bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2909: Correlation between the expression of Sox2, Oct4 and deltaNp63 in clinical samples of resected esophageal cancers
Authors: Inokawa Yoshikuni , Inaoka Kenichi , Sonohara Fuminori , Oya Hisaharu , Hayashi Masamichi , Niwa Yukiko , Iwata Naoki , Kobayashi Daisuke , Koike Masahiko , Kodera Yasuhiro , Nomoto Shuji
Published: July 2016

Background. DeltaNp63 is the oncogenic isoform in several squamous cell carcinomas including esophageal cancer (EC), however, the mechanisms of regulation of the expression are still unclear. Sox2 and Oct4 are well known transcription factors which function in cancer stemness. We supposed that these two molecules work as transcription factors on deltaNp63, and verified the expression of these genes in cases of resected squamous cell EC.

Methods. In 78 surgical specimens of squamous cell EC with no prior neoadjuvant therapy, messenger RNA (mRNA) expression of deltaNp63, Sox2 and Oct4 in EC tissues and corresponding normal tissues was analyzed by quantitative real-time reverse transcription-polymerase chain reaction assay. The mRNA expression was standardized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The expressions were compared in terms of clinicopathological factors and prognosis of postoperative survivals.

Results. Peason correlation analysis showed significant correlations between expression of deltaNp63 and Sox2 in tumor tissues (r = 0.67, P<0.001), and between deltaNp63 and Oct4 (r = 0.71, P<0.001). The deltaNp63 expressions in tumor tissues were significantly higher than that in corresponding normal tissues (P<0.001), whereas Sox2 and Oct4 did not show significant differences between cancer and non-cancerous tissues. Univariate analysis identified poor differentiation, lymphatic involvement and higher ratio (Tumor/Normal) of expression of Oct4 as significant prognostic factors for poor overall survival, however, lymphatic involvement was the only independent prognostic factor in multivariate analysis (hazard ratio: 5.34; P<0.001).

Conclusions. Expression of deltaNp63 in EC tumor tissues showed positive correlation with expression of Sox2 and Oct4. Therefore, these two transcription factors may regulate the expression of deltaNp63. The deltaNp63 is not a strong prognostic factor, however, Oct4 might be a significant predictor for poor prognosis for overall survival in squamous cell EC.

Citation Format: Yoshikuni Inokawa, Kenichi Inaoka, Fuminori Sonohara, Hisaharu Oya, Masamichi Hayashi, Yukiko Niwa, Naoki Iwata, Daisuke Kobayashi, Masahiko Koike, Yasuhiro Kodera, Shuji Nomoto. Correlation between the expression of Sox2, Oct4 and deltaNp63 in clinical samples of resected esophageal cancers. [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 2909.
Abstract 291: Single cell analyses reveal resistance heterogeneity in EGFR mutant NSCLC
Authors: Niederst Matt J. , Zhang Huidan , Hu Haichuan , Cui Naiwen , Cai Yamei , Mulvey Hillary , Piotrowska Zofia , Sequist Lecia , Weitz David A. , Engelman Jeffrey A.
Published: July 2016

In EGFR mutant lung adenocarcinomas, the activating mutation to EGFR is the founder alteration, present in every cancer cell. Most patients with this genetic alteration respond to EGFR tyrosine kinase inhibitors, although the average duration of the response is only one year before the cancer develops acquired resistance. Efforts to identify the mechanisms underlying the resistance in patients have led to the identification of a resistance mechanism in the majority of cases. However, preliminary data is emerging indicating that multiple resistance mechanisms can be present within an individual patient's disease, and thus, classifying resistant disease with a single mechanism is an oversimplification. Here, utilizing single cell approaches on patient samples, we demonstrate heterogeneity within individual biopsies with respect to resistance mechanisms, epigenetic state, and sensitivity to TKI. Additionally, we observe that the fraction of T790M-positive cells is predictive of response to 3rd generation EGFR inhibitor treatment, suggesting that the degree of heterogeneity has important clinical implications. As therapies designed to target singular resistance mechanisms become more widely implemented, assessing heterogeneity will become increasingly critical in order to choose the most effective therapy for the patient.

Citation Format: Matt J. Niederst, Huidan Zhang, Haichuan Hu, Naiwen Cui, Yamei Cai, Hillary Mulvey, Zofia Piotrowska, Lecia Sequist, David A. Weitz, Jeffrey A. Engelman. Single cell analyses reveal resistance heterogeneity in EGFR mutant 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 291.
Abstract 2921: The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells
Authors: Takhar Mandeep K. , Labrecque Mark P. , Tam Kevin J. , Haegert Anne , Bell Robert H. , Altamirano-Dimas Manuel , Collins Colin C. , Prefontaine Gratien G. , Cox Michael E. , Bennewith Kevin L. , Beischlag Timothy V.
Published: July 2016

