bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4781: Changes in intracellular signaling following chronic FGFR inhibition in urothelial bladder cancer models
Authors: Jerchel Isabel S. , Kamburov Atanas , Lesche Ralf , Zitzmann-Kolbe Sabine , Walter Alexander , Ellinghaus Peter , Mumberg Dominik , Politz Oliver , Gruenewald Sylvia
Published: August 2018

Preclinical and clinical studies have validated the therapeutic potential of FGFR inhibition in urothelial bladder cancer (UBC) patients with FGFR genetic aberrations. Rogaratinib is a potent small-molecule pan-FGFR inhibitor being studied in phase I trials in UBC. Here, we studied the effects of chronic exposure of bladder cancer cells in vitro to FGFR inhibition by rogaratinib to identify changes in signaling and gene expression patterns that may identify possible drug combinations that may enhance efficacy of rogaratinib and help overcome inherent and/or acquired treatment resistance. Cell proliferation in response to rogaratinib was evaluated using crystal violet staining in a panel of 13 UBC cell lines. Continuous culture of two cell lines–JMSU1 and RT112–with either constant or increasing concentrations of rogaratinib in several independent approaches generated the resistant sublines, JMSU1-R1 to -R4 and RT112-R1 to -R4. In both cellular models rogaratinib resistance (defined as >30-fold [JMSU1] or >100-fold [RT112] difference in absolute IC50 of sublines vs. parental lines) arose reproducibly and with various treatment schedules. Morphologic changes were also observed. Transcriptomic (RNAseq) and proteomic (R&D Systems proteome profiler arrays) analyses of these cell lines or rogaratinib-treated versus untreated parental cells, respectively, revealed changes that co-occur with the development of resistance. Analysis of the phospho-proteome showed increased phosphorylation of several receptor tyrosine kinases compared to the parental cell lines. Phosphorylation levels varied among the 4 resistant sublines that were derived from the same parental line. This resulted in alteration of downstream signaling pathways in rogaratinib-treated sublines compared to parental cell lines. In conclusion, exposure of selected bladder cancer cell lines to rogaratinib resulted in development of resistance and changes in FGFR signaling and gene expression pathways that may identify strategies to optimize treatment with FGFR inhibitors.

Citation Format: Isabel S. Jerchel, Atanas Kamburov, Ralf Lesche, Sabine Zitzmann-Kolbe, Alexander Walter, Peter Ellinghaus, Dominik Mumberg, Oliver Politz, Sylvia Gruenewald. Changes in intracellular signaling following chronic FGFR inhibition in urothelial bladder cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4781.
Abstract 4788: Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor
Authors: Xile Liu , Hu Lihong , Ding Charles Z , Mao Weifeng , Wang Liqing , Shi Xiaohe , Long Chaofeng , Chen Xiaoxin , Li Haijun , Liu Xing , Liu Zhuowei
Published: August 2018

Objective: Patients of non-small cell lung cancer (NSCLC) with activating EGFR mutations initially respond to the first generation reversible EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. However, most of the patients acquire resistance within 10~16 months. The most common mechanism of resistance is the EGFR T790M mutation. In addition, selectivity of the EGFR inhibitors is important, as inhibition of EGFR wild type can lead to dose limiting toxicities, including rash and diarrhea. This may be the reason why the second generation irreversible EGFR inhibitors, such as afatinib, which also inhibit EGFRWT, is not widely used. Here, we disclose our clinical candidate compound E-191 as a novel irreversible inhibitor that potently inhibits the resistance mutant forms of EGFR with a good selectivity sparing EGFRWT.

Method: The anti-proliferative activity of E-191 was evaluated in NCI-H1975 (EGFRT790M/L58R), HCC827 (EGFRd746-750), and A431 (EGFRWT) cell lines. Daily oral administration of E-191 at 3 doses was used to evaluate the in vivo anti-tumor activity in three cell-derived tumor xenograft (CDX) models, NCI-H1975, HCC827, and Ba/F3 (EGFRT790M /d746-750). The in vivo selectivity was evaluated in the A431 (EGFRWT) CDX model. Effects on EGFR signaling pathway were assessed by western blot analysis of the downstream effector proteins p-EGFR (Y1068), p-AKT (Ser473) and p-ERK (Thr202/Tyr204).

Result: E-191 displayed potent anti-proliferative activity in the EGFR mutant cell lines NCI-H1975 (IC50 = 22 nM) and HCC827 (IC50=1.9 nM), and a much less potent activity in the EGFRWT A431 cell line (IC50 = 531 nM). E-191 has about 24 fold selectivity for EGFRT790M over EGFRWT. E-191 potently inhibited tumor growth at all doses tested (P<0.001) in the NCI-H1975, HCC827 and Ba/F3 (EGFRT790M /d746-750) models, while significantly less so in the A431 model. Western blot analysis of the tumor samples in the NCI-H1975 model study showed that E-191 inhibited p-EGFR (Y1068), p-AKT (Ser473), and p-ERK (Thr202/Tyr204), indicating that the tumor growth inhibition was through inhibition of the resistant mutant EGFR.

Conclusion: We have demonstrated with in vitro and in vivo preclinical models that E-191 is a selective inhibitor of the resistance mutant forms of EGFR, potently inhibiting EGFRT790M but not EGFRWT. These results make us believe that E-191 has a good potential in the clinic for the treatment of resistant EGFR driven NSCLC.

