bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1721: Hypoxia penetrating ultra-small nanoparticle for anti-cancer effect in Glioblastoma
Authors: Rauf Mohd A. , Tatiparti Katyayani , Sau Samaresh , Iyer Arun K.
Published: September 2020

Glioblastoma multiforme (GBM) is a lethal brain tumor currently without successful treatment options. Chemotherapeutic agents are the frontline in the management of GBM. Nonetheless, accessibility of the brain tumor is greatly impeded by the poor blood-brain barrier (BBB) penetration of drugs, which needs immediate attention. Ultra-small multifunctional nanoparticle offers a great promise in overcoming the delivery barriers to GBM. The primary goal of our research is to develop BBB targeted nanoparticle that can selectively deliver drugs to GBM and trigger cell death. We developed Anti-GBM drug, such as temozolomide (TMZ) encapsulated biocompatible, ultra-small nanoparticle using Lactoferrin (LAC) protein. Further, we chemically conjugated LAC with tumor hypoxia biomarker homing molecule, Acetazolamide (ATZ) to obtain LAC-ATZ nanoparticle. To validate our hypothesis of hypoxia targeting, we evaluated the carbonic anhydrase-IX (CAIX) receptor expression in human brain tumor section. The IHC data demonstrated significant overexpression of CAIX in tumor tissue compared to healthy human brain tissue section. Literatures have reported overexpression of LRP-1 in endothelial blood vessel of GBM tumor. Thus, use of LAC-ATZ will enhance its penetration across the BBB and delivery of TMZ in hypoxic core of GBM. To achieve the effective BBB penetration, we engineered LAC-ATZ@TMZ nanoparticle that demonstrated homogenous ultra-small particle size of average diameter of 20-30 nm using TEM analysis. The SDS-PAGE analysis demonstrated the conjugation of LAC with ATZ. Our preliminary data in GBM cells, U87MG suggested the dose dependent cell uptake of DiD-fluorescence labeled LAC-ATZ nanoparticle. The nanoparticles are selective in targeting the U87MG cell when compared to healthy microglial BV2 cells. Thus, it is supporting the potential of LAC-ATZ nanoparticle for targeting the GBM tumor with limited effect in healthy brain tissue. We developed conditioned BBB cell culture model to demonstrate the penetration of LAC-ATZ across the BBB. Based on the exciting preliminary outcome in this study builds a strong rational of using LAC-ATZ nanoparticle for overcoming the bottle neck barrier of BBB with improved anti-GBM response.

Keywords:

Glioblastoma multiforme (GBM), blood-brain barrier (BBB), Lactoferrin (LAC), Acetazolamide (ATZ)

Citation Format: Mohd A. Rauf, Katyayani Tatiparti, Samaresh Sau, Arun K. Iyer. Hypoxia penetrating ultra-small nanoparticle for anti-cancer effect in Glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1721.
Abstract 1740: Transformable amyloid-beta mimetic peptide amphiphiles for lysosomal disruption and cisplatin sensitization in non-small cell lung cancer
Authors: Baehr Christopher M. , Zhang Lu , Lam Kit
Published: September 2020

In the United States, non-small cell lung cancer (NSCLC) is the largest contributor to cancer mortality. Traditional chemotherapies are toxic, and drug-resistance development is common. Recently, several drug candidates were shown to selectively induce lysosomal membrane permeabilization in aggressive tumors. One of these agents, chloroquine and its derivatives, has been shown to sensitize NSCLCs to cisplatin in human clinical trials. This has led to the interest in lysosome dysregulation as a cancer therapeutic target. However, approaches are needed to overcome two limitations of current lysosomal inhibitors: low lysosomal specificity and low potency. Here, we report on the development of a transformable nanomaterial which may be triggered to induce the mechanical lysosomal membrane permeabilization in vitro and in mouse xenograft models. The nanomaterial consists of peptide amphiphiles, which self-assemble into nanoparticles, colocalize with the lysosome, and due to lysosomal pH shift switch conformation to nanofibrils, leading to mechanical disruption of the lysosome, cisplatin sensitization and cell death. We show that the nanomaterial is cytotoxic to NSCLC cells in the low-micromolar range, but is able to sensitize NSCLC cells to cisplatin in the nanomolar range. Moreover, we demonstrate promising antitumor effect in mouse xenograft models with limited toxicity. This is the first example of enhanced LMP via transformable peptide nanomaterial and offers a promising new strategy for cancer therapy.

Citation Format: Christopher M. Baehr, Lu Zhang, Kit Lam. Transformable amyloid-beta mimetic peptide amphiphiles for lysosomal disruption and cisplatin sensitization in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1740.
Abstract 1782: N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target
Authors: Mackey John R. , Lai Justine , Chauhan Utkarsh , Dong Wei-Feng , Glubrect Darryl , Ghosh Sunita , Bigras Gilbert , Lai Raymond , Berthiaume Luc G.
Published: September 2020

