4th international conference on tumor progression and therapeutic resistance: meeting report.

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4th international conference on tumor progression and therapeutic resistance: meeting report a

Varun V Prabhu & Wafik S El-Deiry

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Laboratory of Translational Oncology and Experimental Cancer Therapeutics, Department of Hematology/Oncology and Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA Accepted author version posted online: 17 Mar 2015.

Click for updates To cite this article: Varun V Prabhu & Wafik S El-Deiry (2015) 4th international conference on tumor progression and therapeutic resistance: meeting report, Cancer Biology & Therapy, 16:3, 363-376 To link to this article: http://dx.doi.org/10.1080/15384047.2015.1004928

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MEETING REPORT Cancer Biology & Therapy 16:3, 363--376; March 2015; © 2015 Taylor & Francis Group, LLC

4th international conference on tumor progression and therapeutic resistance: meeting report Varun V Prabhu and Wafik S El-Deiry* Laboratory of Translational Oncology and Experimental Cancer Therapeutics; Department of Hematology/Oncology and Molecular Therapeutics Program; Fox Chase Cancer Center; Philadelphia, PA USA

Downloaded by [ECU Libraries] at 19:02 25 April 2015

Keywords: cancer therapy, cancer stem cell, metastasis, driver mutation, therapeutic resistance, translational medicine, tumor heterogeneity, tumor microenvironment, tumor progression

The fourth international conference on tumor progression and therapeutic resistance organized in association with GTCbio was held in Boston, MA from March 9 to 11, 2014. The meeting attracted a diverse group of experts in the field of cancer biology, therapeutics and medical oncology from academia and industry. The meeting addressed the current challenges in the treatment of cancer including tumor heterogeneity, therapy resistance and metastasis along with the need for improved biomarkers of tumor progression and clinical trial design. Keynote speakers included Clifton Leaf, Editor at Fortune Magazine, Dr. Mina Bissell from the Lawrence Berkeley National Laboratory and Dr. Levi Garraway from the Dana Farber Cancer Institute. The meeting featured cutting edge tools, preclinical models and the latest basic, translational and clinical research findings in the field.

Keynote Presentations Dr. Wafik El-Deiry, conference chair, provided opening remarks by highlighting the purpose of the conference and introducing the Keynote speakers. Clifton Leaf from Fortune magazine spoke about the inspiring story of the one eyed surgeon Denis Parsons Burkitt and his pioneering work on Burkitt’s lymphoma. Clifton Leaf himself is a cancer survivor and author of the provocative book, ‘The Truth in Small Doses-Why We’re Losing the War on Cancer and How to Win It’. Clifton Leaf described Dr. Burkitt’s struggle as a surgeon in Uganda trying to unravel the epidemiology and biology of the lymphoma. The disease was previously unrecognized and occurred as a sarcoma involving the jaw along with intestinal tumors in African children. Dr. Burkitt tried to create an awareness of the disease within the scientific community by publishing his findings. He undertook a safari ride spanning 10 weeks, 12 countries and 56 hospitals to gather epidemiological data about the disease. He concluded from his findings that the disease was prevalent in a large population and *Correspondence to: Wafik S El-Deiry; Email: wafi[email protected] Submitted: 12/30/2014; Accepted: 12/30/2014 http://dx.doi.org/10.1080/15384047.2015.1004928

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occurred mostly in areas with high rainfall and temperatures similar to the occurrence of malaria. Dr. Burkitt published his epidemiological findings in the British Journal of Cancer, gave multiple talks about his findings and was later contacted by Prof M.A. Epstein from the Bland-Sutton Institute. Dr. Epstein was an expert at Electron microscopy. He requested Dr. Burkitt for the pediatric tumor specimens and collaborated with a team of virologists to conclude that the herpes virus (Epstein-Barr virus) was observed in several Burkitt’s lymphoma specimens. Meanwhile, Dr. Burkitt along with Dr. Peter Clifford tried several experimental therapeutic combinations in patients to achieve long-term remission in several cases. All of this progress was achieved over a span of 12 y (1957–1969). However, the molecular basis of the disease still remained unclear. It was later in 1982, that the translocated c-myc oncogene was observed in classic Burkitt’s lymphoma.1 Clifton Leaf pointed out that progress in the treatment and prevention of Burkitt’s lymphoma has been slow since 1982. He postulated that the lack of data sharing within the oncology community and slow motion collaborations have adversely affected progress. Although the number of publications has risen sharply in the last decade involving millions of dollars of government funding, limited meaningful clinical outcomes have been achieved. He advocated for a major overhaul of the current funding system and the focus on publication. Instead he advocated for an alternative system to be developed to encourage data, resource and biospecimen sharing, allowing the establishment of meaningful collaborations. He concluded by highlighting the need to translate academic caution to medical urgency. Dr. Mina Bissell from the Lawrence Berkeley National Laboratory spoke about the importance of the tissue microenvironment in regulating cellular function. The body is composed of 10–70 trillion cells, all of which have the same genetic material but still give rise to several tissues with remarkable specificity. She elaborated on the differential effects of the Rous sarcoma virus oncogene in chickens, human embryos and cells in tissue culture. Growth of cells on plastic does not account for microenvironment and tissue architecture specific regulation. Her work focused on the matrix or ground substance in tissues forming a part of the extracellular matrix (ECM) as well as 3-dimensional (3D) culture models of breast epithelial cells helped overcome the limitations of 2D culture.2 Normal mammary cells form

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acini in 3D cultures while malignant cells lose the potential for acinus formation and form tumors perhaps due to dysregulation of the extracellular matrix and microenvironment. Dysregulation of the ECM can destroy mammary tissue organization. Her previous work has also demonstrated that treatment of breast tumor cells with an EGFR inhibitor or antibody against b1-integrin can revert tumor cells to a normal phenotype in 3D culture and in vivo. The reversion occurs irrespective of the genetic hits in the tumor cells clearly demonstrating that tissue phenotype dominates over cellular genotype.3 Thus, growth and malignant behavior are regulated at the level of tissue organization and this in turn is regulated by the ECM and basement membrane. Cell polarity is also important for tissue integrity. Dr. Bissell spoke about some of her work that helped determine the signals that regulate cell polarity of luminal epithelial and myoepithelial cells in the breast in vivo. In 3D cultures when luminal epithelial cells are grown in collagen cell polarity is reversed while addition of myoepithelial cells corrects the inverse polarity. This function of myoepithelial cells was dependent on the production of laminin1. Breast tumors had reduced staining of laminin-1 and breast cancer derived myoepithelial cells were unable to produce laminin-1 and reverse cell polarity. Thus, loss of microenvironment mediated regulation of cell polarity is an important determining factor during tumor formation.4 Next, Dr. Bissell provided evidence for the regulation of the cellular genome by ECM signaling. The Laminin-nuclear actin axis plays a key role in the regulation of cellular quiescence and growth in human breast cells. Laminin depletes nuclear actin, reducing transcription and DNA synthesis resulting in growth arrest and cellular quiescence.5 She mentioned that the Laminin nuclear actin axis is disrupted in cancer cells. Next, she pointed out the importance of metabolic pathways in cellular functions. Small pH changes (such as opening the door of an incubator) can affect cellular function, which prompted her to design a steady state apparatus to maintain pH to study the effects of radiation treatment. She also spoke about her recently published work that adds a new dimension to the “Warburg effect”. Dr. Bissell explained that mitochondrial dysfunction need not be the only reason for increased aerobic glycolysis during tumorigenesis. Cells can high levels of aerobic glycolysis despite a normal TCA cycle. Her recent work shows that high levels of extracellular glucose could potentially be an oncogenic event and determine the malignant phenotype. Increased glycolytic activation by overexpression of GLUT-3 transporters in non-malignant human breast cells activated oncogenic signaling mechanisms such as EGFR and b1integrin. These studies in a 3D culture model show that EGFR and b1-integrin are not only regulated by genetic events but also by glucose in blood.6 Finally, she provided evidence to close the genotype/phenotype loop explaining how cell-ECM interactions are regulated by microRNAs. Laminin-5 regulates p53 and p53 in-turn determines Laminin-5 levels. She showed data on a p53 regulated microRNA that governs breast cells and the ECM. She concluded with the idea that genetic mutations, signaling pathways, ECM, cell polarity alone cannot define tissue organization and tumorigenesis, instead they are defined by a dynamic interaction and integration of all these events.

