bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1745: Exploiting vulnerabilities to nanoparticle induced lipid peroxidation and proteotoxicity to treat drug resistant lung cancer
Authors: Snyder Christina , Rohde Monica , Singh Ravi N.
Published: June 2022

Protein quality control plays an essential role in cell survival. Clinically translated strategies to target protein quality control in cancer show some efficacy, but off-target effects are severe. We are investigating a new approach using nanomaterials to induce lipid peroxidation to trigger a cascade of protein oxidation, protein misfolding, and decreased protein degradative capacity in cancer cells without affecting normal cells. Our studies show that silver nanoparticles (AgNPs) cause dramatic increases in lipid peroxidation, 4-hydroxynonenal adducts (4-HNE), protein oxidation, protein aggregation, and proteotoxic stress signaling in a mesenchymal-like subset of non-small cell lung (NSCLC) and other cancers at doses that do not affect normal cells in vitro or vivo. Notably, AgNP sensitive NSCLCs are inherently resistant to clinically relevant therapies including epidermal growth factor receptor tyrosine kinase inhibitors ((EGFR-TKIs) e.g. erlotinib, gefitinib, olfatinib) and cisplatin. We identified two defining characteristics causing some cancers to be sensitive to AgNPs. The first is enrichment of cell membranes in polyunsaturated fatty acids (PUFAs), especially arachidonic (AA) and adrenic acid (AdA), which are prone to oxidation. The second is high-level synthesis and secretion of extracellular matrix (ECM), especially fibronectin and collagen, which places AgNP sensitive cancers under substantial baseline endoplasmic reticulum stress, making them susceptible to agents that increase proteotoxicity. Mechanistically, we find that in PUFA-enriched cancers, signaling from ECM sustains the AgNP sensitive phenotype by stimulating Akt activity and stabilizing cytoplasmic β-catenin. Transcriptionally active β-catenin increases expression of fibronectin and collagen, resulting in a positive feedback loop locking cells into the AgNP sensitive phenotype. Use of AgNPs to targeting vulnerabilities associated with ECM synthesis and signaling downstream of β-catenin is a novel approach to overcoming multiple EGFR-TKI resistance mechanisms. Ultimately, our goal is to elucidate the precise mechanism by which AgNPs overcome resistance to clinically available EGFR-TKIs. Identification of the specific drivers of sensitivity to AgNPs (and potentially other metal nanoparticles) will guide development of therapeutic metal nanoparticles, enable selection of cancer patients who will benefit most or are at greatest risk to nanomaterial exposure, and will spur innovation of other treatments that exploit the vulnerabilities we identify.

Citation Format: Christina Snyder, Monica Rohde, Ravi N. Singh. Exploiting vulnerabilities to nanoparticle induced lipid peroxidation and proteotoxicity to treat drug resistant 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 1745.
Abstract 1787: Statin therapy enhances the efficacy of HER2 directed antibody-drug conjugates in breast cancer
Authors: Liu Bo , Drago Joshua Z. , Rao Yi , Pereira Patricia R. , Safonov Anton , Marra Antonio , Ahmed Mehnaj S. , Modi Shanu , Reis-Filho Jorge S. , Montemurro Filippo , Razavi Pedram , Lewis Jason S. , Chandarlapaty Sarat
Published: June 2022

Antibody-drug conjugates (ADCs) targeting human epidermal growth factor receptor 2 (HER2) are a widely successful strategy for the treatment of HER2-positive breast cancer. Despite the demonstrated efficacy, intrinsic and acquired resistance to anti-HER2 ADCs remains a major challenge. The activity of ADCs is dependent upon the internalization of the HER2-ADC complex into specific subcellular compartments permissive for release of the chemotherapeutic payload. Previous studies have demonstrated that statins, a commonly used cholesterol-lowering medication, could increase plasma membrane-bound HER2 and improve trastuzumab efficacy in HER2-positive gastric cancer. In this study, we sought to characterize the impact of statins on the efficacy of HER2 ADCs. We performed in vitro internalization assays with trastuzumab emtansine (T-DM1) labeled with a pH-sensitive pHrodo fluorogenic dye to monitor T-DM1 entering lysosome whereupon payload DM1 is released, and found that combined treatment of T-DM1 and lovastatin potently enhanced T-DM1 internalization of T-DM1 into lysosome in HER2-amplified and HER2-low models. Consistent with the internalization assays, lovastatin increased cell death caused by T-DM1 and sensitized the HER2-low ZR75-1 cells to T-DM1 treatment in vitro. Using HER2-positive xenograft models, we found orally administrated lovastatin promoted T-DM1 uptake in tumors and enhanced T-DM1 efficacy in vivo. To investigate whether these results might be observed in the clinic, we conducted retrospective analyses on a cohort of 164 HER2 positive metastatic breast cancer patients treated at MSKCC who received T-DM1. Among these patients, 21 (12.8%) were taking statins concurrently with T-DM1, and the median progression-free survival in the patients who received statins was 14 months (95% confidence interval, 3.5-24 months) compared to 5.4 months (95% confidence interval, 3.9-7.0 months) in those who had no record of statin use (p=0.1). Overall, our findings demonstrate that statins potentiate the susceptibility of breast cancer cells to anti-HER2 ADCs by modulating HER2 membrane dynamics and HER2-ADC internalization, suggesting statin as a rational therapeutic partner for anti-HER2 ADC in HER2-positive breast cancer, especially those with relatively low HER2 expression.

