bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 299: Synergistic activity of mebendazole and chemotherapeutic agents for rare cancers
Authors: Samizadeh Mahta , Chakrabarti Debashree , Zhu Yuhan , Treuting Robert Louis , Kaplan Johanne , Siders William , Barber Jamie D.
Published: August 2019

Depending upon the definition used, as many as half of all cancers diagnosed are considered rare cancers, and most rare cancer patients would greatly benefit from additional therapies. Repurposing of drugs for use in oncology represents an attractive strategy for the rapid development of therapies that address the significant unmet medical need that remains for rare cancers. The FDA-approved anti-helminthic drug mebendazole (MBZ) has been reported to affect several biological pathways involved in tumor growth and metastasis. Based on this activity, multiple rare cancer tumor cell lines including neuroblastoma, carcinoid tumors, esophageal adenocarcinoma, gastrointestinal stromal tumors, leiomyosarcoma, pheochromocytoma, and mesothelioma were analyzed for their susceptibility to MBZ treatment alone or in combination with standard chemotherapeutic agents in vitro. Cultured cells exposed to increasing concentrations of MBZ alone showed a significant decrease in cell viability with IC50s ranging from 0.09-2.2 μM. To evaluate efficacy of MBZ in an additional model system, an ex vivo 3D assay using gastric cancer primary cells isolated from two patient-derived xenograft (PDX) models was performed. Cells were seeded in 3D cultures and treated for seven days with MBZ at nine different doses in triplicate. IC50s were determined to be 0.74 and 0.76 μM indicative of significant potency. These results along with the in vitro data generated using cell lines suggest that MBZ may represent an effective anti-cancer agent across multiple rare cancer indications. However, since tumor cells can often evade or develop resistance to single agent therapy, the potential for synergistic combinations with chemotherapeutic agents was also explored in vitro. Cells were plated in triplicate and treated with a range of concentrations of MBZ and relevant chemotherapeutic agents in a complete Latin square. Combination indexes (CI) were determined based on the method described by Chou and Talalay. Several synergistic drug combinations were identified with CI values ranging from 0.008 to 0.428 (CI < 0.9 indicates synergy). Overall, our results indicate that synergistic combinations of MBZ and chemotherapy agents represent a promising strategy for the treatment of rare cancers.

Citation Format: Mahta Samizadeh, Debashree Chakrabarti, Yuhan Zhu, Robert Louis Treuting, Johanne Kaplan, William Siders, Jamie D. Barber. Synergistic activity of mebendazole and chemotherapeutic agents for rare cancers [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 299.
Abstract 2993: Impaired PARP1 DNA repair defines chemo-sensitivity in IDH1 mutant cell
Authors: Yang Chunzhang , Lu Yanxin , Liu Yang , Gilbert Mark , Wu Jing
Published: August 2019

INTRODUCTION: Mutations in isocitrate dehydrogenase (IDH1/2) are the most prevalent genetic deficiency in lower grade gliomas. IDH-mutated in glioma exhibits better clinical outcomes with longer patient survival, as well as greater sensitivity to chemotherapy. In the present study, we explored the molecular mechanisms that determine the chemo-sensitivity in IDH1-mutated cells, and seek for potential therapeutic strategy by targeting PARP/BER DNA repair pathway.

METHODS: We investigated transcriptomic profile from WHO grade II/III glioma based on their IDH1/2 mutation status. We established IDH1-mutated cells and investigated the alterations in nicotinamide adenine dinucleotide (NAD+), Poly (ADP-ribose) polymerase (PARP)-associated DNA repair and DNA damage in these cells in respond to temozolomide (TMZ). Moreover, we evaluated a PARP inhibitor olaparib and its synergistic effect on TMZ-associated cytotoxicity.

RESULTS: Our results showed that the IDH1-mutated cells are more vulnerable to genotoxic agent, which recapitulate the disease phenotype in IDH1-mutated glioma. TMZ treatment resulted in over 20-fold increase of cell death in IDH1-mutated cells compared with wild type counterpart. IDH1-mutated cells exhibited over 1.3-fold DNA damage and over 1.42-fold cellular apoptosis under TMZ treatment. Mechanistically, IDH1-mutated cells exhibited compromised NAD+ metabolism, as well as concomitant PARP/BER DNA repair pathway. IDH1-mutated cells produced 83.8% less poly (ADP-ribose) polymer (pADPR), an important substrate for PARP-associated DNA repair, during TMZ treatment, suggesting their incompetence to maintain genomic integrity due to decreased availability of NAD+. Targeting the PARP-associated DNA repair pathway using olaparib, an FDA-approved PARP inhibitor, remarkably potentiated TMZ-induced cytotoxic effects by 2.16-fold in IDH1-mutated cells.

CONCLUSION: Our findings demonstrate that metabolic defects in IDH1-mutated cells affect PARP-associated DNA repair pathway via NAD+ depletion, and therefore prompt the sensitivity to chemotherapies. Targeting PARP-associated DNA repair pathway suggests a novel therapeutic avenue for IDH1-mutated gliomas.