Citation Format: Liu Xile, Lihong Hu, Charles Z Ding, Weifeng Mao, Liqing Wang, Xiaohe Shi, Chaofeng Long, Xiaoxin Chen, Haijun Li, Xing Liu, Zhuowei Liu. Preclinical evaluation of E-191, a highly selective EGFR T790M inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4788.
Abstract 4792: Preclinical evaluation of GST-HG161, a potent and highly selective c-MET inhibitor
Authors: Xu Xiongbin , Li Gang , Hu Lihong , Hao Junguo , Ding Charles Z. , Chen Shuhui
Published: August 2018

Objective: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Studies indicate that aberrant activation of mesenchymal-epithelial transition factor (c-MET) is associated with HCC development. Blocking c-MET signaling shows antitumor efficacy in patients with HCC. Thus, c-MET has been an attractive therapeutic target for HCC. We disclose here for the first time a potent and selective c-MET inhibitor GST-HG161 and show the antitumor efficacy data in preclinical models of HCC.

Method: MHCC97H HCC cell line with c-MET overexpression was used to evaluate the anti-proliferative activity of GST-HG161 in a 120-hour viability assay. Selectivity was evaluated with a panel of cell lines without overexpression of c-MET. Daily oral administration of GST-HG161 doses of 1, 3, 10 and 30mg/kg was used to evaluate its in vivo antitumor efficacy in two animal models of HCC with c-MET overexpression, MHCC97H xenograft (CDX) model and LI-03-0317 xenograft (PDX) model. The c-MET protein levels and gene copy number in LI-03-0317 PDX were assessed by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. High c-MET expression in LI-03-0317 PDX was determined by the presence of tumor cells with membrane staining of 3+ intensity. Effect of GST-HG161 on c-MET signaling inhibition was assessed by western blot analysis of downstream effector proteins including p-MET, p-AKT and p-ERK.

Result: GST-HG161 displayed potent anti-proliferative activity in MHCC97H HCC cell line (IC50 = 5 nM) while showed little activity in the cell lines without c-MET overexpression. It significantly inhibited tumor growth at tested doses (3, 10 and 30mg/kg) (P<0.05) in both MHCC97H CDX model and LI-03-0317 PDX model. In MHCC97H CDX model, western blot analysis showed that GST-HG161 potently inhibited p-MET and downstream effector proteins p-AKT and p-ERK in the tumor tissue, indicating that the tumor growth inhibition in this model was mediated through inhibition of c-MET.

Conclusion: GST-HG161 is a potent and highly selective c-MET inhibitor. Its excellent antitumor efficacy as shown in

the HCC mouse models provide us with confidence that GST-HG161 has high potential in the clinic for treating patients of HCC with high c-MET activity.

Citation Format: Xiongbin Xu, Gang Li, Lihong Hu, Junguo Hao, Charles Z. Ding, Shuhui Chen. Preclinical evaluation of GST-HG161, a potent and highly selective c-MET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4792.
Abstract 4794: Preclinical evaluation of WX-0593, a potent orally active ALK inhibitor
Authors: Xile Liu , Ding Charles Z , Mao Weifeng , Qin Yuxin , Zheng Shansong , Yang Yingying , Zheng Qingmei , Zhang Long , Zhao Shuyong
Published: August 2018

Objective: ALK is a tyrosine kinase belonging to the insulin receptor super-family. The constitutively active ALK fusions are oncogenic and linked with multiple types of human cancer. The EML4-ALK fusion has been proven to be the driver of approximately 5% of NSCLC patients. We disclose here for the first time a novel ALK inhibitor WX-0593 and its preclinical evaluation data in preclinical models of ALK driven NSCLC.

Method: The enzyme activities of WX-0593 for ALKWT, ALKL1196M, ALKC1156Y and EGFRL858R/T790M were measured with corresponding kinase assays. The anti-proliferative activity was evaluated in Karpas-299, Ba/F3, Ba/F3 (EML4-ALK-WT), Ba/F3 (EML4-ALK-L1196M), Ba/F3 (EML4-ALK-C1156Y), and NCI-H1975 cell lines. The specific effect (PD) of WX-0593 on ALK signaling was analyzed in NCI-H3122 cells. Antitumor activity of WX-0593 was evaluated in three patient-derived xenograft (PDX) models of NSCLC (LU-01-0015, LU-01-0319, and crizotinib resistant LU-01-0319R) and one cell-derived xenograft (CDX) model of NSCLC (NCI-H3122). PKPD was assessed by western blot analysis of the treated tumor tissues for p-ALK (Tyr1604), p-STAT3 (Tyr705), p-STAT5 (Tyr694), p-AKT, and p-ERK.

Result: WX-0593 displayed potent enzymatic activities for ALKWT, ALKL1196M, ALKC1156Y and EGFRL858R/T790M with IC50 of 5.38, 9.26, 9.28, and 16.74 nM respectively. It also showed expected anti-proliferative activity in six cell lines, Karpas-299, Ba/F3, Ba/F3 (EML4-ALK-WT), Ba/F3 (EML4- ALK-L1196M), Ba/F3 (EML4-ALK-C1156Y), and NCI-H1975, with IC50 of 4.5, 2208, 4, 9.5, 9, and 508 nM, respectively. Western blot study in NCI-H3122 cells showed that WX-0593 could fully inhibit p-ALK, p-ERK,p-STAT5 at 11.1 nM and p-AKT at 100 nM. WX-0593 significantly inhibited tumor growth at all doses tested (P<0.001) in the LU-01-0015 (HIP1-ALK), LU-01-0319 (EML4-ALK), and LU-01-0319R NSCLC PDX models as well as in the NCI-H3122 CDX model. Western blot analysis of the tumor samples showed that WX-0593 inhibited p-ALK (Tyr1604), p-STAT3 (Tyr705), p-STAT5 (Tyr694), p-AKT, and p-ERK, indicating that the tumor growth inhibition was mediated by inhibition of ALK signaling.

