ERBB3 mutations, together with ERBB2 V956R, survived and expanded in the absence of IL-3. In contrast, control cells expressing ERBB2 V956R together with wild-type ERBB3 rapidly died in the absence of IL-3. The cell background with ERBB2 V956R expression was subsequently used as a target for retroviral expression of a cDNA library of randomly mutated ERBB3 constructs. The cells harboring activating ERBB3 mutations were allowed to evolve for 15-48 days. The identity of the mutations was determined from the surviving cell population by ERBB3-targeted next generation sequencing. The discovered activating mutations were validated by cloning them into expression vectors and addressing their activity by Western analyses and growth assays in Ba/F3, NIH-3T3, and MCF10A cell backgrounds. As a demonstration of the validity of the protocol, the well-characterized activating ERBB3 mutation ERBB3 E928G was identified as one of the major hits. These analyses are expected to identify activating ERBB3 mutations that are directly actionable or that provide predictive value for the use of drugs targeting ERBB signaling.

Citation Format: Marika K. Koivu, Deepankar Chakroborty, Kari J. Kurppa, Klaus Elenius. Screen for actionable ERBB3 mutations [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 828.
Abstract 868: Splicing factor mutant lung cancers: A comprehensive molecular and clinicopathologic characterization
Authors: Harada Guilherme , Falcon Christina J. , Santini Fernando C. , Chang Jason C. , Yang Soo R. , Arcila Maria E. , Rekhtman Natasha , Drilon Alexander
Published: June 2022

Background: Splicing factor mutations can drive oncogenesis in different cancer types. More importantly, strategies to target splicing-dependent cancers have been developed, including PRMT5 inhibition which regulates alternative splicing and targets posttranslational modification; PRMT5 inhibitors are in ongoing phase I clinical trials. The characterization of splicing alterations in non-small cell lung cancer (NSCLC) has not been extensively studied and represents an unmet need.

Methods: Based on previously known genes associated with splicing alterations, genomic and clinicopathologic data from NSCLC patients with hotspot or loss of function mutations identified by next-generation sequencing in the splicing factors SF3B1, SRSF2, U2AF1, ZRSR2, RBM10, and FUBP1 were explored.

Results: Among 626 NSCLC patients with at least one splicing mutation, 85% (529/626) also harbored a co-occurring oncogenic driver, such as alterations involving KRAS (60%; 319/529), EGFR (25%;132/529), MET (5%; 27/529), BRAF(4%; 24/529), and ERBB2 (3%; 17/529). After excluding patients with concurrent drivers, 97 splicing-factor-mutant NSCLCs were identified, with 80 (82%) adenocarcinomas and 6 (6%) squamous cell carcinomas. The median age was 71 years (43-89). Forty-nine patients (51%) were female. Sixty patients (62%) were diagnosed with stage I-III disease. Eleven (11%) were never smokers, while 86 (89%) were former/current smokers, with a median of pack years of 30. The median tumor mutational burden was 7.9 mut/Mb (0.9-90.4). Regarding splicing factor mutations, sixty-two patients (64%) had nonsense or splice-site RBM10 mutations, 17 (17%) had missense hotspot SF3B1 mutations, 16 (16%) had hotspot missense U2AF1 mutations, and 7 (7%) had nonsense, splice-site, or hotspot missense FUBP1 mutation. No patients with ZRSR2 or SRSF2 were identified. Only three patients presented concurrent splicing mutations: one with two SF3B1 mutations; one with RBM10 and SF3B1 mutations; and one with U2AF1 and SF3B1 mutations.

Conclusion: Most patients with splicing factor mutant NSCLCs are smokers whose cancers commonly harbor RBM10 mutations and higher TMB compared to other driver-positive cancers. The characterization of this profile is key to identifying appropriate patients for ongoing targeted therapy trials for this population.

Citation Format: Guilherme Harada, Christina J. Falcon, Fernando C. Santini, Jason C. Chang, Soo R. Yang, Maria E. Arcila, Natasha Rekhtman, Alexander Drilon. Splicing factor mutant lung cancers: A comprehensive molecular and clinicopathologic characterization [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 868.
Abstract 876: Proximity labeling reveals a role for ADAR and DDX54 in suppressing dsRNA sensing in breast cancer
Authors: Cottrell Kyle A. , Ryu Sua , Torres Luisangely Soto , Schab Angela , Weber Jason D.
Published: June 2022

