bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2915: Penfluridol suppresses glioblastoma tumor growth by inhibiting sonic hedgehog signaling
Authors: Ranjan Alok , Srivastava Sanjay K.
Published: July 2016

Glioblastoma (GBM) is the most common brain tumor with poor survival rate. The main obstacle in the treatment of glioblastoma patients is the presence of blood brain barrier, which restricts the movement of the drugs to reach the brain. Penfluridol (PF) is known to cross blood brain barrier and is a clinically approved drug for schizophrenia patients. It is demonstrated and established by us that penfluridol suppresses the growth of metastasized breast cancer cells in brain giving us the rationale to evaluate it against glioblastoma (Alok Ranjan, Parul Gupta and Sanjay Srivastava, Cancer Research 2015). Penfluridol significantly reduced the viability of U87-MG, T98G and U251 MG glioblastoma cells with an IC50 of 6μM, 5.5μM and 9μM respectively after 24 h of treatment and induced apoptosis as exhibited by FITC/Annexin assay and cleavage of caspase 3 as well as PARP. It has been shown that GLI1, a transcription factor belonging to sonic hedgehog signaling is overexpressed in GBM and responsible for tumor progression. Our results demonstrated that penfluridol treatment reduced the expression of GLI1 in U87MG, T98G and U251 MG cells in concentration-dependent manner. In addition, overexpression of MGMT, a DNA repair enzyme has been linked to temozolomide (TMZ) resistance in GBM therapy. Combination of PF and TMZ induced more apoptosis and reduced the expression of GLI1 and MGMT as compared to PF or TMZ treatment alone. Our results further demonstrated that oral administration of PF inhibited the growth of GBM tumors by 66% and 72% in subcutaneous and intracranial GBM tumor model respectively. Immunohistochemical analysis of tumor tissue and western blot analysis of tumor lysate indicated down regulation of GLI1, MGMT and increase in cleavage of caspase 3, confirming in vitro finding. Apoptosis induction effect of PF treatment in tumor tissues was further confirmed by Tunel staining. Taken together, these results indicate that overall GBM tumor growth suppression by penfluridol was associated with inhibition of Gli1 and induction of apoptosis

Citation Format: Alok Ranjan, Sanjay K. Srivastava. Penfluridol suppresses glioblastoma tumor growth by inhibiting sonic hedgehog signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2915.
Abstract 2917: Therapeutic targeting of the mitochondria: An evaluation of the transcriptional link between the antioxidant response and autophagy
Authors: Pokrzywinski Kaytee L. , Mascia Francesca , Rao. V. Ashutosh
Published: July 2016

Mitoquinone (MitoQ), a mitochondrially targeted chemotherapy induces autophagy via excess reactive oxygen species (ROS) production. Elevations in ROS stimulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in breast cancer cell lines. Nrf2 is a nuclear transcription factor that activates the antioxidant response element when under oxidative stress. The aim of this study was to evaluate transcriptional and protein changes associated with Nrf2 activation to identify potential links between oxidative stress, autophagy and cell death. By suppressing Nrf2 and its targeted antioxidant enzymes we should induce elevated ROS levels, which can thereby alter cellular homeostasis and impact the ultimate fate of cancer cells. In the breast cancer cell line MDA-MB-231 RNA was extracted after 24 hours of Nrf2 or non-target pool (NTP) silencing and 18 hours of MitoQ treatment. RNA was sequenced using an Illumina HiSeq and differential expression was analyzed using DESeq in the statistical program R. To avoid bias, raw read counts were filtered to remove low counts possibly due to constraints of the instrumentation. To validate efficient silencing of Nrf2 we confirmed that Nrf2 transcript levels decreased and that there were observed reductions in transcripts containing the Nrf2 transcriptional promoter, the antioxidant response element (ARE) including FTH1, FTL, HMOX1, SRXN1, TXNRD1, NQO1, GCLC, GCLM, GSR and SQSTM1. From there we validated RNAseq data with qPCR using 25 random transcripts and found significant positive correlation between the two methods. Comparisons were first made between the MitoQ and DMSO treatment by silencing (NTP or Nrf2). 2775 genes were differentially expressed (DE) for MitoQ vs DMSO NTP siRNA and 1869 genes were DE for MitoQ vs DMSO Nrf2 siRNA both at an α < 0.05 and Log2 fold change cut off > |1|. We then compared the degree of overlap in DE gene lists between the NTP and Nrf2 siRNA comparisons to identify genes affected only by Nrf2 silencing in combination with MitoQ treatment. 1405 mRNAs were common between silenced and non-silenced sample sets, only 464 mRNAs were due to Nrf2 silencing alone. We are currently performing pathway analysis on these transcripts to determine the role of Nrf2 silencing with redox-active therapies. We are also evaluating autophagic protein levels at later time points, 24 to 48 hours, to further evaluate the potential signaling between oxidative stress and the autophagic response driven by Nrf2.