Citation Format: Bo Liu, Joshua Z. Drago, Yi Rao, Patricia R. Pereira, Anton Safonov, Antonio Marra, Mehnaj S. Ahmed, Shanu Modi, Jorge S. Reis-Filho, Filippo Montemurro, Pedram Razavi, Jason S. Lewis, Sarat Chandarlapaty. Statin therapy enhances the efficacy of HER2 directed antibody-drug conjugates 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 1787.
Abstract 1809: Engineered destabilized ARE on the 3UTR of MYC degrades oncogenic c-MYC transcript and protein and induces apoptosis in multiple cancer types
Authors: Awah Chidiebere , Dong Fu , Glemaud Yana , Levine Fayola , Weiser Daniel , Ogunwobi Olorunseun
Published: June 2022

The c-MYC oncogene is overexpressed in more than 18 human cancers, including triple negative breast cancer (TNBC), prostate cancer, and osteosarcoma, yet there is no clinically approved drug that targets this major oncogenic driver. We discovered that the 3’UTR of c-MYC in many cancers is enriched with stabilizing poly U tract sequences that can be destabilized with our novel engineered stabilizing AU Rich Elements (ARE), leading to degradation of c-MYC. We hypothesize that c-MYC destabilizing constructs will degrade c-MYC transcript and proteins across a range of cancers that overexpress this oncogene. We used heterogeneous panels of TNBC, prostate cancer, and osteosarcoma cell lines that are reflective of the heterogeneity of human cancers. We introduced our c-MYC destabilizing construct and assessed cell viability. We then performed immunofluorescence and western blot for assessment of c-MYC protein expression. Vector constructs were used as controls. Introduction of destabilizing elements achieved the degradation of both c-MYC transcript and proteins within four days, as well as a subsequent loss in cell viability. We observed that treated cells disintegrated from their nucleus, leading to increased active caspase 3/7 and cell death. Taken together, we have developed a novel technology that reliably targets and degrades the c-MYC oncogene across a range of highly aggressive and difficult to treat cancers

Citation Format: Chidiebere Awah, Fu Dong, Yana Glemaud, Fayola Levine, Daniel Weiser, Olorunseun Ogunwobi. Engineered destabilized ARE on the 3UTR of MYC degrades oncogenic c-MYC transcript and protein and induces apoptosis in multiple cancer types [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 1809.
Abstract 1821: SPTBN1 Is a potential target to prevent progression of steatohepatitis to hepatocellular carcinoma
Authors: Yang Xiaochun , Rao Shuyun , Xiang Xiyan , Ohshiro Kazufumi , Latham Patricia , Shetty Kirti , Mishra Lopa
Published: June 2022

Background: Nonalcoholic steatohepatitis (NASH) is a risk factor for the development of hepatocellular carcinoma (HCC). β-spectrin, encoded by SPTBN1, is associated with HCC. The goal of this study was to evaluate the hepatocyte-specific roles of SPTBN1 in NASH and HCC using mouse models.

Methods: We investigated the effect of knocking out Sptbn1 specifically in the liver in mice (LSKO mice). We examined the effect of diets that promote NASH—a high-fat diet (HFD) or Western diet (WD)— and the outcomes of HCC induced by diethylnitrosamine (DEN). Based on the phenotypes of these mice, we examined the therapeutic effects of knocking down Sptbn1 using siRNA.

Results: We found LSKO mice were protected from HFD-induced obesity and NASH. Control mice administered siRNA targeting Sptbn1 were protected from developing NASH when placed on a HFD, and NASH phenotypes triggered by WD were reduced by administration of siRNA targeting Sptbn1. LSKO mice appeared to be protected from HFD-induced HCC: One of four HFD control mice developed liver tumors, but none of the three LSKO mice did. HFD-induced HCC is rare in mice, therefore we evaluated DEN-induced HCC. LSKO mice subjected to DEN with or without WD had less severe cancer than did wild-type mice. Compared to the HCC in the control mice, the number of liver tumors in the LSKO mice was lower, the size of the tumors was smaller, and the tumors were less proliferative based on KI67 staining intensity. Furthermore, overall liver pathology was reduced.

Conclusions: These data suggest that SPTBN1 is a potential target for therapeutic intervention to treat NASH and prevent progression to HCC. Because other injected siRNA therapies are approved to treat liver diseases, a siRNA-based therapy targeting SPTBN1 could translate into clinical trials.

