bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A20: Deregulation of cell cycle and apoptotic mechanisms in UVB-irradiated p16-mutant inducible melanoma cell lines
Authors: Ouhtit Allal , Gupta Ishita , Elmageed Zakariya Y. Abd , Becker Therese M.
Published: July 2015

Melanoma is the deadliest skin cancer as it progresses to a highly aggressive and metastatic tumor during melanomagenesis. However, its underlying mechanisms regulating melanoma-genesis remain nascent. Our previous studies have demonstrated that the p53 and p16INK4a sub-pathways components of the Rb-signaling pathway, plays a critical role in UVB-induced skin cancer. The aim of this study was to study the molecular changes in the expression/function of the different components of the Rb signaling pathway associated with UVB-induced cell cycle and apoptotic programs. Pursuant to this goal, normal melanocytes, and a panel of p16-induced cell lines (wild-type p16INK4a-inducible clone p16wt-A4, and the N-terminal 8 amino-acid duplication p16INK4a-inducible clone p16Dp1 (+24 base pairs) were used to elucidate the early molecular events especially associated with the p16INK4a/Rb-signaling pathway regulating cell-cycle and apoptotic responses. Our results showed that: 1) in the menlanocytes high UVB dose induced p16 but not p53, and the Bax/Bcl2 ratio was also increased, suggesting that the cells had selected to undergo apoptosis in a p53-independent manner; 2) In the wt-p16 melanoma cells however, while low UVB irradiation upregulated p16, high dose reduced p16 expression and abrogated Cdk6 but not Cdk4; 3) Interestingly, mutant-p16 melanoma cells over-expressed cell cycle proteins Cdk4/6 and pRb proteins upon p16 induction, however, UVB exposure did not affect this increase; and 4) The anti-apoptotic properties of mutant cells were predominant at high dose of exposure associated with a decrease in Bax and an increase in Bcl2 expression. These data put together, suggest that the mutant p16 dictate cell cycle deregulation and causes resistance of melanoma cells to apoptosis. In conclusion, our study shows that UVB radiation-induced deregulation of p16INK4a/Cdk4 sub-pathway is a critical early event in melanomagenesis.

Citation Format: Allal Ouhtit, Ishita Gupta, Zakariya Y. Abd Elmageed, Therese M. Becker. Deregulation of cell cycle and apoptotic mechanisms in UVB-irradiated p16-mutant inducible melanoma cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A20.
Abstract A26: Protein phosphatase 4 (PP4) as a potential therapeutic target gene for BRAF wild type melanoma
Authors: Essner Richard , Gong Ke Wei , Kaufman David , Chen Hsiaowang , Ginther Charles , Dering Judy , Euw Erika von , Chmielowski Bartosz , Finn Richard , Slamon Dennis
Published: July 2015

Protein phosphatase 4 (PP4) participates in cell cycle progression and regulation of centrosome maturation. Gene expression of PP4 is elevated in cancerous tissues of various origins and its overexpression in pancreatic cancer is of prognostic importance. Microarray analysis from primary and metastatic melanoma tissues (N=89) showed that PP4 mRNA expression was up-regulated in metastatic melanoma as compared to primary melanoma of any thickness (relative expression: 1.12 ±0.14, p<0.01). Expression levels from thin (<2 mm) were less than thicker primary melanoma (>2 mm), and varied for the site of metastases from lung> intestine> skin and subcutaneous tissue. Human melanoma cell lines also demonstrate PP4 gene expression with variable levels. We hypothesized that PP4 may be a novel target for treatment of metastatic melanoma through a BRAF and MEK independent pathway.

We performed western blot analysis of human melanoma cell lines: M275 (BRAF wild-type) that had a high level of PP4 gene expression and demonstrated its resistant to BRAF inhibitor (PLX4032) and MEK inhibitor (MEK162) treatment. M275 were chosen for PP4 siRNA gene silencing because of its high level of PP4 protein expression. PP4 protein level was reduced by 20% and 60%, respectively in this cell line at day 4 and 6, respectively; and PP4 siRNA gene silencing reduced cell proliferation by 19.9% and 63.6%. Flow cytometry demonstrated there was higher percentage of apoptotic cells after PP4 siRNA gene silencing as compared to controls (54.4% vs. 15.5%, p, 0.05). PP4 is overexpressed in melanoma as it advances from the skin primary to metastases, and varies to the site of the metastases. We demonstrated that PP4 gene silencing leads to cell cycle arrest and apoptosis. PP4 may be a novel target for BRAF wild type melanoma.

Note: This abstract was not presented at the conference.