The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. This process is mediated by the hypoxia inducible factor 1α/2α (HIF1α/2α) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT/ HIF1β). Rb modulates HIF activity by virtue of its association with the thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230), an essential cofactor of the HIF1α/ARNT transcriptional complex. We used short hairpin RNA (shRNA) technology and microarray analysis to interrogate the Rb-negative and wild-type MCF7 cell transcriptomes and generated lists of genes that were either up- or downregulated in response to both loss of Rb and hypoxia. We found that loss of Rb enhances the expression of hypoxia-regulated genes involved in invasion and epithelial-to-mesenchymal transition and significantly decreases the expression of genes involved in cell anchoring and differentiation in MCF7 and MDA-MB-231 breast cancer cells. Genes were validated using both qRT-PCR and immuno-blot analysis. Additionally, gene ontology analysis revealed that AKT and ERK1/2 are downstream effectors of hypoxic gene programs that are sensitive to loss of Rb. Furthermore, these factors regulate the acquisition of a more invasive phenotype in breast cancer cells. Finally, we found that Rb knockdown in combination with pre-treatment of cells with hypoxia increased growth of tumor foci in the lungs after i.v. injection and increased the development of spontaneous metastases from orthotopically implanted breast tumor cells in female NOD-SCID mice. Primary tumors lacking Rb demonstrated enriched protein levels of genes identified in our arrays when compared to negative control tumors. These results show that Rb is a negative modulator of hypoxia-regulated genetic programs by virtue of its direct effects on the HIF-complex. Understanding the HIF complex and the molecular mechanisms controlling the progression from benign tumors to metastasized and lethal forms will allow us to develop more specific breast cancer therapies.

Citation Format: Mandeep K. Takhar, Mark P. Labrecque, Kevin J. Tam, Anne Haegert, Robert H. Bell, Manuel Altamirano-Dimas, Colin C. Collins, Gratien G. Prefontaine, Michael E. Cox, Kevin L. Bennewith, Timothy V. Beischlag. The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human 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 2921.
Abstract 2927: miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cell
Authors: Rasmussen Mads H. , Lyskjær Iben , Jersie-Christensen Rosa R. , Tarpgaard Line S. , Primdal-Bengtson Bjarke , Nielsen Morten M. , Pedersen Jakob S. , Hansen Tine P. , Hansen Flemming , Olsen Jesper V. , Pfeiffer Per , Ørntoft Torben F. , Andersen Claus L.
Published: July 2016

Oxaliplatin (oxPt) resistance in colorectal cancers (CRC) is a major medical problem, and predictive markers are urgently needed. Recently, miR-625-3p was reported as a promising predictive marker. Here, we have used in vitro models to show that miR-625-3p functionally induces oxPt resistance in CRC cells, and have identified signalling networks affected by mir-625-3p. The p38 MAPK activator MAP2K6 was shown to be a direct target of miR-625-3p, and, accordingly, was downregulated in patients not responding to oxPt therapy. miR-625-3p resistance could be reversed in CRC cells by anti-miR-625-3p treatment and by ectopic expression of a miR-625-3p insensitive MAP2K6 variant. In addition, by reducing p38 MAPK signalling using either siRNA technology, chemical inhibitors to p38 or by ectopic expression of dominant negative MAP2K6 protein we induced resistance to oxPt. Transcriptome, proteome and phosphoproteome profiles revealed inactivation of MAP2K6-p38 signalling as a possible driving force behind oxPt resistance.

Our study shows that miR-625-3p induces oxPt resistance by abrogating MAP2K6-p38 regulated apoptosis and cell cycle control networks, and corroborates the predictive power of miR-625-3p.

Citation Format: Mads H. Rasmussen, Iben Lyskjær, Rosa R. Jersie-Christensen, Line S. Tarpgaard, Bjarke Primdal-Bengtson, Morten M. Nielsen, Jakob S. Pedersen, Tine P. Hansen, Flemming Hansen, Jesper V. Olsen, Per Pfeiffer, Torben F. Ørntoft, Claus L. Andersen. miR-625-3p regulates oxaliplatin resistance by targeting MAP2K6-p38 signalling in human colorectal adenocarcinoma cell. [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 2927.
Abstract 2938: Role of CD73 in promoting metastasis and resistance to 5-fluorouracil of colorectal cancer
Authors: Liu Xuan-hui , Wu Xian-rui , Chen Yu-feng , Wang Feng-wei , Zheng Xiao-bin , Xie Dan , Shen Bo , Wang Jian-ping , Wu Xiao-jian , Lan Ping
Published: July 2016

Background and aim: Our previous study showed that high expression of CD73 (ecto-5’-nucleotidase) was a poor prognostic biomarker in patients with colorectal cancer (CRC). However, the underlying mechanism is unclear. Metastasis and chemo-resistance are among the main causes for the poor outcomes of CRC. The aim of this study is to evaluate the role of CD73 in the metastasis and chemo-resistance in CRC.

Methods: The expression levels of CD73 were detected using quantitative RT-PCR and Western blot in 9 CRC cell lines and 3 5-FU-resistant cell lines (HCT8-FR, HCT116-FR, and RKO-FR). CD73 was overexpressed via lentiviral vector system and silenced using specific small interference RNA (si-CD73) in HCT8 and RKO. We then performed migration, invasion and MTS assays and wound healing assessment to evaluate the function of CD73 in cell progression and chemo-resistance. We conducted Cignal Finder Reporter Array to assess the main downstream signaling pathways of CD73.

Results: Increased expression of CD73 was found in all 3 5-FU-resistant cell lines. Inhibition of CD73 expression in HCT8 and RKO by si-CD73 attenuated the capacity of cell migration and invasion but increased the sensitivity to 5-FU. On the other hand, overexpression of CD73 in HCT8 and RKO increased the capacity for cell migration but decreased the sensitivity to 5-FU. CD73-induced migration and chemo-resistance was found to be associated with the activation of MAPK/ERK pathway.

