bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1001: Treatment of RB1-intact cancers with CDK4/6 inhibitor combination therapy
Authors: Takahashi Chiaki
Published: July 2021

Synthetic CDK4/6 inhibitors exert anti-tumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis and increased immunogenicity. These agents currently have an indication in advanced breast cancers, and are in clinical trials for many other solid tumors. Hepatocellular carcinoma (HCC) is one of promising targets of CDK4/6 inhibitors. RB family function is often aberrated in the initiation of HCC, however, this is revivable as RB family genes are rarely mutated or deleted in this malignancy. Loss of all Rb family members in Trp53-/- mouse liver resulted in hepatoma reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into HepG2 cells induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an IKKβ inhibitor. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/β phosphorylation and NF-κB activation. Combination therapy using palbociclib with KKβ inhibitor was significantly more effective in HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact K-Ras mutated lung and colon cancers. These findings provide proof of principle for the treatment of a wide variety of RB1-intact cancers using CDK4/6 inhibitors in combination with an appropriate kinase inhibitor. In this presentation, we will propose a new therapeutic approach to RB1-intact solid tumors.

Citation Format: Chiaki Takahashi. Treatment of RB1-intact cancers with CDK4/6 inhibitor combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1001.
Abstract 1013: Mechanistic role of nelfinavir as drug-repurposing strategy against high-grade serous ovarian cancer
Authors: Subeha Mahbuba R. , Goyeneche Alicia A. , Telleria Carlos M.
Published: July 2021

Platinum-based therapy following tumor-debulking surgery has been the backbone of treatment for high-grade serous ovarian cancer (HGSOC) since the 1970s; however, high recurrence of platinum-resistant disease necessitates the development of improved alternative therapies. Repurposing market-available drugs as cancer therapeutics carries the prospect of reducing the timeframe and cost of drug development, posing potential benefits to drug-resistant as well as financially underprivileged patients. Nelfinavir (NFV), an orally available anti-HIV drug, has shown promising effects against diverse cancers as demonstrated through a myriad of pre-clinical studies and clinical trials; however, its remedial benefits against HGSOC are still unclear. In this study, we explored the therapeutic efficacy of NFV on HGSOC cells generated from patients when platinum sensitive or resistant. Acute drug toxicity was assessed by total cell count, percent viability, and the level of hypo-diploid DNA content following 72 h of treatment with NFV. Living cells that tolerated 72 h of NFV treatment were subjected to further drug-free re-incubation for 14-21 days, to assess the residual anti-clonogenic potential of the drug. NFV triggered a dose-depended reduction of total cell number and viability, with a parallel increased hypo-diploid DNA content in both platinum-sensitive and resistant HGSOC cells. A dose-dependent reduction in the number of colonies - originating from cells that evaded acute toxicity - suggested long-term residual toxicity of NFV. Western blot analysis of underlying molecular mechanisms revealed phosphorylation of γH2AX, which suggested NFV-mediated induction of DNA damage. NFV also inhibited proliferation signals by reducing the phosphorylation of Akt and ERK. Moreover, NFV stimulated endoplasmic reticulum (ER) stress in a dose-dependent manner, which was evident from the enhanced expression of GRP78, IRE1α, ATF4, CHOP, and phosphorylated eIF2α. A time-dependent steady increase of ATF4 and CHOP suggested a progressive temporal apoptotic shift during NFV treatment. Finally, the cleavage of caspases-3 and -7 and their downstream effector PARP, in association with increased pro-apoptotic protein BAX, indicated caspase-associated cell-death during NFV-mediated cytotoxicity. In summary, we demonstrated that HGSOC cells of differential platinum sensitivities could be therapeutically targeted by NFV via multipronged mechanistic approaches, encouraging its prospective repurposing benefit against HGSOC.

Citation Format: Mahbuba R. Subeha, Alicia A. Goyeneche, Carlos M. Telleria. Mechanistic role of nelfinavir as drug-repurposing strategy against high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1013.
Abstract 1017: Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells
Authors: Tchounwou Paul B. , Kumar Sanjay
Published: July 2021

Arsenic trioxide (ATO) has been used for the treatment of acute promyelocytic leukemia (APL). Although ATO modulates cell cycle progression and apoptosis in APL cells, its exact mechanism of action remains elusive. In this research, we investigated its effects on E2F1, cyclin E, p53, pRb and PI3K signaling molecules by western blotting, immunocytochemistry and/or confocal imaging. We found that ATO inhibited the proliferation of APL cells through down-regulation of E2F1 and cyclin E expression, and stimulation of pRb. It also reduced the interaction of pRb and E2F1with binding to the E2F1 promoter, by stimulating pRb association. ATO also effected the phosphorylation of pRb at S608 and T373 residues and association of E2F1, pRb, and p53, simultaneously. However, in p53-knockdown NB4 cells, ATO did not significantly reduce E2F1 and cyclin E expression. Our findings demonstrate that ATO inhibits APL cell growth through reduced expression of E2F1, cyclin E, and stimulation of pRb. It also effected both interaction and association of E2F1, pRb and p53 by phosphorylation of pRb at T373 and S608 residues and reduced phosphorylation of PI3K signaling molecules. This novel mode of action of ATO in APL cells may be useful for designing new APL drugs.

