bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2252: Predicting second breast cancers among women diagnosed with primary breast cancer using patient-level data and machine learning algorithms
Authors: Syleouni Maria Eleni , Karavasiloglou Nena , Manduchi Laura , Wanner Miriam , Korol Dimitri , Rohrmann Sabine
Published: June 2022

In 2020 2.3 million women were diagnosed with breast cancer. About 7.4% of women who have been diagnosed with primary breast cancer will have a second primary breast cancer within 10 years. This study builds a prediction model for second breast cancer for women who have had primary breast cancer. Readily available cancer registry data with machine learning methods for classification are employed. The best-performing model is selected based on the area under the receiver operator curve, and the key characteristics contributing to a high risk for second breast cancer are identified based on the prediction model. Using extreme gradient boosting (XGBoost) with limited patient features we find an area under the curve of 0.65-0.70 for the testing set. Among the most important features are days from incidence to treatment, size of primary tumor based on the pathology report, and oestrogen receptor status.This research is a step towards the development of a tool that will help doctors identify women very likely to develop second breast cancer, which will prioritize their follow-up or inform their course of treatment depending on their characteristics.

Citation Format: Maria Eleni Syleouni, Nena Karavasiloglou, Laura Manduchi, Miriam Wanner, Dimitri Korol, Sabine Rohrmann. Predicting second breast cancers among women diagnosed with primary breast cancer using patient-level data and machine learning algorithms [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 2252.
Abstract 2275: Transcription factor landscape cataloguing highlights key insights in inflammatory breast cancer (IBC)
Authors: Rosado-Tristani Diego A. , Morales Carlos S. , Peterson Esther A. , Rodríguez-Martínez Jose A.
Published: June 2022

Transcription factors are proteins that bind to DNA in a sequence-specific manner to regulate gene expression for normal cellular functions. Cancers have been shown to have aberrant transcriptional regulation. Therefore, identifying transcription factor landscapes will aid in characterizing complex diseases. As an example, breast cancer has many subtypes that are phenotypically and molecularly distinct. Inflammatory Breast Cancer (IBC) is rare, and the most aggressive form of breast cancer currently known. It is a poorly characterized subtype, and diagnosis often results in poor prognosis for patients. As such, we decided to explore the transcription factor landscape in IBC. This was accomplished employing the following strategy: 1) We assembled de novo transcriptomes for SUM149, an IBC cancer line and MCF7, a non-IBC cancer line using Trinity; 2) We translated the assembled transcriptomes using getORF from EMBOSS; 3) We identified putative transcription factors in the translated transcriptomes using CREPE. We then identified differentially expressed genes between SUM-149 and MCF-7 using DEseq2. Our results highlight differences in the transcription factor catalogues between IBC and non-IBC cancers. Of interest in SUM-149 is the gene BNC1, a member of the C2H2 zinc finger family of transcription factors. Dysregulation of this gene is associated with brain metastasis in breast cancers which is a common phenotype in IBC. BNC1-associated genes are linked to GO terms such as, epithelial cell differentiation, and regulation of alternative mRNA splicing, via spliceosome; however, BNC1 functions in IBC are yet to be determined. This analysis will bridge the gap in knowledge of key transcriptional regulators in IBC.

Citation Format: Diego A. Rosado-Tristani, Carlos S. Morales, Esther A. Peterson, Jose A. Rodríguez-Martínez. Transcription factor landscape cataloguing highlights key insights in inflammatory breast cancer (IBC) [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 2275.
Abstract 2278: Expanding cancer therapy options by leveraging synthetic lethal interactions between druggable paralogs
Authors: Parrish Phoebe C. , Thomas James D. , Gabel Austin M. , Kamlapurkar Shriya , Bradley Robert K. , Berger Alice H.
Published: June 2022

Synthetic lethal therapies are a promising approach to expand therapeutic options for cancer patients. Since synthetic lethal therapies target tumor cells specifically, they have fewer off-target effects than oncogene targeted approaches. To identify new cancer drug targets, we focused on paralogs, ancestrally-duplicated genes that frequently retain redundant or overlapping functions.

To find synthetic lethal human paralogs, we developed paired guide RNAs for Paralog gENetic interaction mapping (pgPEN), a pooled CRISPR-Cas9 single and double knockout approach targeting over 2,000 paralogs. We applied pgPEN to two cancer cell lines and found that 12% of human paralogs exhibit synthetic lethality in at least one context. To our knowledge, pgPEN represents the largest experimental assessment of human paralog synthetic lethality to date.

We next identified paralog pairs to prioritize for follow-up study. A key drawback to synthetic lethal therapies is that many genetic interactions are context-dependent. To identify likely penetrant interactions, we compared our data to other published paralog screens and computational predictions of paralog synthetic lethality. We found that over 75% (n=96) of pgPEN hits were predicted to be broadly synthetic lethal, and nearly 10% (n=10) of pairs were synthetic lethal in multiple paralog screens.

Finally, we prioritized paralogs targeted by existing small molecule therapies. Of the 122 synthetic lethal pairs identified by pgPEN, 16% (n=20) are currently druggable. We mined drug repurposing data from DepMap to find pairs where a paralog-targeting drug showed a stronger effect in cell lines with low target gene copy number or expression relative to cell lines with normal target gene copy number or expression. These drugs could selectively target cancer cells in cases where one or both paralogs is lost in tumors but retained in normal tissue. We also leveraged The Cancer Genome Atlas tumor sequencing data to find paralog pairs where one member is recurrently lost in cancer.