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Dr. Levi Garraway from the Dana Farber Cancer Institute spoke about precision medicine in oncology using the approach of genetic analysis and allele-based strategies for clinical profiling of tumor mutations. He described an optimized high-throughput cancer gene mutation profiling platform called OncoMap. Actionable mutations in tumors can be identified with massively parallel targeted sequencing at reduced cost in the clinic allowing informed clinical decision making and optimal use of targeted therapy.7-9 Dr. Garraway mentioned about an alternative approach for determining genomic abnormalities in patient tumors called CanSeq that involves prospective clinical whole exome sequencing. He outlined a step wise process for precision cancer medicine: patient encounter - fresh tumor biopsy - OMIC profiling - data interpretation - management decision by clinical team - rational therapy - fresh biopsy - clinical response achieved? – drug resistance? – fresh biopsy – new therapy options?. The treatment of cancer based on anatomic origin in the clinic needs to be combined with targeted therapeutics selected based on the genetics and genomics data. For example combination of RAF and MEK inhibitors improves progression-free survival as compared to monotherapy in patients with BRAFV600E mutations.10 But resistance in response to targeted therapies is prevalent. Along with tumor heterogeneity, resistance heterogeneity is a major problem in the clinic. He described a case in which multiple RAF inhibitor resistance genetic alterations were observed in a single tumor biopsy.11 Continued accumulation of genetic hits occurs even after combination therapy. There is a need for both DNA- and RNA-based analysis of human tumors in the clinic by combining RNA-seq with DNA-based sequencing. Dr. Garraway mentioned about a study employing both whole exome sequencing and whole transcriptome sequencing to identify genomic alterations resulting in resistance to combined RAF/MEK inhibition.12 Considering the large number of resistance drivers, lack of post resistance tumor sampling is a major problem resulting in clinical undersampling. However, one has to be aware that repeat clinical tumor sampling requires massive data analysis and robust algorithms. Dr. Garraway provided an example of repeat sampling of the bone metastasis of a prostate cancer patient. Wholeexome sequencing revealed several genomic alterations including a homozygous PTEN deletion and a BRCA2 non-sense mutation.13 Finally, he described identification of gene/signaling networks involved in resistance mechanisms using large scale resistance screens with lentiviral ORF collections.14

Session 1: Tumor Microenvironment/Progression and Metastasis Mechanisms The session was chaired by Dr. Ramon Parsons from the Mount Sinai School of Medicine. He spoke about the tumor suppressor PTEN that antagonizes the PI3K/Akt pathway. PTEN has been previously shown to inhibit cell migration.15 PREX2, a guanine nucleotide exchange factor (GEF) is a PTEN interacting protein that regulates PTEN activity. P-REX2 inhibits PTEN phosphatase activity and P-REX2 deletion in fibroblasts increases PTEN activity.16 P-REX2-mediated PTEN


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inhibition consequently stimulates the PI3K pathway driving tumorigenesis and cell migration.15 Dr. Parsons and colleagues investigated whether PTEN can suppress cell movement by inhibition of P-REX2, independent of PI3K pathway. P-REX2 has been previously described as an important regulator of Purkinje cell dendrite morphology.17 Dr. Parsons described new findings that PTEN regulates cell migration upstream of PREX2. In BT549 cells PTEN was unable to suppress invasion in P-REX2 knockdown cells. PTEN suppressed P-REX2 independent of the PI3K pathway. P-REX2 binds the PTEN PDZ domain while the PTEN C-terminal tail antagonizes P-REX2. Dr. Parsons provided further evidence that mutations in the PTEN C-terminal tail impair its ability to antagonize P-REX2mediated migration. In addition, P-REX2 mutants escape PTEN C-terminal tail-mediated inhibition of migration. PTEN does not interact with PREX1 and is unable to block PREX1 driven invasion. Dr. Parsons mentioned that the PTEN C-terminal tail inhibits P-REX2 GEF activity, in experiments that used a fluorescence polarization assay to monitor GEF activity. In patient tumors, P-REX2 is frequently mutated (25% of melanoma, 15% of stomach cancers and 10% of lung cancers harbor mutations). P-REX2 is located on human chromosome 8q which is a site of frequent amplification in cancer. P-REX2, particularly its short isoform P-REX2C is frequently amplified in breast cancer. PTEN interacts much more strongly with PREX2A than with the short isoform P-REX2C. The P-REX2 short isoform lacks the high affinity PTEN binding site and likely escapes PTEN-mediated regulation of migration. P-REX2 and PTEN mRNA expression is associated with tumor metastasis. Although P-REX2 is located on the same chromosome as myc, but myc expression did not correlate with metastasis. Thus P-REX2 is a potential therapeutic target that is PI3K independent. Another observation was that P-REX2 knockout cells are resistant to insulin activation. Dr. Sandra McAllister from Harvard Medical School spoke about her work on various systemic factors that regulate breast cancer progression and metastasis. Breast cancer is often detected in patients having advanced disease with disseminated tumors that eventually develop into clinical metastasis, while most tumors remain indolent. However, the cause for conversion of clinically undetectable micrometastasis into life-threatening metastatic recurrence is unknown. Dr. McAllister showed that distant primary tumors can promote such a metastatic conversion through systemic effects on tumor promoting bone marrow cells and platelets. In luminal breast cancer, cytokines secreted from the primary tumor are absorbed by platelets and the platelets are recruited to distant disseminated indolent tumors. Here, the platelets serve as pro-angiogenic factors by increasing the expression of CD24 and VEGFR2 and promoting tumor growth. Tumor cells at metastatic sites become enriched for CD24 expression that functions as a ligand for p-selectin that is expressed on platelets and that may recruit them. Tumor growth can be prevented by treatment with aspirin that blocks platelet activation.18 Primary tumors Dr. McAllister referred to as ‘instigators’ can also secrete endocrine factors such as osteopontin to functionally activate bone-marrow cells that are recruited to sites of distant


metastasis promoting tumor growth.19 These functionally activated bone-marrow cells are Sca1CcKit- and highly express granulin. The granulinC bone-marrow cells promoted a cancerassociated fibroblast phenotype promoting tumor growth. High granulin expression in tumor tissues of breast cancer patients correlated with poor survival and the aggressive triple-negative tumor subtype.20 The tumor supportive microenvironment at the distant indolent tumor sites was enriched in EGF and IGF-1. Genes associated with cellular reprogramming and EMT such as Oct4, c-myc and zeb1 were highly expressed in the metastatic tumors. Treatment with EGF and IGF-1 inhibitors prevented such metastasis.21 Dr. McAllister’s work could help predict which breast cancer patients are more susceptible to relapse and provide potential avenues for therapy. Dr. Andrei Thomas-Tikhonenko from University of Pennsylvania explained his work related to the mechanisms of malignant transformation by myc via myc-regulated microRNAs. In KRAS mutated, p53-null colonocytes, transduction with myc promoted tumor angiogenesis and growth. However, VEGF levels were not changed, instead an inhibitor of tumor angiogenesis thrombospondin-1 (tsp-1) was downregulated. Dr. Thomas-Tikhonenko provided evidence that myc-regulated miR-17-92, a micro-RNA cluster containing miR 17, 18, 19a, 19b, 20, and 92, was responsible for downregulation of tsp-1.22 Dr. Thomas-Tikhonenko has developed a luciferase sensor assay to monitor miR expression. Myc-mediated tsp-1 downregulation also occurred through increased turnover of mRNA.23 Clusterin, another member of the thrombospondin type I repeat (TSR) superfamily was also negatively regulated by miR-17-92 via reduced TGFb signaling.24 miR-17-92 downregulated multiple effectors in the TGFb pathway including TGFb RII receptor, smad3 and smad4.25 Finally, Dr. Thomas-Tikhonenko spoke about his recently published work evaluating the relationship between myc and TGFb pathways in colorectal cancer angiogenesis, and their possible epistatic relationship. Both myc activation and TGFb inactivation individually increased tumor growth and angiogenesis. But combining these 2 genetic events did not lead to an additive effect indicating that the pathways are functionally redundant in regulation of tumor growth and angiogenesis. Further, comprehensive molecular characterization of human colon and rectal cancer was performed using the Cancer Genome Atlas. In non-hypermutated patient tumors using the cBioportal platform it was determined that myc overexpression or TGFb pathway inactivation are mutually exclusive events.26 Dr. Thomas-Tikhonenko discussed efforts for drug development targeting c-Myc, including BET bromodomain inhibitors in clinical trials. Dr. Michael Andreeff from the MD Anderson Cancer Center spoke about the role of bone marrow microenvironment in leukemia drug resistance and the hypoxic niche. Bone marrow stromal cells growing in co-culture with primary AML blasts induced resistance to chemotherapy in the AML cells. Within the bone marrow stromal niche, it was the mesenchymal cells that mediated the chemoresistance. Dr. Andreeff performed leukemia cell versus stromal cell proteomic as well as principle component analysis. Gene expression profiling revealed that upon coculture with AML cells genes related to NFkB signaling were


