bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1211: Anti-LSR mAb with carboplatin shows the synergistic effect in epithelial ovarian cancer
Authors: Funauchi Masashi , Hiramatsu Kosuke , Serada Satoshi , Fujimoto Minoru , Naka Tetsuji
Published: July 2021

Objectives : Previously, we demonstrated anti-cancer effect of our newly developed monoclonal antibody (mAb) targeting lipolysis-stimulated lipoprotein receptor (LSR) in LSR-positive epithelial ovarian cancer (EOC) in vitro and in vivo. In EOC treatment, platinum agents which induce apoptosis are the key drugs over the decades, therefore, we aimed to demonstrate the synergistic effect of anti-LSR mAb and platinum agent against LSR-positive EOC cells.

Methods : We administrated anti-LSR mAb and carboplatin to RMG-I cells (platinum resistant and LSR-positive EOC cell line) and evaluated cell proliferation in vitro and in vivo. We also investigated whether anti-LSR mAb reverse platinum resistance of RMG-I in vitro. In addition, we analyzed changes in protein expression related to MAPK/ERK and apoptosis using western blotting after treatment.

Results : In RMG-I, anti-LSR mAb inhibited phosphorylation of MAPK pathway-related proteins including MEK and ERK, and activated apoptosis-related proteins. Although RMG-I showed the refractory for carboplatin, anti-LSR mAb reversed its platinum resistance and decreased IC50 value of carboplatin. As a result, anti-LSR mAb induced the apoptotic effect of carboplatin in RMG-I. Finally, the combination therapy of anti-LSR mAb and carboplatin significantly inhibited the tumor growth of RMG-I in a synergistic manner in vitro and in vivo (p<0.05). The combination therapy caused strong apoptotic changes in tumor.

Conclusions : We confirmed the synergistic effect of the combination therapy of anti-LSR mAb and platinum agent against LSR-positive EOC cells via apoptotic pathway. Our findings demonstrated that this combination therapy might be a new treatment option of EOC.

Citation Format: Masashi Funauchi, Kosuke Hiramatsu, Satoshi Serada, Minoru Fujimoto, Tetsuji Naka. Anti-LSR mAb with carboplatin shows the synergistic effect in epithelial 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 1211.
Abstract 1224: Discovery of novel BAF inhibitors for the treatment of transcription factor-driven cancers
Authors: Centore Richard C. , Soares Luis M. , Vaswani Rishi G. , Ichikawa Kana , Li Zhifang , Fan Hong , Setser Jeremy , Lahr David L. , Zawadzke Laura , Chen Xueying , Barnash Kimberly D. , Muwanguzi Jordana , Anthony Neville , Sandoval Gabriel J. , Feldman Katharine , Adam Ammar , Huang David , Schiller Shawn , Wilson Kevin , Voigt Johannes , Hentemann Martin , Millan David S. , Chan Ho Man , Bellon Steven F. , Decicco Carl P. , Xu Lan
Published: July 2021

The BRG/Brahma-associated factors (BAF) family of chromatin remodeling complexes (also referred to as the mSWI/SNF complex) regulates the chromatin landscape of the genome. Through its ATP-dependent chromatin remodeling activity, BAF regulates the accessibility of gene-control elements, allowing for the binding of transcription factors. Thus, BAF is a major regulator of lineage- and disease-specific transcriptional programs. We have discovered and developed a novel series of compounds that potently and selectively inhibits the ATPase components of the BAF complex, SMARCA4 and SMARCA2 (also called BRG1 and BRM, respectively). Mutational, structural, and biochemical studies demonstrated that these SMARCA4/SMARCA2 inhibitors act through a unique allosteric mechanism. Pharmacologic inhibition of the BAF complex resulted in lineage-specific changes in chromatin accessibility in cancer cell lines from diverse origins. Phenotypic screening of cancer cell lines showed that uveal melanoma and hematological cancer cell lines were exquisitely sensitive to BAF inhibition. In the example of uveal melanoma, BAF inhibition resulted in the loss of accessibility at the binding sites of the SOX10 and MITF transcription factors, two essential proteins in supporting the proliferation and survival of uveal melanoma cells. Enhancer occupancy of SOX10 and MITF was reduced upon BAF inhibition, and subsequently, the melanocytic and pigmentation gene expression program regulated by these master transcription factors was suppressed. Finally, in a mouse xenograft model of uveal melanoma, BAF inhibition was well tolerated and resulted in dose-dependent tumor regression that correlated with pharmacodynamic modulation of BAF-target gene expression. These data provide the foundation for first-in-human studies of BAF ATPase inhibition as a novel therapeutic to treat uveal melanoma.