Citation Format: Chunzhang Yang, Yanxin Lu, Yang Liu, Mark Gilbert, Jing Wu. Impaired PARP1 DNA repair defines chemo-sensitivity in IDH1 mutant cell [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 2993.
Abstract 2995: Thymoquinone attenuates breast cancer cell resistance to paclitaxel via targeting tumor associated stem cell population
Authors: Bashmail Hanan A. , Alamoudi Aliaa A. , Hegazy Gehan A. , Agabnoor Ghada M.A. , Noorwali Abdulwahab , Al-Abd Ahmed M.
Published: August 2019

Breast cancer remains the most common type of cancer in females, and remains to be one of the most leading causes of death worldwide. This led to increasing the interest to use adjuvant anti-cancer therapy to improve the efficacy of in-clinical use chemotherapeutic agents. Thymoquinone (TQ) is a naturally occurring bioactive compound which showed cumulative evidences of anticancer properties. Herein, we assessed the potential chemomodulatory effect of TQ as an adjuvant therapy in combination with paclitaxel (PTX) against breast cancer cells. Cytotoxic profiles of TQ alone or in combination with PTX were evaluated in MCF7 and T47D breast cancer cell lines. TQ showed cytotoxic effect against MCF7 and T47D cells with IC50’s of 64.93±14 µM and 165±2 µM, respectively. In MCF7, TQ did not change PTX’s IC50, and the combination index was indicative of antagonism (CI-value = 1.3). Similarly in T47D, combination of TQ with PTX increased PTX’s IC50 from 90±8 nM to 140±20 nM with CI-value of 1.5 indicating antagonism. However, it was found that TQ completely depleted PTX resistant fractions in both cancer cell lines under investigation. Moreover, apoptotic and autophagic cell death due to combination of PTX with TQ were significantly increased in T47D compared to PTX treatment alone by 5.6 and 1.7 folds, respectively. In MCF7, combination of TQ with PTX significantly increased autophagic cell death compared to PTX treatment alone. Furthermore, tumor associated stem cell clone (CD44(+)/CD24(-)/(low) was assessed by flow cytometry in MCF7 and T47D cells. TQ alone significantly decreased CD44+/CD24- tumor associated stem cell clone by 12.4±0.8%. In addition, combination of PTX with TQ further depleted CD44+/CD24- cell clone by 32.3±0.08%. Finally, TQ alone significantly downregulated TWIST-1, and upregulated SNAIL2 gene expression as an evidence to decreased cell resistance to PTX. In conclusion, TQ enhances PTX anticancer profile against breast cancer cells via depleting tumor associated stem cells clone rather than potentiating its direct cell killing effect.

Citation Format: Hanan A. Bashmail, Aliaa A. Alamoudi, Gehan A. Hegazy, Ghada M.A. Agabnoor, Abdulwahab Noorwali, Ahmed M. Al-Abd. Thymoquinone attenuates breast cancer cell resistance to paclitaxel via targeting tumor associated stem cell population [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 2995.
Abstract 2996: Systemic depletion of L-methionine with an engineered human enzyme for the treatment of melanoma
Authors: Wilder Carly S. , Saha Achinto , Georgiou George , Stone Everett , DiGiovanni John
Published: August 2019

Metastatic melanoma is an aggressive form of cancer responsible for the majority of skin cancer related deaths. While treatment for metastatic melanoma has improved in recent years with the introduction of targeted therapies and immunotherapies, the five-year survival rate for stage IV melanoma remains only 15-20%. To address the need for alternative options for melanoma treatment, we have begun exploring use of an engineered human enzyme called methionine-gamma-lyase (hMGL). Many cancers, including melanoma, have a high requirement for methionine in comparison with non-cancerous cells. The hMGL enzyme works to exploit this difference by degrading extracellular L-methionine resulting in cancer cell starvation. The hypothesis tested in this research is that the systemic depletion of L-methionine using hMGL will provide significant benefits for melanoma treatment alone or in combination with other therapeutic agents. Treatment of several melanoma cell lines (A375, Hs294T, Skmel28, and B16F10) with various concentrations of hMGL demonstrated that L-methionine depletion reduced survival and led to a G2 cell cycle arrest. To investigate the mechanism of action, analyses of melanoma cells treated with hMGL have shown that signal transducer and activator of transcription 3 (STAT3) tyrosine phosphorylation is significantly reduced. Additionally, hMGL treatment leads to activation of the uncharged tRNA amino acid sensing pathway as shown by an increase in p-eIF2α and Sestrin 2 and an increase in autophagy shown by a decrease in p-ULKSer757 and an increase in cleaved LC3B. Because L-methionine depletion affects levels of glutathione through reduction of cysteine synthesis, preliminary experiments were conducted to evaluate hMGL treatment combined with a thioredoxin reductase inhibitor. This combination, targeting two different intracellular antioxidant pathways, resulted in synergistic inhibition of survival in multiple melanoma cell lines. The current data support the hypothesis that L-methionine depletion using hMGL may have therapeutic efficacy either alone or in combinations for melanoma treatment. Research funded by CPRIT grant RP180590.