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upregulated in the mesenchymal stromal cells. Primary mesenchymal stromal cells from leukemia patients had an elevated expression of NFkB target genes compared to normal stromal cells. Blocking NFkB activation in the mesenchymal stromal cells enhanced the pro-apoptotic effects of standard chemotherapy and reduced leukemia burden in vivo in mouse experiments. Mesenchymal stromal cells with overexpressed IkB (repressor of NFkB signaling) lost the ability to protect the tumor cells from chemotherapy. Blockade of NFkB signaling via overexpression of IkB in mesenchymal stromal cells in vivo in NOD/SCID/IL-2rg null mice resulted in a decrease in leukemia chemotherapy resistance.27 Dr. Andreeff mentioned an association between VCAM1 and consequent NFkB activation. Hypoxia and HIF1a are prevalent in the leukemic microenvironment with oxygen levels of < 1%. Dr. Andreeff demonstrated that widespread hypoxia occurs upon AML engraftment into human extramedullary bone marrow in immunodeficient mice by use of pimonidazole staining and reporters in vivo including CA-IX staining. Silencing HIF1a in the mesenchymal stromal cells of the human extramedullary bone marrow reduced AML engraftment in the immunodeficient mice.28 He mentioned that both HIF1a and HIF2a are involved in leukemia and that a hypoxia gene expression signature is elevated in AML patient bone marrow. Further, the hypoxic microenvironment enriches for a stem cell-like phenotype as demonstrated with side population analysis and Aldefluor assay. Oct-4, Nanog, Sox-2 and c-Met were also elevated upon hypoxia. Finally, he spoke about the relationship of microRNAs and AML chemotherapy resistance. CXCR4 is a major prognostic factor in AML with high levels correlating with poor prognosis. Stromal derived factor-1a mediated CXCR4 activation resulted in down regulation of miR-let-7a. Downregulation of miR-let-7a resulted in chemoresistance in AML and overexpression of miR-let-7a in AML resulted in enhanced chemosensistivity in vitro and in vivo. The transcription factor Yin Yang 1 was involved in CXCR4 mediated downregulation of miR-let-7a and chemotherapy resistance.29

Session 2: Cancer Stem Cells/Tumor Heterogeneity The session was chaired by Dr. Meenhard Herlyn from the Wistar Institute. Dr. Robert Weinberg from the Whitehead Institute at the Massachusetts Institute of Technology spoke about his work related to breast cancer stem cell (CSC) biology in the context of resistance to therapy. The epithelial-mesenchymal transition (EMT) has an important role in embryogenesis and cancer metastasis and is stimulated by stromal signals. A number of factors including Slug, Snail, Twist, Sox9, Zeb and others appear to orchestrate the EMT phenotype. Dr. Weinberg described that human mammary epithelial cells and cells from mammary carcinomas in a mesenchymal state via EMT acquire a stem-cell like phenotype. Transient expression of EMT-associated transcription factors Snail or Twist led to an enrichment of cells with stem cell properties as determined by mammosphere formation, soft agar assays and in vivo tumorigenicity.30 He explained that primary tumor cells that undergo an EMT are

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equipped for metastasis and do not require additional genetic hits. In particular, Snail expressing cells are highly metastatic compared to Slug expressing cells while Slug and Sox9 increase the metastatic potential of Ras-transformed cells. Next, he showed data that differentiated basal human mammary epithelial cells can spontaneously convert to stem-like cells while mammary epithelial cells of luminal origin do not give rise to stem cells. Thus, basal cells express Slug while luminal cells do not, but in mammary hyperplasia both Snail and Zeb are expressed in luminal cells. There is also an MET that occurs such that distant metastases look more epithelial in showing the MET. Also, dedifferentiation was also observed with transformed epithelial cells that could give rise to CSC-like cells in vitro and in vivo.31 Mechanistically, the EMT-associated transcription factor Zeb1 drives dedifferentiation of non-CSCs to CSCs. Co-existing inductive and repressive histone marks in basal CD44 low cells allows conversion to CD44 high CSCs while luminal cancers have only repressed histone marks.32 In relation to cancer therapy, CSCs undergoing an EMT are resistant to chemotherapeutic agents such as doxorubicin and paclitaxel. He emphasized that cells that have passed through the EMT are intrinsically more resistant to therapy. Dr. Weinberg mentioned a high-throughput screening approach to identify agents that can selectively target breast CSCs enriched via EMT. The screening led to the identification of salinomycin that selectively targets breast CSCs in vitro and in vivo at lower doses than it targets non CSCs.33 Finally Dr. Weinberg elaborated on work related to signaling pathways that govern breast tumor initiation and metastases. He described the phenomenon that highly metastatic cells put out filopodia-like projections. Tumor cells that extravasate into metastatic sites must proliferate to form micrometastases. The formation of filopodium-like structures via b1 integrin is important for interaction with the extracellular matrix. This leads to the activation of focal adhesion kinase important for the proliferation of the metastatic cells. Dr. Weinberg showed data that EMT promotes formation of filopodium-like structures by CSCs. Thus, the EMT program promotes micrometastases formation by promoting tumor-initiating capability via b1 integrin-mediated filopodiumlike structures and FAK activation.34,35 Dr. Weinberg showed that knockdown of Zeb1 prevents inter-conversion of non-CSCs into CSCs. He provided evidence that cells respond to TGFb in the interconversion and that luminal cells are different from basal cells. He has been looking at chromatin signatures in various cell populations and finding that repressive marks impact expression of Zeb1. Dr. Meenhard Herlyn spoke about melanoma cell plasticity and heterogeneity. He described that melanoma cells arise from melanocytes in the epidermis and likely also from stem cells in the dermis and hair follicle.36 He spoke about the importance of Notch signaling which is elevated in dermal neural crest like stem cells and in melanoma cells but not in differentiated melanocytes. Keratinocytes in contact with melanocytes can control melanocyte growth. Melanocytes that give rise to melanoma escape from this keratinocyte control through various switches such as loss of proteins important for intercellular interactions such as E-cadherin, dysregulation of the Wnt and Notch pathway, growth


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factor activation and dominant genetic hits in tumor suppressor genes and oncogenes.37 He mentioned that metastatic melanoma cells can mimic the functions of endothelial cells and fibroblasts to promote invasion and metastasis.38 He mentioned that fibroblasts share the molecular tools for migration and invasion with melanoma cells via ‘mesenchymal mimicry’. He further stated that melanoma cells share molecular markers with monocytes, B and T cells and referred to this as part of their plasticity. He described that there are various sub-groups of melanoma that may require targeting therapy. 45–50% melanoma tumors have mutations in BRAF, and these fall into 2 groups based on PTEN status. Tumor heterogeneity and resistance in response to BRAF inhibitor therapy in melanomas with the BRAFV600E mutation is a major problem in the field.39 There are several drugs that have been identified to target the MAPK, PI3K-Akt and receptor tyrosine kinase RTK pathways to overcome resistance but currently there is little rational strategy to combine them. There are limitations on use of drugs provided by companies under material transfer agreements and this can slow progress in the field. Dr. Herlyn also explained that currently the major limiting factor in improving patient survival is identifying the best rational combinations of these drugs since patients relapse within 6–12 months. Next he elaborated about the sub-populations within melanoma cells and why we should refrain from using the term melanoma stem cells. Every melanoma cell can act as a CSC capable of selfrenewal and tumorigenicity. Several recent studies have demonstrated that single melanoma cells from patients are tumorigenic and so therapeutic strategies need to focus on killing all the tumor cells. Dr. Herlyn described his recently published work on the slow-cycling JARID1BC subpopulation of melanoma cells that is enriched after therapy with cisplatin and vemurafenib. JARID1B is an H3K4 methylase that is turned on by hypoxia. Mitochondrial enzymes involved in oxidative phosphorylation were upregulated in the JARID1BC subpopulation and blocking mitochondrial respiration with phenformin eliminated JARID1BC cells.40 Dr. Herlyn mentioned that BRAF inhibitors or the combination of BRAF and MEK inhibitors can induce senescence in melanoma cells but it is not terminal. He suggested that using a mitochondrial HSP90 inhibitor may kill these cells and that such a strategy may be desirable. Dr. Charlotte Kuperwasser from Boston University spoke about pluripotency, cellular plasticity and stem cells, and the reprogramming events that contribute to breast tumor formation and heterogeneity. The more committed cells lose plasticity although it has been demonstrated that reprogramming iPS cells is possible and can lead to cellular transformation. Metaplastic breast cancer is a rare but deadly subtype of human breast cancer. Dr. Kuperwasser explained that it is the basal cells within the human breast that give rise to such metaplastic tumors. The same metaplastic basal cells from the breast can form skin tissues.41 Cellular plasticity within breast tissues also involves trans-differentiation of luminal progenitors into basal cells. TAZ/WWTR1 was identified as a critical regulator of lineage commitment of luminal and basal progenitors within breast epithelial cells. Overexpression of TAZ promoted luminal-to-basal lineage switching while knockdown of TAZ in basal cells promoted the luminal