Conclusions: High expression of CD73 was found to be associated with the metastasis and resistance to 5-FU via the activation of MAPK/ERK signaling pathway. This finding provides a molecular basis for the role of CD73 in the progression of CRC, suggesting a novel target for the treatment of CRC.

Citation Format: Xuan-hui Liu, Xian-rui Wu, Yu-feng Chen, Feng-wei Wang, Xiao-bin Zheng, Dan Xie, Bo Shen, Jian-ping Wang, Xiao-jian Wu, Ping Lan. Role of CD73 in promoting metastasis and resistance to 5-fluorouracil of colorectal 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 2938.
Abstract 2939: Heregulin-induced resistance against HER2-targeted therapies in HER2 positive breast and gastric cancer in vitro and in vivo
Authors: Nonagase Yoshikane , Yonesaka Kimio , Watanabe Satomi , Haratani Koji , Takahama Takayuki , Takegawa Naoki , Ueda Hiroto , Kawakami Hisato , Hayashi Hidetoshi , Takeda Masayuki , Sakamoto Haruka , Tamura Takao , Nakagawa Kazuhiko , Tsurutani Junji
Published: July 2016

Background

HER2-targeted therapies have been shown clinical benefits in patients with HER2 positive breast and gastric cancers. However, resistance against such targeted therapies eventually develops in most cases. Overexpression of heregulin, a ligand for HER3, has been reported as one of the resistant mechanism against HER2-targeted therapies. Upregulation of heregulin activates HER3-PI3K-AKT signaling, leading to the resistance against HER2-targeted therapies. We investigated the effects of heregulin in cell lines and evaluate its clinical impacts in HER2 positive breast and gastric cancers.

Materials and methods

We utilized transfected or recombinant heregulin to investigate the effect of heregulin on proliferation and apoptosis of SK-BR-3 and NCI-N87 cell lines treated with lapatinib, trastuzumab, trastuzumab-emtansine and paclitaxel. Clinical data and specimens were obtained from patients with HER2 positive breast and gastric cancers, evaluating their heregulin value from both serum and tissues using ELISA and RT-PCR, respectively.

Results

Heregulin transfection and recombinant heregulin conferred resistance against treatment with lapatinib, trastuzumab, and weak resistance against trastuzumab-emtansine, but no resistance against paclitaxel in HER2 positive cell lines. Clinical data of heregulin among patients treated with HER2-targeted therapies revealed that patients with relatively high heregulin value tended to have worse prognosis.

Conclusions

Together these data, overexpression of heregulin may at least in part play a role in resistance against HER2-targeted therapies in HER2 positive cancer patients. Further investigation is warranted to elucidate the relevance of treatments targeting heregulin-HER3-PI3K signaling.

Citation Format: Yoshikane Nonagase, Kimio Yonesaka, Satomi Watanabe, Koji Haratani, Takayuki Takahama, Naoki Takegawa, Hiroto Ueda, Hisato Kawakami, Hidetoshi Hayashi, Masayuki Takeda, Haruka Sakamoto, Takao Tamura, Kazuhiko Nakagawa, Junji Tsurutani. Heregulin-induced resistance against HER2-targeted therapies in HER2 positive breast and gastric cancer in vitro and in vivo. [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 2939.
Abstract 2947: MicroRNA 10b, 27a and 27b are involved in the resistance to treatment with epidermal growth factor tyrosine inhibitor (EGFR-TKI) in EGFR mutant lung cancer cell line
Authors: Montes Hannah , Reyes Emma B D , Sharma Mukut , Huang Chao H.
Published: July 2016

EGFR TKIs are now standard of care in lung cancer patients with EGFR mutation. Invariably, these patients develop resistance and require treatment with another EGFR TKI or chemotherapy. The gate-keeper mutation T790M is responsible for about 50% of resistance. Rocelitinib(R) is a third generation EGFR TKI active in lung cancer cells with T790M mutation. Other mechanisms may also confer resistance in these patients. High expression of microRNA (miR)-10 is associated with worse prognosis in resected lung cancer patients with EGFR mutation. Both miR-27a and 27b are associated with increased expression of c-met which is a potential mechanism of resistance to EGFR TKI. We hypothesized that miR 10b and 27a and 27b modulate resistance in E-resistant lung cancer cells.

Material and Methods

Lung cancer cell lines with EGFR mutation HCC827 and HCC4006 were treated with 5 nM of Erlotinib (E) for 72 hours. E-resistant cells were then treated with R at 21.5 nM for 72h. The cells were harvested and microRNA profiling was performed by real time PCR. HTB177 cell line without EGFR mutation was used as control.

Results

We observed increased expression of miR-10b and miR-27a in HCC4006 compared to control cells HTB 177 cells; miR-27b is upregulated in HCC827 cell line. The increased expression of miR-10b and miR 27a were higher in CRL-2871 than the control cells 9-fold and 8-fold, respectively. MicroRNA- 27b was increased 300-fold in HCC827 compared with control.

