bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2054: A novel Bcl-2/Bcl-XL inhibitor APG-1252-12A as a potential therapeutic strategy for gastric carcinoma
Authors: Yan Xianglei , Yi Hanjie , Luo Qiuyun , Yuan Luping , Zhou Suna , Pan Wentao , Zhang Lin , Qiu Miaozhen , Yang Dajun
Published: August 2019

Background: Gastric cancer (GC) is the fourth most common type of malignant tumor and is the second leading cause of cancer-related mortality. Surgery and chemotherapy play important role in the treatment of GC, however the overall survival of gastric cancer patients is not promising. APG-1252-12A specifically binding to Bcl-2 and Bcl-xl has shown efficacy in some solid tumor. We intend to investigate the anticancer effect and combination enhancement of Fluorouracil (5-FU) in gastric cancer.

Materials and methods: Human gastric cancer cell lines (AGS, NCI-N87, SGC7901, BGC823, MKN45 and NUGC3) were treated with different concentrations and time of APG-1252-12A. CCK-8, survival assays, flow cytometry and western blot were used to assess cell survival, cell cycle progression and apoptosis. In vivo, xenograft mouse models (NCI-N87) were established to analyze the anticancer effect and combination enhancement of 5-FU.

Results: In vitro, APG-1252-12A inhibited proliferation of gastric cancer cell lines, without cell-cycle arrest. Meanwhile, APG-1252-12A increased protein expression of apoptosis signal pathway. The combination of APG-1252-12A and 5-FU resulted in the accumulation of apoptosis protein such as PARP and cleaved caspase3, which indicating an increasing of chemosensitivity. In xenograft mouse models, single therapy with APG-1252-12A obviously suppressed tumor growth, especially in 100mg/kg group. Combination group delayed tumor growth more significantly compared with APG-1252-12A and 5-FU single therapy. By analyzing proliferation marker Ki67 in xenograft tumor specimens, we found that the number of proliferating cells significantly decreased in tumors receiving combination treatment of APG-1252-12A and 5-FU compared with tumors receiving single treatment. The results strongly confirmed the antitumor capacity of APG-1252-12A combined with 5-FU.

Conclusions: The results strongly suggest that Bcl-2 and Bcl-xl inhibitor APG-1252-12A may serve not only as a single therapy, but also as an effective adjunct to current chemotherapeutic regimens for treating gastric cancer through apoptosis signal pathway.

Citation Format: Xianglei Yan, Hanjie Yi, Qiuyun Luo, Luping Yuan, Suna Zhou, Wentao Pan, Lin Zhang, Miaozhen Qiu, Dajun Yang. A novel Bcl-2/Bcl-XL inhibitor APG-1252-12A as a potential therapeutic strategy for gastric carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2054.
Abstract 2057: Small molecule CB001 induces extrinsic apoptosis via downregulation of c-FLIP expression in mutant p53-expressing cancer cells
Authors: Zhang Shengliang , Zhou Lanlan , Ralff Marie D. , El-Deiry Wafik S.
Published: August 2019

Cellular FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP) is a master anti-apoptotic regulator in cancer cells. c-FLIP inhibits caspase 8 activation and suppresses cell apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) in malignant cells. Targeting c-FLIP is an attractive strategy for cancer therapy. We previously identified a small-molecule CB001 as a p53-restoring compound and found that CB001 induces cell death with little or no genotoxicity in human colorectal cancer cells. We now show that CB001 downregulates c-FLIP expression in cancer cells at the post-translational level via protein degradation. CB001 treatment induces an increase in caspase 8 activity which correlates with the decrease in c-FLIP at the protein level in mutant p53-expressing cancer cells. Knockdown of caspase 8 rescues cancer cells from CB001-induced apoptosis in mutant p53-expressing cancer cells, suggesting that downregulation of c-FLIP is one of the major mechanisms by which CB001 induces extrinsic cell apoptosis. Furthermore, our studies reveal a synergy between CB001 and TRAIL to induce apoptosis in TRAIL-resistant cancer cells, but not in normal cells at the tested doses. These results taken together suggest that CB001 is a promising lead for drug development with a strong capability to suppress tumor growth through targeting c-FLIP in cancer cells.

Citation Format: Shengliang Zhang, Lanlan Zhou, Marie D. Ralff, Wafik S. El-Deiry. Small molecule CB001 induces extrinsic apoptosis via downregulation of c-FLIP expression in mutant p53-expressing cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2057.
Abstract 2062: Evaluating the effect of tumor necrosis on glycolytic flux and cell survival in glioblastoma
Authors: Noch Evan , Yim Isaiah , Cantley Lewis
Published: August 2019