Conclusion: We have identified a potent orally active second generation ALK inhibitor, WX-0593. It can inhibit the activity of both wide type and crizotinib resistant ALK and showed strong antitumor activity in in vitro and in vivo preclinical models. These results are considered highly promising and warrant moving the compound forward to clinical investigation.

Citation Format: Liu Xile, Charles Z Ding, Weifeng Mao, Yuxin Qin, Shansong Zheng, Yingying Yang, Qingmei Zheng, Long Zhang, Shuyong Zhao. Preclinical evaluation of WX-0593, a potent orally active ALK inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4794.
Abstract 4795: Potency of a new ALK/ROS1 inhibitor TPX-0005 to ALK G1202R mutation and ROS1 G2032R mutation
Authors: Yoda Satoshi , Dardaei Leila , Prutisto-Chang Kylie , Cui Jean , Shaw Alice T. , Hata Aaron N.
Published: August 2018

ALK rearrangements and ROS1 rearrangements are important therapeutic targets in non-small cell lung cancer (NSCLC). ALK-positive NSCLC is currently treated with the first-generation ALK inhibitor crizotinib followed by more potent, second-generation ALK inhibitors, such as ceritinib, alectinib, or brigatinib, and ROS1-positve NSCLC is currently treated with crizotinib. Even though these drugs produce responses in most patients, drug resistance eventually develops. Notable mechanisms of resistance are kinase domain solvent front mutations, such as ALK G1202R and ROS1 G2032R. Genomic analysis of relapsed cases show ALK G1202R in 21%, 29%, or 43% of ceritinib-, alectinib-, or brigarinib-resistant cases, and ROS1 G2032R in 41% of crizotinib-resistant cases.

The novel ALK/ROS1 inhibitor TPX-0005 was designed to overcome clinical resistance mutations, especially solvent front mutations, and is currently being evaluated in a phase 1/2 study. Engineered preclinical models have demonstrated the potency of TPX-0005 against ALK G1202R and ROS1 G2032R. For example, Ba/F3 cells expressing EML4-ALK G1202R or CD74-ROS1 G2032R resistant to second-generation ALK inhibitors or crizotinib, respectively, but were sensitive to TPX-0005. Here we investigate the potency of TPX-0005 to overcome solvent front mutations in resistant patient-derived models. We generated cell lines and patient-derived xenograft mouse models from ALK G1202R and ROS1 G2032R tumors at the time of relapse on second-generation ALK inhibitors or crizotinib, respectively. As expected, these models were resistant to second-generation ALK inhibitors or crizotinib. In contrast, TPX-0005 suppressed ALK or ROS1 phosphorylation, and led to decreased cell viability and tumor regression in vivo. These data demonstrate that TPX-0005 overcomes solvent front mutations in clinically relevant models and provides rationale for the clinical development of TPX-0005 for patients harboring ALK G1202R or ROS1 G2032R resistance mutations.

Citation Format: Satoshi Yoda, Leila Dardaei, Kylie Prutisto-Chang, Jean Cui, Alice T. Shaw, Aaron N. Hata. Potency of a new ALK/ROS1 inhibitor TPX-0005 to ALK G1202R mutation and ROS1 G2032R mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4795.
Abstract 480: Regulation of crosstalk between AR and Wnt/Beta-catenin pathways by miR-644a in prostate cancer
Authors: Plaga Alexis R. , Shukla Girish C.
Published: August 2018

Prostate Cancer (PCa) is the most commonly diagnosed cancer in males with 161,360 people diagnosed in the United States and is predicted to cause 26,730 deaths in 2017. The current treatment of androgen deprivation therapy (ADT) initially depletes circulating androgens; however, intratumoral androgens rescue androgen receptor (AR) signaling and promotes the development of castration-resistant prostate cancer (CRPC). The dysregulation of the Wnt/B-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of B-catenin which upregulates many genes involved in tumorigenesis. Additionally, B-catenin acts as AR cofactor. Our previous studies show that AR is a direct target of miR-644a. We hypothesized that targeting the expression of both the B-catenin pathway and AR by conventional drugs and/or tumor suppressor miR-644a would have synergistic therapeutic benefits. In this study, we are investigating miR-644a mediated posttranscriptional downregulation of GSK3-B and B-catenin in the wnt/B-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (B-catenin or GSK3-B) was also assessed using viability assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/B-catenin pathway as well as assess if miR-644a can inhibit the cross-talk of these pathways.