phenotype. TAZ expression is restricted to basal myoepithelial cells in breast tissues. Mice with homozygous TAZ loss exhibited an imbalance in ratios of basal and luminal cells. TAZ lacks a DNA-binding domain but is capable of binding to other transcription factors. Mechanistic studies revealed that TAZ interacts with SWI/SNF complex members and recruits BRM ATPase to regulate gene expression, lineage commitment and plasticity. Analysis of data from the cancer genome atlas showed that WWTR1 gene is amplified in breast cancer, associated with a basal-like phenotype and poor prognosis of basal-like breast cancers.42 Finally, Dr. Kuperwasser showed that transcription factor Slug is required for breast stem cell activity and tumorigenesis.43 A Slug knockout mouse model revealed that Slug is involved in reprogramming of luminal and basal progenitors to bi-potent mammary stem cells. Thus, she showed that plasticity is required and influenced by the microenvironment along with gene mutations and epigenetics. Dr. Vivek M. Rangnekar from the University of Kentucky spoke about an approach utilizing p53 wild type normal cells to target cancer cells via stimulating secretagogues. The Par-4 tumor suppressor is secreted by both normal and cancer cells and selectively induces apoptosis in various types of cancer cells. Par-4 is mutated or inactivated in endometrial, renal and prostate cancer. Par4 binds to the cell surface receptor GRP78 via its SAC domain and is involved in TRAIL-mediated apoptosis.44 Dr. Rangnekar explained that p53 activation in normal cells with wild-type p53 can also induce apoptosis in p53-deficient cancer cells via a Par-4 mediated paracrine effect. Small molecule Nutlin-3a was used for p53 activation.45 NFkB signaling has been previously shown to block Par-4 mediated apoptosis.46 Nutlin3a was combined with the NFkB pathway inhibitor PS-1145 to further augment cell death. Nutlin-3a induced apoptosis in p53deficient cancer cells co-cultured with p53 wild type mouse embryonic fibroblasts (MEFs) but not p53 null cell MEFs. Conditioned medium from treated MEFs also induced apoptosis in p53-deficient cancer cells. Activation of p53 in normal mice with Nutlin-3a plus PS-1145 induced systemic elevation of Par-4 but not in p53 null or Par-4 null mice. Par4 is located on human chromosome 12q21 and is downregulated in diverse cancer subtypes. Regulation by Akt in prostate cancer sequesters Par4 in the cytoplasm and prevents secretion. Par4-null mice get spontaneous tumors and when crossed with PTEN-null mice they get aggressive prostate cancer. Dr. Rangnekar created Par-4 expressing transgenic mice that live longer than normal mice. p53 does not directly regulate Par-4, instead it is governed by an intermediate regulator UACA affected by both p53 and NFkB signaling. UACA, a binding partner of Par-4, sequesters it and prevents Par-4 secretion. UACA is suppressed by p53 but induced by NFkB. p53 causes direct repression of UACA via its consensus binding motif. The p53 dependent secretion of Par-4 occurs through the classical endoplasmic reticulum-golgi secretory pathway.45 Dr. Maria Nagai from the University of Sao Paolo spoke about experiments evaluating the prognostic value of Secreted Protein Acidiic in Rich in Cysteine (SPARC) in breast cancer. She generated SAGE libraries for transcriptomic profiling before


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and after docetaxel treatment. She further described the effects of SPARC expression on chemosensitivity to docataxel in MCF7 breast cancer cells. SPARC is an extracellular matrix protein that plays a role in several biological processes such as cell adhesion, migration and tissue remodeling, however its exact role in tumor progression remains largely unknown. Loss of SPARC expression correlated with poor prognosis in primary breast cancer.47 Transcriptome and proteome analysis with metacore pathway analysis were used to further elucidate the role of SPARC in the context of chemosensitivity. GTP metabolism was associated with effects of SPARC. Dr. Suresh Mohla from the National Cancer Institute conducted a special lunch session on NIH research funding updates. Dr. Mohla highlighted a special NIH workshop for newly funded investigators to guide them and help address the challenges for their first competing grant renewal. Dr. Mohla explained that NIH budget cuts have affected new investigator funding. Early stage investigators (within 7 y of PhD) have an 18% success rate on grant applications while new investigators have a 12% success rate. Considering these challenges the NIH has tried to maintain a fine balance between the number of RO1 and R21 grants available. For RO1 awards, funding is guaranteed for applications within the top 9 percentile, after that various factors determine whether the grant is awarded. Dr. Mohla said that the success rate for RO1 grants is not just 10% as is commonly assumed, it is actually 14.2% due to the funding provided for grants that score in the top 10–30 percentile. Interestingly, 19% of all RO1 applications are within the 19 percentile. PO1 funding has remained the same in 2012 and 2013. Considering the large amount of funding provided, the PO1 has the most stringent review among all funding mechanisms involving highly experienced evaluators and applicants. Dr. Mohla also spoke about the provocative question (PQ) grants for topics that address the most important research areas as decided by the research community. Unlike the RO1, the PQ grant should not be the main focus of the lab and no resubmissions are allowed. If the PQ application is not funded, it can be submitted for the RO1. For the NCI cleared concepts grants approved by the NCI board of scientific advisors, RFA’s are announced 3–4 months ahead of the submission deadline.

Session 3: Mechanisms of Cancer Therapy Resistance The session was chaired by Dr. Keith Flaherty from the Massachusetts General Hospital. Dr. Flaherty spoke about mechanisms of melanoma resistance in response to BRAF inhibitor therapy and lineage specific opportunities for the development of combination therapies. BRAFV600E mutation occurs in 52%, NRAS in 28%, and NF1 in 14% thereby defining mutually exclusive subsets among melanoma patients. TCGA analysis shows that among the BRAF subtype there is considerable heterogeneity with secondary events. Treatment with BRAFV600E inhibitors such as vemurafenib and dabrafenib resulted in disease progression within a year despite an initial dramatic reduction in

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tumor volume in melanoma patients. During tumor progression upon resistance, the status of ERK phosphorylation observed is variable. ERK either continues to remain inhibited or is reactivated but below or above pretreatment levels. Despite resistance, no new activating BRAF mutations have been observed. Various mechanisms of resistance have been identified in response to BRAF inhibitors such as COT overexpression, MEK/ERK activation, NRAS mutation, PI3K-Akt activation, PTEN loss and RAF dimerization. Several approaches are currently being explored to overcome BRAF inhibitor resistance. The MEK inhibitor trametinib received FDA approval for use in melanoma patients with BRAF mutations either alone or in combination with dabrafenib. Combination therapy improved progression free-survival and response rates as compared to single agent dabrafenib therapy. There was a 19% response rate with adding MEK inhibitor trametinib after progression on BRAF inhibitor therapy.48 Resistance has also been observed in patients treated with combination therapy by mechanisms such as activating MEK2 mutations.12 Oncogenic BRAF upregulates the MITF oncogene and BRAF inhibitor therapy causes MITF downregulation.48 MITF upregulation leads to elevated prosurvival Bcl2 family member BCL2A1 that promotes melanoma growth.49 MITF upregulation leads to enhanced melanoma antigen expression providing an opportunity for the use of immunotherapy in combination with BRAF inhibition. MITF upregulation in response to BRAF inhibition also has metabolic consequences. MITF upregulates PGC1a that leads to increased oxidative phosphorylation and mitochondrial energy metabolism promoting melanoma survival. Thus, inhibitors of oxidative phosphorylation could potentially help overcome resistance to BRAF inhibition.50,51 Mutation in PTEN or CDKN2A in addition to BRAF leads to reduced response rates to targeted therapy. Dr. Bruce Zetter from the Boston Children’s Hospital spoke about the role of Prohibitin1 (PHB1) in paclitaxel resistance. In order to identify differentially expressed proteins in taxol sensitive (A549, MES-SA) and resistant cell lines (A549TR, MES-SATR, always growing on drug, selected in vitro) a proteomicsbased approach was used. Proteomics and sequencing by mass spectrometry revealed that PHB1, Topo2A, MDR and GSTp were upregulated in the microsomal fraction of resistant cells. In most cells, upregulated PHB1 is mostly localized on the inner mitochondrial membrane where it acts as a chaperone. Thus no differences were seen in whole cell expression between taxol sensitive and resistant cells. PHB1 is observed more on the cell surface of taxol-resistant cells. High PHB1 levels correlate with poor patient survival in non-small cell lung cancer. PHB1 was expressed at equal levels in the mitochondria of sensitive and resistant cells. Using a fluorescently tagged Prohibitin-binding peptide it was determined that PHB1 is localized on the cell surface of resistant but not sensitive cells in vitro and in tumor xenografts. Silencing of PHB1 or GSTp improved taxol sensitivity of resistant cells in vitro and in vivo. PHB1 also mediates resistance to other taxanes such as docataxel. Interestingly, silencing PHB1 increased paclitaxel-mediated apoptosis in resistant cells but the same was not observed with GSTp silencing.52 Resistance to etoposide or doxorubicin was conferred by prohibitin but the same