Treatment with R reduced the proliferation in HCC827 to 30% and in HCC4006 to 50% compared to control. It did not inhibit the proliferation of HTB177 cells. R increase miR-10b in HCC4006 by 3-fold, miR-27a by 1.5-fold; in HCC827 it reduced miR-10b to 0.4, miR-27a to 0.4 and miR-27B 0.5.

Conclusion

We observed upregulation of miR-10b, 27a and 27b in lung cancer cells lines with EGFR mutation that were resistant to E treatment. Treatment of E resistant cells with R reduced proliferation and changed the expression of miR-10b, 27a and 27b. Our results suggest an epigenetic mechanism of resistance in addition to the T790M mutation. MicroRNAs may be studied as targets for developing novel approach for to treating patients with EGFR TKI resistance.

Citation Format: Hannah Montes, Emma B D Reyes, Mukut Sharma, Chao H. Huang. MicroRNA 10b, 27a and 27b are involved in the resistance to treatment with epidermal growth factor tyrosine inhibitor (EGFR-TKI) in EGFR mutant lung cancer cell line. [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 2947.
Abstract 2948: Glucose-6-phosphate dehydrogenase mediates drug resistance in diffuse large B-cell lymphoma
Authors: Maxwell Steve A. , Mousavi-Fard Seyed , Davis Timothy , Wallils Deeann , Sacchettini Jim
Published: July 2016

A leading cause of mortality in diffuse large B-cell lymphoma patients is the development of resistance to the CHOP chemotherapy regimen. We have discovered that CHOP-resistant DLBCL cells express higher G6PDH activities and lower reactive oxygen (predominantly superoxide) levels than CHOP-sensitive cells. Glucose-6-phosphate dehydrogenase (G6PDH) is the rate-limiting enzyme of the NADPH-producing pentose phosphate shunt (PPP). The PPP is the most important pathway for recycling of cellular glutathione (GSH), a key scavenger of reactive oxygen species (ROS). In the PPP oxidative phase, glucose 6-phosphate is irreversibly converted into ribulose 5-phosphate and CO2 leading to the synthesis of NADPH, a redox cofactor for many antioxidant enzymes. The NADPH produced is used by glutathione reductase to reduce GSSG (glutathione disulfide) to GSH. We hypothesized that increased activity of the G6PDH pathway leads to higher GSH production, lower oxidative state (ROS), and CHOP-resistance. In support of our hypothesis, downregulation of G6PDH lowered NADPH and GSH levels, increased ROS, and reversed CHOP-resistance in DLBCL cells. These results indicate an important role for the G6DPH pathway in mediating CHOP resistance in DLBCL.

Citation Format: Steve A. Maxwell, Seyed Mousavi-Fard, Timothy Davis, Deeann Wallils, Jim Sacchettini. Glucose-6-phosphate dehydrogenase mediates drug resistance in diffuse large B-cell lymphoma. [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 2948.
Abstract 2966: Preclinical combinations of the antibody-drug conjugate SGN-LIV1A with chemotherapies show increased activity
Authors: Li Fu , Anderson Martha , Hunter Joshua , Miyamoto Jaime , Ulrich Michelle , Kostic Ana , Law Che-Leung , Sussman Django
Published: July 2016

SGN-LIV1A is an antibody-drug conjugate (ADC) currently being evaluated in a phase 1 clinical trial for metastatic breast cancer. SGN-LIV1A consists of the microtubule disrupting agent, monomethyl auristatin E (MMAE), conjugated to the anti-LIV-1 humanized monoclonal antibody hLIV22. LIV-1, as a downstream target of STAT3, promotes the epithelial to mesenchymal transition that is important in the malignant progression to metastasis. We have previously shown that as a single agent, SGN-LIV1A displays target specific internalization and cytotoxic activity against a breast cancer cell line in vitro and also demonstrates antitumor activity in in vivo preclinical xenograft models with significant delay of tumor growth. We report here additive and synergistic effects when combining SGN-LIV1A with current chemotherapeutic modalities used in the treatment of metastatic breast cancer. Specifically, we show synergy between SGN-LIV1A in combination with either doxorubicin or Abraxane in MCF-7 breast cancer tumor model. In addition, we show additive effects when carboplatin or protein kinase inhibitors are dosed in combination with SGN-LIV1A in this tumor model. These findings support further evaluation and development of SGN-LIV1A in combination with standard of care chemotherapeutic agents for the treatment of breast cancer.

Citation Format: Fu Li, Martha Anderson, Joshua Hunter, Jaime Miyamoto, Michelle Ulrich, Ana Kostic, Che-Leung Law, Django Sussman. Preclinical combinations of the antibody-drug conjugate SGN-LIV1A with chemotherapies show increased activity. [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 2966.
Abstract 2968: A novel bispecific CD3/CDH19 antibody construct (CDH19 BiTE) directs potent killing of melanoma cells in vitro and in vivo and is enhanced by blockade of PD-L1
Authors: Moody Gordon E. , Moriguchi Jodi , Li Shyun , Lee Fei , Frank Brendon , Gilbert Amy , Case Ryan , Dang Khue , Hinkle Beth , Coberly Suzanne , Rottman James , Merriam Kim , Bailis Julie , Beltran Pedro J.
Published: July 2016

CDH19 is an unconventional type 2 cadherin widely expressed in malignant melanoma with limited expression in normal tissues. RNAseq and immunohistochemical analysis of