Taken together, these studies identify druggable, highly penetrant synthetic lethal paralog interactions. In combination with tumor sequencing data, we identify high-priority paralog drug targets that can be further tested and translated to the clinic. Paralog synthetic lethal therapies could provide a relatively low-toxicity therapeutic approach to improve the efficacy of cancer treatments and prevent the emergence of acquired resistance.

Citation Format: Phoebe C. Parrish, James D. Thomas, Austin M. Gabel, Shriya Kamlapurkar, Robert K. Bradley, Alice H. Berger. Expanding cancer therapy options by leveraging synthetic lethal interactions between druggable paralogs [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 2278.
Abstract 2287: Dissecting the heterogeneity of central nervous system hemangioblastomas by single-cell and single-nuclei RNA sequencing
Authors: Konstantakou Eumorphia , Perez Elizabeth , Barbera Alex , Haradhvala Nicholas , Stanzione Marcello , Barker Fred , Nahed Brian , Stemmer-Rachamimov Anat , Dyson Nick , Getz Gad , Suva Mario , Iliopoulos Othon
Published: June 2022

Hemangioblastomas (HBs) are highly vascular tumors of the Central Nervous System that are commonly associated with the familial von Hippel-Lindau (VHL) disease and contribute significantly to the morbidity and mortality of VHL patients. They are characterized by high heterogeneity but the cell origin of HB tumors is still unclear, and the genomic landscape and signaling pathways underlying the tumor formation are unknown. We performed single-cell and single-nuclei RNA sequencing of cerebellar and spinal HBs surgically removed from VHL patients, in order to identify the lineage of the tumor cells and to profile the tumor microenvironment (TME). The percentage of VHL-/- tumor cells ranges from 8-80% of the cell population; the TME is reactive, contributing to a high percentage of the total tumor volume and playing a critical role in HB development. Single-cell and single-nuclei HB analysis provided significant insights into the cell of origin as well as the oncogenic pathways that drive the growth of HB. Whole exome sequencing (WES) in a cohort of HB patients revealed that biallelic VHL inactivation is necessary for the tumor formation of VHL disease-related HBs.

Citation Format: Eumorphia Konstantakou, Elizabeth Perez, Alex Barbera, Nicholas Haradhvala, Marcello Stanzione, Fred Barker, Brian Nahed, Anat Stemmer-Rachamimov, Nick Dyson, Gad Getz, Mario Suva, Othon Iliopoulos. Dissecting the heterogeneity of central nervous system hemangioblastomas by single-cell and single-nuclei RNA sequencing [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 2287.
Abstract 2310: Histone methyltransferase-independent function of EZH2 in the cell cycle regulation
Authors: Colon Tania Marlyn , Choi Byeong Hyeok , Dai Wei
Published: June 2022

Enhancer of zeste homologue 2 (EZH2) is an evolutionarily conserved histone methyltransferase that mediates the methylation of lysine 27 of histone H3 (H3K27me3). EZH2 is frequently mutated or overexpressed in many types of cancer. Extensive research in the past has revealed that EZH2 forms part of the polycomb repressive complex 2 (PRC2), an essential multi-protein complex required for gene silencing and chromatin compaction. EZH2 also displays additional cellular functions that include transcriptional activation of certain gene expression and methylation of non-histone substrates, the latter modulating protein stability or actin polymerization. Moreover, EZH2 is known to play a role in regulating cell cycle progression. However, it remains unclear whether EZH2 has an enzyme-independent function in the cell cycle. Here we show that EZH2 knockdown by small interfering RNAs (siRNAs) results in an increased expression of cyclin-dependent kinase inhibitors including p21, p27, and p57, which is associated with G1/S arrest. We have also observed that HeLa cells treated with Tazemestostat, a specific EZH2 inhibitor, display a significant reduction in H3K27me3. However, Tazemestostat treatment neither increases expression of p21, p27, and p57 nor induces cell cycle arrest. Taken together, our findings suggest a novel mechanism by which EZH2 regulates cell cycle progression through its enzyme-independent function.

Citation Format: Tania Marlyn Colon, Byeong Hyeok Choi, Wei Dai. Histone methyltransferase-independent function of EZH2 in the cell cycle regulation [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 2310.
Abstract 2348: Contribution of the GR/LEDGFp75 axis to prostate cancer chemoresistance
Authors: Hernandez Evelyn S. Sanchez , Hernandez Greisha L. Ortiz , Ochoa Pedro T. , Gomez Christian R. , Casiano Carlos A.
Published: June 2022

