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Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow–derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here, we report that chemokine CCL9 was highly induced in Gr-1+CD11b+ immature myeloid cells and in premetastatic lung in tumor-bearing mice. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. Importantly, CCL9 overexpression in myeloid cells lacking TGFβ signaling rescued the tumor metastasis defect observed in mice with myeloid-specific Tgfbr2 deletion. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. In addition, targeting CCL9 may avoid the adverse effects of TGFβ-targeted therapy. Cancer Res; 75(24); 5283–98. ©2015 AACR.

Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers; however, the precise mechanistic basis for its role remains unknown. Here, we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biologic characteristics to full-length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism. Cancer Res; 72(4); 1001–12. ©2011 AACR.

Among the mechanisms implicated in the tumor-promoting effects of obesity, signaling by insulin-like growth factor-I (IGF-I) and insulin has received considerable attention. However, the emerging realization that obesity is associated with chronic inflammation has prompted other consideration of how the IGF-I axis may participate in cancer progression. In the present study, we used two mouse models of chronic (LID) and inducible (iLID) igf-1 gene deficiency in the liver to investigate the role of IGF-I in regulating the host microenvironment and colorectal carcinoma growth and metastasis in obese mice. Obese mice had a heightened inflammatory response in the liver, which was abolished in mice with chronic IGF-I deficiency (LID). In control animals changes to the hepatic microenvironment associated with obesity sustained the presence of tumor cells in the liver and increased the incidence of hepatic metastases after intrasplenic/portal inoculation of colon carcinoma cells. These changes did not occur in LID mice with chlonic IGF-1 deficiency. In contrast, these changes occured in iLID mice with acute IGF-1 deficiency, in the same manner as the control animals, revealing a fundamental difference in the nature of the requirement for IGF-1 on tumor growth and metastasis. In the setting of obesity, our findings imply that IGF-1 is critical to activate and sustain an inflammatory response in the liver that is needed for hepatic metastasis, not only through direct, paracrine effect on tumor cell growth, but also through indirect effects involving the tumor microenvironment. Cancer Res; 70(1); 57–67

Nm23-H1 transcriptionally down-regulates expression of the lysophosphatidic acid receptor EDG2 and this down-regulation is critical for Nm23-H1–mediated motility suppression in vitro. We investigated the effect of altered EDG2 expression on Nm23-H1–mediated metastasis suppression in vivo. Clonal MDA-MB-435–derived tumor cell lines transfected with Nm23-H1 together with either a vector control or EDG2 had similar anchorage-dependent and anchorage-independent growth rates in vitro. However, a 45- and 300-fold inhibition of motility and invasion (P < 0.0001), respectively, was observed in Nm23-H1/vector lines, whereas coexpression of EDG2 restored activity to levels observed in the parental line. Using fluorescently labeled cells and ex vivo microscopy, the capacity of these cells to adhere, arrest, extravasate, and survive in the murine lung over a 24-h time course was measured. Only 5% of Nm23-H1/vector–transfected cells were retained in the murine lung 6 h following tail vein injection; coexpression of EDG2 enhanced retention 8- to 13-fold (P < 0.01). In a spontaneous metastasis assay, the primary tumor size of Nm23-H1/vector and Nm23-H1/EDG2 clones was not significantly different. However, restoration of EDG2 expression augmented the incidence of pulmonary metastasis from 51.9% to 90.4% (P = 2.4 × 10−5), comparable with parental MDA-MB-435 cells. To determine the relevance of this model system to human breast cancer, a cohort of breast carcinomas was stained for Nm23-H1 and EDG2 and a statistically significant inverse correlation between these two proteins was revealed (r = −0.73; P = 0.004). The data indicate that Nm23-H1 down-regulation of EDG2 is functionally important to suppression of tumor metastasis. [Cancer Res 2007;67(24):11751–9]

Human tumor xenografts have been used extensively for rapid screening of the efficacy of anticancer drugs for the past 35 years. The selection of appropriate xenograft models for drug testing has been largely empirical and has not incorporated a similarity to the tumor type of origin at the molecular level. This study is the first comprehensive analysis of the transcriptome of a large set of pediatric xenografts, which are currently used for preclinical drug testing. Suitable models representing the tumor type of origin were identified. It was found that the characteristic expression patterns of the primary tumors were maintained in the corresponding xenografts for the majority of samples. Because a prerequisite for developing rationally designed drugs is that the target is expressed at the protein level, we developed tissue arrays from these xenografts and corroborated that high mRNA levels yielded high protein levels for two tested genes. The web database and availability of tissue arrays will allow for the rapid confirmation of the expression of potential targets at both the mRNA and the protein level for molecularly targeted agents. The database will facilitate the identification of tumor markers predictive of response to tested agents as well as the discovery of new molecular targets. [Cancer Res 2007;67(1):32–40]

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia. (Cancer Res 2006; 66(14): 7216-24)

It has been shown previously that the suppression of tumor immunosurveillance may be a mechanism by which tumors resist immune detection and elimination. In this study, we evaluated the role of the immunoregulatory natural killer T (NKT) cells in the biology of immunosurveillance of osteosarcoma. The K7M2 mouse osteosarcoma cell line was implanted orthotopically into wild-type and NKT cell–deficient CD1d knockout (KO) BALB/c mice, and mice were monitored for growth of primary tumors. Further, we examined the role of CD4+ and/or CD8+ cells by depleting the cells in vivo and measuring CTL activity in vitro. We also asked the role of interleukin (IL)-4 receptor α (IL-4Rα)-signal transducer and activator of transcription 6 (STAT6) signaling, including IL-13, and transforming growth factor β (TGF-β) by using gene-disrupted mice or treating mice with cytokine antagonists. We were surprised to find a high rate of rejection of osteosarcoma primary tumors in 88% (14 of 16) of CD1d KO mice compared with syngeneic wild-type BALB/c mice that showed rejection of tumor in

Osteosarcoma is the most frequent primary malignant tumor of bone with a high propensity for metastasis. We have previously showed that ezrin expression is necessary for metastatic behavior in a murine model of osteosarcoma (K7M2). In this study, we found that a mechanism of ezrin-related metastatic behavior is linked to an Akt-dependent mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (S6K1)/eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) pathway. Suppression of ezrin expression either by antisense transfection or by small interfering RNAs or disruption of ezrin function by transfection of a dominant-negative ezrin-T567A mutant led to decreased expression and decreased phosphorylation of both S6K1 and 4E-BP1. Proteosomal inhibition by MG132 reversed antisense-mediated decrease of S6K1 and 4E-BP1 protein expression, but failed to affect the effect of ezrin on phosphorylation of S6K1 and 4E-BP1. Blockade of the mTOR pathway with rapamycin or its analog, cell cycle inhibitor-779 led to significant inhibition of experimental lung metastasis in vivo. These results suggest that blocking the mTOR/S6K1/4E-BP1 pathway may be an appropriate target for strategies to reduce tumor cell metastasis.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in signature enhancer-histone marks between near-isogenic pairs of high and low lung-metastatic osteosarcoma cells. We term these regions Metastatic Variant Enhancer Loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster non-randomly, indicating that activity of these enhancers and their associated gene targets is positively selected. Osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors whose motifs are enriched in Met-VELs, and by knockdown of individual genes activated by Met-VELs. These observations have implications for the discovery and development of targeted anti-metastatic therapies. Citation Format: James J. Morrow, Tyler E. Miller, Alina Saiakhova, Michael M. Lizardo, Cynthia F. Bartels, Ian Bayles, Stevephen Hung, Arnulfo Mendoza, Jay T. Myers, Frederick Allen, Analisa DiFeo, Brian P. Rubin, Alex Y. Huang, Paul S. Meltzer, Lee J. Helman, Chand Khanna, Peter C. Scacheri. Positively selected enhancer elements endow tumor cells with metastatic competence. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-151.