Citation Format: Paul B. Tchounwou, Sanjay Kumar. Arsenic trioxide reduces the expression of E2F1, cyclin E, and phosphorylation of PI3K signaling molecules in acute leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1017.
Abstract 1036: Identification of Tofacitinib, Filgotinib and Ruxolitinib on experimental Sjögren's syndrome model in mice
Authors: Yan Ruiqing , Yuan Yanping , Luo Chao , Yangmeng Chuanzhi , Fang Li , Lin Qing
Published: July 2021

Sjögren's syndrome, which is affected hundreds of millions of people worldwide, is a chronic autoimmune disorder characterized by leukocytic infiltration into the exocrine glands, such as the salivary and lacrimal glands. However, the pathological mechanism remains to be elucidated, and urgent need for the novel treatment should still meet. Over the past decades, an extensive progress has been made in establishment of Sjögren's syndrome model in mice, which offers us an invaluable tool to understand SjS pathogenesis and makes it possible to develop the novel drug and treatment. Here, we identified the potential effect of Tofacitinib, Filgotinib and Ruxolitinib on the experimental Sjögren's syndrome model in C57/B6L mice from both In-life study and ex-vivo study at the endpoint. Our data suggest that Tofacitinib, Filgotinib and Ruxolitinib, three JAK inhibtors exhibits the effect on reducing the severity of Sjögren's syndrome in mice, reduces autoantibodies producion and promotes the saliva secretion in mice, which also provides the direction of novel JAK inhibitor on treatment for Sjögren's syndrome.

Citation Format: Ruiqing Yan, Yanping Yuan, Chao Luo, Chuanzhi Yangmeng, Li Fang, Qing Lin. Identification of Tofacitinib, Filgotinib and Ruxolitinib on experimental Sjögren's syndrome model in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1036.
Abstract 1040: Imipridones exhibit synergy with sorafenib, HDAC inhibition, PARP inhibition, and proteasome inhibition in liver cancer cell lines
Authors: Honeyman Joshua N. , Chang Wen-I , Prabhu Varun V. , Allen Joshua E. , Zhou Lanlan , El-Deiry Wafik S.
Published: July 2021

Liver cancer is a significant cause of cancer-related death worldwide. We previously showed that the imipridone class of small molecule therapeutics is active against hepatocellular malignancies. ONC201, the first imipridone to reach clinical trials, antagonizes the G protein-coupled receptor DRD2 and is an agonist of mitochondrial caseinolytic protease ClpP. ONC206 also antagonizes DRD2 and is a ClpP agonist. ONC212 is an agonist of both the orphan G protein-coupled receptor GPR132 and ClpP. We investigated the efficacy of ONC201, ONC206, and ONC212 in combination with histone deacetylase inhibition (HDACi), poly ADP ribose polymerase inhibition (PARPi), proteasome inhibition, and current standard of care multi-kinase inhibitor sorafenib. Liver cancer cell lines Hep3B and HepG2 were treated with ONC201, ONC206, and ONC212 to determine dose-response effect. Both cell lines were also treated with each imipridone in combination with sorafenib. Combination treatment effect was also tested between ONC201 and the HDACi vorinostat, PARPi nariparib, and the proteasome inhibitor marizomib. Cell viability was measured with CellTiter-Glo. Synergy was quantified using the combination index (CI). Protein expression was assessed using Western blot. As has been previously reported, both HepG2 and Hep3B cell lines were sensitive to ONC201 (Hep3B IC50 = 2.29μM; HepG2 IC50 = 2.69μM), ONC206 (Hep3B IC50 = 0.11μM; HepG2 IC50 = 0.15μM), and ONC212 (Hep3B IC50 = 0.07μM; HepG2 IC50 = 0.05μM) administered as single-agent therapy with ONC212 demonstrating the greatest potency. In combination with sorafenib, all three imipridones demonstrated synergy in both cell lines. ONC201 and ONC206 exhibited stronger synergy with CI < 0.3 at multiple dose combinations. ONC201 was also found to exert a synergistic reduction in cell viability when Hep3B and HepG2 cells were treated with vorinostat, nariparib, and marizomib. These results suggest that the imipridone family of small molecules is a novel therapy for liver cancer with efficacy as a single agent and in combination with existing targeted agents. Future studies will clarify the mechanisms of synergy and expand the findings into small animal models.

Citation Format: Joshua N. Honeyman, Wen-I Chang, Varun V. Prabhu, Joshua E. Allen, Lanlan Zhou, Wafik S. El-Deiry. Imipridones exhibit synergy with sorafenib, HDAC inhibition, PARP inhibition, and proteasome inhibition in liver cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1040.
Abstract 1043: Enhancing cytotoxic chemotherapy effects by nintedanib in gastric cancer preclinical models
Authors: Awasthi Niranjan , Schwarz Margaret A. , Hilberg Frank , Schwarz Roderich E.
Published: July 2021

Background: Gastric adenocarcinoma (GAC) remains the 3rd most common cause of cancer mortality in the world. Systemic chemotherapy is a preferred treatment option for advanced and recurrent GAC, but response rates and survival extension remain limited. Tumor angiogenesis plays a critical role in GAC growth, invasion and metastasis. In this study, we determined the antitumor efficacy of nintedanib, a potent triple angiokinase inhibitor for VEGFR-1/2/3, PDGFR-α/β and FGFR-1/2/3, alone or in combination with chemotherapy, in preclinical models of GAC.

Methods: Animal survival and tumor growth studies were performed in 4-6 week-old female NOD/SCID mice with human GAC cell xenografts (MKN-45, KATO-III, SNU-5). The mechanistic evaluation involved Immunohistochemistry and Immunoblot analyses of subcutaneous tumors. In vitro cell viability assays were performed using colorimetric WST-1 reagent.

