Establishment and Thorough Characterization of Xenograft (PDX) Models Derived from Patients with Pancreatic Cancer for Molecular Analyses and Chemosensitivity Testing.

Patient-derived xenograft (PDX) tumor models are essential for identifying new biomarkers, signaling pathways and novel targets, to better define key factors of therapy response and resistance mechanisms. Therefore, this study aimed at establishing pancreas carcinoma (PC) PDX models with thorough molecular characterization, and the identification of signatures defining responsiveness toward drug treatment. In total, 45 PC-PDXs were generated from 120 patient tumor specimens and the identity of PDX and corresponding patient tumors was validated. The majority of engrafted PDX models represent ductal adenocarcinomas (PDAC). The PDX growth characteristics were assessed, with great variations in doubling times (4 to 32 days). The mutational analyses revealed an individual mutational profile of the PDXs, predominantly showing alterations in the genes encoding KRAS, TP53, FAT1, KMT2D, MUC4, RNF213, ATR, MUC16, GNAS, RANBP2 and CDKN2A. Sensitivity of PDX toward standard of care (SoC) drugs gemcitabine, 5-fluorouracil, oxaliplatin and abraxane, and combinations thereof, revealed PDX models with sensitivity and resistance toward these treatments. We performed correlation analyses of drug sensitivity of these PDX models and their molecular profile to identify signatures for response and resistance. This study strongly supports the importance and value of PDX models for improvement in therapies of PC.

Somatic Epigenetic Silencing of RIPK3 Inactivates Necroptosis and Contributes to Chemoresistance in Malignant Mesothelioma.

PURPOSE: Receptor-interacting protein kinase 3 (RIPK3) phosphorylates effector molecule MLKL to trigger necroptosis. Although RIPK3 loss is seen in several human cancers, its role in malignant mesothelioma is unknown. This study aimed to determine whether RIPK3 functions as a potential tumor suppressor to limit development of malignant mesothelioma. EXPERIMENTAL DESIGN: RIPK3 expression was examined in 66 malignant mesothelioma tumors and cell lines. Promoter methylation and DNMT1 siRNA studies were performed to assess the mode of RIPK3 silencing in RIPK3-deficient malignant mesothelioma cells. Restoration of RIPK3 expression in RIPK3-negative malignant mesothelioma cells, either by treatment with 5-aza-2'-deoxycytidine or lentiviral expression of cDNA, was performed to assess effects on cell viability, necrosis, and chemosensitization. RESULTS: Loss of RIPK3 expression was observed in 42/66 (63%) primary malignant mesotheliomas and malignant mesothelioma cell lines, and RT-PCR analysis demonstrated that downregulation occurs at the transcriptional level, consistent with epigenetic silencing. RIPK3-negative malignant mesothelioma cells treated with 5-aza-2'-deoxycytidine resulted in reexpression of RIPK3 and chemosensitization. Ectopic expression of RIPK3 also resulted in chemosensitization and led to necroptosis, the latter demonstrated by phosphorylation of downstream target MLKL and confirmed by rescue experiments. Mining of RIPK3 expression and survival outcomes among patients with malignant mesothelioma available from The Cancer Genome Atlas repository revealed that promoter methylation of RIPK3 is associated with reduced RIPK3 expression and poor prognosis. CONCLUSIONS: These data suggest that RIPK3 acts as a tumor suppressor in malignant mesothelioma by triggering necroptosis and that epigenetic silencing of RIPK3 by DNA methylation impairs necroptosis and contributes to chemoresistance and poor survival in this incurable disease.

Cholesterol Pathway Inhibition Induces TGF-ß Signaling to Promote Basal Differentiation in Pancreatic Cancer.

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by KrasG12D expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor ß signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients.

CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma.