Citation Format: Richard C. Centore, Luis M. Soares, Rishi G. Vaswani, Kana Ichikawa, Zhifang Li, Hong Fan, Jeremy Setser, David L. Lahr, Laura Zawadzke, Xueying Chen, Kimberly D. Barnash, Jordana Muwanguzi, Neville Anthony, Gabriel J. Sandoval, Katharine Feldman, Ammar Adam, David Huang, Shawn Schiller, Kevin Wilson, Johannes Voigt, Martin Hentemann, David S. Millan, Ho Man Chan, Steven F. Bellon, Carl P. Decicco, Lan Xu. Discovery of novel BAF inhibitors for the treatment of transcription factor-driven 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 1224.
Abstract 1225: LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of cancer
Authors: Natale Chris , Garyantes Tina , Ridky Todd
Published: July 2021

Women have a lower incidence of and improved clinical outcomes for most cancer types, but the mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that many cancer types lack classic nuclear estrogen receptors. The GPER agonist G-1, a racemate, has been demonstrated to have anticancer activities by many independent laboratories, though there are some contradictory findings in the literature. We resolved the enantiomers that compose G-1 and determined that LNS8801 is the sole enantiomer responsible for G-1's anticancer effects. Using CRISPR to deplete GPER, we determined that the anticancer activity of LNS8801 is dependent upon GPER expression within the tumor cells. Previously, we determined that the anticancer mechanism of GPER activation is through a rapid proteasomal degradation of c-Myc. The PRISM screen was performed on LNS8801 at the Broad Institute, and it was determined that this compound has universal anticancer activity across multiple cancer types, consistent with our inhouse data. Sensitivity to LNS8801 was highly correlated with expression of c-Myc target genes, which is consistent with the known mechanism of action. Lineage analysis identified two specific cancer types that were uniformly more sensitive to LNS8801 than other cancer types: diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL). DLBCL has historically been considered to be highly dependent on c-Myc, which is consistent with the known mechanism of action of LNS8801; however, the identification of ALCL was unexpected. Deeper analysis determined that high expression of anaplastic lymphoma kinase (ALK) fusion genes tightly correlated with sensitivity to LNS8801 in ALCL and other ALK+ cancer types. Using non-small cell lung cancer cell lines engineered to express EML4-ALK, we validated that ALK fusion expression results in increased sensitivity to LNS8801 relative to isogenic controls. This work has identified an enantiomerically pure GPER agonist that has potent activity across many cancer types, with c-Myc-dependent and ALK+ cancer types being enriched for heightened sensitivity.

Citation Format: Chris Natale, Tina Garyantes, Todd Ridky. LNS8801: A novel, enantiomerically pure, small molecule agonist of the G protein-coupled estrogen receptor (GPER) for the treatment of 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 1225.
Abstract 1226: Targeting OSCC fitness genes YAP1 and WWTR1 via synergistic killing of CA3 in combination with cisplatin
Authors: Chai Annie Wai Yeeng , Yee Pei San , Yee Shi Mun , Cheong Sok Ching
Published: July 2021

Oral squamous cell carcinoma (OSCC) is one of the most common forms of head and neck cancer, with more than half of the global mortality occurring in Asia. The current treatment regimens for OSCC are of limited benefit to the patients and the development of new targeted therapies is very much needed. In our previous study using genome-wide CRISPR/Cas9 screen to identify the genetic vulnerabilities for OSCC, we have successfully identified two key fitness genes - YAP1 and WWTR1 (TAZ) of the Hippo signaling pathway as potential therapeutic targets for OSCC. We discovered that CRISPR/Cas9-mediated knockout of either YAP1 or WWTR1 in the YAP1- or WWTR1-dependent OSCC cell lines resulted in the down-regulation of pro-proliferative genes such as the CTGF and CYR61. Further, both YAP1 and WWTR1 have separately been implicated as potential mediators of resistance to cisplatin, a common chemotherapy used for the treatment of OSCC patients. Inhibition of the interaction between either YAP1 or WWTR1 with their downstream binding partners, such as the transcription factor, TEAD, has been one of the main focus for therapeutic intervention. However, this has not been examined in the context of OSCC. We, therefore, aim to i) identify and evaluate the anti-proliferative effect of existing YAP1/WWTR1 inhibitors on YAP1/WWTR1-dependent OSCC, ii) evaluate the synergistic effect of combining the YAP1/WWTR1 inhibitors with cisplatin in controlling OSCC growth. We identify two putative inhibitors of YAP/WWTR1/TEAD - Verteporfin and CA3. We show that compared to Verteporfin, the new inhibitor, CA3 exerts a more prominent anti-proliferative effect on the OSCC models that are dependent on either YAP1 or WWTR1, with IC50 in the nanomolar range. We then test the effect of combining CA3 (at 0.5µM and 1µM) with various dosages of cisplatin and demonstrated strong synergistic effects in OSCC across most combinations. We show that CA3 sensitizes OSCC cells to cisplatin at concentrations as low as 0.5µM. Our results further confirm that YAP1/TAZ are important therapeutic targets in OSCC and the treatment of YAP1/TAZ inhibitors could sensitize the OSCC cells to cisplatin. Further investigation is warranted to uncover the mechanism underlying YAP/TAZ-mediated cisplatin sensitivity in OSCC to improve the proportion of OSCC patients who could respond to cisplatin.

