bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4029: BET inhibitor suppresses neutrophil-dependent lung metastasis of inflammatory renal cancer cells
Authors: Ehata Shogo , Nishida Jun , Miyakuni Kosuke , Koinuma Daizo , Miyazono Kohei
Published: September 2020

Recent studies have shown that cancer cells alter the immune landscape by secreting inflammation-related cytokines or chemokines through a process known as cancer cell-intrinsic inflammation. Cancer cell-intrinsic inflammation facilitates both the initial progression of cancers and cancer metastasis via tumor microenvironment reconstitution, immune surveillance, and premetastatic niche formation. However, in the case of clear cell renal carcinoma (ccRCC), the role of cancer cell-intrinsic inflammation is not fully uncovered. In the present study, we explored the function and regulatory mechanisms of cancer cell-intrinsic inflammation in ccRCC.

First, highly malignant derivatives were obtained through the repetitive exposure of human ccRCC cells into mouse renal microenvironment. Of note, these derivatives elicited cancer cell-intrinsic inflammation in the host mice, which facilitated lung metastasis in neutrophil-dependent manner. Thus, we defined these derivatives as inflammatory ccRCC cells and characterized in the following study.

Next, the regulatory mechanisms for the transcription of genes related to neutrophil dynamics were explored. RNA-sequencing analysis revealed that mRNA-expression levels of four CXC chemokines were up-regulated in all inflammatory ccRCC derivatives. ChIP-sequencing analysis of H3K27ac or bromodomain-containing 4 (BRD4) indicated that super enhancers were recruited at each gene in inflammatory ccRCC derivatives.

Finally, we evaluated the effect of the bromodomain and extra-terminal motif (BET) inhibitor on neutrophil-dependent lung metastasis of inflammatory ccRCC cells. Although the sizes of primary tumors in inflammatory ccRCC derivative-bearing mice were not altered by the treatment of BET inhibitor, those of metastatic lung tumors were significantly decreased.

Overall, our findings revealed a functional role of cancer cell-intrinsic inflammation, which primes neutrophils and drives metastasis of ccRCC cells. The primary tumor microenvironment enriched inflammatory ccRCC cells via epigenetic remodeling. We also showed the utility of inflammation management in suppressing metastasis by utilizing a BET inhibitor.

Citation Format: Shogo Ehata, Jun Nishida, Kosuke Miyakuni, Daizo Koinuma, Kohei Miyazono. BET inhibitor suppresses neutrophil-dependent lung metastasis of inflammatory renal 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 4029.
Abstract 4044: Pharmacological targeting of the circadian clock, a new frontier for cancer therapy
Authors: Sulli Gabriele , Rich Jeremy
Published: September 2020

The circadian clock imposes daily rhythms in cell proliferation, metabolism, inflammation and DNA damage response. Perturbations of these processes are hallmarks of cancer. This raises the hypothesis that pharmacological modulation of the circadian machinery may be an effective therapeutic strategy for combating cancer. Indeed, targeting a circadian regulator could affect multiple cancer vulnerabilities, thus mimicking combination therapy. Here we show that pharmacological modulation of clock genes is specifically lethal to cancer cells and oncogene-induced senescent cells and have no effect on the viability of normal cells or tissues. This novel strategy can target multiple oncogenic drivers (such as HRAS, BRAF, PIK3CA, lack of p53 and others) and is effective under hypoxic conditions. Autophagy and de novo lipogenesis two crucial metabolic nodes for cancer survival are simultaneously blocked by chemical clock modulators. Notably, this approach impairs glioblastoma growth in vivo and improves survival without causing overt toxicity in mice. These results indicate that pharmacological modulation of circadian regulators is a novel antitumor strategy, identifying a class of anticancer agents with a wide therapeutic window. This new therapeutic paradigm could offer promising tools for the treatment of highly heterogeneous tumors that are unaffected by targeted therapy. The integration of circadian biology into cancer research therefore offers new options for making cancer treatment more effective and selective.

Citation Format: Gabriele Sulli, Jeremy Rich. Pharmacological targeting of the circadian clock, a new frontier for cancer therapy [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 4044.
Abstract 4064: Optimization of the transposon expression system for electroporation based gene delivery
Authors: Chen Jian , Chen Youbang
Published: September 2020

Stable protein expression in therapeutic cells such as T cells and NK cells is important for long-lasting clinical effects in cell therapy such as CAR-T therapy. Stable protein expression can be achieved by the viral vector expression method, transposon based expression method and CRISPR/Cas9 mediated knock-in method. Transposon based expression method has the advantage of being relatively safe and cost-effective compared with other methods. However, in the actual application of the transposon systems such as the piggyBac (PB) system and the Sleeping Beauty (SB) system, it is quite challenging to achieve a high delivery efficiency and a high cell viability. We found that in addition to the choice of electroporation methodology, vector design and cell culture are two most important aspects that need to be optimized. Vector design dictates protein expression level as commonly anticipated, it also dictates the expression timing. We found that two distinct designs give two very different expression timing, one with initial high efficiency of expression which tapers down to about half, and another one with very low initial expression level and it goes up to a very high level after two weeks. We propose that the differential expression profiles of the vector systems can be applied to different circumstances to achieve better therapeutic effects.