N-myristoyltransferases (NMTs) catalyze the addition of 14-carbon fatty acids to the N-terminus of proteins. This activity regulates numerous membrane-bound signal transduction pathways important in cancer biology, and the pan-NMT inhibitor PCLX-001 is in clinical development as a cancer therapy. The physiologic distribution and relative contributions of the two human NMTs, NMT1 and NMT2, remain poorly understood as previous studies used polyclonal antibodies with potential cross-reactivity. We generated and validated mutually exclusive monoclonal antibodies (mAbs) specific to the human isotypes of NMT1 and NMT2. These mAbs were used to perform an immunohistochemical (IHC) analysis of the abundance and distribution of NMT1 and NMT2 in normal human breast epithelial samples and a large (n=703) cohort of primary breast adenocarcinomas from the BCIRG001 study. While NMT1 protein was readily identifiable in most normal and transformed breast epithelial tissue, NMT1 abundance was associated with higher overall histologic grade, higher Ki67, and lower hormone expression. While NMT2 protein was readily detected in normal breast epithelial tissue, NMT2 protein was undetectable in the majority of malignant breast cancers, but detectable NMT2 protein correlated with significantly poorer overall survival outcomes (hazard ratio for death 1.36; p < 0.029) and significantly worse biological features including younger age, higher histologic grade, lower hormone receptor expression, higher Ki67, and p53 positivity. NMT status was unrelated to HER2 status. NMT1 and NMT2 protein abundances were positively correlated with each other. Treatment of cultured breast cancer cells with the pan-NMT inhibitor PCLX-001 reduced cell viability in vitro. Daily oral administration of PCLX-001 to immunodeficient mice bearing human MDA-MB-231 breast cancer xenografts induced significant dose-dependent tumor growth inhibition in vivo. These results support the further evaluation of NMT immunohistochemistry for selection of patients for NMT inhibitor therapy, and clinical trials of NMT inhibition in breast cancer patients.

Citation Format: John R. Mackey, Justine Lai, Utkarsh Chauhan, Wei-Feng Dong, Darryl Glubrect, Sunita Ghosh, Gilbert Bigras, Raymond Lai, Luc G. Berthiaume. N-myristoyltransferase proteins in breast cancer: Prognostic relevance and validation as a new drug target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1782.
Abstract 1817: C/EBPβ antagonist peptide, ST101, attenuates oncogenic gene transactivation in cancer cells to drive antitumor activity
Authors: Rotolo Jim A. , Ramirez Rick , Koester Mark , Gallagher Erin , Leong Siok , Ghamsari Lila , Merutka Gene , Kappel Barry J.
Published: September 2020

Transcription factor dysregulation is common in cancer, resulting in aberrant gene expression that drives oncogenesis. Antagonism of oncogenic transcription factor activity, by disrupting essential protein-protein interactions needed for activation of downstream effector molecules or association with DNA, represents a powerful approach to target this previously ‘undruggable' class of proteins. CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is a transcription factor overexpressed in many cancers that regulates expression of factors that promote tumor survival, proliferation and inhibit differentiation. Here, we decribe the anti-tumor activity of ST101, a cell-penetrating peptide antagonist of C/EBPβ. To demonstrate ST101 disruption of the interaction of C/EBPβ with co-factor activating transcription factor 5 (ATF5), a competition ELISA assay was performed. Recombinant ATF5 associated with plate-bound C/EBPβ with a binding affinity of ∼1 nM. Addition of ST101 inhibited this interaction in dose-dependent manner, resulting in an IC50 of 25 nM. To demonstrate ST101 disruption of C/EBPβ phosphorylation and gene transactivation in cancer cells, western blot analysis and quantitative polymerase chain reaction (qPCR) were performed on U251 glioblastoma, MCF7 breast adenocarcinoma and A549 lung adenocarcinoma cells. Administration of ST101 resulted in a dose-dependent decrease in C/EBPβ activation, as evidenced by a decrease in Thr189 phosphorylation. Anatgonism of C/EBPβ activity resulted in a dose-dependent decrease in mRNA expression of genes involved in survival (BCL2 and the baculoviral inhibitor of apoptosis factors BIRC3, BIRC5), inhibition of differentiation (Inhibitor of DNA binding proteins ID1, ID2 and ID3) and proliferation (cyclins CCNB1 and CCNA2 and cyclin-dependent kinases CDK1 and CDK2). Finally, in a mouse xenograft model, 25mg/kg ST101 administered three times per week for three weeks resulted in significant and sustained tumor growth inhibition in U251 subcutaneous tumors, both when ST101 administration was initiated early (day 2, 200 mm3 tumors, p<0.05) or late (day 16, >500mm3 tumors, p<0.05). These data demonstrate the therapeutic potential of systemic administration of ST101 and support clinical development of ST101 as a potent peptide therapeutic for a variety of solid tumor malignancies.

Citation Format: Jim A. Rotolo, Rick Ramirez, Mark Koester, Erin Gallagher, Siok Leong, Lila Ghamsari, Gene Merutka, Barry J. Kappel. C/EBPβ antagonist peptide, ST101, attenuates oncogenic gene transactivation in cancer cells to drive antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1817.
Abstract 1829: Olaparib antiproliferative effect in pancreatic cancer and correlation with the response to other anticancer agents
Authors: Leo Elisabetta , Illuzzi Giuditta , Bentouati Sabrina
Published: September 2020

AstraZeneca obtained positive results from POLO, the first Phase III randomised, double-blind study that evaluated the efficacy of the PARP inhibitor (PARPi) olaparib, for maintenance monotherapy in patients with gBRCA mutated metastatic pancreatic cancer whose disease has not progressed on first-line platinum-based chemotherapy.

To support the application of olaparib for the proposed indication in these patients, a series of in vitro pharmacological tests were performed and will be here presented.