Citation Format: Annie Wai Yeeng Chai, Pei San Yee, Shi Mun Yee, Sok Ching Cheong. Targeting OSCC fitness genes YAP1 and WWTR1 via synergistic killing of CA3 in combination with cisplatin [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 1226.
Abstract 123: A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation
Authors: Lo Yuan-Hung , Kolahi Kevin S. , Du Yuhong , Chang Chiung-Ying , Krokhotin Andrey , Nair Ajay , Sobba Walter D. , Karlsson Kasper , Jones Sunny J. , Longacre Teri A. , Mah Amanda T. , Sockell Alexandra , Seoane Jose A. , Chen Jin , Weissman Jonathan S. , Curtis Christina , Califano Andrea , Fu Haian , Crabtree Gerald R. , Kuo Calvin J.
Published: July 2021

Mutations in ARID1A, encoding a subunit of the BAF chromatin remodeling complex, rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward human genetic model systems. Here, CRISPR/Cas9 knockout of ARID1A in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation but impaired Wnt/β-catenin signaling. Genetic Wnt pathway activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative essential pathways of ARID1A-mediated transformation. ARID1A loss induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1 regulation of multiple mitotic regulatory genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to BIRC5/survivin inhibition, functionally implicating BIRC5/survivin as an essential mediator of proliferation following initial ARID1A loss. However, CRISPR inactivation of ARID1A in established gastric cancer cell lines did not elicit sensitivity to BIRC5/survivin inhibitors, suggesting that this pathway may be selectively required during the early establishment of tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, indicating the general utility of organoid-based forward genetic cancer analysis in human cells.

Citation Format: Yuan-Hung Lo, Kevin S. Kolahi, Yuhong Du, Chiung-Ying Chang, Andrey Krokhotin, Ajay Nair, Walter D. Sobba, Kasper Karlsson, Sunny J. Jones, Teri A. Longacre, Amanda T. Mah, Alexandra Sockell, Jose A. Seoane, Jin Chen, Jonathan S. Weissman, Christina Curtis, Andrea Califano, Haian Fu, Gerald R. Crabtree, Calvin J. Kuo. A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation [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 123.
Abstract 125: First report of a full spectrum of de novo induced human breast cancer subtypes generated from oncogene-transduced freshly isolated normal human mammary cells
Authors: Tan Susanna , Lefort Sylvain , El-Naggar Amal , Pellacani Davide , Sorensen Poul H. , Eaves Connie
Published: July 2021

Human breast cancers are genetically and biologically heterogeneous with variable responses to current treatments. As an alternative strategy to identify causal and shared mechanisms, we have been exploring the utility of creating de novo models of human breast cancers from freshly isolated, purified subsets of normal human mammary cells that are lentivirally transduced with defined genes of interest and then transplanted subcutaneously in Matrigel into highly immunodeficient adult female Nonobese-diabetic/Rag1-/--IL2Rγc-/- (NRG) mice. In an initial study using this approach, we discovered that forced expression of KRASG12D alone rapidly generates small (growth-arrested), but polyclonal and serially transplantable invasive ductal carcinomas (IDCs) from both normal basal and luminal progenitor subsets of normal human mammary cells. This outcome is neither replaced nor influenced by transduction of the cells with mutant forms of TP53 or PI3KCA (Nguyen et al Nature 2015). More recently, we have found that transduction of these normal human mammary cells with a cDNA encoding a myristylated form of AKT1 (myrAKT1) produces a model of ductal carcinoma in situ (DCIS), whereas forced expression of BMI1, MYC and TP53R273C with KRASG12D (KBMT) rapidly produces large and aggressive tumors within 6 weeks post-transplant, with intermediate outcomes obtained when either MYC or TP53R273C is omitted. Immunohistochemical staining for estrogen, progesterone and epidermal growth factor receptor 2 suggests that the KBMT tumors are triple negative, and that the proportion of proliferating cells in the KBMT tumors is higher than in the tumors induced by just KRASG12D. The latter finding is consistent with the known role of BMI1 as a positive upstream regulator of proliferation via its ability to inhibit expression of p16INK4a, although the relevance of this activity in the KBMT model has yet to be established. At the opposite end of the spectrum, we have also shown that co-induced suppression of Y-Box binding protein-1 (YBX1) expression blocks the in vivo growth of KRASG12D-induced IDCs and its forced increased expression enhances the growth of myrAKT-induced DCIS cells. Taken together, these findings illustrate the advantages of creating and characterizing de novo models of human breast cancers to identify and manipulate pathways that are required for the acquisition of malignant properties.