The RNA editing enzyme ADAR has been identified as a therapeutic target for multiple cancers. Through its A-to-I editing activity the p150 isoform of ADAR suppresses activation of dsRNA sensors involved in the innate immune response and translational repression. Recently, our laboratory has shown that p150-ADAR is required for the viability of a subset of triple-negative breast cancer cell lines, thus making p150-ADAR a strong therapeutic target for a disease that lacks targeted therapies. Unlike the p150 isoform, which is interferon inducible, the p110 isoform of ADAR is ubiquitously expressed and has not been shown to suppress dsRNA sensing. To date there is much less known about the role of p110-ADAR in cancer, though it is known to be highly expressed in breast cancer. To begin to explore the role of p110-ADAR in breast cancer, we turned to proximity labeling by APEX2 to identify proteins that may interact with p110-ADAR. Mass-spectrometry of proximity labeled proteins revealed numerous nuclear and nucleolar proteins with roles in multiple aspects of RNA biology, including processing of rRNA and ribosome biogenesis. Among the list of enriched proteins were eight members of the DEAD-box RNA helicase family, including the nucleolar localized DDX54. Co-immunoprecipitation confirmed that p110-ADAR and DDX54 interact, furthermore both localize to nucleoli. Knockdown of DDX54 in non-TNBC cell lines (SK-BR-3 and MCF-7) caused apoptosis and reduced proliferation. Additionally, knockdown of DDX54 caused accumulation of dsRNA within the nucleolus and activation of the dsRNA sensor PKR. Knockdown of ADAR, which alone did not affect proliferation or phosphorylation of PKR in the above cell lines, synergized with DDX54 knockdown to cause increased cell death and further activation of PKR. ADAR knockdown did not synergize with knockdown of another ADAR-interacting DEAD-box helicase, DDX17, suggesting that the observed phenotype is not common to combined knockdown of ADAR and any RNA helicase. These findings suggest that combined inhibition/depletion of ADAR and DDX54 may serve as therapeutic strategy for breast tumors, including those that are refractory to ADAR inhibition/depletion alone.

Citation Format: Kyle A. Cottrell, Sua Ryu, Luisangely Soto Torres, Angela Schab, Jason D. Weber. Proximity labeling reveals a role for ADAR and DDX54 in suppressing dsRNA sensing in 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 876.
Abstract 879: SLFN12-mediated depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and represses global translation initiation
Authors: Hoyt Stephanie , Lee Sooncheol , McGaunn Joseph , Cherniack Andrew , Meyerson Matthew , Greulich Heidi
Published: June 2022

Velcrin compounds kill cancer cells expressing high levels of PDE3A and SLFN12 by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A-SLFN12 complex is not fully understood. We previously determined that SLFN12 is an RNase, that PDE3A binding upregulates SLFN12 RNase activity, and that SLFN12 RNase activity is required for velcrin response.

Here, we report that the depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. We performed ribosome profiling in HeLa cells treated with DMSO or DNMDP and found that ribosome footprints containing Leu-TTA codons at the A-site of the ribosome were significantly more abundant in DNMDP-treated cells than in cells treated with DMSO, indicating a pausing or stalling of ribosomes at Leu-TTA upon velcrin treatment. Although the overall number of ribosome pausing sites across the transcriptome was doubled by DNMDP treatment, ribosome pausing at Leu-TTA was increased > 1000-fold, also resulting in an increased number of genes with TTA pause sites.

Even though Leu-TTA is a rare codon, over 70% of all transcripts contain at least one TTA codon, implying that ribosome pausing at TTA codons could directly impact many genes. Genes with TTA pause sites most enriched upon velcrin treatment prominently include those involved in control of mRNA translation and protein homeostasis. These results imply that, in addition to direct effects on mRNA translation due to ribosome pausing, indirect effects could occur due to decreased availability of particular components of the translation machinery, resulting in a widespread impact on protein synthesis. To test our hypothesis, we measured the effects of velcrin treatment on nascent protein abundance. Treatment of HeLa cells with DNMDP resulted in downregulation of newly synthesized proteins, whereas no significant changes in total protein content were observed. Downregulation of protein translation was observed only in velcrin-sensitive cell lines, indicating that inhibition of protein translation could be the mechanism of action of cancer cell killing by PDE3A-SLFN12 complex formation.