Citation Format: Richard Essner, Ke Wei Gong, David Kaufman, Hsiaowang Chen, Charles Ginther, Judy Dering, Erika von Euw, Bartosz Chmielowski, Richard Finn, Dennis Slamon. Protein phosphatase 4 (PP4) as a potential therapeutic target gene for BRAF wild type melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A26.
Abstract A30: Small molecule kinase inhibitor mediated modulation of immunotherapy in melanoma
Authors: Suriano Robert , Tuli Neha , Geliebter Jan , Tiwari Raj K. , Wallack Marc
Published: July 2015

As the BRAFV600E mutation is observed in over 60% of melanomas, the development of the small inhibitor, PLX4032, which specifically targets mutated BRAF, represents a novel therapeutic approach. This targeted inhibition of BRAF effectively blocks activation of key signaling molecules of the mitogen activated protein kinase (MAPK) pathway, such as Mek and Erk, thus halting cell proliferation. In addition, since MAPK signaling targets the micropthalmia transcription factor (MITF), which is linked to the expression of various melanoma associated antigens (MAAs), we hypothesize that the inhibition of BRAFV600E can modulate the expression of MAAs thus serving as a viable target for immunotherapy of melanoma. Our lab has developed a vaccinia virus based immunotherapeutic vaccine using patient derived melanoma cell lines which express various MAAs. The melanoma cell lines have been characterized as far as the status of the BRAFV600E mutation and were found to be either homozygous or heterozygous for the mutation and are responsive to PLX4032, as evidenced by inhibition of two key MAPK pathway molecules, namely Mek and Erk. Phenotypically, the cells invasive and migratory properties were also modulated upon treatment with PLX4032. Interestingly was the observation that upon treatment with PLX4032, the expression of various MAAs was enhanced. Expression of gp100, MART-1, and tyrosinase increased both on the RNA level as well as on the protein level. The expression of MMAs were well maintained in vivo following the analysis of melanoma cell xenografts in BALB/c nude mice. We conclude that an effective multivalent immunotherapeutic vaccine requires the use of various MAAs and since PLX4032 upregulates expression of MAAs, further studies are warranted to determine the clinical effectiveness of combining BRAFV600E inhibitors with our melanoma cell line derived preparation.

Citation Format: Robert Suriano, Neha Tuli, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Small molecule kinase inhibitor mediated modulation of immunotherapy in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A30.
Abstract A35: Integration of melanoma genotyping in clinical care
Authors: Salhi Amel , Silva Ines Pires Da , Lui Kevin P. , Ismaili Naima , Wu Chaowei , Miera Eleazar C. Vega-Saenz de , Shapiro Richard L. , Berman Russell S. , Pavlick Anna C. , Zhong Judy , Heguy Adriana , Osman Iman
Published: July 2015

Background: Molecularly targeted therapy is improving response rates and overall survival in subsets of melanoma patients. However, targeted therapy for triple negative patients (BRAF, NRAS and c-KIT wild type) is not yet defined. In addition, new evidence suggests that germline variants may have an impact in melanoma progression and response to therapy. In this study, we attempt to define the utility of a recently developed clinical assay that encompasses targeted sequencing of 50 genes with known impact on cancer progression.

Methods: We used the AmpliSeq Cancer Panel HotSpot.V2 from Ion Torrent. The panel targets sequencing of Hotspot regions including 2,800 COSMIC mutations within 50 oncogenes and tumor suppressor genes. Tumor and blood germline DNAs were studied. All identified mutations were independently validated by Sanger sequencing. We then linked the molecular profile to extensive clinicopathological information including treatment and prospective clinical follow-up.

Results: We examined 35 tumor samples from 35 melanoma patients (2 patients had 2 specimens). 14/35 (40%) had BRAF mutations, 15/35 (34%) had NRAS, KRAS or HRAS mutations and 1 had c-KIT mutant tumor. Eight patients were triple negative and presented with mutations in TP53, ERBB4, PIK3CA, NOTCH, EZH2 and VHL. We detected germline variants in TP53 (49%), KDR (VEGFR2 - 34%), APC (31%), KIT (23%) and PIK3CA (14%) genes. Notably, KDR Q472H has been shown to affect VEGFR2 function. Patients with KDR Q742H had higher microvessels density and higher VEGF production. In addition, melanoma cell lines with KDR Q472H variant proliferate and invade more when compared with WT cell lines. More important, only cell lines with that variant respond to anti-VEGFR2 treatment. Finally, the presence of the KDR Q472H variant is associated with a higher prevalence of brain metastases and with a shorter survival (15.1 months compared to 54.6 months).

Conclusions: Sequencing using a validated clinical assay was informative of several targetable mutations in triple negative melanoma. Inhibitors targeting some of these mutations are already FDA approved for non-melanoma and others are currently tested in clinical trials. Our data also revealed a role of germline variant KDR Q472H in melanoma progression, that was not reported before, suggesting that further functional studies are warranted.

Citation Format: Amel Salhi, Ines Pires Da Silva, Kevin P. Lui, Naima Ismaili, Chaowei Wu, Eleazar C. Vega-Saenz de Miera, Richard L. Shapiro, Russell S. Berman, Anna C. Pavlick, Judy Zhong, Adriana Heguy, Iman Osman. Integration of melanoma genotyping in clinical care. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A35.
Abstract A37: The signet ring cell melanoma - rare morphological variant of melanoma: Case report
Authors: Maksimovic Sinisa
Published: July 2015

Introduction: Melanoma is a malignant tumor of melanocytes of skin. The histopathological variants of malignant melanoma include the common type (lentigo maligna (5%), superficial spreading melanoma (65%), nodular melanoma (15%), acrolentiginous melanoma (5%). The signet ring cell melanoma is a rare form melanoma, being seen in some 0.5% of melanomas.