BACKGROUND: Multi-targeted tyrosine kinase inhibitors (TKIs) are the standard of care for patients with advanced clear cell renal cell carcinoma (ccRCC). However, a significant number of ccRCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilisation nor clinical benefits. METHODS: We used CRISPR/Cas9-based high-throughput loss of function (LOF) screening to identify cellular factors involved in the resistance to sunitinib. Next, we validated druggable molecular factors that are synthetically lethal with sunitinib treatment using cell and animal models of ccRCC. RESULTS: Our screening identified farnesyltransferase among the top hits contributing to sunitinib resistance in ccRCC. Combined treatment with farnesyltransferase inhibitor lonafarnib potently augmented the anti-tumour efficacy of sunitinib both in vitro and in vivo. CONCLUSION: CRISPR/Cas9 LOF screening presents a promising approach to identify and target cellular factors involved in the resistance to anti-cancer therapeutics.

Anti-tumor effects of ONC201 in combination with VEGF-inhibitors significantly impacts colorectal cancer growth and survival in vivo through complementary non-overlapping mechanisms.

BACKGROUND: Small molecule ONC201 is an investigational anti-tumor agent that upregulates intra-tumoral TRAIL expression and the integrated stress response pathway. A Phase I clinical trial using ONC201 therapy in advanced cancer patients has been completed and the drug has progressed into Phase II trials in several cancer types. Colorectal cancer (CRC) remains one of the leading causes of cancer worldwide and metastatic disease has a poor prognosis. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents. METHODS: We investigated the potential combination of VEGF inhibitors such as bevacizumab and its murine-counterpart; along with other anti-angiogenic agents and ONC201 in both CRC xenograft and patient-derived xenograft (PDX) models. We utilized non-invasive imaging and immunohistochemistry to determine potential mechanisms of action. RESULTS: Our results demonstrate significant tumor regression or complete tumor ablation in human xenografts with the combination of ONC201 with bevacizumab, and in syngeneic MC38 colorectal cancer xenografts using a murine VEGF-A inhibitor. Imaging demonstrated the impact of this combination on decreasing tumor growth and tumor metastasis. Our results indicate that ONC201 and anti-angiogenic agents act through distinct mechanisms while increasing tumor cell death and inhibiting proliferation. CONCLUSION: With the use of both a murine VEGF inhibitor in syngeneic models, and bevacizumab in human cell line-derived xenografts, we demonstrate that ONC201 in combination with anti-angiogenic therapies such as bevacizumab represents a promising approach for further testing in the clinic for the treatment of CRC.

Targeted delivery of chemotherapy using HSP90 inhibitor drug conjugates is highly active against pancreatic cancer models.

The lack of effective treatment modalities is a major problem in pancreatic cancer (PCa), a devastating malignancy that is nearly universally driven by the "undruggable" KRAS and TP53 cancer genes. Poor tumor tissue penetration is the major source of resistance in pancreatic cancer where chemotherapy is the mainstay of treatment. In this study we exploited the selective tumor-targeting properties of the heat shock 90 protein inhibitors as the vehicle for drug delivery to pancreatic tumor tissues. STA-12-8666 is a novel esterase-cleavable conjugate of an HSP90i and a topoisomerase I inhibitor, SN-38. STA-12-8666 selectively binds activated HSP90 and releases its cytotoxic payload resulting in drug accumulation in pancreatic cancer cells in vivo. We investigated the preclinical activity of STA-12-8666 in patient derived xenograft and genetic models of pancreatic cancer.Treatment with STA-12-8666 of the KPC mice (knock-in alleles of LSL-KrasG12D, Tp53fl/fl and Pdx1-Cre transgene) at the advanced stages of pancreatic tumors doubled their survival (49 days vs. 74 days, p=0.008). STA-12-8666 also demonstrated dramatically superior activity in comparison to equimolar doses of irinotecan against 5 patient-derived pancreatic adenocarcinoma xenografts with prolonged remissions in some tumors. Analysis of activity of STA-12-8666 against tumor tissues and matched cell lines demonstrated prolonged accumulation and release of cytotoxic payload in the tumor leading to DNA damage response and cell cycle arrest.Our results provide a proof-of-principle validation that HSP90i-based drug conjugates can overcome the notorious treatment resistance by utilizing the inherently high affinity of pancreatic cancer cells to HSP90 antagonists.