Citation Format: Alexis R. Plaga, Girish C. Shukla. Regulation of crosstalk between AR and Wnt/Beta-catenin pathways by miR-644a in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 480.
Abstract 481: QKI, a direct target of miRNA-200, inhibits epithelial-to-mesenchymal transition
Authors: KIM Eun Ju , Sun Der Sheng , Won Hye Sung , Ko Yoon Ho , Ahn Young-Ho
Published: August 2018

MicroRNA (miR)-200 family members are well-known regulators of epithelial-to-mesenchymal transition (EMT) in epithelial tumors. To identify novel miR-200 targets, we analyzed TargetScan and TCGA data sets. Among 20 candidate genes, we selected QKI gene and studied its role during EMT process in human head and neck squamous cell carcinoma (HNSCC) and non-small cell lung carcinoma (NSCLC) cells. The expression of QKI was proved to negatively correlate with those of miR-200 members in TCGA data sets. miR-200 suppressed QKI expression directly, which was confirmed by 3'-UTR reporter assays. Interestingly, shRNA-mediated knockdown of QKI promoted migration, invasion, and EMT in cancer cells and increased tumor growth in a xenograft mouse model. These results indicate that QKI inhibits EMT and tumorigenesis in HNSCC and NSCLC cells. Even though further studies are necessary to elucidate the underlying mechanism, we suggest that QKI and miR-200 form a feedback loop to maintain homeostatic responses to EMT-inducing signals.

Citation Format: Eun Ju KIM, Der Sheng Sun, Hye Sung Won, Yoon Ho Ko, Young-Ho Ahn. QKI, a direct target of miRNA-200, inhibits epithelial-to-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 481.
Abstract 4814: Adaptation of uveal melanoma cells to MEK inhibition can be overcome through HDAC inhibition
Authors: Faião-Flores Fernanda , Maria-Engler Silvya S. , Eroglu Zeynep , Smalley Keiran S.
Published: August 2018

Uveal melanoma is the most common adult cancer of the eye. Although uveal melanoma has a low incidence, it has a high capability to develop metastases, mainly to the liver. Mortality rates are high and new therapeutic strategies are urgently needed. 85-90% of all uveal melanomas harbor driver mutations in GNAQ or GNA11 leading to constitutive activation of many signaling pathways, including the MAPK pathway. Although MEK inhibitors have been evaluated clinically for metastatic uveal melanoma, responses are short-lived, and the mechanisms of adaptation are not well delineated. In the current study we performed mass spectrometry-based proteomics to define the adaptive response of uveal melanoma cells to MEK inhibition at a systems level with the hope of identifying the key escape pathways. These analyses identified MEK inhibition to lead to increased histone phosphorylation and histone deacetylase (HDAC) activity, as well as reorganization of the cytoskeleton associated with cortactin phosphorylation. MEK inhibition also increased receptor tyrosine kinase (RTK) signaling (through ROR1 and IGFIR) and enhanced AKT phosphorylation. Use of pan-HDAC inhibitors sensitized the uveal melanoma cells to MEK inhibition leading to enhanced apoptosis and decreased cell invasion. Mechanistic studies showed the MEK+HDAC inhibitor combination inhibited MAPK, PI3K/AKT and the Hippo signaling, an effect associated with increased cell death inhibition of cell invasion. Chronic MEK inhibitor treatment led to acquired resistance that was associated with increased RTK signaling and the adoption of an invasive phenotype. Studies are ongoing to identify the key HDAC isoform required for the adaptation to MEK inhibitor therapy as well as the anti-invasive potential of the cortactin knockdown with the goal of developing novel combination therapies that can be evaluated clinically.

Citation Format: Fernanda Faião-Flores, Silvya S. Maria-Engler, Zeynep Eroglu, Keiran S. Smalley. Adaptation of uveal melanoma cells to MEK inhibition can be overcome through HDAC inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4814.
Abstract 4841: Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex
Authors: Song Jin H. , Luevano Libia A. , Kang Hyunjin , Gokhale Vijay , Hurley Laurence H. , Kraft Andrew S.
Published: August 2018

Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is widely overexpressed in human cancers including prostate cancer. Along with its regulation of the telomere, hTERT was also shown to regulate various signal transduction mechanisms involving DNA damage response, cell cycle checkpoint, and apoptosis. The majority of human solid tumors display telomerase activity, which was found to be well correlated with hTERT expression, and hTERT inhibition led to reduced telomerase activity and telomere length. However, approaches that directly inhibit the action of telomerase have not produced a therapeutically useful response because of long lag times for subsequent cell division to produce telomere shortening. Alternatively, recent studies demonstrated that this delay problem can be solved through downregulation of hTERT transcription. It was shown that telomeric DNA is capable of folding into four-stranded guanine quadruplex (G4) structures, which then lock the hTERT promoter activity that leads to robust telomere shortening. Our new approach has focused on the discovery of small molecules that could stabilize G-quadruplexes by interacting with the telomeric G-rich overhang stem loop, producing a lead candidate compound that enhances the kinetic folding rate of the G4 silencer element. Our in vitro studies demonstrate that this singular mechanism of action permitted reduced telomerase production and telomere shortening. Significantly, hTERT downregulation with the identified molecule caused prostate cancer cell death, which was characterized by activation of the caspase cascade and increased Annexin V- positive cells. In contrast, this compound did not harm normal prostate epithelial cells. Similarly, mouse prostate cancer cells that do not contain a G-quadruplex were not killed, demonstrating that the unique mechanism of action is mediated through the hTERT promoter G-quadruplex element. Our in vivo study demonstrated that administration of this significantly delayed tumor growth on xenografted mice. The rapid onset of cell death by G4 stands in contrast to the delayed action of classic telomerase inhibition. Therefore, our current study provides important data to support developing inhibitors of hTERT as a cancer drug target.