1. In a panel of pancreatic cancer cell lines, a striking correlation was observed between the responses to platinum and to olaparib. This indicates that it is predictable that olaparib will show efficacy in treatment of platinum-sensitive pancreatic cancer, as it has been previously demonstrated in other cancer indications.

2. Tests in isogenic cell line pairs and non-tumour cell lines revealed that platinum treatments lead to cytotoxic effects that are only ~10-20-fold more selective for BRCAm versus BRCAwt and non-tumour models; in contrast, olaparib is able to selectively kill BRCAm cells, with ~500-1000-fold sensitivity versus the BRCAwt cells.

3. In the panel of pancreatic cancer cells, the sensitivity to other DNA damaging agents used as standard of care, such as irinotecan (and, to some extent, 5FU, other key components of FOLFIRINOX as well as gemcitabine), correlated with sensitivity to olaparib. In contrast, no correlation was observed with paclitaxel (mitotic inhibitor), or with other agents.

4. The antiproliferative effects to olaparib treatments in the cancer cell panel were compared to that of other 5 clinical PARPi. High correlations were observed in the responses to all the PARPi except for veliparib, which inhibits the catalytic activity of PARP but does not act as PARP-trapper, confirming that the efficacy of PARPi in monotherapy depends on their ability to stabilise the interaction between PARP and the DNA.

Citation Format: Elisabetta Leo, Giuditta Illuzzi, Sabrina Bentouati. Olaparib antiproliferative effect in pancreatic cancer and correlation with the response to other anticancer agents [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1829.
Abstract 1855: Statins decrease the expression of c myc in cancer cell lines
Authors: Rao U.S. , Mullins Randy , Rao Prema
Published: September 2020

The widely used statin drugs for the prevention of cardiovascular diseases are envisioned to have the potential to control cancer. Repurposing these cardiovascular drugs for cancer prevention and/or treatment is an attractive proposition as these drugs are commonly prescribed in the clinic. However, there was no clinical evidence for statins being used as chemopreventive agents and the reasons for this are unclear. To further establish the effectiveness of statins as chemopreventive agents, we investigated the effects of three most common statins, simvastatin, atorvastatin and lovastatin on six different cancer cell lines including an ovarian cancer cell line that overexpresses P-glycoprotein, an ABCB1 gene product. Incubation of cells with statins (at both 12.5 microM and 25 microM) resulted in decreased cell growth in a time-dependent manner. The decreased cell growth could be correlated with decreased cellular NADPH levels. Measurement of viable and dead cells in statin treatments indicated that statins induced cell proliferation arrest without inducing cell death. Removal of statins from culture medium resulted in robust cell growth, identical to that observed in controls. Further, cell cycle analysis clearly indicated that statins induced cell cycle arrest at G0/G1 phase without inducing apoptosis. Analysis of the mechanism of statins in these cancer cells indicated that these drugs inhibit HMG CoA reductase, without affecting the transcript of the HMGCR. The HMG CoA reductase inhibition could be relieved with mevalonate. Analysis of key target proteins important for cancer cell proliferation identified that exposure of cancer cells to statins significantly decreased c-Myc protein without affecting its transcription. Importantly, removal of statins reversed the c-Myc expression to the normal level. Finally, incubation of cells with statins increased phosphorylation of H2AX, suggesting that statins promote cellular senescence. These data together suggested that statins are cytostatic, and eliminate c-Myc expression. These observations are consistently observed in the multidrug-resistant cells as well. These data lend strong support that statins could serve as excellent agents for cancer chemoprevention.

Citation Format: U.S. Rao, Randy Mullins, Prema Rao. Statins decrease the expression of c myc in cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1855.
Abstract 1859: Elucidating the role of KMT2D as a novel therapeutic target for resistant HER2+ breast cancer
Authors: MA EMILY , Zlobin Andrei , Wyatt Debra , Ng Jeffrey , Dingwall Andrew , Osipo Clodia
Published: September 2020

HER2 targeted agents have improved clinical outcomes for women with HER2+ breast cancer. However, drug resistance remains a critical challenge for curing HER2+ disease. Cancer stem cells (CSCs)-enriched during anti-HER2 therapy promote resistance and recurrence. Therefore, novel targets that drive survival of CSCs are needed. Recently, the histone-lysine N-methyltransferase 2D (KMT2D) has been shown to be a prognostic marker for poor recurrence free survival in women with HER2+ breast cancer. Preliminary data showed that KMT2D RNA was increased in HER2+ CSC-enriched mammospheres compared to bulk cells. Further, KMT2D protein is increased in resistant compared to sensitive cells. These data suggest that KMT2D is necessary for survival of CSCs and resistance to anti-HER2 therapy. To address this hypothesis, RNAi-mediated knockdown of KMT2D was performed in trastuzumab sensitive and resistant cells. KMT2D depletion in sensitive cells resulted in almost complete blockade of proliferation in 2-D culture and CSC survival in 3-D culture. In contrast, proliferation and CSC survival was only moderately inhibited by KMT2D depletion in resistant cells. ChIP studies confirmed that KMT2D is enriched on the MYC enhancer in sensitive cells but only moderately in resistant cells. Based on these data, KMT2D is necessary for growth of HER2+ breast cancer and is possibly inactivated during acquisition of resistance. To determine the mechanism, RNA sequencing was performed in both sensitive and resistance cells expressing or depleted for KMT2D. Several differentially expressed genes that were positively or negatively regulated by KMT2D were identified. Specifically, transcript levels of ITGB6 and CLIC3 were decreased upon KMT2D knockdown, while PGR and AR transcripts were increased. Initial results showed that ITGB6 depletion resulted in decreased bulk cell proliferation and survival of CSCs similar to KMT2D knockdown. These results suggest that ITGB6 could be a direct target gene of KMT2D and thus mediates the phenotypic effects of KMT2D. Overall, our results suggest that KMT2D is necessary for both proliferation and CSC survival in HER2+ breast cancer sensitive to anti-HER2 therapy and a combination approach of targeting both HER2 and KMT2D could prevent enrichment of CSCs and acquired resistance.