Aim of work: That showed young women with signet ring cell melanoma is a rare form melanoma.

Material and methods: We describe a 39-year-old patient with a primary signet-ring cell melanoma of the skin located on the left upper quadrant of abdominal wall.

Results: Diagnosis the tumour of skin was in department of surgery and histhopatology in General hospital in Bijeljini in December of the 2007. We performed clinical and histhopatologic examination. Physical examination revealed a well circumscribed and firm mass measuring 150x 100x 80 mm of the skin located on the upper quadrant of abdominal wall and solid mass in pelvis. Because of high suspect melanoma in patient performed surgical excision of skin tumor. Pathohysthology diagnosis was: Melanoma nodularis achromaticum. Immunohistochemistry showed positive reaction of the tumor cells for S-100, HMB-45 protein and vimentin, confirming their melanocytic differentiation. Tumor cells were negative for cytokeratins, epithelial membrane antigen (EMA), and carcinoembryonic antigen (CEA). After immunohistochemistry analysis definitive pathohystology was: Signet ring cell melanoma. The signet-ring cell melanoma disclosed an invasion to Clark Level V and tumor thickness Breslow level III.

Tumor from pelvis was appertain uterus and histopathologic diagnosis is a leomyoma.

Conclusion: Signet-ring cell melanoma is a rare morphologic variant of melanoma. Its recognition is important for differentiation from other tumors featuring signet ring cells.

Note: This abstract was not presented at the conference.

Citation Format: Sinisa Maksimovic. The signet ring cell melanoma - rare morphological variant of melanoma: Case report. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A37.
Abstract B09: Phosphorylation of BRAF by AMPK impairs BRAF-KSR1 association and cell proliferation
Authors: Shen Che-Hung , Yuan Ping , Perez-Lorenzo Rolando , Zhang Yaqing , Lee Sze Xian , Ou Yang , Cai Li , Asara John M. , Cantley Lewis C. , Zheng Bin
Published: July 2015

BRAF is an oncogenic protein kinase that drives cell growth and proliferation through the MEK-ERK signaling pathway. BRAF inhibitors have demonstrated antitumor efficacy in melanoma therapy but have also been found to be associated with the development of cutaneous squamous cell carcinomas (cSCCs) in certain patients. Here, we report that BRAF is phosphorylated at Ser729 by AMP-activated protein kinase (AMPK), a critical energy sensor. This phosphorylation promotes the association of BRAF with 14-3-3 proteins and disrupts its interaction with the KSR1 scaffolding protein, leading to attenuation of the MEK-ERK signaling. We also show that phosphorylation of BRAF by AMPK impairs keratinocyte cell proliferation and cell-cycle progression. Furthermore, AMPK activation attenuates BRAF inhibitor-induced ERK hyperactivation in keratinocytes and epidermal hyperplasia in mouse skin. Our findings reveal a mechanism for regulating BRAF signaling in response to energy stress and suggest a strategy for preventing the development of cSCCs associated with BRAF-targeted therapy.

Citation Format: Che-Hung Shen, Ping Yuan, Rolando Perez-Lorenzo, Yaqing Zhang, Sze Xian Lee, Yang Ou, Li Cai, John M. Asara, Lewis C. Cantley, Bin Zheng. Phosphorylation of BRAF by AMPK impairs BRAF-KSR1 association and cell proliferation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B09.
Abstract B12: Synergistic anticancer activity of Aurora A kinase and MDM2 antagonists in melanoma
Authors: Vilgelm Anna E. , Pawlikowski Jeff S. , Liu Yan , Oriana E. Hawkins , Tyler A. Davis , Weller Kevin P. , Horton Linda W. , McClain Colt M. , Ayers Gregory D. , Turner David , Essaka David C. , Stewart Clinton F. , Sosman Jeffrey A. , Kelley Mark C. , Ecsedy Jeffrey A. , Johnston Jeffrey N. , Richmond Ann
Published: July 2015

Senescence-inducing therapies can block proliferation of malignant cells and promote anti-tumor immune activity. However, the risk of tumor relapse remains high due to the long lifespan of senescence cells with potential to escape senescence. Here our preclinical studies demonstrate that combining a senescent-inducing aurora kinase A (AURKA) inhibitor alisertib (MLN8237) with an MDM2 antagonist [(-)-nutlin 3a] effectively induces robust p53 activation in senescent Tp53WT tumors accompanied by: 1) tumor cell proliferation arrest; 2) mitochondrial depolarization and tumor cell apoptosis; and 3) tumor cell clearance via CCL5-, CCL1- and CCL9-mediated recruitment of anti-tumor leukocytes. This combined therapy shows adequate bioavailability and low toxicity to the host in the mouse model. Moreover, the prominent preclinical response of patient-derived melanoma tumors to the co-targeting of MDM2 and AURKA provides rationale for further investigation of alisertib and MDM2 inhibitors.