Insulin-like growth factor I receptor (IGF-IR) and its ligands are overexpressed by tumors, mediating proliferation and protecting against stress-induced apoptosis. Accordingly, there has been a considerable amount of interest in developing therapeutic agents against IGF-IR. IGF-IR is believed to be ubiquitously expressed without detectable mutation or amplification in cancer. We explored the determinants of cellular response to a humanized anti–IGF-IR antibody. Our results showed a large variation in IGF-IR levels in rhabdomyosarcoma tumor specimens that were comparable with those in rhabdomyosarcoma cell lines. In vitro analysis revealed a direct and very significant correlation between elevated IGF-IR levels and antiproliferative effects of the antibody and defined a receptor number that would predict sensitivity. Our data further suggested a strong dependence on IGF-IR for AKT signaling in cells with elevated IGF-IR. The sensitivity of the high IGF-IR–expressing cells was blocked with a constitutively active AKT. The extracellular signal-regulated kinase pathway was not affected by the antibody. In vivo studies showed that anti–IGF-IR had single-agent antitumor activity; furthermore, predictions of responses based on IGF-IR levels were accurate. In vivo biomarker analysis suggested that h7C10 down-regulated both IGF-IR and p-AKT initially, concordant with antitumor activity. Subsequent progression of tumors was associated with reactivation of p-AKT despite sustained suppression of IGF-IR. These results identified the first predictive biomarker for anti–IGF-IR therapies in cancer. [Cancer Res 2008;68(19):8039–48]

Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin β1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin β1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin β1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch. [Cancer Res 2008;68(15):6241–50]

The role of Gemin5 in alternative mRNA splicing, tumor cell motility, and proteomic instability was investigated. Isotope Capture Affinity Tag proteomic analysis was conducted on MDA-MB-435 tumor cells transfected with either a control vector (C-100) or the Nm23-H1 metastasis suppressor (H1-177). Ingenuity pathway analysis revealed that RNA posttranscriptional processing was the most prominent class of differentially expressed proteins. Within this category, overexpression of Acinus1, Poly(a) binding protein, HNRPA2B1, Bop1, and Gemin5 was confirmed in less metastatic H1-177 cells. Overexpression of the latter four proteins was also observed in the lower metastatic antisense Ezrin transfectant of a murine osteosarcoma model system, confirming the general relevance of the trends. Gemin5, a component of the spliceosomal complex, was chosen for further study. Analysis of global mRNA splicing by SpliceArray chips revealed that 16 genes were differentially spliced in C-100 compared with H1-177 cells; transient transfection of gemin5 into C-100 cells restored the splice pattern to that of H1-177 cells. Alternative splicing patterns for the engulfment and cell motility 1 and thrombospondin 4 genes were confirmed by semiquantitative reverse transcription-PCR. Gemin5 overexpression coordinately reduced C-100 cell motility by 50%, and siRNA-mediated reduction of Gemin5 expression increased the motility of H1-177 cells by 2-fold (P < 0.004). The data provide the first demonstration that alterations in the expression of a spliceosome protein can effect both specific splicing events and tumor cell motility. The data also show that changes in mRNA splicing patterns accompany metastatic progression, which may contribute to proteome instability. [Cancer Res 2008;68(3):639–44]

Tumor cell survival in the hostile distant organ is a rate-limiting step in cancer metastasis. Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor-promoting microenvironment. However, it is unclear whether these myeloid cells in the premetastatic site have any direct effect on tumor cell survival. Here we report that chemokine CCL9 was highly induced in Gr-1+CD11b+ immature myeloid cells and in premetastatic lung of mice bearing 4T1 mammary tumor and B16 melanoma. Knockdown of CCL9 in myeloid cells decreased tumor cell survival and metastasis. The expression level of CCL23, the human orthologue for CCL9, in peripheral blood mononuclear cells correlated with progression and survival of cancer patients. Our studies identify CCL9 as a key pro-survival factor for cancer cells in the premetastatic organ. Interestingly, the expression of CCL9 in myeloid cells is regulated by TGF-β signaling as overexpression of CCL9 in myeloid cells lacking TGF-β signaling rescued the tumor metastasis defect observed in mice with myeloid-specific deletion of TGF-β signaling. These data together with our previous report of a fundamental role of myeloid TGF-β signaling in host compromised immune surveillance (Cancer Discovery 2013); demonstrate that myeloid-specific TGF-β signaling constitutes an essential component of the metastasis-promoting puzzle of TGF-β. This finding is different from TGF-β signaling in fibroblasts and a variety of epithelial cells, as well as T cells in which the TGF-β signaling functions as a tumor suppressor. This new understanding of TGF-β at the interface of tumor and host may provide insight into how TGF-β switches from a tumor suppressor to a tumor promoter, a long-standing challenge in cancer biology field. Our study demonstrates that CCL9 could serve as a good candidate for anti-metastasis treatment by targeting the rate-limiting step of cancer cell survival. Additionally, targeting CCL9 may avoid the adverse effects of TGF-β-targeted therapy. Citation Format: Hannah HY Yan, Yanli Pang, Kent Hunter, Chand Khanna, Li Yang. CCL9 induction in myeloid cells of the premetastatic niche enhances tumor cell survival and metastatic colonization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3172. doi:10.1158/1538-7445.AM2015-3172

Introduction: LMP744 (NSC706744) is one of 3 indenoisoquinolines being evaluated in pet dogs with lymphoma to define their safety, pharmacokinetics, and pharmacodynamic modulation (COTC007b). LMP744 forms stable DNA-topoisomerase I (Top1) cleavage complexes and induces unique DNA cleavage sites relative to approved Top1 poisons; LMP744 is not a substrate for ABC transporters and does not have the stability issues inherent to camptothecins. Here we report the plasma and tumor pharmacokinetics of LMP744 administered IV daily x 5 to patient dogs. Methods: Eligibility included: dogs >15 kg with histologically confirmed non-Hodgkin's lymphoma with nodal presentation (stage 2 or greater) and minimal nodes size for biopsy of 3 cm in the longest dimension, performance status of Grade 0 or 1 and informed owner consent. Dose levels (DL) explored were 25, 50, 75, 100, and 125 mg/m2/day through a 3+3 dose escalation study design. LMP744 was administered over 1 h IV daily x 5, Q28D. LMP744 was quantitated with a validated LC-MS/MS assay, and plasma pharmacokinetic parameters determined non-compartmentally with PK Solutions and plasma and tumor parameters compartmentally with ADAPT5 through iterated two stage (ITS) and maximum likelihood solution with the expectation maximization algorithm (MLEM). Results: All 18 dogs had useable pharmacokinetic data. Non-compartmental analysis suggested linear relationships between plasma Cmax and tumor C2h vs. dose. LMP744 distributed extensively to tumor tissue and mean tumor concentrations were at least 2 orders of magnitude higher than plasma concentrations at equivalent time points of 2, 6 and 120 h. Compartmental modeling with a 3 compartment linear model resulted in a good fit to the plasma data. The parameters and%CVs respectively for Clt, Vc, Cld1, Vp1, Cld2, Vp2 were 57.5 L/h/m2 (29%), 136 L/m2 (24%), 234 L/h/m2 (24%), 1180 L/m2 (27%), 30.8 L/h/m2 (63%), 5140 L/m2 (67%). Sequential plasma half-lives were 16.5 min, 13 h, and 183 (31%) h, respectively. In line with the long t1/2, plasma accumulation was observed on this daily times-5 schedule. Conclusion: The plasma pharmacokinetics of LMP744 in dogs display triphasic behavior, a relatively high total body clearance at 57.5 L/h/m2, and a terminal half-life of 183 h. The favorable biodistribution to tumor may be a valuable distinction to consider in the translation of this novel indenoisoquinoline to the clinic. Support: N01-CM-2011-00015, N01-CM-42202 and P30-CA-47904 Note: This abstract was not presented at the meeting. Citation Format: Julie L. Eiseman, Julianne Holleran, David L. McCormick, Miguel Muzzio, Joseph M. Covey, Chand Khanna, Christina Mazcko, Yves Pommier, Melissa Paolini, Amy Leblanc, Jenna H. Burton, James H. Doroshow, Joseph E. Tomaszewski, Jan H. Beumer. Plasma and tumor pharmacokinetics of IV LMP744, a novel indenoisoquinoline topoisomerase I inhibitor, in a canine phase I study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2015-4517