Citation Format: Susanna Tan, Sylvain Lefort, Amal El-Naggar, Davide Pellacani, Poul H. Sorensen, Connie Eaves. First report of a full spectrum of de novo induced human breast cancer subtypes generated from oncogene-transduced freshly isolated normal human mammary 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 125.
Abstract 1269: Pharmacological targeting of transcription-replication conflict leads to anti-cancer efficacy with minimal side effects in preclinical models
Authors: Gu Long , Li Caroline , Lingeman Robert , Hickey Robert J. , Malkas Linda H.
Published: July 2021

Unchecked growth of cancer cells places simultaneous demands on DNA replication and transcription. Targeting the transcription-replication conflict (TRC), a major source of genome instability, could have important therapeutic implications. However, testing this hypothesis is difficult without agents directly targeting TRC. Here, we report a small molecule ligand (AOH1996) of the replisome component PCNA, which stabilizes interaction between PCNA and the large subunit (rpb1) of RNA polymerase II through the APIM motif, resulting in proteasome-dependent degradation of rpb1 and lethal DNA damages. Orally administrable, AOH1996 suppresses tumor growth without discernable toxicity at more than 10 time the compound's effective dose. In addition, AOH1996 works synergistically with known DNA damage agents to kill cancer cells. These results reveal TRC as cancer-selective vulnerability and demonstrated the therapeutic potential of AOH1996.

Citation Format: Long Gu, Caroline Li, Robert Lingeman, Robert J. Hickey, Linda H. Malkas. Pharmacological targeting of transcription-replication conflict leads to anti-cancer efficacy with minimal side effects in 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 1269.
Abstract 1289: Conjugation of indomethacin with novel oleanolic acid oximes increases its Nrf2 and NF-κB signaling pathways modulating effect in pancreatic cancer cells
Authors: Baer-Dubowska Wanda , Narozna Maria , Krajka-Kuźniak Violetta , Bednarczyk-Cwynar Barbara
Published: July 2021

Chronic inflammation is a key factor in the etiology of neoplastic diseases, including pancreatic cancer. Nonsteroidal anti-inflammatory drugs (NSAIDs) were approved for the chemoprevention of colon tumors and suggested for pancreas cancers prophylaxis. Anti-inflammatory and anti-tumorigenic activities are also exerted by naturally occurring and synthetic triterpenoids. Coupling of triterpenoid analogues with NSAIDs may enhance this effect and prevent the unfavorable side effects related to NSAIDs long-term use. In this study novel oleanolic acid oxime (OAO) derivatives conjugated with indomethacin (IND) differing in substitution group at position C-17 were evaluated in the context of their possible modulating effect of Nrf2-ARE and NF-κB signaling pathways in pancreatic cancer cells.PSN-1 cells were incubated for 24h with IND and OAO-IND derivatives at the concentrations of 10µM and 20µM selected based on the results of the MTT assay. The activation of Nrf2 and NF-κB was assessed by the evaluation of its translocation into the nucleus and binding to specific DNA sequences by the ELISA assay. Expression of Nrf2, SOD-1, NF-κBp50, NF-κBp65 and COX-2 was evaluated by RT-PCR and Western blot methods.Cell viability was affected mostly by 3-indomethacinoxyiminoolean-12-en-28-oic acid morpholide and 3-indomethacinoxyiminoolean-en-28-oic acid benzyl ester. The level of Nrf2 in the nucleus and binding to ARE sequence was decreased in PSN-1 cells, similarly as was NF-κBp50 and NF-κBp65 binding and nuclear accumulation. As result of diminished activation of these transcription factors decreased expression of SOD-1 and COX-2 i.e. mRNA and protein levels were found. These results indicate that the OAO-IND derivatives, particularly morpholide and benzyl ester conjugates, are more potent suppressors of NF-κB signaling pathway than IND alone. Moreover, conjugation of IND with novel OAO may protect cancer cells against chemoresistance through inhibition of the Nrf2-ARE pathway. Those novel compounds might be considered the potential modulators of hepatocellular carcinoma therapy and chemopreventive agents.

Funding: This work was supported by Polish National Science Centre, grant 2016/21/B/NZ7/01758.

Citation Format: Wanda Baer-Dubowska, Maria Narozna, Violetta Krajka-Kuźniak, Barbara Bednarczyk-Cwynar. Conjugation of indomethacin with novel oleanolic acid oximes increases its Nrf2 and NF-κB signaling pathways modulating effect in pancreatic 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 1289.
Abstract 1297: Synthesis and structural analysis on an ionic diphenyltin complex as a potential anticancer agent
Authors: Witter Jade , Song Xueqing , Henry Tajera , Li William
Published: July 2021

Triorganotins are popular among chemists due to their potential biological properties against bacteria, fungi, and cancer cell lines. However, they have limited applications due to their poor water solubility. We hypothesize that solubility can be improved when the complex exhibits partial ionic characteristics. Therefore, it is our research goal to synthesize triphenyltin complexes that possess ionic characteristics. Syntheses of the ionic triphenyltin complex involved the reaction of a diprotic carboxylic acid, with triphenyltin chloride in the presence of di-isopropylamine. However, a tin-carbon cleavage reaction was observed, as IR/NMR spectroscopy and X-ray Crystallography studies showed that an ionic diphenyltin complex was successfully obtained in the reaction. The ionic complex consists of a diphenyltin anionic moiety, two oxalate ligands are observed coordinating to the tin atom in the bidentate mode, and two diisopropylammonium as the counterions. The anionic diphenyltin moieties has a distorted octohedral geometry with two carbons and two oxygens atom occupying the equatorial positions and two oxygen atoms occupying the axial positions. The ionic complex is essentially a 1-D polymer chain through an extensive hydrogen-bonding network between the oxalate groups (OCO) and the N atom from the ammonium cations. Compared to the similar reaction with triphenyltin hydroxide, which resulted in ionic triphenyltin complexes, cleavage of one phenyl group was observed for the first time in the reaction with triphenyltin chloride. Future studies will be focused on the mechanistic studies on the Sn-C cleavage.