Citation Format: Anna E. Vilgelm, Jeff S. Pawlikowski, Yan Liu, E. Hawkins Oriana, A. Davis Tyler, Kevin P. Weller, Linda W. Horton, Colt M. McClain, Gregory D. Ayers, David Turner, David C. Essaka, Clinton F. Stewart, Jeffrey A. Sosman, Mark C. Kelley, Jeffrey A. Ecsedy, Jeffrey N. Johnston, Ann Richmond. Synergistic anticancer activity of Aurora A kinase and MDM2 antagonists in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B12.
Abstract B18: Non-thermal plasma, tirapazamine, and gap junctions: A novel approach to melanoma therapy through ROS induction
Authors: Zucker Shoshanna N. , Higley Cory , Koch Zethan , Goli Haneesha , Casey Peter , Francis Akeem , Burke Kevin , Zirnheld Jennifer
Published: July 2015

Melanoma cells typically have high levels of superoxide anion which may increase their susceptibility to cell death by reactive oxygen species (ROS). We have used this information to establish a system whereby melanoma cells are saturated with ROS through treatment with non-thermal plasma (NTP). NTP is formed from a mixture of highly charged helium and atmospheric gases to emit a total of 45 molecular species, many of which are ROS. We have recently demonstrated that NTP causes selective apoptotic cell death of melanoma cells as compared to normal skin keratinocytes when plated in co-culture. Since tumors flourish under low oxygen (hypoxic) conditions, we tested the effects of NTP under hypoxia. In order to enhance the effects of NTP, we combined this therapy with the experimental anticancer drug, tirapazamine, which promotes cell toxicity through DNA damage only under hypoxic conditions. The question then remains as to how to increase the target area of melanoma therapy. Our previous studies have demonstrated that gap junctions act via the bystander effect to enable the passage of cell-death promoting signals by NTP. We utilized stably transfected 1205Lu metastatic melanoma cells that express three different levels of gap junctions: endogenous low levels, increased levels through expression of connexin 43, or an absence of gap junctions due to expression of a dominant negative mutant connexin 43. We have shown that the cells with the mutant connexin 43 showed increased invasive potential as well as enhanced expression of HIF1α. The combination therapy with NTP and tirapazamine under hypoxic conditions caused a synergistic effect which increased cell death in all cells, yet was most apparent in the 1205Lu cells overexpressing the functional gap junctions. In addition, the target area of the NTP torch was dramatically increased under these conditions. This suggests that the ROS generated by NTP and tirapazamine induced the bystander effect and promoted the spreading of cell death signals well beyond the area of the plasma treatment. These results are indicative a novel future approach to melanoma therapy through selective ROS induction and modification of gap junction phenotype.

Citation Format: Shoshanna N. Zucker, Cory Higley, Zethan Koch, Haneesha Goli, Peter Casey, Akeem Francis, Kevin Burke, Jennifer Zirnheld. Non-thermal plasma, tirapazamine, and gap junctions: A novel approach to melanoma therapy through ROS induction. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B18.
Abstract B20: Design, synthesis and SAR studies of novel survivin inhibitors with potent antiproliferative properties
Authors: Xiao Min , Wang Jin , Lu Yan , Miller Duane D. , Li Wei
Published: July 2015

A series of novel survivin inhibitors were designed and synthesized based our recently reported compound UC-112. All the new analogs were tested against several cancer cell lines including multidrug-resistant melanoma cancer cell lines. The in vitro results showed that several compounds had excellent antiproliferative activity with IC50 values within nanomolar range. Mechanism of action of those new analogs was investigated by Western blot assay. The study confirmed that the new analogs acted by selectively suppressing survivin with the similar mechanism as the parent compound UC-112. Those new analogs represent promising agents that can be further developed for selective anticancer treatment.

Citation Format: Min Xiao, Jin Wang, Yan Lu, Duane D. Miller, Wei Li. Design, synthesis and SAR studies of novel survivin inhibitors with potent antiproliferative properties. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B20.
Abstract B24: A phosphoproteomic comparison of oncogenic BRAF and MKK1/2 inhibition in human melanoma
Authors: Stuart Scott , Houel Stephane , Wang Nan , Old William , Ahn Natalie
Published: July 2015

The purpose of this study was to use phosphoproteomics to gain insight into the different clinical responses to BRAF inhibition, MKK1/2 inhibition, and combined BRAF/MKK inhibition. Experiments were designed to test whether BRAF inhibition may affect a distinct subset of targets that MKK1/2 inhibition does not and whether BRAF and MKK1/2 inhibitors work additively or synergistically when used in combination. To this end, we employed a SILAC-based method to quantify changes in the phosphoproteome of WM239A melanoma cells treated with a clinically relevant BRAF inhibitor (PLX4032; 10μM) or MEK1/2 inhibitor (AZD6244; 10μM) inhibitor. Phosphopeptides were enriched by titanium dioxide prior to fractionation on an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) column. In a set of three biological replicate experiments, we quantified changes at 23,986 phosphosites on 4,722 proteins. We identified 1,356 phosphosites that decreased in abundance, and 341 that increased in abundance, in response to treatment with either PLX4032 or AZD6244. The overwhelming majority of phosphosites were responsive to both inhibitors. Of the phosphosites that decreased in abundance, 13 responded only to PLX4032, suggesting that certain targets are regulated by BRAF but not MKK1/2. In contrast, we failed to identify any phosphosites that responded only to AZD6244. To determine whether the therapeutic benefit of combined BRAF and MKK1/2 inhibition may be a result of drug synergy, we treated cells either with a single inhibitor or with a combination of AZD6244 and PLX4032, each at submaximal doses (30 nM). We then compared the magnitude of the responses observed with the combination treatment to those observed for each drug alone. Comparison of observed versus predicted changes at 6,463 phosphosites demonstrated that the primary effect of the two inhibitors is additive. Taken together, we show that the combination of BRAF and MKK1/2 inhibitor leads to predominantly additive interactions, and that the BRAF inhibitor has a potential for regulating phosphorylation events that are not targeted by MKK1/2 inhibitor.