Osteosarcoma, the most common primary bone malignancy, has a bimodal age distribution, with a primary peak in adolescence and a smaller peak in the elderly. The etiologic contribution of germline genetic variation to osteosarcoma is not well-understood. It occurs at higher than expected frequencies in individuals with the Li-Fraumeni syndrome (LFS) cancer predisposition syndrome. Approximately 70% of classic LFS families have germline TP53 mutations. Two previous studies reported that 3% of young osteosarcoma cases ( We determined the prevalence of germline TP53 mutations in 765 unselected osteosarcoma cases. DNA was extracted from blood and TP53 sequenced using custom Ampliseq panels. Variants were validated with Sanger sequencing. The IARC germline TP53 database was used to identify TP53 mutations reported in families with LFS (LFS-associated mutations). Variants were considered “likely LFS-associated mutations” if absent from publically available databases (ESP and 1,000 Genomes Project) and predicted non-functional or deleterious using in silico algorithms. Variants were considered “rare exonic variants” if their minor allele frequency (MAF) was There were 32 LFS-associated or rare TP53 variants in 62 osteosarcoma cases. The frequency of cases with an LFS or likely LFS-associated mutation and/or rare exonic variant was 8.1%. Notably, all 32 TP53 variants were present in cases A logistic regression case-case analysis identified a novel significant association between a rare TP53 variant, rs1800372 (p.R213R), and metastasis at diagnosis in cases of European ancestry (odds ratio [OR] 4.27, 95% CI 1.2-15.5, P = 0.026). We additionally confirmed that a common exonic variant, rs1042522 (p.P72R), was significantly associated with osteosarcoma risk (OR 1.22, 95% CI 1.1-1.4, P = 0.0098) and poorer survival (HR 1.35, 95% CI 1.00-1.83, P = 0.048). Our data suggest that genetic susceptibility to young onset osteosarcoma is distinct from adult onset osteosarcoma. The high TP53 mutation prevalence we identified in osteosarcoma cases aged Citation Format: Lisa J. Mirabello, Meredith Yeager, Phuong L. Mai, Julie Gastier-Foster, Richard Gorlick, Chand Khanna, Ana Patiño-Garcia, Luis Sierrasesúmaga, Fernando Lecanda, Irene L. Andrulis, Jay S. Wunder, Nalan Gokgoz, Donald A. Barkauskas, Xijun Zhang, Aurelie Vogt, Kristine Jones, Joseph F. Boland, Stephen J. Chanock, Sharon A. Savage. High prevalence of germline TP53 mutations in young osteosarcoma cases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5574. doi:10.1158/1538-7445.AM2015-5574

The antitumor efficacy of the synthetic benzamide derivative MS-27-275 (MS-275), an inhibitor of histone deacetylation [T. Suzuki et al., J. Med. Chem., 42: 3001-3003, 1999], was evaluated in a series of pediatric solid tumor cell lines, including neuroblastoma, rhabdomyosarcoma, Ewing's sarcoma (EWS), retinoblastoma, medulloblastoma, undifferentiated sarcoma (US), osteosarcoma, and malignant rhabdoid tumors. Treatment with MS-275 results in an increase in acetylation of histones within 4 h of drug exposure. The cell lines were treated with various concentrations of MS-275 for 3 days and incubated with [(3)H]thymidine for 20 h before cell harvest. MS-275 inhibited [(3)H]thymidine uptake in a dose-dependent manner in all tumor cell lines examined. The IC(50) ranged from 50 nm in the D283 medulloblastoma cell line to 1.3 micro M in the US. A common feature of MS-275 treatment of pediatric tumor cell lines was induction of p21mRNA. However, the effects on cell cycle were diverse because in some cases MS-275 induced an increase in G(1) or G(2), whereas in others, there was an induction of apoptosis. In EWS, the EWS/fli chimeric transcription factor created by the t(11;22) suppresses transforming growth factor (TGF) betaRII transcription, however, MS-275 was able to induce an increase in TGF-betaRII mRNA and restore TGF-beta signaling. Using xenograft orthotopic models of US, EWS, and neuroblastoma, we find that the growth of established tumors is inhibited in mice treated with MS-275.

Despite remarkable therapeutic advances in malignant melanoma, an incomplete understanding of melanoma's extraordinary genetic diversity, complex disease course and ability to resist treatment continues to impede rational, curative clinical intervention. This is particularly true for the understudied “dark matter” of the melanoma genome, i.e. cases that lack mutations in the three most commonly-mutated melanoma genes (BRAF, NRAS, NF1) and that display extensive mutational heterogeneity. Clearly a pressing need exists for transformative approaches to melanoma medicine. We have therefore broadened our traditional focus on melanoma in human patients to include canine melanoma as a model of human disease. Given canine melanoma's clinicopathological resemblance to human non-cutaneous triple-negative subtypes, study of this disease in each species may inform the other. Further, the accelerated clinical course of canine melanoma and flexibility in veterinary clinical trial design may allow for evaluation of novel treatment designs that would not be possible in human cancer trials. Thus, we have undertaken a first-in-dog genomic characterization study of canine melanomas from a cohort of 37 dogs to further explore the landscape of this comparative model. This analysis combines comprehensive genomics (whole genome, whole genome long-insert, RNA sequencing, and array comparative genomic hybridization) in 7 matched canine melanoma/normal tissues with targeted analysis in an additional 27 tumors and 3 cell lines. These canine tumors display striking patterns of mutation and subtype heterogeneity resembling that of their non-cutaneous human counterparts with the majority being BRAF, NRAS, and NF1 wild type and lacking recurrent single nucleotide variants. Despite heterogeneity at the single nucleotide level, shared patterns of structural variation and gene expression exist and these patterns suggest core pathways that may define therapeutic vulnerabilities in these disease subtypes. These data provide the genomic framework for further clinical pursuit of melanoma in the canine model as a paradigm for drug development. Citation Format: William P. Hendricks, Victoria Zismann, Berry Button, Christophe Legendre, Waibhav Tembe, Holly Yin, Michael Bittner, Donald Berry, Matthew Huentelman, Chand Khanna, Patricia LoRusso, Matthew Breen, Aleksandar Sekulic, Jeffrey Trent. The genomic landscape of canine melanoma reveals broad mutational heterogeneity and recurrent patterns of structural variation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3417. doi:10.1158/1538-7445.AM2014-3417

