Abstract While de-regulated MYC signaling has been identified in a wide variety of human malignancies, no targeted therapeutic strategies have been established to target tumors with elevated MYC expression. We have previously reported that receptor “triple negative” (TN) breast cancers, which lack predictive biomarkers of response (i.e., estrogen/progesterone receptors, human epidermal growth factor receptor 2), exhibit significantly elevated MYC expression as well as MYC pathway activation (Horiuchi, D., et al. 2012 JEM). In an effort to uncover the potential Achilles’ heels of TN tumors, we performed a kinome, synthetic-lethal shRNA screen in human mammary epithelial cells in which MYC activity can be controlled (i.e., HMEC-MycER cells). The screen yielded 13 potential synthetic lethal partners of MYC, including recently described AMPK-related kinase 5 (Liu, L., et al. 2012. Nature). One of our top hits is the proto-oncogene PIM1, previously shown by others to be a genetic enhancer of MYC in transgenic mouse models of lymphomas and prostate cancer. Our bioinformatics studies performed on four independent clinical cohorts (n: 146 ∼ 683 patients each) show that in all the cohorts both MYC and PIM1 are significantly enriched in the TN populations. MYC and PIM1 expression are correlated in three out of the four cohorts. Furthermore, we find that PIM1 overexpression is a poor prognostic factor associated with diminished recurrence-free survival. Our in vivo efficacy studies using conventional as well as novel “human-in-mouse” orthotopic xenograft models demonstrate that PIM1 is required for the growth of MYC-overexpressing human TN tumors. We are currently investigating the cellular mechanisms by which PIM kinase inhibition causes such growth defects. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B232. Citation Format: Dai Horiuchi, Alicia Y. Zhou, Alexandra N. Corella, Christina Yau, Devon A. Lawson, Alexey V. Bazarov, Paul Yaswen, Michael T. McManus, Alana L. Welm, Zena Werb, Andrei Goga. PIM1 kinase is essential for the growth of MYC-overexpressing triple-negative breast tumors and is an efficacious therapeutic target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B232.

Bone destruction occurs in aging and numerous diseases, including osteoporosis and cancer. Many cancer patients have bone osteolysis that is refractory to state-of-the-art treatments, which block osteoclast activity with bisphosphonates or by inhibiting the receptor activator of nuclear factor κB ligand (RANKL) pathway. We previously showed that macrophage-stimulating protein (MSP) signaling, which is elevated in about 40% of breast cancers, promotes osteolytic bone metastasis by activation of the MSP signaling pathway in tumor cells or in the bone microenvironment. We show that MSP signals through its receptor, RON tyrosine kinase, expressed on host cells, to activate osteoclasts directly by a previously undescribed pathway that is complementary to RANKL signaling and converges on proto-oncogene, non-receptor tyrosine kinase SRC (SRC). Genetic or pharmacologic inhibition of RON kinase blocked cancer-mediated bone destruction and osteoporosis in several mouse models. Furthermore, the RON kinase inhibitor BMS-777607/ASLAN002 altered markers of bone turnover in a first-in-human clinical cancer study, indicating the inhibitor's potential for normalizing bone loss in patients. These findings uncover a new therapeutic target for pathogenic bone loss and provide a rationale for treatment of bone destruction in various diseases with RON inhibitors.

Ewing sarcoma is the second-most-common bone cancer in children. Driven by an oncogenic chromosomal translocation that results in the expression of an aberrant transcription factor, EWS/FLI, the disease is typically aggressive and micrometastatic upon presentation. Silencing of EWS/FLI in patient-derived tumor cells results in the altered expression of hundreds to thousands of genes and is accompanied by dramatic morphological changes in cytoarchitecture and adhesion. Genes encoding focal adhesion, extracellular matrix, and actin regulatory proteins are dominant targets of EWS/FLI-mediated transcriptional repression. Reexpression of genes encoding just two of these proteins, zyxin and α5 integrin, is sufficient to restore cell adhesion and actin cytoskeletal integrity comparable to what is observed when the EWS/FLI oncogene expression is compromised. Using an orthotopic xenograft model, we show that EWS/FLI-induced repression of α5 integrin and zyxin expression promotes tumor progression by supporting anchorage-independent cell growth. This selective advantage is paired with a tradeoff in which metastatic lung colonization is compromised.