Glioblastoma continues to rank as one of the most lethal primary human tumors. The degree of necrosis in glioblastoma remains one of the most powerful predictors of poor prognosis, but the relationship between necrosis and poor prognosis is not known. It is unclear whether endogenous tumor necrosis is merely an indicator of an aggressive phenotype or whether tumor necrosis itself propagates the aggressive phenotype. Determining the role of necrosis in mediating the malignant potential of glioblastoma therefore represents an important aspect of therapy. We describe a model of patient-derived glioblastoma spheroids expressing a genetically encoded fluorescent NADH/NAD+ biosensor. This biosensor reports glycolytic flux and can be imaged with fluorescent microscopy. We imaged spheroids using multiphoton microscopy and differentiated cells by high versus low NADH/NAD+ levels. FACS analysis of dissociated spheroids followed by RT-PCR analysis demonstrated increased expression of genes known to be upregulated in peri-necrotic regions, including procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 (PLOD2), activating transcription factor 5 (ATF5), and protein O-fucosyltransferase 2 (POFUT2). These cells also had increased expression of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and jumonji domain-containing protein 6 (JMJD6). We created an intracranial glioblastoma patient-derived xenograft model, with cells expressing this NADH/NAD+ biosensor. When imaging these tumors in vivo, we were able to demonstrate that cells with high NADH/NAD+ levels are more likely to survive within the peri-necrotic environment. These findings indicate that increased glycolytic flux facilitates survival from expanding regions of necrosis. Future experiments will focus on biopsy of cellular populations with high versus low NADH/NAD+ levels within the peri-necrotic niche. We will then perform RNAseq and metabolomics analysis to determine the genetic and metabolic adaptations that facilitate survival within this microenvironment. This model to study cell survival within the peri-necrotic region will improve awareness of necrosis-mediated growth proliferation and may lead to treatments targeted to tumor necrosis that could improve patient prognosis.

Citation Format: Evan Noch, Isaiah Yim, Lewis Cantley. Evaluating the effect of tumor necrosis on glycolytic flux and cell survival in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2062.
Abstract 2081: NUC 3373 induces endoplasmic reticulum stress in colorectal cancer cells
Authors: McKissock Fiona G. , Prabhakaran Asmithaa , Mullen Peter , Harrison David J.
Published: August 2019

Background

Although 5-fluorouracil-based (5-FU) chemotherapies remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. This includes the intracellular conversion of 5-FU into its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), before core anti-cancer activity can be exerted via inhibition of the enzyme thymidylate synthase (TS). NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass the key resistance mechanisms associated with 5-FU, resulting in a potentially more effective and safer treatment option. In a Phase I dose-finding study, plasma obtained from patients with advanced cancers revealed that NUC-3373 has a longer half-life than 5-FU (9.7 hours vs 8-14 minutes). Herein, we aimed to investigate the potential role of endoplasmic reticulum (ER) stress pathways in the cancer cell response to NUC-3373.

Methods IC50 values for NUC-3373 were established in nine CRC cell lines by sulforhodamine B assay, after which one sensitive (HCT116) and one resistant (SW480) cell line were selected for further characterization. Control experiments were conducted with thapsigargin and tunicamycin. Cells were examined by transmission electron microscopy and, using stereological principles, changes in cell ultrastructure were quantified. Expression of the ER chaperone protein binding immunoglobulin protein (BiP) was detected by Western blot analysis.

Results Ultrastructural analysis revealed that NUC-3373 had differential effects on ER stress that were dependent on cell line sensitivity to the drug. Following treatment, NUC-3373-sensitive HCT116 cells responded by dilation of their ER, while the SW480 resistant cells responded by lengthening of ER but without dilation. These changes were reflected with the two classical ER stress inducers, thapsigargin and tunicamycin. NUC-3373 activated the unfolded protein response (UPR), as evidenced by upregulation of BiP. Both the magnitude and the duration of the BiP response were higher in SW480 cells.

Conclusions This study supports a novel mechanism of action for NUC-3373-induced anti-cancer activity through modulation of cell stress. NUC-3373 induces UPR and ER stress in colorectal cancer cells. Results of the ultrastructural analysis suggest that the sensitivity of cells to NUC-3373 may be determined by the differential adaptation of ER to stress conditions.

Citation Format: Fiona G. McKissock, Asmithaa Prabhakaran, Peter Mullen, David J. Harrison. NUC 3373 induces endoplasmic reticulum stress in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2081.
Abstract 2088: Comprehensive CRISPR screening unravel the different mechanisms of IMiDs resistance
Authors: Zhou Wenrong , Song Tianyu , Long Min , Liu Jiye , Ji Qunsheng , Cang Yong
Published: August 2019

Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. To have a comprehensive understanding of mechanism underlying sensitivity of MM cells to IMiDs, we performed genome-wide CRISPR-Cas9 screening in three MM cell lines: MM.1S, NCI-H929 and OPM2, which showed resistance to IMiDs at different extent. In the published screening in MM.1S cell, we found that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, regulates MM sensitivity to IMiDs by modulating CRBN expression via SCFFbxo7 ubiquitin ligase. With the newly outcome from H929 and OPM2 cells, we identified 5 top ranking genes with more than 2 sgRNAs ranking in the top 5% sgRNAs in all the three cell lines. CRBN was the top ranking gene with all CRBN-targeting sgRNAs ranking in the top 1% enriched sgRNAs. UbE2G1, reported as an E2 of CRL4-CRBN E3 ligase recently, was another top ranking gene in all the three cell lines. DEPDC5, a component of the GATOR1 complex which inhibits mTORC1 pathway, and SLC35G2, classified as a member of nucleotide sugars transporters (NSTs), were also identified in the top list of all the three cell lines, indicating that mTOR pathway as well as Glycosylation may play an important role in the MM cytotoxicity induced by IMiDs. We also identified 76 top genes shared in two of the three cell lines with more than 2 sgRNAs. Taken together, these results provide a research framework leading to distinguish the cancer patients who become resistant to IMiDs therapy.