Citation Format: Jin H. Song, Libia A. Luevano, Hyunjin Kang, Vijay Gokhale, Laurence H. Hurley, Andrew S. Kraft. Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4841.
Abstract 4850: The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells
Authors: Guo Huizi , Kaur Harpreet , Akhtarkhavari Sepehr , Eberhart Charles G. , Raabe Eric H.
Published: August 2018

Diffuse intrinsic pontine glioma (DIPG) is an invasive, incurable and aggressive pediatric brain tumor found in the brainstem. Improved understanding of the biology of DIPG tumors is urgently needed to develop novel treatments. Our previous studies have shown that DIPG tumors and cell lines express high levels of the DNA-binding stem cell factor high mobility group AT-hook 2 (HMGA2).. Targeting HMGA2 using lentiviral shRNA decreased DIPG cell invasion, proliferation and increased apoptosis in our studies. We hypothesized that inhibiting HMGA2 using DNA minor groove binding drugs like quinacrine would decrease DIPG proliferation and increase apoptotic cell death. We used three DIPG cell lines (JHHDIPG1, JHHDIPG16A, and SUDIPG13) to test the effect of quinacrine. Quinacrine has traditionally been used as an anti-malarial drug and is known to strongly bind to DNA. We used BrdU incorporation as a measure of proliferation and expression of cleaved caspase-3 (CC-3) as a measure of apoptosis. Treatment of DIPG cells with quinacrine showed a dose dependent increase in cell death (CC-3 expression) from 3uM to 20uM, with the most effective doses being from 3uM to 5uM. In all three DIPG cell lines, treatment with 1uM to 5uM quinacrine showed a significant reduction in cell proliferation (BrdU) from 3uM to 5uM compared to vehicle-treated cells (P<0.0001). Additionally, treatment of DIPG cell lines with 1uM to 5uM quinacrine showed a significant increase in apoptotic cell death (CC-3 expression) compared to vehicle control cells from 3uM to 5uM (P<0.001). Our data suggests that minor groove binding agents like quinacrine could be effective therapies for this lethal brain tumor. Our future studies are aimed at testing the efficacy of quinacrine in inhibiting DIPG tumorigenicity and elucidating the mechanism of action.

Citation Format: Huizi Guo, Harpreet Kaur, Sepehr Akhtarkhavari, Charles G. Eberhart, Eric H. Raabe. The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4850.
Abstract 4853: Regulation of proteolytic repair of Top2 covalent complexes
Authors: Sun Yilun , Nitiss Karin C. , Nitiss John L.
Published: August 2018

DNA topoisomerases (topos) play an essential role in nuclear processes such as replication, transcription, and chromosome segregation. These enzymes regulate DNA topology by introducing transient breaks in DNA with formation of transient protein/DNA covalent complexes. These complexes can be trapped by aberrant DNA structures, and are also the basis for anti-cancer drugs that target topos. Since topos can be trapped on DNA, leading to strand breaks with protein adducts at the site of the breaks, cells require DNA repair pathways to process protein adducts. We have examined the repair of the type II topos (Top2) in yeast and mammalian cells. We developed a sensitive assay to detect ubiquitylation and other post-translational modifications (PTMs) of Top2 trapped on DNA in S. cerevisiae and have shown that the proteasome plays a key role in repair of Top2 damage induced by the anti-cancer drug etoposide. We demonstrated that sumoylation of Top2 is induced by etoposide, and that deletion of the SUMO ligase Siz2 prevents etoposide induced sumoylation. Deletion of either of the genes encoding the ubiquitin ligase Slx5/Slx8 led to decreased ubiquitylation of trapped Top2. The mammalian ortholog of Slx5/Slx8 is Rnf4, and it too plays a role in ubiquitylation and degradation of Top2 trapped by etoposide. A human cell line completely lacking Rnf4 is defective in ubiquitylation of Top2β, one of the two mammalian Top2 isoforms. Interestingly, repair of the Top2α isoform is unaffected in an Rnf4 knockout cell line. Proteolysis of trapped Top2 is also subject to negative regulation. The UBA domain protein UBAP2L, (also termed NICE4) is amplified in various cancers. Knockout of the UBAP2L increases proteolysis of trapped Top2. Levels of trapped Top2α and Top2β are decreased in the absence of UBAP2L, and the level of trapped Top2 returns to the wild type level when MG132 is added, indicating UBAP2L regulates the proteasome mediated degradation of Top2. UBAP2L physically interacts with Top2β as determined by co-immunoprecipitation, and the interaction is not seen in Rnf4 knockout cells. Finally, knockout of UBAP2L confers high level resistance to etoposide. Our results provide insights to the response of cancer cells to Top2 targeting agents.

Citation Format: Yilun Sun, Karin C. Nitiss, John L. Nitiss. Regulation of proteolytic repair of Top2 covalent complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4853.
Abstract 4855: Indotecan (LMP400), Imidotecan (LMP776) and LMP744: A new class of non-camptothecin TOP1 inhibitors selective for cancer cells with homologous recombination deficiencies and high SLFN11 expression
Authors: Pommier Yves , Marzi Laetitia , Ohler Zoe Waever , Szabova Ludmila , Sharan Shyam , Murai Junko , Cushman Mark
Published: August 2018