Citation Format: Scott Stuart, Stephane Houel, Nan Wang, William Old, Natalie Ahn. A phosphoproteomic comparison of oncogenic BRAF and MKK1/2 inhibition in human melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B24.
Abstract B29: Differential effects of BRD4 inhibition on uveal melanoma cells with Gnaq/11 mutations
Authors: Ambrosini Grazia
Published: July 2015

Uveal melanoma (UM) represents the most common intraocular malignancy, and there are no effective treatments for this aggressive disease. Nearly 80% of UM is characterized by oncogenic mutations in G protein subunits Gnaq and Gna11, and 70% have extra copies of the oncogene MYC.

Chromatin regulators have become attractive targets for cancer therapy. In particular, the BET-bromodomain inhibitor JQ1 has shown selective inhibition of c-Myc expression, followed by the suppression of c-Myc-dependent target genes. Treatments of UM cell lines with JQ1 showed that the Gnaq/Gna11 mutant cells were the most sensitive to BET inhibition with induction of cell death, while the cells without the mutations underwent cell cycle arrest in G1.

Microarray analysis of UM cells revealed that several genes were differentially regulated by JQ1 treatment in Gnaq/11 mutant cells, with the downregulation of c-Myc, of the anti-apoptotic protein Bcl-2, and DNA repair proteins Rad51 and Brca1. While depletion of each of these genes did not affect cell viability, the concomitant silencing of Rad51 and Bcl-2 represented the minimal requirement to mimic the apoptotic effects of JQ1. As opposed to hematopoietic cancer cell lines, c-Myc depletion did not seem to play a major role in inhibiting the growth of these cells.

Furthermore, the treatment with JQ1 resulted in significant antitumor activity in a mouse xenograft model of uveal melanoma with Gnaq mutation.

Our results suggest the potential therapeutic use of BET bromodomain inhibitors for treating uveal melanoma with Gnaq/Gna11 mutations.

Citation Format: Grazia Ambrosini. Differential effects of BRD4 inhibition on uveal melanoma cells with Gnaq/11 mutations. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B29.
Abstract B39: Exome sequencing in primary melanoma identifies novel drivers of melanoma progression
Authors: Montagnani Valentina , Benelli Matteo , Apollo Alessandro , Gerlini Gianni , Borgognoni Lorenzo , Stecca Barbara
Published: July 2015

Melanoma is the most malignant and lethal among skin cancers, due to its high infiltration and invasion ability and resistance to therapy. Whereas early stages melanoma can be cured in the majority of cases by surgical excision, metastatic melanoma is a highly lethal condition. The most frequent known oncogenic mutation in melanoma is BRAF-V600E and several full exome sequencing studies have revealed numerous other alterations (Wei et al, 2011; Nikolaev et al, 2011; Stark et al, 2011; Krauthammer et al, 2012). A crucial issue in understanding melanoma progression is to identify which mutations are specifically involved in making an individual melanoma competent for metastatic spread. It is well established that this behavior is highly correlated with histological features, such as the thickness of the primary tumor and the mitotic index. Here we performed full exome sequencing of 5 thin (<1mm in thickness) and 5 thick (>4mm in thickness) primary melanomas compared to matched-normal DNA. We confirmed recurrent somatic mutations in known melanoma-related genes, including BRAF, c-KIT, EGFR, PPP6C, MLL3 and several components of the glutamate signaling. In addition, we discovered mutations in genes not previously linked to this tumor, such as CSMD1, FGFR4 and components of the Hedgehog (HH) signaling pathway. In particular, in a thick melanoma we found an novel activating mutation in the transcription factor GLI1, one of the final effectors of the HH signaling. Notably, in the only 3 thick melanomas that produced metastasis, we identified candidate metastasis-driving mutations in six genes (ADAMTS6, ADAMTS7, CHD9, MLL3, NALCN and TSC2). Interestingly, we identified several regions of focal somatic copy-number alterations (SCNAs) that were altered at significantly higher frequency in thick compared to thin melanomas. Several gene families are comprised among these regions of focal SCNAs, including components of Notch, HH and Wnt/ß-catenin signaling pathways, BRAF, c-MYC and its cofactor PIM1, several ADAMs, EGFR and the HOX genes. Our preliminary results identify potential drivers of melanoma progression, enhancing our understanding of the genomic complexity underlying melanoma.