Citation Format: Jade Witter, Xueqing Song, Tajera Henry, William Li. Synthesis and structural analysis on an ionic diphenyltin complex as a potential anticancer agent [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 1297.
Abstract 1310: Peptide delivery of siRNA in GBM
Authors: Boulos Jessica , Alexander-Bryant Angela
Published: July 2021

Glioblastoma Multiforme (GBM), a grade VI glioma, is an aggressive malignant tumorwith a poor prognosis. With an incidence rate of 3.19 per 100,000, GBM has a median survivalrate of 12-15 months and a five-year survival rate of less than 5%. Typical treatments involvesurgical removal, chemotherapy, and radiation; however, these treatments still result in a lowsurvival rate and can lead to potential harm to healthy cells. Therefore, a new therapeutic isneeded for GBM treatment to improve patient outcomes. RNAi, a naturally found process,utilizes siRNA to target specific mRNA for degradation to inhibit protein translation and hasshown clinical potential for targeting and silencing oncogenes. However, barriers to siRNAdelivery, such as cellular uptake, non-specificity, and lack of endosomal escape hinder clinicaltranslation of RNAi therapeutics. Peptides are a promising carrier for siRNAs and havedemonstrated the potential to overcome barriers to delivery. The purpose of this study is todesign a novel tandem peptide for siRNA delivery, evaluate ability to mediate knockdown of atarget oncogene, and compare the efficacy of the tandem peptide to the individual peptidesalone. The tandem peptide is a novel combination of a fusogenic peptide and a targetingpeptide. STAT3, the target gene, is an oncogene that plays a role in cell proliferation,differentiation, apoptosis, and angiogenesis in GBM. Initial results have confirmed basal expression of EGFR and STAT3 in all three GBM cell lines, LN18, U118, and T98G.Characterization of the fusogenic, targeting, and tandem peptides indicated formation of monodispere nanoparticles with diameters less than 150 nm when complexed with siRNA. Furthermore, gel shift assays were conducted and confirmed that the tandem peptide protectedthe siRNA from degradation in 50% FBS. MTS assays conducted in all three cell lines for eachpeptide confirmed that there was no significant cytotoxicity due to the vehicles alone. Finally, flow cytometry confirmed uptake of the tandem peptide in T98G cell line. Therefore, it ishypothesized that the tandem peptide composed of both fusogenic targeting peptide willenhance cell-specific uptake of STAT3 siRNA into glioblastoma cells for mediating endosomalescape, resulting in more effective gene silencing and associated downstream effects comparedto the fusogenic and targeting peptides alone. Future studies include immunofluorescence for uptake and endosomal escape, knockdown studies (MTS, Western Blot, and qPCR assays), and assays to examine the downstream effect of silencing STAT3, such as migration and invasion assays.

Citation Format: Jessica Boulos, Angela Alexander-Bryant. Peptide delivery of siRNA in GBM [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 1310.
Abstract 1325: Overexpression of Tribbles 2 enhances prostate cancer cell growth and contributes to enzalutamide resistance
Authors: Monga Jitender , Sanghvi Parshva , Ghosh Jagadananda
Published: July 2021

Background: Enzalutamide, an FDA-approved androgen receptor blocker, is commonly prescribed for advanced prostate cancer which slows down prostate tumor growth. However, therapeutic benefit of enzalutamide is temporary because enzalutamide-resistant prostate cancer (ERPC) invariably develops which is incurable, primarily because currently available therapies cannot effectively kill ERPC cells. Molecular basis behind enzalutamide-resistance is not clearly understood, which is delaying progress towards development of new therapies. Thus, proper molecular characterization of ERPC cells is an urgent and unmet medical need to find appropriate targets to improve the current state of ERPC therapy.

Methods: We developed an ERPC model by chronically treating prostate cancer cells to mimic the clinical conditions of Enzalutamide resistance. These ERPC cells were used for comprehensive gene expression analysis by Illumina Hi-Seq whole genome gene-expression array. Hits were confirmed by RT-PCR and Western blot. Regulation of expression and pro-cancer effects of new hits were determined by overexpression and/or treating cells with shRNA and analyzing cell viability, soft-agar colony formation, apoptosis, and effects on downstream targets.

Results: Transcriptional profiling revealed that Tribbles 2 (Trib2), a pseudokinase, is overexpressed in ERPC cells. Elevated level of Trib2 was also observed in enzalutamide treated PDX tumors in mice, and in prostate tumors from patients who were treated with enzalutamide. Inhibition of Trib2 by shRNA effectively kills ERPC cells, suggesting that Trib2 plays an important role in ERPC. We found that the Trib2 protein level is especially higher in prostate cancer cells where the AR activity is either low, or absent due to genetic changes. Moreover, transfection and re-expression of the AR gene in AR-negative cells decreases Trib2 protein level, indicating that the expression of Trib2 is negatively regulated by androgenic signaling. Interestingly, forced overexpression of Trib2 results in faster prostate cancer cell growth and makes the cells completely resistant to enzalutamide, and inhibition of Trib2 by shRNA or chemical inhibitor decreases viability and re-sensitizes these cells to enzalutamide.

