Although metastasis is the leading cause of cancer-related death, it is not clear why some patients with localized cancer develop metastatic disease after complete resection of their primary tumor. Such relapses have been attributed to tumor cells that disseminate early and remain dormant for prolonged periods of time; however, little is known about the control of these disseminated tumor cells. Here, we have used a spontaneous mouse model of melanoma to investigate tumor cell dissemination and immune control of metastatic outgrowth. Tumor cells were found to disseminate throughout the body early in development of the primary tumor, even before it became clinically detectable. The disseminated tumor cells remained dormant for varying periods of time depending on the tissue, resulting in staggered metastatic outgrowth. Dormancy in the lung was associated with reduced proliferation of the disseminated tumor cells relative to the primary tumor. This was mediated, at least in part, by cytostatic CD8 + T cells, since depletion of these cells resulted in faster outgrowth of visceral metastases. Our findings predict that immune responses favoring dormancy of disseminated tumor cells, which we propose to be the seed of subsequent macroscopic metastases, are essential for prolonging the survival of early stage cancer patients and suggest that therapeutic strategies designed to reinforce such immune responses may produce marked benefits in these patients. IntroductionMetastatic disease is the major cause of death by cancer (1, 2). Metastasis is a complex multistage process that requires cancer cells within the primary tumor to invade the local tissue and enter the blood or lymphatic vessels. Tumor cells need to survive in the circulation and migrate across vessel walls in order to colonize new sites and grow to form secondary tumors (3). The traditional view has been that tumor cell dissemination occurs late in cancer development (4-6); however, this notion has recently been challenged. Several expression profiling studies (7-10) suggest that the propensity of cancer cells to metastasize is acquired relatively early during tumor progression (reviewed in ref. 11). In addition, examination of bone marrow from early stage cancer patients without overt metastases (reviewed in refs. 12 and 13) and tumorbearing mice (14) revealed that disseminated tumor cells (DTCs) are present at much earlier time points than expected. We now need to understand the significance of these DTCs. Specifically, we must determine how early DTCs contribute to clinically relevant macrometastases and identify the mechanisms involved in the development, maintenance, and breakdown of dormancy.Transplanted tumor models in rodents are often used to study metastasis, with most of our current knowledge of cancer cell dissemination being drawn from xenograft models. However, these models often fail to recapitulate the gradual process of tumorigenesis that is observed in humans, and, in the case of immuno-

Animal studies have shown that autophagy is essential in the process of obesity. Here, we performed daily injection of the autophagy inhibitor chloroquine (CQ) in mice and found that systemic administration of CQ blocks high-fat diet-induced obesity. To investigate the potential underlying molecular mechanism, we employed genetic and pharmacological interventions in cultured preadipocytes to investigate the role of autophagy in the control of the expression of the adipogenic regulator peroxisome proliferatior-activated receptor-γ (PPARγ). We show that adipogenic differentiation of 3T3-L1 preadipocytes is associated with activation of autophagy and increased PPARγ2 protein level. Treatment with CQ, shRNA-mediated knockdown, or genetic engineering-induced deletion of autophagy-related gene 5 (Atg5) promoted proteasome-dependent PPARγ2 degradation and attenuated adipogenic differentiation. Therefore, activated autophagy increases PPARγ2 stability and promotes adipogenic differentiation, and inhibition of autophagy may prevent high-fat diet-induced obesity and the consequential type 2 diabetes.

Introduction and Purpose. Monitoring solid tumor growth and metastasis in small animals is important for cancer research. Noninvasive techniques make longitudinal studies possible, require fewer animals, and have greater statistical power. Such techniques include FDG positron emission tomography (FDG-PET), magnetic resonance imaging (MRI), and optical imaging, comprising bioluminescence imaging (BLI) and fluorescence imaging (FLI). This study compared the performance and usability of these methods in the context of mouse tumor studies. Methods. B16 tumor-bearing mice (n = 4 for each study) were used to compare practicality, performance for small tumor detection and tumor burden measurement. Using RETAAD mice, which develop spontaneous melanomas, we examined the performance of MRI (n = 6 mice) and FDG-PET (n = 10 mice) for tumor identification. Results. Overall, BLI and FLI were the most practical techniques tested. Both BLI and FDG-PET identified small nonpalpable tumors, whereas MRI and FLI only detected macroscopic, clinically evident tumors. FDG-PET and MRI performed well in the identification of tumors in terms of specificity, sensitivity, and positive predictive value. Conclusion. Each of the four methods has different strengths that must be understood before selecting them for use.

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC 50 ¼ 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC 50 : PI3Ka ¼ 16 nmol/L; PI3Kb ¼ 68 nmol/L; PI3Kg ¼ 25 nmol/L; PI3Kd ¼ 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials. Mol Cancer Ther; 12(2); 151-61. Ó2012 AACR.