Citation Format: Xiaochun Yang, Shuyun Rao, Xiyan Xiang, Kazufumi Ohshiro, Patricia Latham, Kirti Shetty, Lopa Mishra. SPTBN1 Is a potential target to prevent progression of steatohepatitis to hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1821.
Abstract 1824: EZH2 inhibition induces blast differentiation in acute myeloid leukemia
Authors: Fobare Sydney , Elgamal Ola A. , Stahl Emily H. , Mehmood Abeera , Truxall Jean , Johnson Mariah L. , Abdul-Aziz Amina , Byrd John C. , Hertlein Erin
Published: June 2022

Enhancer of zeste homolog 2 (EZH2) is a part of the polycomb repressive complex and catalyzes the trimethylation of lysine 27 on histone H3 (H3K27me3). EZH2 inhibition has a complex role in the pathogenesis of acute myeloid leukemia (AML), in that it has been shown to be either a tumor suppressor or an oncogene depending on the stage of AML development and the genes that EZH2 is regulating during each stage. Unlike follicular and diffuse large B-cell lymphoma where EZH2 mutations result in gain of function, EZH2 mutations are typically loss of function in myeloid diseases. However, we hypothesized that in AML patients without EZH2 mutations, loss of EZH2 function may produce a phenotype that would allow for therapeutic targeting without influencing normal hematopoiesis.

We used EPZ011989 (EPZ), an EZH2 inhibitor tool compound, to inhibit H3K27me3 in our studies. We started by treating the MOLM-13 AML cell line with EPZ and confirmed a decrease in H3K27me3. This reduction in H3K27me3 resulted in a slight decrease in metabolic activity via MTS assays as well as decreased colony formation in methocult. These studies were followed up with EPZ inhibition in primary AML samples in vitro. We found that EZH2 inhibition resulted in decreased self-renewal of primary AML samples but not of CD34+ bone marrow cells from normal donors. Furthermore, we found that after 7-day treatment with EPZ, primary AML samples undergo moderate differentiation as suggested by an increase in CD11b surface expression via flow cytometry. These results are further supported by the morphological changes seen after 14-days of EPZ treatment in vitro. Based on these results, we hypothesize that EZH2 inhibition in primary AML samples promotes the differentiation of AML blasts. Furthermore, our preliminary data suggests that daily treatment with 150 mg/kg of EPZ results in a survival advantage and reduced disease burden in the MOLM-13-luciferase murine xenograft model.

Despite loss of function EZH2 mutations portending poor outcomes in myeloid malignancies, we demonstrate that pharmacologic EZH2 inhibition reduces AML blast stemness and promotes differentiation into mature myeloid cells. In contrast, no change in normal CD34+ stem cells occurs with EZH2 inhibition, offering the opportunity to selectively target myeloid leukemia.

Citation Format: Sydney Fobare, Ola A. Elgamal, Emily H. Stahl, Abeera Mehmood, Jean Truxall, Mariah L. Johnson, Amina Abdul-Aziz, John C. Byrd, Erin Hertlein. EZH2 inhibition induces blast differentiation in acute myeloid leukemia [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 1824.
Abstract 1825: Romidepsin therapy results in p53 mediated apoptosis in hepatoblastoma
Authors: Espinoza Andres F. , Whitlock Richard , Srivastava Rohit , Patel Roma , Govindu Sai , Shah Aayushi , Larson Samuel , Woodfield Sarah , Vasudevan Sanjeev
Published: June 2022

Purpose: Relapsed and treatment refractory hepatoblastoma (HB) has a survival rate less than 50% due to limited treatment options. We have previously shown that HB and treatment refractory HB has upregulation of class I histone deacetylase (HDAC) genes (HDAC1-4) through microarray, confirmed by RT-PCR. The highest consistent upregulation noted was with HDAC 2 and 3. Given this upregulation, we tested established and treatment refractory HB cell lines with HDAC 2/3 inhibitor, Romidepsin. We sought to characterize Romidepsin efficacy and mechanism of action.

Methods: Romidepsin was tested with cytotoxic assays (MTT). Tumor derived cell lines were treated with Romidepsin and evaluated for gene expression of p53 pathway via qRT-PCR. Immunoblotting assays were used to assess changes in expression of Acetyl-p53 at time points of 0, 4, 8, and 24 hours after 0.01 µM concentrated Romidepsin treatment. Evaluation of the specific cell death pathway involved was performed via cell viability assays using 0.01 µM concentration of Romidepsin in combination with cell death reversal agents including Z-VAD for apoptosis, 3-Methyladenine for autophagy, Necrostatin-1 for necroptosis, and Ferrostatin-1 for ferroptosis at 48 hours.

Results: Romidepsin (IC50 of 0.001-0.02 µM) showed strong in vitro effects for viability. HB cell lines treated with HDAC inhibitor had increased p21 and PUMA expression on RT-PCR, most notably at the 24-hour mark (p = < 0.05). Acetyl p-53 was noted to increase in HUH-6 and Hep G2 throughout the 24 hours of treatment on western blots, while Hep T1 was noted to have no changes in expression. All cell lines showed the highest level of cell death reversal with ZVAD in cell viability assay after Romidepsin treatment. Hep T1 showed the highest cell death reversal with Romidepsin: relative cell viability of 52% (±5%) versus 95% (±2%) in ZVAD/Romidepsin.

Conclusions: Romidepsin demonstrates strong in vitro efficacy and suggest HDAC 2/3 inhibition may act through p53 mediated mechanism, notably acetylated p53. Cell death via apoptosis is further supported by cell death reversal with ZVAD/Romidepsin treatment in HB cell lines.