Citation Format: Wenrong Zhou, Tianyu Song, Min Long, Jiye Liu, Qunsheng Ji, Yong Cang. Comprehensive CRISPR screening unravel the different mechanisms of IMiDs resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2088.
Abstract 2098: IFIT1 and IFIT3 modulate the drug response in human oral squamous cell carcinoma through interaction and activation of Hsp90
Authors: Pidugu Vijaya Kumar , wu Meei-Maan , Pidugu Hima Bindu , Lee Te-Chang
Published: August 2019

Interferon-induced protein with tetratricopeptide repeats (IFITs) family, well-known interferon-stimulated genes, have multiple tetratricopeptide repeats with helix-turn-helix structural motifs that mediate a variety of protein-protein interactions. Four IFIT genes have been identified in humans: IFIT1, IFIT2, IFIT3, and IFIT5. While the importance of IFIT1 and IFIT3 in the prognosis of cancer has been reported, the molecular basis of IFIT1 and IFIT3 in cancer progression remains unexplored. We have previously shown that high IFIT1 and IFIT3 expression associated with advanced T-stage, lymph node metastasis, perineural invasion, lymphovascular invasion, extranodal extension, and poor overall survival rate in oral squamous cell carcinoma (OSCC) patients. Also, we have demonstrated that high IFIT1 or IFIT3 expression promoted OSCC invasion and metastasis by EGFR signaling via enhancing EGFR endocytic recycling. Intriguingly, our recent studies have revealed the involvement of IFIT1 and IFIT3 in the resistance to chemotherapeutic agents in OSCC cells. The failure of chemotherapy or targeted therapy was often associated with the intrinsic or acquired drug resistance in cancer cells. In the present study, we intended to explore the biological role of IFIT1 and IFIT3 on drug resistance in OSCC cells. Our results showed that ectopic expression of IFIT1 or IFIT3 proteins in OSCC cells significantly increased the resistance to various therapeutic drugs such as cisplatin, carboplatin, 5-FU, and doxorubicin. Whereas, silencing of IFIT1 and IFIT3 by shRNA enhanced the sensitivity to these drugs. We further demonstrated that enhanced expression of IFIT1 and IFIT3 significantly increased the levels of C-terminal phosphorylation of Hsp90 and activation of its client proteins, such as EGFR, AKT, p38, and SAPK/JNK. Using mass spectrophotometric, immunoprecipitation and immunofluorescence analyses, we identified the interactions of IFIT1 and IFIT3 with Hsp90. Suppression of Hsp90 by a specific inhibitor resulted in decreased activation of its downstream targets in IFIT1- or IFIT3-overexpressing cells. These results suggested a novel mechanism of IFIT1 and IFIT3 in activating Hsp90 and several downstream signaling regulators which are crucial for OSCC tumor progression and drug resistance. Thus, IFIT1 and IFIT3 may act as co-chaperones and serve as potentially important prognostic biomarkers for OSCC progression.

Citation Format: Vijaya Kumar Pidugu, Meei-Maan wu, Hima Bindu Pidugu, Te-Chang Lee. IFIT1 and IFIT3 modulate the drug response in human oral squamous cell carcinoma through interaction and activation of Hsp90 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2098.
Abstract 2101: Targeting the root of cancer persister cells using an expressed barcode library
Authors: Oren Yaara , Thakore Pratiksha , Cuoco Mike S. , Cabanos Heidie Frisco , Hata Aaron , Brugge Joan S. , Regev Aviv
Published: August 2019

Despite a favorable initial response, many cancer patients will experience recurrence of disease within months or years after diagnosis. Recurrence largely arises as a result of the growth of residual cancer cells that remain after treatment. The ability of a subset of cells to survive is attributed frequently to genetic heterogeneity, however recently it was shown that in multiple cancer types relapse can arise due to the presence of persister cells. Persisters are a subpopulation of transiently drug-tolerant cells that are able to survive therapy through reversible, non-mutational mechanisms. Tumor dormancy, stochastic cell state shifts and stem cell-like populations are amongst the mechanisms hypothesized to underlie persister phenotype. However, given the lack of high-throughput methods to concurrently track cell state and lineage, it is not currently feasible to distinguish the relative contribution of each of these factors. To address this need, we generated the Watermelon library. The Watermelon library is a high-complexity expressed barcode library that enables simultaneous tracing of lineage as well as the transcriptional and proliferative state of each cell in the population during drug treatment. We have applied the watermelon system to study the mechanisms underlying the ability of a small population of cells to regain proliferative capacity under constant treatment with EGFR tyrosine kinase inhibitors. We combine time-lapse imaging with single-cell RNA sequencing to show that early drug-cyclers do not acquire a facilitating resistance mutation but rather transition to a new cell state. We find that this non-genetic drug-proliferative state is not restricted to a certain clonal lineage and can be reached by distinct transcriptional paths. We anticipate that this unique library, which can be applied to other systems, would facilitate a better understanding of the cellular and molecular pathways that affect non-inherited drug resistance.