Conclusion: Collectively, our findings reveal that Trib2 is overexpressed in ERPC cells and tumors, and suggest that Trib2 may turn out to be a novel, promising molecular target for therapy of enzalutamide-resistant, lethal prostate cancer.

Citation Format: Jitender Monga, Parshva Sanghvi, Jagadananda Ghosh. Overexpression of Tribbles 2 enhances prostate cancer cell growth and contributes to enzalutamide resistance [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 1325.
Abstract 1331: Synthesis and evaluation of FPR1 inhibitors as a novel treatment for cancers
Authors: Aghamohammadi Amin , Lu Di , Salem Anwar , Cooper Patricia A. , Shnyder Steven D. , Vinader Victoria , Afarinkia Kamyar
Published: July 2021

The formyl peptide receptor-1 (FPR1) is a member of 7TM GPCRs. It was first identified in phagocytic leukocytes and has been found to play a role in innate immune response to bacterial infection. Recently FPR1 has been shown to be overexpressed in many malignant cancers and particularly in glioblastoma. Our more recent investigation has shown that increased expression of FPR1 in cancer is most notably prominent in the periphery of the necrotic foci. We have hypothesised that FPR1 activation by molecules released from the hypoxic/necrotic core of tumours (including Annexin-A1 and short chain-N-formylated peptides such as fMLF) leads to increased proliferation, invasion, resistance, and angiogenesis. Therefore, antagonism of FPR1 should prevent these key drivers of malignancy in cancer. Here we present the synthesis and biological evaluation of a series of pyrazole compounds that potently inhibit FPR1. In addition, we provide a computational rationale for their relative potency based on docking of the molecules inside a homology model of FPR1. We will also show the efficacy of our lead compound, ICT12035 (IC50 =30nM in calcium mobilisation assay) in a number 2D and 3D proliferation and invasion assays in vitro, and in an in vivo animal model where it arrests tumour growth. Furthermore, we demonstrate the significance of FPR1 in tumour resistance by showing that ICT12035 increases the efficacy of cytotoxic agents TMZ and BCNU, as well as radiation in vitro. ICT12035 significantly abrogates the effects fMLF induced resistance to TMZ and BCNU in in U87 cell lines. A similar effect is also observed in a cell viability assay where in the addition of ICT12035 significantly increased cell death following radiation. Our findings suggest that inhibiting FPR1 in combination with chemotherapy and radiotherapy could be a new treatment strategy against cancers.

Citation Format: Amin Aghamohammadi, Di Lu, Anwar Salem, Patricia A. Cooper, Steven D. Shnyder, Victoria Vinader, Kamyar Afarinkia. Synthesis and evaluation of FPR1 inhibitors as a novel treatment for 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 1331.
Abstract 1332: Antiretroviral agent inhibits proliferation and motility of BT-20 cells through inhibition of human endogenous retrovirus type K
Authors: Lin John , Farris Mason , Robbins Lauren , Lee Matthew , Nong Serratt , Liu Yingguang
Published: July 2021

The purpose of the study was to establish the role of human endogenous retrovirus K (HERV-K) in cancer development and to explore the possibility of repurposing antiretroviral agents for cancer treatment. We screened different categories of antiretroviral agents and several breast cancer cell lines to study the ability of antiretroviral agents to inhibit cancer cell proliferation and to inhibit the natural expression of HERV-K in cancer cells. Cells were transduced with exogenous HERV-K genes or with HERV-K knockdown plasmids to investigate the relationship between cytotoxic effect of antiretroviral agents and HERV expression. We found that an integrase strand-transfer inhibitor, dolutegravir, inhibited HERV-K expression on both the RNA and protein levels in BT-20 cells. Transduction with both HERV-K env and pol genes rendered the cell line more resistant to dolutegravir. Knockdown of HERV-K with shRNA also increased resistance to dolutegravir compared to cells transduced with random double-stranded RNA. The cytotoxic effect of dolutegravir correlated with enhanced expression of E-cadherin and a reduction in cell motility. Interestingly, an HERV-K knockdown cell line also exhibited enhanced expression of E-cadherin and reduced motility. As a potential carcinogenic role of HERVs has raised interest in using antiretroviral agents as a new approach to treat cancer, we provide the first evidence that one antiretroviral agent actually inhibits the proliferation and motility of a cancer cell line through inhibiting an HERV.