Citation Format: Carly S. Wilder, Achinto Saha, George Georgiou, Everett Stone, John DiGiovanni. Systemic depletion of L-methionine with an engineered human enzyme for the treatment of melanoma [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 2996.
Abstract 3004: Anticancer effects and associated molecular changes of Carica papaya against prostate cancer
Authors: Singh Surya Pratap , Mathan Sivapar V. , Dheeraj Arpit , Tailor Dhanir , Singh Rana P. , Acharya Arbind
Published: August 2019

Carica papaya (papaya), from the family of Caricaceae, is a perennial plant originating from the Southern part of Mexico. Carica papaya leaf extract (CPE) has been traditionally used to treat various diseases, including infectious diseases and cancer. However, the anticancer effects and molecular mechanism of CPE are elusive. The aim of our study is to examine the anticancer effects of aqueous leaf extract of Carica papaya against prostate cancer (PCa). We investigated the effect of CPE on LNCaP, DU145 and PC-3 PCa cells. CPE treatment (5, 10, 25µl/ml) significantly reduced the cell proliferation and induced cell death in PCa cells. Furthermore, we found that CPE induced G1, S and G1 as well as G2/M phase cell cycle arrest in LNCaP, DU145, and PC-3 cells, respectively. At molecular level, the cell cycle arrest was associated with decreased expression of CDK 4, cyclin D1, cyclin B1, and PCNA. CPE induced cell death was associatedwith depolarization of mitochondrial membrane potential, increase in ratio of Bax/Bcl2, cleavage of caspase-3 and cleaved -Poly(ADP-ribose) polymerase (PARP). CPE treatment also reduced mitochondrial fission and induced mitochondrial fusion by reducing the level of Drp1 protein. Further, we observed that CPE increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin. We checked the CPE toxicity in C57BL/6 male mice. We found that oral administration of CPE(0.25%, 0.5% and 1% v/v) in drinking water did not show any significant changes in body weight, water consumption and food intake as compared to control group. Collectively, CPE inhibited cell proliferation, induced cell death via apoptosis, mitochondrial fusion, epithelial marker and reduced mesenchymal markers.In vivo toxicity study with CPE treatment via drinking water was found to be non-toxic. These findings suggest that CPE has potential as anticancer therapeutic option for prevention and treatment of prostate cancer.

Citation Format: Surya Pratap Singh, Sivapar V. Mathan, Arpit Dheeraj, Dhanir Tailor, Rana P. Singh, Arbind Acharya. Anticancer effects and associated molecular changes of Carica papaya against prostate 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 3004.
Abstract 3005: High density lipoprotein mimics inhibit prostate cancer exosome-mediated communication with myeloid cells
Authors: Henrich Stephen E. , McMahon Kaylin M. , Plebanek Michael P. , Thaxton C. Shad
Published: August 2019

Introduction: Exosomes are 30-150 nm membrane-bound vesicles that mediate intercellular communication and are secreted in abundance by neoplastic cells. Tumor-derived exosomes are capable of transmitting pro-malignant signals to target cells at local or distant sites to enhance tumor invasiveness and promote metastasis. A sub-set of this pro-malignant intercellular communication is dependent upon target cell cholesterol homeostasis. Here, we show that prostate cancer exosome communication with myeloid cells can be inhibited by treatment with high density lipoprotein mimetic nanoparticles (HDL NPs), which reduce cholesterol in myeloid cells in a targeted fashion.

Methods: Exosomes were isolated by ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells and used for all experiments. In vitro assays were conducted using murine bone marrow macrophages obtained from culture of total murine bone marrow in M-CSF for 7 days. Confocal microscopy and flow cytometry were used for in vitro and in vivo uptake assays. Osteoclastogenesis assays were performed using a commercially available TRAP staining kit (Sigma-Aldrich). NF-kB signaling experiments were performed using a reporter human monocyte cell line (THP1-Dual). HDL NPs were synthesized using 5 nm gold nanoparticle templates, apolipoprotein A-1, and phospholipids.

Results: HDL NPs were found to inhibit the cellular uptake of prostate cancer exosomes in mouse bone marrow macrophages in vitro. Furthermore, HDL NPs inhibited exosome-induced osteoclastogenesis and exosome-induced monocyte NF-kB signaling. Finally, HDL NP-mediated inhibition of exosome communication was found to be dependent upon scavenger receptor type B-1 (SR-B1). SR-B1 was shown to be expressed ubiquitously in mouse bone marrow macrophages; and HDL NPs were unable to inhibit exosome communication in bone marrow macrophages derived from SR-B1-/- mouse bone marrow.

Conclusion: These data demonstrate that HDL NPs inhibit prostate cancer exosome communication with murine myeloid cells, as evidenced by cellular uptake, osteoclastogenesis, and NF-kB signaling. Moreover, SR-B1 is required for HDL NP inhibition of PCa exosome communication. In sum, these results indicate that target cell cholesterol homeostasis may be important for exosome-mediated signaling in prostate cancer, particularly in immune cells and in the bone microenvironment, and that HDL NPs are potent inhibitors of PCa exosome-mediated signaling.

Citation Format: Stephen E. Henrich, Kaylin M. McMahon, Michael P. Plebanek, C. Shad Thaxton. High density lipoprotein mimics inhibit prostate cancer exosome-mediated communication with myeloid 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 3005.
Abstract 3007: Validation of an NGS panel assay for detection of hotspot cancer somatic mutations
Authors: Li Wenzhi , She Henry , Tian Xiaohua , Neja Margaret , Harris Adrian , Li Yuhua
Published: August 2019

Background

The gene tests to predict whether cancers respond to specific targeted therapies or prediction of prognosis has quickly increased over the last decade. Next-generation sequencing (NGS) has become a powerful tool for high-throughput and sensitive gene mutation testing, which is required for development of targeted therapies, clinical trials, and further research. The aim of this study was to validate the AmpliSeq for Illumina Cancer Hotspot Panel v2 on the Illumina MiSeq platform and simultaneously analyze 2800 somatic mutations across the hotspot regions of 50 genes, including SNP and small INDEL, with known associations to cancer, as identified in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. This panel enables the study of the genes associated with different cancer types, including the cancers of the lung, colon, breast, thyroid, oral, bladder, prostate, liver, kidney, ovarian and cervix, etc.