CDH19 in human samples confirmed its over-expression in >60% of melanoma, whereas normal expression primarily occurred in tissues derived from the neural crest such as nerve fibers and autonomic ganglia. CD3 based bi-specific T-cell engagers (BiTEs) were used to determine if redirected lysis of T-cells against CDH19 would drive anti-tumor efficacy. In vitro, the CDH19 BiTEs were high affinity binders to both human and cyno CDH19, and were able to induce specific T-cell activation and cytotoxicity against a panel of melanoma cell lines. In addition, soluble CDH19 levels were detected in human serum and the effects of BiTE cytotoxicity toward melanoma cells in the presence of soluble CDH19 was investigated. In vivo studies were conducted to confirm the specificity and activity of CDH19 BiTEs in xenograft models of melanoma. The CDH19 BiTE AMG-CDH19X was able to cause tumor growth inhibition in models expressing as few as 250 receptors per cell, and inhibition of tumor growth was enhanced by the addition of a blocking antibody against PD-L1. Immunohistochemical analysis of post-treatment xenograft samples suggested that anti-PD-L1 the persistence of tumor reactive T cells, and provided rationale for combining a BiTE against CDH19 with a PD-1 or PD-L1 blocking antibody in melanoma. In summary, targeting CDH19 presents a promising novel opportunity for BiTEs in the treatment of melanoma, both alone and in combination with current standard of care.

Citation Format: Gordon E. Moody, Jodi Moriguchi, Shyun Li, Fei Lee, Brendon Frank, Amy Gilbert, Ryan Case, Khue Dang, Beth Hinkle, Suzanne Coberly, James Rottman, Kim Merriam, Julie Bailis, Pedro J. Beltran. A novel bispecific CD3/CDH19 antibody construct (CDH19 BiTE) directs potent killing of melanoma cells in vitro and in vivo and is enhanced by blockade of PD-L1. [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 2968.
Abstract 2971: Optides (optimized knottin peptides) computationally designed to target the oncogenic HIPPO pathway
Authors: Crook Zachary R. , Bradley Philip , King Chris , Mhyre Andrew J. , Baker David , Olson James M.
Published: July 2016

The HIPPO pathway plays a critical role in contact inhibition, a pathway that is commonly dysregulated in many human cancers (including liver, colon, ovarian, and lung) and which relies on the intranuclear interaction of the transcriptional coactivator YAP and the transcription factor TEAD(1-4). This pathway also plays a crucial role in recovery from injury; for example, its regulated repression allows hepatocytes to divide and replace tissue lost to a partial hepatectomy, after which its activation suppresses cell growth and prevents tissue overgrowth. While small molecule enzyme inhibitors have proven to be a revelation in cancer therapy, cell growth signaling via protein-protein interactions has proven much more difficult to drug. While Antibodies can be effective in targeting extracellular or cell surface epitopes, intracellular targets, such as the interaction between YAP and TEAD, are not amenable to antibody-based therapeutics. Optides are small disulfide-knotted peptides (knottins) and serve to bridge these capabilities; they are large enough to interfere with protein-protein interactions, but small enough to penetrate into the cytosol. Examples include imperatoxin, an activator of mitochondrial ryanodine receptors, and Tumor Paint, which contains an optimized variant of chlorotoxin conjugated to a fluorescent probe and is capable of accumulating in a wide range of tumor types.

To test whether Optides can abrogate oncogenic signaling mediated by protein-protein interactions, we created libraries of computationally designed candidates to target the TEAD/YAP interface. The library is expressed on the surface of mammalian cells, chosen for the improved fidelity of disulfide bridge connectivity observed in the mammalian secretory pathway as compared to that found in yeast. By repetitive screening against soluble TEAD protein, we are optimizing the pool of candidates for targeting TEAD. The lead Optides will be characterized for their ability to reduce YAP-TEAD interaction, and to impair YAP-mediated cell growth. Owing to the wide variety of knottin scaffolds, both natural and in silico designed, this flexible technology could be applied to other targets in order to impair oncogenic protein-protein interactions.

Citation Format: Zachary R. Crook, Philip Bradley, Chris King, Andrew J. Mhyre, David Baker, James M. Olson. Optides (optimized knottin peptides) computationally designed to target the oncogenic HIPPO 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 2971.
Abstract 300: Inhibition of oncogenic MAPK signaling in melanoma triggers SOX2-dependent adaptive drug resistance
Authors: Flueckiger Stefanie , Aloia Andrea , Frischknecht Lukas , Tuozzo Amanda , Croci Davide , Shakhova Olga , Britschgi Christian , Dummer Reinhard , Levesque Mitchell Paul , Krek Wilhelm
Published: July 2016

