The Public Repository of Xenografts enables discovery and randomized phase II-like trials in mice

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease

The Public Repository of Xenografts (ProXe) enables discovery and randomized phase II-like trials in mice

Over 90% of drugs with preclinical activity fail to ultimately benefit patients. We hypothesized that adequately sized and appropriately powered, randomized phase II-like trials of patient-derived xenografts (PDX) could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment and made them available through an open-source web portal (www.PRoXe.org) that includes all de-identified information relevant to the xenografted specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs designed to mimic human randomized phase II trials are feasible and can generate transcriptional, functional and proteomic biomarkers as well as large numbers of in vivo models with acquired resistance. Investigators from >50 institutions have already accessed PRoXe.org and models have been shared between >20 centers

Targeting Vascular Pericytes in Hypoxic Tumors Increases Lung Metastasis via Angiopoietin-2

Strategies to target angiogenesis include inhibition of the vessel-stabilizing properties of vascular pericytes. Pericyte depletion in early-stage non-hypoxic tumors suppressed nascent angiogenesis, tumor growth, and lung metastasis. In contrast, pericyte depletion in advanced-stage hypoxic tumors with pre-established vasculature resulted in enhanced intra-tumoral hypoxia, decreased tumor growth, and increased lung metastasis. Furthermore, depletion of pericytes in post-natal retinal blood vessels resulted in abnormal and leaky vasculature. Tumor transcriptome profiling and biological validation revealed that angiopoietin signaling is a key regulatory pathway associated with pericyte targeting. Indeed, pericyte targeting in established mouse tumors increased angiopoietin-2 (ANG2/Angpt2) expression. Depletion of pericytes, coupled with targeting of ANG2 signaling, restored vascular stability in multiple model systems and decreased tumor growth and metastasis. Importantly, ANGPT2 expression correlated with poor outcome in patients with breast cancer. These results emphasize the potential utility of therapeutic regimens that target pericytes and ANG2 signaling in metastatic breast cancer

An F876l mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (Enzalutamide)

Castration-resistant prostate cancer (CRPC) is the most aggressive, incurable form of prostate cancer. MDV3100 (enzalutamide), an antagonist of the androgen receptor (AR), was approved for clinical use in men with metastatic CRPC. Although this compound showed clinical efficacy, many initial responders later developed resistance. To uncover relevant resistant mechanisms, we developed a model of spontaneous resistance to MDV3100 in LNCaP prostate cancer cells. Detailed characterization revealed that emergence of an F876L mutation in AR correlated with blunted AR response to MDV3100 and sustained proliferation during treatment. Functional studies confirmed that ARF876L confers an antagonist-to-agonist switch that drives phenotypic resistance. Finally, treatment with distinct antiandrogens orcyclin-dependent kinase (CDK)4/6 inhibitors effectively antagonized ARF876L function. Together, these findings suggest that emergence of F876L may (i) serve as a novel biomarker for prediction of drug sensitivity, (ii) predict a "withdrawal" response to MDV3100, and (iii) be suitably targeted with other antiandrogens or CDK4/6 inhibitors. SIGNIFICANCE: We uncovered an F876L agonist-switch mutation in AR that confers genetic and phenotypic resistance to the antiandrogen drug MDV3100. On the basis of this finding, we propose new therapeutic strategies to treat patients with prostate cancer presenting with this AR mutation. © 2013 American Association for Cancer Research

Design and Discovery of N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide (LXH254), A selective, efficacious, well-tolerated RAF inhibitor targeting RAS mutant cancers: The path to the clinic

RAS and BRAF oncogenes are mutated in more than one-third of human cancers and exquisite dependency on CRAF, MEK1/2 and ERK1/2 has been demonstrated in preclinical models of RAS mutant cancer. Direct pharmacological inhibition of RAS has remained elusive and efforts to target CRAF have been challenging due to the nature of the RAF signaling complex downstream of activated RAS and the poor overall kinase selectivity profile of putative RAF inhibitors such as sorafenib and RAF265. Herein, we describe 15 (LXH254), a selective B/C RAF inhibitor, which has been developed through a hypothesis-driven approach focusing on drug-like properties. We have previously disclosed the discovery of 3 (RAF709), a preclinical tool compound which was potent, selective, efficacious, and well-tolerated in preclinical models, but the high intrinsic clearance [HLM Cl(int) = 94] precluded further development.X The high clearance of 3 by HLM prompted the medicinal chemistry team to further investigate close analogs as well as novel scaffolds. While keeping drug-like properties in mind, the team identified multiple cell-potent scaffolds with low-to-moderate human clearance and progressed them into in-vivo pharmacology studies. Unexpectedly, the majority of novel scaffolds caused significant body weight loss in mice for unknown reasons, with the 2-pyridine series emerging as the only scaffold which was not plagued by this problem. A structure-based approach led to the realization that an alcohol side-chain in the 2-position of the pyridine could interact with the DFG loop and significantly improve cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition of CYP3A4 (TDI) led to the discovery of 15, which had favorable PK and proved to be efficacious in multiple xenograft models such as Calu-6 (KRASQ61K),with a favorable therapeutic index. Due to its excellent in vitro/ in vivo properties, it has progressed through preclinical toxicology studies and now being tested as a single agent and as a combination partner in phase 1 clinical trial