Citation Format: Stephanie Hoyt, Sooncheol Lee, Joseph McGaunn, Andrew Cherniack, Matthew Meyerson, Heidi Greulich. SLFN12-mediated depletion of tRNA-Leu-TAA by velcrin treatment induces ribosome pausing at Leu-TTA codons and represses global translation initiation [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 879.
Abstract 894: CXCR1: A novel therapeutic avenue for CSC-like phenotypes in pancreatic ductal adenocarcinoma
Authors: Molczyk Caitlin , Thomas Elizabeth , Zaman Lubaba , Goel Paran , Singh Rakesh K.
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the United States. Often diagnosed late in disease progression, PDAC is notorious for chemotherapy resistance as well as having metastases. A cell population of interest aiding in this progression is the cancer stem cell (CSC). These cells are known for having high resistance to chemotherapy, along with propagating and re-building the tumor after most non-CSCs have been therapeutically targeted. Previous studies have determined CXCR4, ALDH1, CD24, CD44, and CD133 are markers of PDAC CSCs. In the present study, we investigated CXCR1 as a marker and therapeutic target for PDAC CSCs. CXCR1 is a G-coupled transmembrane protein receptor with downstream effects known to aid in anti-apoptosis, proliferation, and angiogenesis via binding CXCL8 and CXCL6. Already known to be a CSC marker and target in triple-negative breast cancer, initial studies by Chen et al. of CXCR1 in PDAC demonstrate CXCL8 induces increased tumorsphere formation in vitro, leading us to investigate CXCR1 in PDAC CSCs. Considering these findings, we hypothesized that PDAC cells with high CXCR1 activity exhibit increased CSC-like characteristics, and targeting CXCR1 will reduce those characteristics. To investigate the role of CXCR1 in PDAC CSC-like phenotype, we used two PDAC cell lines, CD18/HPAF and T3M4, and developed gemcitabine resistant (GemR) counterparts. These GemR cell lines were shown to have over 10-fold higher resistance than their respective parent cell lines. We treated with the CXCR1/2 antagonist navarixin at concentrations known to inhibit CXCR1. Using the parent cell lines’ relative IC50 concentrations for each drug, we treated cells for 72 hours. We used qRT-PCR and ELISAs for analysis of several known CSC markers and CXCR1 axis expression. From our results, we see the beginning trends of GemR cells having increased expression of CSC markers as well as gemcitabine-treated parent and resistant cells having increased expression. Using flow cytometry, we evaluated the CXCR1+ cell populations post-control and gemcitabine treatment. The population of CXCR1+ cells was higher in the gemcitabine-treated groups. Together, our observations suggest an association between CXCR1 and the CSC-like phenotype in PDAC.

Citation Format: Caitlin Molczyk, Elizabeth Thomas, Lubaba Zaman, Paran Goel, Rakesh K. Singh. CXCR1: A novel therapeutic avenue for CSC-like phenotypes 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 894.
Abstract 907: CD133 stimulates cell proliferationviaupregulation of amphiregulin in melanoma
Authors: Simbulan-Rosenthal Cynthia M. , Islam Nusrat , Haribabu Yoga , Alobaidi Ryyan , Kuo Li-Wei , Shalamzari Azadeh , Leibowitz Adam , Moulton Joanna , Rosenthal Dean S.
Published: June 2022

Melanoma, a malignant transformation of melanocytes, accounts for 90% of skin cancer deaths due to its ability to efficiently metastasize to other organs. CD133, also known as Prominin1, is a pentaspan glycosylated transmembrane protein that is overexpressed in cancer stem cells from various tumors, including melanoma, and has been associated with tumor recurrence, chemoresistance, and metastasis. To characterize and target CD133-positive cancer stem cells, patient-derived melanoma cell lines BAKP and POT, harboring BRAFWT/NRASQ61K and BRAFWT/NRASQ61R driver mutations, respectively, were co-transduced with a Tet activator (rtTA3) and a Tet-on vector expressing CD133 (TRE3G-CD133) to produce stable doxycycline (Dox)-inducible cell lines. These cell lines were exposed to Dox (1µg/mL) to induce CD133 expression for 24 h; mRNA was extracted and subjected to RNA-seq analysis, followed by bioinformatic analyses, which revealed genes that are significantly up- or down-regulated by Dox-induced CD133 expression. Among the most significantly altered genes was amphiregulin (AREG), which increased 7-fold with Dox induction. These results were validated in both BAKP and POT cell lines by qPCR and immunoblot analysis. AREG binds EGFR and activates RAS/RAF/MEK/ERK and PI3K/AKT signaling pathways, leading to cell proliferation, survival, and migration. Consistently, cell cycle and cell growth analyses revealed that Dox-induced CD133 expression increases the percent of cells in S-phase of the cell cycle, leading to increased cell growth in BAKP cells. The role of CD133 in cell proliferation via AREG upregulation was further studied with immunofluorescent and flow cytometry analysis of proliferation markers PCNA, Ki67, as well as BrdU incorporation into newly synthesized DNA in Dox-induced BAKP cells, with or without siRNA knockdown of AREG. AREG may play an integral role in melanoma progression due to its vital function in cell proliferation, serving as an attractive target for novel therapeutics in melanoma and cancers exhibiting overexpression of both CD133 and AREG. Further studies using specific AREG inhibitors or RNAi knockdown in melanoma cell lines is currently ongoing, and can further elucidate molecular mechanisms highlighting the role(s) of CD133 and AREG in melanoma cancer stem cells.