Citation Format: EMILY MA, Andrei Zlobin, Debra Wyatt, Jeffrey Ng, Andrew Dingwall, Clodia Osipo. Elucidating the role of KMT2D as a novel therapeutic target for resistant HER2+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1859.
Abstract 1903: PTEN loss mediates clinical cross-resistance to CDK4/6 and PI3Ká inhibitors in breast cancer
Authors: Costa Carlotta , Wang Ye , Ly Amy , Hosono Yasuyuki , Murchie Ellen , Walmsley Charlotte S. , Huynh Tiffany , Healy Christopher , Peterson Rachel , Yanase Shogo , Jakubik Charles T. , Henderson Laura E. , Damon Leah J. , Timonina Daria , Sanidas Ioannis , Pinto Christopher J. , Mino-Kenudson Mari , Stone James , Dyson Nicholas J. , Ellisen Leif W. , Bardia Aditya , Ebi Hiromichi , Benes Cyril H. , Engelman Jeffrey A. , Juric Dejan
Published: September 2020

The combination of CDK4/6 inhibitors with anti-estrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Since PTEN loss also causes resistance to PI3Kα-inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment sequencing strategies.

Citation Format: Carlotta Costa, Ye Wang, Amy Ly, Yasuyuki Hosono, Ellen Murchie, Charlotte S. Walmsley, Tiffany Huynh, Christopher Healy, Rachel Peterson, Shogo Yanase, Charles T. Jakubik, Laura E. Henderson, Leah J. Damon, Daria Timonina, Ioannis Sanidas, Christopher J. Pinto, Mari Mino-Kenudson, James Stone, Nicholas J. Dyson, Leif W. Ellisen, Aditya Bardia, Hiromichi Ebi, Cyril H. Benes, Jeffrey A. Engelman, Dejan Juric. PTEN loss mediates clinical cross-resistance to CDK4/6 and PI3Ká inhibitors in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1903.
Abstract 1924: Genetic analysis of responses to eribulin versus vinorelbine and paclitaxel in 100 cancer cell lines from the cancer cell line Encyclopedia (CCLE)
Authors: Sachdev Pallavi , Ronen Roy , Dutkowski Janusz , Littlefield Bruce A.
Published: September 2020

Background: Eribulin mesylate, a synthetic analog of the marine sponge natural product halichondrin B, is a microtubule dynamics inhibitor that acts via both cytotoxic antimitotic mechanisms as well as non-cytotoxic effects on tumor vasculature, tumor phenotype and the tumor microenvironment. Eribulin is approved in the US for treatment of certain patients with advanced breast cancer or liposarcoma. Here we examined in vitro antiproliferative responses to eribulin versus vinorelbine and paclitaxel in 100 human cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE), followed by analysis of CCLE baseline gene expression data with the goal of assessing overlapping and non-overlapping pathways and genes associated with drug response and resistance.

Methods: In vitro proliferation assays were conducted on 100 CCLE cell lines. Cancer types represented by ≥5 cell lines include breast, lung, blood, colorectum, ovary, pancreas, skin and kidney. Comparative systems-level analyses of genes, networks and pathways associated with drug response and/or resistance were performed to identify gene and pathway-level determinants of drug response that were either unique to eribulin or shared with vinorelbine and/or paclitaxel.

Results: We evaluated genes and molecular pathways driving sensitivity to eribulin versus vinorelbine and paclitaxel in a panel of 100 CCLE cell lines and identified key molecular determinants of response and resistance unique to eribulin. Pathway expression analysis revealed key pathways significantly associated with response to eribulin, including those associated with oxidative stress-induced senescence, histone deacetylation, DNA methylation and Notch signaling. The oxidative stress-induced senescence pathway was significantly enriched for genes whose pretreatment/basal mRNA expression levels were positively associated with response to eribulin but not vinorelbine or paclitaxel. Expression levels of various tubulin isoforms were also identified as positively correlated with eribulin response. Higher and lower pretreatment expression of E-cadherin and N-cadherin, respectively, were also found in cell lines sensitive to eribulin treatment.

Conclusion: Pretreatment gene expression analysis demonstrated key gene sets and molecular pathways that may drive response and resistance to eribulin and may differentiate this drug from vinorelbine and paclitaxel. Detailed analysis will be presented at the meeting.