Citation Format: Yaara Oren, Pratiksha Thakore, Mike S. Cuoco, Heidie Frisco Cabanos, Aaron Hata, Joan S. Brugge, Aviv Regev. Targeting the root of cancer persister cells using an expressed barcode library [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2101.
Abstract 2112: Structural characterization of transmembrane protein TMEM205
Authors: Bai Xiaoyun , Xia Di
Published: August 2019

Cisplatin resistance is notorious in leading to therapeutic failure of many solid malignancies. Various resistance mechanisms have been proposed while many more are being added, based on the growing number of resistance-inducing genes identified. TMEM205 is an integral membrane protein with unknown function, but its intracellular level was found markedly elevated in cisplatin resistance cells, the mechanism of which is unknown. Here for the first time we characterized the molecular mechanism of TMEM205-mediated cisplatin resistance through structural and functional studies of TMEM205. The crystal structure of the full-length TMEM205 was obtained at 2.5 Å resolution and shows a 4-helix bundle with a highly hydrophilic tunnel. Strong anomalous signals were observed indicating platinum binding sites. Our structural and functional study of TMEM205 provides novel insight into the complexity of cisplatin resistance.

Citation Format: Xiaoyun Bai, Di Xia. Structural characterization of transmembrane protein TMEM205 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2112.
Abstract 2113: KRAS amplification mediates resistance to osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR
Authors: Yamaoka Toshimitsu , Motoi Ohba , Kishino Yasunari , Kusumoto Sojiro , Tsurutani Junji , Ohmori Tohru
Published: August 2019

Background: The second mutation of T790M in epidermal growth factor receptor (EGFR) exon 20 mediates resistance to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib, and afatinib). The critical T790M mutation in EGFR has facilitated the development of third-generation mutation-selective EGFR TKIs (rociletinib and osimertinib). We previously reported heterogeneous afatinib resistance in lung adenocarcinoma PC-9 cells, harboring 15bp deletions in EGFR exon 19. Among the afatinib-resistant PC-9 cells, we noted the emergence of T790M in EGFR exon 20 and responses to third-generation EGFR-TKIs. Here, we used afatinib-resistant lung adenocarcinoma PC-9 cells, called AfaR, harboring the exon 19 deletion/T790M in EGFR.

Purpose: To identify novel resistance mechanisms in post-afatinib treatment, two osimertinib-resistant cell lines, OsiR1 and OsiR2, were established using increasing concentrations of osimertinib by stepwise dose escalation up to 1 μmol/L over 10 to 12 months.

Results: A linear and reversible KRAS amplification and overexpression with increased osimertinib concentrations in OsiR1 and OsiR2 cells, respectively, was detected. OsiR1 cells maintained osimertinib resistance with KRAS amplification after osimertinib withdrawal for 2 months. After osimertinib withdrawal for 2 months, OsiR2 cells exhibited KRAS attenuation and osimertinib sensitivity was entirely recovered in vitro and in vivo. Phospho-EGFR (Y1068) and growth factor receptor-bound protein 2 (GRB2)/son of sevenless homolog 1 (SOS1) complex was found to mediate osimertinib resistance in OsiR1 cells with sustained KRAS activation. The association of phosphorylated EGFR with the GRB2/SOS1 complex leads to KRAS activation. After 2 months of osimertinib withdrawal, the GRB2/SOS1 complex was dissociated, and the EGFR signal but not the GRB2/SOS1 signal was activated. Concomitant inhibition of mitogen-activated protein kinase kinase and EGFR could overcome osimertinib-resistance in OsiR1 cells.

Conclusion: We characterized novel acquired resistance mechanisms for third-generation EGFR-TKI, osimertinib, providing insights into the development of novel treatment strategies.