Results: In MKN-45 cell-derived peritoneal dissemination xenografts, animal survival was improved by nintedanib (33%), docetaxel (100%) or irinotecan (181%), while oxaliplatin, 5-FU and epirubicin had no effect. The addition of nintedanib to docetaxel (157%) or irinotecan (214%) led to a further extension in animal survival. In KATO-III cell-derived xenografts carrying FGFR2 gene amplification, nintedanib monotherapy extended survival much more (209%). Docetaxel and irinotecan effects were again further enhanced by nintedanib. In MKN-45 subcutaneous xenografts, nintedanib, epirubicin, docetaxel or irinotecan reduced tumor growth (range: 68-87%), while 5-FU and oxaliplatin had a smaller effect (40%). The addition of nintedanib to all chemotherapy groups demonstrated a further reduction in tumor growth. Subcutaneous tumor analysis revealed that nintedanib attenuated tumor cell proliferation, reduced tumor vasculature and increased tumor cell death. Nintedanib (10 μM) inhibited in vitro cell proliferation of mutationally different GAC cells by 25% (MKN-45), 75% (KATO-III) or 82% (SNU-5), and confirmed additive inhibitory effects in combinations with chemotherapy agents.

Conclusion: Nintedanib showed notable antitumor efficacy and significantly improved taxane or irinotecan chemotherapy responses. Combination regimens with nintedanib have the potential for improving clinical GAC therapy.

Citation Format: Niranjan Awasthi, Margaret A. Schwarz, Frank Hilberg, Roderich E. Schwarz. Enhancing cytotoxic chemotherapy effects by nintedanib in gastric cancer preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1043.
Abstract 1055: Novel and highly potent ATR inhibitor M4344 kills cancer cells with replication stress and enhances the chemotherapeutic activity of widely used DNA damaging agents
Authors: Jo Ukhyun , Senatorov Ilya , Zimmermann Astrid , Saha Liton Kumar , Murai Yasuhisa , Kim Se Hyun , Rajapakse Vinodh N , Elloumi Fathi , Takahashi Nobuyuki , Schultz Christopher , Thomas Anish , Zenke Frank , Pommier Yves
Published: July 2021

Purpose: Although several ATR inhibitors are in development, there are unresolved questions regarding their differential potency, molecular signatures of cancer patients for predicting activity and most effective therapeutic combinations. Here, we elucidate how to improve ATR-based chemotherapy with the newly developed ATR inhibitor, M4344 using in vitro and in vivo models.

Experimental design: The potency of M4344 was compared with the clinically developed ATR inhibitors BAY1895344, berzosertib, and ceralasertib. The anticancer activity of M4344 was investigated as monotherapy and combination with clinical DNA damaging agents in multiple cancer cell lines, patient-derived tumor organoids and mouse xenograft models. We also elucidated the anticancer mechanisms and potential biomarkers for M4344.

Results: M4344 is highly potent among the clinically developed ATR inhibitors. Replication stress (RepStress) and neuroendocrine (NE) gene expression signatures are significantly associated with a response to M4344 treatment. M4344 kills cancer cells by inducing cellular catastrophe and DNA damage. M4344 is highly synergistic with a broad range of DNA-targeting anticancer agents. It significantly synergizes with topotecan and irinotecan in patient-derived tumor organoids and xenograft models.

Conclusions: M4344 is a promising and highly potent ATR inhibitor. It enhances the activity of clinical DNA damaging agents commonly used in cancer treatment including topoisomerase inhibitors, gemcitabine, cisplatin and talazoparib. RepStress and NE gene expression signatures can be exploited as predictive markers for M4344.

Citation Format: Ukhyun Jo, Ilya Senatorov, Astrid Zimmermann, Liton Kumar Saha, Yasuhisa Murai, Se Hyun Kim, Vinodh N Rajapakse, Fathi Elloumi, Nobuyuki Takahashi, Christopher Schultz, Anish Thomas, Frank Zenke, Yves Pommier. Novel and highly potent ATR inhibitor M4344 kills cancer cells with replication stress and enhances the chemotherapeutic activity of widely used DNA damaging agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1055.
Abstract 1059: Combination of gemcitabine and cytidine deaminase inhibitor: A breakthrough cocktail for the treatment of pancreatic ductal adenocarcinoma
Authors: Patki Manali , Saraswat Aishwarya , Bhutkar Shraddha , Dukhande Vikas , Patel Ketankumar
Published: July 2021

Pancreatic ductal adenocarcinoma is a very common type of pancreatic cancer and is one of the deadliest cancers. Gemcitabine (Gem), is still the mainstay drug for the treatment of PDAC. Gem is a hydrophilic drug which requires nucleoside transporters for its efficient uptake. Once inside the cell, Gem is converted to an active triphosphate form which inhibits DNA synthesis and causes apoptosis. However, Gem gets inactivated by cytidine deaminase (CDA). Long term treatment with Gem leads to increased CDA production thereby developing resistance. We hypothesize that combination of Gem with CDA inhibitor like Zebularine (Zeb), could prevent the intracellular degradation of Gem. Combination can potentially result in enhancing the anti-cancer activity of Gem, reduced dose and dosing frequency and minimizing side effects. The objectives of our study were; (a) to investigate the cytotoxic interaction of Gem and Zeb alone and in combination (b) Evaluating effect of various apoptosis and proliferation markers using flow cytometry and western blot assay. Initially we explored the anti-proliferative effects of Gem and Zeb as a single drug and in combination on MiaPaca-2 cells using MTT assay. Combination index was calculated using chou talalay method and Combenefit software. Effect of Gem, Zeb, and combination treatment on the cell viability of 3D tumor spheroids and its area was carried out using fluorescence microscopy and ImageJ software respectively. Apoptosis study was carried out using Annexin V apoptosis kit.Gem and Zeb exhibited dose-dependent inhibition in viability of MiaPaca-2 cells with an IC50 of 0.48 and 67.82 µM, respectively. The combination index of 0.09 suggest “very strong synergism” (Table 1). 3D spheroid treated with combination exhibited a highly distorted surface with significantly higher number of dead cells. Our research confirms that this synergistic combination can be a breakthrough approach for the treatment of PDAC.