Recurrent metastatic disease many years after initial therapy is a major cause of breast cancer (BC) mortality and strongly suggests that disseminated cells survive long periods in a growth-arrested state that is refractory to chemotherapy, known as “cellular dormancy”. Alterations in the components of the extracellular matrix (ECM) are associated with increased metastasis and poor prognosis for BC patients. This suggests that ECM alterations may have a role in influencing the behavior of dormant BC cells. We have previously shown both in vitro (using 3-dimensional Col-1-supplemented Matrigel culture) and in vivo (preclinical model of fibrosis at the lung metastatic site) that alterations in the ECM surrounding disseminated, dormant BC cells can induce signaling pathways requiring Src and ERK1/2 activation that switch dormant D2.0R cells into a proliferative state. We now demonstrate, using the Src inhibitor AZD0530 or Src shRNA, that the proliferative outbreak of dormant BC cells in Col-1-enriched microenvironments is prevented through the induction of a reversible G1-phase arrest and the upregulation of nuclear cyclin dependent kinase inhibitor p27, without affecting the viability of the dormant cells. Src inhibition, however, is not efficacious in regressing already established metastatic lesions, suggesting that Src signaling is critical to engage the dormant-to-proliferative switch but is not necessary for survival once the cells have begun to proliferate, which potentially explains its modest success in BC patients with advanced metastatic disease. Since ERK1/2 activation is also required for the dormant-to-proliferative switch, we determined the effect of the MEK1/2 inhibitor AZD6244 that prevents ERK1/2 activation on the proliferative switch alone and in combination with the Src inhibitor. Whereas AZD6244 is capable of reducing the proliferation of outbreaking cells, combination treatment with AZD0530 and AZD6244 of dormant cells undergoing the dormant-to-proliferative switch induces apoptotic cell death in a significant fraction of the dormant cells, which is not seen with either inhibitor alone, both in vitro and in vivo, and significantly delays metastatic outgrowth in the lung metastatic site. Induction of apoptotic cell death is also observed in a number of human and mouse cancer cell lines treated with AZD0530+AZD6244 but not with either inhibitor alone as they were induced to proliferate in 3-dimensional matrices, suggesting the potential applicability of this combination beyond BC. In conclusion, inhibition of Src alone prevents the proliferative response of dormant cells to external stimuli without affecting survival, but the addition of a MEK1/2 inhibitor suppresses survival of dormant cells, indicating that this combination may have clinical value in preventing breast cancer recurrence. Citation Format: Lara H. El Touny, Anthony Vieira, Arnulfo Mendoza, Chand Khanna, Mark J. Hoenerhoff, Jeffrey E. Green. Identification of a novel combination therapy that prevents the metastatic outgrowth and reduces the viability of dormant breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4028. doi:10.1158/1538-7445.AM2014-4028

The tumor suppressor p53 plays important roles in bone density regulation and osteosarcoma suppression. However, the relationship between p53-mediated bone density regulation and osteosarcoma suppression is unclear mainly because the underlying molecular and cellular mechanisms are elusive. Here, we reported that p53 controls differentiation of mouse mesenchymal stem cells (mMSCs) in a cell cycle and apoptosis independent manner. The loss of p53 predisposes mMSCs towards osteogenic lineage but not adipogenic and chondrogenic lineages. This pro-osteogenic effect caused by p53 loss is associated with the up-regulation of Runt related transcription factor 2, a factor required for the osteogenic differentiation of mMSCs. Without extrinstic stress, studies show that p53 does not directly repress the transcription of Runt related transcription factor 2. Instead, it indirectly represses Runt related transcription factor 2 through activating microRNAs (miRs), which suppress Runt related transcription factor 2 at a post-transcriptional level. In osteosarcoma cells, the loss of p53 correlates with high levels of Runt related transcription factor 2 and the oncogene addiction of these cells to Runt related transcription factor 2. Together, our results discover a p53 and miR-mediated pathway regulating the differentiation of MSCs and this pathway is dysregulated in osteosarcoma cells. This lineage controlling role links the role of p53 in the osteogenic differentiation of MSC to its suppressive functions in osteosarcoma. Citation Format: Yunlong He, Shunlin Jiang, Wendy Dubois, Ling Ren, Chand Khanna, Jing Huang. A p53 and microRNA-mediated pathway regulates mesenchymal stem cell differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2468. doi:10.1158/1538-7445.AM2014-2468

Introduction: LMP400 is one of 3 indenoisoquinolines that are being evaluated in pet dogs with lymphoma to define their safety, pharmacokinetics, and pharmacodynamic modulation (COTC007b). LMP400 forms stable DNA-topoisomerase I (Top1) cleavable complexes and induces unique DNA cleavage sites relative to approved Top1 poisons; LMP400 is not a substrate for ABC transporters and does not have the stability issues that most camptothecin analogs do. Here we report the plasma and uncoupled tumor pharmacokinetics of LMP400 administered IV daily x 5 to patient dogs. Methods: Eligibility included: dogs >15 kg with histologically confirmed non-Hodgkin's lymphoma with nodal presentation (stage 2 or greater) and minimal nodes size for biopsy of 3 cm in the longest dimension, performance status of Grade 0 or 1 and informed owner consent. Dose levels (DL) explored were 8, 16, 24, and 40 mg/m2/day through a 3+3 dose escalation study design. LMP400 was administered over 1 h IV daily x 5, Q28D. LMP400 was quantitated with a validated LC-MS/MS assay (PMID: 20236781), and plasma pharmacokinetic parameters determined non-compartmentally with PK Solutions and plasma and tumor parameters compartmentally with ADAPT5 through iterated two stage (ITS) and maximum likelihood solution with the expectation maximization algorithm (MLEM). Results: All fifteen dogs had useable pharmacokinetic data. Non-compartmental analysis suggested linear relationships between plasma Cmax and AUC vs. dose and tumor C2h vs. dose. Plasma terminal half-life was 11.9 ± 5.0 h and the CL was 26.7 L/h/m2. In line with the t1/2, slight plasma accumulation was observed on this daily times 5 schedule. Compartmental modeling with a 2 compartment linear model resulted in a good fit to the data. The parameters and %CVs respectively for Ke, Vc, Kcp, Kpc, Kct and Ktc were 0.181 h-1 (42.6%), 108 L/m2 (32.7%), 0.250 h-1 (30.1%), 0.0602 h-1 (67%), 0.0632 h-1 (3.65%), and 0.649 h-1 (42.2%). Plasma compartmental CL and half-life were 19.5 L/h/m2 and 29.7 h. Conclusion: The plasma pharmacokinetics of LMP400 in dogs display biphasic behavior, a relatively high total body clearance at 19.5 L/h/m2, and a terminal half-life of 29.7 h. LMP400 distributed to the tumors and mean tumor concentrations were at least 10-fold higher than plasma concentrations at equivalent time points of 2, 6 and 120 h. This favorable biodistribution to tumor (lymph node) may be a valuable distinction to consider in the translation of this novel indenoisoquinoline to the clinic. Support: N01-CM-2011-00015, N01-CM-42202 and P30-CA-47904 Citation Format: Julie L. Eiseman, Julianne Holleran, David L. McCormick, Miguel Muzzio, Joseph M. Covey, Chand Khanna, Christina Mazcko, Yves Pommier, Melissa Paoloni, James D. Doroshow, Joseph E. Tomaszewski, Jan H. Beumer. Plasma and tumor pharmacokinetics of IV LMP400, a novel indenoisoquinoline topoisomerase I inhibitor, in a canine phase I study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4632. doi:10.1158/1538-7445.AM2014-4632

Recurrent metastatic disease many years after initial therapy is a major cause of breast cancer (BC) mortality and strongly suggests that disseminated cells survive long periods in a growth-arrested state that is refractory to chemotherapy, known as “cellular dormancy.” Alterations in the components of the extracellular matrix (ECM), such as fibrotic foci and collagen-1 (Col-1) deposition, are associated with more aggressive breast cancer, increased metastasis and poor prognosis for BC patients. This suggests that ECM alterations may have a role in influencing the behavior of dormant BC cells. In fact, we have previously shown both in vitro (using 3-dimensional culture) and in vivo (preclinical model of fibrosis at the lung metastatic site) that alterations in the ECM surrounding disseminated, dormant BC cells can induce signaling pathways requiring Src and ERK1/2 activation that switch dormant cells into a proliferative state. We now demonstrate using the Src inhibitor AD0530 or Src shRNA, that the proliferative outbreak of dormant BC cells in Col-1-enriched microenvironments is prevented through the induction of a reversible G1-phase arrest and the upregulation of nuclear cyclin dependent kinase inhibitor p27Kip1, without affecting the viability of the dormant cells. Src inhibition, however, is not efficacious in regressing already established metastatic lesions, suggesting that Src signaling is crucial to engage the dormant-to-proliferative switch but is not necessary for survival once the cells have begun to proliferate. Since ERK1/2 activation is also required for the dormant-to-proliferative switch, we determined the effect of the MEK1/2 inhibitor AZD6244 that prevents ERK1/2 activation on the proliferative switch alone and in combination with the Src inhibitor. Importantly, we show that combination treatment with AZD0530 and the MEK1/2 inhibitor AZD6244 of dormant cells undergoing the dormant-to-proliferative switch induces apoptotic cell death in a significant fraction of the dormant cells, which is not seen with either inhibitor alone, and significantly delays metastatic outgrowth in the lung metastatic site. In conclusion, inhibition of Src alone prevents the proliferative response of dormant cells to external stimuli without affecting survival, but the addition of a MEK1/2 inhibitor suppresses survival of dormant cells, indicating that this combination may have clinical value in preventing breast cancer recurrence. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-18.