Citation Format: Cynthia M. Simbulan-Rosenthal, Nusrat Islam, Yoga Haribabu, Ryyan Alobaidi, Li-Wei Kuo, Azadeh Shalamzari, Adam Leibowitz, Joanna Moulton, Dean S. Rosenthal. CD133 stimulates cell proliferationviaupregulation of amphiregulin in melanoma [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 907.
Abstract 918: Development of a platform for programmable in vivo oncogene activation and screening using CRISPRa technology
Authors: Thege Fredrik Ivar , Rupani Dhwani N. , Barathi Bhargavi B. , Maitra Anirban , Rhim Andrew D. , Wörmann Sonja M.
Published: June 2022

Conventional genetically engineered mouse models (GEMMs) are time consuming, laborious and offer limited spatio-temporal control. We have developed a streamlined platform for in vivo gene activation using CRISPR activation (CRISPRa) technology. Our model system allows for flexible, sustained and timed activation of one or more target genes, in vitro or in vivo, using single or pooled lentiviral guides. Using Myc and Yap1 as model oncogenes, we implemented this platform to study the effect of oncogene activation on the tumorigenic potential of primary pancreatic organoids, as well as in an autochthonous model of lung adenocarcinoma. We found that Myc-activation in pancreatic organoids increased their tumorigenic potential and resulted in significantly shorter survival relative to controls when transplanted orthotopically. In vivo Myc activation in the lung accelerated tumor progression and resulted in significantly shorter overall survival relative to non-targeted tumor controls. Furthermore, we found that Myc-activation drives the acquisition of an immune suppressive “cold” tumor microenvironment. Through cross-species validation of our results, we were able to link MYC to a previously described, immunosuppressive transcriptomic subtype in patient tumors, thus identifying a patient cohort that may benefit from combined MYC/immune-targeted therapies. Our work demonstrates how CRISPRa can be used for rapid functional validation of putative oncogenes and may allow for the identification and evaluation of potential metastatic and oncogenic drivers through competitive screening.

Citation Format: Fredrik Ivar Thege, Dhwani N. Rupani, Bhargavi B. Barathi, Anirban Maitra, Andrew D. Rhim, Sonja M. Wörmann. Development of a platform for programmable in vivo oncogene activation and screening using CRISPRa technology [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 918.
Abstract 946: Utility of intestinal organoids to model serrated colon cancer in vitro
Authors: Premsrirut Prem K. , Wang Huaien , Reino Eduardo J. Garcia , Breen Isabella , Vasileva Ana A.
Published: June 2022

Serrated colorectal cancer (CRC) is an aggressive and treatment-resistant form of colorectal carcinogenesis that accounts for one-fourth of the patients with CRC1. At the molecular level, serrated CRC is characterized by epigenetic modifications termed the CpG Island Methylator Phenotype (CIMP) and the presence of somatic mutations that activate the mitogen-activated protein kinase (MAPK) pathway1,2,3. The BRAFV600E is an early genetic change in serrated polyps, but it is not sufficient to drive tumor formation. Combinations of mutations and epigenetic modifications in P16INK4A, MLH1, ZNFR3, RNF43, and TGFBR2 are often seen in most patients4. Therefore, a combinatorial approach to develop BrafV600E-driven serrated CRC organoids harboring a variety of the above mutants will enable in vitro studies of the tumorigenic potential of each combination as well as allow engraftment into animals. In turn, the latter will allow for applying preclinical in-vivo pharmacology modalities in vitro, thereby bypassing the time required to create a specific knock-in/out strain for each genetic modification desired. Here, we describe a pipeline to directly genetically manipulate specific organoid systems in vitro. We first established organoid cultures from the small and large intestine of unique Tet-inducible RNAi mice where efficient gene knock-down can contribute to microsatellite instability. Using CRISPR/Cas9, we then edited these to generate CRC organoid models containing the BrafV600E mutation in combination with knock outs or knock-ins of cell cycle regulators and negative regulators of the Wnt pathway. These deficiencies mimic the progressive mutational changes upon acquisition BrafV600E. Our data lay the groundwork for future use of RNAi technology in combination with sequential gene editing for in vitro pharmacology and modeling diseases with complex genetic architecture both in vitro and in vivo.

Citation Format: Prem K. Premsrirut, Huaien Wang, Eduardo J. Garcia Reino, Isabella Breen, Ana A. Vasileva. Utility of intestinal organoids to model serrated colon cancer in vitro [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 946.
Abstract 990: Functional genomic screen of kinases in high-grade serous ovarian cancer metastasis
Authors: Kotnik Emilee Nicole , Lomonosova Elena , Wilke Daniel , Longmore Gregory , Fuh Katherine C.
Published: June 2022