Table 1.IC50 of Gem, Zeb and combination of Gem+Zeb in Miapaca2 cellsDrugMolar RatioIC50 of GemIC50 of ZebCombination IndexSingle-0.4867.82Gem1:100.0440.40.09

Citation Format: Manali Patki, Aishwarya Saraswat, Shraddha Bhutkar, Vikas Dukhande, Ketankumar Patel. Combination of gemcitabine and cytidine deaminase inhibitor: A breakthrough cocktail for the treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1059.
Abstract 1065: Concomitant dexamethasone treatment and tumor treating fields induced cell death in glioblastoma
Authors: Schiesl Abigail , Linder Benedikt , Rödel Franz , Hehlgans Stephanie , Güllülü Ömer , Seifert Volker , Koegel Donat , Senft Christian , Dubinski Daniel
Published: July 2021

Objective: Patients suffering from Glioblastoma (GBM) usually present with perilesional edema which is the major contributor of neurologic deficit. Dexamethasone (Dex) has become the corticoid of choice due to its fast and effective alleviation of perilesional edema. The impact of concomitant Dex treatment on TTFields efficacy is unexplored to date.

Methods: Human GBM cell lines MZ54 and U251 were treated with TTF in combination with Dex and the effects on cell counts and cell death were determined via flow cytometry. We furthermore investigated the influence of Dex on the cell cycle distribution/cell cycle arrest with and without double thymidine block for synchronisation via flow cytometry in both cell lines.

Results: TTF treatment (200 kHz/250 kHz) for 72 h was able to induce massive cell death (70 %) in the cultures of both cell lines. Cell cycle analysis performed after synchronization showed that Dex treatment (65 µM/165 µM) evoked an accumulation of both cell lines in the G0/G1 phase. Concurrent Dex treatment had no significant influence on the extent of apoptosis induced by TTFields.

Conclusions: Despite the partial G0/G1 arrest induced by Dex, our results show no interference of Dex with the antitumorous capacity of TTF in vitro. These data support the hypothesis that concomitant therapy with Dex does not negatively interfere with TTF efficacy in primary GBM.

Citation Format: Abigail Schiesl, Benedikt Linder, Franz Rödel, Stephanie Hehlgans, Ömer Güllülü, Volker Seifert, Donat Koegel, Christian Senft, Daniel Dubinski. Concomitant dexamethasone treatment and tumor treating fields induced cell death in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1065.
Abstract 1071: Studying the ability of retinoids to inhibit growth of CRC cells based on their retinoid pathway genotype
Authors: Hunsu Victoria O. , Facey Caroline O. , Opdenaker Lynn M. , Boman Bruce M.
Published: July 2021

Colorectal cancer (CRC) tumorigenesis arises through acquisition of mutations in a colonic stem cell (SC) that transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC because CSCs appear to be resistant to chemotherapy and/or radiation. Because all-trans retinoic acid (ATRA) is a highly effective and often curative treatment for acute promyelocytic leukemia patients, we conjecture that retinoids may be an efficacious treatment for CRC. Retinoic acid (RA) is a metabolite of vitamin A which is known to induce differentiation of SCs and inhibit cell proliferation. Our goal is to discover how to therapeutically target human CRC CSCs using the differentiation-inducing effects of retinoid agents. Our previous study on the RA metabolizing enzyme aldehyde dehydrogenase (ALDH) shows that ALDH marks normal and malignant colonic SCs and that RA signaling mainly occurs through colonic ALDH+ SCs. Hypothesis: The ability of retinoid agents, ATRA, 9-cis retinoic acid, 13-cis retinoic acid and Liarozole, to induce differentiation of CSCs depends on the RA pathway genotype according to mutations in RA signaling components in CRC cells. Indeed, our bioinformatics analysis shows that many RA pathway genes are overexpressed and often mutated in CRC. We have also evaluated the ability of the different RA ligands to inhibit cell growth and induce differentiation using a panel of CRC cell lines that have specific RA signaling mutations. Further investigation of RA signaling mechanisms that regulate colon SCs and how dysregulation contributes to the SC overpopulation that drives CRC growth should provide insight into strategies for designing new SC-targeted therapies for CRC.

Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the ability of retinoids to inhibit growth of CRC cells based on their retinoid pathway genotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1071.
Abstract 1079: A synthetic lethal strategy using PARP and ATM inhibition for overcoming trastuzumab-resistance in HER2-positive cancers
Authors: Oh Kyoung-Seok , Nam Ah-Rong , Bang Ju-Hee , Seo Hye-Rim , Kim Jae-Min , Yoon Jeesun , Kim Tae-Yong , Oh Do-Youn
Published: July 2021

Background: DNA damage response (DDR) pathway is essential for maintaining genomic stability, cell survival, and tumorigenesis. However, the roles of DDR pathway in HER2-positive tumors remain unclear. This study aims to discover the function of DDR in trastuzumab-resistant (TR) cells to better understand HER2-biology and to suggest novel treatment strategy using DDR-targeting agents in HER2-positive TR tumors.

Methods: Four HER2-positive TR cell lines (NCI-N87TR, SNU-216TR, SNU-2670TR, and SNU-2773TR) and their corresponding parental cells (NCI-N87, SNU-216, SNU-2670, and SNU-2773) were subject to immunoblotting to identify expression profiles of DDR proteins. Proteasome inhibitor MG-132 and cycloheximide chase assay were used for understanding PARP1 dynamics in TR cells. MTT assay and colony-forming assay were performed to evaluate antiproliferative effect of PARP inhibitor (PARPi, olaparib), ATM inhibitor (ATMi, AZD0156) and ATR inhibitor (ATRi, Ceralasertib). DNA damage and homologous recombination repair (HRR) efficiency were measured by comet assay and immunofluorescence respectively. Cell cycle analysis using flow cytometry was performed to assess drug-induced apoptosis.

Results: Increase of PARP1 expression was observed in all four TR cells. Decreased proteasomal degradation of PARP1 was found in TR cells compared to parental cells. Olaparib showed better anti-proliferative effect in TR cells compared to parental cells. After treating with olaparib, there was a defect in formation of Rad51 foci, instead, there was induction of phosphorylation of ATM and ATR in TR cells, which might attenuate the effects of olaparib in TR cells. Combination of olaparib with AZD0156 showed stronger anti-tumor effect than olaparib monotherapy in TR cells. Moreover, synthetic lethal interaction was observed by olaparib/AZD0156 combination in TR cells. Interestingly, this synthetic lethal interaction was exclusively occurred in TR cells not in parental cells. Even though olaparib and ceralasertib combination showed better anti-tumor effect than each single drug in some TR cells, synthetic lethal interaction was not observed by olaparib/ceralasertib combination in TR cells.

Conclusion: Dual inhibition of PARP1 and ATM could be a good strategy to overcome the resistance to trastuzumab in HER2-positive cancers using its synthetic lethal interaction.

Citation Format: Kyoung-Seok Oh, Ah-Rong Nam, Ju-Hee Bang, Hye-Rim Seo, Jae-Min Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. A synthetic lethal strategy using PARP and ATM inhibition for overcoming trastuzumab-resistance in HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1079.
Abstract 1085: PD-L1-mediated resistance to chemotherapy is overcome by an irreversible JNK inhibitor in colorectal cancer cells
Authors: Sinicrope Frank A. , Sun Lei , Patai Arpad , Hogenson Tara L. , Fernandez-Zapico Martin E. , Qin Bo
Published: July 2021

Background Colorectal cancers (CRCs) are intrinsically drug resistant tumors with frequent low or absent tumor cell PD-L1 expression. Previously, we reported that PD-L1 depletion in CRC cells can confer chemoresistance. Given that JNK signaling has been implicated in cell survival and drug resistance in CRC cells, we examined the role of JNK and its regulation in chemoresistance in CRC cells with loss of PD-L1.

Materials & MethodsHuman CRC cell lines (RKO, DLD1 and HCT15) were utilized and parental and PD-L1 knockout/knockdown cells were generated. Cells were treated with a JNK1-3 inhibitor (JNK-IN-8) alone or combined with a MEK1/2 inhibitor (cobimetinib), CPT-11 or oxaliplatin. Protein/protein interaction was analyzed by immunoprecipitation (IP); protein/RNA interaction was analyzed by RNA IP assay. RNA level and half life were measured by QRT-PCR. Apoptosis was measured by cleaved caspase-3 and quantified by Annexin V labeling using FACS. Colony formation assay was performed.

ResultsPD-L1 depletion in CRC cells was shown to enhance JNK activity that was due to reduced mRNA stability of the upstream CYLD deubiquitinase. Since PD-L1 competes with the ribonuclease EXOSC10 for binding to CYLD mRNA, loss of PD-L1 increases the interaction of EXOSC10 and CYLD mRNA resulting in its ribonuclease-mediated degradation and activation of JNK signaling. JNK activation increased BIM phosphorylation at Thr116 that promotes its sequestration by MCL-1 and BCL-2. Notably, a selective and irreversible JNK1-3 inhibitor (JNK-IN-8) reduces BIMThr116 phosphorylation and can release BIM from its sequestration by MCL-1 and BCL-2 to promote apoptosis. Furthermore, use of JNK-IN-8, an MCL-1 antagonist (AZD5991), or their combination in tumor cells lacking PD-L1 is shown to promote apoptosis and reduce long-term clonogenic survival induced by multiple anti-cancer drugs. As confirmation, knockdown of PD-L1 can confer drug resistance in a human colon cancer-derived organoid model that can be reversed by JNK-IN-8.

Conclusion Tumor cells with low or absent PD-L1 expression show increased JNK activity that promotes BIM sequestration by MCL-1/BCL-2 to confer multiple drug resistance that can be reversed by a JNK inhibitor.