Citation Format: Kaytee L. Pokrzywinski, Francesca Mascia, V. Ashutosh Rao. Therapeutic targeting of the mitochondria: An evaluation of the transcriptional link between the antioxidant response and autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2917.
Abstract 2931: Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in non small cell lung cancer cells with oncogenic EGFR mutation
Authors: Kim Nayoung , Cho Ahye , Watanabe Hideo , Choi Yoon-La , Aziz Meraj , Kassner Michelle , Joung Je-Gun , Park Angela KJ , Francis Joshua , Bae Joon Seol , Ahn Soo-min , Kim Kyoung-Mee , Park Joon-Oh , Park Woong-Yang , Ahn Myung-Ju , Park Keunchil , Yin Hongwei Holly , Cho Jeonghee
Published: July 2016

Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic alterations responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the resistant clones and RNAi-based systematic synthetic lethal screening revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, we detected increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs refractory patients by immunohistochemistry. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs in a subset of lung cancer patients and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients.

Citation Format: Nayoung Kim, Ahye Cho, Hideo Watanabe, Yoon-La Choi, Meraj Aziz, Michelle Kassner, Je-Gun Joung, Angela KJ Park, Joshua Francis, Joon Seol Bae, Soo-min Ahn, Kyoung-Mee Kim, Joon-Oh Park, Woong-Yang Park, Myung-Ju Ahn, Keunchil Park, Hongwei Holly Yin, Jeonghee Cho. Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in non small cell lung cancer cells with oncogenic EGFR mutation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2931.
Abstract 2933: Long-term BRAF(V600E) inhibition results in a spontaneous KRAS(G12D) mutation and increased epithelial to mesenchymal transition (EMT) in papillary thyroid cancer cells (PTC)
Authors: Danysh Brian P. , Cabanillas Maria E. , Hofmann Marie-Claude
Published: July 2016

Objective:

Activating mutations in BRAF(V600E) are commonly observed in PTC and result in a functional dependence upon the constitutively active MAPK pathway. While targeted inhibitors are initially effective, inevitably cells develop alternative mechanisms of pathway activation. Mechanisms of primary resistance have been described in thyroid cancer cell lines, however acquired resistance has not. Our study investigates adaptive mechanisms of BRAF(V600E) inhibitor resistance and accompanying metastatic phenotypes in PTC cells following long-term vemurafenib exposure.

Materials & Methods:

KTC1 sub-cell lines (PTC cells, BRAFV600E) were developed following treatment with either 0.25 or 1.0 μM of vemurafenib, a BRAF V600E inhibitor, for >20 passages. Western blot and qRT-PCR were used to assess EMT marker expression and pathway activation. 2D and 3D invasion assays, immunofluorescence microscopy, and direct cell count growth assays were used to assess inhibitor resistance and metastatic phenotypes. Sequenom was used to detect acquired mutations.

Results:

A vemurafenib resistant (VR) subline of KTC1 cells was derived following long-term treatment with the drug. Resistance coincided with spontaneous acquisition of a KRAS(G12D) activating mutation. Our data show KRAS(G12D) driven vemurafenib-resistance significantly enhanced expression of distinct EMT markers and translated into increased growth, migration, and invasion, when compared to the control sub-lines. This gain in metastatic phenotype coincided with increase activation of both the AKT and ERK pathways.

Conclusion:

Our results suggest an acquired KRAS mutation confers BRAF V600E inhibitor resistance and a more aggressive metastatic phenotype in vitro. Our hope is that further study of the mechanisms of resistance will add to potential markers for early detection of BRAF inhibitor resistance, leading to improved patient outcomes.

Citation Format: Brian P. Danysh, Maria E. Cabanillas, Marie-Claude Hofmann. Long-term BRAF(V600E) inhibition results in a spontaneous KRAS(G12D) mutation and increased epithelial to mesenchymal transition (EMT) in papillary thyroid cancer cells (PTC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2933.
Abstract 2935: Systematic drug testing and RNA-sequencing of tamoxifen resistant breast cancer cell lines
Authors: Hultsch Susanne , Kangaspeska Sara , Kankainen Matti , Pietiäinen Vilja , Kallioniemi Olli
Published: July 2016

Tamoxifen, as a standard treatment of estrogen receptor (ER)-positive breast cancer, reduces breast cancer mortality by 31%. In 50% of advanced ER-positive cancers, however, de novo resistance exists and in 40% of patients with initial response acquired resistance frequently evolves. In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new opportunities to treat patients, we created seven tamoxifen-resistant breast cancer cell line models that represent the luminal A subtype (MCF-7, T-47D, ZR-75-1), which expresses ER, and the luminal B subtype (BT-474), which additionally expresses the HER2 oncoprotein. We then performed drug sensitivity and resistance testing (DSRT), exome-sequencing and network analysis on all these cancer cell lines to determine the molecular and drug response profiles specific to tamoxifen resistance. As the cells became tamoxifen-resistant, we observed increasing sensitivity towards drugs like ERK1/2-, proteasome- and BCL-family inhibitors, but each of the isogenic cell line pairs had its distinct genomic and drug response profile. We then studied the molecular profiles of the 7 drug-resistant variants by RNA-sequencing in comparison to their 4 isogenic parental cells. We could not detect any common significantly differentially expressed genes (more than 2 fold change) across all the cell lines. However, the cell lines could be grouped into two different categories: The ones with low amount of differentially expressed genes, < 800 genes in the BT-474 and ZR-75-1 cell line pairs (with a 1,2% overlap), and the ones with high amount of differentially expressed genes, >1800 genes in T-47D and MCF-7 (with 7,3% overlap). Further analysis with the Ingenuity pathway analysis tool revealed an involvement of “Fatty Acid Activation” as well as “Stearate Biosynthesis I” in this group. Additionally, we discovered that genes involved in iron metabolism (TFRC, IREB2 and FTL) or iron-regulated genes like CP and NDRG1 are deregulated. These genes and pathways thereby provide avenues to identify new drug vulnerabilities for the tamoxifen resistant cancer cells, which we are now investigating at gene and protein level e.g. by image-based phenotyping. In summary, by combining drug testing data with the RNA-sequencing results, we hope to provide a number of potential drugs as well as matching biomarkers for planning clinical trials for patients with tamoxifen-resistant breast cancer.