High-Grade Serous Ovarian Cancer (HGSC) is a highly metastatic cancer with the majority of patients presenting in advanced stages. Currently there are limited targeted treatment options for patients, especially for women with high metastatic tumor burden. In order to improve patient outcomes, we have aimed to identify and characterize kinases that may regulate the steps of metastasis in HGSC. Kinases in particular serve as actionable targets because we can design inhibitors of the kinases and their ligands. Several kinases, such as tyrosine kinases and aurora kinases, have previously been implicated in ovarian cancer and seem to influence tumor cell metastasis. Our lab performed a functional genomic screen on 719 kinases in a tumor cell attachment and proliferation assay. In this screen, we cultured primary fibroblasts from patient omentum and plated them on top of a collagen and fibronectin matrix, to mimic the tumor microenvironment. We used siRNA transfection reagents, which were plated atop the fibroblasts along with GFP-labeled ovarian cancer cells that were then incubated with the other cell layers for 72 hours. Based on the florescence intensities, we calculated median average deviations (MAD) values normalized to controls to be a measure of ovarian cancer cell attachment and proliferation. This screen was replicated in 3 ovarian cancer cell lines, including OVCAR8, ES2, and A2780, across 9 panels for 2 libraries. Both libraries had 2 distinct siRNAs for each kinase, in total 4 siRNAs per kinase. From this initial screen, we have identified 20 candidate kinases to validate based on having significantly negative MAD values in at least two cells lines in the screen. Candidate kinases have been validated by knock-down in two ovarian cancer cell lines, ES2 and OVCAR8, and assessed on their ability to migrate in a wound-healing assay. The top kinases will be further studied for their ability to regulate the steps of tumor cell metastasis and the mechanisms that allow them to promote metastasis. Our study aims to identify actionable targets that influence these steps of metastasis, so we can develop drugs that target metastatic tumors and serve as maintenance therapies to help prevent further metastasis, mediate chemoresistance, and improve patient outcomes.

Citation Format: Emilee Nicole Kotnik, Elena Lomonosova, Daniel Wilke, Gregory Longmore, Katherine C. Fuh. Functional genomic screen of kinases in high-grade serous ovarian cancer 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 990.
Abstract 991: OVO-like genes are master regulators of the epithelial fate in breast cancer
Authors: Garcia Diana Drago , Yarden Yosef
Published: June 2022

The OVO-like genes are evolutionary conserved C2H2 zinc finger transcription factors (TFs) with SNAG repressor domains involved in the maintenance of epithelial homeostasis. From the three Ovo-like family genes identified in mammals, Ovol-like1 (Ovol1) and Ovol-like2 (Ovol2) are the most studied members with multiple reports pinpointing relevant roles in development and disease. In cancer, one of the hallmarks of invasive carcinoma involves the loss of E-cadherin expression by the repression of SNAG transcription factors. Interestingly, contrary to the other SNAG domain family members the OVO-like genes regulate epithelial cell fate. We performed data analysis in breast cancer cell lines and patient data obtained from GEO, CCLE, TCGA and METABRIC to assess the potential of the OVO-like genes as relevant regulators of gene expression in breast carcinoma. In addition, we stablished breast cancer models with inducible expression or repression of the OVO-like genes. We also analyzed the cellular systems we generated using naïve high-throughput methods such as RNA-Seq. Likewise, we studied the effect of OVO-like genes on tumor progression and metastasis using inducible expression systems in both xenograft and syngeneic breast cancer models. We observed that Ovol1 and Ovol2 are constitutively expressed in human breast cancer cell lines, and they are upregulated due to chromosomal gains in breast cancer patients. We observed how the introduction of Ovol1 and Ovol2 into mesenchymal-like breast cancer cell lines modified their transcriptome in a way that promoted expression of epithelial marker genes (e.g., Amphiregulin) and downregulated mesenchymal markers (e.g., Vimentin). In addition, introducing Ovo-like genes in mesenchymal-like cells greatly affected their viability and function. Surprisingly, we observed a similar effect by downregulating the Ovo-like genes in epithelial-like cells. We propose a model where a common epigenetic machinery is shared between structurally related transcription factors with opposing effects, such as OVO-like proteins and other SNAG-containing transcription factors, such as Snail and Slug, such that the abundance of the transcription factors and their co-repressors defines the state of the system as either epithelial or mesenchymal.

Citation Format: Diana Drago Garcia, Yosef Yarden. OVO-like genes are master regulators of the epithelial fate in 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 991.
Abstract CT231: Digital phenotyping in young breast cancer patients treated with neoadjuvant chemotherapy
Authors: Delrieu Lidia , Hamy Anne-Sophie , Coussy Florence , Kassara Amyn , Asselain Bernard , Antero Juliana , De Villèle Paul , Dumas Elise , Forstmann Nicolas , Guérin Julien , Hotton Judicael , Jouannaud Christelle , Milder Maud , Leopold Armand , Sedeaud Adrien , Soibinet Pauline , Toussaint Jean-François , Vercamer Vincent , Laas Enora , Reyal Fabien
Published: June 2022