Screening of Conditionally Reprogrammed Patient-Derived Carcinoma Cells Identifies ERCC3-MYC Interactions as a Target in Pancreatic Cancer.

PURPOSE: Even when diagnosed prior to metastasis, pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with almost 90% lethality, emphasizing the need for new therapies optimally targeting the tumors of individual patients. EXPERIMENTAL DESIGN: We first developed a panel of new physiologic models for study of PDAC, expanding surgical PDAC tumor samples in culture using short-term culture and conditional reprogramming with the Rho kinase inhibitor Y-27632, and creating matched patient-derived xenografts (PDX). These were evaluated for sensitivity to a large panel of clinical agents, and promising leads further evaluated mechanistically. RESULTS: Only a small minority of tested agents was cytotoxic in minimally passaged PDAC cultures in vitro Drugs interfering with protein turnover and transcription were among most cytotoxic. Among transcriptional repressors, triptolide, a covalent inhibitor of ERCC3, was most consistently effective in vitro and in vivo causing prolonged complete regression in multiple PDX models resistant to standard PDAC therapies. Importantly, triptolide showed superior activity in MYC-amplified PDX models and elicited rapid and profound depletion of the oncoprotein MYC, a transcriptional regulator. Expression of ERCC3 and MYC was interdependent in PDACs, and acquired resistance to triptolide depended on elevated ERCC3 and MYC expression. The Cancer Genome Atlas analysis indicates ERCC3 expression predicts poor prognosis, particularly in CDKN2A-null, highly proliferative tumors. CONCLUSIONS: This provides initial preclinical evidence for an essential role of MYC-ERCC3 interactions in PDAC, and suggests a new mechanistic approach for disruption of critical survival signaling in MYC-dependent cancers. Clin Cancer Res; 22(24); 6153-63. ©2016 AACR.

A Novel HSP90 Inhibitor-Drug Conjugate to SN38 Is Highly Effective in Small Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is a highly aggressive disease representing 12% to 13% of total lung cancers, with median survival of <2 years. No targeted therapies have proven effective in SCLC. Although most patients respond initially to cytotoxic chemotherapies, resistance rapidly emerges, response to second-line agents is limited, and dose-limiting toxicities (DLT) are a major issue. This study performs preclinical evaluation of a new compound, STA-8666, in SCLC. EXPERIMENTAL DESIGN: To avoid DLT for useful cytotoxic agents, the recently developed drug STA-8666 combines a chemical moiety targeting active HSP90 (concentrated in tumors) fused via cleavable linker to SN38, the active metabolite of irinotecan. We compare potency and mechanism of action of STA-8666 and irinotecan in vitro and in vivo RESULTS: In two SCLC xenograft and patient-derived xenograft models, STA-8666 was tolerated without side effects up to 150 mg/kg. At this dose, STA-8666 controlled or eliminated established tumors whether used in a first-line setting or in tumors that had progressed following treatment on standard first- and second-line agents for SCLC. At 50 mg/kg, STA-8666 strongly enhanced the action of carboplatin. Pharmacokinetic profiling confirmed durable STA-8666 exposure in tumors compared with irinotecan. STA-8666 induced a more rapid, robust, and stable induction of cell-cycle arrest, expression of signaling proteins associated with DNA damage and cell-cycle checkpoints, and apoptosis in vitro and in vivo, in comparison with irinotecan. CONCLUSIONS: Together, these results strongly support clinical development of STA-8666 for use in the first- or second-line setting for SCLC. Clin Cancer Res; 22(20); 5120-9. ©2016 AACR.