Citation Format: Susanne Hultsch, Sara Kangaspeska, Matti Kankainen, Vilja Pietiäinen, Olli Kallioniemi. Systematic drug testing and RNA-sequencing of tamoxifen resistant breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2935.
Abstract 2937: Expression of osteopontin splicing isoforms in prostate cancer cells resistant to docetaxel
Authors: Menezes Mariana Concentino , O’Neil Amanda , Guimaraes Isabella , Rangel Letícia , Gimba Etel Rodrigues Pereira
Published: July 2016

Resistance to chemotherapeutic drugs corresponds to main causes of treatment failure. Total osteopontin (OPN) has been described as a gene product involved on chemoresistance, besides activating several aspects of tumor progression. OPN regulates the expression of P-glycoprotein (P-gp) in prostate cancer (PCa) cells, besides promoting apoptosis evasion induced by chemotherapy using paclitaxel and estramustin. These data point OPN as a potential therapeutic target to approaches that could reduce chemotherapy tumor resistance. However, most of this data are related to total OPN. Once OPN suffers alternative splicing, producing 3 isoforms, further investigation should demostrate their specific involvement on chemoresistance. Recent data from our group indicated that OPNb and OPNc splicing isoforms activate PCa tumor progression features. Besides, PCa cells that overexpress these splice variants are more resistant to apoptosis induced by docetaxel (DXT). The current work aims to investigate the expression pattern of OPN splicing isoforms (OPN-SIs) and their relationships in DXT-PCa resistant cells. Total RNA has been extracted from PCa cell lines resistant to DXT (PC3-D8 and PC3-D12) and PC3 control cells with similar cell passages (PC3-Ag). Then, cDNa has been synthesized. OPN-SI expression levels were analyzed by quantitative real time PCR (qRT-PCR) using SYBR Green and isoform specific oligonucleotides. Relative expression levels were calculated using ΔΔCT method. Among the 3 OPN-SIs, we observed that OPNb and OPNc variants are overexpressed in relation to OPNa in DXT-PCa resistant cells PC3-D8 and PC3-D12, when compared to PC3-Ag. PC3-D12 cell line, resistant to higher DXT concentrations, expresses higher levels of OPNc, when compared to the other cell lines. Overall, our data further evidence that OPNb and OPNc overexpression in DXT-PCa resistant cells could be involved on resistance to DXT. Functional assays will be performed in order to investigate the molecular mechanisms by which these OPN-SIs mediate resistance to this chemotherapeutic drug.

Citation Format: Mariana Concentino Menezes, Amanda O’Neil, Isabella Guimaraes, Letícia Rangel, Etel Rodrigues Pereira Gimba. Expression of osteopontin splicing isoforms in prostate cancer cells resistant to docetaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2937.
Abstract 294: The role of the histone deacetylase HDAC 8 in mediating BRAF/MEK inhibitor metastatic dissemination and resistance
Authors: Emmons Michael , Rab Rashed , Sharma Ritin , Koomen John , Smalley Keiran
Published: July 2016

BRAF/MEK inhibitor resistance frequently results in increased phosphorylation of EphA2 at S897, which increases the invasive potential of melanoma cells in a ligand-independent manner. Through use of an unbiased phosphoproteomic screen, we identified HDAC phosphorylation as also being increased following the acquisition of BRAF/MEK inhibitor resistance. Treatment with the pan-HDAC inhibitor LBH 589 inhibited EphA2 phosphorylation at S897 and reduced tumor invasion, indicating a role for HDACs in the regulation of the resistance-associated metastatic phenotype. A comparison of HDAC protein levels in matched BRAF inhibitor sensitive/resistant cell lines showed that HDAC6, HDAC8, and HDAC11 protein levels were more highly expressed in BRAF resistant cell lines with HDAC8 being upregulated in 5 of 6 cell line pairs. shRNA knockdown of HDAC8 in the BRAF/MEK inhibitor resistant cell line WM164RR resulted in a decreased in pEphA2 as well as a decrease in pAKT at S473, which is known to directly bind to and phosphorylate EphA2 at S897. Reduced expression of HDAC8 in WM164RR cells resulted in a decrease in the invasive potential of the resistant phenotype as well as a resensitization of these cells to the BRAF inhibitor vemurafenib. HDAC8 overexpression in drug naïve cells led to increased melanoma cell invasion in a 3D spheroid and a matrigel invasion model. Overexpression of HDAC8 was also found to decrease the level of apoptosis following BRAF inhibitor treatment and was associated with increased number of clones in a colony formation assay. Conversely, knocking down HDAC8 with shRNA led to an increase in vemurafenib-mediated apoptosis. Mechanistically, it was found that HDAC8 overexpression increased the speed of adaptive MAPK signaling following BRAF inhibition and that this in turn limited the expression of pro-apoptotic BIM. Overall, these results indicate a dual role for HDAC8 in mediating BRAF/MEK inhibitor resistance and metastatic dissemination. Increased levels of HDAC8 caused an increase in invasion as well as a decrease in vemurafenib-induced apoptosis in BRAF inhibitor sensitive cell lines. Our studies suggest that selective HDAC8 inhibition may be one strategy to limit adaptation to both BRAF and BRAF/MEK inhibitor therapy.