Prostate cancer (PCa) is the second leading cause of cancer deaths in the U.S., disproportionally affecting African American (AA) men. Glucocorticoids (GCs) are administered to PCa patients and have been implicated in therapy resistance. This may be critical to AA men with PCa since they have elevated endogenous GCs levels compared to Caucasian American (CA) men. GCs bind to the glucocorticoid receptor (GR) to exert their actions. The mechanisms of GR-mediated chemoresistance, and its possible contribution to PCa mortality disparities are unknown. We demonstrated that GCs upregulate the chemoresistance-associated protein and transcription co-activator LEDGF/p75 in PCa cells and identified consensus GR binding sites in the promoter region of this protein. Given that both GR and LEDGF/p75 are components of the RNA polymerase II transcription complex, we hypothesized that GR transcriptionally upregulates LEDGF/p75 and then interacts with it to enhance taxane resistance in PCa cells. Pharmacological and genetic inhibition of GR in a panel of docetaxel (DTX)-sensitive and -resistant PCa cells decreased the expression of LEDGF/p75, confirming its status as a candidate GR target gene. However, silencing of LEDGF/p75 had no effects on GR expression. Immunoprecipitation studies revealed that GR and LEDGF/p75 interact in DTX-resistant PCa cells. This interaction was confirmed by confocal microscopy. Immunohistochemical analysis of GR and LEDGF/p75 expression in normal and tumor prostate tissues was performed, and the results are currently being analyzed. Our studies use a mechanistic approach to evaluate the potential contribution of the GR-LEDGF/p75 axis to PCa chemoresistance. Evaluating the co-expression of these proteins in racially diverse PCa tissues may also reveal race-related differential expression, providing insights into the potential contribution of this axis to PCa chemoresistance and mortality disparities.

Citation Format: Evelyn S. Sanchez Hernandez, Greisha L. Ortiz Hernandez, Pedro T. Ochoa, Christian R. Gomez, Carlos A. Casiano. Contribution of the GR/LEDGFp75 axis to prostate cancer chemoresistance [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 2348.
Abstract 2364: ABI1 regulates transcriptional activity of STAT3
Authors: Li Xiang , Porter Baylee , Kemraj Allysa , Bah Alaji , Kortylewski Marcin , Kotula Leszek
Published: June 2022

Background: Prostate cancer (PCa) is characterized by the complexity of oncogenic signaling and heterogeneity of transcriptional landscapes. Adaptor protein ABI1 is a tumor suppressor in PCa as evidenced by its loss or downregulation in high grade and metastatic tumors. STAT3 activation is a hallmark of high-risk prostate tumors. ABI1 loss is associated with STAT3 activation leading to transcriptional reprogramming and epithelial-mesenchymal-transition (EMT) of prostate cancer cells (Nath, Li et al. 2019 Cell Commun Signal). EMT changes involve several homeobox transcription factors. The fact that ABI1 contains a homeobox homology region (HHR) suggested the possibility that it plays a role in EMT through directly regulating transcriptional activity by DNA binding.

Methods: To examine this hypothesis we set out to analyze ABI1-DNA binding. We purified ABI1 HHR and demonstrated its in vitro interactions with different homeobox DNA-binding consensus of double-stranded DNA sequences. We created DU145 ABI1 CRISPR KO cell line and rescue ABI1 expression using retroviral transfection with either ABI1 wild type or Abi1 HHR mutants. DU145 cell lines STAT3 cellular localization was studied by Immunofluorescence staining and the STAT3 transcription activities was studied by Firefly Luciferase Assays with STAT3 specific luciferase reporter plasmid.

Results: We found that the presence of alternatively spliced Exon 4-encoded sequence, located in the C-terminus of HHR, regulates binding of ABI1 to DNA. Structural NMR studies confirmed ABI1 binding to DNA. Subsequent functional studies using DU145 CRISPR KO cell lines expressing wild type or HHR mutants of ABI1 demonstrated that HHR regulates the nuclear localization and transcriptional activity of STAT3.

Conclusion: We propose that ABI1 is a critical regulator of STAT3 activity during prostate cancer progression by the ABI1 HHR mediated DNA binding mechanism.

Funding resources: NCI R01 CA161018; R21 CA260381

Conflicts of Interest Disclosure Statement: The authors declare no conflicts of interest.

Citation Format: Xiang Li, Baylee Porter, Allysa Kemraj, Alaji Bah, Marcin Kortylewski, Leszek Kotula. ABI1 regulates transcriptional activity of STAT3 [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 2364.
Abstract 2384: Investigating Fis1 upregulation as a stress response in glioblastoma cells
Authors: Buckley Yuli , Brett-Morris Adina , Stoll Maria , Mears Jason
Published: June 2022

Dysfunctional mitochondrial dynamics contribute to many pathologies, and elevated levels of mitochondrial fission have been observed in glioblastoma (GBM). Mitochondrial fission 1 protein (Fis1) plays a minor role in basal fission, but has been shown to be more critical for pathological fission. Further, it has been shown to facilitate peripheral fission events, generally occurring after cellular exposure to stress, as a means to prepare damaged mitochondria for degradation through mitophagy. Therefore, my goal is to elucidate the role of Fis1, an integral outer membrane partner protein, in mitochondrial fission within GBM cells. I propose that Fis1 plays a protective role against therapeutic treatment in GBM. This is demonstrated through experiments showing that ionizing radiation (IR) leads to upregulation of Fis1 protein levels, while GBM cell viability is simultaneously unaffected. Further, this increase in Fis1 results in an increase in mitochondrial fission, which likely reflects an increase in mitophagy as a response to IR stress. Thus, I anticipate that GBM cells upregulate Fis1 as a protective measure and that targeting Fis1 function could have therapeutic benefit. In this regard, I have found that depletion of Fis1 alters mitochondrial morphology, and I am studying changes in mitochondrial bioenergetics to correlate ultrastructural changes with mitochondrial function. Preliminary data suggests that Fis1 knockdown in Gli36 cells leads to decreased oxidative phosphorylation and ATP production, with deficits observed specifically in Complex I and III. Concurrently, I am assessing potential synergistic effects of combined radiation and Fis1 inhibition on GBM cell viability and proliferation. To do this, I will use genetic and small molecule approaches to inhibit Fis1-dependent mitochondrial fission. Overall, these studies will demonstrate whether Fis1 drives pathological fission to promote cancer cell growth and proliferation. Additionally, the selective inhibition will evaluate Fis1 as a novel therapeutic target in GBM.