Pericyte Depletion Results in Hypoxia-Associated Epithelial-to-Mesenchymal Transition and Metastasis Mediated by Met Signaling Pathway

The functional role of pericytes in cancer progression remains unknown. Clinical studies suggest that low numbers of vessel-associated pericytes correlated with a drop in overall survival of patients with invasive breast cancer. Using genetic mouse models or pharmacological inhibitors, pericyte depletion suppressed tumor growth but enhanced metastasis. Pericyte depletion was further associated with increased hypoxia, epithelial-to-mesenchymal transition (EMT), and Met receptor activation. Silencing of Twist or use of a Met inhibitor suppressed hypoxia and EMT/Met-driven metastasis. In addition, poor pericyte coverage coupled with high Met expression in cancer cells speculates the worst prognosis for patients with invasive breast cancer. Collectively, our study suggests that pericytes within the primary tumor microenvironment likely serve as important gatekeepers against cancer progression and metastasis.National Institutes of Health (U.S.) (NIH Grant CA125550)National Institutes of Health (U.S.) (NIH grant CA155370)National Institutes of Health (U.S.) (NIH Grant CA151925)National Institutes of Health (U.S.) (NIH Grant DK81576)National Institutes of Health (U.S.) (NIH Grant CA163191)National Institutes of Health (U.S.) (NIH grant DK55001)Champalimaud Foundation (Champalimaud metastasis programme)Champalimaud Foundation (Champalimaud investigator)National Institutes of Health (U.S.) (NRSA F32 Ruth Kirschstein Postdoctoral Fellowship from NIH/NIDDK (5F32DK082119-02))National Institutes of Health (U.S.) (NIH Research Training Grant in Gastroenterology (2T32DK007760-11))National Institutes of Health (U.S.) (NIH Research Training Grant in Cancer Biology (5T32CA081156-08))United States. Dept. of Defense (DoD Breast Cancer Research Predoctoral Traineeship Award (W81XWH-09-1-0008))National Institutes of Health (U.S.) (NIH Research Training Grant in Cardiovascular Medicine (5T32HL007374-30))United Negro College Fund/Merck (Postdoctoral Science Research Fellowship)National Institutes of Health (U.S.) (NIH supplemental grant (CA125550)

VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization

Increased numbers of S100A4+ cells are associated with poor prognosis in patients who have cancer. Although the metastatic capabilities of S100A4+ cancer cells have been examined, the functional role of S100A4+ stromal cells in metastasis is largely unknown. To study the contribution of S100A4+ stromal cells in metastasis, we used transgenic mice that express viral thymidine kinase under control of the S100A4 promoter to specifically ablate S100A4+ stromal cells. Depletion of S100A4+ stromal cells significantly reduced metastatic colonization without affecting primary tumor growth. Multiple bone marrow transplantation studies demonstrated that these effects of S100A4+ stromal cells are attributable to local non–bone marrow-derived S100A4+ cells, which are likely fibroblasts in this setting. Reduction in metastasis due to the loss of S100A4+ fibroblasts correlated with a concomitant decrease in the expression of several ECM molecules and growth factors, particularly Tenascin-C and VEGF-A. The functional importance of stromal Tenascin-C and S100A4+ fibroblast-derived VEGF-A in metastasis was established by examining Tenascin-C null mice and transgenic mice expressing Cre recombinase under control of the S100A4 promoter crossed with mice carrying VEGF-A alleles flanked by loxP sites, which exhibited a significant decrease in metastatic colonization without effects on primary tumor growth. In particular, S100A4+ fibroblast-derived VEGF-A plays an important role in the establishment of an angiogenic microenvironment at the metastatic site to facilitate colonization, whereas stromal Tenascin-C may provide protection from apoptosis. Our study demonstrates a crucial role for local S100A4+ fibroblasts in providing the permissive “soil” for metastatic colonization, a challenging step in the metastatic cascade

Inhibition of wild-type p53-expressing AML by novel small molecule HDM2 inhibitor, CGM097

The tumor suppressor, p53, is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (wt) p53, the Human Double Minute 2 (HDM2) protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell cycle arrest. We have characterized the novel HDM2 inhibitor, CGM097, as having significant activity against wt p53-expressing acute myeloid leukemia (AML). Specifically, CGM097 potently and selectively inhibited the proliferation of human AML cell lines and primary AML cells expressing wt p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against mutant FLT3-expressing cells, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. Taken together, these data suggest that CGM097 might be a promising treatment for AML characterized as harboring wt p53 as a single agent, as well as possibly in combination with another targeted therapy using tyrosine kinase inhibitors (TKIs) against oncogenes that drive AML

Inhibition of wild-type p53-expressing AML by the novel small molecule HDM2 inhibitor CGM097

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cellbased proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient-derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors

Purpose: Targeting RAF for anti-tumor therapy in RAS-mutant tumors holds promise. Herein we describe in detail novel properties of the type II RAF inhibitor LXH254. Experimental Design: LXH254 was profiled in biochemical, in vitro, and in vivo assays including examining the activities of the drug in a large panel of cancer-derived cell lines, and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations co-expressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS mutant lines loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Lastly, in vivo, LXH254 drove complete regressions of isogenic variants of RAS mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. Conclusions: LXH254 is a novel RAF-inhibitor able to inhibit dimerized BRAF and CRAF as well as monomeric BRAF while largely sparing ARAF