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was not observed with camptothecin. Dr. Zetter mentioned that he is also interested in unraveling the interactome of prohibitin. Dr. Constantinos Koumenis from the University of Pennsylvania spoke about the role of the unfolded protein response (UPR) in cancer progression and therapy resistance. The unfolded protein response (UPR) occurs during endoplasmic reticulum (ER) stress upon the accumulation of unfolded proteins in the ER lumen. UPR activates the ER kinase PERK that in turn activates eIF2a through phosphorylation to overcome ER stress by blocking general protein translation. PERK-eIF2amediated cell survival during ER-stress is mediated by the transcription factor ATF4 which upregulates amino acid biosynthesis, transport and anti-oxidant genes. PERK can also activate transcription factor Nrf2 for anti-oxidant response and cell survival. Under conditions of prolonged ER stress, apoptosis can be activated via pro-apoptotic protein CHOP. Other than PERK, ER stress proteins such as IRE1 signal to XBP1 via mRNA splicing to activate the UPR. ATF6 is also involved in the UPR program through nuclear events. The UPR program is activated in hypoxic regions in human tumors and during oncogenic stress. The UPR is important for tumor cell survival under hypoxia and tumor growth in vitro and in vivo.53 Melanoma resistance to oncogenic BRAF-inhibition or combined BRAF and MEK inhibition involves cytoprotective therapy-induced autophagy. Autophagy mediated resistance to BRAF inhibition is mediated via ER-stress in a PERK-dependent manner.54 Thus targeting ER stress-induced autophagy can overcome BRAF resistance. Similarly, ATF4 is highly expressed in tumor cells as compared to normal cells and inhibition of ATF4 prevents tumor growth in vivo. ATF4 stable knockdown also prevented metastatic colonization to the lung of fibrosarcoma HT1080 cells. However, ATF4 did not affect cell migration or invasion. Anoikis is a form of apoptotic cell death that occurs in normal cells upon ECM detachment. ATF4 mediates resistance to anoikis in tumor cells through induction of autophagy. Detachment of HT1080 cells from the ECM under suspension culture activated the eIF2aATF4-mediated UPR program and knockdown of ATF4 in suspension cultures increased anoikis-induced cell death. Autophagy also mediates cell survival under non-adherent conditions. Interestingly, ATF4 knockdown prevents autophagy-mediated survival in suspension tumor cells. ATF4 promotes resistance to anoikis-mediated cell death via ATG5 and ULK1 mediated-cytoprotective autophagy. In addition, heme oxygenase1 (HMOX1) was identified as a key mediator of ATF4-dependent resistance to anoikis. HMOX1 contributed to tumor metastasis in vivo and correlated with poor patient survival. Thus, loss of matrix attachment in tumor cells activates the PERK-eIF2a-ATF4-mediated UPR program, maintaining cell survival and anoikis resistance. Dr. Edward Gunther from the Pennsylvania State College of Medicine spoke about the role of cooperating subclones in maintaining tumor heterogeneity within breast tumors. Peter C Nowell first described the clonal evolution model in cancer.55 Genetic hits do not accumulate in a single clone within the tumor, multiple subclones are known to be present within a single breast tumor.56 Traditionally, subclones are thought to battle it out for tumor dominance, however subclones can also cooperate to


maintain a tumor. In the mouse mammary tumor virus (MMTV)-Wnt1 transgenic mouse model with constitutive Wnt1 expression (cWnt), mixed lineage mammary tumors are observed that arise from bipotent mammary progenitors. Both basal and luminal cells are observed in these tumors that can be separated by flow cytometry sorting. Dr. Gunther showed data that mutations in HRAS are restricted to the basal clones in half of the tumors analyzed, while only luminal cancer cells expressed Wnt1. In a transgenic mammary tumor mouse model with doxycycline inducible Wnt1 expression (iWnt), tumors regressed in the absence of doxycycline. When tumor cells from the iWnt model were injected into the mammary fat pads of the cWnt model, iWnt initiated tumors grew even in the absence of doxycycline. This was due to the recruitment of host cWnt luminal cells as a replacement source of Wnt1. The transplanted tumors retained the biclonal configuration of parental tumors indicating a selective pressure to maintain the clones. Sorted basal or luminal subsets from biclonal iWnt tumors were unable to efficiently initiate tumors individually, but a mixture of the subsets gave rise to tumors. Dr. Gunther provided evidence that the model can also be used to study acquired resistance to cancer therapy. Upon doxycycline withdrawal iWnt tumors regress and then relapse by acquiring a doxycycline-independent phenotype. This occurs via 2 mechanisms, rtTA mutations that rescue tumor growth via doxycycline-independent expression of the transgene or mutations in the downstream Wnt effector b-catenin. Interestingly, the rtTA mutations potentially occur in HRAS wild-type luminal cells while b-catenin mutations probably originate in HRAS mutant basal subclones. Thus, tumor heterogeneity could arise via 2 potential mechanisms, hierarchical vs. clonal model, and these are indistinguishable by immunohistochemistry.57 However, the study raises several important questions: How does a biclonal tumor undergo metastasis? Is the biclonal metastatic tumor more or less likely to relapse after Wnt withdrawal? When do these cooperating subclones emerge during tumorigenesis? Does this phenomenon occur in primary human tumors? Does this phenomenon occur only when tumors are initiated by secreted signaling molecules?

Session 4: Personalized/Targeted Therapeutics The session was chaired by Dr. Alex Adjei from the Roswell Park Cancer Institute. Dr. Ryan Young from the National Institutes of Health spoke about the molecular pathogenesis and therapy of Diffuse large B cell lymphoma (DLBCL). There are 2 major subtypes of DLBCL namely activated B cell-like (ABC) and germinal center B cell-like, with a worse clinical outcome for ABC DLBCL. ABC DLBCL tumors rely on B cell receptor (BCR) signaling and constitutive NFkB activation for survival. Recent evidence suggests that BCR signaling mediates NFkB activation in ABC DLBCL. There are 2 forms of BCR signaling in lymphoma: tonic and chronic active. BCR signaling in ABC DLBCL involves chronic active rather than tonic signaling. CD79A and CD79B are critical mediators of BCR signaling linked to the BCR. Phosphorylation of the immunoreceptor