Methods and findings

We validated this assay using the genomic DNA extracted from human paraffin-embedded (FFPE) samples and peripheral blood with negative control DNA (NA12878), positive control DNA (HD701), blank control as well as the global proficiency testing samples. Following two lab scientists parallel NGS library preparation, sequenced on Miseq platform (2x150 PE) and analyzed using a pipeline centered on Illumina Basespace, the somatic mutations were called at an average number of reads coverage >2500X, variant frequency cutoff 1%. An integrated test report can be generated within five working days. Panel sequencing analysis showed that the mutations are 100% concordance with known mutations and consistent in four runs, between lab personal performances, different input DNA concentrations (1ng-100ng), etc. Briefly, 25 mutations were detected in the FFPE samples and 20 mutations detected in proficiency testing samples. The variant types, HGVS ID and frequency are 100% match with its standard results which are a prove of precision and sensitivity.

Conclusions

A sensitive, high-throughput, pan-cancer mutation panel for NGS analysis of cancer hot-spot mutations in 50 genes was highly repeatable and reproducible and validated for routine application in a gene testing lab.

Note: This abstract was not presented at the meeting.

Citation Format: Wenzhi Li, Henry She, Xiaohua Tian, Margaret Neja, Adrian Harris, Yuhua Li. Validation of an NGS panel assay for detection of hotspot cancer somatic mutations [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 3007.
Abstract 301: Concurrent targeting of BMI1 and CDK4/6 inhibited breast cancer tumorigenicity in vitro and in vivo
Authors: Alajez Nehad M.
Published: August 2019

Despite recent advances in cancer management and therapy, resistance to cytotoxic medications remains a major clinical challenge. Intratumoral heterogeneity appears to play crucial role in drug resistance; therefore, combination-based approaches are currently gaining momentum. In the current study, we investigated the ramifications of concurrent targeting of tumor-initiating cells (using BMI1 specific inhibitor, PTC-209) and cell cycle progressiontargeting CDK4/CDK6 using palbociclib on breast, colorectal and prostate cancer cell growth in vitro and in vivo. PTC-209 reduced BMI1 protein expression, while palbociclib (PB) inhibitedCDK4, Rb, and PRb Ser795 phosphorylation. PTC-209 and palbociclib exhibited dose-dependent cytotoxic effects against MDA-MB-231, HCT-116, and PC-3 cell models, which was more profound in the combination group. Global gene expression profiling in MDA-MB-231 treated with PTC-209 revealed multiple affected cellular pathways including insulin signaling, focal adhesion, DNA damage response, and Wnt/pluripotency. On the other hand, PB treatment ofMDA-MB-231 led to substantial changes in gene expression affecting mainly cell cycle progression. PTC-209 and palbociclib reduced colony formation potential, sphere formation, cell migration, and cell viability, which was further enhanced in the combination group. Consistent with our results in vitro, combination treatment of PTC-209 and palbociclib reduced tumor growth more profoundly than either of these drugs alone in in vivo model of MDA-MD-231 cells. Therefore, our data suggest concurrent targeting of BMI1 and CDK4/CDK6 might provide novel therapeutic opportunity for breast, colorectal, and prostate cancer.

Citation Format: Nehad M. Alajez. Concurrent targeting of BMI1 and CDK4/6 inhibited breast cancer tumorigenicity in vitro and in vivo [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 301.
Abstract 3016: Dicer overexpression contributes to chemoresistance in colorectal cancer via up-regulating a set of miRNAs
Authors: Lin Li Jyuan , Hung Liang-Yi
Published: August 2019

Chemotherapy has a good success rate in the treatment of colorectal cancer; however, recurrence of colorectal cancer is still frequent due to acquired resistance. Aurora-A, a cell cycle-regulated kinase, plays an important role in colorectal cancer development and drug resistance. Dicer, one of the key enzymes of the microRNA (miRNA) biogenesis pathway, may be involved in chemoresistance through regulating the expression of miRNAs. According to the TCGA database, the expression levels of Dicer and Aurora-A were increased in colorectal cancer, which may contribute to chemoresistance. Our results showed that the protein levels of Dicer and Aurora-A are increased in oxaliplatin resistant cell lines. Knocked-down expression of Dicer or Aurora-A can enhance drug sensitivity in oxaliplatin-resistant cells; in contrast, overexpression of Dicer or Aurora-A increased drug resistance in parental drug sensitive cells. By next-generation sequencing, we found that the expression of a set of miRNAs is increased in oxaliplatin resistant cells. Dicer overexpression enhances the expression of those miRNAs in drug sensitive parental cells. Clinical evaluation further confirmed the increased expression of those miRNA in the plasma of CRC patients with a poor response to oxaliplatin. Our results suggest that those miRNAs may act as biomarkers to predict oxaliplatin response in colorectal cancer by detecting their plasma miRNAs. The molecular mechanism of those miRNAs in regulating the drug response is currently under our investigation.