Citation Format: Yuli Buckley, Adina Brett-Morris, Maria Stoll, Jason Mears. Investigating Fis1 upregulation as a stress response in glioblastoma cells [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 2384.
Abstract 2392: Adapted tumor suspension cells rewire metabolic pathways for anchorage independent survival
Authors: Yang Young , Ju Hyun Jeong
Published: June 2022

Metastatic spread of cancer cells is the main cause of cancer-related death. As cancer cells adapt themselves in a suspended state in the blood stream before penetration and regrowth at distal tissues, understanding their survival strategy in an anchorage-independent condition is important to develop appropriate therapeutics. We have previously generated adapted suspension cells (ASCs) from parental adherent cancer cells to study the characteristics of circulating tumor cells. In this study, we explored metabolic rewiring in MDA-MB-468 ASCs to adapt to suspension growth conditions through extracellular flux analyses and various metabolic assays. We also determined the relationship between protein kinase B (AKT) activation and metabolic rewiring in ASCs using the AKT inhibitor, MK2206. ASCs reprogramed metabolism to enhance glycolysis and basal oxygen consumption rate. RNA-sequencing analysis revealed the upregulation in the genes related to glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. The changes in the metabolic program led to a remarkable dependency of ASCs on carbohydrates as an energy source for proliferation as compared to parental adherent cells (ADs). AKT activation was observed in ASCs and those generated from pancreatic and other breast cancer cells, and AKT activation inhibition in ASCs decreased glycolysis and oxygen consumption. AKT activation is an important strategy for obtaining energy through the enhancement of glycolysis in ASCs. The regulation of AKT activity and/or glycolysis may provide a new therapeutic strategy to prevent the metastatic spread of cancer cells.

Citation Format: Young Yang, Hyun Jeong Ju. Adapted tumor suspension cells rewire metabolic pathways for anchorage independent survival [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 2392.
Abstract 2408: Elevated phosphate promotes a pro-migration phenotype in lung cancer
Authors: Arnst Jamie , Beck George
Published: June 2022

Despite advances in treatment, metastasis remains a major cause of cancer-associated death in the US. Thus, identification of intrinsic and extrinsic factors that promote or inhibit tumor migration and invasion are critical. Increasingly, the dysregulation of cellular metabolism is being linked not only to tumor growth but tumor cell migration. Phosphorus (Pi) is an essential micronutrient necessary for energy metabolism, cell signaling via ATP, formation of nucleic acids and the cell membrane, and mineralization of the skeleton. Unsurprisingly, high levels of Pi are observed in tumor tissue compared to normal tissue, consistent with a demand for this metabolite in rapidly growing cells. Further, our lab has reported that high extracellular Pi increases cell growth, alters gene expression and protein translation, and promotes angiogenesis. High dietary Pi levels has also been shown to increase tumorigenesis in both lung and skin cancer models. However, it remains unknown how phosphate metabolism affects tumor migration and invasion. Here we report that high levels of Pi increases gene expression of EMT-related genes such as Twist, Vimentin, CD44, and OPN in the lung cancer cell line, A549. Further, increased Pi promotes tumor cell migration in a transwell assay and decreased Pi levels or inhibition of phosphate transport by phosphonoformic acid (PFA) impaired migration. Suggesting that high Pi can promote a pro-migration phenotype by upregulating EMT in cancer cells and by increasing directed cell migration. Ongoing research will focus on how intracellular Pi flux changes during cell invasion using a novel FRET-based phosphate sensor in vitro and the effect of Pi inhibition on metastasis in vivo. These findings provide further evidence for the role dysregulated Pi metabolism in promoting cancer progression and the potential for therapeutic intervention with broad impacts on patient outcomes.

Citation Format: Jamie Arnst, George Beck. Elevated phosphate promotes a pro-migration phenotype in lung 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 2408.
Abstract 2419: Understanding the role of DCLK1 mediated invadopodia regulation in HNSCC
Authors: Arnold Levi K. , Standing David , Ramamoorthy Prabhu , Saunders Harmony , Ly Thuc , Anant Shrikant , Thomas Sufi
Published: June 2022