Osteosarcoma (OS), the most common primary malignant bone tumor, has a peak incidence during the pubertal growth spurt. OS occurs at increased frequency in several inherited cancer syndromes (e.g., Li-Fraumeni and Rothmund Thomson syndromes). A limited number of common SNPs associated with OS have been identified in biologically plausible pathways (e.g., growth or DNA repair). We developed an international, multi-institutional study in order to conduct the first genome-wide association study (GWAS) of OS. Participating subjects provided informed consent through local IRBs. Control subjects were cancer free adults derived from large studies in prior GWAS at the NCI. Genotyping of all cases was conducted using the Illumina OmniExpress SNP microarray chip platform in 2 phases. The first phase analyzed 910 osteosarcoma cases. After quality control filtering and assessment of population substructure, 596 cases of European (EUR) ancestry were advanced to the primary GWAS analysis (Phase 1A). We combined data from 2,703 previously genotyped controls drawn from 3 EUR cohorts. There was no evidence for significant population substructure differences between OS cases and controls. The association analysis (1-degree of freedom trend test) was adjusted for study, sex and 4 eigenvectors. The top 30 SNPs (P The fixed-effects meta-analysis of all 941 OS cases and 3,291 controls identified 2 regions that achieved genome-wide significance. The first, rs1906953 on 6p21.3, is associated with susceptibility to OS (P = 8.1x10-9, odds ratio [OR] 1.57, 95% confidence interval [CI] 1.35-1.83). rs1906953 is in intron 7 of GRM4. GRM4 is expressed in osteoblasts and osteoclasts and involved in glutamate signaling, which is suggested to be involved in cell differentiation and regulation of bone formation. The second signal resides in an intergenic region on chromosome 2p25.2; rs7591996 (P = 1.0 x 10-8, OR 0.72, 95% CI 0.65-0.81). Another SNP in this region, rs10208273, is moderately correlated with the first signal (r2= 0.32) and approached genome-wide significance (P = 2.9 x 10-7, OR 1.35, 95% CI 1.21-1.52). A third locus in the ADAMTS6 gene on 5q12.3 is promising but does not yet achieve genome-wide significance: rs17206779 (P = 5.1x10-7, OR 0.75, 95% CI 0.68-0.84). Notably, variants in genes encoding members of the ADAMTS protein family have been associated with height, a known OS risk factor. We have conducted the first GWAS of OS and identified several novel loci associated with OS risk. Further investigation of these loci will advance understanding of OS etiology and biology. Citation Format: Sharon A. Savage, Lisa Mirabello, Zhaoming Wang, Julie Gastier-Foster, Richard Gorlick, Chand Khanna, Adrienne M. Flanagan, Roberto Tirabosco, Irene L. Andrulis, Jay Wunder, Nalan Gokgoz, Ana Patino-Garcia, Luis Sierrasesumaga, Fernando Lecanda, Nilgun Kurucu, Inci Ergurhan Ilhan, Neriman Sari, Massimo Serra, Claudia Hattinger, Piero Picci, Logan Spector, Donald A. Barkauskas, Neyssa Marina, Siliva Caminada de Toledo, Sergio Petrilli, Maria Fernanda Amary, Dina Halai, David Thomas, Chester Douglass, Paul Meltzer, Kevin Jacobs, Charles C. Chung, Sonja I. Berndt, Mark P. Purdue, Neil E. Caporaso, Margaret Tucker, Nathaniel Rothman, Maria Teresa Landi, Debra T. Silverman, Peter Kraft, David J. Hunter, Nuria Malats, Manolis Kogevinas, Sholom Wacholder, Rebecca Troisi, Lee Helman, Joseph F. Fraumeni, Jr., Meredith Yeager, Robert Hoover, Stephen J. Chanock. Genome-wide association study identifies novel loci associated with osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4593. doi:10.1158/1538-7445.AM2013-4593

Metastasis continues to be the most significant problem in the field of cancer. The biological complexity that characterizes metastasis requires relevant experimental systems for its study, necessitating the use of animal models. When using murine models for pulmonary metastasis, it is valuable to conduct longitudinal studies during which progression of metastatic burden in the lung occurs. Current methods for evaluating pulmonary metastatic burden in experimental mice are limited to advanced imaging approaches or to the development of morbidity linked to pulmonary metastasis. Unfortunately, clinical signs of morbidity are often vague and unpredictable. A novel, inexpensive, clinical method to detect advanced metastatic burden in mice before the development of significant morbidity or mortality would be beneficial to the study of metastasis in mice. With this goal in mind we have developed a novel, non-invasive technique for assessing pulmonary metastatic burden in laboratory mice. The PAMM (Pulmonary Assessment of Metastatic Morbidity) test is performed by restraining an awake mouse and gently applying pressure with the index finger under the xiphoid process. This pressure reduces the diaphragmatic component to respiration. Mice with advanced lung metastases will show signs of respiratory distress within 3 seconds of applying this pressure. Application of this method in 4 models of pulmonary metastasis (n=27 mice including sarcoma and mammary carcinoma histologies), amongst 3 independent “users,” yielded 100% positive and 100% negative predictive values that were validated by post mortem examination. In summary, the PAMM method is a simple, inexpensive, reproducible and efficient method to assist in the detection of advanced pulmonary metastasis. This method contributes to the humane care of mice included in longitudinal studies of pulmonary metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1365. doi:1538-7445.AM2012-1365

Our previous studies have demonstrated the association between Ezrin and the metastatic biology of pediatric sarcomas including osteosarcoma (OS) and rhabdomyosarcoma. Mechanistic studies exploring this association have shown that Ezrin expression enhances the survival of metastatic cells upon their arrival to the secondary metastatic site. In order to better understand this role in metastasis, we undertook two non-candidate analyses of Ezrin function including a microarray subtraction of high and low Ezrin expressing cells and a proteomic approach to identify proteins that bind the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis we found Ezrin to be part of a ribonucleoprotein complex and to bind with poly A binding protein 1 (PABA1; PABPC1). Using luciferase reporter-based assays, we have shown that OS cells expressing high levels of Ezrin are able to translate mRNAs containing a stem loop 5′ UTR structure, so called “weakly translated mRNAs,” more efficiently than low Ezrin OS cells. This finding suggests that Ezrin's contribution to the metastatic phenotype may be due in part to enhanced translation of specific mRNAs during metastasis that are normally expressed at low levels outside of the metastatic context. Ongoing studies will now assess the ability of Ezrin to enhance the expression of specific mRNAs in 3-dimensional contexts more relevant to cancer cell growth in vivo. We have developed stable high and low Ezrin osteosarcoma cell lines that express a GFP reporter with or without a complex 5′ UTR stem loop structure in the mRNA transcript and with and without protein destabilizing elements. Initial studies have shown that the addition of the 5′ UTR structure and destabilizing elements progressively limits GFP reporter expression. We plan to confirm that this reduction in expression is due to reduced translation and to assess whether 3-D culture conditions or the in vivo environment enhance expression of these tunable reporters of translation in an Ezrin-dependant manner. We expect these results to provide a novel mechanistic basis to consider how Ezrin may contribute to metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4190. doi:1538-7445.AM2012-4190