HSP90 Inhibitor-SN-38 Conjugate Strategy for Targeted Delivery of Topoisomerase I Inhibitor to Tumors.

The clinical benefits of chemotherapy are commonly offset by insufficient drug exposures, narrow safety margins, and/or systemic toxicities. Over recent decades, a number of conjugate-based targeting approaches designed to overcome these limitations have been explored. Here, we report on an innovative strategy that utilizes HSP90 inhibitor-drug conjugates (HDC) for directed tumor targeting of chemotherapeutic agents. STA-12-8666 is an HDC that comprises an HSP90 inhibitor fused to SN-38, the active metabolite of irinotecan. Mechanistic analyses in vitro established that high-affinity HSP90 binding conferred by the inhibitor backbone could be exploited for conjugate accumulation within tumor cells. In vivo modeling showed that the HSP90 inhibitor moiety was required for selective retention of STA-12-8666, and this enrichment promoted extended release of active SN-38 within the tumor compartment. Indeed, controlled intratumoral payload release by STA-12-8666 contributed to a broad therapeutic window, sustained biomarker activity, and remarkable degree of efficacy and durability of response in multiple cell line and patient-derived xenograft models. Overall, STA-12-8666 has been developed as a unique HDC agent that employs a distinct mechanism of targeted drug delivery to achieve potent and sustained antitumor effects. These findings identify STA-12-8666 as a promising new candidate for evaluation as novel anticancer therapeutic.

Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints.

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

A two-hybrid approach to identify inhibitors of the RAS-RAF interaction.

MCP compounds were developed with the idea to inhibit RAS/RAF interaction. They were identified by carrying out high-throughput screens of chemical compounds for their ability to inhibit RAS/RAF interaction in the yeast two-hybrid assay. A number of compounds including MCP1, MCP53, and MCP110 were identified as active compounds. Their inhibition of the RAS signaling was demonstrated by examining RAF and MEK activities, phosphorylation of ERK as well as characterizing their effects on events downstream of RAF. Direct evidence for the inhibition of RAS/RAF interaction was obtained by carrying out co-IP experiments. MCP compounds inhibit proliferation of a wide range of human cancer cell lines. Combination studies with other drugs showed that MCP compounds synergize with MAPK pathway inhibitors as well as with microtubule-targeting chemotherapeutics. In particular, a strong synergy with paclitaxel was observed. Efficacy to inhibit tumor formation was demonstrated using mouse xenograft models. Combination of MCP110 and paclitaxel was particularly effective in inhibiting tumor growth in a mouse xenograft model of colorectal carcinoma.

Genetic and functional characterization of putative Ras/Raf interaction inhibitors in C. elegans and mammalian cells.

BACKGROUND: Activation of the mammalian Ras-Raf-MEK-ERK MAPK signaling cascade promotes cellular proliferation, and activating Ras mutations are implicated in cancer onset and maintenance. This pathway, a therapeutic target of interest, is highly conserved and required for vulval development in C. elegans. Gain-of-function mutations in the Ras ortholog lead to constitutive pathway signaling and a multivulva (Muv) phenotype. MCP compounds were identified in a yeast two-hybrid screen for their ability to disrupt Ras-Raf interactions. However, this had not been confirmed in another system, and conflicting results were reported regarding selective MCP-mediated blockade of Ras- and Raf-mediated biological activities in mammalian cells. Here we used the easily-scored Muv phenotype as an in vivo readout to characterize the selectivity of MCP110 and its analogs, and performed biochemical studies in mammalian cells to determine whether MCP treatment results in impaired interaction between Ras and its effector Raf. RESULTS: Our genetic analyses showed significant dose-dependent MCP-mediated reduction of Muv in C. elegans strains with activating mutations in orthologs of Ras (LET-60) or Raf (LIN-45), but not MAP kinases or an Ets-like transcription factor. Thus, these inhibitors selectively impair pathway function downstream of Ras and upstream of or at the level of Raf, consistent with disruption of the Ras/Raf interaction. Our biochemical analyses of MCP110-mediated disruption of Ras-Raf interactions in mammalian cells showed that MCP110 dose-dependently reduced Raf-RBD pulldown of Ras, displaced a fluorescently-tagged Raf-RBD probe from plasma membrane locations of active Ras to the cytosol and other compartments, and decreased active, phosphorylated ERK1/2. CONCLUSIONS: We have effectively utilized C. elegans as an in vivo genetic system to evaluate the activity and selectivity of inhibitors intended to target the Ras-Raf-MAPK pathway. We demonstrated the ability of MCP110 to disrupt, at the level of Ras/Raf, the Muv phenotype induced by chronic activation of this pathway in C. elegans. In mammalian cells, we not only demonstrated MCP-mediated blockade of the physical interaction between Ras and Raf, but also narrowed the site of interaction on Raf to the RBD, and showed consequent functional impairment of the Ras-Raf-MEK-ERK pathway in both in vivo and cell-based systems.