Citation Format: Jian Chen, Youbang Chen. Optimization of the transposon expression system for electroporation based gene delivery [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 4064.
Abstract 4072: Analysis of clinical BRCA1/2 reversions identifies hotspot mutations and predicted neoantigens associated with therapy resistance
Authors: Pettitt Stephen , Frankum Jessica , Punta Marco , Lise Stefano , Alexander John , Chen Yi , Haider Syed , Tutt Andrew N. , Lord Christopher J.
Published: September 2020

Although PARP inhibitors and platinum salts form part of the standard-of-care for cancers with homologous recombination (HR) defects, drug resistance does emerge and is often caused by reversion mutations in BRCA1 or BRCA2. Reversion mutations are secondary mutations that restore functional protein or compensate for frameshift mutations. To better understand the nature and aetiology of these mutations, we collated, codified and analysed over 200 reversion mutations from 24 published studies. Most reversions have been reported as case reports or small clinical series - we have collected all published reports into a single freely-accessible database to which further cases can be submitted.

The majority of reported reversion mutations are from BRCA1/2 mutant serous ovarian cancer, reflecting the longer use of platinum and PARP inhibitors in this disease. Our analysis identified reversion “hotspots” and “deserts” in the N- and C-terminal coding regions (respectively) of BRCA2, suggesting that pathogenic mutations in these domains may be at higher or lower chance of reversion, an effect not seen for BRCA1. Missense and splice-site pathogenic mutations in BRCA1/2 also appeared less likely to revert than frameshift-causing mutations. Unexpectedly, whilst most reversions were <100 bp “second site” deletions, microhomology use was not universal, especially in BRCA1-mutant cancers, suggesting that multiple DNA repair processes cause reversion and these vary in BRCA1 vs. BRCA2 mutant tumours. Reversions in BRCA1 mutant tumours were less likely to be mediated by deletions.

Many reversions contain novel protein sequence not found in the wild type protein, for example where a stretch of out-of-frame protein sequence is retained between the pathogenic and reversion mutations. We modelled MHC binding affinities for these sequences and found that many reversions were predicted to encode potentially immunogenic neopeptides, suggesting a route to the treatment of reverted disease. These observations have implications for how drug resistance might be managed in BRCA-mutant cancers.

Citation Format: Stephen Pettitt, Jessica Frankum, Marco Punta, Stefano Lise, John Alexander, Yi Chen, Syed Haider, Andrew N. Tutt, Christopher J. Lord. Analysis of clinical BRCA1/2 reversions identifies hotspot mutations and predicted neoantigens associated with therapy resistance [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 4072.
Abstract 4074: Reversing acquired PARPi resistance of TNBC through combined inhibition of cMet and EGFR
Authors: Chu Yu Yi , Yam Clinton , Chen Mei-Kuang , Chan Li-Chuan , Nie Lei , Hung Mien-Chie
Published: September 2020

Front line therapeutic failure happened to half of triple-negative breast cancer (TNBC) patients represent the effective treatment strategies for these patients are urgently needed. Poly (ADP-ribose) polymerase (PARP) is currently the most promising drug target for BRCA-mutated TNBC, and PARP inhibitors (PARPi), olaparib and talazoparib, were recently approved for the treatment of metastatic breast cancer (including TNBC) in patients with germline BRCA1/2 mutations. Despite impressive response rates of ~60%, the prolongation in median progression-free survival with a PARPi is modest, suggesting the emergence of resistance. Several studies have reported that receptor tyrosine kinases (RTKs), such as c-MET (also known as hepatocyte growth factor receptor), are involved in resistance to various anti-neoplastic agents, including PARPi. However, the mechanism by which c-MET contributes to acquired resistance to PARPi in TNBC is not fully understood. In this study, we analyzed acquired PARPi resistant TNBC cells and found c-Met is hyperactivated in these cells with PARPi treatment. We further treated these cells with talazoparib and crizotinib (a multi-kinase inhibitor that inhibits c-MET) and found synergistic inhibition effects of cell proliferation. Unexpectedly, limited effects of talazoparib sensitivity showed while depleting c-MET in PARPi-resistant cells. Interestingly, we found epidermal growth factor receptor (EGFR) is also hyperactivated and interaction of EGFR/c-Met in these cells. Notably, combination of c-MET and EGFR inhibitors increased sensitivity to talazoparib in TNBC cells with acquired resistance to PARPi. Combining EGFR and PARP inhibitors also resulted in greater inhibition of proliferation in c-MET-depleted TNBC cells. Collectively, our findings suggested a potential combination therapy of inhibiting c-MET and EGFR to overcome acquired PARPi resistance in TNBC.