Despite therapeutic advances, head and neck squamous cell carcinoma (HNSCC) is marked by high mortality, particularly with increasing stage. Locoregional invasion, an early step in metastasis, is driven by the development of finger-like processes on the tumor cell known as invadopodia. Mature, active invadopodia secrete matrix metalloproteases (MMP) that enable extension of the cell into the extracellular matrix (ECM). Kinesins haul RAB-bound vesicles containing MMPs along microtubules the length of invadopodia for secretion to the most distal aspect of the invadopodia. It is unclear how these kinesins are modulated within invadopodia. In preliminary studies on patient samples, we identified high levels of doublecortin like kinase (DCLK1) at the invasive front of HNSCC. We hypothesize DCLK1 supports the function of kinesins as a mechanism of invasion. DCKL1 was knocked down in HNSCC lines with a short hairpin RNA (shDCLK1). Invasion and migration assays were performed in shDCLK1 and shcontrol tumor cells by utilizing a transwell assay. Confocal microscopy using Leica LAS X, was performed to assess colocalization of DCLK1 and markers of mature invadopodia (cortactin, TKS5, MT1-MMP) in HNSCC lines for. . To validate the association of DCLK1 with Kif16B and RAB40b, we carried out a co-immunoprecipitation analysis. Finally, conditioned media from shDCLK1 and shControl HNSCC cells was used to assess MMP activity using gelatin zymography and MMP profiler arrays. Transwell assay demonstrates reduced movement in shDCLK1 compared to shcontrol tumor cells (p <0.0001 invasion, p <0.001 migration). Confocal microscopy shows colocalization of MMP 9 and DCLK1 to invadopodia. DCLK1 immunoprecipitation show RAB40b and Kif16b complex together. Additionally, DCLK1, RAB40b, and Kif16b colocalize to invadopodia in confocal studies. We found that DCLK1 colocalizes with markers of mature invadopodia, and that DCLK1 colocalizes to Rab40b, Kif16b, and MMP9 within invadopodia. DCLK1 and MMP9 co-localize to invadopodia as well. Cells expressing DCLK1 had increased MMP 1, and 9 (p <0.041, p <0.001) expression compared to shDCLK1 cells. Further, shDCLK1 cells have significantly attenuated MMP 1 and 9 activity as measured by gelatin zymography. These studies reveal a novel role of DCLK1 in the molecular mechanism driving invadopodia, MMP trafficking and signaling. These early studies indicate that targeting DCLK1 in HNSCC may reduce locoregional invasion and help prevent early metastatic spread.

Citation Format: Levi K. Arnold, David Standing, Prabhu Ramamoorthy, Harmony Saunders, Thuc Ly, Shrikant Anant, Sufi Thomas. Understanding the role of DCLK1 mediated invadopodia regulation in HNSCC [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 2419.
Abstract 2475: Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells
Authors: Xiao Zebin , Hopper Leslie A. , Kopp Meghan C. , McMillan Emily , Li Yue , Barrett Richard L. , Puré Ellen
Published: June 2022

The efficacy of chimeric antigen receptor (CAR) T-cell therapy in solid tumors is limited by inefficient T-cell infiltration, localization and hypofunctionality within the tumor microenvironment. Prior studies demonstrated that stromal cell targeted CAR T cells, in particular CAR T cells that target fibroblast activation protein (FAP), can effectively infiltrate, and inhibit tumor growth in mouse models of pancreatic ductal adenocarcinomas (PDAC) due to their capacity to deplete stromal cells and extracellular matrix (ECM) that otherwise present a barrier to adoptive cell therapies. However, the kinetics of tumor infiltration by FAP-CAR T cells, and the cell-cell and cell-matrix interactions that contribute to their anti-tumor activity are poorly understood. A better understanding FAP-CAR T-cell kinetics and intratumoral interactions should provide insights into the requirements for successful adoptive cell therapies in solid tumors. By combining immunostaining and real-time two-photon microscopy in tumor fragments of PDAC, we found that stromal cell targeted FAP-CAR T cells can efficiently traffic into tumors. Initially (days 1-3) FAP-CAR T cells were enriched in the stroma surrounding tumor nests but excluded from the tumor nests per se. While associated with the stroma rich region, the CAR T cells appeared activated and T cell motility was observed in loose collagen regions, but T cells migrated poorly in dense matrix areas. Aligned collagen fibers in the tumor boarder dictated the migratory trajectory of T cells and restricted them from entering tumor nests. By day 5 we observed depletion of immunosuppressive stromal cells, dissolution of the fibrillar collagen network surrounding tumor nests and infiltration of FAP-CAR T cells into tumor nests. Treatment with hyaluronidase increased the ability of T cells to infiltrate the tumor nest. Thus, FAP-CAR T cell-mediated ablation of immunosuppressive FAP+ stromal cells and disruption of the desmoplastic stroma they generate, can enhance accumulation and functionality of CAR-T cell therapy in the context of highly desmoplastic solid tumors.

Citation Format: Zebin Xiao, Leslie A. Hopper, Meghan C. Kopp, Emily McMillan, Yue Li, Richard L. Barrett, Ellen Puré. Visualization of cell to cell and cell to matrix interactions in the disruption of tumor-promoting desmoplasia by adoptive transfer of fibroblast activation protein targeted chimeric antigen receptor (CAR) T cells [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 2475.
Abstract 2544: Preoperative exercise inhibits hepatic metastasis by suppressing PMN-MDSC formation of NETs
Authors: Cheng Xiang , Zhang Hongji , Tsung Allan , Huang Hai
Published: June 2022

Introduction: Under surgical trauma, the pathological amplification and activation of neutrophils with potent immunosuppressive functions, also known as polymorphonuclear myeloid-derived suppressive cells (PMN-MDSCs), can promote metastasis by forming neutrophil extracellular traps (NETs) that are web-like structures consisting of nuclear DNA, histones, and granule proteins. Preoperative exercise (PEx) reduces postoperative complications with improved clinical outcomes in cancer patients undergoing surgeries. are. Our preliminary data show that PEx suppresses liver ischemia/reperfusion (I/R)-induced colorectal cancer (CRC) liver metastasis. Our recent studies have shown I/R-induced NETs promote liver CRC metastasis by capturing tumor cells and preventing infiltration and cytotoxicity of CD8+ T cells in tumor microenvironment (TME). We hypothesize that PEx remodels the TME by suppressing PMN-MDSCs formation of NETs.