Citation Format: Toshimitsu Yamaoka, Ohba Motoi, Yasunari Kishino, Sojiro Kusumoto, Junji Tsurutani, Tohru Ohmori. KRAS amplification mediates resistance to osimertinib in acquired afatinib-resistant NSCLC harboring exon 19 deletion/T790M in EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2113.
Abstract 2116: Cariprazine is a potent chemo-adjuvant against multidrug resistant colon and lung cancer
Authors: Hussein Noor , Nyinawbera Angelique , Amawi Haneen , Tiwari Amit
Published: August 2019

Multidrug resistance (MDR) is considered a continuing clinical problem, reducing the efficacy of cancer chemotherapy. The over expression of the ATP - binding cassette (ABC) family G2 (ABCG2) transporter is considered to be one of the main mechanisms responsible for mediating MDR in cancer. Molecular modeling studies have shown the significant affinity and binding mode of cariprazine, a dopamine D3-preferring D2/D3 receptor partial agonist, to the drug binding cavity of ABCG2 with a Glide XP score of the -5.761. Therefore, in this in vitro study, we determined the effect of cariprazine in increasing the efficacy of MX (an ABCG2 substrate) in the ABCG2 overexpressing cancer cell lines H460-MX20 and S1M1-80. Also, studies conducted to determine whether cariprazine is a reversal compound that specifically targets the ABCG2 protein. Alone, cariprazine, at concentrations up to 20 μM, did not significantly decrease cell viability. Cariprazine, at concentrations ranging from 1 to 20 μM, did not significantly alter the cytotoxicity of mitoxantrone (MX) in the parental non-small cell cancer cell line, H460 and colon cancer cell S1. However, cariprazine (1-20 μM) significantly enhanced the efficacy of ABCG2 substrate antineoplastic drug MX in the ABCG2- overexpressing MDR cell line, H460-MX20 and S1M1-80, by reducing the resistance fold from 28 to 1 and from 93 to 1.33, respectively. The intracellular accumulation of rhodamine 123 in the ABCG2-overexpressing cells, S1M1-80 was significantly increased by the addition of cariprazine, in a concentration-dependent manner (1 - 20 μM). Interestingly, 10 or 20 μM of cariprazine significantly decreased the expression levels of the ABCG2 protein in the colon and lung cancer cell lines, suggesting that cariprazine inhibits both the function and

expression of ABCG2 transporters at non-toxic concentrations. Overall, our results suggest that cariprazine, via several distinct mechanisms, can re-sensitize resistant cancer cells to mitoxantrone.

Citation Format: Noor Hussein, Angelique Nyinawbera, Haneen Amawi, Amit Tiwari. Cariprazine is a potent chemo-adjuvant against multidrug resistant colon and lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2116.
Abstract 2120: Human epidermal growth factor receptor 3 might be a targetable molecule for treatment of chemotherapy resistant breast cancer
Authors: Kotake Takeshi , Suzuki Eiji , Pu Fengling , Kataoka Tatsuki , Hirata Masahiro , Hohokabe Eri , Toi Masakazu
Published: August 2019

Introduction: Disease control of chemotherapy refractory breast cancer patients (CRBC), who do not respond to conventional treatment strategies, is important. In the setting of neoadjuvant chemotherapy (NAC), susceptibility of the chemotherapeutic agents could be confirmed with clinical relevance. Doxorubicin, docetaxel and paclitaxel have been used as conventional NAC. Eribulin and platinum may also be added in the future. When the drug sensitivity is evaluated by NAC and it is not sensitive to the drugs, there is currently no effective therapy expected to be improved. This is one of the recent unmet medical needs in breast cancer treatment strategy. Materials and Methods: In this study, in order to establish a novel therapeutic strategy for CRBC, we established chemotherapy resistant breast cancer cell lines against doxorubicin, paclitaxel, docetaxel and eribulin using BT549, Hs578t, MDA-MB231, MDA-MB468, and HCC70. We evaluated human epidermal growth factor receptor 3 (HER3) expression in these parental and resistant cell lines. We used U3-1402 (a novel anti-HER3 antibody-drug conjugate) to test if HER3 targeting treatment inhibits the cell proliferation of the chemotherapy resistant cell line with positive conversion of HER3 expression. Results: We found that the change to positive expression of HER3 in paclitaxel resistant BT549 cell line (BT549/PTX) and eribulin resistant Hs578t cell line (Hs578t/ERI) while these two cell lines initially showed negative expression of HER3. In the cell proliferation assay, as we expected, there was no inhibition of proliferation by treatment with HER3 targeting antibody agent U3-1402 in these two cell lines. However, BT549/PTX and Hs578t/ERI with positive conversion of HER3 expression were clearly inhibited their cell proliferation by treatment with U3-1402. Conclusions: It suggests that even in cases whose tumor does not express HER3 initially and show no response for chemotherapy in the NAC, U3-1402 could be effective for the patients with positive conversion of HER3 expression after NAC. We are studying for the role of positive conversion of HER3 expression in the process of acquired chemo-resistance and the elucidation of the molecular mechanism of positive conversion of HER3 expression in these unique treatment condition.

Citation Format: Takeshi Kotake, Eiji Suzuki, Fengling Pu, Tatsuki Kataoka, Masahiro Hirata, Eri Hohokabe, Masakazu Toi. Human epidermal growth factor receptor 3 might be a targetable molecule for treatment of chemotherapy resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2120.
Abstract 2126: Development of a precision chordoma research program
Authors: Groth Marie , Kress Jana , Hajnic Matea , Marinho Joana , Mellert Kevin , Barth Thomas F. , Möller Peter , Dreier Birgit , Plückthun Andreas , Scholl Claudia , Fröhling Stefan
Published: August 2019

Chordomas are malignant tumors of the axial skeleton that originate from remnants of the embryonic notochord. With an incidence of one per million per year, chordoma is a rare disease that accounts for 1-4% of all bone tumors and approximately 20% of primary spinal tumors. As first-line treatment, patients undergo surgical resection followed by adjuvant radiotherapy, whereas most chordomas are resistant to conventional cytotoxic drugs and there are no clinically validated molecular drug targets.