Citation Format: Frank A. Sinicrope, Lei Sun, Arpad Patai, Tara L. Hogenson, Martin E. Fernandez-Zapico, Bo Qin. PD-L1-mediated resistance to chemotherapy is overcome by an irreversible JNK inhibitor in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1085.
Abstract 1135: Antitumor activity of novel reversible LSD1 inhibitors in preclinical models of AML and SCLC
Authors: Byun Joo-Yun , Lee Won Jong , Kim Won jung , Kim Minjeong , Bae Inhwan , Hong Seokhyun , Kim Yu-Yon , Park Hyunjin , Kim Eunyoung , Kim Young Hoon , Ahn Young Gil , Suh Kwee Hyun
Published: July 2021

Lysine-specific demethylase 1 (LSD1) has become a biologically important and validated epigenetic target for cancer therapy since its identification in 2004. LSD1 mediates many cellular signaling pathways and is involved in the initiation and development of cancers. Aberrant overexpression of LSD1 has been observed in different types of cancers, and such epigenetic dysregulation plays a critical role in pathogenesis of various types of cancers such as acute myeloid leukemia (AML) or small cell lung cancer (SCLC). In this study, we introduce HM97346 and its derivatives as novel LSD1 inhibitors, and report a therapeutic potential for AML and SCLC patients based on several preclinical characterization and assessment. HM97346 and its derivatives were designed and synthesized as novel reversible inhibitors of LSD1. Standard cell proliferation assay, immunoblotting, biomarker expression, and apoptosis analysis were carried out to validate their potency in vitro studies using different AML and SCLC cell lines. The in vivo antitumor activity was evaluated in AML and SCLC cell xenograft mouse models. Tumor sizes were measured and tumor samples were analyzed to define the mechanisms of action. HM97346 and its derivatives showed strong inhibition on LSD1 enzymatic activity in biochemical assay and promoted the methylations of histone H3 lysine 4 (H3K4) in both SCLC and AML cells. They reduced the expression of neuroendocrine factors ASCL1 in SCLC cells, effectively and dose-dependently. Moreover, in human AML cell lines, they stimulated cellular differentiation by disrupting LSD1-GFI1 complex and induced transcription factor PU.1 as well as myeloid differentiation markers CD86 and CD11b. As a result, they significantly inhibited cellular proliferation and induced apoptosis in studies using AML and SCLC cells. Oral administration of HM97346 and its derivatives inhibited tumor growth in human AML and SCLC cell xenograft mouse models studies. These antitumor effects dramatically increased in combination with standard chemotherapies. In conclusion, HM97346 and its derivatives, new synthesized reversible LSD1 inhibitors, showed potential anticancer activity in preclinical AML and SCLC models. Moreover, these compounds could provide a new insight and possibility in cancer therapy through epigenetic modulation as not only monotherapy but also combination therapy.

Citation Format: Joo-Yun Byun, Won Jong Lee, Won jung Kim, Minjeong Kim, Inhwan Bae, Seokhyun Hong, Yu-Yon Kim, Hyunjin Park, Eunyoung Kim, Young Hoon Kim, Young Gil Ahn, Kwee Hyun Suh. Antitumor activity of novel reversible LSD1 inhibitors in preclinical models of AML and SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1135.
Abstract 1162: Monitoring COVID-19 RNA methyltransferases activities for developing therapeutic drugs
Authors: Goueli Said A. , Hsiao Kevin
Published: July 2021

The recent pandemic outbreak of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-Cov-2) was first reported in Wuhan, China in late 2019 resulting in infection of over 20 million and death of over one million world-wide as of October 2020. The infection by this virus is highly transmissible and can lead to severe pneumonia and death. The SARS-CoV-2 is an enveloped virus and belongs to the beta coronavirus family containing a large and complex positive-sense single stranded RNA genome (~30kb).It binds to human ACE2 receptor with high affinity, compared to the other CoVs, via its spike protein (S) and membrane fusion leading to the release of its genomic RNA that replicates and produce higher number of subgenomic RNAs. The viral RNA is then capped by nsp 14 and nsp10/16, two nonstructural proteins that catalyze the methylation of RNA to limit its degradation by cellular 5'-3'exonuclease and evade host immune system. These enzymes play critical role in translation of the viral RNA and its replication and thus leading to viral survival. We have used a bioluminescent high throughput enzyme assay for monitoring the activity of these enzymes in order to develop novel inhibitors which can be advanced for therapeutic treatment. We show that we can determine the activity of nanogram amounts of these enzymes and test the activity of these enzymes using different inhibitors, some of which are competitive with S-adenosyl methionine the canonical substrate for these enzymes. We also show the effect of inhibitors that compete with the oligonucleotide substrate. Finally, we tested the effect of a peptide modulator of COVID-2 on the activity of both nsp10/16 and nsp14. The assay is simple, homogenous, fast, and bioluminescent which provides a very robust platform for testing novel compounds on the viral methyltransferases leading to the generation of new drugs towards inhibiting the replication of COVID19

Citation Format: Said A. Goueli, Kevin Hsiao. Monitoring COVID-19 RNA methyltransferases activities for developing therapeutic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1162.
Abstract 1163: Histone deacetylase inhibitors as potential differentiation therapy in lung adenocarcinoma
Authors: Chong Wai Chin , Vaghjiani Vijesh , Cain Jason Edward
Published: July 2021

Impaired differentiation is a common feature of cancer. In contrast to chemotherapy, the goal of differentiation therapy is to exhaust the malignant potential of tumour cells through the reactivation of developmental programs rather than cell death. The Switch/Sucrose Non-Fermentable (SWI/SNF) DNA remodelling complexes play a vital role in regulating cellular differentiation and have been strongly implicated in cancer. SWI/SNF complexes bind to the DNA-histone scaffold and initiate transcription via the recruitment of vital cellular transcriptional machinery including histone acetyltransferases. Next-generation sequencing has identified mutations in at least 10 genes encoding subunits of the SWI/SNF complexes that are recurrently mutated in cancers originating in nearly every body tissue, collectively occurring in >25% of all human malignancies. Lung adenocarcinoma (LAC) accounts for >40% of all lung cancers and has an 18% of overall 5-year survival. Mutations in SMARCA4 are believed to occur in >12% of LAC.