Citation Format: Yu Yi Chu, Clinton Yam, Mei-Kuang Chen, Li-Chuan Chan, Lei Nie, Mien-Chie Hung. Reversing acquired PARPi resistance of TNBC through combined inhibition of cMet and EGFR [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 4074.
Abstract 4102: Genome-wide CRISPRa screen reveals new drivers of Enzalutamide resistance in prostate cancer
Authors: Rodriguez Yara , Vatapalli Rajita , Han Huyin , Unno Kenji , Sagar Vinay , Chalmers Zachary , Lysy Barbara , Yoo Young A , Truica Mihai , Abdulkadir Sarki
Published: September 2020

Potent anti-androgens such as Enzalutamide (Enza) have been approved for the treatment of mCRPC. Despite initial response, nearly all patients eventually develop resistance to Enza. Although a few mechanisms of Enza resistance have been identified, none have been translated into a clinical benefit for recurrent patients. Our goal is to identify new therapeutic targets to overcome Enza resistance. We performed a genome-wide CRISPR activation screen using deactivated Cas9 (dCas9) fused to VP64 transcriptional activator in the androgen-dependent prostate cancer cell line (LNCaP). We used the Calabrese sgRNA library which targets 18,885 genes using 3 different sgRNAs per gene (56,762 sgRNAs). Cells expressing the gRNA library were treated with Enza (20µM) for 7 weeks to allow for enrichment of potential resistance genes. We identified 10 genes which were targeted by at least 2 sgRNAs with a Fold Change (FC) > 4 in the Enza vs control group. Notably, AR was the most enriched gene. AR up-regulation is one of the main drivers of Enza resistance in patients. Pathway enrichment analysis using all the sgRNAs with FC> 5 revealed an over-representation of the RB1, TP53 and TGF-β pathways. Importantly, these pathways are also known to drive Enza resistance. Our screen also identified new candidate genes that have never been linked to Enza resistance. Interestingly, most of these genes are transcription factors. We validated the screen results in vitro. Further analysis of this target genes in vitro and in vivo will allow us to identify novel genes and potential therapeutic targets for Enza resistance.

Citation Format: Yara Rodriguez, Rajita Vatapalli, Huyin Han, Kenji Unno, Vinay Sagar, Zachary Chalmers, Barbara Lysy, Young A Yoo, Mihai Truica, Sarki Abdulkadir. Genome-wide CRISPRa screen reveals new drivers of Enzalutamide resistance in prostate 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 4102.
Abstract 4103: Polyphenols from Artemisia annuaL (pKAL) exhibit anticancer effects on radio-resistant MDA-MB-231 cells by suppressing stemness phenotype and EMT process
Authors: Lee Won Sup , Ko Yougn Shin , paramanantham Anjugam , Kim GonSup , Shin Sung Chul , Kim Hye Jung
Published: September 2020

Artemisia annua L., (Gaddongsook in Korean) has been used as Korean folk medicine for anti-inflammatory, and anticancer activities. However, the molecular mechanisms for anti-cancer activities of Artemisia annua L. still need elucidating. Here, we determine whether pKAL exhibit the anti-cancer effects on RT-R breast cancer cells, and the molecular characteristics for the radio-resistant (RT-R) breast cancer. In addition, we further explore the molecular mechanisms for anticancer effects of pKAL on stemness and epithelial-mesenchymal transition (EMT). MTT and colony forming assay revealed that pKAL inhibited cell proliferation on both RT-R-MDA-MB-231 cells and MDA-MB-231 cells in a dose- and time-dependent manner. GO enrichment analysis of differentially expressed proteins between RT-R-MDA-MB-231 cells and MDA-MB-231 cells with the use of KEGG pathway analysis and PANTHER functional analysis revealed that MAPK signaling pathway might be used as a survival mechanism for the RT R-MDA-MB-231 cells to attain the resistance. The detailed western blot analysis for anti-cancer effects of pKAL revealed that it inhibited the expression of CD44 and Oct 3/4 (stem cell markers), and β-catenin and MMP-9 (EMT markers) of RT-R-MDA-MB-231 cells. In addition, pKAL inhibited STAT 3 activity that is upregulated in RT-R-MDA-MB-231 cells compared to in parental MDA-MB-231 cells. JNK and JAK2 inhibitors also inhibited STAT3 activity on both RT-R-MDA-MB-231 cells and MDA-MB-231 cells. However, JNK or JAK2 inhibitor could not suppress nor augment the anti-cancer effects of pKAL on CD44, and Oct 3/4 expression while JAK2 inhibitor could inhibit the expression of β-catenin, and MMP-9 expression. These findings suggest that pKAL-induced anticancer effects are not closely associated with JNK and JAK2/STAT3 signaling. In conclusion, this study suggested pKAL exhibit anticancer effects on radio-resistant MDA-MB-231 cells by suppressing stemness phenotype and EMT.

Citation Format: Won Sup Lee, Yougn Shin Ko, Anjugam paramanantham, GonSup Kim, Sung Chul Shin, Hye Jung Kim. Polyphenols from Artemisia annuaL (pKAL) exhibit anticancer effects on radio-resistant MDA-MB-231 cells by suppressing stemness phenotype and EMT process [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 4103.
Abstract 4108: The use of conditionally reprogrammed cells for high throughput screening for novel drug combinations to treat drug resistant triple negative breast cancers
Authors: Aguilar-Mahecha Adriana , Chabot Catherine , Santos-Martinez Nancy , Darini Cedric , Hajira Midhet , Kong Tim , Morin Genevieve , Huang Sidong , Park Morag , Basik Mark
Published: September 2020

Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer, with prognosis tightly dependent on its responsiveness to cytotoxic chemotherapy. Indeed, overcoming chemoresistance is a major unmet need in this disease. Patient derived xenografts (PDXs) are now widely used in research since they faithfully represent the patient's tumor. However, testing novel drug combinations is a challenge in these models since it is time consuming and quite expensive to test each novel combination. The development of conditionally reprogrammed cells (CRCs) offers the opportunity to perform high throughput screens on clinically relevant models in a more timely and less expensive manner. To this end, we developed a 7 CRCs from PDXs of drug-resistant TNBC tumors. We characterized these CRCs at the molecular level and validated drug resistance to confirm they matched treatment response in both clinical and PDX tumors from which they were derived. These CRCs underwent high-throughput screens of both compound and shRNA libraries of genes targeted by FDA-approved drugs to identify novel drug combinations as well as genetic vulnerabilities that could re-sensitize these cells to carboplatin or paclitaxel chemotherapy. We found that drugs such as mitomycin C and oxfendazole were putative chemo-sensitizers while genes such as ATR and CDK2 were identified as novel genetic vulnerabilities. Treatment of these drug-resistant cells with combinations of carboplatin with mitomycin C or with an ATR inhibitor resulted in synergistic growth inhibition in proliferation and colony formation assays. We are further validating these combinations in organoid models derived from PDXs. These results have the potential to lead to novel therapeutic strategies against chemoresistant TNBCs.

Citation Format: Adriana Aguilar-Mahecha, Catherine Chabot, Nancy Santos-Martinez, Cedric Darini, Midhet Hajira, Tim Kong, Genevieve Morin, Sidong Huang, Morag Park, Mark Basik. The use of conditionally reprogrammed cells for high throughput screening for novel drug combinations to treat drug resistant triple negative breast cancers [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 4108.
Abstract 4112: OTULIN promotes Wnt/β-Catenin activation by genotoxic treatments and enhances drug-resistance in breast cancer
Authors: Wang Wei , Mingqi Li , Chi Yayun , Xue Jingyan , Fahmy Beshoy , Wu Jiong , Wu Zhaohui
Published: September 2020

Patients with basal-like breast cancer and triple-negative breast cancer (TNBC) generally have a worse prognosis than other breast cancer subtypes due to rapid-arising therapeutic resistance accompanied by aggressive metastasis. The drug-resistant tumor cells, including cancer stem-like cells with intrinsic resistance or tumor cells acquired resistance during treatments, could adopt a drug-tolerant state and serve as seeds for recurrent metastatic lesions. However, the molecular events orchestrating the innate and adaptive therapeutic resistance are not completely understood. We found that a linear ubiquitin-specific deubiquitinase OTULIN is overexpressed in breast tumors, especially in the basal-like subtype. Increased OTULIN levels were also observed in PDX samples from chemo-refractory TNBC patients compared to those were responsive to chemotherapy. OTULIN overexpression significantly increased TNBC cell resistance to chemotherapy and is associated with shorter OS and DFS in breast cancer patients. Mechanistically, we found genotoxic treatments induce OTULIN phosphorylation, which is required for the activation of Wnt/β-Catenin signaling in TNBC cells. Inhibiting Wnt/β-Catenin signaling or OTULIN sensitized TNBC xenografts to Doxorubicin treatment in vivo. In TNBC patients, increased OTULIN levels are higher in subtypes enriched with Wnt/β-catenin signaling signature. Our findings suggest that OTULIN may increase TNBC therapeutic resistance and aggressiveness by promoting Wnt/β-Catenin activation in response to chemotherapy. Targeting OTULIN and the chemotherapy-induced Wnt/β-Catenin activation may serve as a promising strategy to mitigate drug resistance and reduce metastasis in TNBC patients.

Citation Format: Wei Wang, Li Mingqi, Yayun Chi, Jingyan Xue, Beshoy Fahmy, Jiong Wu, Zhaohui Wu. OTULIN promotes Wnt/β-Catenin activation by genotoxic treatments and enhances drug-resistance 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 4112.
Abstract 4123: ORIC-101 overcomes glucocorticoid receptor-mediated chemoresistance in pancreatic cancer models
Authors: Sun Jessica D. , Zhou Haiying , Kong Wayne , Yuen Natalie , Duong Frank L. , Barkund Shravani , Pankov Aleksandr , McWeeney Dan , Ye Qiuping , Kabbarah Omar , Friedman Lori S. , Daemen Anneleen , Junttila Melissa
Published: September 2020

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and one of the most lethal cancers, with a 5-year survival rate of 8%. Chemotherapy remains the main treatment option for patients with advanced and metastatic tumors. However, intrinsic resistance to chemotherapeutics and lack of effective targeted therapies are major factors contributing to the dismal prognosis. Activation of glucocorticoid receptor (GR) signaling confers resistance to chemotherapy in solid tumors, making GR antagonism an attractive combination treatment strategy to overcome chemotherapeutic resistance. ORIC-101 is a novel selective GR antagonist under clinical evaluation in combination with anticancer therapies. We previously reported that ORIC-101 can sensitize triple-negative breast cancer cells to chemotherapy by reversing multiple GR-modulated pathways. Here, we investigate the effects of ORIC-101 in preclinical models of PDAC. Employing in vitro functional assays and gene expression analysis, ORIC-101 exhibited chemo-potentiation in a panel of PDAC cell lines. Biomarker analysis of transcription activity following ORIC-101 treatment in vitro confirmed pharmacodynamic (PD) modulation of core GR target genes, indicative of suppressed GR signaling. Extending these findings in vivo across several PDAC xenograft models (SW1990, BxPC3, and HPAC) revealed that ORIC-101 blocks GR transcriptional activity induced by dexamethasone in a dose-dependent manner. The PD modulation observed in vivo correlated with ORIC-101 plasma exposure. Consistent with ORIC-101's ability to suppress GR activity, in vivo efficacy studies in combination with paclitaxel significantly inhibited PDAC tumor growth compared to the paclitaxel control group. These results in PDAC preclinical models support the clinical development of ORIC-101 in overcoming GR-driven chemoresistance. A phase 1b study of ORIC-101 in combination with nab-paclitaxel in patients with advanced or metastatic solid tumors is ongoing (NCT03928314).