Methods: Male mice were subjected to exercise for 4 weeks, before MC38 colorectal cancer cells were injected through portal vein, and then subjected to 70% partial liver warm I/R as surgical stress. The single-cell RNA sequencing (scRNA-seq) was performed on CD45+leukocytes from TME 3 weeks after surgery. Immunofluorescence staining and Western Blot were used to determine NETs formation in the TME. Flow cytometry was used to determine the infiltration of PMN-MDSCs and CD8+ T lymphocytes in the tumors.

Results: Our scRNA-seq data reveal that PEx leads to a remarkable transcriptomic shift in most immune cells types, such as PMN-MDSCs, M-MDSCs, T cells, Kupffer cells, B cells, NK and NKT cells, in the TME. PEx decreases the percentages of PMN-MDSCs in the TME and inhibits theNETs formation by PMN-MDSCs. scRNA-seq shows a significant increase of the chemokines CXCL10 in PEx-trained PMN-MDSCs obtained 3 weeks after I/R-induced hepatic metastasis, compared with sedentary controls. In addition, PEx increases the number of CD8+ T lymphocytes infiltrating the TME.

Summary: Our study shows that PEx downregulates NETs formation and increases the CXCL10 expression in PMN-MDSCs, thus promoting the recruitment more cytotoxic CD8 T lymphocytes to suppress tumor metastasis.

Citation Format: Xiang Cheng, Hongji Zhang, Allan Tsung, Hai Huang. Preoperative exercise inhibits hepatic metastasis by suppressing PMN-MDSC formation of NETs [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 2544.
Abstract 2568: ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification
Authors: Liang jiaqi , Cheng Dai , Guo Yali , zhang xiaobin , Liu Xipan , Fu Jia , Ding Qiang , Xiong Wen , Li Fang , Chen Yaoyu , Sheng Qing
Published: June 2022

Cyclin E1 amplification is prevalent in cancers with high unmet medical need such as high grade serous ovarian cancer, stomach cancer and esophageal cancer. Cancer cell lines with high expression level of cyclin E1 gene exhibited profound sensitivity to CDK2 gene depletion, suggesting CDK2 selective inhibitors have the potential to treat patients harboring Cyclin E1 alterations. Here we report the development and preclinical characterization of ARTS-021, an orally bioavailable small molecule CDK2 selective inhibitor. In enzymatic assay against a panel of different CDKs, ARTS-021 potently inhibits the activity of CDK2/CyclinE1 complex with 50% inhibitory concentration (IC50) in the sub-nanomolar range, displaying superior selectivity against other CDKs (CDK1,4,6,7,9). Very low hit rate (S(10)=0.022, at 1uM) is observed when ARTS-021 is assessed in whole Kinome profiling, further revealing the selectivity of this compound. ARTS-021 also demonstrates potent CDK2 inhibition and selectivity against other CDK family members (CDK1,4,6,9) in a series of cellular assays. Consistent with genetic CDK2 dependence data, cell lines with CCNE1 amplification are particularly sensitive to ARTS-021. Double nanomolar ARTS-021 inhibits Rb phosphorylation and blocks G1/S transition, leading to cell growth arrest specifically in CCNE1 amplified cell lines. This CDK2 dependent anti-tumor activity is further validated in vivo, where daily ARTS-021 administration leads to tumor stasis in CCNE1 amplified but not wild type xenograft models. Taken together, these data demonstrate ARTS-021 as a promising CDK2 selective inhibitor with strong potential towards the development of effective therapies for CCNE1 altered cancer.

Citation Format: jiaqi Liang, Dai Cheng, Yali Guo, xiaobin zhang, Xipan Liu, Jia Fu, Qiang Ding, Wen Xiong, Fang Li, Yaoyu Chen, Qing Sheng. ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification [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 2568.
Abstract 257: Normalization of tumor stroma improves the anti-tumor activity of anti-PD-L1 peptides in pancreatic cancer
Authors: Lin Chien-Yu , Cheng Kun
Published: June 2022

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the abundant fibrotic stroma surrounding malignant epithelial cells. The tumor stroma is composed of extracellular matrix proteins, myofibroblasts, stellate cells, and immune cells, and comprises up to 80% of the tumor mass. The stroma promotes the progression and metastasis of tumors and the development of chemoresistance. However, the strategies of completed depletion of the stroma resulted in a more aggressive tumor and a poor survival rate in clinical trials. We discovered that silencing the RNA binding protein αCP2, which is encoded by the poly(rC)-binding protein 2 (PCBP2) gene, could destabilize the type I collagen mRNA in human pancreatic stellate cells and mouse NIH/3T3 fibroblasts, leading to the normalization of the tumor stroma. We recently discovered an anti-PD-L1 peptide that effectively blocks the PD1/PD-L1 pathway. We hypothesize that silencing αCP2 modulates the PDAC stroma, thus improving the therapeutic efficacy of immunotherapy. Therefore, the objective of this study is to develop a combination therapy strategy to treat PDAC with PCBP2 siRNA nanoparticles and the anti-PD-L1 peptide. A mixture of 1×106 PANC02 tumor cells and NIH/313 fibroblast cells was subcutaneously implanted into C57BL/6 mice to establish the desmoplastic PDAC model. The mice were treated with saline, PCBP2 siRNA nanoparticles, the anti-PD-L1 peptide, and PCBP2 siRNA nanoparticles plus the anti-PD-L1 peptide. The results showed that the combination of PCBP2 siRNA nanoparticles with the anti-PD-L1 peptide reduced tumor growth. PCBP2 siRNA decreased type I collagen expression in the tumor. The normalized stroma showed increased infiltration of effective immune cells. This approach provides a new strategy to improve the therapeutic efficacy of immunotherapy in PDAC by normalizing the tumor stroma with the PCBP2 siRNA nanoparticle.