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tyrosine-based activation motifs (ITAMs) of CD79A and CD79B is an important step in BCR signaling. Mutations in the ITAM motif of CD79A and CD79B were frequently observed in ABC DLBCL and knockdown of CD79A and CD79B was toxic to ABC DLBCL cells. Bruton’s tyrosine kinase (BTK) is an important BCR signaling component that mediates NFkB activation and survival in ABC DLBCL. The BTK inhibitor ibrutinib was toxic to ABC DLBCL cells and targeted BCR signaling by covalently binding to the active site of BTK. In a Phase 2 study of ibrutinib in relapsed/refractory DLBCL, ibrutinib selectively benefitted ABC subtype patients. Upon ibrutinib treatment enrichment for the CD79A ITAM mutation was observed in ABC DLBCL patients. A CD79A deletion mutant increased BCR expression and decreased ibrutinib sensitivity. Constitutive oncogenic myeloid differentiation primary response protein 88 (MYD88) signaling is also observed in ABC DLBCL and regulates proximal BCR signaling. MYD88 signaling also activates NFkB via an alternative mechanism involving the interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4. Interestingly, patients with CD79B and MYD88 mutations responded to ibrutinib while patients with MYD88 mutation alone were resistant. The drug lenalidomide targeted MYD88 signaling via IRAK4 and synergized with ibrutinib for killing ABC DLBCL cells.58 In another study, a high throughput screen was performed to identify drugs for combination therapy with ibrutinib for ABC DLBCL therapy. Several potential combinations were identified with chemotherapeutic agents and drugs targeting the PI3K pathway.59 Dr. Mone Zaidi from the Mount Sinai School of Medicine spoke about the anti-cancer effects of bisphosphonates (BIS) for EGRF-driven cancers. BIS are used to treat osteoporosis and act on osteoclasts on the resorptive surface of the bone. BIS can directly kill cancer cells, prevent bone metastasis and improve survival of breast cancer patients in combination with conventional therapy.60 Dr. Zaidi mentioned that gene expression profiling was performed to understand the cellular response to bisphosphonates and a bisphosphonate gene signature was created. The connectivity map is a genome-wide collection of expression profiles that allows simultaneous analysis of drugs, diseases and genes to identify potential targets for novel drugs. Connectivity mapping showed that BIS act similar to cancer drugs such as PARP inhibitors and EGFR inhibitors. The expression profiling data was also analyzed using KEGG pathway analysis which revealed that cellular effects of BIS involved effects on the EGFR signaling pathway. Dr. Zaidi spoke about the anti-tumor effects of BIS in lung cancer. BIS selectively prevented cell survival of cancer cells dependent on EGFR signaling. Docking studies revealed that BIS directly bound to the ATP binding site in the kinase domain of EGFR and consequently inhibited EGFR kinase activity. Various BIS compounds were tested for their effects on lung cancer cells. Structure activity relationship studies revealed that only BIS compounds with a cyclical ring structure and at least one nitrogen atom (imidazole ring) exerted anti-cancer effects. The T790M EGFR mutation in lung cancer is known to confer resistance to EGFR inhibitors such as erlotinib. Dr. Zaidi highlighted that BIS can bind to both wild-type

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and mutant EGFR and can help target lung cancer cells resistant to erlotinib. Farnesyl diphosphate synthase (FPPS) inhibition is known to be important for the effects of BIS on osteoclasts. Interestingly, FPPS inhibition did not contribute to the EGFR-mediated anti-cancer effects of BIS. BIS were also observed to inhibit the survival of EGFR L858R mutant-expressing H3255 cancer cells. BIS compounds such as zoledronic acid also synergized with EGFR inhibitors like erlotinib for targeting EGFR-dependent lung cancer cells in vitro and in vivo. Thus H1975 with T790M and L858R EGFR mutations responded to zoledronic acid. Dr. Zaidi explained that the doses of BIS used in the study are physiologically relevant as the same doses are being used for osteoporosis treatment. Current work is focused on testing the effects of BIS on other EGFR driven tumor types such as breast and colon cancers. Jonathan Pachter from Verastem, Inc. spoke about targeting CSCs with small molecule inhibitors of FAK, PI3K/mTOR and Wnt. Tumor recurrence can be prevented via targeting of the CSC population to achieve a durable anti-tumor response. The occurrence of ALDH1C CSCs in lymph nodes after neoadjuvant chemotherapy predicts poor prognosis in breast cancer patients.61 FAK pathway is important for the tumor-initiating ability of CSCs and tumor progression including metastasis.62 He showed that FAK depletion reduces tumor-initiating ability. FAK inhibition using the compound VS-4718 preferentially reduced CSCs in multiple assays such as the Aldefluor assay, sphere assay and side population analysis. Oral administration of VS-4718 reduced CSCs in MDAMB231 triple-negative breast tumors. VS-4718 is being tested in Phase 1/1b clinical trials for various solid tumors. CSCs predict poor survival in ovarian cancer.63 FAK is an important target in ovarian cancer and elevated FAK expression predicts poor survival.64 Combination of another FAK inhibitor VS-6063 with paclitaxel reduced tumor-initiating capability in ovarian cancer. VS-6063 is currently being tested in a Phase 1/1b clinical trial for ovarian cancer in combination with paclitaxel. Dr. Pachter showed promising initial data from the clinical trial in which one of the patients had a complete response on the regimen and a good safety profile. CSCs mediate chemotherapy resistance in mesothelioma.65 Loss of merlin protein function is an important step in mesothelioma tumorigenesis. Mesothelioma cells lacking merlin were highly sensitive to VS-6063. FAK inhibition reduced AldefluorC CSCs and prevented enrichment of CSCs upon treatment with chemotherapeutic agent pemetrexed or cisplatin in vitro and in vivo. VS-6063 is currently being tested in a Phase 2 study for mesothelioma in which patients are stratified according to merlin status. Dr. Pachter also mentioned about a CSC-targeting PI3K and mTORC1/2 inhibitor VS5584 currently being tested in Phase 1/1b for solid tumors and lymphomas. VS-5584 has been found to inhibit CSCs in contrast to everolimus. Dr. Pachter mentioned an opportunity to use the inhibitor in small cell lung cancer after cisplatin plus etoposide treatment. FAK inhibitors as well as PI3K/mTOR inhibitors are potent agents as they potentially target both bulk tumor cells and CSCs. He suggested that time to new lesions may be a CSC related clinical end-point.


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Dr. Alex Adjei provided an update from on-going clinical trials for various promising agents currently in early development including Wnt inhibitors, SMAC mimetics, and immune checkpoint inhibitors. He mentioned that tumor sequencing has identified finite actionable genetic targets and cited that in 3284 tumors, 138 drivers were found including 64 oncogenes and 74 tumor suppressor genes. Some changes are rare such as ROS1. Dr. Adjei described the canonical, noncanonical and calcium Wnt pathways. Vantictumab (OMP-18R5; OncoMed pharmaceuticals) is an antibody that inhibits canonical Wnt signaling by targeting the frizzled receptor. Phase I study has been completed after preclinical activity of the monoclonal antibody was observed in various tumor types such as breast, colon, pancreatic, non-small cell lung cancer (NSCLC) and teratocarcinoma as a single agent and in combination with chemotherapy. Vantictumab downregulated Wnt pathway and CSC genes and upregulated differentiation genes in hair follicles and tumors of patients in a phase 1a study. There were no single agent responses although some stable disease was observed with neuroendocrine tumors. Currently Vantictumab is being combined with paclitaxel and platinum-based drugs in lung cancer. Next, Dr. Adjei spoke about the small molecule Smac mimetics including CUDC-427 (Curis), AT-406 (Ascenta), LCL-161 (Novartis) and TL32711 (Birinapant, TetraLogic pharmaceuticals) that target the inhibitor of apoptosis proteins (IAPs). Potent in vivo efficacy was observed with multiple tumor types in single agent studies as well as in combination with chemotherapy, TRAIL or TNF-a. However, differential response was observed in some cell lines and on-going work involves development of a predictive signature of response. In a Phase 1a single agent study, Birinapant was well tolerated in patients, showed favorable pharmacokinetic profile, suppressed cIAP1 and activated caspase 3 in peripheral blood mononuclear cells and tumors. There was a small 15% response rate due to stable disease in patients. In a phase 1b/2a combination study Birinapant showed clinical activity in a 5-arm study with chemotherapy such as carboplatin/paclitaxel, docetaxel, irinotecan and gemcitabine in relapsed/refractory patients. The liposomal doxorubicin and gemcitabine arms were dropped. Bell’s palsy was seen in 1 of 50 patients in the single agent study as a dose limiting toxicity that was mitigated using an altered dosing regimen. In the combination therapy trial, this DLT was seen with irinotecan and docetaxel combinations but not with the carboplatin/paclitaxel combination. Dr. Adjei mentioned a study of Birinapant plus irinotecan in colorectal cancer. Dr. Adjei also provided an update on Programmed cell death 1 (PD-1) and Programmed death-ligand 1 (PD-L1) inhibitors currently being tested in the clinic. PD-1 upregulation in the tumor microenvironment enables cancer cells to escape a T cell response. In phase 1 studies for melanoma and NSCLC, Nivolumab (anti-PD1 antibody; BristolMyers Squibb) showed impressive clinical activity with tumor regression and an acceptable safety profile. The anti-tumor response persisted for several weeks even after treatment was suspended. Nivolumab was also tested in colorectal cancer patients. Colitis, hepatitis, hypophysitis and thyroiditis were observed. Dr. Adjei mentioned a NSCLC patient with a response with T790M EGFR mutation who earlier took erlotinib plus an HDAC inhibitor, then premetrexate. This patient received a 10 mg/kg dose and the lung lesion was bigger at 2 months but at 4 months it was much smaller. Thus with


checkpoint blockade, patients may appear to have progressive disease early in the course of therapy by conventional criteria but then the tumors shrink subsequently. Dr. Adjei shared excitement that patients are continuing to respond to checkpoint blockade. PD-L1 ligand blockade prevents its binding to receptors such as PD-1 and inhibits cancer immune evasion. In a phase 1 study for monoclonal antibody MPDL3280A (Roche) that targets PD-L1 tumor regression and acceptable toxicity was observed with multiple tumor types in patients with advanced disease. PD-L1 expression appeared to predict clinical response.