Citation Format: Jessica D. Sun, Haiying Zhou, Wayne Kong, Natalie Yuen, Frank L. Duong, Shravani Barkund, Aleksandr Pankov, Dan McWeeney, Qiuping Ye, Omar Kabbarah, Lori S. Friedman, Anneleen Daemen, Melissa Junttila. ORIC-101 overcomes glucocorticoid receptor-mediated chemoresistance in pancreatic cancer models [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 4123.
Abstract 4138: Glycogen synthase kinase-3 inhibition impairs DNA damage response through ubiquitin ligase hHYD-mediated TopBP1 degradation
Authors: Ding Li , Roeck Kaely R. , Alekhina Olga , Schmitt Daniel M. , Billadeau Daniel D.
Published: September 2020

Glycogen synthase kinase-3 (GSK-3) is overexpressed and accumulates in the nucleus in several human malignancies including pancreatic cancer. We recently demonstrated that GSK-3 inhibition sensitizes pancreatic cancer cells to chemotherapy by abrogating the TopBP1/ATR-mediated DNA damage response. Specifically, GSK-3 kinase activity is required to stabilize TopBP1 protein. Herein, we investigated the mechanism by which GSK-3 phosphorylation of TopBP1 prevents its degradation. Interestingly, GSK-3 inhibition not only decreased the total levels of TopBP1, but also reduced the ability of TopBP1 to bind chromatin following gemcitabine treatment. Mutation of a putative GSK-3 phosphorylation site in TopBP1 (S860A) resulted in decreased TopBP1 stability and chromatin recruitment, whereas a phosphomimetic mutant (S860E) had the opposite effect. Furthermore, we found enhanced TopBP1 ubiquitination after GSK-3 inhibition as well as an increased interaction between TopBP1 and hHYD, an ubiquitin ligase that has been shown to ubiquitylate TopBP1 resulting in its degradation. Significantly, the S860A mutation showed a stronger interaction with hHYD compared to WT or the S860E mutant of TopBP1. In conclusion, our study identifies GSK-3 phosphorylation of TopBP1 as a mechanism to prevent the interaction with and ultimate ubiquitylation-mediated degradation by hHYD.

Citation Format: Li Ding, Kaely R. Roeck, Olga Alekhina, Daniel M. Schmitt, Daniel D. Billadeau. Glycogen synthase kinase-3 inhibition impairs DNA damage response through ubiquitin ligase hHYD-mediated TopBP1 degradation [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 4138.
Abstract 4139: Investigations of drug synergy reveal promising efficacy of EphA2 inhibition combined with CDK inhibition in triple-negative breast cancer
Authors: Wilson Kalin L. , Brantley-Sieders Dana M.
Published: September 2020

Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor prognosis relative to other breast cancers. Due to its lack of expression of commonly targeted cell surface markers and hormone receptors, there is a dearth of precision treatment options for this malignancy, with chemotherapy and radiation therapy remaining the major mainstays of care. Our group has previously reported that the receptor tyrosine kinase EphA2 is enriched in the basal-like/triple-negative breast cancer subtype and that this expression is associated with reduced recurrence-free survival. Furthermore, genetic knockdown or inhibition of EphA2 with the small molecule ALW-II-41-27 (ALW) reduces cancer cell growth in vitro and in vivo. In these studies, we sought to investigate whether the antitumor properties of ALW can be augmented by addition of other compounds to further reduce cancer cell growth and induce apoptosis. To this end, TNBC lines MDA-MB-231, BT-549, HCC1395, and HCC1187 were seeded in 96-well plates and exposed to ALW alone or in combination with chemotherapeutic agents doxorubicin and paclitaxel, CDK inhibitor SCH-727965, and MCL-1 inhibitor S63845 and cell viability assessed by MTT assay at 48 hours. While there was no significant reduction in viability for cells treated with ALW plus chemotherapy and no consistent improvement with addition of S63845 across cell lines, the combination of ALW with SCH-727965 performed significantly better than either drug alone (p < 0.05) in HCC1395 and BT-549 cells. To evaluate drug synergy, cells of all lines above were exposed to variable concentrations of SCH-727965, ALW, and the combination in a fixed ratio of their respective IC50 values to generate dose response curves, and these data analyzed via CompuSyn software. Combination indices (CI) were <1 for the majority of conditions across all cell lines, supporting synergistic interaction. Effects were most pronounced in HCC1395 cells with average CI 0.82. Furthermore, TUNEL assays show that addition of SCH-727965 to ALW significantly increases the induction of apoptosis by HCC1395 and HCC1187 cells. These preclinical studies demonstrate that combination of ALW and SCH-727965 potently reduces TNBC cell growth and promotes cell death, representing promising early data on the effects of EphA2 inhibition as part of a combination targeted therapeutic approach for triple-negative breast cancer.