Citation Format: Pallavi Sachdev, Roy Ronen, Janusz Dutkowski, Bruce A. Littlefield. Genetic analysis of responses to eribulin versus vinorelbine and paclitaxel in 100 cancer cell lines from the cancer cell line Encyclopedia (CCLE) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1924.
Abstract 1932: Effect of the C677T MTHFR polymorphism on toxicity in patients with colon adenocarcinoma treated with fluoropyrimidine based chemotherapy
Authors: Ramos-Esquivel Allan , Valle-Cano Marta
Published: September 2020

Background: Fluoropyrimidines (5FU and capecitabine) are the backbone of treatment for metastatic colorectal cancer. Their mechanism of action mainly consists on the inhibition of tymidylate synthase in a reaction where the 5,10-methylenetetrahydrofolate (MTHF) acts as a cofactor. This cofactor is highly regulated by the MTHF reductase (MTHFR) activity. It has been previously shown that C677T and A1298C polymorphisms of MTHFR are associated with a low activity of this enzyme, which can result in a higher therapeutical activity and toxicity from these compounds. The aim of this study was to assess the relationship between C677T and A1298 MTHFR polymorphisms and adverse events in first-line patients treated with fluoropyrimidine-based chemotherapy for stage IV colorectal adenocarcinoma. (NCT registration number: 03852290)

Methods: Patients with metastatic colorectal cancer were prospectively recruited in the San Juan de Dios Hospital (San José, Costa Rica). First line therapy consisted in any fluoropyrimidine used alone or in combination with oxaliplatin and/or irinotecan +/- bevacizumab, panitumumab or cetuximab. Germline DNA extraction was done from blood samples to determine the C677T (rs1801133) and A1298C (rs18011131) polymorphisms of MTHFR. Patients were followed by their treating physician every 12 weeks in order to assess the response and tolerability of their treatment. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria version 4.0.

Results: A total of fifty patients were enrolled. C677T MTHFR genotypic frequencies were distributed as follows: CC (30%), CT(50%), and TT (20%). The C allele frequency was 55%, following the Hardy-Weinberg equilibrium (HWE) (p=0.91). A1298C MTHFR genotypic frequencies were: AA (70%), AC (26%), and CC (4%). The A allele frequency was 80%, also following the HWE (p=0.58). MTHF 677 TT genotype was associated to high risk of grade III or IV anaemia (p=0.004), neutropenia (p<0.001), thrombocytopenia (p=0.001), neuropathy (p=0.03), diarrhea (p=0.015), and hand-foot syndrome (p=0.045). There was no statistically significant association between the A1298 MTHFR polymorphism and side effects.

Conclusion: The MTHF 677 TT genotype can potentially predict toxicity in a subset of Hispanic patients treated with fluoropyrimidine-based therapy for metastatic colorectal cancer.

Citation Format: Allan Ramos-Esquivel, Marta Valle-Cano. Effect of the C677T MTHFR polymorphism on toxicity in patients with colon adenocarcinoma treated with fluoropyrimidine based chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1932.
Abstract 206: The landscape of oncogenic gene fusions in Chinese colorectal cancer patients
Authors: Lu Zhenhai , Song Rongfeng , Li Kunsong , Cheng Ying , Wang Wenjing , Wang Weifeng
Published: September 2020

Background: Oncogenic gene fusions have been reported in patients with colorectal cancer (CRC) as promising actionable targets, but recognition of rare molecular subgroups is a challenge for precision oncology. We aimed to comprehensively characterize the clinical, pathological, and molecular landscape of Chinese CRC patients with oncogenic gene fusions.

Methods: A total of 1226 patients with CRC underwent genomic testing between Aug. 2017 and Sep. 2019 using a tissue-based next-generation sequencing (NGS) assay. All the coding exons of 450 cancer-related genes and selected introns were captured by the custom hybridization capture panel. Microsatellite instability (MSI) and tumor mutational burden (TMB) were assessed in all patients.

Results: Oncogenic gene fusions were found in 1.6% (20/1226) of Chinese CRC tumors, which was higher than that in the western population (0.68%, R. Madison, ESMO 2018 Congress). The most frequently detected fusions were NTRK1 (N = 5, 25%), BRAF (N = 5, 25%), ALK (N = 3, 15%), NTRK3 (N = 2, 10%), and RET (N = 2, 10%). FGFR1, ROS1, and EGFR fusions each occurred in one patient. Compared with patients without fusions, those with fusions were older (median age 69 vs 59 years, respectively, P=0.009), had right-sided CRC (75.0% vs 27.4%, respectively, P<0.001), MSI-high (45.0% vs 8.7%, respectively, P<0.001), and a higher TMB (median TMB of 9.8 vs 5.6 muts/mb, respectively). All MSI-high patients with fusions were TMB-high with a median value of 73.5 muts/Mb (range 51.8-180.3). The most frequently co-mutated genes in fusion cases were TP53 (70%), APC (60%), KMT2D (50%), ACVR2A (45%), RNF43 (45%), and TGFBR2 (40%). KRAS, NRAS, BRAF, POLE, and mismatch repair gene mutations were all mutually exclusive with oncogenic gene fusions. One patient harboring an EGFR-SEPT14 fusion, who had been heavily treated for right colon cancer metastasis, achieved a partial response to erlotinib.

Conclusions: This study revealed that oncogenic gene fusions were found in 1.6% of Chinese CRC patients by using a tissue-based NGS assay. As oncogenic gene fusions are mutually exclusive with common oncogenic mutations, using NGS to detect fusions as actionable targets or resistance mechanisms has notable implications for precision therapy.