Introduction: Breast cancer (BC) has particular characteristics in young women, with diagnosis at more advanced stages, a poorer prognosis and highly aggressive tumors. In NeoFit, we will use an activity tracker to identify and describe various digital profiles (heart rate, physical activity, and sleep patterns) in women below the age of 45 years on neoadjuvant chemotherapy for BC.Methods/Analysis: NeoFit is a prospective, national, multicenter, single-arm open-label study. It will include 300 women below the age of 45 years treated with neoadjuvant chemotherapy for BC. Participants will be asked to wear a Withing Steel HR activity tracker round the clock for 12 months. The principal assessments will be performed at baseline, at the end of neoadjuvant chemotherapy and at 12 months. We will evaluate clinical parameters, such as toxicity and the efficacy of chemotherapy, together with quality of life, fatigue, and parameters relating to lifestyle and physical activity. The women will complete REDCap form questionnaires via a secure internet link.Discussion: In this study, the use of an activity tracker will enable us to visualize changes in the lifestyle of young women on neoadjuvant chemotherapy for BC, over the course of a one-year period. This exploratory study will provide crucial insight into the digital phenotypes of young BC patients on neoadjuvant chemotherapy and the relationship between these phenotypes and the toxicity and efficacy of treatment. This trial will pave the way for interventional studies involving sleep and physical activity interventions.

Citation Format: Lidia Delrieu, Anne-Sophie Hamy, Florence Coussy, Amyn Kassara, Bernard Asselain, Juliana Antero, Paul De Villèle, Elise Dumas, Nicolas Forstmann, Julien Guérin, Judicael Hotton, Christelle Jouannaud, Maud Milder, Armand Leopold, Adrien Sedeaud, Pauline Soibinet, Jean-François Toussaint, Vincent Vercamer, Enora Laas, Fabien Reyal. Digital phenotyping in young breast cancer patients treated with neoadjuvant 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 CT231.
Abstract CT546: A phase 2, multicenter study to evaluate the efficacy and safety of TACE sequential tislelizumab as adjuvant therapy in patients with HCC at high risk of recurrence after curative resection
Authors: Liang Tingbo , Zhang Zongli , Zheng Jinfang , Wang Yijun , Zhang Jialin , Li Bo , Ma Tao , Xu Yunfei , Wu Changxiong , Sun Quan , Meng Yiman , Yang Xiaoli
Published: June 2022

Background: Surgical resection is one of the most important radical treatment in hepatocellular carcinoma (HCC). However, the 5-year recurrence rate of HCC after surgery is up to 70%, hampering further improvement in the prognosis of HCC patients. Risk factors for recurrence include microvascular invasion (MVI), multiple lesion and solitary tumor ≥ 5 cm, etc. Postoperative adjuvant treatment with transarterial chemoembolization (TACE) can reduce the recurrence rate and improve overall survival (OS) for patients with high risk of recurrence to some extent. TACE treatment promotes the release of tumor related antigen and the recruitment of immune cells by killing tumor cells, thereby promoting the anti-tumor immune response; it may have a potential synergistic effect with PD-1 blockade. Currently, there is no ongoing study on TACE combined with immunotherapy as postoperative adjuvant therapy for HCC. This is an open-label, multicenter, phase II study to evaluate the efficacy and safety of TACE sequential tislelizumab as adjuvant therapy in HCC patients with high risk of recurrence after curative resection (NCT04981665).

Trial design: Eligible patients are ≥18 y, ECOG PS 0 or 1, Child-Pugh class A, diagnosed with HCC confirmed by histopathology or cytology, with no prior systematic treatment and locoregional therapy for HCC, and have received a curative resection with high risk of recurrence fulfilling one of the following criteria: (1) a solitary tumor > 5 cm, (2) solitary tumor > 2 cm and ≤ 5 cm with MVI, (3) 2-3 lesions. 50 pts will receive TACE treatment once (4±1 w) after curative resection, then followed by tislelizumab 200 mg IV Q3W (5±2 d). Tislelizumab will be administered until disease recurrence, intolerable toxicity, death, withdrawal of consent or completion of a maximum of 17 cycles of tislelizumab. Primary endpoint is 2-year recurrence-free survival rate (2-year RFS rate). Secondary endpoints include RFS, time to recurrence, OS, 1-year RFS rate, 1-year and 2-year OS rate, and safety. Exploratory endpoints include biomarkers related to the response and prognosis of adjuvant TACE sequential tislelizumab therapy.