Citation Format: Kalin L. Wilson, Dana M. Brantley-Sieders. Investigations of drug synergy reveal promising efficacy of EphA2 inhibition combined with CDK inhibition in triple-negative 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 4139.
Abstract 4165: Stable RUNX1p.(Leu56Ser) kinetics during the course of myeloid neoplasms disagree with putative RUNX1 poor prognosis status
Authors: Rai Lena , Phyo Sai , Nacheva Elisabeth
Published: September 2020

‘De novo Acute Myeloid Leukemia (AML) with mutated RUNX1' is a provisional WHO entity with poor prognosis. Missense variant, RUNX1c.167T>C, NP_001745.2:p.(Leu56Ser), with COSMIC reference and FATHMM score of 0.90 suggests it is ‘Pathogenic'; while dbSNP reference and population frequencies of >1.5% suggests ‘Benign'. To address this discrepancy, we studied RUNX1p.(L56S) kinetics in patients with primary or secondary Myeloid neoplasms receiving varied chemotherapy regimen. Bone marrow (BM) samples received were subjected to routine diagnostic algorithm including targeted next-generation sequencing. ¶Out of 227 patients with RUNX1 mutated Myeloid neoplasms, 75 (33%) had RUNX1p.(L56S) making it the most frequent RUNX1 variant (Table-1). RUNX1p.(L56S) was present at ≈55% (46.6%-62.4%) Variant Allele Frequency (VAF) in all cases except one where VAF was 99%. Paired T-test (95% CI) analysis of RUNX1p.(L56S) VAF at diagnosis and remission (p-value=0.89) or relapse/AML transformation (p-value=0.7017) showed no significant difference; similar to that of single nucleotide polymorphisms (SNPs) identified in these cases (remission samples, p-value=0.15; disease evolution, p-value=0.79). In contrast, VAF of other coexisting gene variants was significantly different between diagnostic and remission (p-value=0.02) or relapse/transformation (p-value=0.01) samples, largely owing to acquisition or loss of pathogenic variants. RUNX1p.(L56S) VAF remained stable in sequential samples despite variable BM blasts; nor was it significantly different amongst patients with adverse risk cytogenetics versus those with favourable or intermediate risk (p-value=0.60). In the MDS cohort, RUNX1p.(L56S) was not associated with morphological subtypes. ¶Our data demonstrates RUNX1p.(Leu56Ser) kinetics remain stable during Myeloid disease course, similar to co-occurring SNPs. Our data strongly recommend considering clinical relevance of individual variant when assessing gene mutation-based disease entities.

Table-1:Patient characteristics [includes only those with RUNX1p.(Leu56Ser)]AML (1o or 2o) [No./Median (Range)]MDS (1o or 2o)/MPN [No./Median (Range)]Fisher Exact (2-tailed; 95% CI)No. of patients1758No. of samples76105Genderp-value=0.27Male631Female1127Age (years)57.5 (13.6- 71.4)65 (20.8- 88.2)Cytogenetic risk groupp-value=1.00Favourable228Intermediate1215Adverse33Unavailable012Clinical outcomep-value= 0.24No. of patients in remission (immunophenotypic)79No. of patients with relapse/transformation63Time to event (months)21.0 (8-68)24.0 (10-49)

Citation Format: Lena Rai, Sai Phyo, Elisabeth Nacheva. Stable RUNX1p.(Leu56Ser) kinetics during the course of myeloid neoplasms disagree with putative RUNX1 poor prognosis status [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 4165.
Abstract 4169: The novel thioredoxin reductase inhibitor A-Z2 triggers intrinsic apoptosis and shows efficacy in the treatment of acute myeloid leukemia
Authors: Zhou Fuling , Zhang Dongdong
Published: September 2020

Chemoresistance and high incidence of relapse in acute myeloid leukemia (AML) patients are associated with thioredoxin (Trx) overexpression. Thus, targeting the Trx system has emerged as a promising approach to treating AML. Both arsenicals and azelaic acid (AZA) are thioredoxin reductase inhibitors (TrxR) and possess antileukemic effects. In this study, to exploit agents with higher potency and lower toxicity, we got some organic arsenicals and further synthesized a series of targeted compounds by binding AZA to organic arsenicals, and then screened the most effective one, N-(4-(1, 3, 2-dithiarsinan-2-yl) phenyl)-azelamide (A-Z2). A-Z2 showed a stronger inhibitory effect against TrxR activity and in AML cell lines than did AZA or arsenicals. Additionally, A-Z2 was less toxic to healthy cells compared with traditional chemotherapeutic drugs. A-Z2 induces apoptosis by collapsing of mitochondrial membrane potential, reducing ATP level, releasing of cytochrome c and TNF-α, activating of caspase 9, 8 and 3. Analysis of the mechanism revealed that A-Z2 activates the intrinsic apoptotic pathway by directly selectively targeting TrxR/Trx and indirectly inhibiting NF-κB. A-Z2's better efficacy and safety profile against arsenicals and azelaic acid were also evident in vivo. A-Z2 had better plasma stability and biological activity in rats. A-Z2-treated mice displayed significant symptom relief and prolonged survival in a patient-derived xenograft (PDX) AML model. Herein, our study provides a novel antitumor candidate and approach for treating AML.