Citation Format: Zhenhai Lu, Rongfeng Song, Kunsong Li, Ying Cheng, Wenjing Wang, Weifeng Wang. The landscape of oncogenic gene fusions in Chinese colorectal cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 206.
Abstract 2081: Intestinal estrogen receptor beta suppresses colon inflammation and tumorigenesis in both sexes
Authors: Hases Linnea , Indukuri Rajitha , Birgersson Madeleine , Nguyen-Vu Trang , Lozano Rodrigo , Saxena Ashish , Hartman Johan , Frasor Jonna , Gustafsson Jan-Åke , Katajisto Pekka , Archer Amena , Williams Cecilia
Published: September 2020

Estrogen has been shown to protect women against colorectal cancer (CRC). In this study, using tissue-specific knockout mice, we characterize the role for intestinal estrogen receptor beta (ERβ) and, for the first time, demonstrate a direct preventive role against colitis-induced colon tumors in both sexes. By inducing colitis and CRC using azoxymethane and dextran sodium sulfate in intestine-specific ERβ knockout mice, we observe an increase in colon ulceration in addition to an enhanced development of neoplasms, with increased number of tumors in males and increased tumor size in females. We identify a local increase in TNFα expression and deregulation of NFκB targets. We note that males exhibit especially strong effects following intestinal ERβ knockout. The protection by intestinal ERβ against TNFα-induced damage is corroborated using ex vivo organoid culture, where we observe that activation of ERβ protects against TNFα-induced epithelial cell damage. Further, we characterize an underlying mechanism of action, using CRC cell lines with re-introduced ERβ, where ERβ binds to cis-regulatory chromatin areas of key NFκB regulators BCL3, BIRC3 and ATF3, and thereby modulates TNFα-induced signaling. With these results we define a clear role for intestinal ERβ and demonstrate an underlying mechanism, strongly supporting the notion that selective activation of intestinal ERβ has potential to prevent CRC in both sexes.

Citation Format: Linnea Hases, Rajitha Indukuri, Madeleine Birgersson, Trang Nguyen-Vu, Rodrigo Lozano, Ashish Saxena, Johan Hartman, Jonna Frasor, Jan-Åke Gustafsson, Pekka Katajisto, Amena Archer, Cecilia Williams. Intestinal estrogen receptor beta suppresses colon inflammation and tumorigenesis in both sexes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2081.
Abstract 2083: Alternative splicing regulation by the androgen receptor in prostate cancer cells
Authors: Germain Lucas , Lafront Camille , Beaudette Jolyane , Poluri Raghavendra Tejo Karthik , Weidmann Cindy , Audet-Walsh Etienne
Published: September 2020

The androgen receptor (AR) is a transcription factor that drives prostate cancer (PCa) by modulating the expression of thousands of genes to promote proliferation and survival and to reprogram metabolism. However, how AR activation affects alternative splicing is mostly unknown. Our objective was to define its role in the transcriptome-wide regulation of alternative splicing. Three human PCa models–LNCaP, LAPC4, and 22Rv1 cells–were treated with and without androgens, and RNA was purified for deep-sequencing analyses (RNA-seq). Several bio-informatic tools were then used to study alternative splicing. We demonstrate that in the absence of androgens, alternative splicing complexity is similar among AR-positive PCa cells, with 48% of all transcripts having various levels of alternative splicing. We also describe splicing differences among cell lines, such as specific splicing of AR, REST, TSC2, and CTBP1. Interestingly, AR activation changed the alternative splicing of thousands of transcripts in all the cell lines tested. Overlap between AR-sensitive alternative splicing events revealed that genes linked to PCa metabolism are major targets for this specific modulation. These genes encode metabolic enzymes such as the prostate-specific membrane antigen (FOLH1), malate dehydrogenase 1 (MDH1), and malic enzyme 2 (ME2). Enzymatic assays revealed that AR alters their activities without changing their total gene expression levels, demonstrating that AR-driven regulation of alternative splicing has a direct effect on cancer cell metabolism. Overall, our study presents a comprehensive analysis of PCa cell transcriptome and its modulation by AR, revealing cell metabolism as a key target for AR-dependent regulation of alternative splicing.

Citation Format: Lucas Germain, Camille Lafront, Jolyane Beaudette, Raghavendra Tejo Karthik Poluri, Cindy Weidmann, Etienne Audet-Walsh. Alternative splicing regulation by the androgen receptor in prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2083.
Abstract 2097: AI powered platform to identify primary prostate cancer patients with high risk of recurrence
Authors: Huang Wei , Jain Parag , Randhawa Raman , Jaiswal Bijay , Hubbard Samuel , Basu Hirak , Roy Rajat
Published: September 2020

Background: To-date, there is no approved adjuvant therapy for primary prostate cancer (PCa). Long time of recurrence and a lack of a strong biomarker predicting recurrence after the first line of therapy is prohibitory to short clinical trials within a reasonable time. Artificial intelligence (AI) offers a unique opportunity for extracting critical morphological features from PCa tissues as prognostic marker that have evaded human eyes. We have built a AI-based platform to analyze H&E stained histological images of PCa. Our platform can accurately detect, grade and quantify PCa in patient tissue images. Here, we show how this platform can identify morphometric features that indicate biochemical recurrence within 3 years after radical prostatectomy (RP).

Methods: Our dataset comprised of a cohort of 169 PCa patients who underwent RP. Whole slide H&E images (WSI) of their tissue sections were scanned at 40x magnification. corresponding pathologic parameters such as Gleason grade, tumor volume, TNM staging, margin status and post-treatment follow-up data were also obtained. Among the 169 patients, 89 patients recurred within 3 years of RP.