Citation Format: Li Jyuan Lin, Liang-Yi Hung. Dicer overexpression contributes to chemoresistance in colorectal cancer via up-regulating a set of miRNAs [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 3016.
Abstract 3040: The expressional effects of RBBP6 in breast cancer cells are p53-dependent
Authors: Moela Pontsho , Motadi Lesetja , Lekganyane Marcia
Published: August 2019

The incidence rate of breast cancer has increased beyond that of lung cancer, making it the most common malignancy among women. Breast tumor progression is partly because of p53 inactivation by overexpressed that possess p53 binding domain. RBBP6 forms a member of ubiquitous regulatory proteins because its RING finger-like domain has an E3 ligase activity. The overexpression of RBBP6 in several malignancies makes it a potential target in cancer management. However, there is no evidence on whether the effect of RBBP6 on cell growth and apoptosis is cell line dependent, more especially in breast cancer cell lines that have distinct p53 expression profiles. Therefore, the aim of this study was to evaluate the RBBP6 effects on cell growth and apoptosis in breast cancer cell lines with different p53 expression profiles. RBBP6 expression was successfully manipulated using gene silencing and protein overexpression techniques in MCF-7 and MDA-MB-231 cell lines. Transfected cells were co-treated with anti-cancer agents followed by apoptosis detection using confocal microscopy and flow cytometry, which was further confirmed by caspase 3/7 activity and quantification of apoptotic genes. RBBP6 was overexpressed in breast cancer tissues that were classified as stage 3 and 4 while in stage 1 it was expressed but at much lower levels. The wt. p53-expressing MCF-7 cell line was more susceptible to apoptosis induction as opposed to the mt. p53-expressing MDA-MB-231. RBBP6 silencing led to a significant accumulation of p53 expression in MCF-7 as compared to MDA-MB-231. Co-treatment with GABA and camptothecin seemed to sensitize the cells to apoptosis induction. These data suggest that RBBP6 silencing triggers significant levels of intrinsic apoptosis and over-expression appears to promote cell proliferation in wild-type p53-expressing MCF-7 rather than in MDA-MB-231 cells. In conclusion, the effect of RBBP6 on cell proliferation and apoptosis induction in breast cancer seem to be cell line dependent based on p53 status.

Note: This abstract was not presented at the meeting.

Citation Format: Pontsho Moela, Lesetja Motadi, Marcia Lekganyane. The expressional effects of RBBP6 in breast cancer cells are p53-dependent [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 3040.
Abstract 3053: Development of novel cell-based bioassays for the development of biologics targeting LAG-3 and PD-1xLAG-3
Authors: Grailer Jamison , Garvin Denise , Hartnett Jim , Fan Frank , Cong Mei , Cheng Zhi-jie Jey
Published: August 2019

Programmed cell death protein 1 (PD-1) and Lymphocyte-activation gene 3 (LAG-3) are immune checkpoint receptors that bind to PD-L1/PD-L2 or MHCII, respectively, on antigen presenting cells or tumor cells. PD-1 or LAG-3 activation results in the inhibition of T cell receptor (TCR) signaling and reduced T cell activation. However, co-blockade of PD-1 and LAG-3 leads to a synergistic enhancement in T cell responses, suggesting functionally divergent inhibitory signaling mechanisms between the two receptors. Preclinical studies suggest that blocking LAG-3 re-activates the immune system to kill cancer cells, and co-blockade of both PD-1 and LAG-3 lead to a synergistic increase in tumor control and survival in preclinical models. Current methods for measuring the potency of LAG-3 and PD-1xLAG-3 blocking antibodies measure cytokine production from human PBMCs upon Staphylococcal Enterotoxin B (SEB) superantigen stimulation. These protocols are tedious, highly variable, and require the use of highly toxic superantigen. Herein, we describe the development of reporter-based bioassays for the development of LAG-3 and PD-1xLAG-3 combination-targeted biologics. Engineered T effector cell lines expressing LAG-3 alone or in combination with PD-1 are activated in an antigen-dependent manner by APC cell lines. The assays are specific for LAG-3 or PD-1xLAG-3-targeted biologics and are robust as demonstrated by Design of Experiments analyses. In addition, the assays can be used to measure the relative potencies of test samples compared with reference sample in a detection range of 50% to 200% and show appropriate precision, accuracy and linearity. The assays can detect potency changes for heat-stressed samples, and are tolerant to human serum, demonstrating the potential to be further developed into neutralizing antibody (NAb) assays. Therefore, these bioassays can serve as valuable tools for the development of biologics targeting LAG-3 and PD-1xLAG-3.

Citation Format: Jamison Grailer, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Development of novel cell-based bioassays for the development of biologics targeting LAG-3 and PD-1xLAG-3 [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 3053.
Abstract 3055: Genome-wide CRISPR/Cas9 screen for the identification of novel YAP1/TAZ modulators
Authors: Naujoks Jan , Potze Lisette , Anurin Anna , Kuehnlenz Julia , Lesche Ralf , Kamburov Atanas , Nevedomskaya Ekaterina , Steffen Andreas , Lange Martin , Nicke Barbara
Published: August 2019

Aberrant activation of the Hippo pathway effectors YAP1/TAZ promotes cell proliferation and tumorigenesis. To identify novel regulators of YAP1/TAZ as a possible means to treat cancer, we established a novel, FACS-based screening system monitoring YAP1/TAZ activity in MDA-MB-231 breast cancer cells. Using these cells, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. We identified approximately 50 genes potentially activating YAP1/TAZ with functions in the Actin Cytoskeleton signaling, p53 signaling, cell polarity or ER stress, amongst others. Moreover, we identified about 30 potential targets which when knocked out induce activity of YAP1/TAZ. The list of hits included genes known to affect the YAP1/TAZ activity such as AJUBA, LATS2 and TEAD, demonstrating the validity of the screen. Functional validation of the novel potential YAP1/TAZ modulators will aid to the further understanding of YAP1/TAZ biology and may open the door to new therapeutic avenues for targeting YAP1/TAZ in cancer.