Nm23 was the first metastasis suppressor gene characterized. One of the best correlates of the metastasis suppressive effect of Nm23 is its inhibition of tumor cell motility, but the mechanistic basis for this phenotype have not been elucidated. Our data in this abstract describe a novel interaction of Nm23-H1 with the FERM domain of Ezrin, which competitively inhibits the ability of FAK to bind Ezrin and induce motility. We hypothesized that Nm23-H1 may suppress motility and metastasis through the binding and inactivation of proteins that promote aggressive behavior. An unbiased survey of Nm23-H1 binding proteins was conducted in a 4T1 murine mammary carcinoma system. Flag tagged Nm23-H1 and its mouse variant, M1, were transfected into 4T1 cancer cells. Primary tumor size was unaffected by over-expression of Nm23-H1 or M1. Histological examination revealed a 75% decrease in liver metastasis by Nm23-H1 or M1 over-expression (P < 0.001). To characterize Nm23-H1/M1 binding proteins, Flag tagged Nm23 from both H1 and M1 clones were immunoprecipitated (IP) from cell lines in vitro as well as isolated primary tumors and metastases. Mass spectrometry analysis of the IP samples identified Ezrin as a potential binding partner. Ezrin is a member of the ERM family of proteins, which have been described to link the actin cytoskeleton to membrane associated proteins. Using co-IP, Ezrin was validated as a binding partner of Nm23-H1 and M1 in both the 4T1 and human MDA-MB-231 cell lines. Nm23-H1 and M1 were found to bind to the N terminal of Ezrin, which contains the highly conserved FERM domain. Deletion mutants established the interaction of Nm23 in both cell lines with the FERM-C portion, corresponding to Ezrin amino acids 205 to 300. Ezrin exists as a folded circular protein that, when activated by phosphorylation at amino acid 567 unfurls, exposing the FERM domain. Nm23-H1 co-immunoprecipitated constitutively active (T567D) but not inactive (T567A) mutants of Ezrin, suggesting that the active “open” structure permitted access to the FERM domain. As focal adhesion kinase (FAK) binds to Ezrin also through the FERM domain, it was hypothesized that Nm23-H1 would competitively prevent this association, confirmed in triple co-IP experiments. Expression of the T567D mutant of Ezrin increased cell motility of the 4T1 cells by an average of 148% (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3421. doi:1538-7445.AM2012-3421

Though advancements in chemotherapy and surgical techniques have ameliorated treatment of primary osteosarcoma (OS), the metastatic phenotype remains a clinical challenge. Overall five-year survival rates for patients with localized OS have improved to 60-70%, yet survival rates of patients with metastasis remains at 20-30% with mortality linked to metastatis-induced respiratory failure. Therefore, targeting fundamental molecular events that lead to metastasis may yield significant benefit to patients with OS. Accumulating evidence from clinical samples and pre-clinical animal models suggests that ezrin is a key regulator in the metastasis of OS cells. Ezrin is a multifunctional protein that connects the actin cytoskeleton to extracellular matrix through transmembrane proteins and a critical component for cell motility, adhesion and shape. We have recently identified that a small molecule, NSC 668394 acts as a potent and selective inhibitor of ezrin function and inhibits migration in both in-vitro and in-vivo models. Moreover, suppression of ezrin phosphorylation by NSC 668394 significantly reduced the metastatic behavior in cellular and animal models and has thus emerged as an important lead inhibitor. Consequently, we conducted a series of structure-activity-relationship (SAR) studies that monitored direct binding; OS cell migration and HUVEC monolayer invasion in ‘real-time’ with surface-plasmon resonance and electrical impedance technology. From our 2nd generation library, we have designed novel candidate inhibitors which feature enhanced ezrin-binding affinity, and improved anti-migration and anti-invasion activities. In addition to their potency against the different stages of OS metastasis, these compounds possess other desirable attributes for development including good druggable physical-chemical characteristics and are currently undergoing further preclinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3906. doi:1538-7445.AM2012-3906

Melanoma is an aggressive human cancer whose incidence is increasing more rapidly than any other cancer in the United States. It is characterized by an extremely high potential to develop metastases and poor response to current therapy. Although the genetic studies indicated that melanoma genesis related with molecular lesions in c-MET, INK4a, Ras/Raf, p53 and PTEN pathways, the molecular mechanism inciting metastatic melanoma are poorly understood. PTEN is a major tumor suppressor in humans. To study the role of PTEN in progression of human melanoma, we first used tissue microarrays to examine PTEN expression in 30 lymph node melanoma metastases, 30 primary melanomas, and 9 normal skin tissues. Our results showed that PTEN loss was significantly correlated with progression to the metastatic state. Moreover, analysis of various human melanoma cell lines demonstrated that PTEN expression was related to metastatic behavior, supporting the notion that PTEN is involved in the metastatic regulation of melanoma. PTEN has phosphatase activity that can act upon both polypeptide and phosphoinositide substrates. The PTEN lipid phosphatase is a ubiquitous regulator of PI3K signaling pathway. The loss of expression or mutational inactivation of PTEN protein enhances the phosphorylation of PKB/AKT and thus influences cell survival signaling. Interestingly, a PTEN mutant, which loses lipid but not protein phosphatase activity, lacks the ability to induce either G1 arrest or apoptosis, but nonetheless retains the ability to inhibit cell spreading and motility, suggesting that PTEN protein phosphatase activity is important for these behaviors. To further dissect the role of this PTEN activity in metastasis, we have established a model system containing exploiting mutant PTEN proteins with various phosphatase activities. We found that wild type PTEN can inhibit cell motility and invasiveness, while PTEN mutants cannot. Notably, wild type PTEN was able to reduce the metastatic potential of melanoma cells, whereas phosphatase deficient PTEN could promote the melanoma cell metastatic behavior. We found that PTEN mediates metastatic activity through the AKT pathway. We further determined that Akt 3 was key mediators of metastatic potential in melanoma cells in vivo. Akt3 could also rescue PTEN-mediated inhibition of metastasis. PTEN loss and Akt3 overexpression functioned synergistically to promote metastasis. Our data demonstrate that metastatic behavior in melanoma cells is dependent on the status of PTEN protein phosphatase activity and downstream Akt3, and suggest that AKT/PTEN signaling should be considered as a therapeutic target in metastatic melanoma Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1446. doi:10.1158/1538-7445.AM2011-1446

The most common cause for mortality in osteosarcoma (OS) is respiratory failure due to metastasis to lungs. Thus targeting underlying molecular events that lead to metastasis can provide a significant benefit to patients with fatal metastatic disease. Accumulating evidence from experimental animal models and human cases suggests that ezrin is a key factor in the metastasis of OS cells. Ezrin is a multifunctional protein that connects the actin cytoskeleton to extracellular matrix through transmembrane proteins. We screened small molecule libraries for compounds that directly interact with ezrin protein. Two small molecules that directly bind to ezrin with low micromolar affinity were selected based on inhibiting ezrin function in multiple assays. They inhibited ezrin phosphorylation, ezrin-actin interaction, and ezrin mediated motility of OS cells in culture. These compounds were unique in their mechanism of action such that they inhibited in vitro phosphorylation of ezrin by protein kinase C (PKC) by binding to the substrate not by inhibiting the kinase activity of the enzyme. PKC was able to phosphorylate other substrates in the presence of these novel ezrin inhibitors. They also mimicked the ezrin morpholino phenotype and caused a unique developmental defect consistent with reduced cell motility in zebrafish. Following tail vein injection of osteosarcoma cells to mice, both molecules inhibited lung metastasis of ezrin-sensitive cells, but not ezrin-resistant cells. These two molecules demonstrate a novel targeted therapy that directly inhibits ezrin protein as an approach to prevent tumor metastasis in osteosarcoma and other tumors with elevated ezrin in their metastatic subclones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4505. doi:10.1158/1538-7445.AM2011-4505