Citation Format: Michael Emmons, Rashed Rab, Ritin Sharma, John Koomen, Keiran Smalley. The role of the histone deacetylase HDAC 8 in mediating BRAF/MEK inhibitor metastatic dissemination and resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 294.
Abstract 2945: Cell stress during HIPEC causes heat shock protein induction and reduced chemosensitivity in human colon cancer
Authors: Grimmig Tanja , Kloos Kerstin , Thumm Rebecca , Moench Romana , Germer Christoph T. , Waaga-Gasser Ana Maria , Gasser Martin
Published: July 2016

Background: Hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising procedure for the treatment of peritoneal carcinomatosis (PC). Heat shock proteins (HSPs) and other proteins involved in cellular repair mechanisms seem to induce cytoprotective processes during HIPEC therapy. Therefore, the aim of this study was to analyze the effects of HIPEC-related conditions on tumor cell proliferation and the expression of HSPs in human colon cancer.

Methods: Human colon cancer cell lines HT29, SW480 and SW620 were exposed to different temperatures (37°C, 41°C, and 43°C) as well as defined cytostatic agents (Oxaliplatin, Mitomycin C, and 5-Fluorouracil). After cellular regeneration (30 min, 24 h, 48 h and 72 h) RNA isolation and whole cell extraction was performed. Gene and protein expression analysis of HSP27, 70, 72 and 90 as well as PCNA, Ki-67, BCl-2 and BCl-Xl were carried out using RT-qPCR and Western blot. Additionally, MTS cell proliferation assays were performed 24 h, 48 h, 72 h and 96 h post treatment. Moreover, AnnexinV apoptosis assays were conducted.

Results: All colon cancer cells exposed to hyperthermic conditions showed initially up-regulated HSP gene expression. Highest expression was found after exposure to 43°C. Combined cytostatic and hyperthermic treatment demonstrated additional increase in HSP27 expression and in other HSPs to a lesser degree. Tumor cells exposed to cytostatic agents showed overall higher HSP gene expression compared to cells without chemotoxic treatment. Similar effects were detected for the expression of the proliferation marker PCNA and anti-apoptotic protein BCl-Xl. Apoptosis assay demonstrated decreased numbers of apoptotic cells at 43°C compared to normothermia. Additionally, proliferation assays revealed reduced chemosensitivity in cells treated with hyperthermia.

Conclusion: Desired effects of hyperthermia used in HIPEC therapy to achieve anti-proliferative and apoptosis inducing effects seem to be negatively influenced by cell stress mediated repair mechanisms in colon cancer. Our in vitro findings suggest analyzed HSPs to be significantly involved in this hyperthermia and chemotoxicity mediated cellular repair mechanisms. While initial increase in HSP expression can counteract cytotoxic effects during HIPEC therapy their prolonged expression may promote lasting resistance to cellular stress.

Citation Format: Tanja Grimmig, Kerstin Kloos, Rebecca Thumm, Romana Moench, Christoph T. Germer, Ana Maria Waaga-Gasser, Martin Gasser. Cell stress during HIPEC causes heat shock protein induction and reduced chemosensitivity in human colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2945.
Abstract 2949: REV3L, the catalytic subunit of pol ζ, is involved in the progression and chemoresistance of esophageal squamous cell cancer
Authors: Zhou Jundong , Zou Sitao , Ding Wei-Qun , Wu Jinchang
Published: July 2016

REV3L, the catalytic subunit of DNA ploymerse zeta, is well known to participate in error-prone translesion synthesis (TLS) with less stringent and lower processivity. Recent evidence demonstrated that REV3L is involved in carcinogenesis and tumor progression. However, the function of REV3L remains unclear in esophageal squamous cell cancer (ESCC). In this study, we examined REV3L expression in ESCC tissues and its association with clinicopathological parameters. REV3L was found to be significantly up-regulated in ESCC tissues, which is correlated with lymph node metastasis and clinical stages of the patients. To further investigate the potential role of REV3L in esophageal cancer, stable ESCC cell lines with the suppression of REV3L expression were established. Down regulation of REV3L expression led to a decrease in cell proliferation and invasive tendency partly through suppressing cyclinD1 and survivin expression, and an increase in cellular sensitivity to 5-Fu by inducing G1 phase arrest and apoptosis. Therefore, REV3L plays an important role in ESCC progression and chemoresistance, and is a potential diagnostic marker and therapeutic target for ESCC.