Citation Format: Jan Naujoks, Lisette Potze, Anna Anurin, Julia Kuehnlenz, Ralf Lesche, Atanas Kamburov, Ekaterina Nevedomskaya, Andreas Steffen, Martin Lange, Barbara Nicke. Genome-wide CRISPR/Cas9 screen for the identification of novel YAP1/TAZ modulators [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 3055.
Abstract 3059: Identification and validation of an Aspergillus nidulanssecondary metabolite derivative as an inhibitor of the Musashi1-RNA interaction
Authors: Lan Lan , Liu Jiajun , Amith Amber , Appelman Carl , Wu Xiaoqing , Li Ke , Roy Anuradha , Gowthaman Ragul , Karanicolas John , Somoza Amber , Wang Clay , Oakley Berl , Guzman Roberto De , Neufeld Kristi , Xu Liang
Published: August 2019

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. It is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb, APC and P21WAF-1, key regulators of Notch, Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1-mRNA interaction thus blocking the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA binding site. Two clusters of hit compounds have been identified, one of which is composed of derivatives of secondary metabolites from Aspergillus nidulans. The top hit, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance which revealed that Aza-9 binds to MSI1 directly, in the RNA binding pocket. Next, we tested whether Aza-9 has anti-cancer potential in cells and whether such activities work through MSI1. Due to poor cellular uptake, we used PEGylated liposomes to facilitate Aza-9 entry into the cells. Aza-9-lipo inhibits colon cancer cell proliferation, induces apoptosis and autophagy, and down-regulates Notch/Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1 function while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

Citation Format: Lan Lan, Jiajun Liu, Amber Amith, Carl Appelman, Xiaoqing Wu, Ke Li, Anuradha Roy, Ragul Gowthaman, John Karanicolas, Amber Somoza, Clay Wang, Berl Oakley, Roberto De Guzman, Kristi Neufeld, Liang Xu. Identification and validation of an Aspergillus nidulanssecondary metabolite derivative as an inhibitor of the Musashi1-RNA interaction [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 3059.
Abstract 3066: The expression of carcinoembryonic antigen-related cell adhesion molecule 6 and 8 in breast cancer
Authors: Iwabuchi Erina , Miki Yasuhiro , Takagi Kiyoshi , Onodera Yoshiaki , Shibahara Yukiko , Ishida Takanori , Suzuki Takashi , Sasano Hironobu
Published: August 2019

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 6 is a protein reported to be involved in tumor cell proliferation in breast cancer. The presence of intratumoral CEACAM8 positive neutrophils has been reported as an independent prognostic factor in both renal cell and hepatocellular cancer. In addition, CEACAM6 and 8 form a heterodimer; however, the possible interactions of these two proteins remain unclear in breast cancer. Therefore, in this study we first immunolocalized CEACAM6 and 8 in surgical pathology specimens of breast cancer. We examined 109 breast cancer and 20 metastatic breast cancer patients in this study. We then established MCF-7 cells (CEACAM6 positive) stably transfected with CEACAM8 and analyzed the interaction between CEACAM6 and CEACAM8 using proximity ligation assay (PLA) and further evaluated cell proliferation, invasion, and adhesion. Results of immunohistochemical analysis revealed that the number of CEACAM6 positive breast cancer cells was significantly higher in patients with low histological grade and stage also in CEACAM8 positive cells. The number of CEACAM6 and 8 double positive breast cancer cells was significantly higher in patients with low stage. In addition, CEACAM6 and 8 interaction was detected using the PLA through high expression of both in MCF-7 cells. Furthermore, their co-expression promoted cell adhesion of MCF-7 cells to the monolayer of endothelial cells but suppressed both cell invasion and proliferation. In metastatic breast cancer, CEACAM6 status was significantly higher in bone metastases than in lung metastases. To the best of our knowledge, this is the first study demonstrating the expressions of CEACAM6 and 8 in breast cancer. Results of our present study also indicated that both CEACAM6 and CEACAM8 could be involved in less aggressive subtypes of breast cancer possibly trough inhibition of vascular invasion and proliferation. In addition, results indicated that CEACAM6 was involved in breast cancer metastases to the bone but further studies are required to clarify the clinical significance of their interactions in breast cancer patients.

Citation Format: Erina Iwabuchi, Yasuhiro Miki, Kiyoshi Takagi, Yoshiaki Onodera, Yukiko Shibahara, Takanori Ishida, Takashi Suzuki, Hironobu Sasano. The expression of carcinoembryonic antigen-related cell adhesion molecule 6 and 8 in 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 3066.
Abstract 3072: GFH018, a novel TGF-βRI inhibitor, for the treatment of advanced solid tumors
Authors: Hu Gang , Zhao Rong , Zhou Fusheng , Lan Jiong , Zheng Biao , LU Qiang , Lu Jianyu , Wu Lifang , Sun Fei , Hu Lihong , Han Shuhua , Tong Haijun , Xia Yuanfeng , Li Wei , Ma Liping , Chen Ying , Ding Charles , Chan Chichung , Chen Shuhui
Published: August 2019

Background: The transforming growth factor-β (TGF-β) signaling pathway has been shown to play an important role in various cellular processes including growth inhibition, cell migration, tumor invasion, epithelial-mesenchymal transition, and immune-suppression. TGF-βs are often overexpressed in various types of cancers, and the anti-TGF-β therapy has been pursued intensively for cancer treatment.