Ras-driven transformation of human nestin-positive pancreatic epithelial cells.

Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the means by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this signaling biology. This chapter describes the establishment and characterization of telomerase-immortalized human pancreatic duct-derived cells to study mechanisms of Ras growth transformation. An important strength of this model system is the ability of mutationally activated K-Ras to cause potent growth transformation in vitro and in vivo. We have utilized this cell system to evaluate the antitumor activity of small molecule inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase cascade. This model will be useful for genetic and pharmacologic dissection of the contribution of downstream effector signaling in Ras-dependent growth transformation.

Isoquinolin-1-one inhibitors of the MDM2-p53 interaction.

p53 has been at the centre of attention for drug design since the discovery of its growth-suppressive and pro-apoptotic activity. Herein we report the design and characterisation of a new class of isoquinolinone inhibitors of the MDM2-p53 interaction. Our identification of druglike and selective inhibitors of this protein-protein interaction included a straightforward in silico compound-selection process, a recently reported NMR spectroscopic approach for studying the MDM2-p53 interaction, and selectivity screening assays using cells with the same genetic background. The selected inhibitors were all able to induce apoptosis and the expression of p53-related genes, but only the isoquinolin-1-one-based inhibitors stabilised p53. Our NMR experiments give a persuading explanation for these results, showing that isoquinolin-1-one derivates are able to dissociate the preformed MDM2-p53 complex in vitro, releasing a folded and soluble p53. The joint application of these methods provides a framework for the discovery of protein interaction inhibitors as a promising starting point for further drug design.

Selective Raf inhibition in cancer therapy.

Over the past 5 years, the Raf kinase family has emerged as a promising target for protein-directed cancer therapy development. The goal of this review is to first provide a concise summary of the data validating Raf proteins as high-interest therapeutic targets. The authors then outline the mode of action of Raf kinases, emphasizing how Raf activities and protein interactions suggest specific approaches to inhibiting Raf. The authors then summarize the set of drugs, antisense reagents and antibodies available or in development for therapeutically targeting Raf or Raf-related proteins, as well as existing strategies combining these and other therapeutic agents. Finally, the authors discuss recent results from systems biology analyses that have the potential to increasingly guide the intelligent selection of combination therapies involving Raf-targeting agents and other therapeutics.

Context-dependent roles of mutant B-Raf signaling in melanoma and colorectal carcinoma cell growth.