Citation Format: Andres F. Espinoza, Richard Whitlock, Rohit Srivastava, Roma Patel, Sai Govindu, Aayushi Shah, Samuel Larson, Sarah Woodfield, Sanjeev Vasudevan. Romidepsin therapy results in p53 mediated apoptosis in hepatoblastoma [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 1825.
Abstract 1831: 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR
Authors: Haw Jason L. , Moore Elizabeth M. , McKinney Kimberly , Dykema Karl , Nagulapally Abhinav , Steuerwald Nury , Sholler Giselle S.
Published: June 2022

Purpose: The study aims to determine the activity of the blood brain barrier permeable glycogen synthase kinase 3B (GSK3B) inhibitor, 9-ING-41, in pediatric brain tumors: embryonal tumor with multilayered rosettes (ETMR) and atypical rhabdoid tumor (ATRT).

Methods: ETMR cell line, BT183, was grown in ultralow attachment flasks and ATRT cell lines: ATRT-7316-2187, ATRT-787197, and CHLA02 were grown in suspension flasks. Cells were treated with 9-ING-41 at dose range (0-20µM). After 24 and 48 hours of 9-ING-41 cells were analyzed by western blot. After 72 hours of 9-ING-41 cell viability was assessed with Cell Titer Glo 2.0 and IC50s were analyzed by GraphPad. RNA seq was performed on cells after 12, 24, and 48 hours of 9-ING-41 treatment. Total RNA was isolated with miRNeasy Mini Kit. RNA integrity was evaluated by an Agilent 2100 Bioanalyzer profile. rRNA was selectively depleted by employing RiboZero Globin. Next generation sequencing libraries were prepared using an Illumina TruSeq Stranded Total RNA kits. Read mapping was performed using STAR in BaseSpace. DESeq2 was used for normalization and differential expression analysis. Ingenuity Pathway Analysis was performed.

Results: The IC50s of 9-ING-41 in the cell lines BT183, CHLA02-ATRT, ATRT-787197, and ATRT-2187 are 145.5nM, 481.2nM, 503.1nM, and 528.3nM, respectively. In BT183 cells treated with 150nM 9-ING-41 for 48 hours, XIAP decreased significantly by 55% and cleaved caspase 3 increased significantly by 29-fold, quantified by densitometry. In addition, western blot showed a decrease in LIN28A and an increase in TP53 protein levels. RNA-seq data show an increase in the p53 signaling pathway in the ETMR BT183 cell line with z-scores of 2.53 and 3 (p=3.73x10-13 and 2.35x10-14) after 12 and 24 hours of 150nM 9-ING-41, respectively. In ATRT-787197 cells p53 signaling increased with z-scores of 1.0 and 2.236 (p=2.62x10-8 and 2.89x10-5) after 24 and 48 hours of 400nM 9-ING-41, respectively. RNA seq data also show the ability of 9-ING-41 to significantly increase the expression of CDK inhibitor, CDKN1A, and pro-apoptotic genes BBC3, DRAM1, and BAX in ETMR and ATRT while significantly decreasing stemness associated genes: SMO and GLI1 in ETMR.

Conclusions: 9-ING-41 decreases ETMR and ATRT cell viability at clinically relevant concentrations and these doses increase the p53 pathway and induce apoptosis while reducing the expression of stemness associated genes.

Citation Format: Jason L. Haw, Elizabeth M. Moore, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Nury Steuerwald, Giselle S. Sholler. 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR [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 1831.
Abstract 1842: Drug repurposing screen reveals glioblastoma cell line susceptibility to statins
Authors: Harwood Dylan S. , Michaelsen Signe R. , Kristensen Bjarne W.
Published: June 2022

Background: The standard therapy for glioblastoma patients is tumor resection followed by radiotherapy and temozolomide chemotherapy. Although glioblastoma has been extensively molecularly profiled along with other cancers, this knowledge has not yet been translated into improved survival outcomes. We used a bioinformatics approach to identify potential novel therapeutic strategies for glioblastoma. Objectives: Comprehensive online datasets which have assessed up to 1376 cancer cell lines in multiple ways were interrogated to identify potential drug candidates for glioblastoma.

Methods: Datasets included were from the cancer cell line encyclopedia (mRNA expression), the Achillies project (cell viability following Crispr-Cas9 knockout) and PRISM (drug treatment). A t-test comparing cell viability of glioblastoma cell lines versus other cancers was used to identify potential drug candidates, followed by the use of multiple statistical tools to investigate potential mechanism of action and status of biomarkers.

Results: Fluvastatin and pitavastatin were amongst the drugs with the most significant effects against glioblastoma cell lines while also being FDA approved. These effects were found in both glioblastoma cells and other cancer types with a mesenchymal-like expression phenotype. The anti-cancer properties of statins have previously been attributed to the inhibition of HMG-Coa reductase. Here, we found their effects correlated with the gene knockout of UBIAD1, which participates in non-mitochondrial ubiquinone and menaquinone-4 synthesis. We tested the effects of statins on patient-derived glioblastoma cell lines with a mesenchymal (n = 2) and non-mesenchymal phenotype (n=2). Mesenchymal-like glioblastoma cells lines were found to be particularly susceptible to multiple statins.