Treatment of metastatic melanoma with BRAFV600E-specific small molecule inhibitors (BRAFi) provides dramatic clinical relief response but fails to suppress long-term tumor growth in the long-term. Extensive studies have uncovered identified a number of resistance-causing genetic alterations that, which are acquired upon continued drug exposure. However, little is known about the changes in gene expression programs changes during the critical time before drug resistance is manifested on at the genomic level. These changes may lead to adaptive drug resistance and hence, represent vital mechanisms by which tumor cells survive the acute drug insult prior to acquisition of resistance-causing mutations. Using a combination of RNAseq and molecular analyses, we find that acute inhibition of oncogenic MAPK signaling by BRAFi or MEKi in established and primary patient-derived melanoma cell cultures, leads to a rapid induction of the stem cell maintenance and pluripotency factor SOX2. Immunohistochemical analysis of Sox2 expression in melanoma sections derived from BrafV600E/Pten−/−-mice treated for 36 hours with BRAFi/MEKi confirmed the emergence of a Sox2-positive melanoma subpopulation in vivo. SOX2 Ectopic ectopic expression of SOX2 protected melanoma cells from the anti-proliferative effects of BRAFi indicative ofindicating a role for SOX2 in mediating drug resistance. Furthermore, siRNA-mediated depletion or overexpression experiments of SOX2 unveiled revealed that it is necessary and sufficient for the expansion of a drug-tolerant SOX2+CD24+ cell population. This subpopulation is characterized by increased migratory and invasive behavior as well as an increased proliferative potential in the presence of BRAFi when compared to SOX2+CD24− cells. These results imply that BRAFi treatment induces a SOX2-dependent cellular program with features of adaptive drug resistance. Suppression of drug-dependent induction of SOX2 or its downstream effector pathways could potentially extend the clinical efficacy of BRAFi.

Citation Format: Stefanie Flueckiger, Andrea Aloia, Lukas Frischknecht, Amanda Tuozzo, Davide Croci, Olga Shakhova, Christian Britschgi, Reinhard Dummer, Mitchell Paul Levesque, Wilhelm Krek. Inhibition of oncogenic MAPK signaling in melanoma triggers SOX2-dependent adaptive drug 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 300.
Abstract 3005: The impact of mitochondrially targeted oncology agents on mitochondrial DNA (mtDNA) integrity
Authors: Pokrzywinski Kaytee L. , Biel Thomas G. , Kryndushkin Dmitry , Rao. V. Ashutosh
Published: July 2016

Reactive oxygen species (ROS) are natural byproducts of mitochondrial oxidative phosphorylation. Dysfunction in electron shuttling leads to enhanced ROS production that is notorious for inflicting damage to macromolecules. Mitochondrial DNA (mtDNA) is particularly sensitive to ROS as it is located in close proximity to the respiratory chain. Additionally, mtDNA lack histones which are known to provide protection from ROS. Mitochondria also have limited DNA repair mechanisms making damage to mtDNA more detrimental to mitochondrial physiology. Recent evidence suggests that oxidative insult, leads to mtDNA degradation to prevent the accumulation of mutagenic base lesions, which is enabled by the high redundancy of mtDNA. The primary objectives of this study were to determine the impact of redox-active mitochondrial-targeted chemotherapeutic agents on mitochondrial physiology and mtDNA integrity. All experiments were performed in two cancer cell lines [MDA-MB-231 (breast) and H23 (lung)] that were treated with 2 uM of the mitochondrial-targeted redox-active compounds: MitoTempol [C4 (MT4)], MitoQuinone (MQ) and MitoChromanol-Acetate (MCA) for 1 or 24 hrs. Mitochondrial status was determined by measuring mitochondrial membrane potential, superoxide production, oxygen consumption rate (OCR) and protein levels of the respiratory chain complexes using the JC-1 and MitoSOX assays, Seahorse XF analysis, and immunoblotting, respectively. Next, mtDNA integrity was analyzed by quantifying the degree of mtDNA fragmentation using PCR amplification of a 10kb fragment compared to a short 100bp fragment. Moreover, changes in the mtDNA replication machinery were assessed using immunoblotting. MQ and MCA induced a loss in mitochondrial function and mtDNA replication machinery as indicated by a reduction in membrane potential, OCR and protein levels. Furthermore, increases in superoxide levels and mtDNA fragmentation were observed with MQ and MCA treatment. Finally, we assessed mitochondrial content using two methods. The first method being the ratio of mitochondrial to nuclear DNA, determined by qPCR of short (∼100bp) nuclear and mitochondrial sequences. The second method was the ratio of mitochondrial outer membrane proteins to cytoplasmic housekeeping proteins, determined by immunoblotting. Both mitochondria DNA and outer membrane proteins decreased with MQ and MCA treatment suggesting that redox-active mitochondrial-targeted agents may reduce the mitochondrial content in cancer cells.

Citation Format: Kaytee L. Pokrzywinski, Thomas G. Biel, Dmitry Kryndushkin, V. Ashutosh Rao. The impact of mitochondrially targeted oncology agents on mitochondrial DNA (mtDNA) integrity. [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 3005.
Abstract 3018: Cotargeting MEK and CDK4/6 to treat pancreatic adenocarcinoma
Authors: Maust Joel D. , Simeone Diane , Sebolt-Leopold Judith
Published: July 2016