Keywords: Acute myeloid leukemia, Thioredoxin reductase, TrxR inhibitor, organic arsenicals, NF-κB

Citation Format: Fuling Zhou, Dongdong Zhang. The novel thioredoxin reductase inhibitor A-Z2 triggers intrinsic apoptosis and shows efficacy in the treatment of acute myeloid leukemia [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 4169.
Abstract 4206: EUD-GK-001 is a novel kinase inhibitor with in vitro anti-lymphoma activity
Authors: Scalise Lorenzo , Tarantelli Chiara , Zuccotto Fabio , Spriano Filippo , Mascia Michele , Cascione Luciano , Gaudio Eugenio , Angiolini Mauro , Bertoni Francesco
Published: September 2020

Introduction. EUD-GK compounds, and in particular EUD-GK-91, show selectivity for a restricted number of kinases (ALK, RET, TRK, DDR2, ROS1) and have shown anti-tumor activity in specific solid tumor models (Zuccotto et al, EORTC-NCI-AACR 2016). Here, we have assessed a novel derivative of this class of compounds, EUD-GK-001, in a panel of lymphoma models.

Methods. Lymphoma cell lines, including both B- and T-cells lymphomas, were exposed to a large range of concentrations of EUD-GK-001 as single agent for 72h, followed by MTT proliferation assay and IC50 calculation. Cell cycle were assessed in four cell lines (FARAGE, DOHH2, TMD8, OCI-LY-10) treated with 500 nM of EUD-GK-001 for 72h. Baseline protein expression was assessed by mass spectrometry. Baseline gene expression was obtained by using the Illumina HumanHT 12 Expression BeadChips.

Results. EUD-GK-001 presented a median IC50 of 1.65 μM and 3.75 μM across 14 B- and 4 T- cell lines, respectively. Five out of 14 DLBCL cells lines, including both ABC- and GCB- subtypes, presented IC50 values lower than 1 μM, A dose-depended increase of the cells in the sub-G0 phase (15-55%) was observed in two GCB-DLBCL and two ABC-DLBCL cell lines after EUD-GK-001 exposure for 72h.

Taking advantage of available omics data for the tested cell lines (Cascione at al, AACR 2019), functional annotation of RNA and protein levels showed that TNF/NFKB, inflammation, IL6/JAK/STAT3, IL2/STAT5 signatures were enriched among the transcripts more expressed in the sensitive cell lines, while E2F, DNA repair, MYC signatures were enriched in the resistant cell lines. No genes coding for kinases were more expressed in both GCB (n.=2) and ABC (n=2) sensitive than in resistant DLBCL cell lines (GCB, n=2; ABC, n=3 R). However, three kinases, namely SRC, JAK2 and LCK, were significantly more expressed in EUD-GK-001 GCB-DLBCL sensitive cell lines (DOHH2, FARAGE) than in EUD-GK-001 resistant cells (SU-DHL-8, TOLEDO) and merging the top-50 mRNA and top-50 proteins identified by limma, SRC appeared as a central hub of the top genes associated with sensitivity to EUD-GK-001. The pattern of activity of EUD-GK-001 was however different from what reported for the SRC inhibitor dasatinib in the same lymphoma models (Scuoppo et al, PNAS 2019).

Conclusion. EUD-GK-001 is a novel small molecule with in vitro anti-tumor activity in subsets of lymphoma models, which needs to be further explored.

Citation Format: Lorenzo Scalise, Chiara Tarantelli, Fabio Zuccotto, Filippo Spriano, Michele Mascia, Luciano Cascione, Eugenio Gaudio, Mauro Angiolini, Francesco Bertoni. EUD-GK-001 is a novel kinase inhibitor with in vitro anti-lymphoma 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 4206.
Abstract 4234: Machine learning-driven label-free cell sorting for cell therapy
Authors: Ochiai Hiroshi , Sugimoto Keiki
Published: September 2020

Ghost cytometry (GC) is a recently realized approach for machine learning-driven analysis of image information without image production and enabled high throughput cell sorting through its integration with a microfluidic sorting technology. When a supervised machine learning is adopted in GC, a classifier model is first trained using a data set comprising of simultaneously measured imaging waveform signals and cell specific (ground truth) labels indicating cell types and functions. This trained model, in turn, is able to infer the cell specific label directly from the imaging waveforms at a speed of several orders of magnitude faster than the other methods, thus enabling subsequent cell sorting at high speed. Here, we investigate the applicability of label-free GC to manufacture and isolate the effective therapeutic cells.First, we tested classification of live T cells from dead cells to understand the potential of GC for monitoring the healthiness of live cells in cell manufacturing processes. A model for classifying the label-free information was trained using labels based on the positivity of annexin V and PI staining and applied to test data. Next, we assessed classification of T cells from live blood samples after hemolysis process. In this experiment, labels of a CD3 marker was used to construct a training data set to develop the T cell classifier. Finally, we applied the label-free GC to identify cells with low glycolytic activity because the cell metabolic status has a big impact on their therapeutic potency and persistency. Selection of the low glycolytic T cell may address current challenges on CAR-T cell therapy such as sustained tumor elimination. As a result, GC was found to classify not only live and dead cells but also apoptotic cells. Moreover, GC was able to discriminate CD3 positive T cells from PBMC with a good accuracy. These results indicate that our label-free GC can detect the healthy live cells without using any surface markers and could be beneficial for monitoring the quality of T cell products. Furthermore, the label-free GC may have a potential to classify the cells based on their phenotypic properties such as metabolic status. We would like to share our recent data at the meeting and discuss the potential of label-free GC for delivering sophisticated therapeutic cells, especially CAR-T cells, with good efficacy and persistency.