Citation Format: Jundong Zhou, Sitao Zou, Wei-Qun Ding, Jinchang Wu. REV3L, the catalytic subunit of pol ζ, is involved in the progression and chemoresistance of esophageal squamous cell cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2949.
Abstract 295: The cytosine deaminase APOBEC3B affects responses to therapy in estrogen receptor positive breast cancer cells
Authors: LaPara Kelly S. , Law Emily , Harris Reuben , Yee Douglas
Published: July 2016

Endocrine therapy, chemotherapy, or both represent the mainstay of therapy for estrogen receptor positive (ER+) breast cancer. However, acquired (secondary) drug resistance is a major clinical problem. We identified APOBEC3B (A3B), a DNA cytosine deaminase, as a source of DNA mutagenesis driving tumor evolution and contributing to poor clinical outcomes in several cancers by catalyzing genetic changes required for drug resistance and metastasis. A3B accounts for up to half of the mutational load in breast carcinomas expressing this enzyme. High levels of A3B correlate with poor clinical outcomes in tamoxifen treated patients. To evaluate the role for A3B in resistance to therapy, we used the ER+ MCF-7L breast cancer cell line. These cells have modestly elevated levels of endogenous A3B and we suppressed A3B levels by shRNA and enhanced levels by transduction. In vitro, alterations in A3B levels do not affect monolayer growth or cell doubling times. However, in vitro response to tamoxifen was prolonged in MCF-7L cells with suppressed A3B. In a xenograft model, MCF-7 cells with suppressed A3B remained responsive to tamoxifen for a longer period of time compared to wild type cells. Cells with overexpression of A3B had a very short period of tamoxifen suppression compared to wild-type cells. These data suggest high A3B levels are associated with a shorter period of response to tamoxifen. To evaluate the role for A3B in chemotherapy resistance, we treated cells with 5-fluorouracil. In contrast to tamoxifen therapy, cells with elevated A3B levels remained responsive to fluorouracil for longer periods of time compared to wild-type cells. Since A3B converts cytosine to uracil (C-to-U), we speculate that A3B enzymatic activity might enhance sensitivity to a fluoropyrimidine. Thus, the ability of A3B to affect therapeutic resistance in breast cancer may be dependent on the type and mechanism of drug treatment.

Citation Format: Kelly S. LaPara, Emily Law, Reuben Harris, Douglas Yee. The cytosine deaminase APOBEC3B affects responses to therapy in estrogen receptor positive breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 295.
Abstract 2952: Generation and molecular profiling of drug-resistant cancer cell lines
Authors: He Fang , Pan Pengwei , Yuan Zengjin , Huang Jingqi
Published: July 2016

Drug resistance is the main cause of ineffective chemotherapy in patients. Many cancers respond initially well to therapy but eventually the cancer recurs as drug-resistant disease leading to the patient death. Therefore, a better understanding of the molecular basis of the drug resistance is warranted in order to elucidate the mechanisms and markers underlying this drug-resistant phenotype In this study, we aimed to generate and characterize a panel of drug resistant cancer cell lines, providing a valuable tool with which to investigate the molecular pathways and putative markers that may be associated with this resistance phenotype in cancer.

Citation Format: Fang He, Pengwei Pan, Zengjin Yuan, Jingqi Huang. Generation and molecular profiling of drug-resistant cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2952.
Abstract 2956: Optimal PEGylation of an auristatin linker provides ADCs with improved pharmacological properties
Authors: Burke Patrick J. , Hamilton Joseph Z. , Jeffrey Scott C. , Hunter Joshua H. , Cochran Julia H. , Chemuturi Nagendra , Anderson Martha E. , Senter Peter D. , Lyon Robert P.
Published: July 2016

As antibody-drug conjugates (ADCs) continue to emerge as an important therapeutic modality for the treatment of cancer, there is an increased effort to elucidate critical design parameters and devise improved linker technologies. The impact of drug-to-antibody ratio (DAR) on conjugate plasma pharmacokinetics (PK) is known to be an important attribute, and accelerated clearance induced by high levels of drug loading has served as a barrier to translating increased in vitro potency to in vivo xenografts. We have recently demonstrated that the incorporation of a discrete PEG24 unit into an auristatin drug-linker can greatly diminish the impact of drug loading on ADC PK. In an effort to optimize the antibody-mediated delivery of monomethylauristatin E (MMAE) as a homogeneous DAR 8 conjugate, we prepared a series of MMAE linkers using PEG units of varying lengths to identify constructs that preserve antibody PK properties and provide enhanced in vivo activity. The extent of PEGylation and linker chemistry was found to impact conjugate PK properties, biodistribution, antitumor activity, and tolerability. From that effort, a cleavable MMAE linker incorporating the glucuronide-based trigger, a self-stabilizing maleimide, and 12 PEG units emerged as the optimal design. ADCs prepared with this linker have now undergone further preclinical characterization in activity and toxicology models in which they have demonstrated an increase in therapeutic index relative to other MMAE-based ADCs.