Conclusion: Statins appeared to be especially effective against glioblastoma lines and the effect could be linked to the direct or indirect inhibition of UBIAD1. In vitro validation of this finding is ongoing.

Citation Format: Dylan S. Harwood, Signe R. Michaelsen, Bjarne W. Kristensen. Drug repurposing screen reveals glioblastoma cell line susceptibility to statins [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 1842.
Abstract 1852: G protein-coupled estrogen receptor-1 agonist LNS8801 induces mitotic arrest and cell death in uveal melanoma cells
Authors: Ambrosini Grazia , Natale Christopher A. , Schwartz Gary K.
Published: June 2022

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. Nearly 50% of the patients develop hepatic metastasis, and when this occurs the 1-year survival rate is 12 months. While the primary tumor can be treated with radiotherapy and surgery, current treatments for metastatic disease have shown limited clinical activity in UM patients and there is an urgent need for new effective therapies. Recent reports have shown that male patients have more metastases than female patients, and the time from the diagnosis until development of metastases was shorter, suggesting a potential role of female hormones in disease progression. The G-protein-coupled estrogen receptor-1 (GPER) has distinct functions from those of the classic estrogen receptors ERα/β and its activation with a specific agonist has tumor-suppressive roles in several cancers. Here, we demonstrate that the novel clinical GPER agonist, LNS8801 (Linnaeus Therapeutics), has potent anticancer activities in UM cells including inhibition of cell migration and cell proliferation. LNS8801 treatment induced a G2/M cell cycle arrest due to disruption of tubulin polymerization and defective mitotic spindle formation. There was a time-dependent increase of mitotic proteins, such as phosphorylated aurora-A, aurora-B and histone-3, while c-Myc and phospho-Rb were downregulated. The drug treatment also induced significant cell death, as detected by caspase 3/7 activation and PARP cleavage. Finally, LNS8801 inhibited tumor growth in a UM xenograft model in vivo. In conclusion, our studies suggest that GPER agonists may be a novel treatment for uveal melanoma, and clinical trials with the GPER agonist LNS8801 are warranted and currently underway.

Citation Format: Grazia Ambrosini, Christopher A. Natale, Gary K. Schwartz. G protein-coupled estrogen receptor-1 agonist LNS8801 induces mitotic arrest and cell death in uveal melanoma 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 1852.
Abstract 1856: Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206
Authors: Zhang Yiqun , El-Deiry Wafik S.
Published: June 2022

ONC201 (originally discovered as TRAIL-Inducing Compound #10 or TIC10) and analogue ONC206 have been found to induce an integrated stress response with suggested primary targets and mechanisms involving agonist activity against mitochondrial ClpP and antagonism of dopamine receptors D2/3 (DRD2/3). We hypothesized that dopamine, the agonist of DRD2, may compete with ONC201 or ONC206 for DRD2/3 and impair the anti-cancer effect of ONC201 or ONC206. We therefore pre-treated cancer cells from different tissue origins including breast cancer, pancreatic cancer, colorectal cancer, lung cancer, GBM, and DIPG with dopamine for 12 hours, 24 hours or 48 hours, respectively, followed by treatment of ONC201 or ONC206 for 72hours. Cell viability was determined by the CellTiter Glo assay. Single treatment with dopamine did not increase cell viability. We observed that 48 hours of pre-treatment with dopamine at 50 μM, 100 μM or 200 μM impaired the cell viability suppression effect of ONC201 at 4 μM and 8 μM, while ONC206 at 0.5 μM, 1 μM and 2 μM led to similar protection for loss of cell viability in pancreatic cancer cells. The impairment of the anti-cancer effect by dopamine was also observed in colorectal cancer cells with 48 hours pre-treatment with dopamine at 50 μM, 100 μM, 200 μM or 400 μM and treatment with ONC201 at 4 μM and 8 μM, or ONC206 at 0.5 μM, 1 μM, or 2 μM. In addition, we treated pancreatic cancer and colorectal cancer cells with ONC212 after 48 hours pre-treatment with dopamine and observed a similar impairment of the anti-cancer effect, even though ONC212 is not a DRD2 antagonist. We pre-treated pancreatic cancer and colorectal cancer cells with specific DRD2 agonist, sumanirole, for 48 hours followed by treatment of ONC201, ONC206, or ONC212 for 72 hours, and no impairment was seen. We pre-treated multiple cancer cell lines with 2 μM or 10 μM dopamine for one week followed by ONC201, ONC206, or ONC212 treatment and performed colony assays. Pre-treatment with dopamine at 10 μM impaired the anti-cancer effect of ONC201 or ONC206 in pancreatic cancer. Pre-treatment with dopamine at 2 μM or 10 μM impaired the anti-tumor effect of ONC201 in GBM cells. By contrast to the cell viability result, no impairment of ONC212 effect from dopamine was seen in pancreatic cancer cells. Western blotting was conducted to investigate whether dopamine pre-treatment impacted signaling pathways reported to be affected by ONC201. Dopamine pre-treatment did not impact changes induced by ONC201 in ATF4, CHOP, DR5, ClpX, clpP, pERK, pAkt and pS6. We are pursuing whether the protective effect of dopamine is due to the agonism of DRD2 or alternative pro-survival mechanisms.