Citation Format: Chien-Yu Lin, Kun Cheng. Normalization of tumor stroma improves the anti-tumor activity of anti-PD-L1 peptides in pancreatic 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 257.
Abstract 2630: Heterogeneity inducible tumor stem cells relate distant metastasis and the treatment resistance
Authors: Miyoshi Norikatsu , Fujino Shiki , Sekido Yuki , Hata Tsuyoshi , Ogino Takayuki , Takahashi Hidekazu , Uemura Mamoru , Yamamoto HIrofumi , Doki Yuichiro , Eguchi Hidetoshi
Published: June 2022

Regarding metastatic potential of cancer cells and the refractory status, regulatory and epigenetic factors underlie the cancer treatment resistance by inducing cellular heterogeneity. Circulating tumor cells (CTCs) and their stemness are critical for metastases formation associated with poor survival rates. In colorectal cancer (CRC), several stem-cell markers, such as CD44v9 and LGR5, the positive cells cause intertumoral heterogeneity and promote refractory potential. However, the mastermind of the cells which is critical for the treatment resistance remains unknown. Here, we aimed to identify the mechanisms of the treatment resistance of the CRC by focusing on typical stem cells. We selected several stem cell-related genes such as pluripotency related genes, based on the expression profile analysis of CRC patients along with drug susceptibility assays using patient-derived organoids. Despite the small number of cancer stem cells, the single cell produced heterogeneous cell populations in vitro and in vivo, expressing various differentiation and stem biomarkers, and confirmed by the knockdown analysis. Single-cell trajectory analysis suggested that heterogeneous populations including cancer stem and some persister cells were derived from the stem cells following administration of anti-cancer drugs. Furthermore, the stem cells formed heterogeneous liver metastases at a high rate, and these unique cells were observed in the bloodstream of CRC patients. This present study unveils a mechanism of CRC treatment resistance and highlights the potential of stem marker-expressing cells as CTCs.

Citation Format: Norikatsu Miyoshi, Shiki Fujino, Yuki Sekido, Tsuyoshi Hata, Takayuki Ogino, Hidekazu Takahashi, Mamoru Uemura, HIrofumi Yamamoto, Yuichiro Doki, Hidetoshi Eguchi. Heterogeneity inducible tumor stem cells relate distant metastasis and the treatment resistance [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 2630.
Abstract 2680: PCAI-induced phosphorylation of MAP kinase pathway enzymes is associated with cell death of black American lung adenocarcinoma cell line NCI-H23
Authors: Gregory Matthew D. , Tawfeeq Nada , Lazarte Jassy M. , Jin Yonghao , Puente Pablo , Belony Nadine , Offringa Ite A. , Huang Yong , Lamango Nazarius S.
Published: June 2022

Lung cancer is a leading cause of cancer-related mortality accounting for 23% of cancer deaths in the US. Non-small cell lung cancer (NSCLCs) comprises 84% of all lung cancer cases, and of these, 40% are lung adenocarcinoma (LUAD). Moreover, black Americans are underrepresented in scientific clinical trials but have a lung cancer incidence rate that is 37% higher than their white counterparts. To address this disparity and lack of effective treatment options, we determined the potency of polyisoprenylated cysteinyl amide inhibitors (PCAIs) on cell viability and its effects on KRAS and its downstream mediators in NCI-H23, a black American cell line that harbors a KRAS mutation. Among the 17 PCAIs that were tested, compound NSL-YHJ-2-27 showed the most potency with an EC50 of 2.7 µM. Western blot analysis was performed to determine the effect of the PCAIs on the levels of Mitogen-Activated Pathway Kinase (MAPK)-related proteins. After 48 h of exposure to 5 μM of NSL-YHJ-2-27, we observed increases of 150, 64.5, and 77% in phosphorylation levels of ERK1/2, MEK1/2, and 90RSK. While the particular 90RSK enzymes whose phosphorylation is/are elevated are not known at this time, 90RSK 3 and 4 are known to be proapoptotic. These findings suggest that the PCAIs may be used to target cancers with KRAS mutations that currently lack effective therapies. In the future, we will use a collection of isogenic cell lines to examine the potency of the PCAIs in LUAD driven by other RAS and related monomeric G-proteins, epidermal growth factors, and their receptors as well as RAS-GAPs. Therapies emanating from these studies have the potential to contribute to mitigating the lung cancer therapeutic inequities.