To relax DNA supercoiling, topoisomerase I (TOP1) induces DNA cleavage complexes (TOP1cc). These TOP1cc can be trapped by camptothecin, leading to replication induced DNA double-strand breaks (DSB). Widely used, camptothecin derivatives have known limitations. It is now possible to overcome these limitations with the non-camptothecin indenoisoquinolines currently in clinical trials (LMP400, LMP776 and LMP744). Homologous recombination (HR) and its key components BRCA1, BRCA2 and PALB2 are needed to repair DSB induced by TOP1 inhibitors (Maede, Y et al. 2014). It has been shown that BRCA1 is a determinant of response to camptothecins in addition to Olaparib. Also, Schlafen11 (SLFN11) expression has been demonstrated to be a highly penetrant determinant of response to camptothecin derivatives (Zoppoli, G et al. 2012). To rationally select patients for phase 2 clinical trials based on cancer-specific genomic alterations, we have determined whether HR components deficiency or SLFN11 expression are determinants of LMPs response. Using isogenic cell lines, we assessed the survival after treatment with the LMPs. We found that SLFN11-positives cells are 10-times hypersensitive to LMPs compare to WT cells. Also, BRCA1-, BRCA2- and PALB2-deficient cells are 3- to 5- times hypersensitive to the LMPs. Adding Olaparib led to a greater cell death, especially in HR-deficient (HRD) cells. Our results show that the indenoisoquinolines are active at nanomolar concentrations and that HR deficiency and SLFN11 expression are strong drug response determinants. They also demonstrate that the LMPs synergize with Olaparib. These findings provide a rationale for personalized treatment and further clinical trials with the indenoisoquinolines in HR-deficient cancers.

Citation Format: Yves Pommier, Laetitia Marzi, Zoe Waever Ohler, Ludmila Szabova, Shyam Sharan, Junko Murai, Mark Cushman. Indotecan (LMP400), Imidotecan (LMP776) and LMP744: A new class of non-camptothecin TOP1 inhibitors selective for cancer cells with homologous recombination deficiencies and high SLFN11 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4855.
Abstract 486: MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes
Authors: Croset Martine , Pantano Francesco , Kan Casina , Bonnelye Edith , Descotes Francoise , Alix-Panabières Catherine , Lecellier Charles , Bachelier Richard , Allioli Nathalie , Hong Saw See , Bartkowiak Kai , Pantel Klaus , Clézardin Philippe
Published: August 2018

MicroRNAs (miRNAs), which are master regulators of gene expression, have emerged as key players in cancer metastasis. Here, we established miR-30 family members (miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) as breast cancer bone metastasis suppressor genes. Low expression levels of miR-30s in primary tumors of breast cancer patients were associated with poor relapse-free survival. Additionally, miR-30s levels were significantly lower in estrogen receptor (ER)-negative/progesterone receptor (PR)-negative tumors than in their hormone-responsive counterparts. Overexpression of miR-30s in ER/PR-negative breast cancer cells resulted in the reduction of bone metastasis burden in vivo. MiR-30s overexpression also reduced tumor outgrowth, when tumor cells were implanted subcutaneously in animals. In vitro, miR-30s did not affect tumor cell proliferation, but inhibited tumor cell invasion. Furthermore, miR-30s restored bone homeostasis by reversing the in vitro effects of the tumor cell conditioned medium on stimulation of osteoclastogenesis and inhibition of osteoblastogenesis. We identified a number of genes associated with osteoclastogenesis stimulation (IL-8, IL-11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11) and invasiveness (CTGF, ITGA5, ITGB3) that were direct and/or indirect targets for repression by miR-30s. Among these genes, integrin ITGA5 was a previously unknown miR-30 target. By silencing ITGA5 in ER-/PR-negative breast cancer cells, colonization of the bone marrow by tumor cells was drastically reduced in vivo. Overall, our data indicate that miR-30s employ multiple mechanisms to impede breast cancer bone metastasis. These findings may pave the way to a new field of therapeutic interventions in breast cancer patients with bone metastasis.

Citation Format: Martine Croset, Francesco Pantano, Casina Kan, Edith Bonnelye, Francoise Descotes, Catherine Alix-Panabières, Charles Lecellier, Richard Bachelier, Nathalie Allioli, Saw See Hong, Kai Bartkowiak, Klaus Pantel, Philippe Clézardin. MicroRNA-30 family members inhibit breast cancer invasion, osteomimicry, and bone destruction by directly targeting multiple bone metastasis-associated genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 486.
Abstract 4860: A novel class of small-molecule inhibitors of galectin-1 for prostate cancer therapy
Authors: Liu Ruiwu , Shih Tsung-Chieh , Kiss Sophie , Li Xiaocen , Wang Ting , Huynh Jonathan S. , Wu Chun-Te , Duan Yong , Ghosh Paramita M. , Lam Kit S.
Published: August 2018

Galectin-1 (Gal-1) is a multifunctional carbohydrate-binding protein and plays a key role in cancer cell proliferation, apoptosis, cell cycle, tumor angiogenesis and evasion of immune responses. It is also directly involved in the process of angiogenesis and tumor immune escape. An elevated level of Gal-1 has been found in many cancers, including prostate cancer. We show by immunohistochemistry in human prostate tissue that Gal-1 expression level was very low in all non-tumor samples and highly expressed in prostate cancer tissues. In addition, Gal-1 was progressively upregulated from low- , intermediate- to high-grade prostate cancer. Taken together, Gal-1 is an excellent therapeutic target against cancer. Yet, no inhibitors against Gal-1 have been approved by the FDA for cancer treatment due to lack of potent, specific and in vivo effective molecules. We recently discovered a novel small-molecule inhibitor of Gal-1, named LLS2, through a high-throughput one-bead two-compound combinatorial library approach. LLS2 exhibits cytotoxic activity against prostate cancer and ovarian cancer cells. We synthesized a series of LLS2 analogs and performed structure-activity relationship studies, leading to a more potent inhibitor LLS30 that induced apoptosis in castration-resistant prostate cancer cells expressing high levels of Gal-1. Molecular modeling study showed LLS30 bound to the sugar-binding groove of Gal-1. In enzalutamide (ENZ)-resistant 22Rv1 prostate cancer cells expressing high levels of Gal-1 and androgen receptor (AR) splice variants, LLS30 could inhibit expression of AR splice variants and sensitize the cells to ENZ in a dose-dependent manner. Downregulation of Gal-1 by siRNA prevented the expression of alternatively spliced AR variants. More importantly, LLS30 showed potent in vivo antitumor efficacy and synergistic activities with docetaxel and ENZ against PC-3 and 22Rv1 prostate cancer models, respectively. Blood chemistry test from the treated animal showed minimum effect of LLS30 on the liver and kidney functions, implying low toxicity. LLS30 is an excellent drug candidate for preclinical development as a novel prostate cancer therapeutic.