To overcome the lack of effective medical treatment and to better understand chordoma biology, we have established a chordoma precision research program. Here, whole-exome or genome and transcriptome sequencing are used to determine mutational landscapes, DNA copy number profiles, and transcriptome changes in chordoma cell lines and patient samples. Additionally, large-scale functional genomic screens and (phospho-)proteomic analyses have been performed in multiple chordoma cell lines to identify new chordoma-specific vulnerabilities. Based on these multilayered datasets, we selected a panel of previously unrecognized candidate chordoma driver genes, which are currently being validated as novel therapeutic targets using genetic and pharmacologic approaches.

In addition to this unbiased approach, we seek to inhibit the embryonic transcription factor brachyury, an established driver of chordomagenesis. Although brachyury represents, in principle, a promising therapeutic target, inhibition of transcription factors remains difficult. To circumvent this challenge, we are developing Designed Ankyrin Repeat Proteins (DARPins) that specifically prevent the binding of brachyury to DNA. These DARPins are not only a valuable tool for studying chordomagenesis, but also represent a new class of molecularly targeted agents that might be used for the treatment of chordoma patients.

In summary, using multi-omics data, functional genetics, and inhibition of an established chordoma driver, we are working towards a better understanding of chordoma biology and the development of new therapeutic approaches. Updated results of the precision chordoma research program will be presented.

Citation Format: Marie Groth, Jana Kress, Matea Hajnic, Joana Marinho, Kevin Mellert, Thomas F. Barth, Peter Möller, Birgit Dreier, Andreas Plückthun, Claudia Scholl, Stefan Fröhling. Development of a precision chordoma research program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2126.
Abstract 2162: ActivSignal technology for multiplex analysis of signaling protein activation profiles
Authors: Alexandrov Ilya , Mackenzie Malcolm , Brandina Irina
Published: August 2019

The IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways in multiplex by examining phosphorylation status or expression of 70 protein targets from canonical signaling pathways. The technology uses antibody pairs for each protein target to ensure high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The ActivSignal IPAD platform can be used to profile cell culture samples, tissues and different blood fractions. Human and mouse samples can be analyzed. The IPAD assay requires only a very small amount of your precious sample. Applications include:

- Direct analysis of protein activity of the signaling cascades and not subsequent downstream events.

- Testing physiological effects of compounds of interest.

– Influence on specific protein targets, as well as the signaling pathways as a whole.

– Secondary and/or off-target effects.

- Similarly, analysis of signaling pathways activation status in response to different stimuli, such as: CRISPR, shRNA, hormones, stresses and other treatments.

- Medium-throughput analysis (96-well format).

- Tissue sample profiling from patients and animals (murine).

- Profiling of tumor biopsies.

Our collaborators analyzed the mechanism of action of JG-98, an Hsp70 inhibitor that exhibits anti-cancer activities that affect both cancer cells and tumor associated macrophages. Using gene expression and ActivSignal protein profiling assay, the signaling pathways influenced by JG-98 were identified, which predicted potential drug combinations that can be used with JG-98 for further preclinical development. ActivSignal is a breakthrough, low-cost tool for the analysis of signaling pathway activation profiles. It enables researchers to rapidly and efficiently gain insight into function of their favorite drug molecules, genes etc.

Citation Format: Ilya Alexandrov, Malcolm Mackenzie, Irina Brandina. ActivSignal technology for multiplex analysis of signaling protein activation profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2162.
Abstract 2169: Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis
Authors: Berry Jameson T. , Mainou Bernardo A.
Published: August 2019

Breast cancer remains the most diagnosed type of cancer and second leading cause of cancer-related deaths in women in the United States. Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancer cases. TNBCs are characterized by lack of estrogen (ER) and progesterone (PR) receptors and growth factor receptor HER2/Neu on the cell surface. Therapies that target ER, PR, or HER2 are thus ineffective against TNBC. TNBC is primarily treated with cytotoxic chemotherapy or radiation therapy, which can cause significant damage to healthy cells and tissue. Therefore, there is need for therapies that selectively kill TNBC cells with reduced off-tumor effects. The concept of oncolytic virotherapy has existed since the early 1900s. However, oncolytic viruses have limited efficacy as single agents. Mammalian orthoreovirus (reovirus) is a non-enveloped, segmented double-stranded RNA virus with tropism for transformed cells. Reovirus is in Phase I - III clinical trials to test its oncolytic efficacy against a variety of cancers. Success has been limited and few trials have tested reovirus against breast cancer. In a high-throughput screen of small molecule inhibitors, we identified the topoisomerase II inhibitor doxorubicin hydrochloride (dox) as an enhancer of reovirus infectivity in the TNBC cell line MDA-MB-231. To better control dox delivery and enhance reovirus oncolytic potential, we chemically conjugated dox to reovirus virions (reo-dox). While reo-dox attachment to MDA-MB-231 cells is slightly impaired, viral replication is largely unaffected. Infection of MDA-MB-231 cells with reo-dox resulted in increased cytopathicity and faster induction of cell death than virus alone. MDA-MB-231 cells infected with reo-dox, but not virus alone, induced DNA double-strand breaks and activation of DNA damage response. Together, our findings show that reo-dox exhibits superior toxicity in TNBC cells than virus alone. Future studies will define the mechanism of enhanced cytopathicity of reo-dox and oncolytic efficacy of dox-conjugated reovirus in vivo. Delivery of small molecule inhibitors via conjugation to reovirus particles may provide an effective new method to directly target and kill cancer cells while minimizing toxicity to healthy cells and tissues.