Using a panel of characterized human LAC cell lines we show a marked sensitivity of the HDACi, HDACi, exclusively to SMARCA4-null LAC only. This is preceded by increased H3K27ac, decreased H3K27me3 and decreased EZH2 expression. Sustained low-dose panobinostat treatment of SMARCA4-mutant LAC cell lines, results in unique gene expression changes consistent with differentiation, not observed in SMARCA4 wildtype lines. To further demonstrate the specific dependence of loss of SMARCA4 on response to HDACi, we generated three isogenic cell lines using CRISPR KO. H358, H441 and H2009 SMARCA4 KO LAC cell lines were markedly more sensitive to panobinostat than their wildtype parental lines. Lastly, panobinostat treatment results in tumour growth arrest and increased survival in SMARCA4-deficient LAC cell line xenografts and PDX models in vivo but not in SMARCA4-intact LAC models. Together, these data suggest that SMARCA4-deficient LAC are dependent on HDAC activity to maintain an undifferentiated state and are therefore highly susceptible to epigenetic differentiation therapy.

Citation Format: Wai Chin Chong, Vijesh Vaghjiani, Jason Edward Cain. Histone deacetylase inhibitors as potential differentiation therapy in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1163.
Abstract 1165: Complementary diagnostics for precision targeting of cellular pathways
Authors: Siddaway Robert , Nobre Liana , Johnson Monique , Milos Scott , Ryall Scott , Sheth Javal , Tabori Uri , Hawkins Cynthia
Published: July 2021

Precision medicine aims to tailor treatment to an individual patient through understanding the molecular basis of their disease. Targeted therapies have the potential to optimize responses and minimize side effects, while also offering savings to the healthcare system through tailoring treatment and avoiding futile therapies. Regimes are usually designed through identifying DNA-level alterations in the tumour and selecting drugs tailored to that mutation. However, cancer is not a one-pathway disease: not all patients with a particular mutational profile will respond to the same treatment, and patients without a canonical pathway-activating alteration will be excluded from potentially life-saving treatment. To address this, we developed a NanoString assay that combines proteomic and transcriptomic profiling of 4 key cancer-related, actionable pathways (MAPK, PI3K, NF-κB and JAK/STAT). Using RNA-Seq data from gold standard cell lines with defined changes in each pathway we generated minimal sets of genes indicative of pathway activation, and integrated this with total and phospho protein measurements to show pathway activation levels. The combined panel was run on a series of isogenic cell lines and glioma samples with both known and unknown driving alterations. Overall, pathway activation is more variable than would be predicted based on DNA alterations alone, with alternate pathway activation emerging as a potential new target and implying that consideration of proteomic and/or transcriptomic-level information will be important for future therapeutic decision-making.

Citation Format: Robert Siddaway, Liana Nobre, Monique Johnson, Scott Milos, Scott Ryall, Javal Sheth, Uri Tabori, Cynthia Hawkins. Complementary diagnostics for precision targeting of cellular pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1165.
Abstract 1176: CHD4 predicts aggressiveness in PTC patients and promotes cancer stemness and EMT in PTC cells
Authors: Poyil Pratheeshkumar , Siraj Abdul K. , Divya Sasidharan Padmaja , Parvathareddy Sandeep Kumar , Diaz Roxanne , Begum Rafia , Al-Obaisi Khadija , Thangavel Saravanan , Al-Sobhi Saif S. , Al-Dayel Fouad , Al-Kuraya Khawla S.
Published: July 2021

Papillary thyroid carcinoma (PTC) is the most common histologic subtype, accounting for nearly 90% of thyroid cancer. Chromodomain-helicase-DNA-binding protein 4 (CHD4), a core subunit of the nucleosome remodeling and deacetylation (NuRD) complex is highly expressed in several cancers. However, its role in the pathogenesis and progression of PTC has not been investigated. Immunohistochemistry was performed to assess CHD4 protein expression in PTC using a tissue microarray. We also performed in vitro analysis to investigate the role of CHD4 in regulating cancer stemness and EMT in PTC cells. CHD4 overexpression was observed in 45.3% (650/1436) of PTCs, and was associated with aggressive clinico-pathological parameters and worse outcome. Functional analysis using PTC cell lines showed that forced expression of CHD4 promoted cell proliferation, spheroid growth, migration, invasion, and progression of epithelial to mesenchymal transition (EMT) in PTC cells whereas its knockdown reversed the effect. Methylation of E-cadherin was associated with loss of expression in CHD4 expressing cells, while CHD4 depletion reactivated E-cadherin expression. Most importantly, knockdown of mesenchymal transcriptional factors, Snail1 or Zeb1, attenuated the spheroid growth in CHD4 expressing PTC cells, showing a potential link between EMT activation and stemness maintenance in PTC. These findings suggest that CHD4 might be a promising therapeutic target in the treatment of patients with an aggressive subtype of PTC.