Citation Format: Patrick J. Burke, Joseph Z. Hamilton, Scott C. Jeffrey, Joshua H. Hunter, Julia H. Cochran, Nagendra Chemuturi, Martha E. Anderson, Peter D. Senter, Robert P. Lyon. Optimal PEGylation of an auristatin linker provides ADCs with improved pharmacological properties. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2956.
Abstract 2959: Peptide-linked indolino-benzodiazepine DNA-alkylating agents for use in antibody-drug conjugates (ADCs)
Authors: Shizuka Manami , Wilhelm Alan , Archer Katie , Reid Emily , Yoder Nicholas C. , Bai Chen , Fishkin Nathan , Harris Luke , Maloney Erin , Hong Erica , Wu Rui , Ab Olga , Lai Kate , Sikka Surina , Jin Shan , Pinkas Jan , Chari Ravi , Miller Michael L.
Published: July 2016

As part of our efforts to expand the available toolbox of cytotoxic payloads for use in antibody-drug conjugates (ADCs), we have recently described the development of the mono-imine-containing indolino-benzodiazepine dimer, IGN-P1 (Miller, et al., AACR 2014 #652). IGN-P1 was conjugated via a protease-cleavable peptidic L-alanine-L-alanine linker to a folate receptor α (FRα)-binding antibody, and an EGFR-binding antibody. The resulting ADCs demonstrated high in vitro potency (IC50 ∼4-100 pM) and specificity towards several cancer cell lines. In vivo, anti-FRα-IGN-P1 induced complete regressions in NCI-H2110 non-small cell lung cancer xenografts following a single dose of 3 μg/kg (linked payload dose, equivalent to 0.18 mg/kg Ab dose).

Here we describe the structure-activity relationship of a number of peptide-linked IGN ADCs, leading to the selection of Ab-IGN-P1 for further advancement. The cytotoxic activities of these peptide-IGN conjugates were evaluated in vitro in cancer cell lines with both high and low target antigen expression. We also assessed bystander activity and identified in vitro and in vivo catabolites. We found that the stereochemistry of the peptide linker was crucial for bystander activity and in vivo efficacy. The IGN-P1 ADC exhibited strong bystander activity, which we believe is dictating the strong antitumor activity in vivo. Treatment of low to moderate antigen-expressing models with Ab-IGN-P1 further resulted in tumor regressions at doses as low as 3-10 μg/kg linked payload.

These data underscore the potential therapeutic benefit of highly active and specific IGN-P1 conjugates, even for the challenging subset of patients with solid tumors where the target antigen is expressed at lower levels.

Citation Format: Manami Shizuka, Alan Wilhelm, Katie Archer, Emily Reid, Nicholas C. Yoder, Chen Bai, Nathan Fishkin, Luke Harris, Erin Maloney, Erica Hong, Rui Wu, Olga Ab, Kate Lai, Surina Sikka, Shan Jin, Jan Pinkas, Ravi Chari, Michael L. Miller. Peptide-linked indolino-benzodiazepine DNA-alkylating agents for use in antibody-drug conjugates (ADCs). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2959.
Abstract 2964: An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies
Authors: Prudent James R. , Hall Chad A. , Marshall David J. , Murphy John , Harried Scott
Published: July 2016

Purpose:

CD20 is known to be a therapeutic antibody drug target as it is expressed on the surface of most B-cell neoplasms. Here, we assessed the anti-tumor activity of EDC9, a novel extracellular drug conjugate composed of Rituximab (a well know and FDA approved anti-CD20 monoclonal antibody) conjugated to a steroidal glycoside via a long, flexible and stable linker.

Experimental Design:

The anti-cancer activity and safety of EDC9 were examined and compared to Rituximab using in vitro and in vivo techniques.

Results:

We found that EDC9 showed picomolar cytotoxic activity that was independent of effector functions and exhibited cytotoxic activity through a mechanism that was dependent on CD20 expression, as cells not expressing CD20 were resistant and Rituximab alone could compete with EDC9, rendering it inactive. After 72 hours of EDC9 treatment at levels between 100 and 200 picomolar, no cells were determined to be viable by CellTiter-Glo or high definition phase contrast imaging. Importantly, when compared on the basis of toxicity to PBMC, EDC9 was found not to be more toxic than Rituximab and the activity of EDC9 was dependent on specific steroid chemistry. In a tumor xenograft model, EDC9 provided complete long-term remission of diffuse large B-cell human lymphoma line SU-DHL-8 tumors, while Rituximab alone did not.

Conclusion:

These results support efforts to further evaluate EDC9 (a novel CD20 specific antibody drug conjugate) and may lead to phase I clinical trials in patients with certain B-cell related malignancies.

Citation Format: James R. Prudent, Chad A. Hall, David J. Marshall, John Murphy, Scott Harried. An anti-CD20 extracellular antibody-drug conjugate for the treatment of B-cell malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2964.
Abstract 2981: DNA damaging agent-induced apoptosis is controled by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex
Authors: Nakajima Wataru , Lee June Y. , Maxim Nicolas , Sharma Kanika , Hicks Mark A. , Vu Thien-Trang , Luu Angela , Yeudall William Andrew , Tanaka Nobuyuki , Harada Hisashi
Published: July 2016

DNA damaging agents, such as cisplatin and etoposide, are employed for the treatment of a wide array of solid tumors, but the prolonged use of chemotherapeutic drugs is limited by their toxicity and by the development of resistance. To overcome these major roadblocks to improved prognosis requires mechanism-based therapeutic strategies that maximize the antitumor effect of drugs while limiting their toxicities. These agents exert anticancer effects via multiple mechanisms, yet their most prominent mode of action involves the generation of DNA lesions followed by activation of the DNA damage response and induction of the BCL-2 family-dependent mitochondrial apoptosis. However, the signaling pathways that connect to BCL-2-family-regulated cell death are not entirely clear.