The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency in vitro thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.

Blood vessels support tumours by providing nutrients and oxygen, while also acting as conduits for the dissemination of cancer¹. Here we use mouse models of breast and lung cancer to investigate whether endothelial cells also have active 'instructive' roles in the dissemination of cancer. We purified genetically tagged endothelial ribosomes and their associated transcripts from highly and poorly metastatic tumours. Deep sequencing revealed that metastatic tumours induced expression of the axon-guidance gene Slit2 in endothelium, establishing differential expression between the endothelial (high Slit2 expression) and tumoural (low Slit2 expression) compartments. Endothelial-derived SLIT2 protein and its receptor ROBO1 promoted the migration of cancer cells towards endothelial cells and intravasation. Deleting endothelial Slit2 suppressed metastatic dissemination in mouse models of breast and lung cancer. Conversely, deletion of tumoural Slit2 enhanced metastatic progression. We identified double-stranded RNA derived from tumour cells as an upstream signal that induces expression of endothelial SLIT2 by acting on the RNA-sensing receptor TLR3. Accordingly, a set of endogenous retroviral element RNAs were upregulated in metastatic cells and detected extracellularly. Thus, cancer cells co-opt innate RNA sensing to induce a chemotactic signalling pathway in endothelium that drives intravasation and metastasis. These findings reveal that endothelial cells have a direct instructive role in driving metastatic dissemination, and demonstrate that a single gene (Slit2) can promote or suppress cancer progression depending on its cellular source.

Development of candidate cancer treatments is a resource-intensive process, with the research community continuing to investigate options beyond static genomic characterization. Toward this goal, we have established the genomic landscapes of 536 patient-derived xenograft (PDX) models across 25 cancer types, together with mutation, copy number, fusion, transcriptomic profiles, and NCI-MATCH arms. Compared with human tumors, PDXs typically have higher purity and fit to investigate dynamic driver events and molecular properties via multiple time points from same case PDXs. Here, we report on dynamic genomic landscapes and pharmacogenomic associations, including associations between activating oncogenic events and drugs, correlations between whole-genome duplications and subclone events, and the potential PDX models for NCI-MATCH trials. Lastly, we provide a web portal having comprehensive pan-cancer PDX genomic profiles and source code to facilitate identification of more druggable events and further insights into PDXs' recapitulation of human tumors.

We created the PDX Network (PDXNet) portal (https://portal.pdxnetwork.org/) to centralize access to the National Cancer Institute-funded PDXNet consortium resources, to facilitate collaboration among researchers and to make these data easily available for research. The portal includes sections for resources, analysis results, metrics for PDXNet activities, data processing protocols and training materials for processing PDX data. Currently, the portal contains PDXNet model information and data resources from 334 new models across 33 cancer types. Tissue samples of these models were deposited in the NCI's Patient-Derived Model Repository (PDMR) for public access. These models have 2134 associated sequencing files from 873 samples across 308 patients, which are hosted on the Cancer Genomics Cloud powered by Seven Bridges and the NCI Cancer Data Service for long-term storage and access with dbGaP permissions. The portal includes results from freely available, robust, validated and standardized analysis workflows on PDXNet sequencing files and PDMR data (3857 samples from 629 patients across 85 disease types). The PDXNet portal is continuously updated with new data and is of significant utility to the cancer research community as it provides a centralized location for PDXNet resources, which support multi-agent treatment studies, determination of sensitivity and resistance mechanisms, and preclinical trials.