Citation Format: Pratheeshkumar Poyil, Abdul K. Siraj, Sasidharan Padmaja Divya, Sandeep Kumar Parvathareddy, Roxanne Diaz, Rafia Begum, Khadija Al-Obaisi, Saravanan Thangavel, Saif S. Al-Sobhi, Fouad Al-Dayel, Khawla S. Al-Kuraya. CHD4 predicts aggressiveness in PTC patients and promotes cancer stemness and EMT in PTC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1176.
Abstract 1181: Neratinib induces synthetic lethality with PARP inhibitors in triple negative breast cancer cells in vitro and in vivo
Authors: Xing Chuan , Zhang Zhuo , Manna Deborah Della , Diala Irmina , Eli Lisa , Lalani Alshad S. , Yang Eddy Shih-Hsin
Published: July 2021

Background: We previously showed preclinical and clinical activity of combined inhibition of EGFR and PARP with lapatinib and veliparib in triple negative breast cancer (Nowsheen et al. PLoS One 2012;7(10):e46614; Reasor et al. J Clin Onc 2018; 36(15_suppl):1095), due to an induced homologous recombination repair defect with EGFR inhibition sensitizing cells to PARP inhibition. In this study, we tested the in vitro and in vivo activity of the irreversible pan-HER tyrosine kinase inhibitor, neratinib, with the PARP inhibitor olaparib or niraparib.

Methods: Human triple negative breast cancer (TNBC) cells MDA-MB231 (BRCA wt), MDA-MB453 (BRCA wt), and MDA-MB436 (BRCA1 mutant) and Mouse TNBC 4T1 cells (BRCA wt) were used. Cell proliferation and colony formation assays were performed to assess the in vitro activity of various concentrations of neratinib and olaparib. Markers of DNA damage were measured via gamma-H2AX western blot analysis. In vivo effects of combination treatments on tumor metastasis were tested using intracardiac injection of tumor cells labelled with luciferase and mice were monitored for survival.

Results: Combining neratinib with olaparib or niraparib significantly decreased proliferation compared to either agent alone in MDA-MB231, MDA-MB453, MDA-MB436 and 4T1 cells. Similar results were observed using colony formation assays. Cytotoxicity was associated with increased gamma-H2AX levels, indicating persistent DNA damage. Mice treated with 20mg/kg/day of neratinib and 100mg/kg/day of olaparib after MDA-MB231-Luc intracardiac injection exhibited reduced tumor metastases compared to either agent alone, resulting in better survival (p=0.0088).

Conclusions: Combining neratinib with PARP inhibition induces synthetic lethality resulting in increased anti-tumor activity in TNBC in cell lines and in xenografts. Future clinical trials testing these combinations are warranted.

Citation Format: Chuan Xing, Zhuo Zhang, Deborah Della Manna, Irmina Diala, Lisa Eli, Alshad S. Lalani, Eddy Shih-Hsin Yang. Neratinib induces synthetic lethality with PARP inhibitors in triple negative breast cancer cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1181.
Abstract 1200: Long-duration term TTFields treatment of glioblastoma cells induces cell death
Authors: Castiglione Stefania , Ranucci Matteo , Cannavale Gaetano , Cianci Francesca , Verduci Ivan , Mazzanti Michele
Published: July 2021

Glioblastoma is the most aggressive among brain tumors. Radiotherapy and chemotherapy, as adjuvanttreatments after surgical removal of the tumor mass, are often accompanied by undesirable symptoms. Evenwith these available treatment options, patient prognosis is extremely poor. The only approved treatmentoption demonstrating improved life expectancy in the last twenty years has been the application of TumorTreating Fields (TTFields). However, despite different therapeutic actions, tumor relapse occurs inexorably inmost patients. It is essentially unanimously agreed upon that tumor recurrence is due to a small populationof cells named glioblastoma stem cells (GSCs). GSCs represent a restricted cellular subpopulation in the tumormass composed mainly of bulk cells. The main characteristic of GSCs is their tumorigenic capacity even if asmall number of cells are left after surgery. Our working hypothesis is based on evidence that TTFieldsexposure destabilizes the mitotic spindle of rapidly dividing cells to elicit cell death. Thus, TTFields stimulationshould preferentially target bulk cells due to their higher proliferation rates compared to GSCs. Preliminaryexperiments performed using glioblastoma primary cultures obtained from patients' biopsies demonstratedthe presence of two different cell populations. In glioblastoma primary cultures maintained in stem-cellselective medium, more than 90% of glioblastoma cells did not exhibit stemness characteristics. After 72hours of TTFields stimulation, cell death was ascertained in approximately 50% of cells. The remaining viablecells were more than 20% positive for three different stemness markers. Long duration TTFields treatmentexperiments (144 and 288 hours) showed that TTFields at 200 kHz is able to increase the percentage of GSCin glioblastoma cells, yet did exhibit a propensity to reestablish cellular proliferation.In conclusion, long duration TTFields (200 kHz) treatment demonstrated efficacy by inducing cell death anddelaying glioblastoma cell expansion. However, to prevent glioblastoma relapse it is important to combineTTFields with different combination partners that specifically target glioblastoma stem cells.

Citation Format: Stefania Castiglione, Matteo Ranucci, Gaetano Cannavale, Francesca Cianci, Ivan Verduci, Michele Mazzanti. Long-duration term TTFields treatment of glioblastoma cells induces cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1200.