Citation Format: Valentina Montagnani, Matteo Benelli, Alessandro Apollo, Gianni Gerlini, Lorenzo Borgognoni, Barbara Stecca. Exome sequencing in primary melanoma identifies novel drivers of melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B39.
Abstract IA12: Preclinical models for targeting oncogenic Ras signaling in cancer
Authors: Shannon Kevin
Published: July 2015

Somatic RAS mutations occur in 30% of human cancers, and are common in both melanoma and hematopoietic cancers. Importantly, whereas KRAS mutations predominate in epithelial cancers, NRAS is mutated much more often in melanoma and hematopoietic cancers. The most obvious therapeutic strategy for cancers with RAS mutations – developing specific inhibitors of the mutant protein – is extremely challenging due to the very high affinity of Ras for GTP, structural considerations of the Ras/GTPase activating protein (Ras/GAP) molecular switch, and the loss of normal enzymatic activity as a consequence of oncogenic RAS mutations. Given these issues, most recent drug discovery efforts have focused on inhibiting downstream biochemical targets of mutant Ras such as Raf, MEK, and Akt with many compounds undergoing clinical evaluation. We have exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human hematologic cancers and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable myeloid and lymphoid leukemias characterized by hyperactive Ras signaling. We utilize cohorts of mice that are engrafted with the same primary leukemias for preclinical testing. In this presentation, I will focus on three biologic and therapeutic questions related to NRAS in tumorigenesis: (1) loss of the normal NRAS allele in cancers with oncogenic NRAS mutations; (2) responses of primary Nras mutant leukemias to MEK and PI3 kinase inhibitors; and, (3) potential strategies for selectively targeting oncogenic N-Ras in cancer.

Note: This abstract was presented as part of the Joint Session: Clinical Science Interactions between Melanoma and Hematologic Malignancies with the AACR Special Conference: Hematologic Malignancies: Translating Discoveries to Novel Therapies held September 20-23, 2014 in Philadelphia, PA.

Citation Format: Kevin Shannon. Preclinical models for targeting oncogenic Ras signaling in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA12.
Abstract IA15: Adoptive T cell therapy with CAR modified T cells: We have a model A Ford, can we build a Ferrari?
Authors: Brentjens Renier
Published: July 2015

Recently published reports support the novel approach of treating cancer with patient derived T cells genetically modified to express artificial T cell receptors, termed chimeric antigen receptors (CARs), targeted to tumor associated antigens. To this end, initial clinical trial outcomes of patients with relapsed B cell acute lymphoblastic leukemia (B-ALL) treated with T cells genetically modified to express a CAR specific to the CD19 antigen demonstrate that this approach is a very promising therapeutic intervention which potentially may dramatically alter the standard of care in this disease. In contrast far more modest clinical responses were seen in patients with relapsed low grade B cell malignancies including chronic lymphocytic leukemia (CLL) and B cell non-Hodgkins lymphomas (NHL). The etiologies of discordant clinical outcomes between B-ALL and CLL/NHL patients treated with CD19 targeted CAR T cells remains a subject of conjecture although the tumor microenvironment is a strong focus for further investigation. To this end, we will present novel data on a next generation of CAR T cells, termed “armored CAR” T cells genetically designed to enhance persistence, anti-tumor efficacy, and an ability to overcome an immune suppressive tumor microenvironment through further genetic modification. Promising preclinical studies utilizing various “armored CAR” T cell approaches and their role in future clinical trials will be discussed as well as the relevance of these findings to the extrapolation of this technology from hematologic tumors to far more prevalent solid tumor malignancies.

Note: This abstract was presented as part of the Joint Session: Clinical Science Interactions between Melanoma and Hematologic Malignancies with the AACR Special Conference: Hematologic Malignancies: Translating Discoveries to Novel Therapies held September 20-23, 2014 in Philadelphia, PA.

Citation Format: Renier Brentjens. Adoptive T cell therapy with CAR modified T cells: We have a model A Ford, can we build a Ferrari? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA15.
Abstract IA19: Response and resistance PD-1 pathway blockade
Authors: Topalian Suzanne L.
Published: July 2015

Melanoma is a genetically complex cancer harboring a multitude of somatic mutations for potential targeting by the immune system. However, melanoma cells can co-opt normal immune tolerance mechanisms to evade elimination. The immunoglobulin-like molecule PD-L1 (B7-H1), expressed by many melanomas in situ, creates a protective shield against immune attack. PD-L1 is a ligand for the programmed death-1 (PD-1) receptor expressed on activated T and B cells, and it delivers an inhibitory signal to suppress antitumor immunity. Blocking the PD-1:PD-L1 interaction can rejuvenate anti-melanoma immunity and mediate tumor regression. Multicenter clinical trials of blocking antibodies against PD-1 (nivolumab, pembrolizumab) or PD-L1 (BMS-936559, MPDL3280A, MEDI4736) have been undertaken. In an early phase trial of biweekly nivolumab administration, durable objective tumor regressions and disease stabilization were observed among 31% and 7%, respectively, of 107 patients with advanced treatment-refractory melanoma. Median overall survival in this trial was 17 months. Partial tumor regressions that are durable and persist after drug discontinuation suggest that PD-1 blockade can reset the balance between the immune system and melanoma. In some patients, “mixed” tumor regressions or initial disease progression followed by regression suggest that tumor lesions separated anatomically or chronologically may be immunologically heterogeneous. Durable objective tumor regressions reported in clinical trials of various antibodies blocking the PD-1 and PD-L1 immune checkpoints validate the importance of targeting this pathway in melanoma therapy. Ongoing investigations into pharmacodynamics and potential biomarkers of response are designed to reveal the basis for immunological heterogeneity among different patients, and among diverse tumor sites in individual patients, to enable the optimal clinical development of these drugs.