Mutational activation of Ras and a key downstream effector of Ras, the B-Raf serine/threonine kinase, has been observed in melanomas and colorectal carcinomas. These observations suggest that inhibition of B-Raf activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK) and the extracellular signal-regulated kinase MAPK cascade may be an effective approach for the treatment of RAS and B-RAF mutation-positive melanomas and colon carcinomas. Although recent studies with interfering RNA (RNAi) and pharmacologic inhibitors support a critical role for B-Raf signaling in melanoma growth, whether mutant B-Raf has an equivalent role in promoting colorectal carcinoma growth has not been determined. In the present study, we used both RNAi and pharmacologic approaches to further assess the role of B-Raf activation in the growth of human melanomas and additionally determined if a similar role for mutant B-Raf is seen for colorectal carcinoma cell lines. We observed that RNAi suppression of mutant B-Raf(V600E) expression strongly suppressed the anchorage-dependent growth of B-RAF mutation-positive melanoma, but not colorectal carcinoma, cells. However, the anchorage-independent and tumorigenic growth of B-RAF mutation-positive colorectal carcinomas was dependent on mutant B-Raf function. Finally, pharmacologic inhibition of MEK and Raf was highly effective at inhibiting the growth of B-RAF mutation-positive melanomas and colorectal carcinoma cells, whereas inhibitors of other protein kinases activated by Ras (AKT, c-Jun NH(2)-terminal kinase, and p38 MAPK) were less effective. Our observations suggest that Raf and MEK inhibitors may be effective for the treatment of B-RAF mutation-positive colorectal carcinomas as well as melanomas.

K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

In vitro and in vivo synergy of MCP compounds with mitogen-activated protein kinase pathway- and microtubule-targeting inhibitors.

An important clinical task is to coherently integrate the use of protein-targeted drugs into preexisting therapeutic regimens, with the goal of improving treatment efficacy. Constitutive activation of Ras-dependent signaling is important in many tumors, and agents that inhibit this pathway might be useful in numerous therapeutic combinations. The MCP compounds were identified as inhibitors of Ras-Raf interactions and previously shown to inhibit multiple Ras-dependent transformation phenotypes when used as monoagents in cell culture analyses. In this study, we investigate the ability of the MCP110 compound to synergistically enhance the activity of other therapeutic agents. In both a defined K-Ras-transformed fibroblast model and in human tumor cell lines with mutationally activated Ras, MCP110 selectively synergizes with other agents targeting the mitogen-activated protein kinase pathway, and with multiple agents (paclitaxel, docetaxel, and vincristine) targeting the microtubule network. The synergistic activity of MCP110 and paclitaxel was further established by experiments showing that in Kaposi's sarcoma oncogenically transformed cell lines, cellular models for tumors treated with taxanes in the clinic and in which Raf-dependent signaling plays an important role, MCP110 synergizes with paclitaxel and limit growth. Finally, in vivo testing indicate that MCP110 is bioavailable, inhibits the growth of LXFA 629 lung and SW620 colon carcinoma cells in xenograft models, and again strongly synergizes with paclitaxel. Together, these findings indicate that MCP compounds have potential to be effective in combination with other anticancer agents.

Yeast screens for inhibitors of Ras-Raf interaction and characterization of MCP inhibitors of Ras-Raf interaction.

Because of the central role of Ras in cancer cell signaling, there has been considerable interest in developing small molecule inhibitors of the Ras signaling pathways as potential chemotherapeutic agents. This chapter describes the use of a two-hybrid approach to identify the MCP compounds, small molecules that disrupt the interaction between Ras and its effector Raf. We first outline the reagent development and selection/counter selection methods required to successfully apply a two-hybrid approach to isolation of MCP compounds. Separately, we describe the collateral benefits of this screening approach in yielding novel antifungal compounds. We then discuss secondary physiological validation approaches to confirm the MCP compounds specifically target Ras-Raf signaling. Finally, we develop a decision tree for subsequent preclinical characterization and optimization of this class of pathway-targeted reagent.

Development of a yeast two-hybrid screen for selection of human Ras-Raf protein interaction inhibitors.

A yeast two-hybrid screening system was developed to screen for small molecules that inhibit the interaction of the Ras and the Raf proteins. Hyperpermeable yeast strains useful for high-throughput screening (HTS) for the two-hybrid system were created. Differential inhibition of the Ras-Raf vs the hsRPB4-hsRPB7 interaction allowed the identification of selective inhibitors.