Our platform technology involved two different deep CNN (Convolutional Neural Network) architectures. The platform first divided each WSI into multiple tiles. Each tile was analyzed using a CNN that graded the tile and generated a high dimensional vector to provide a mathematical equivalent of the morphology. The combination of high dimensional vectors across the WSI was then fed into a second CNN that generated a morphological score. The clinical parameters were combined with the morphologic score using a logistic classifier to predict outcome.

Results: Thus far, we have obtained 84% AUC (Area under the Curve) for the ROC (Receiver operating characteristics) curve, using 10-fold cross-validation. In contrast, the most predictive clinical covariate was the T stage with 71% AUC.

Conclusion: Our AI based platform can identify high risk PCa patients early for tumor progression post RP and may be selected for more cost effective and much shorter Clinical Trials necessary to develop adjuvant therapy.

Citation Format: Wei Huang, Parag Jain, Raman Randhawa, Bijay Jaiswal, Samuel Hubbard, Hirak Basu, Rajat Roy. AI powered platform to identify primary prostate cancer patients with high risk of recurrence [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2097.
Abstract 2145: Ex vivo phenotyping and potency monitoring of CD19 CAR T cells with a combined flow cytometry and impedance-based real time cell analysis workflow
Authors: Zhao Li , Lei Ming , Ye Peifang , Lu Yan , Ji Xiaoping , Yan Yu , Jachimowicz Lauren , Guenther Garret , Li Nancy
Published: September 2020

Immunotherapy harnesses the immune system to attack cancer cells. While immunotherapy holds tremendous promise for cancer treatment, significant challenges remain in clinical translation. Further development is still required to expand treatment to more cancers and minimize adverse effects. T-cell differentiation and exhaustion influence the establishment and persistence of CAR-expressing T cells. Evaluation of the potency of CAR T cells is essential for effective immunotherapies. Here, we study the potency of CAR-T cells using a combined impendence-based Real-Time Cell Analysis (RTCA) and flow cytometry cell analysis workflow for ex vivo cytolytic potency monitoring of CD19 CAR T cells (CART19). CART19 cell killing of CD19 expressing HEK-293 cells with different E:T (Effector to Target) ratio was monitored by the xCELLigience RTCA system. Concurrently, T cell cytokine production was measured on the NovoCyte Quanteon. This workflow provides a thorough examination of CART cell function, RTCA data demonstrates that CART19 killing is highly potent as well as being antigen-specific and dose-dependent. In conjunction, more cytokine was produced with higher numbers of effector CART19 cells. It was also observed that CART19 cells express stem memory cell (TSCM) markers, expressing activation and exhaustion markers after expansion. Co-culture with HEK-293-CD19 also results in increased expression of CD25, decreased expression of CD127 and upregulation of exhaustion markers, PD-1, TIM-3, and LAG-3. In summary, this study demonstrates the combination of RTCA and flow cytometry cell analysis is a powerful workflow for immunotherapy research and can be employed for QC in CAR T cell manufacturing.

Citation Format: Li Zhao, Ming Lei, Peifang Ye, Yan Lu, Xiaoping Ji, Yu Yan, Lauren Jachimowicz, Garret Guenther, Nancy Li. Ex vivo phenotyping and potency monitoring of CD19 CAR T cells with a combined flow cytometry and impedance-based real time cell analysis workflow [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2145.
Abstract 2159: IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signaling pathway in malignant melanoma
Authors: Yang Jilong
Published: September 2020

Immunotherapy targeting the PD-1/PD-L1 receptor in melanoma patients has achieved great success. Although many studies have addressed the underlying mechanisms involved in the blockade of PD-1/PD-L1 and the consequent modulation of the immune system, the mechanisms of PD-L1 upregulation and reliable biomarkers to predict the efficacy of anti-PD-1/PD-L1 therapy remain unknown. The present study demonstrates the correlation between IGFBP2 and PD-L1, reveals a novel immune-associated tumour function of IGFBP2 in facilitating nuclear accumulation of EGFR and activation of the EGFR/STAT3/PD-L1 signalling pathway in melanoma cells. Our results also suggest that combined IGFBP2 and PD-L1 expression have the potential to predict the efficacy of anti-PD-1 treatment for malignant melanoma; because the combination of high IGFBP2 and PD-L1 expression characterizes melanoma patients with worse overall survival and is associated with a better immune ecosystem. These characteristics have been confirmed by both in vitro and in vivo data. So, IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signalling pathway and its function as a PD-L1 regulator might suggest novel therapeutic approach for melanoma.

Citation Format: Jilong Yang. IGFBP2 regulates PD-L1 expression by activating the EGFR-STAT3 signaling pathway in malignant melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2159.
Abstract 2162: KIM–1 mediatesimmune evasioninrenal cell carcinoma
Authors: Yotis Demitra M. , Shrum Bradly , Sarabusky Marie , Gunaratnam Lakshman
Published: September 2020