Keywords: Polyisoprenylated Cysteinyl Amide Inhibitors (PCAIs), Mitogen Activated Pathway Kinases (MAPK), KRAS, Lung Adenocarcinoma (LUAD), Non-small cell lung cancer (NSCLCs)

Citation Format: Matthew D. Gregory, Nada Tawfeeq, Jassy M. Lazarte, Yonghao Jin, Pablo Puente, Nadine Belony, Ite A. Offringa, Yong Huang, Nazarius S. Lamango. PCAI-induced phosphorylation of MAP kinase pathway enzymes is associated with cell death of black American lung adenocarcinoma cell line NCI-H23 [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 2680.
Abstract 2687: The estrogenic activities of endocrine disruptors alter the extracellular matrix and tissue stiffness in the mammary gland
Authors: Wormsbaecher Clarissa , Hindman Andrea R. , Avendano Alex , Cortes-Medina Marcos , Song Jonathan W. , Burd Craig J.
Published: June 2022

In utero exposure to estrogenic endocrine disrupting compounds (EDCs) increases a woman’s lifetime risk of breast cancer. Similarly, mice exposed in utero to the estrogenic EDC bisphenol A (BPA) have increased susceptibility to mammary gland tumors. It is unclear which BPA-induced alterations predispose the mammary gland to cancer transformation. There is a critical need to understand the mechanisms that drive increased cancer risk in order to assess the impact of BPA and BPA alternatives that retain estrogenic activity. We have utilized in utero BPA exposure as a model system for in utero estrogenic endocrine disruption to study the long-term consequences to the mouse mammary stroma. We found that BPA exposed fibroblasts showed significant transcriptional deregulation, with the extracellular matrix being the most altered cellular component and multiple collagen genes being more highly expressed. The fibroblasts from the BPA exposed mice decreased fluid permeability of the extracellular matrix, indicative of an increased density in the extracellular matrix. Also, in utero BPA exposure increased mammary gland stiffness. Changes to breast density, stiffness, and collagen deposition are all associated with breast cancer risk. Further, we test BPA alternative compounds with varying affinities for the estrogen receptor in our in utero model to assess the phenotypes in the mouse mammary stroma which are associated with breast cancer risk. Additionally, we use a mesenchymal estrogen receptor alpha (ERα) knockout mouse model to dissect the in utero cellular target of EDCs.

Citation Format: Clarissa Wormsbaecher, Andrea R. Hindman, Alex Avendano, Marcos Cortes-Medina, Jonathan W. Song, Craig J. Burd. The estrogenic activities of endocrine disruptors alter the extracellular matrix and tissue stiffness in the mammary gland [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 2687.
Abstract 2691: Expression pattern and prognostic significance of HER family members and EGFRvIII in pancreatic cancer
Authors: Khan Tanzeel , Khelwatty Said A. , Bagwan Izhar , Seddon Alan , Dalgleish Angus , Mudan Satvinder , Modjtahedi Helmout
Published: June 2022

Pancreatic cancer is one of the most aggressive, heterogeneous, and fatal types of human cancers. There is urgent need for discovery of biomarkers of prognostic significance, predictive value and as well as therapeutic targets in patients with pancreatic cancer. In this study, we examined the relative expression and prognostic significance of all HER family members and the type III EGFR mutant (EGFRvIII) in patients with pancreatic ductal adenocarcinoma (PDC). The expression levels of wild-type EGFR (wtEGFR), HER2, HER3, HER4 and EGFRvIII were determined in tumor specimens from 43 patients by immunohistochemistry using antibodies DAK-H1-WT, Neu 3B5, SP71, HFR1 and NBP2-50599 respectively. The staining was scored based on the percentage of positive tumor cells, the intensity (i.e. strong 3+, moderate 2+, weak 1+) and the cellular location of immunostaining (i.e. membranous, cytoplasmic or nuclear). The association between the expression level of the biomarkers and patient overall survival was evaluated using Chi-squared, Kaplan-Meier survival curves and log rank test. At cut-off value of above 5%, we found weak to moderate expression of wtEGFR, HER2, HER3, HER4 and EGFRvIII in 4.7%, 14%, 14%, 20.9% and 2.3% of the cases examined respectively. The cellular location of wtEGFR and HER2 was found to be predominantly cytoplasmic, HER-3 membranous and cytoplasmic whereas HER4 expression was found to be mainly nuclear. There was no statistically significant association between the expression level of these markers and patient overall survival. The expression levels of the above biomarkers were also evaluated in tissue microarrays (TMAs) from 40 cases of pancreatic ductal adenocarcinoma. Interestingly, at the same cut-off value, 62.5%, 75%, 0%, 55% and 5% of the TMAs were positive for wtEGFR, HER2, HER3, HER4 and EGFRvIII respectively. Overall, we found that the expression pattern of HER family members was heterogenous, and with EGFRvIII expression being very low in both whole tumor sections and TMAs from patients with pancreatic cancer. Taken together, our results provide support for more detailed studies of co-expression of all HER family members in a larger group of pancreatic cancer patients. Such studies should help to unravel their potential as targets for therapeutic interventions as well as their prognostic significance and predictive value for response to therapy.

Citation Format: Tanzeel Khan, Said A. Khelwatty, Izhar Bagwan, Alan Seddon, Angus Dalgleish, Satvinder Mudan, Helmout Modjtahedi. Expression pattern and prognostic significance of HER family members and EGFRvIII in pancreatic 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 2691.