Citation Format: Tingbo Liang, Zongli Zhang, Jinfang Zheng, Yijun Wang, Jialin Zhang, Bo Li, Tao Ma, Yunfei Xu, Changxiong Wu, Quan Sun, Yiman Meng, Xiaoli Yang. A phase 2, multicenter study to evaluate the efficacy and safety of TACE sequential tislelizumab as adjuvant therapy in patients with HCC at high risk of recurrence after curative resection [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 CT546.
Abstract LB151: Deep learning - based protein therapeutics design to treat HER2+ cancers
Authors: Liang Ziwei , Murray Tom , Bluestone Max , Zoumadakis Chris , Martin Luke , Vlasenko Alexey , LaFrence Jeffrey , Lazar John , Denecke Matthias , Longo David
Published: June 2022

Background: HER2 amplification and activation occurs in approximately 15~25% of all breast cancers and is further indicated in other cancer types with high un-met medical needs, such as cholangiocarcinoma, ovarian and esophageal cancer. Further improvement of current HER2 targeting therapeutics on safety and efficacy remains to be desired. Deep learning - based computational approach, such as AlphaFold, has been successfully applied to the understanding of protein structure. However, its applications to protein therapeutic design remain largely underdeveloped. Method: We have developed an end-to-end protein design engine Apollo, which can be used to design target-binding protein therapeutics in silico. Apollo uses recurrent neural networks (RNNs), transformers and graph convolution neural networks (GCNs) to represent proteins, protein structures and protein-protein interactions. This is further improved by multi-head and hierarchical attention layers. Using generative AI, Apollo can either generate putative binding proteins de novo, or to optimize protein lead molecules. Candidate proteins are evaluated in silico for binding affinity and specificity, secondary and tertiary structure, isoelectric point, solubility, and other drug-like properties. Generated designs are iteratively improved by Apollo via a reinforcement learning loop. Results: Apollo has achieved high prediction accuracy by in silico evaluations, using metrics such as AUC and the confusion matrices. Using Apollo, we then designed mini protein binders of 50~100 aa to target HER2 extracellular domain (ECD). These binders can be produced with high yield and purity using mammalian protein expression systems. From de novo generation, the designed candidates are shown to bind to human HER2 with EC50 in the nanomolar range by ELISA. From optimizations based on lead molecules, the designed candidates have shown improved production yield and thermostability while maintaining binding affinity. Conclusions: We have shown the proof-of-concept success of using machine learning to design protein therapeutics.

Citation Format: Ziwei Liang, Tom Murray, Max Bluestone, Chris Zoumadakis, Luke Martin, Alexey Vlasenko, Jeffrey LaFrence, John Lazar, Matthias Denecke, David Longo. Deep learning - based protein therapeutics design to treat HER2+ cancers [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 LB151.
Abstract LB161: Whole mammogram image improves breast cancer prediction
Authors: Jiang Shu , Colditz Graham A.
Published: June 2022

To efficiently capture data from mammographic breast images and classify long term risk of breast cancer, we developed methods that use the extensive existing data that are currently ignored in the context of breast cancer risk stratification. More than 20 studies support texture features add value to risk prediction beyond breast density. However, the entire mammogram imaging data has a high dimension of pixels (~13 million per image), greatly exceeding the number of women in a cohort. We apply functional principal component analysis methods to predict 5-years breast cancer incidence using baseline mammograms. We applied these methods onto women participating in the Joanne Knight Breast Health Cohort which is comprised of over 10,000 women undergoing repeated mammography screening at Siteman Cancer Center and followed since 2010. All women had baseline mammogram at entry, provided a blood sample and completed a risk factor questionnaire. Mammograms are all using the same technology (Hologic). During follow-up through October 2020, we identified 246 incident breast cancer cases (pathology confirmed) and matched them to controls from the perspective cohort based on month of mammogram and age at entry. In a baseline model we controlled for age, menopause, BMI, and mammographic breast density (BIRADs). We then added the full image (characterized by the FPC) to the base model and further compared the AUC of the new model vs the base model using the likelihood ratio test. AUC is validated with internal 10-fold cross validation. The AUC for 5-year breast cancer risk classification increased significantly from a median of 0.61 (sd 0.09 for estimated AUCs across 10-fold internal validation) for the baseline model to 0.70 (0.10) when the full image is added, p < 0.001. We conclude that using full mammogram images for breast cancer risk prediction captures additional information on breast tissue characteristics that relate to cancer risk, and improves prediction classification. This prediction algorithm can run efficiently in real time (in seconds) with processing of digital mammograms. Thus, this model can be easily implemented in mammography screening services and other clinical settings to guide real-time risk stratification to improve precision prevention of the leading cancer in women world-wide. Further analysis will quantify the value of adding other breast cancer risk factors, including polygenic risk scores. Addition of repeated mammogram images over time should further increase classification performance. This approach has the potential to improve risk classification by using data already available for the vast majority of women already having repeated screening mammograms.