Citation Format: John Lin, Mason Farris, Lauren Robbins, Matthew Lee, Serratt Nong, Yingguang Liu. Antiretroviral agent inhibits proliferation and motility of BT-20 cells through inhibition of human endogenous retrovirus type K [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 1332.
Abstract 1346: Direct targeting of MAPK/ERK signaling pathway is a novel therapeutic approach for high-risk neuroblastoma
Authors: Chilamakuri Rameswari , Sharma Bharti , Rouse Danielle C. , Agarwal Saurabh
Published: July 2021

Neuroblastoma is a highly heterogenous pediatric tumor that develops during the early embryonic stages, and accounts for almost 15% of pediatric cancer related deaths. Despite advanced and intensive therapeutic approaches that combine surgery, radiation, and chemotherapy, high-risk neuroblastoma (NB) patients' long-term survival is still less than 50%. Tumor metastasis, relapse, drug-resistance, and treatment related toxicities mandate the development of novel therapeutic approaches to treat NB patients. MAPK/ERK pathway plays an important oncogenic role in different types of cancers, including NB. Activation of the MAPK/ERK pathway is known to induce cancer cell proliferation, progression, metabolism, and resistance to drug-induced apoptosis. In the present study, we investigated the effect of a small-molecule MEK inhibitor that targets MEK1 and MEK2 to block MAPK/ERK pathway in NB. Cytotoxicity assays using Cell-Titer One solution in different MYCN-amplified (NGP, LAN-5, CHLA-255-MYCN) and MYCN-non-amplified (SH-SY5Y, SK-N-AS, CHLA-255) NB cell lines show that MEK inhibitor significantly reduce the NB cell proliferation in a dose-dependent manner. We further performed clonogenic assays and 3D spheroidal assays to mimic the NB tumor growth, and our results show that MEK inhibitor significantly inhibit NB colony formation, inhibit 3D spheroidal tumor size by inducing cell death in 3D spheroidal tumors, in comparison to controls. Additionally, MEK inhibitor in a dose-dependent manner induces apoptosis and blocks cell cycle progression in NB cells in comparison to control, as determined by Annexin V apoptosis assays and Click-iT EdU cell proliferation assay respectively. Furthermore, gene expression analysis show that MEK inhibitor significantly reduce the mRNA expression of specific MAPK/ERK pathway targets such as MEK1, MEK2, and ERK2. Western blot assays further confirm the efficacy of MEK inhibitor in blocking MAPK/ERK pathway by significantly inhibiting the phosphorylation of key pathway proteins. Taken together, our results highlight that: a) MAPK/ERK pathway is implicated in NB growth, and b) direct targeting of MAPK/ERK signaling pathway by using a novel small molecule MEK inhibitor inhibit NB proliferation and 3D tumor growth. We will further combine this MEK inhibitor with current chemotherapy drugs to develop effective therapeutic approaches for NB patients.

Citation Format: Rameswari Chilamakuri, Bharti Sharma, Danielle C. Rouse, Saurabh Agarwal. Direct targeting of MAPK/ERK signaling pathway is a novel therapeutic approach for high-risk neuroblastoma [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 1346.
Abstract 1388: ATM inhibitor AZD1390 sensitizes breast cancer brain metastasis to radiation therapy
Authors: Tew Ben Yi , Durant Stephen , Salhia Bodour
Published: July 2021

Metastasis to the central nervous system (CNS) has remained a major source of morbidity and mortality for patients with breast cancer. This is mainly driven by the lack of therapeutic options, as traditional chemotherapy and targeted therapies have been shown to have limited efficacy in the brain. Ataxia-telangiectasia mutated (ATM) protein orchestrates the cellular DNA damage response (DDR) to cytotoxic DNA double-strand breaks induced by ionizing radiation (IR). ATM genetic ablation or pharmacological inhibition results in tumor cell hypersensitivity to IR.AZD1390 is a potent orally bioavailable ATM inhibitor, specifically optimized for blood brain barrier penetration and has shown to be an effective radiosensitizer in gliomas. We have shown that there are frequent alterations in DNA damage response (DDR) pathways in CNS metastasis (CM), which include frequent mutations in TP53. Incorrect repair of DNA lesions often leads to genomic instability. ATM, a core component of the DNA repair system, is activated to enhance the homologous recombination (HR) repair pathway upon DNA double-strand breaks. Here we show that AZD1390, when used in combination with radiation therapy, can inhibit the growth of breast cancer CM in patient derived xenograft (PDX) tumors. Three PDX models (2 Her2+ and one triple negative), all of which harbor TP53 mutations and other DDR alterations, were implanted into the flank or brain of immunocompromised mice. Pre-treatment with AZD1390 followed by a fractionated regime of radiation therapy (2Gy/day for 5 days) significantly inhibited growth of flank PDX tumors by an average of 81% (85% and 80% in the HER2+ and 77% in the triple negative PDX, p-value < 0.05). This is compared to an average of 7% (8%, 13% and 0% respectively) for tumors treated with AZD1390 alone or 40% (39%, 21% and 58% respectively) for radiation therapy alone in all PDX models. Growth inhibition was sustained until the experiment endpoint at 90 days in 75% of the tumors, and 25% of the tumors regressed below their size at the beginning of treatment. About 31% of tumors across three models started to grow back and gene expression analysis comparing these tumors with complete responders demonstrated changes in oxidative phosphorylation, sirtuin signaling, and ERK/MAPK signaling. Adjuvant therapy of AZD1390 did not further decrease tumor growth. Our results demonstrate that AZD1390 can sensitize breast cancer CM with DDR pathway alterations to radiation therapy. This points to the potential of AZD1390 as a potential novel therapeutic agent for patients with breast cancer CM.