Session 5: Identifying and Therapeutic Targeting of Driver Mutations The session was chaired by Dr. William Hahn from the Dana Farber Cancer Institute. Dr. Hahn spoke about uncovering new biology related to the KRAS pathway using functional genomics. Gain and loss of function approaches are commonly used to identify novel cancer drivers.66 A genetic screen for open reading frames that rescue KRAS mutant cancer cells from KRAS suppression identified YAP1. The results of the screen were validated in a mouse model of acquired KRAS resistance in which YAP1 was activated. YAP1 is known to function as a transcriptional co-activator downstream of the Hippo pathway. YAP transcriptional activity was required for the KRAS rescue activity. Expression profiling revealed that KRAS and YAP regulate EMT markers. A second mechanism of KRAS rescue mediated by FOS was also identified. Dr. Hahn found that YAP1 and FOS interacted at the promoters of EMT genes. Dr. Hahn described another study that identified TBK1 as a selective mediator of survival in oncogenic KRAS expressing cells in a RNA interference (RNAi) screen. TBK1 is an IkB kinase that mediates its effects by regulating NFkB signaling.67 The TBK1 inhibitor CYT387 impaired tumor growth in a KRAS-driven genetic mouse model of lung cancer. The anti-tumor effects of the TBK1 inhibitor were improved in combination with a MAPK pathway inhibitor.68 Thus, these studies identified 2 new aspects of TBK1 and YAP1 mediated signaling in the KRAS pathway. Next, Dr. Hahn explained that genome wide RNAi screens do not lead to identification of all genetic hits because of a lack of genetic saturation. A comprehensive approach would be to combine RNAi based screening with massively parallel exome sequencing, gene expression profiling and copy number analyses. Dr. Hahn described such a study that identified YAP1 as a critical mediator of tumorigenesis and survival in b-catenin driven cancer cells.69 RNAi screening has the disadvantage of off target effects on other mRNA’s and microRNAs. Recently the CRISPR-Cas9 system has been described as an alternative approach that is target specific and can be adapted for loss of function screening.70 Dr. Hahn showed data for CRISPR-Cas9 screening based validation of hits from a RNAi screen. A CRISPR pooled screen was performed in 6 Wnt-dependent colorectal cancer cell lines along with RNAi screening. The CRISPR screen validated 83 of 177 (46%) Wnt-dependent genes identified by the shRNA screen. Dr. Hahn further explained that ingenuity pathway analysis relies on existing biology and cannot help uncover


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new biology. His group worked on building signaling networks for Wnt-dependent genes based on high throughput expression profiling and identified that PIK3CA and CTNNB1 show a strong genetic interaction. His current work focuses on building similar networks of protein-protein interactions. Dr. Wafik El-Deiry spoke about mutant p53 as a therapeutic target for cancer therapy. The p53 tumor suppressor is one of the most commonly mutated genes in all of human cancer. p53 has both transcription-dependent and -independent mechanisms of action. Several mechanisms other than p53 mutations are involved in the inactivation of the p53 pathway such as mutations in ARF and Chk2 as well as MDM2 overexpression.71,72 p53 status is an important predictor of therapy response.73 The Li-Fraumeni syndrome involving germ-line mutations of the TP53 gene is a rare hereditary disorder and not many treatments are available to prevent tumors in this disease. Dr. El-Deiry mentioned about his work that led to the discovery of the p53 target gene p21/WAF1 involved in cell cycle arrest.74 However, the relative contribution of p53 mediated effects on apoptosis by various target genes is more complex and not completely understood. He described multiple strategies currently available to promote the tumor suppressor activity of p53 in tumors with p53 pathway defects. The MDM2-inhibiting Nutlins activate the p53 pathway without DNA damage while classical chemotherapy relies on DNA damage. However, these approaches are useful only for tumors with wild-type p53. Examples of mutant p53 restoring drugs include CP-31398 and PRIMA-1.71,72 PRIMA-1 was recently evaluated in a phase-1 study for hematological and prostate cancers.75 Dr. El-Deiry described a cell-based functional screening approach with a p53-responsive luciferase reporter.76 Using this approach several p53 pathway-restoring compounds have been identified and characterized such as a nongenotoxic derivative of ellipticine for targeting mutant p53 expressing tumor cells.77 He highlighted a recently published study about small molecule Prodigiosin that restores the p53 pathway through stimulation of p73 and disruption of the mutant p53:p73 complex.78 Finally, he mentioned about his work related to therapeutic targeting of CSCs using p53 pathway restoration. Using a single cell p53-regulated green fluorescent protein (EGFP)-reporter system in colon tumor cells expressing mutant p53 his group demonstrated p53 restoration by ellipticine in putative colon CSCs. Putative colon CSCs were depleted by ellipticine in combination with 5-fluorouracil.79 He also showed data that p53 pathway restoration via p73 activation can target CSCs. Restoring p53 pathway function in human cancer represents an important albeit challenging goal. The approach involving activation of p73 along with the targeting of mutant p53 may represent a general paradigm for p53 pathway restoration that can restore pathway function in tumors with a broad range of p53 mutations. The targeting of CSCs by p53 pathway restoring compounds provides an advantage over classical chemotherapy and overcomes a formidable drug resistance mechanism. Dr. Todd Waldman from the Georgetown Lombardi Cancer Center spoke about aneuploidy and the functional characterization of tumor suppressor STAG2 in cancer cells including glioblastoma where it is mutated. Tumor cells frequently harbor an

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abnormal number of chromosomes known as aneuploidy. The molecular mechanisms of aneuploidy in cancer cells are largely unknown. STAG2 is a member of the cohesin complex and is responsible for sister chromatid cohesion and chromosome segregation during DNA replication. STAG2 is located on the X chromosome and so requires only a single hit. Dr. Waldman’s group identified deletions and mutations of the STAG2 gene along with loss of STAG2 expression in diverse human tumor types using cell lines, primary tumors and xenografts. Loss of STAG2 was observed in tumor types such as glioblastoma, Ewing’s sarcoma, melanoma, colorectal cancer, lymphoma and medulloblastoma. To determine the role of STAG2 in aneuploidy associated with tumor cells, adeno-associated virus vectors were used to correct the mutant allele of STAG2 in glioblastoma cell lines. Similarly, a nonsense mutation was introduced into wild-type STAG2 HCT116 colorectal cancer cells. STAG2 mutation caused loss of sister chromatid cohesion and increased chromosome number while correction of the STAG2 mutation prevented these defects and reduced chromosomal instability.80 Dr. Waldman described another study that revealed STAG2 is one of the most commonly mutated bladder cancer genes. Bladder cancer is the 5th most common cancer and 90% cases are urothelial cancers. A large scale immunohistochemistry screen was performed with 2214 tumor samples of various tumor types. Loss of STAG2 expression was observed in 18% of urothelial carcinoma samples. STAG2 was robustly expressed in normal urothelium. Sanger sequencing revealed 25 STAG2 mutations that occurred in various stages of urothelial carcinoma. STAG2 mutation frequently correlated with p53 overexpression or mutation. Depletion of STAG2 resulted in aneuploidy while STAG2 reexpression did not affect cell proliferation. Interestingly, STAG2 mutant tumors and wild-type tumors had similar levels of copy number aberrations indicating that other mechanisms also contribute to aneuploidy. Also, loss of STAG2 expression had differential effects on clinical outcomes for non-muscle invasive versus invasive carcinoma.81 Thus, he found a paradoxical effect with fewer STAG2 mutations in the more invasive bladder cancers.