Citation Format: Jameson T. Berry, Bernardo A. Mainou. Doxorubicin conjugation to reovirus enhances tumor cell-directed oncolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2169.
Abstract 2173: Developing a high-throughput, high content screening technology to identify new anticancer drug treatments
Authors: Milcarek Andrew T. , Daus Kevin , Alpaugh Mary L.
Published: August 2019

The high attrition rate of new chemotherapeutic drugs represents the single most important challenge in anticancer drug development. With only one drug out of 10, 000 test compounds gaining FDA-approval, attrition is often attributed to failure of in vitro drug efficacy to translate to clinical success. The overall drug discovery and development pipeline can be dramatically improved with new, innovative assays of high pharmacological relevance that can shorten the discovery/preclinical phase and ensure rapid translation to clinic. We report the development of a three-dimensional phenotypic drug screening platform based on spheroidsMARY-X. The tight, compact spheroidsMARY-X form spontaneously due to innate molecular determinants with proliferative cells on the outer periphery and a dormant tumor cell core. These features mimic the intratumoral biological complexities of tumor tissue and thus provide a relevant in vitro model for drug discovery with a high degree of predictive value. Significantly, this model allows analysis of drug targeting of dormant tumor cells, the difficult to treat resistant or highly refractory cell population. Drug response can be reliably assessed using brightfield microscopy and can be quantified by measuring spheroid circularity (i.e., dissolution index). Evaluation of soluble factor concentration is performed using molecular rotors through restricted intramolecular rotation upon protein binding with a subsequent quantum yield. Overall, we present a 3-dimensional anticancer assay that provides a rapid, efficient, relevant platform for the evaluation of drug response and allows identification of small molecule hits in both proliferative and more importantly, dormant tumor cells, the major cause of relapse and recurrent metastatic disease.

Citation Format: Andrew T. Milcarek, Kevin Daus, Mary L. Alpaugh. Developing a high-throughput, high content screening technology to identify new anticancer drug treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2173.
Abstract 2175: Synthetic antigen receptor mesenchymal stem cells (SAR-MSCs) targeting perlecan for drug delivery to ovarian cancer
Authors: Nethi Susheel Kumar , Sehgal Dristhi , Cheng Shen , Panyam Jayanth , Prabha Swayam
Published: August 2019

Mesenchymal stem cells (MSCs) can be engineered with polymeric nanoparticles for tumor-targeted delivery of small molecule drugs. Such nano-engineered MSCs have demonstrated exciting anticancer activity in multiple ovarian cancer models. Despite significantly improved delivery of chemotherapeutics to tumor tissues, non-specific accumulation of MSCs in clearance organs remains a concern. Paralleling the concept of CAR-T cells, we advance here a strategy for synthetic modification of MSCs with antibodies targeting specific antigens overexpressed on cancer cells. Our approach consists of stably incorporating recombinant protein G (PG) on the surface of MSCs, followed by binding of a full-length IgG to the PG handle. Because protein G binds to the Fc region of IgG, antigen-binding affinity of the antibody is conserved. We have previously shown the overexpression of perlecan (HSPG2) on ovarian cancer cells and its correlation to poor patient survival. In the current study, we investigated the incorporation of anti-perlecan antibody on the surface of nano-engineered MSCs. The anti-perlecan IgG antibody was first derivatized with palmitic acid (PA), which was then used to insert PG on MSCs cell membrane. We characterized the PAPG derivative by LC/Q-TOF/MS. Flow cytometry and confocal microscopy were used to confirm the incorporation of fluorescently labeled PAPG handle on MSC surface. Similarly, the binding of fluorescently labeled anti-perlecan IgG to PAPG-functionalized MSCs was confirmed by flow cytometry. We are currently investigating the anticancer efficacy of paclitaxel-loaded, anti-perlecan SAR-MSCs using in vitro and mouse models of ovarian cancer.