Citation Format: Yiqun Zhang, Wafik S. El-Deiry. Adaptation to dopamine impairs the anti-cancer effect of ONC201 and ONC206 [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 1856.
Abstract 1861: Trophoblast cell surface antigen 2 (TROP2) expression in human tumors: A tissue microarray study on 18,563 tumors
Authors: Dum David , Hube-Magg Claudia , Simon Ronald , Bady Elena , Mandelkow Tim , Blessin Niclas C. , Lennartz Maximilian , Sauter Guido , Minner Sarah , Luebke Andreas M.
Published: June 2022

Trophoblast cell surface antigen 2 (TROP2) is the target of sacituzumab govitecan (SG), an antibody-drug conjugate that was recently approved for previously treated triple negative breast cancer and urothelial carcinomas. To comprehensively determine TROP2 expression in normal and neoplastic tissues, a tissue microarray containing 18,563 samples from 150 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. TROP2 positivity was found in most normal epithelial cell types and in 109 of 150 tumor categories, including 92 of 95 epithelial tumor categories. Particularly high rates of TROP2 positivity and highest expression levels were seen in squamous cell carcinomas of various origins and various categories of urothelial, breast, prostate, pancreatic, and ovarian cancers (>95% positive). High TROP2 expression was linked to advanced stage (p=0.0069) and nodal metastasis (p<0.0001) in colorectal cancer as well as to nodal metastasis in gastric adenocarcinoma (p=0.0246) and papillary thyroid cancer (p=0.0013). Low TROP2 expression was linked to advanced stage in urothelial carcinoma (p<0.0001), high pT (p=0.0024) and high grade (p<0.0001) in breast cancer, as well as with high grade (p=0.0005) and pT stage (p=0.0009) in papillary renal cell carcinomas. Associations between TROP2 expression and clinicopathological features were not found in clear cell renal cell carcinomas, high grade serous ovarian carcinomas, pancreatic adenocarcinomas and in endometroid endometrium carcinomas. In summary, TROP2 is abundantly expressed in a broad range of epithelial neoplasms. Both TROP2 upregulation and downregulation can be associated with cancer progression in a tumor type dependent manner. As anti-TROP2 cancer drugs have demonstrated efficiency and induce tolerable side effects they may be applicable to a broad range of tumor entities in the future.

Citation Format: David Dum, Claudia Hube-Magg, Ronald Simon, Elena Bady, Tim Mandelkow, Niclas C. Blessin, Maximilian Lennartz, Guido Sauter, Sarah Minner, Andreas M. Luebke. Trophoblast cell surface antigen 2 (TROP2) expression in human tumors: A tissue microarray study on 18,563 tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1861.
Abstract 1898: SNAF: Accurate and compatible computational framework for identifying splicing derived neoantigens
Authors: Li Guangyuan , Salomonis Nathan
Published: June 2022

Patient outcome with immune checkpoint blockade (ICB) therapy relies largely on the presence and detection of tumor neoantigens. Neoantigens can be produced from a broad spectrum of genetic (SNV, fusion) and non-genetic (splicing, alternative promoter) sources. While alternative splicing is a broad source of neoantigens, concerns regarding their tumor specificity have hindered their evaluation as therapeutic targets relative to SNVs. To systematically define tumor-specific and likely immunogenic neoantigens, we developed SNAF (Splicing Neo Antigen Finder), an easy-to-use and open-source computational workflow. SNAF automates the prediction of high-confidence patient-specific and shared splicing derived neoantigens from RNA-Seq data. This workflow leveraged deep learning and hierarchical Bayesian models to prioritize potential splicing-neoantigen candidates by their tumor specificity (inferred off-target effects) and immunogenicity (T cell reactivity). SNAF is compatible with all major splicing identification algorithms and can be easily integrated into any existing splicing analysis workflows. SNAF predicted splicing neoantigens were validated with an improved accuracy over alternative approaches in ovarian cancer HLA targeted MS profiles. Applied to 825 breast cancer patients in TCGA, we find that splicing neoantigens are frequently shared among patients and overall burden is highly associated with overall survival and specific clinical subtypes. These data suggest alternative genomic monitoring of splice-derived neoantigen burden can be used to prioritize selection of patients for ICB.

Citation Format: Guangyuan Li, Nathan Salomonis. SNAF: Accurate and compatible computational framework for identifying splicing derived neoantigens [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 1898.
Abstract 191: Establishment and characterization of patient-derived cancer cell models for pharmaceutical drug screening
Authors: Griffiths Gareth J. , Dodd Abbie , Nee Olivia , Thuringer Tilly , Singh Subir , Howard-Jones Rachel , Fan Yuen Ngan , Bezman Albert , Bell Rachel , Sloma Ido , Noakes Chris , James Dominic I.
Published: June 2022