Citation Format: Ruiwu Liu, Tsung-Chieh Shih, Sophie Kiss, Xiaocen Li, Ting Wang, Jonathan S. Huynh, Chun-Te Wu, Yong Duan, Paramita M. Ghosh, Kit S. Lam. A novel class of small-molecule inhibitors of galectin-1 for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4860.
Abstract 4863: Targeting the NFAT1-MDM2-MDMX network for prostate cancer therapy
Authors: Qin Jiang-Jiang , Li Xin , Wang Wei , Zhang Ruiwen
Published: August 2018

The MDM2 and MDMX oncogenes are overexpressed in human prostate cancer, promoting tumor growth and metastasis. We recently demonstrate that the transcription factor NFAT1 activates MDM2 oncogene, independent of p53. The present study was designed to examine the effectiveness of targeting the NFAT1-MDM2-MDMX network for prostate cancer therapy. We identified a natural product NFAT1-MDM2 inhibitor, Inulanolide A (InuA) in the present study. The anticancer activity of InuA and its effects on the expression and stability of NFAT1, MDM2, and MDMX were examined in human prostate cancer cell lines with different genetic background, including LNCaP (p53 wild-type, AR positive), PC3 (p53 null, AR negative) and DU145 (p53 mutant, AR negative) cells. Our molecular docking studies predicted the binding ability and binding sites of InuA on NFAT1, MDM2, and MDMX. Its binding affinity and specificity were investigated using recombinant proteins and biotinylated InuA. To further demonstrate the specificity of InuA in targeting NFAT1-MDM2-MDMX network and the importance of NFAT1-MDM2-MDMX network in InuA's anticancer activity, the transient overexpression (OE) and knockdown (KD) experiments of these genes, as well as the pharmacological activators and inhibitors of NFAT1 signaling, were used. Our results indicated that InuA directly bound to the DNA binding domain of NFAT1, leading to a marked inhibition of MDM2 transcription. The compound also bound to the RING domains of MDM2 and MDMX with high affinity, inhibited the MDM2-MDMX binding, and enhanced MDM2 protein degradation. InuA inhibited the proliferation, migration, and invasion of prostate cancer cells, regardless of their p53 status and AR responsiveness. The expression of NFAT1, MDM2, and MDMX were important for InuA's anticancer activity, as demonstrated using the cancer cell lines with transient OE and KD of these genes. In conclusion, InuA represents a novel class of NFAT1-MDM2-MDMX inhibitor, and inhibiting the NFAT1-MDM2-MDMX network may be a promising strategy for prostate cancer therapy. (Supported by NIH R01 CA186662 and R01CA214019 and ACS RSG-15-009-01-CDD.)

Citation Format: Jiang-Jiang Qin, Xin Li, Wei Wang, Ruiwen Zhang. Targeting the NFAT1-MDM2-MDMX network for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4863.
Abstract 4868: BI 907828: A novel, potent MDM2 inhibitor that is suitable for high-dose intermittent schedules
Authors: Rudolph Dorothea , Gollner Andreas , Blake Sophia , Rinnenthal Jörg , Wernitznig Andreas , Weyer-Czernilofsky Ulrike , Haslinger Christian , Garin-Chesa Pilar , Quant Jens , McConnell Darryl B. , Moll Jürgen , Norbert Kraut
Published: August 2018

MDM2 inhibitors block the interaction between the Tumor Protein p53 (TP53) and MDM2, its key negative regulator, and represent a new therapeutic concept for cancer therapy. MDM2 inhibitors are designed to restore p53 activity in TP53 wild-type tumors. Several MDM2 inhibitors are currently being evaluated in early clinical development with mainly daily dosing regimens. However, recent clinical data suggest myelosuppression as an on-target, dose-limiting toxicity for this class of inhibitors. Particularly, thrombocytopenia could limit the clinical utility of MDM2 inhibitors. Hence there is a need to mitigate these side effects and to improve the therapeutic window. One approach is less frequent dosing to allow bone marrow recovery while still maintaining efficacious exposure levels and clinical activity. Here we present data on BI 907828, a novel and potent MDM2 inhibitor with optimized drug-like properties including a reliable, dose-linear PK across species with good bioavailability after oral dosing that allows various dose schedules. Remarkably, a single oral dose of 2 mg/kg led to tumor regressions in a SJSA-1 xenograft model in all treated mice, as did treatment on a daily low dose schedule. Data on PK, PD and efficacy relationships in this xenograft model will be presented including dose-dependent induction of TP53 target genes and markers of apoptosis. Moreover, BI 907828 significantly prolonged the survival by more than 50 days with a daily oral dose of 2.5 mg/kg in a difficult to treat disseminated MOLM-13 AML model, as noted by profiling of a clinical-stage MDM2 inhibitor. In summary, BI 907828 is a novel, potent, orally bioavailable MDM2 inhibitor that shows excellent efficacy with daily low dose but also intermittent high dose schedules in preclinical models of cancer.