We hypothesized that Nm23-H1 may suppress metastasis through binding and inactivation of proteins that promote aggressive behavior. An unbiased survey of Nm23-H1 binding proteins was conducted in a 4T1 murine mammary carcinoma model system. Flag tagged Nm23-H1 and its mouse variant, -M1, were transfected into 4T1 mouse mammary cancer cells. Clonal lines were inoculated into the number four mammary fat pad of mice and allowed to grow for 10 days prior to excision due to size of the primary tumor. Mice were then imaged weekly for the remainder of the experiment, 8 weeks to follow the metastatic spread of the 4T1 cells from each group. Primary tumor size was unaffected by over-expression of Nm23-H1 or -M1. Histological examination revealed a 75% decrease in liver metastasis by Nm23-H1 or -M1 over-expression (P < 0.001). To characterize Nm23-H1/M1 binding proteins, Flag tagged Nm23 from both H1 and M1 clones were immunoprecipitated (IP) from in vitro cell lines as well as isolated primary tumors, lymph nodes, spleen and liver tissue after verification of metastasis by histological examination of the tissue. Mass spectrometry analysis of IP samples from cell lines and primary tumors showed 99 proteins of interest including Ezrin. Co-IP of Ezrin and Nm23-H1/M1 has been confirmed using both Nm23 and Ezrin as the IP antibodies, implicating Ezrin as a potential binding partner of NM23-H1 and M1. Ezrin belongs to a family of widely expressed proteins known as ERMs which have been described to link the actin cytoskeleton to membrane signaling pathways. To rule out potential interaction due to F-actin, cells were exposed to latrunculin B and co-IP of Nm23-H1 and Ezrin was again verified, indicating that the Ezrin and Nm23-H1/M1 interaction is not due to F-actin binding. To further elucidate this interaction plasmid constructs expressing either the N terminal FERM domain or C terminal F-actin binding domain of Ezrin coupled to GFP were constructed. Co-IP showed a binding of Nm23-H1 to the N terminal domain of Ezrin. In addition the expression of the N terminal overcame the motility and invasive inhibitory phenotype of Nm23-H1 expression in both 4T1 and MDA-MB-231 cells. The data identify a novel interaction of the metastasis suppressor Nm23-H1 and the cytoskeletal linker Ezrin. Suppression of metastasis by Nm23-H1 may be due, in part, to the abrogation of a pro-invasive function of the N-terminus of Ezrin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1447. doi:10.1158/1538-7445.AM2011-1447

GD2 is a disialoganglioside that is expressed on the cell surface in specific normal and malignant human tissues. Tumor specific cell surface antigens serve as important targets in cancer therapy. Anti-GD2 therapy using antibodies targeted to the cell surface antigen GD2 to induce antibody-dependent cellular cytotoxicity has been proven effective in treating GD2 expressing tumors like neuroblastoma and melanoma. Prior studies of osteosarcoma have shown GD2 to be expressed in malignant cells of osteosarcoma patients. In this study, the expression of GD2 in osteosarcoma will be evaluated further. A previously created and described tissue microarray was stained with 14.G2a, a murine monoclonal anti-GD2 antibody that was obtained from the National Cancer Institute. The intensity and location of tissue staining were assessed by a comparison between the positive and negative control slides. To evaluate GD2 expression quantitatively a cell-based ELISA was performed on different osteosarcoma patient derived cell cultures. The results of the tissue microarray showed that 34.8% of patient samples stained positive for GD2 while 60.9% of samples showed intermediate staining and only 4.3% of samples showed negative staining for GD2 (n=88). Nearly all of the osteosarcoma cell lines (n=24) showed levels of GD2 expression comparable to the neuroblastoma and fibrosarcoma cell lines and in some cases GD2 expression was significantly greater. Anti-GD2 therapy has been proven effective in treating patients with neuroblastoma and melanoma. The findings in this paper indicate that osteosarcomas express GD2 to a similar extent as neuroblastomas. These results would suggest clinical trials are worthwhile to further evaluate the benefit of anti-GD2 therapy in patients with osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5332. doi:10.1158/1538-7445.AM2011-5332

Background: Chromosomal translocations involving the EWS gene and one of the members of the ETS family of transcriptions factors are the hallmark of the Ewings Sarcoma Family of Tumors (ESFT). The most common translocation, EWS-FLI1, is found in 85% of the tumors and plays an important role in malignant transformation and maintenance of the oncogenic phenotype. Multiple groups have demonstrated that continued expression of this transcription factor is required for cellular survival. Methods: High throughput screening of a library of more than 50,000 pure compounds was utilized to identify inhibitors of a known EWS-FLI1 downstream target, NR0B1, luciferase promoter construct. The top compounds were subsequently validated and sorted by using a novel 17 gene multiplex PCR assay that quantitates a gene signature of EWS-FLI1. The results were confirmed by quantitative PCR and western blot analysis. The resulting compounds were analyzed for cytotoxicity and selectivity in a panel of ESFT cell lines and compared to cell lines that do not contain the EWS-FLI1 transcription factor. Results Several compounds have been identified that appear to modulate the activity of the EWS-FLI1 transcription factor. These compounds include microtubule inhibitors, topoisomerase inhibitors, compounds of unknown mechanism and an sp1 inhibitor. One of the most potent inhibitors identified was Mithramycin, a natural product known to inhibit sp1. Mithramycin inhibits expression of luciferase in a dose dependent fashion in the NR0B1 promoter reporter construct at concentrations between 10 and 100 nM without decreasing expression in a constitutively active CMV promoter construct. This effect translates into an LD50 of 10 to 25 nM in a panel of ESFT cell lines and increased selectivity relative to osteosarcoma, breast carcinoma and ovarian carcinoma cell lines. Mithramycin also inhibits the expression of the gene signature of EWS-FLI1 as determined by multiplex PCR and confirmed by quantitative PCR. Western blot analysis confirms the inhibition of the downstream target ID2 with a corresponding induction of apoptosis as demonstrated by an increase in cleaved PARP. A review of the literature identified a small panel of patients treated with Mithramycin in the 1960s with at least one documented durable complete response. Conclusion: We have developed a method to screen for transcription factor inhibitors and have utilized it to identify inhibitors of the EWS-FLI1 transcription factor. The most potent inhibitor was Mithramycin which inhibits the expression of a gene signature of EWS-FLI1, has a nanomolar LD50 in ESFT cells and shows some selectivity compared to cells that do not contain EWS-FLI1. Work continues to define the relationship between EWS-FLI1 and sp1 and evaluate the safety and efficacy profile in xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3414.

Ezrin, a linker protein between the cytoskeleton and plasma membrane, plays an important role in the metastatic phenotype of cancers that includes osteosarcoma and rhabdomyosarcoma. To better understand the role of ezrin in metastasis, we have identified proteins that bind the N-terminus of ezrin using tandem mass spectrometry affinity chromatography. One of the mass spectrometry identified proteins is the p53, TNFα, and c-Myc regulated stress-response protein Chloride Intracellular Channel 4 (CLIC4). Under conditions of cell stress, CLIC4 has been shown to translocate to the nucleus and subsequently cause cell-cycle arrest and/or accelerate apoptosis in cancer cells including osteosarcoma. Emerging data from our lab suggests that ezrin may provide metastatic cells with an early survival advantage by helping to resist the stresses of metastasis and resultant apoptosis. Combining these two lines of reasoning, we hypothesized that ezrin prevents CLIC4 induced apoptosis during times of stress by maintaining CLIC4 out of the nucleus. Using cell free conditions we have demonstrated a direct relationship between ezrin and CLIC4 recombinant proteins and through co-immunoprecipitation in osteosarcoma cells. In addition, experiments to define the minimal binding determinants of the ezrin-CLIC4 interaction are currently underway. We have confirmed co-expression of ezrin and CLIC4 in multiple human osteosarcoma cell lines and human sarcomas using both immunohistochemistry and expression microarray. We have recently developed assays and reagents to monitor and manipulate CLIC4 nuclear localization under conditions of stress. The outcome of this work will be to define the nature and biological significance of the ezrin-CLIC4 interaction in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2357.