Hyperactivation of KRAS and inactivation of CDKN2A [p16] play a prominent role in tumor initiation and progression in a broad spectrum of human cancers. In pancreatic adenocarcinoma, KRAS mutation occurs in 90-95% of cases and is often coupled with inactivation of CDKN2A (>90% incidence), typically by homozygous deletion. Based on the high incidence of these genetic events, the present study addresses the hypothesis that dual targeting of MEK and CDK4/6 represents a viable therapeutic strategy for the treatment of pancreatic adenocarcinoma. A panel of primary and high passage xenograft models was screened for in vitro synergy to the antiproliferative effects of the MEK inhibitor trametinib and the CDK4/6 inhibitor palbociclib. Two models that emerged as highly sensitive to this combination treatment strategy, L3.6pl and UM59, both exhibited G1 cell cycle arrest that was significantly more pronounced in response to the combination compared to either single agent. In vivo studies were subsequently carried out to evaluate the efficacy of this combination in tumor-bearing mice. Mice implanted subcutaneously with L3.6pl or UM59 cells were treated daily by oral gavage for ten days with trametinib (3 mg/kg), palbociclib (100 mg/kg), or the combination of these two agents at these doses. Consistent with in vitro synergy observations, both models proved to be exceptional responders in vivo to this combination treatment strategy, showing a robust response to the combination not seen with either single agent. These results suggest that the combined inhibition of MEK and CDK4/6 may offer a valuable therapeutic strategy for the treatment of pancreatic adenocarcinoma. Studies are ongoing to identify molecular determinants of response in pancreatic tumors that are highly sensitive to this combination approach to guide future patient enrichment strategies.

Citation Format: Joel D. Maust, Diane Simeone, Judith Sebolt-Leopold. Cotargeting MEK and CDK4/6 to treat pancreatic adenocarcinoma. [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 3018.
Abstract 3019: High throughput drug screening identified spleen tyrosine kinase as a novel therapeutic target in head and neck cancer with potent in vitro and in vivo activity
Authors: Black Morgan , Ailles Laurie , Sun Ren , Datti Alessandro , Vizeacoumar Frederick , Pinto Nicole , Ruicci Kara , Yoo John , Fung Kevin , MacNeil Danielle , Palma David A. , Winquist Eric , Mymryk Joe S. , Boutros Paul C. , Barrett John W. , Nichols Anthony C.
Published: July 2016

Background and Significance: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, and survival remains poor highlighting the need for novel treatments for the treatment of HNSCC. High throughput drug screening has shown the potential to discover novel therapeutics in other cancers.

Methods: Twenty-eight HNSCC cell lines, including 5 HPV-positive lines, were characterized with whole exome sequencing and copy number arrays and screened with 1505 potential anti-cancer agents on a robotic liquid handling platform at a single dose (4uM). The most potent hits were confirmed with 10-point dose response curves. Novel therapeutic targets were further investigated with mechanistic and xenograft studies.

Results: Drug screening identified 10 agents with broad activity across our cell line panel. One of the most potent agents was ER27319 maleate, reported to be a spleen tyrosine kinase (Syk) inhibitor. We confirmed that this molecule inhibited Syk phosphorylation specifically at tyrosine residues 525/526. Additionally, ER27319 maleate as well as a more clinically relevant Syk inhibitor, Fostamatinib, were observed to significantly impaired cellular migration and invasion. Additionally, siRNA knockdown of Syk in HNSCC cells was found to decrease HNSCC cell line growth. Finally, inhibition of Syk was observed to control HNSCC tumour growth in vivo in cell line-derived xenografts.

Conclusions: High throughput drug screening of HNSCC cell lines identified Syk as a novel target and confirmed potent in vitro and in vivo activity. Further preclinical evaluation is planned with a panel of patient-derived xenografts. Should these results yield significant activity, we will aim to repurpose approved Syk inhibitors to improve outcomes for patients suffering from HNSCC.

Citation Format: Morgan Black, Laurie Ailles, Ren Sun, Alessandro Datti, Frederick Vizeacoumar, Nicole Pinto, Kara Ruicci, John Yoo, Kevin Fung, Danielle MacNeil, David A. Palma, Eric Winquist, Joe S. Mymryk, Paul C. Boutros, John W. Barrett, Anthony C. Nichols. High throughput drug screening identified spleen tyrosine kinase as a novel therapeutic target in head and neck cancer with potent in vitro and in vivo activity. [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 3019.
Abstract 3060: Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib and carfilzomib
Authors: Hasinoff Brian B. , Wu Xing , Patel Daywin
Published: July 2016

Bortezomib and carfilzomib are anticancer drugs that target the proteasomal system. Case reports indicate that the use of these drugs can result in cardiotoxicity. These drugs may also be used in combination with doxorubicin, which is itself cardiotoxic. Thus, in order to determine the mechanism of this cardiotoxicity studies were undertaken using a neonatal rat myocyte primary cell model. Using LDH release as a measure of damage in the myocyte model we showed that even brief exposure (6 h) to submicromolar concentrations of bortezomib resulted in significant myocyte damage. Carfilzomib was slightly less toxic and displayed myocyte damage in the low micromolar concentration range. Utilizing a fluorogenic substrate both bortezomib and carfilzomib inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the submicromolar concentration range. The inhibition kinetics were consistent with formation of an essentially irreversible covalent complex at the active site. Bortezomib irreversibly inhibited proteasomal activity about four-fold faster than carfilzomib. Thus, the reduced toxicity of carfilzomib may be due to its slower inhibition kinetics. A brief pre-exposure (6 h) of myocytes to low non-toxic nanomolar concentrations of bortezomib greatly increased doxorubicin-mediated damage. The doxorubicin cardioprotective agent dexrazoxane partially protected the myocytes from doxorubicin treatment and from bortezomib plus doxorubicin treatment under these conditions. However, at slightly toxic bortezomib concentrations dexrazoxane offered little protection against bortezomib plus doxorubicin treatment. Bortezomib, at least over short times, did not induce oxidative damage in myocytes as measured with a fluorogenic DCF assay. Likewise, a 6 h bortezomib treatment did not affect the mitochondrial membrane potential of myocytes as measured in a JC-1 ratiometric assay. In conclusion both bortezomib and carfilzomib are potently toxic to myocytes at submicromolar concentrations, likely through their ability to irreversibly inhibit myocyte proteasomal activity. Support: CIHR; a Canada Research Chair in Drug Development.