Supported by research grants from Bristol-Myers Squibb, the National Institutes of Health, Stand Up 2 Cancer, the Barney Family Foundation, the Laverna Hahn Charitable Trust, Moving for Melanoma of Delaware, and the Melanoma Research Alliance.

Citation Format: Suzanne L. Topalian. Response and resistance PD-1 pathway blockade. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA19.
Abstract IA20: Targeting AMPK signaling in melanoma
Authors: Zheng Bin
Published: July 2015

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated anti-tumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. We have previously reported that AMPK signalling is attenuated by BRAFV600E in melanoma through phosphorylation of LKB1, a major upstream kinase of AMPK. More recently, we have also discovered that BRAF is directly phosphorylated by AMPK, leading to impaired BRAF-KSR1 association and MEK-ERK signalling (Mol Cell, 2013, 52:161). Based on this crosstalk between the AMPK and BRAF signaling pathways, we investigated the anti-tumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Co-treatment of BRAF mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared to the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells. In contrast, PLX4720 selectively targeted JARID1B negative cells, which represents the majority of cells in the tumors, and spared the JAR1D1B-positive slowing cycling population. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAFV600E/PTENnull-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.

Citation Format: Bin Zheng. Targeting AMPK signaling in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA20.
Abstract IA30: Combined targeted therapies, where to next
Authors: Long Georgina V.
Published: July 2015

Combined BRAF and MEK inhibition has improved patient outcomes beyond single agent BRAF inhibition in BRAF-mutant metastatic melanoma, however 25% of patients do not have a RECIST-response and 30% progress within six months. Analysis of melanoma progressing metastases from combination-treated patients reveals that early resistance via the MAP kinase pathway is more common than that observed with single agent BRAF inhibition, which raises the questions of whether even stronger MAP kinase pathway inhibition would be of benefit to patients, and whether this is achievable therapeutically. However, the combination of MAP kinase inhibitors with immunotherapy may be a more effective strategy. Preliminary results in human tissues suggest that the presence of tumor-infiltrating lymphocytes and tumor-cell PDL1 expression may assist in patient selection for such a strategy. Early-during treatment responding metastases from combined BRAF and MEK inhibition-treated patients demonstrate similar levels of tumor-infiltrating lymphocytes as observed with single agent BRAF inhibition, suggesting that MEK inhibitors may not negatively impact the immunogenicity of targeted therapy early-on, and supports the concept that concurrent targeted- and immuno-therapy strategies may provide a greater opportunity for disease control than in sequence.

Citation Format: Georgina V. Long. Combined targeted therapies, where to next. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA30.
Wnt/β-Catenin and Wnt5a/Ca2+ Pathways Regulate Proliferation and Apoptosis of Keratinocytes in Psoriasis Lesions
Authors: Zhang Yanfei , Tu Chen , Zhang Dingwei , Zheng Yan , Peng Zhenhui , Feng Yiguo , Xiao Shengxiang , Li Zhengxiao
Published: July 2015

Background/Aims: Wnt5a is overexpressed in psoriasis lesions, however the mechanism by which Wnt5a is involved in the pathogenesis of psoriasis is not clear. To address this, the expression of Wnt5a in psoriatic lesions and its effect on keratinocyte cell proliferation and apoptosis was examined in vitro. Methods: The expression levels of WNT5A, and genes encoding its receptors frizzled2 (FZD2) and frizzled5 (FZD5) were examined in samples obtained from individuals with psoriasis and healthy controls. Knockdown of Wnt5a with short interfering (si)RNAs was performed in cultured HaCaT keratinocytes and normal human keratinocytes (NHK), and the expression of Wnt5a, protein kinase C (PKC), and β-catenin were determined, and cell cycle activity, proliferation and apoptosis were assessed. Results: The expression of WNT5A, FZD2 and FZD5 mRNA and protein were increased in psoriatic lesions. Wnt5a knockdown suppressed proliferation and induced apoptosis in HaCaT and NHK cells. Additionally, expression of PCNA, MKI67, CCND1, BCL2, CTNNB1, and genes encoding PKC and survivin were downregulated, whereas CASP3 was upregulated. The mRNA levels of the Wnt pathway inhibitors DKK1 and SFRP1 were upregulated, Western blotting analyses demonstrated reduction in β-catenin and PKC protein levels. Conclusion: Knockdown of Wnt5a suppresses the proliferation of keratinocytes and induces apoptosis by inhibiting the Wnt/β-catenin or Wnt5a/Ca2+ pathways.
Acetylcholine Inhibits LPS-Induced MMP-9 Production and Cell Migration via the a7 nAChR-JAK2/STAT3 Pathway in RAW264.7 Cells
Authors: Yang Yong-Hua , Li Dong-Ling , Bi Xue-Yuan , Sun Lei , Yu Xiao-Jiang , Fang Huan-Le , Miao Yi , Zhao Ming , He Xi , Liu Jin-Jun , Zang Wei-Jin
Published: July 2015