Noxa, a BH3-only pro-apoptotic BCL-2 family protein, causes apoptosis by specifically interacting with the anti-apoptotic BCL-2 family protein MCL-1 to induce its proteasome-mediated degradation. We show here that the DNA damaging agents cisplatin and etoposide upregulate Noxa expression, which is required for the phosphorylation of MCL-1 at Ser64/Thr70 sites, proteasome-dependent degradation, and as a consequence, apoptosis. Noxa-induced MCL-1 phosphorylation at these sites occurs at the mitochondria and is primarily regulated by CDK2. MCL-1 and CDK2 form a stable complex and Noxa binds to this complex to facilitate the phosphorylation of MCL-1. When Ser64 and Thr70 of MCL-1 are substituted with alanine, the mutated MCL-1 is neither phosphorylated nor ubiquitinated, and becomes more stable than the wild-type protein. As a consequence, this mutant can inhibit apoptosis induced by Noxa overexpression or cisplatin treatment. These results indicate that Noxa-mediated MCL-1 phosphorylation followed by MCL-1 degradation is critical for apoptosis induced by DNA damaging agents through regulation of the Noxa/MCL-1/CDK2 complex. Of note, the identified phosphorylation sites are only observed in human MCL-1; thus, the regulatory mechanism shown here may be human-specific. It is of interest in the future to analyze whether CDK2 expression/activity correlates to cisplatin-resistance in patient samples.

Citation Format: Wataru Nakajima, June Y. Lee, Nicolas Maxim, Kanika Sharma, Mark A. Hicks, Thien-Trang Vu, Angela Luu, William Andrew Yeudall, Nobuyuki Tanaka, Hisashi Harada. DNA damaging agent-induced apoptosis is controled by MCL-1 phosphorylation and degradation mediated by the Noxa/MCL-1/CDK2 complex. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2981.
Abstract 299: Patient-derived tumor microenvironment models uncover non-autonomous TKI resistance mechanisms in NSCLC
Authors: Hu Haichuan , Niederst Matthew , Engelman Jeffrey
Published: July 2016

Background: Tyrosine kinase inhibitors (TKI) have yielded great responses in non-small-cell lung cancer (NSCLC) with EGFR mutations and ALK translocations, however these and other targeted therapies are limited by intrinsic and acquired drug resistance. Previous studies looking into relapsed samples largely expanded our understanding of tumor autonomous resistance mechanisms. In this study, we aimed to decipher the non-autonomous mechanisms in vitro by recapitulating the in vivo tumor microenvironment from patient-derived models.

Method: Cancer-associated fibroblast cells are isolated directly from EGFR mutant and ALK translocated NSCLC biopsies. Over 20 patient-derived fibroblast (PDF) models have been established for studying the tumor microenvironment's effect on TKI response.

Result: We found that many, but not all, PDF cells exert significant resistance to TKIs. Furthermore, there is considerable variability in the degree and secreted factor / mechanism by which they are protecting the tumor cells. These variations are further correlated with patient in vivo response.

Conclusion: Together, our results indicate that PDFs are clinically relevant models for deciphering non-autonomous resistance mechanisms, that they are heterogeneous in protecting cancer cells from TKI treatment, and that the resistance mediated by PDFs can be overcome by specific therapeutic combinations.

Citation Format: Haichuan Hu, Matthew Niederst, Jeffrey Engelman. Patient-derived tumor microenvironment models uncover non-autonomous TKI resistance mechanisms in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 299.
Abstract 2991: Inhibition of Rac1 GTPase activity by SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10
Authors: Balhorn Rodney , van Adrichem Arjan J. , Hok Saphon , Balhorn Monique C.
Published: July 2016