Results: GFH018 is a potent, selective inhibitor of TGF-βRI with a kinase inhibition activity IC50 of 70.5 nM. Our studies demonstrated that GFH018 could inhibit TGF-β-induced SMAD phosphorylation and migration of tumor cells. GFH018 is a potent immune regulator in that it could inhibit in vitro Treg cell induction and reverse M2 phenotype to M1, leading to increased pro-inflammatory cytokine production. In addition, GFH018 exhibited a significant inhibitory effect on in vitro angiogenesis formation assay. Importantly, GFH018 has shown promising anti-tumor efficacy in multiple in vivo mouse syngeneic models as a monotherapy as well as in combination with an anti-PD-(L)1 antibody. Following oral administration, GFH018 demonstrated fast absorption, and high oral bioavailability in preclinical animal species including mouse, rat, and dog. Results from the toxicology studies in rat and dog have also supported further development of GFH018.

Conclusion: GFH018 has the potential to be developed as a monotherapy or combinational therapy with checkpoint inhibitors for advanced solid tumor patients. The first-in-human clinical trial is scheduled in 2019.

Citation Format: Gang Hu, Rong Zhao, Fusheng Zhou, Jiong Lan, Biao Zheng, Qiang LU, Jianyu Lu, Lifang Wu, Fei Sun, Lihong Hu, Shuhua Han, Haijun Tong, Yuanfeng Xia, Wei Li, Liping Ma, Ying Chen, Charles Ding, Chichung Chan, Shuhui Chen. GFH018, a novel TGF-βRI inhibitor, for the treatment of advanced solid tumors [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 3072.
Abstract 3081: DZD9008, an oral, wild type selective EGFR inhibitor for the treatment of non-small-cell lung cancer with Exon20 insertion and other activating mutations
Authors: Xu Yan , Zhang Lin , Zhu Lifang , Wang Yingchun , Wang Mei , Yang Zhenfan
Published: August 2019

Background: Several EGFR TKIs have been approved for the treatment of non-small cell lung cancer (NSCLC) with L858R mutation, Exon 19 deletion, and T790M mutations. However, for patients with uncommon mutations, such as EGFR or HER2 Exon20ins, there lacks safe and effective therapy. All Exon20ins inhibitors under clinical development have a negative selectivity profile, inhibiting wild type EGFR more potently than mutant ones. Consequently, severe wild type EGFR-driven toxicities have been observed in patients. DZD9008 is an oral, potent, irreversible, wild type-selective EGFR TKI against EGFR or HER2 Exon20ins and other mutations. The present study shows anti-tumor activity of DZD9008 in tumor cell lines and xenograft models.

Materials and Methods: The enzyme assay was performed by incubating DZD9008 with recombinant enzymes at 2 mM ATP concentrations. The cellular activity of DZD9008, including phosphorylation of EGFR or HER2 and cell proliferation, was evaluated in a panel of cell lines expressing wild type EGFR, mutant EGFR or HER2, using MSD assay and CellTiter-Glo assays. DZD9008 in vivo anti-tumor activity was evaluated in both cell line-derived (CDX) and patient-derived xenograft (PDX) models, carrying EGFR single or double mutations, Exon20ins mutations, and wild type EGFR.

Results: The enzymatic IC50 of DZD9008 in mutant EGFR ranges from 0.4 to 2.1 nM. DZD9008 downregulated pEGFR with IC50 ranging from 1 to 22 nM in a panel of tumor cell lines expressing EGFR L858R, Exon19del, L858R/T790M, various Exon20ins or uncommon mutations. Similar activity against pHER2 was observed in tumor cells with HER2 Exon20ins mutation, with IC50 at 7 nM. In contrast, DZD9008 was less potent in modulating pEGFR in tumor cells expressing wild type EGFR, with IC50 greater than 80 nM. In cell proliferation assays, DZD9008 suppressed cell proliferation with GI50 of 1 to 60 nM in tumor cells carrying EGFR L858R, Exon19del, L858R/T790M, various Exon20ins or uncommon mutations. In CDX and PDX models carrying EGFR Exon19del single mutation, L858R/T790M double mutations, and PDX models harboring G719S/L861Q or Exon20ins, DZD9008 induced dose dependent tumor growth inhibition and regression. Good PK/PD relationship was established across these tumor models.

Conclusion: DZD9008 is a potential EGFR TKI for NSCLC patients with EGFR or HER2 Exon20ins and other activating mutations.