The spleen and its response to parasite infection are important in eliminating parasites in malaria. By comparing P. yoelii parasite lines with different disease outcomes in mice that had either intact spleens or had had their spleens removed, we showed that upon parasite infection, the spleen exhibits dramatic changes that can affect parasite clearance. The spleen itself directly impacts RBC deformability independently of parasite genetics. The data indicated that the changes in the biomechanical properties of malaria parasite-infected RBCs are the result of the complex interaction between host and parasite, and RBC deformability itself can serve as a novel predictor of clinical outcome. The results also suggest that early responses in the spleen are a key factor directing the clinical outcome of an infection.

Hepatocellular carcinoma (HCC) is the fifth most common and third deadliest primary neoplasm. Since HCC is a particularly vascular solid tumor, we determined the antitumor and antiangiogenic activities of sunitinib malate, a potent inhibitor of two receptors involved in angiogenesis - vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). In the present study, we reported that treatment of HepG2 and SK-Hep-1 cells with sunitinib led to growth inhibition and apoptosis in a dose-dependent fashion. Sunitinib inhibited phosphorylation of VEGFR-2 at Tyr951 and PDGFR-beta at Tyr1021 both in vitro and in vivo. Sunitinib also suppressed tumor growth of five patient-derived xenografts. Sunitinib-induced tumor growth inhibition was associated with increased apoptosis, reduced microvessel density and inhibition of cell proliferation. This study provides a strong rationale for further clinical investigation of sunitinib in patients with hepatocellular carcinoma.

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Introduction: Janus kinase 2 (JAK2) and fms-related tyrosine kinase 3 (FLT3) are important tyrosine kinases in hematopoietic progenitor cell differentiation and proliferation. Aberrant signaling of JAK2 or FLT3 is implicated in various myeloid malignancies. SB1518, a new chemical entity, is an inhibitor of both JAK2 (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) as well as FLT3 (IC50 = 22 nM) and its mutant FLT3D835Y (IC50 = 6 nM). It is currently being evaluated in Phase I and II clinical trials for advanced hematologic malignancies and is showing promising therapeutic effects. Here we present a pharmacological evaluation of SB1518 in in vitro and in vivo models of myeloid disease that provide a rationale for its selection for clinical development. Methods: The inhibitory effect of SB1518 on JAK2, FLT3 and STAT signaling was evaluated by Western-blot analysis in cell lines with different JAK2/FLT3 mutation status. Cell proliferation assays using the CellTiterGlo® assay were performed on a panel of different cancer cell lines and primary cells from patients. Selected cells were treated with SB1518 to determine effects on apoptosis (Caspase 3/7 activation) and cell cycle (propidium iodide stain). SET-2 cells (a megakaryoblastic cell line derived from an ET-patient bearing JAK2V617F) and MOLM-13 cells (an AML cell line bearing FLT3-ITD) were used to establish subcutaneous tumors in nude mice to test the anti-tumor efficacy of SB1518 after oral daily dosing. Results: SB1518 inhibits phosphorylation of JAK2 at Y221, FLT3 and of STAT proteins and shows anti-proliferative activity in cell lines driven by mutant or wt JAK2 or FLT3-ITD (IC50 range of 47-348 nM), leading to cell cycle arrest (in G0/1) and induction of apoptosis. Cells bearing JAK2 or FLT-3 mutations or genetic alterations leading to high expression of JAK2, are the most sensitive to the anti-proliferative effects of SB1518, as are primary cells isolated from PV or AML patients. In these cells SB1518 induces cell cycle arrest and apoptosis. SB1518 has favorable physicochemical properties and pharmacokinetics allowing oral dosing. It significantly blocks tumor growth in a subcutaneous model of megakaryoblastic AML (SET-2 cells, TGI = 61% at 150 mg/kg b.i.d.). In a model of late stage, aggressive AML (MOLM-13), SB1518 not only blocked tumor growth (TGI = 81%, at 150 mg/kg b.i.d.), but also significantly reduced lung metastasis. In both models SB1518 was very well tolerated and did not induce cytopenia. Conclusions: SB1518, a novel, orally bio-available small molecule inhibitor of JAK2/FLT3, shows promising activities in various hematological cancer cell lines as well as primary cells from patients. It is efficacious in mouse xenograft models of FLT-3 dependent MOLM-13 cells and JAK2-dependent SET-2 cells. SB1518 is currently undergoing evaluation in Phase 2 clinical trials in myelofibrosis (post-PV, post-ET and primary) and has shown encouraging clinical activity. 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 3570. doi:10.1158/1538-7445.AM2011-3570