Citation Format: Shu Jiang, Graham A. Colditz. Whole mammogram image improves breast cancer prediction [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 LB161.
Abstract LB177: Widespread hypertranscription in aggressive human cancer
Authors: Zatzman Matthew , Fuligni Fabio , Ripsman Ryan , Suwal Tannu , Edward Lisa-Monique , Denroche Rob , Jang Gun Ho , Notta Faiyaz , Gallinger Steven , Selvanathan Saravana P. , Toretsky Jeffrey , Hellmann Matthew D. , Tabori Uri , Huang Annie , Shlien Adam
Published: June 2022

Cancers are often defined by the dysregulation of specific transcriptional programs; however, the importance of global transcriptional changes is less understood. Hypertranscription is the genome-wide increase in RNA output. Hypertranscription’s prevalence, underlying drivers and prognostic significance are undefined in primary human cancer. This is due in part to limitations of expression profiling methods, which assume equal RNA output between samples. Here, we developed a computational method to directly measure hypertranscription in 7,494 human tumors, spanning 31 cancer types. Hypertranscription is ubiquitous across cancer, especially in aggressive disease. It defines patient subgroups with worse survival, even within well-established subtypes. Our data suggest that loss of transcriptional suppression underpins the hypertranscriptional phenotype. Single-cell analysis reveals hypertranscriptional clones, which dominate transcript production regardless of their size. Finally, patients with hypertranscribed mutations have improved response to immune checkpoint therapy. Our results provide fundamental insights into gene dysregulation across human cancers and may prove useful in identifying patients that would benefit from novel therapies.

Citation Format: Matthew Zatzman, Fabio Fuligni, Ryan Ripsman, Tannu Suwal, Lisa-Monique Edward, Rob Denroche, Gun Ho Jang, Faiyaz Notta, Steven Gallinger, Saravana P. Selvanathan, Jeffrey Toretsky, Matthew D. Hellmann, Uri Tabori, Annie Huang, Adam Shlien. Widespread hypertranscription in aggressive human 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 LB177.
Abstract LB521: Antitumor activity of potent RAF inhibitors in solid tumors with activated RAS-RAF axis
Authors: Hwang Kwangwoo , Jo SeoHyun , Choi Jieun , Choi Ga-young , Choi Jiseon , Kwon Ji-Hye , Shin Dong-Guk , Kim Jiyeon , Kim Se-Hyuk , Kim Haelee , Cho Ha Yeon , Son Jung Beom , Kim Nam Doo , Choi Hwan Geun , Kim Daekwon , Kim Sunghwan
Published: June 2022

FDA approved three RAF inhibitors for the treatment of tumors containing BRAFV600 mutations, but one of the major drawbacks of these type I RAF inhibitors is to activate MAPK signaling pathway, instead of inhibiting signaling, which is referred to as paradoxical activation. Such undesired paradoxical activation not only leads to renewed tumor growth but also spurs additional cancer growth in non-cancerous wild-type BRAF tissue. Plus, these first-generation RAF inhibitors targeting BRAFV600 mutants are unable to inhibit oncogenic RAF dimers. This has led to the development of type II RAF inhibitors such as belvarafenib and day101 to block the activity of multiple forms of RAF while avoiding paradoxical activation. Two lead-like stage compounds 1 and 2 were specifically designed as type II RAF inhibitors to have activity across RAF isoforms including BRAFV600E, BRAF wild-type, and CRAF, showing higher potency than competitors. Compounds 1 and 2 potently inhibited the growth of BRAFV600E melanoma cells and NRAS or KRAS mutant cancer cells. Compounds 1 and 2 promoted the formation of BRAF/CRAF heterodimers by directly binding to the RAF kinase domain like other type II RAF inhibitors and inhibited phosphorylation of downstream effectors MEK and ERK in a dose-dependent manner in RAS mutant cancer cells, suggesting less paradoxical activation liability. Compound 2 showed superior on-target inhibitory activity for BRAFV600E, BRAF wild-type, and CRAF than belvarafenib through RAF immunoprecipitation (IP) kinase assay. Inhibition of the RAF downstream signaling was also confirmed by quantifying the level of phospho-ERK in NRAS or KRAS mutant cancer cells. In HCT116 (KRASG13D) subcutaneous xenograft model, compound 1 showed tumor growth inhibition efficacy, suggesting a potential to address RAS mutant driven- as well as BRAFV600 mutant driven tumors. Also, combination treatment with MEK inhibitor and/or immune checkpoint inhibitor would further improve the therapeutic activity and expand target indication for unmet medical needs.

Citation Format: Kwangwoo Hwang, SeoHyun Jo, Jieun Choi, Ga-young Choi, Jiseon Choi, Ji-Hye Kwon, Dong-Guk Shin, Jiyeon Kim, Se-Hyuk Kim, Haelee Kim, Ha Yeon Cho, Jung Beom Son, Nam Doo Kim, Hwan Geun Choi, Daekwon Kim, Sunghwan Kim. Antitumor activity of potent RAF inhibitors in solid tumors with activated RAS-RAF axis [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 LB521.