Renal cell carcinoma (RCC) is the most common and lethal form of kidney cancer. Cancer immune evasion is a major obstacle for effective immunotherapy in RCC. Mechanisms of immune evasion are characterized by three phenotypes: Immune Inflamed; tumor contains infiltrating T cells which are rendered inactive within the tumor microenvironment due to localized inhibition. Immune Desert; tumor is devoid of activate T cells due to defective antigen presentation, and/or T cell activation. Lastly, Immune Excluded; tumor is surrounded by T cells that are unable to penetrate the parenchyma, caused by immunosuppression within the tumor stroma. Kidney Injury Molecule-1 (KIM-1) is a cell-surface glycoprotein aberrantly expressed in >90% of RCC tumors. The purpose of this study was to determine the pathophysiological significance of KIM-1 in RCC pathogenesis. We generated murine RCC cells (Renca) expressing KIM-1 (KIM-1pos) or control vector (KIM-1neg) using lentiviral transduction. We found that KIM-1 expression on RCC cells promoted more rapid tumor growth when injected contralaterally into syngeneic immunocompetent BALB/c mice (KIM-1neg = 263.75mm3, KIM-1pos = 849.72, p = 0.0149 & KIM-1neg = 0.32g, KIM-1pos = 0.58, p = 0.0229), but not in RAG1-/- immunodeficient BALB/c mice suggesting the KIM-1 promotes tumor growth through evasion of the adapt immune system. When analyzing tumor infiltrating lymphocytes (TILs) from both tumor groups, we found a relative scarcity of CD4+ and CD8+ T cells within the KIM-1pos vs. KIM-1neg tumors. To classify the immune evasion phenotype, we analyzed localization of the immune infiltrate using immunofluorescence within KIM-1pos and KIM-1neg RCC tumors. We found significantly fewer CD3+ cells within the KIM-1pos vs. KIM-1neg tumor parenchyma (KIM-1neg = 3625.78%, KIM-1pos = 272.36%, p = 0.0410). Moreover, CD3+ cells of the KIM-1neg tumors were observed in the parenchyma, whereas CD3+ cells of the KIM-1pos tumors were localized to the tumor stroma. In addition, we observed a higher frequency of myeloid derived suppressor cells (MDSCs) within the KIM-1pos vs the KIM-1neg tumor parenchyma (KIM-1neg = 0.05, KIM-1pos = 0.19, p = 0.0266). Transcriptomic profiling of both KIM-1pos and KIM-1neg Renca cells suggests that KIM-1 promotes deposition of extracellular matrix (KIM-1neg = -1.21, KIM-1pos = 1.96-fold change), which may contribute to KIM-1-mediated immune evasion Our data suggests that KIM-1 expression in RCC promotes immune evasion by altering the tumor microenvironment resulting in an Immune Excluded phenotype.

Citation Format: Demitra M. Yotis, Bradly Shrum, Marie Sarabusky, Lakshman Gunaratnam. KIM–1 mediatesimmune evasioninrenal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2162.
Abstract 2206: Degradation of hyaluronan in the tumor microenvironment enhances oncolytic reovirus and anti-PD-L1 therapy in a murine syngeneic breast tumor model
Authors: Lee Jisook , Wilkinson Grey A. , Kimbler Trevor , Blouw Barbara , Thompson Curtis B.
Published: September 2020

The extracellular matrix (ECM) structure heavily influences tumor growth, invasion and various steps of the cancer-immunity cycle. Hyaluronan (HA), a major component of ECM, is a glycosaminoglycan that accumulates in the tumor microenvironment (TME) of many solid tumor types, and HA accumulation can restrict T cell infiltration and promote immunosuppression. We have demonstrated in preclinical tumor models that enzymatic degradation of TME HA by intravenous PEGylated recombinant human hyaluronidase PH20 (Pegvorhyaluronidase alfa, PEGPH20) facilitates delivery of chemotherapeutics to tumors and enhances efficacy of current immunotherapy strategies including checkpoint inhibitors. In this study, we hypothesized that degradation of HA with PEGPH20 treatment may remodel the TME to improve the efficacy of oncolytic reovirus therapy (pelareorep) and checkpoint blockade therapy. To test this hypothesis, EMT6 murine breast tumor cells, which spontaneously accumulate HA and are sensitive to oncolytic activity of pelareorep were implanted orthotopically into the mammary fat pad of BALB/c mice. In this model, PEGPH20 treatment resulted in a reduction of HA measured by immunohistochemistry and induced tumor growth inhibition (30% TGI). Pelareorep plus anti-PD-L1 treatment also resulted in anti-tumor activity (44% TGI), and flow cytometric analysis of tumor infiltrating immune cells in pelareorep + anti-PD-L1-treated tumors demonstrated a significant increase in the numbers of cytotoxic CD8+ tumor infiltrating lymphocytes (TILs, p=0.0004), a decrease in regulatory T cells (T reg, p<0.0001) and an increase in CD8:Treg ratio (p=0.0018), indicating a robust antitumor immune response. Combination of PEGPH20 with pelareorep + anti-PD-L1 resulted in enhanced anti-tumor activity (71% TGI) compared to either PEGPH20 (30%TGI) or pelareorep + anti-PD-L1 (44% TGI). PEGPH20 in combination with pelareorep + anti-PDL1 also extended survival (median survival time, MST- 30 days, p<0.0001) in comparison with PEGPH20 alone (MST-21 days) or pelareorep + anti-PD-L1 (MST-22 days). Our data demonstrate the potential for matrix modeling through degradation of HA as a strategy to improve oncolytic reovirus + anti-PD-L1 immunotherapy. These findings are consistent with prior preclinical studies and support clinical evaluation of combining PEGPH20 with immune checkpoint inhibitors in HA-accumulating tumors.

Citation Format: Jisook Lee, Grey A. Wilkinson, Trevor Kimbler, Barbara Blouw, Curtis B. Thompson. Degradation of hyaluronan in the tumor microenvironment enhances oncolytic reovirus and anti-PD-L1 therapy in a murine syngeneic breast tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2206.