Citation Format: Dorothea Rudolph, Andreas Gollner, Sophia Blake, Jörg Rinnenthal, Andreas Wernitznig, Ulrike Weyer-Czernilofsky, Christian Haslinger, Pilar Garin-Chesa, Jens Quant, Darryl B. McConnell, Jürgen Moll, Kraut Norbert. BI 907828: A novel, potent MDM2 inhibitor that is suitable for high-dose intermittent schedules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4868.
Abstract 4869: Discovery and characterization of highly potent and selective USP7 inhibitors and benchmarking against clinical MDM2 antagonists
Authors: Gavory Gerald , O'Dowd Colin , Arkoudis Elias , Barker Oliver , Cassidy Eamon , Dossang Anthony , Flasz Jakub , Helm Matt , Hughes Caroline , McClelland Keeva , Miel Hugues , Odrzywol Ewa , Page Natalie , Harrison Tim
Published: August 2018

Given the importance of USP7 in known oncogenic pathways and its emerging role in immuno-oncology, the identification of USP7 inhibitors has attracted considerable interest in the scientific community. However, despite substantial efforts over the past 15 years, the development of genuine deubiquitinase (DUB) inhibitors, which exhibit both drug-like properties and a well-defined mechanism of action, has proven particularly challenging.

In this study, we report the application of UbiPlexTM, our purpose-built DUB drug discovery platform, to USP7. In particular, we detail the identification, optimization and detailed characterization of a new class of non-covalent and highly potent USP7 inhibitors (IC50 < 10 nM). In addition, these inhibitors have shown exquisite selectivity for USP7 over other DUBs and proteases. We also disclose high-resolution co-crystal structures of USP7 in complex with these inhibitors that reveal an allosteric mode of binding.

Further profiling in cells demonstrated potent target engagement with endogenous USP7 (EC50 <20 nM). In line with the known biology of USP7, this effect induced the proteasomal degradation of MDM2, concomitant stabilization of p53 and induction of p21 in multiple cell lines. The identification and parallel profiling of inactive enantiomers further validated the on-target effect of our lead molecules. Finally, we report the identification of cell lines hyper-sensitive (EC50 < 30 nM) to USP7 inhibitors in both haematological and solid cancer cell line settings and demonstrate equal or superior activity when compared to clinically relevant MDM2 antagonists.

In summary, this work exemplifies the tractability and druggability of USP7 as a cancer target and provides new insights and directions for the potential clinical development of USP7 inhibitors.

Citation Format: Gerald Gavory, Colin O'Dowd, Elias Arkoudis, Oliver Barker, Eamon Cassidy, Anthony Dossang, Jakub Flasz, Matt Helm, Caroline Hughes, Keeva McClelland, Hugues Miel, Ewa Odrzywol, Natalie Page, Tim Harrison. Discovery and characterization of highly potent and selective USP7 inhibitors and benchmarking against clinical MDM2 antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4869.
Abstract 4884: OXPHOS: A novel target for cancer therapy in oncogene addicted tumor
Authors: Hirpara Jayshree L. , Qing Jie Eu , Wong Andrea , Ohi Naoto , Takeshi Tsunoda , Goh Boon Cher , Pervaiz Shazib
Published: August 2018

Cancer cells are highly dependent on glycolysis over oxidative phosphorylation (OXPHOS). However, there is emerging evidence that (a) dysfunctional OXPHOS may be a major factor driving oncogene addiction and (b) feedback activation of the transcription factor STAT3 is a common surrogate survival pathway in oncogene addicted cancer cells. As such, invasive cancers exhibit enhanced oxidative phosphorylation, mitochondrial biogenesis and mitochondrial oxygen consumption rate. Therefore, inhibiting OXPHOS and STAT3 activity could have potential therapeutic implications for oncogene addicted and OXPHOS-dependent cancers. Here we report that oncogene addicted non-small cell lung carcinoma (NSCLC) cell line HCC827 and its gefitinib resistant clone and malignant melanoma cell line A375 and its vemurafanib resistant clone have significantly higher OXPHOS activity. In addition, these cells express higher level of the mitochondrial electron transport chain complex I protein (NDUFA9) and increased complex I activity as well as higher mitochondrial DNA copy number. Of note, significantly increased activity of STAT3 is detected in oncogene addicted cancer cells, and the increased mitochondrial oxygen consumption and complex I activity could be significantly inhibited by a novel small molecule inhibitor of STAT3, OPB-51602. The latter is shown to be an effect that might be independent of the STAT3 inhibitory activity of the small molecule compound. Most importantly, the novel STAT3 inhibitor showed strong activity in three different murine models of carcinogenesis as well as in patients treated with the STAT3 inhibitor. These data provide evidence that oncogene addicted cancer cells switch to OXPHOS over glycolysis and serve as proof of principle for identifying targeted new OXPHOS inhibitor in recalcitrant cancers.

Citation Format: Jayshree L. Hirpara, Jie Eu Qing, Andrea Wong, Naoto Ohi, Tsunoda Takeshi, Boon Cher Goh, Shazib Pervaiz. OXPHOS: A novel target for cancer therapy in oncogene addicted tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4884.