Background: Excessive activation of matrix metalloproteinase 9 (MMP-9) has been found in several inflammatory diseases. Previous studies have shown that acetylcholine (ACh) reduced the levels of pro-inflammatory cytokines and decreased tissue damage. Therefore, this study was designed to explore the potential effects and mechanisms of ACh on MMP-9 production and cell migration in response to lipopolysaccharide (LPS) stimulation in RAW264.7 cells. Methods: MMP-9 expression and activity were induced by LPS in RAW264.7 cells, and examined by real-time PCR, western blotting and gelatin zymography, respectively. ELISA was used to determine the changes in MMP-9 secretion among the groups. Macrophage migration was evaluated using transwell migration assay. Knockdown of a7 nicotinic acetylcholine receptor (a7 nAChR) expression was performed using siRNA transfection. Results: Pre-treatment with ACh inhibited LPS-induced MMP-9 production and macrophage migration in RAW264.7 cells. These effects were abolished by the a7 nAChR antagonist methyllycaconitine (MLA) and a7 nAChR siRNA. The a7 nAChR agonist PNU282987 was found to have an effect similar to that of ACh. Moreover, ACh enhanced the expression of JAK2 and STAT3, and the JAK2 inhibitor AG490 and the STAT3 inhibitor static restored the effect of ACh. Meanwhile, ACh decreased the phosphorylation and nuclear translocation of NF-κB, and this effect was abrogated in the presence of MLA. In addition, the JAK2 and STAT3 inhibitor abolished the inhibitory effects of ACh on phosphorylation of NF-κB. Conclusions: Activation of a7 nAChR by ACh inhibited LPS-induced MMP-9 production and macrophage migration through the JAK2/STAT3 signaling pathway. These results provide novel insights into the anti-inflammatory effects and mechanisms of ACh.
The Down-Regulation of MicroRNA-497 Contributes to Cell Growth and Cisplatin Resistance Through PI3K/Akt Pathway in Osteosarcoma
Authors: Shao Xue-jun , Miao Mei-hua , Xue Jun , Xue Jian , Ji Xue-qiang , Zhu Hong
Published: July 2015

Background: Down-expression of microRNA-497 (miR-497) was often found in malignancies. The purposes of this study were to determine the expression of miR-497 in human osteosarcoma and to establish the association between miR-497 expression with cell survival and the sensitivity to cisplatin in human osteosarcoma cells. Methods: The effects of ectopic miR-497 expression on the cell survival and cisplatin sensitivity in osteosarcoma cells were measured by the Cell Counting Kit-8 (CCK-8) assay. Quantitative real-time PCR (qRT-PCR) was utilized to determine the expression of miR-497. The effects of ectopic miR-497 expression on the expression of VEGFA, Akt and p-Akt were determined by western blot. Results: Real-time quantitative PCR analysis revealed that miR-497 was significantly down-regulated in osteosarcoma tissues and in the osteosarcoma cell line SAOS-2 compared with adjacent nontumorous osteosarcoma tissues and normal human osteoblasts. Up-regulation of miR-497 inhibited cell survival and enhanced the sensitivity to cisplatin in osteosarcoma cells. In addition, knockdown of miR-497 induced osteosarcoma cells growth and cisplatin resistance. Luciferase reporter assay and western blot confirmed that VEGFA was a direct target of miR-497. PI3K inhibitor LY294002 abrogated miR-497 inhibitors induced cisplatin resistance. Conclusion: Taken together, our results suggest that miR-497 modulates the sensitivity to cisplatin at least in part through PI3K/Akt pathway in osteosarcoma cells.
Galectin-3 Modulates Experimental Colitis
Authors: Lippert Elisabeth , Stieber-Gunckel Manuela , Dunger Nadja , Falk Werner , Obermeier Florian , Kunst Claudia
Published: July 2015

Background: We recently identified galectin-3 (gal-3) as a new and strong fibroblast activator produced by colonic epithelial cells. Very little is known about the influence of gal-3 in inflammatory bowel disease. We, therefore, investigated the impact of gal-3 on dextran sodium sulfate (DSS)-induced colitis in a mouse model. Methods: Colonic lamina propria fibroblasts of healthy controls were used for co-incubation studies of gal-3 with gal-1, TGF-β1, IFNγ, IL-4 and IL-10. Acute and chronic DSS colitis was induced by 3% DSS in drinking water in female Balb/c mice weighing 20-22 g. Recombinant gal-3 was expressed by the pET vector system and used for a 5-day treatment in different concentrations intraperitoneally. The distal third of the colon was used for histologic analysis. Colonic cytokine expression was determined by quantitative RT-PCR. Results: In vitro, gal-3 induced IL-8 secretion was significantly reduced by co-incubation with IL-10 (5 ng/ml) and IL-4 (10 ng/ml). Acute DSS-induced colitis was ameliorated by gal-3 treatment as indicated by increased colonic length and reduced weight loss compared to that of controls. In acute and chronic colitis, gal-3 treatment resulted in a significant suppression of colonic IL-6. Conclusion: Gal-3 significantly reduces inflammation in acute and chronic DSS colitis in mice indicating a potential role in intestinal inflammation.