Session 6: Innovative Technologies for Translational Cancer Medicine The session was chaired by Dr. Wafik El-Deiry. Dr. Mark Sausen from Personal Genome Diagnostics spoke about the applications of circulating cell-free tumor DNA (ctDNA) analysis technology in cancer detection and therapy. The company has a proprietary technology platform that allows genome-wide and massively parallel next generation sequencing analysis of ctDNA. The combination of digital karyotyping and short (>50 bp) paired end libraries along with bioinformatics analysis allows millions of mapped reads per sample and genome-wide coverage with high resolution. Dr. Sausen described that their technology can detect both tumor-specific point mutations as well as structural genomic alterations such as amplifications, translocations, deletions and chromosome-arm loss and gain.82 ctDNA analysis allows a non-invasive liquid biopsy of tumors for early detection


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and monitoring as well as detection of therapy resistance mechanisms. In a recent study, ctDNA was detectable in approximately 50–75% of patients across various tumor types. ctDNA was detectable in patients with early stage localized tumors as well as advanced disease. Interestingly, ctDNA was also detectable in patients without circulating tumor cells indicating that ctDNA analysis is more sensitive than detection of circulating tumor cells. The number of patients with detectable ctDNA as well as levels of ctDNA increased with advanced cancer.83 ctDNA analysis was also used to study the molecular evolution of acquired resistance to anti-EGFR therapy. MET amplification was observed in the ctDNA of relapse patients. Interestingly, MET amplification was detectable at low levels prior to therapy.84 In another study chromosomal copy number changes and rearrangements were observed in ctDNA from colorectal and breast patients. Aneuploid ctDNA was detected, several chromosome arms were gained or lost, and amplifications in known driver genes were observed. These chromosomal aberrations were not detected in the plasma DNA of normal patients.82 Dr. Peng Li from the Pennsylvania State University spoke about the surface acoustic wave (SAW) based technology called acoustic tweezers for several applications such as manipulation of single particles and cells as well as flow cytometry. SAW technology has been widely used in cell phones since they provide the advantages of simplicity, small size and lower price. SAW technology is non-invasive, biocompatible and easy to generate. SAWs are generated when an electric field is applied to a piezoelectric substrate. When the SAW encounters a liquid, longitudinal waves are produced that induces flow in the fluid and any movement of particles or cells in the fluid. The resulting acoustic force on the cells can be controlled to move the particles along with the fluid stream. Next, Dr. Li spoke about SAW-based fluorescence-activated cell sorting (FACS) for biomedical research and clinical diagnostics. With conventional FACS it is very difficult to preserve cellular integrity post sorting due to the high shear of sheath fluid, high voltage and high pressure. The cell viability is reduced by 30–70% and gene expression is significantly altered post-sort with conventional FACS. Dr. Li explained that SAW-based FACS is highly biocompatible and allows more precise 3-dimensional particle focusing without the use of sheath fluid. In comparison to a commercial flow cytometer, a SAWbased flow cytometer is smaller in size, cheaper and yielded similar results when used to detect HeLa cells. The SAW-based FACS machine is amenable to multichannel cell sorting and provides high biosafety as no aerosol is generated. Acoustic tweezers can also be used for cell separation based on physical properties such as size, density and compressibility. Separation of HL-60 and MCF-7 tumor cells from white blood cells in whole blood was performed using acoustic separation. Dr. Li highlighted that acoustic separation could be a useful alternative for isolating circulating tumor cells in blood and demonstrated 90% enrichment of each tumor cell type relative to WBC. Optical tweezers allow cell manipulation but they are complicated expensive systems that can damage cells due to heat generation. SAW-based technology can be used for patterning of single cells such as red blood cells, E. coli cells and organisms such as C. elegans. By controlling


the acoustic field precise control of cell-cell distance can be achieved. By observing intercellular dye transfer via gap junctions it was determined that cells could be positioned at a distance of 3 micron without cell-cell interaction. No changes in cellular metabolic activity, viability, cell death or gene expression were observed with the use of SAW-based technology.85 Dr. Franziska Michor from Harvard University spoke about the evolutionary modeling of cancer for optimization of drug dosage schedules for maximal long-term anti-tumor effects. Tumorigenesis and progression is an evolutionary process involving natural selection of somatic cells based on genetic, genomic and epigenetic aberrations. Tumor genome evolution results in several hallmark phenotypes of cancer such as resistance to cell death, hyper-proliferation, invasion and metastasis.86 Dr. Michor explained that by delineating the evolutionary dynamics of tumor progression and acquired resistance it is possible to design optimal drug dosage schedules for improved anti-cancer therapy.87 She described her study on optimizing the dosing of tyrosine kinase inhibitors (TKI) for the treatment of EGFR-mutant nonsmall cell lung cancer. Evolutionary cancer modeling was applied to the growth kinetics of TKI erlotinib sensitive and resistant cells along with toxicity data from clinical trials of erlotinib. Instead of a traditional high dose pulsed schedule that requires toxicity recovery periods, a low dose continuous strategy combined with high dose pulses was proposed to delay resistance. The results from the mathematical modeling were validated in vitro and in vivo. Currently, such a dosage schedule is being tested in clinical trials at the Memorial Sloan Kettering Cancer Center.87,88 Resistance to radiation therapy is an important problem in glioblastoma and is potentially driven by a stem-cell like population within the tumor. Dr. Michor described another study in which an animal model for PDGF-driven proneural glioma was used to develop a mathematical model of glioma response to radiation. Stem like cells and non-stem like cells respond differently to radiation and conversion between these populations can occur in response to radiation stress. The mathematical model accounted for all dynamic cellular processes that could potentially occur in response to radiation such as differentiation, de-differentiation, cell death and quiescence. Based on the mathematical model an optimized radiation dosage regimen was developed and was tested in a mouse clinical trial. The optimal radiation dosing scheduled improved the survival of proneural glioma mice compared to the standard regimen. The optimal dosing regimen was further optimized based on the dynamic response of stem-like glioma cells to radiation measured from the initial trial.89 The approach is leading to non-intuitive optimized dosing schedules that are being tested in the clinic.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

Wafik S El-Deiry is an American Cancer Society Research Professor.


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Volume 16 Issue 3

Meeting report: Tenth International Conference on Hantaviruses.

4th International Symposium on Sensor Science (I3S2015): Conference Report.

27th International Mammalian Genome Conference meeting report.

Workshop report: 4th European crystal network meeting.

19th International Congress on Rural Health & 4th International Conference Ragusa SHWA 2015 Lodi, Italy.

Information Processing in Computer-Assisted Interventions: 4th International Conference, 2013.

Meeting report on membrane transport and metabolism 4th International Congress on Amino Acids Vienna, Austria, August 7-11, 1995.

Meeting report: 27th International conference on antiviral research, in Raleigh, NC, USA.

Fire raiser or fire accelerant? A meeting report on the 14th International TNF Conference 2013.

Conference Report: THE SIXTH INTERNATIONAL MEETING ON CHEMICAL SENSORS Gaithersburg, MD July 22-25, 1996.

Conference report meeting on the Danube: Report on the annual ESHHS conference Belgrade, 2011.

Meeting report: 3rd international transplant conference: how much risk can you take?

Meeting report of the 8th International Conference on cGMP "cGMP: generators, effectors, and therapeutic implications" at Bamberg, Germany, from June 23 to 25, 2017.

Report on the International Conference on Emergency Health Care Development.

International Conference on Advances in AIDS Vaccine Development. 4th Annual Meeting of the National Cooperative Vaccine Development Group for AIDS. Marco Island, Florida, October 15-19, 1991.

Consensus Report of the 2015 Weinman International Conference on Mesothelioma.

Report on Third International Intensive Aftercare Conference in Norrköping, Sweden.

8th Georg Rajka International Symposium on Atopic Dermatitis: meeting report.

Conference Report: Sixth International Congress of Gerontology.

Meeting report: the 4th symposium on animal models of non-human primates in Kunming, Yunnan, China.

The Seventh q-bio Conference: meeting report and preface.

20th International Chromosome Conference (ICCXX) : 50th Anniversary, University of Kent, Canterbury, 1st-4th September 2014.

Conference report: International Conference on Placenta--Tokyo, 1-3 October 1990.

Breakthrough in cardiac arrest: reports from the 4th Paris International Conference.