Citation Format: Yan Xu, Lin Zhang, Lifang Zhu, Yingchun Wang, Mei Wang, Zhenfan Yang. DZD9008, an oral, wild type selective EGFR inhibitor for the treatment of non-small-cell lung cancer with Exon20 insertion and other activating mutations [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 3081.
Abstract 3083: A novel hedgehog signaling inhibitor for targeting pancreatic ductal adenocarcinoma
Authors: Khire Atul , Zhong Yi , Rajanala Kalpana , Iacobuzio-Donahue Christine , Resh Marilyn D.
Published: August 2019

Sonic Hedgehog (Shh) signaling is deregulated in pancreatic cancer and can drive tumor onset and progression. Previous attempts to block Shh signaling used Smoothened (Smo) inhibitors, which target canonical Shh signaling. These approaches met with limited success, due to development of resistance, and contributions from non-canonical, Smo-independent signaling pathways that operate within tumor epithelial cells. Here, we show that Hedgehog acyltransferase (Hhat), the enzyme responsible for palmitoylation of Shh, is a novel target for inhibition of Shh signaling in human and mouse pancreatic cancer cells and tumors. TDI-003410, a small molecule inhibitor of Hhat recently developed by our group, reduced proliferation of AsPC1 cells, a human pancreatic cancer cell line, in vitroand in subcutaneous xenografts in mice in vivo. The mechanism is via induction of a G2/M cell cycle arrest leading to apoptosis. We next used cell lines isolated from tumors formed in KPC (K-RasG12D/p53R172H) mice, a genetically engineered mouse model of pancreatic cancer.TDI-003410 inhibited the growth of KPC cell linesand also reduced Gli1 levelsin vitro. Neither LDE225, a Smo inhibitor, nor SAG, a Smo activator, had any effect on KPC cell proliferation or Gli1 levels in vitro. In orthotopic xenografts using KPC cells implanted into the pancreas, TDI-3410 treatment significantly reducedin vivotumor burden. No significant changes in CD31 or Trichrome (collagen) stainingwith TDI-3410 were detected in KPC orthotopic tumor tissue. However, Gli-1 levels in tumor tissue from TDI-3410 treated mice were significantly reduced compared to the vehicle controls. These findings provide proof of concept for the potential therapeutic efficacy of Hhat inhibition in pancreatic cancer. The concept of using an Hhat inhibitor is unique: Hhat is a novel target that hits upstream, directly at hedgehog ligands. This approach can inhibit both canonical and non-canonical (Smo-independent) as well as autocrine and paracrine Shh signaling.

Citation Format: Atul Khire, Yi Zhong, Kalpana Rajanala, Christine Iacobuzio-Donahue, Marilyn D. Resh. A novel hedgehog signaling inhibitor for targeting pancreatic ductal adenocarcinoma [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 3083.
Abstract 3088: Farnesyl/geranylgeranyl transferase dual inhibitor thwarts mutant KRas-driven patient-derived pancreatic tumors
Authors: Kazi Aslamuzzaman , Xiang Shengyan , Yang Hua , Chen Liwei , Kennedy Perry , Ayaz Muhammad , Fletcher Steven , Cummings Christopher , Lawrence Harshani , Beato Francisca , Yang Ya'an , Kim Michael P. , Delitto Andrea , Underwood Patrick , Fleming Jason B. , Trevino Jose , Hamilton Andrew D. , Sebti Said M.
Published: August 2019

Although mutant KRas is a significant driver of pancreatic oncogenesis and resistance to therapy, there are no KRas inhibitors available for these patients. Farnesyltransferase inhibitors (FTIs) were developed as potential anticancer drugs because KRas requires farnesylation for its membrane localization and cancer-causing activity. However, KRas becomes geranylgeranylated and active when cancer cells are treated with FTIs. In this study, we designed a Ras C-terminal mimetic dual Farnesyl/geranylgeranyltransferase-1 (GGT-1) inhibitor, FGTI-2734, to overcome the geranylgeranylation-dependent resistance to FTIs. Immunofluorescence, cellular fractionation, and gel shift assays showed that FGTI-2734, but not the selective FTI-2148 and GGTI-2418, inhibited membrane localization of KRas in mt KRas pancreatic, lung, and colon human cancer cells. FGTI-2734 inhibited the growth in mice of mt KRas-dependent but not -independent human tumors, indicating its selectivity for mt KRas-driven cancers. Importantly, FGTI-2734 inhibited the in vivo growth of xenografts derived from four pancreatic cancer patients with mt KRas (two G12D, and two G12V) tumors. In addition, FGTI-2734 was highly effective at inhibiting, in three-dimensional co-cultures with chemotherapy resistance-promoting pancreatic stellate cells, the viability of primary and metastatic mutant KRas (G12D, G13D, and G12V) tumor cells derived from 8 pancreatic cancer patients. Finally, FGTI-2734 suppressed oncogenic pathways mediated by Akt, mTOR, and cMyc while upregulating p53 and inducing apoptosis in patient-derived xenografts in vivo. Thus, the development of this novel dual FT and GGT-1 inhibitor overcomes a major hurdle in KRas resistance, thwarting the growth of patient-derived mutant KRas-driven xenografts from pancreatic cancer patients, and as such it warrants further advanced preclinical and clinical studies.

Citation Format: Aslamuzzaman Kazi, Shengyan Xiang, Hua Yang, Liwei Chen, Perry Kennedy, Muhammad Ayaz, Steven Fletcher, Christopher Cummings, Harshani Lawrence, Francisca Beato, Ya'an Yang, Michael P. Kim, Andrea Delitto, Patrick Underwood, Jason B. Fleming, Jose Trevino, Andrew D. Hamilton, Said M. Sebti. Farnesyl/geranylgeranyl transferase dual inhibitor thwarts mutant KRas-driven patient-derived pancreatic tumors [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 3088.