Citation Format: Hiroshi Ochiai, Keiki Sugimoto. Machine learning-driven label-free cell sorting for cell therapy [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 4234.
Abstract 4244: The efficacy and safety of anti-PD-1 therapy in metastatic sarcomas
Authors: Yang Jilong
Published: September 2020

Sarcomas are a group of rare and malignant tumors originating from mesenchymal tissue with a variety of cellular subtypes. Although several major standard treatments, including neo-adjuvant and adjuvant chemotherapy, surgery and radiation, have made breakthroughs to improve overall survival, there are few additional effective treatments for metastatic sarcomas that have failed traditional therapy. With the encouraging efficacy of PD-1 monoclonal antibodies in nonresectable or metastatic malignant melanomas and lung cancers, immunosuppressive agents provide new hope for the treatment of sarcomas. We retrospectively collected information from 15 patients with stage IV sarcomas who were treated with a PD-1 inhibitor with or without angiogenesis inhibitor after the failure of traditional treatments, from June 15, 2016 to September 30, 2019. The primary observation endpoint was median progression-free survival (mPFS), and the objective response rate (ORR) and the disease control rate (DCR) were secondary endpoints. Treatment-related adverse events (AEs) were evaluated. Patients were observed until death, loss to follow-up, or the end of the PD-1 inhibitor therapy. The average follow-uptime was 8.47 months (1.97-28.12 months). At 12 weeks, the ORR was 23.1% (3/13) and the DCR was 84.6% (11/13). Finally the overall median PFS was 5.06 months, the ORR was 15.4% (2/13) and the DCR was 61.5% (8/13). Common AEs (occurring with 15 patients) included grade 4 hypertension (n=1, 2.6%), grade 3 leukopenia (n=1,2.6%), grade 2 hand and foot syndrome(HFSR) (n=2, 5.1%), grade 1 reactive capillary hyperplasia (RCCEP) (n=7,17.9%), fatigue and weakness (n=5, 12.8%), digestive dysfunction (n=3,7.7%) and fever (n=3,7.7%). The results indicate that PD-1 inhibitors based therapies are effective, safe and well tolerated in patients with advanced sarcomas. The PD-1 inhibitor combined with anti-angiogenesis agents might provide a new and effective strategy to treat metastatic sarcomas.

Citation Format: Jilong Yang. The efficacy and safety of anti-PD-1 therapy in metastatic sarcomas [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 4244.
Abstract 4255: Reliable identification of both known and novel gene fusions using a high-efficiency amplicon-based NGS panel
Authors: LI Chenyu
Published: September 2020

Gene fusions and RNA splicing errors are important events frequently involved in tumorigenesis, and often represent clinically addressable targets for various cancers. The reliable detection of such mutations via NGS requires the development of cutting-edge technologies capable of overcoming many challenges. These include detecting fusion mutations regardless of the complexity of fusion structures, removing the length and quantity constraints of FFPE RNA, reducing ribosomal RNA interference, eliminating false negatives and positives, revealing the existing copies of mutations, and improving the reproducibility and ease of workflow. Here we describe the development and validation of a fast and user-friendly RNA-based fusion detection technology - CleanPlex® Fusion. Adding a universal primer at the 3' end of single-stranded cDNA via template switching allows us to eliminate the constraint of template length, and hence amplify damaged RNA fragments, such as FFPE RNA. The presence of a target-specific primer on one side enables the detection of any mutations on the RNA fragments, including novel ones. With a panel of 61 primers (covering most of fusion mutations of NSCLC) annealing to the 3' end portion of RNA, we demonstrated the high on-target rate, mapping rate, and low rRNA rate and primer-dimer rate. We validated the amplification of each amplicon by using fragmented total RNA. Additionally, we also made a dual-primer multiplex PCR panel covering the same set of fusion mutations as defined by the single-primer panel, confirmed its coverage of all fusion mutations using a synthetic pool of fusion targets, and detected all commercial reference fusion mutations targeted by the panel. We again demonstrated the detection of such reference fusion mutations using the single-primer mPCR panel, and demonstrated that the single-primer method even detected fusion mutations outside the boundary of the dual primers. Moreover, we showed that the detected fusion reads significantly correlate with copy numbers determined by digital PCR. Lastly, we validated detection of fusion mutations in cell lines and FFPE RNA. CleanPlex® Fusion therefore represent a robust and efficient NGS method for detecting fusion mutations.

Citation Format: Chenyu LI. Reliable identification of both known and novel gene fusions using a high-efficiency amplicon-based NGS panel [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 4255.