Patient-derived cell (PDC) models are a tool that bridge the gap between conventional cancer lines that do not faithfully replicate clinical responses and costly and time-consuming patient derived xenograft (PDX) models. Imagen Therapeutics is establishing the world’s largest biobank of PDC models for both patient diagnostic (PredictRx) and biopharma drug development (PredictTx) studies that show a high degree of correlation between PDC and patient drug response. Through our research, we have established unique culture conditions that specifically select for cancer stem cells, and these have been characterized using both 2-D and 3-D short-term high content image (HCI)-based analysis. To better understand the genomic and phenotypic profiles of these models we analyzed Next Generation Sequencing data and phenotypic cell killing using a panel of FDA-approved agents. Our results showed that mRNA expression of several known cancer stem cell markers were upregulated in our PDCs in addition to multidrug resistance (MDR) genes. Drug resistance in the phenotypic assay was then correlated with stemness and MDR expression profiles. Finally, sequential PDCs (models developed from same patient at different stages in disease progression), in addition to PDCs developed from the same patient at multiple lesion sites, were compared in terms of genomic sequencing and drug responses. Together our data supports the premise that PDCs recapitulate the cancer stem cell population and are a useful platform with which to interrogate drug responses in a variety of different cancer types.

Citation Format: Gareth J. Griffiths, Abbie Dodd, Olivia Nee, Tilly Thuringer, Subir Singh, Rachel Howard-Jones, Yuen Ngan Fan, Albert Bezman, Rachel Bell, Ido Sloma, Chris Noakes, Dominic I. James. Establishment and characterization of patient-derived cancer cell models for pharmaceutical drug screening [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 191.
Abstract 1925: Ovarian tumor diagnosis using deep convolutional neural networks and denoising convolutional autoencoder
Authors: Choi Youn Jin , Jung Yuyeon
Published: June 2022

Objective: Discrimination of ovarian tumors is a necessary procedure for proper treatment. In this study, we developed a convolutional neural network model with a convolutional autoencoder (CNN-CAE) to classify ovarian tumors.

Methods: A total of 1,613 ultrasound images of ovarian tumors with the known pathologic diagnosis were pre-processed and augmented for deep learning analysis. We designed the CNN-CAE model that removes the unnecessary information on ultrasound images (e.g. calipers and annotations) and classifies ovarian tumors into five classes. We assessed the CNN-CAE model performance using a validation set of 327 images with the following metrics: accuracy, sensitivity, specificity, and the area under the receiver operating characteristic curve (AUC). Gradient-weighted class activation mapping (Grad-CAM) was applied to visualize and verify the CNN-CAE model results qualitatively.

Results: In classifying normal versus ovarian tumors, the CNN-CAE model showed 96.0% accuracy, 96.5% sensitivity, and 0.9837 AUC with DenseNet161 CNN architecture. In determining malignancy of ovarian tumors, the CNN-CAE model showed 86.85% accuracy, 75.68% sensitivity, and 0.9225 AUC with DenseNet121 CNN architecture. The Grad-CAM results show that the CNN-CAE model finds valid texture and morphology features from the ultrasound images and classifies ovarian tumors from these features.

Conclusion: CNN-CAE is a feasible diagnostic tool that is capable of robustly classifying ovarian tumors by eliminating marks on ultrasound images. CNN-CAE demonstrates a significant application value in clinical conditions.

Citation Format: Youn Jin Choi, Yuyeon Jung. Ovarian tumor diagnosis using deep convolutional neural networks and denoising convolutional autoencoder [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 1925.
Abstract 1931: Multi-cancer classification: An analysis of neural network complexity
Authors: Webber James W. , Elias Kevin M.
Published: June 2022

Background: Often, in analysis of expression data, binary classification problems are considered, with the aim to separate a control group from a single cancer group (e.g., ovarian cancer). Such models lack any cancer specific information, as they are only trained on one cancer type. It is more useful and efficient to train a model on multiple cancer types, and controls, simultaneously, so that a physician can be directed to the correct area of the body for further testing.

Results: We introduce novel, data-driven neural network models to address the multi-cancer classification problem across a number of data types commonly applied in cancer prediction, including circulating miRNA expression (blood draw), protein (tissue sample), and mRNA (tissue sample). In particular, we present an analysis of neural network depth and complexity and investigate how this relates to classification performance. Comparisons of our models are also given to the state-of-the-art neural network models from the literature. The proposed models yield high accuracy (mean AUC greater than 0.95) across all data types considered and are shown to offer greater performance when compared to the models from the literature.

Conclusion: We have introduced a simple neural network framework which can be applied to multiple data types and which yields the best performance when compared to similar models from the literature. Upon analysis of the neural network complexity, we discover that shallow, feed-forward neural net architectures offer greater performance when compared to more complex deep feed-forward, Convolutional Neural Network (CNN), and Graph CNN (GCNN) architectures considered in the literature. The depth analysis indicates that shallow neural nets (e.g., 1 or 2 layers) are more favorable for this problem, when compared to deep architectures (e.g., more than 3 layers). The results show that multiple cancers and controls can be classified simultaneously, and accurately using the proposed models, across a range of expression technologies in cancer prediction.