Citation Format: Brian B. Hasinoff, Xing Wu, Daywin Patel. Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib and carfilzomib. [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 3060.
Abstract 3063: Discovery of a third-generation EGFR inhibitor, which is highly selective and potent against both resistant and activating EGFR mutations for NSCLC therapy
Authors: Bao Rudi , Gao Peng , Zhang Fujun , Tong Zhaolong , Yu Hongping , Xu Yaochang
Published: July 2016

EGFR T790M mutation which renders resistance to the first-generation EGFR inhibitors,accounts for about 50% of all originally responsive non-small cell lung cancer patients. In order to overcome this type of resistance, we have developed a small molecular drug candidate with unique structural features that offer differentiated pharmacological and safety profiles pre-clinically when compared with other leading third-generation EGFR inhibitors. The compound selectively inhibits T790M as well as activating EGFR mutations, with much less inhibition to the wild type EGFR. Enzymatic inhibition IC50 against EGFR T790M,T790M/Del19 and T790M/L858R is less than 1nM. In addition, enzymatic inhibition IC50 against EGFR activating mutations Del19 and L858R is around 1nM, with much less inhibition to the wild type EGFR, a 10-time selection ratio. In cell based assays, the compound potently inhibits proliferation of EGFR T790M mutated NSCLC cell lines H1975(T790M/L858R)and PC9-GR(T790M/Del19)with an IC50 around 3nM. Similarly, it potently inhibits EGFR activating mutant NSCLC cell lines HCC827(Del19)and PC9(Del19). However, the compound displays about 200-fold less sensitivity to the EGFR wild type cancer cell line A431, with an IC50 around 600nM. When dosed to tumor-bearing animals, the compound dose-dependently induces profound regression of xenografts derived from both H1975 and primary human lung cancer LU1868, both of which carry an EGFR T790M mutation. It also induces regression of HCC827 xenografts potently, similarly to the first- and second-generation EGFR inhibitors; whereas it displays much less inhibition to the EGFR wild type A431 xenografts, demonstrating a higher specificity to the mutated EGFR forms when compared with the EGFR inhibitors on market. The drug candidate displays favorable PK and safety profiles. These properties warrant further development of this compound as first- as well as second-line therapy for NSCLC.

Citation Format: Rudi Bao, Peng Gao, Fujun Zhang, Zhaolong Tong, Hongping Yu, Yaochang Xu. Discovery of a third-generation EGFR inhibitor, which is highly selective and potent against both resistant and activating EGFR mutations for NSCLC therapy. [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 3063.
Abstract 3066: Anti-angiogenic properties of PM060184
Authors: Galmarini Carlos M. , Guillen Maria J. , Martinez-Leal Juan F. , Martinez-Diez Marta , Aviles Pablo
Published: July 2016

Vascular supply of tumors is one of the main targets for cancer therapy. Different studies have shown that most of the microtubule-binding agents present antiangiogenic and vascular-disrupting effects. PM060184 is a new marine-derived drug that binds to a new site in β-tubulin inhibiting tubulin polymerization. The compound is currently being evaluated in phase 1/2 studies in patients with advanced malignancies. The present study describes the antiangiogenic and vascular-disrupting properties of PM060184. Treatment of HUVEC endothelial cells with PM060184 resulted in depolymerization of the microtubule network. Differently from untreated cells, PM060184-treated cells showed a rounded morphology with a well-developed actin filament structure and plasma membrane blebs. Using transwell chambers pre-coated with matrigel™ or collagen matrix, we showed that PM060184 inhibited migration and invasion of HUVEC endothelial cells. We finally found that PM060184 interfered with the correct formation of the HUVEC capillary network grown on matrigel™; moreover, drug treatment also resulted in a significant disruption of the previously formed capillary-like network. This rapid collapse of the endothelial tubular network in vitro occurred in a concentration-dependent manner and was observed at concentrations lower than those affecting cells survival. These in vitro findings were confirmed in nude mice bearing MDA-MB-231 (breast) xenografts: after the administration of a single dose of PM060184 (at its MTD, 16 mg/kg,) a very strong reduction in the number of vessels was quantified in serial tumor sections stained with H&E. Moreover, nude mice xenografted with H-460 (NSCLC) tumors and treated with a single dose of either 2 or 16 mg/kg of PM060184, experienced a strong and dose-dependent decrease of the intratumoral fluorescence after the administration of Angiosense™ 680, a fluorescence vascular imaging probe. Altogether, these data suggest that an anti-vascular mechanism is also contributing to the anti-tumor activities of PM060184.

Citation Format: Carlos M. Galmarini, Maria J. Guillen, Juan F. Martinez-Leal, Marta Martinez-Diez, Pablo Aviles. Anti-angiogenic properties of PM060184. [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 3066.