SH7139, the first in a series of selective high affinity ligand (SHAL) therapeutics designed to treat non-Hodgkin's lymphoma, has been shown to be selectively cytotoxic to lymphoma cells over-expressing HLA-DR10. Recent efforts to elucidate the mechanisms of action of SH7139 show that the small molecule drug functions similar to both an antibody drug conjugate and a pro-drug. SH7139 is comprised of three small molecule recognition elements that, when linked together, collectively target the drug to HLA-DR10. Following its binding to HLA-DR10, SH7139 is shuttled into the interior of the lymphoma cell where the subsequent metabolism of these recognition elements releases a series of metabolites that inhibit multiple activities required for tumor cell growth and replication. Studies performed using the Burkitt's lymphoma cell line Raji have shown that SH7139 is metabolized by Raji cells, and the metabolic cleavage of two of the recognition elements (Ct and Dv) produce cytotoxic compounds that contribute to tumor cell killing. While the third recognition element, Cb, is not cleaved off the SHAL scaffold or hydrolyzed to release a cytotoxic metabolite, its structural similarity to known inhibitors of the GTPase activating protein (GAP) MgcRacGAP suggested that it might be active in this pathway. MgcRacGAP functions as a switch that stimulates by many orders of magnitude the activity of the Rac1 GTPase, which is required for cleavage furrow formation, ingression, and the completion of cytokinesis. Experiments conducted with the MgcRacGAP-Rac1 complex have shown that the intact SH7139 molecule (IC50 = 10.6±1.6μM) as well as SH7139 fragments containing the Cb recognition element is effective in inhibiting the GTPase activity of the MgcRacGAP:Rac1 complex. These results confirm that one mechanism action of SH7139 is the inhibition of the Rac1-dependent effector pathways that control the rounding of cells undergoing mitosis, confine Rho activation to the equator of the cell for proper cleavage furrow formation and other processes involved in the completion of cytokinesis. This research was supported by the National Cancer Institute Phase II SBIR award R44CA159843 to SHAL Technologies Inc. Part of this work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

Citation Format: Rodney Balhorn, Arjan J. van Adrichem, Saphon Hok, Monique C. Balhorn. Inhibition of Rac1 GTPase activity by SH7139, a new drug candidate for non-Hodgkin's lymphoma targeting HLA-DR10. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2991.
Abstract 2992: Propranolol induces G0/G1/S phase arrest and apoptosis in melanoma cells via AKT/MAPK pathway
Authors: Zhou Chengfang , Chen Xiang , McLeod Howard L , Knepper Todd C , He Yijing
Published: July 2016

Both preclinical and clinical studies implied that β-adrenoceptor-blockers (β-blockers) could inhibit the growth of melanoma. However, the underline mechanism is still unclear, especially in acral melanoma. MAPK and AKT pathway is involved in tumor survival and apoptosis. Therefore, we investigated the effect of propranolol on melanoma in the A375, two primary acral melanoma cell lines and mice model. In this study, propranolol significantly reduced cell viability and induced apoptosis by activating intrinsic mitochondrial pathway and inhibiting MAPK and AKT pathway in vitro. Meanwhile, we further highlighted that low dose propranolol could slow down the growth of tumor in immunodeficient mice engrafted with human melanoma cells. Thus, our data firstly showed that propranolol may inhibit melanoma by activating intrinsic mitochondrial pathway and regulating MAPK pathway and AKT pathway.

Citation Format: Chengfang Zhou, Xiang Chen, Howard L McLeod, Todd C Knepper, Yijing He. Propranolol induces G0/G1/S phase arrest and apoptosis in melanoma cells via AKT/MAPK pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2992.
Abstract 2994: Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy
Authors: Wysham Weiya Z. , Roque Dario R. , Han Jianjun , Guo Hui , Zhang Lu , Gehrig Paola A. , Zhou Chunxiao , Bae-Jump Victoria L.
Published: July 2016

Objectives: Fatty acid synthase (FAS) is a key enzyme involved in fatty acid synthesis. It also plays an important role in tumorigenesis and is highly expressed in many cancers, including endometrial cancer. Orlistat is a weight loss medication that has been shown to be a potent inhibitor of FAS. The goal of this study was to evaluate the anti-tumorigenic potential of orlistat in endometrial cancer cell lines.

Methods: The endometrial cancer cell lines ECC-1 and KLE were treated with varying doses of orlistat (0.01-500 μM). Cell proliferation was assessed using the MTT assay. Cell cycle progression was evaluated by Cellometer and apoptosis was assessed using the Annexin V assay. Reactive oxygen species (ROS) was measured using the DCFH-DA assay. Western immunoblotting was performed to determine changes in FAS, cellular stress, cell cycle progression and the AMPK/mTOR pathways.

Results: Orlistat inhibited cell proliferation in a dose-dependent manner in both cell lines (IC50 = 81.7 μM in ECC-1; IC50 = 145.5 μM in KLE). Treatment with orlistat resulted in G1 arrest but did not affect apoptosis. Orlistat increased ROS at high doses (500 uM, p = 0.008 for ECC-1 cells and p = 0.012 for KLE cells) and induced expression of BIP and PERK. Western immunoblot analysis demonstrated that orlistat decreased expression of FAS, acetyl-CoA carboxylase (ACC), and carnitine palmitoyltransferase 1A (CPT1A), important proteins in fatty acid metabolism. Treatment with orlistat also increased expression of phosphorylated AMPK and decreased expression of the downstream S6 protein.

Conclusions: Orlistat suppressed cell growth in endometrial cancer cell lines through inhibition of fatty acid metabolism, induction of cell cycle G1 arrest, and modulation of the AMPK/mTOR pathway. Given that patients with endometrial cancer have high rates of obesity, orlistat should be further investigated as a novel strategy for endometrial cancer treatment and prevention.

Citation Format: Weiya Z. Wysham, Dario R. Roque, Jianjun Han, Hui Guo, Lu Zhang, Paola A. Gehrig, Chunxiao Zhou, Victoria L. Bae-Jump. Orlistat decreases endometrial cancer cell proliferation: implications for a novel treatment strategy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2994.