Citation Format: James W. Webber, Kevin M. Elias. Multi-cancer classification: An analysis of neural network complexity [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 1931.
Abstract 1944: Prognostic biomarkers for gallbladder cancer: A machine learning approach
Authors: Jackson Linsey , Allotey Loretta , Kenneth Valles , Oliver Gavin , Nair Asha , O'Brien Daniel , Graham Rondell , Borad Mitesh , Athreya Arjun , Roberts Lewis
Published: June 2022

Gallbladder cancer (GBC) is one of the deadliest cancers, with a 5-year-survival-rate of less than 5 percent for late-stage disease. The response rate to chemotherapy among GBC patients is generally poor. Recent research has attempted to identify diagnostic, prognostic, and predictive biomarkers, however, currently, no biomarkers can accurately diagnose GBC and predict patients’ prognosis. Integrative analysis of molecular and clinical characterization has not been fully established, and minimal improvement has been made to the survival of these patients, in part due to the heterogeneity of GBC. Machine learning techniques have been proven to empower analysis of big data in oncology, allowing for improvement in the generation of biomarkers to predict patient outcomes. Using machine learning, we can utilize high-throughput RNA sequencing with clinicopathologic data to develop a predictive tool for GBC prognosis. Current predictive models for GBC outcomes often utilize clinical data only, with the highest C-statistic reported being 0.71. C-statistic values over 0.7 generally indicate good models, however 0.8 is the threshold for strong predictive models. We aim to build a superior algorithm to predict overall survival in GBC patients with advanced disease, using machine learning approaches to prioritize biomarkers for GBC prognosis. We have identified over 80 fresh frozen GBC tissue samples from Mayo Clinic Rochester, Dongsan Medical Center in Daegu, Korea, University of the Witwatersrand, in Johannesburg, South Africa, Lithuanian University of Health Science in Vilnius, Lithuania, and University of Calgary in Calgary, Canada, from patients enrolled between 2012 and 2021. We will perform next-generation RNA sequencing on these tissue samples. The patients’ clinical, pathologic and survival data will be abstracted from the medical record uniformly across sites. Feature engineering and dimensionality reduction will be performed. Then random forests, support vector machines, and gradient boosting machines will be applied to train the data. Variable importance will prioritize multi-omic markers. Standard 5-fold cross validation will be used to assess performance of each ML algorithm. If overall survival can be better predicted with the addition patients’ transcriptional sequencing data compared to using clinical profiles alone, we can gain a greater understanding of key biomarkers driving the tumor phenotype.

Citation Format: Linsey Jackson, Loretta Allotey, Valles Kenneth, Gavin Oliver, Asha Nair, Daniel O'Brien, Rondell Graham, Mitesh Borad, Arjun Athreya, Lewis Roberts. Prognostic biomarkers for gallbladder cancer: A machine learning approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1944.
Abstract 1956: Perioperative opioid analgesia affects the circulating tumor cells levels in colorectal cancer patients
Authors: Stejskal Pavel , Srovnal Josef , Berta Emil , Rehulkova Alona , Vecera Lubomir , Haiduk Filip , Michalek Pavel , Hajduch Marian
Published: June 2022

Purpose Colorectal cancer (CRC) is one of the most common cancers worldwide and metastases-related death is the main cause of its high mortality. Previous studies showed the poorer survival in colorectal cancer patients treated perioperatively with morphine in comparison to piritramide. Circulating tumor cells (CTCs) are considered as precursors of distant metastatic spread and can act as an independent prognostic and predictive biomarker. The hypothesis that different pain-killers used in perioperative period can affect the CTCs levels was tested.

Patients and methods In total, 100 CRC stage I-III patients undergoing radical surgery were enrolled in this prospective multicentric randomized pilot study (NCT03700411). Three peripheral blood samples were collected into Cell-Free DNA BCT® (Streck, Inc.) preoperatively, one day and one month after surgery respectively. Patients were randomized into three arms using different perioperative analgesia - morphine, piritramide and epidural analgesia. The CTCs were identified using the CytoTrack CT11࣪ (2/C, Denmark), a semi-automated immunofluorescence microscopy detecting the pan-cytokeratin and EpCAM signals. The method recovery rate was analyzed using SW-480 cancer cell line by flow cytometry.

Results The CTCs were found most frequent in the samples obtained at first day after surgery (44,9%) followed by the samples collected before surgery (36%). The lowest levels were observed in control samples one month after surgery (31%). The significant increase of CTCs levels was found in majority of patients treated with morphine (16,3%) compared to piritramide and epidural analgesia. However, the significant decrease of CTCs levels was observed in samples drawn one month after surgery in both, morphine and piritramide arm. There were no significant changes in CTCs levels of patients with epidural analgesia. The CTCs recovery rate of the method was 72%.

Conclusion The significant increase of CTCs levels was found in postoperative blood samples of CRC patients treated with morphine compared to the piritamide and epidural analgesia. The enrollment into the study is ongoing.

Acknowledgements This study was supported by Ministry of Health of the Czech Republic (NV18-03-00470), Ministry of Education, Youth and Sport of the Czech Republic (LM2018132), Palacky University Olomouc (LF 2021_019) and European Regional Development Fund (ENOCH CZ.02.1.01/0.0/0.0/16_019/0000868, ACGT CZ.02.1.01/0.0/0.0/16_026/0008448).

Citation Format: Pavel Stejskal, Josef Srovnal, Emil Berta, Alona Rehulkova, Lubomir Vecera, Filip Haiduk, Pavel Michalek, Marian Hajduch. Perioperative opioid analgesia affects the circulating tumor cells levels in colorectal cancer patients [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 1956.