Citation Format: Ben Yi Tew, Stephen Durant, Bodour Salhia. ATM inhibitor AZD1390 sensitizes breast cancer brain metastasis to radiation 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 1388.
Abstract 1402: HNRNPA2B1 increases the serine synthesis pathway in endocrine-resistant breast cancer cells
Authors: Klinge Carolyn M. , Petri Belinda J. , Piell Kellianne M. , Clem Brian C.
Published: July 2021

Despite the limited success of new combination therapies with fulvestrant improving survival of breast cancer patients with metastatic disease after initial response to endocrine therapies based on primary estrogen receptor alpha (ER+) tumors, mechanisms for acquired endocrine resistance remain to be fully elucidated. The RNA binding protein HNRNPA2B1, a reader of epitranscriptomic N(6)-methyladenosine (m6A) in transcribed RNA, is upregulated in endocrine-resistant, ER+ LCC9 and LY2 cells compared to parental MCF-7 endocrine-sensitive luminal A breast cancer cells. HNRNPA2B1 regulates pri- to pre- miRNA processing as well as splicing and transcript stability, depending on interacting proteins and cellular context. The miRNA-seq transcriptome of MCF-7 cells transiently overexpressing HNRNPA2B1 identified gene ontology (GO) pathways including cellular response to steroid hormone signaling and serine family amino acid metabolic processes, i.e., the serine synthesis pathway (SSP). Stable, modest (4.5-fold) overexpression of HNRNPA2B1 in MCF-7 cells (MCF-7-A2B1 cells) phenocopies the endocrine-resistance of LCC9 and LY2 cells including ablation of growth inhibition by 4-hydroxytamoxifen (4-OHT) and fulvestrant. This was not the result of decreased ER alpha; rather, ER alpha, and androgen receptor (AR), were increased. Conversely, transient knockdown of HNRNPA2B1 in LCC9 and LY2 cells sensitized the cells to growth inhibition by 4-OHT and fulvestrant. MCF-7-A2B1 cells, like LCC9 and LY2 cells, have higher expression of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) transcripts and proteins compared to MCF-7 cells. We identified two miRNAs, miR-424-5p and miR-145-5p downregulated in MCF-7-A2B1 cells that directly target the PSAT1 3'UTR in dual luciferase assays. Lower miR-424-5p and miR-145-5p in endocrine-resistant LCC9 and LY2 correlate with increased PSAT1 and higher PSAT1 is associated with reduced overall and metastasis-free survival in breast cancer patients. The correlation of HNRNPA2B1 and PSAT1 expression was examined by IHC in a breast tumor/tissue microarray. Overall, our data suggest a role for increased HNRNPA2B1 in endocrine-resistance and upregulation of the SSP to promote tumor progression in ER+ breast cancer.

Citation Format: Carolyn M. Klinge, Belinda J. Petri, Kellianne M. Piell, Brian C. Clem. HNRNPA2B1 increases the serine synthesis pathway in endocrine-resistant breast 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 1402.
Abstract 1403: Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss
Authors: Arenas Pamela D. , Maloney Sara M. , Prosperi Jenifer R.
Published: July 2021

Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC is an aggressive subtype of breast cancer that lacks the expression of estrogen and progesterone receptors and HER2 protein. This lack of functioning receptors results in limited treatment options, usually resulting in the use of taxanes, platinums, and anthracyclines. Although TNBC often starts as being sensitive to traditional chemotherapies, tumor relapse and/or resistance often develops. Paclitaxel (PTX) is a commonly used taxane for TNBC, well known to induce mitotic arrest through stabilization of microtubules (MT) leading to an interference with the treadmilling function of MT through the activation of the spindle assembly checkpoint. We have previously shown that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor significantly increases resistance to PTX in APC knockdown (APCKD) cells generated in two human TNBC cell lines, MDA-MB-157 and MDA-MB-231. Given this striking PTX resistance, we were interested in seeing if resistance in APCKD can be seen using other non-taxane MT targeting agents. To accomplish this, we have used an epothilone (Epothilone B) and a vinca alkaloid (Vinblastine), that function by stabilizing and destabilizing the MTs, respectively. Our previous investigations demonstrated that APC loss results in increased CDK1 and CDK6, so these have been used as markers of cell cycle progression after drug treatment. In addition, changes in apoptosis were measured through the use of Annexin V and PI staining, and cleaved caspase 3 expression. Examined together, a better understanding of how APC loss influences chemoresistance in TNBC can lead to more efficient treatments for TNBC patients resistant to taxanes. Given the critical interaction between APC and MTs, this may be an avenue for targeted therapies to overcome taxane resistance.

Citation Format: Pamela D. Arenas, Sara M. Maloney, Jenifer R. Prosperi. Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss [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 1403.