Osteosarcoma (OS) is the most common type of primary bone cancer and the third most common cancer in children and adolescents. Current combination modalities have improved OS treatment, but did not have a major improvement on patient survival. Thus targeting underlying molecular events can provide dramatic benefits especially for the treatment of patients with fatal metastatic disease. Recently it is shown in a mouse model of OS that high ezrin expression is linked to pulmonary metastasis, providing an early survival advantage for cancer cells reaching the lung. Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) family is conserved through evolution both structurally and functionally. By regulating membrane-cytoskeleton complexes, it plays key roles in normal cellular processes like maintenance of membrane dynamics, survival, adhesion, motility, cytokinesis, phagocytosis and integration of membrane transport with signaling pathways. Due to its critical role in these cellular functions and relevance to metastasis, ezrin is a validated molecular target for prevention of metastasis in OS. In order to identify small molecules that directly interact with recombinant ezrin protein, we screened a small molecule library by surface plasmon resonance technology (Biacore). Initial hits from this primary screen were then evaluated in functional assays. We tested our small molecules for inhibiting ezrin mediated chemotaxis in cell culture, mimicking ezrin morpholino phenotype in zebrafish development, inhibiting ezrin phosphorylation and ezrin-actin interaction, effecting xenopus embryo development and inhibiting lung metastasis of osteosarcoma cells in a mouse lung organ culture assay. Results from these secondary functional assays, binding kinetics values from Biacore experiments and drugability of the chemical structures allowed us to pick two lead compounds. In summary, we discovered two lead compounds that directly bind to ezrin protein with ∼10uM affinity and inhibit its function in multiple independent assays. These molecules form the foundation of new potential therapeutic agents for osteosarcoma metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1550.

The development of novel and targeted cancer drugs requires innovative clinical trial designs to define drug (pharmacokinetic) and host (pharmacodynamic) relationships early in the development process. For most new cancer agents there is increased emphasis on pharmacodynamic (PD) endpoints measured in tumor and surrogates as biomarkers of response. Accordingly, novel tissue collection procedures and instruments are needed to ensure that PD endpoint assessments are representative of their actual in vivo “state”. The Cassi Rotational Core Biopsy Device (Cryobiopsy) (Sanarus Medical, Inc, Pleasanton, California) is a biopsy instrument capable of incisional tissue collection and simultaneous freezing. In rodent xenograft models it demonstrated superiority in consistency for tumor analyte detection compared to traditional biopsy techniques. Whether this value would translate to the clinical setting was unknown. Use of naturally occurring cancers in pet dogs provides a model system to validate and optimize instrumentation techniques prior to use in human trials. A prospective clinical trial in dogs with lymphoma was conducted through the National Cancer Institute-Comparative Oncology Trials Consortium (NCI-COTC) to 1) determine if Cyrobiopsy provides sample quality advantages over conventional tru-cut, and open biopsy procedures, and 2) validate a “practice setting” Cryobiopsy Standard Operating Procedure (SOP) that could be followed by multiple users providing consistent high-quality biopsy samples. Ten dogs with newly diagnosed lymphoma enrolled. Cryobiopsy and tru-cut samples were collected in each patient from two lymph nodes in three planes via alternating techniques. A whole lymph node was then removed via excisional biopsy from the same dog. No adverse events were reported with Cryobiopsy or standard biopsy methods. The SOP for cryobiopsy collection of tissues was successfully implemented in a relevant clinical setting. Cryobiopsy collected samples successfully preserved target phospho-proteins (p-AKT: tAKT, p-ERK: tERK, p-MEK: tMEK) collected from tumor tissue. No difference in phospho-protein detection by Western was seen across biopsy techniques. In conclusion, this effort highlights the utility of the comparative approach to model medical device evaluation and PD biomarker assay validation in the clinical theatre. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4207.

Background: Metastasis has previously been described as an inefficient process - that is, many tumor cells do not survive the process of early metastasis. Metastatic cells must survive exposure to a foreign microenvironment, which can induce cell death, and to the circulation, which provides mechanical sheer stress and immune cells. By observing the kinetics of tumor growth in both ex vivo and in vivo models of metastasis, we hope to determine the relative contributions of circulatory flow and a foreign microenvironment to metastatic inefficiency. Methods: We have developed a novel Pulmonary Metastasis Assay (PuMA), in which we observe the progression of fluorescence-tagged tumor cells in an ex vivo whole organ lung culture. The PuMA model exposes metastatic cells to a foreign microenvironment, but not to the circulation. The intravital videomicroscopy (IVVM) model exposes tumor cells to both the circulation and a foreign microenvironment. We performed serial assessments of tumor burden for the first seven days after arrival of metastatic cells to the lungs, to compare kinetics of tumor growth in both circulation-dependent and circulation-independent models of metastasis. Results: Both the PuMA and IVVM models displayed metastatic inefficiency. The kinetics of metastatic inefficiency and recovery in the PuMA is similar to that seen in IVVM, with periods of 1) decline in tumor burden, 2) a plateau of several days, and 3) an escape, in which tumor burden grows beyond its baseline value. The magnitude of the initial decline in tumor burden, however, is greater in the IVVM model (85% decrease from baseline, at 24 hours after metastasis), than in the PuMA model (43% decrease at 24 hours). Conclusions: These data indicate that there is a significant circulation-independent contribution to metastatic inefficiency (as seen in the decrease in tumor burden in our PuMA model), as well a circulation-dependent contribution (as seen in the greater decrease in our IVVM model). We believe that the PuMA model will be a valuable tool in studying signaling pathways that affect survival in foreign environment. Studies are now underway to use the PuMA, in combination with pharmaceutical and genetic targeting, to study the effect of individual signaling pathways on survival during early metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2359.

The MET tyrosine kinase receptor is important during human development for normal cell growth and migration. Activating alterations of MET have also been identified in several carcinomas. Previous immunohistochemistry studies in our laboratory found high levels of MET in 85% of osteosarcoma tumor samples. In addition, pharmacologic modulation of MET resulted in a migratory decrease in some osteosarcoma cell lines. This led us to hypothesize that MET is necessary for osteosarcoma metastasis, and genetic down-regulation of MET expression would reduce the metastatic phenotype of osteosarcoma cells. We used lentiviral shRNA constructs to knockdown MET expression in several osteosarcoma cell lines. These included MNNG-HOS, a metastatic chemically transformed line that expresses constitutively activated MET, MG63.2, a highly metastatic variant of poorly metastatic MG63, AI and LR, which are two newly derived cell lines that have extremely high MET levels. We quantified levels of MET expression using a sensitive and quantitative electrochemiluminescence (ECL) assay and assessed in vitro parameters of metastasis including motility, migration, and invasion. MET expression was assessed by ECL in protein lysates from frozen osteosarcoma tumor samples and cell lines, and 15-20% of samples showed extremely high levels of total MET. AI, LR and MG63.2 had the highest levels of MET expression. MNNG-HOS has activation of MET due to translocation of the 3′end of MET with the 5′end of TPR. We utilized these lines for shRNA knock down of MET. ECL quantification of total MET levels revealed greater than 70% decrease in MET expression with shRNA knockdown in all four cell lines, compared with cells infected with scramble shRNA controls. As predicted for MNNG-HOS, shRNA constructs targeting the 3’ tyrosine kinase domain of MET resulted in marked decreased motility, compared to intermediate decreases using shRNA constructs targeting the 5’ region. Knock down of MET in MG63.2 resulted in inhibition of both motility and migration. AI and LR assays are in progress. Taken together, these data are the first evidence that genetic down-regulation of MET results in a decreased metastatic phenotype in osteosarcoma. This is a promising finding, as several small molecule tyrosine kinase inhibitors targeting MET are in development. We are currently testing our hypothesis in a mouse model to determine the ability of the above genetically knocked-down MET cell lines to decrease metastasis in vivo. Concurrently, in preparation for translational studies, we are also assessing MET specific tyrosine kinase inhibitors utilizing the parental cell lines described above. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3401.