Citation Format: Susheel Kumar Nethi, Dristhi Sehgal, Shen Cheng, Jayanth Panyam, Swayam Prabha. Synthetic antigen receptor mesenchymal stem cells (SAR-MSCs) targeting perlecan for drug delivery to ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2175.
Abstract 2177: Streptavidin-pHast: A readily conjugatable, pH-sensitive dye to screen for internalization
Authors: Shramm Patrick A. , Ancheta Leonardo
Published: August 2019

Quick and efficient screening of targeting agents that internalize effectively is vital for determining their suitability as potential therapeutics. Some of the most recent successes in the treatment of cancers have been from antibodies to cell surface proteins that are responsible for tumor cell proliferation. Examples are Cetuximab (target: EGFR) approved for colorectal cancer, and Trastuzumab (target: HER2) for breast cancer. These antibodies have more than one effect on the cancer cell, but one of the most important is that, upon binding to the cell surface antigen, the complex is internalized. As such, the down-regulated cell surface protein no longer plays a role in cancer cell division. Here we describe a method for determining internalization of cell surface molecules by targeting agents using a pH-dependent fluorescent reporter cross-linked to streptavidin. Streptavidin is a tetrameric protein (molecular weight 53 kDa in its recombinant form), with each subunit able to bind a single biotin molecule. The bond between streptavidin and biotin is rapid and essentially non-reversible, unaffected by most extremes of pH, organic solvents, and denaturing reagents. It is the strongest known noncovalent biological interaction (Ka = 1015 M-1) between protein and ligand. A variety of molecules, including lectins, proteins, and antibodies, can be biotinylated and reacted with streptavidin-labeled probes or other detection reagents for use in biological assays. The fluorescence from this reporter increases intensity as the pH of its surroundings becomes more acidic, as demonstrated when exposed to the environment inside a cell (thereby providing evidence of internalization). Here we describe methods that can be used to explore candidates as cancer therapeutics in a quick, reliable and reproducible manner.

Citation Format: Patrick A. Shramm, Leonardo Ancheta. Streptavidin-pHast: A readily conjugatable, pH-sensitive dye to screen for internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2177.
Abstract 2182: Preclincial in vitro and in vivo combination therapies for mutant IDH1R132 tumors with BAY 1436032
Authors: Kaulfuss Stefan , Zaman Julia , Chaturvedi Anuhar , Hess-Stumpp Holger , Höde Jennifer , Biber Ricarda , Borowicz Renan , Heuser Michael , Deimling Andreas von , Pusch Stefan
Published: August 2019

Background: Isocitrate dehydrogenase (IDH) mutations are drivers of different tumors. The IDH1R132 mutation is frequently found in glioma (80%), chondrosarcoma (CS, 45%), intrahepatic cholangiocarcinoma (ICC, 20%), acute myeloid leukemia (AML, 7%), and with low incidences in other solid tumors. Although clinical therapies of mutant IDH inhibition were recently approved for the treatment of AML, few patients benefit from a single therapy with a mutant IDH inhibitor and most of them relapse. Here we report preclinical in vitro and in vivo data on combination therapies of the mIDH1R132 inhibitor BAY 1436032 with standard of care chemotherapies.

Methods: in vitro: The sarcoma cell line HT1080 (IDH1R132C), together with two IDH1 wildtype sarcoma cell lines, the SW1353 (IDH2R172S) and SW872 (IDHwt) cell lines, were treated with BAY 1436032 alone and in combination with different standard of care drugs. in vivo: The patient derived xenograft (PDX) ICC model LIXFC2084 (IDH1R132L), the melanoma PDX model MEXF1341 (IDH1R132C) (MEXF1341), the PDX AML model 13PB020 (IDH1R132C) and a sarcoma cell line derived xenograft model HT1080 (IDH1R132C) were included. All models were treated with BAY 1436032 alone and in combination with different standard of care (SOC) drugs.

Results: The in vitro treatment with BAY 1436032 unexpectedly increased growth in HT1080 (IDH1R132C) cells with no effect to the control cell lines SW1353 and SW872. In contrast, in vivo, combination of BAY1436032 with everolimus had additive anti-tumor efficacy and was synergistic with cisplatin or the MEK inhibitor refametinib in the treatment of HT1080 xenografts. The combination of BAY 1436032 and refametinib was also additive in the treatment of a melanoma xenograft with IDH1R132C mutation. Another additive efficacy of BAY 1436032 and gemcitabine or cisplatin could be observed in an ICC xenograft model and further in the combination with everolimus in the same model. The combination of BAY 1436032 together with azacitidine in a PDX AML model was also synergistic.

Conclusion: These results highlight the combinatorial potential of the mIDH1R132 inhibitor BAY 1436032 with standard of care therapy and the importance of microenvironmental factors, which has to be taken into account when dealing with mutant IDH tumors.

Citation Format: Stefan Kaulfuss, Julia Zaman, Anuhar Chaturvedi, Holger Hess-Stumpp, Jennifer Höde, Ricarda Biber, Renan Borowicz, Michael Heuser, Andreas von Deimling, Stefan Pusch. Preclincial in vitro and in vivo combination therapies for mutant IDH1R132 tumors with BAY 1436032 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2182.