Pawel K. Mazur

A Drug Repositioning Approach Identifies Tricyclic Antidepressants as Inhibitors of Small Cell Lung Cancer and Other Neuroendocrine Tumors

UNLABELLED Small cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with high mortality. We used a systematic drug repositioning bioinformatics approach querying a large compendium of gene expression profiles to identify candidate U.S. Food and Drug Administration (FDA)-approved drugs to treat SCLC. We found that tricyclic antidepressants and related molecules potently induce apoptosis in both chemonaïve and chemoresistant SCLC cells in culture, in mouse and human SCLC tumors transplanted into immunocompromised mice, and in endogenous tumors from a mouse model for human SCLC. The candidate drugs activate stress pathways and induce cell death in SCLC cells, at least in part by disrupting autocrine survival signals involving neurotransmitters and their G protein-coupled receptors. The candidate drugs inhibit the growth of other neuroendocrine tumors, including pancreatic neuroendocrine tumors and Merkel cell carcinoma. These experiments identify novel targeted strategies that can be rapidly evaluated in patients with neuroendocrine tumors through the repurposing of approved drugs. SIGNIFICANCE Our work shows the power of bioinformatics-based drug approaches to rapidly repurpose FDA-approved drugs and identifies a novel class of molecules to treat patients with SCLC, a cancer for which no effective novel systemic treatments have been identified in several decades. In addition, our experiments highlight the importance of novel autocrine mechanisms in promoting the growth of neuroendocrine tumor cells.

Liver-specific inactivation of Notch2, but not Notch1, compromises intrahepatic bile duct development in mice.

The Notch pathway is an evolutionary conserved, intercellular signaling pathway that plays an important role in cell fate specification and the embryonic development of many organs, including the liver. In humans, mutations in the Notch receptor ligand Jagged1 gene result in defective intrahepatic bile duct (IHBD) development in Alagille syndrome. Developmental abnormalities of IHBD in mice doubly heterozygous for Jagged1 and Notch2 mutations propose that interactions of Jagged1 and its receptor Notch2 are crucial for normal IHBD development. Because different cell types in the liver are involved in IHBD development and morphogenesis, the cell‐specific role of Notch signaling is not entirely understood. We investigated the effect of combined or single targeted disruption of Notch1 and Notch2 specifically in hepatoblasts and hepatoblast‐derived lineage cells on liver development using AlbCre transgenic mice. Hepatocyte differentiation and homeostasis were not impaired in mice after combined deletion of Notch1 and Notch2 (N1N2F/FAlbCre). However, we detected irregular ductal plate structures in N1N2F/FAlbCre newborns, and further postnatal development of IHBD was severely impaired characterized by disorganized ductular structures accompanied by portal inflammation, portal fibrosis, and foci of hepatocyte feathery degeneration in adulthood. Further characterization of mutant mice with single deletion of Notch1 (N1F/FAlbCre) or Notch2 (N2F/FAlbCre) showed that Notch2 but not Notch1 is indispensable for normal perinatal and postnatal IHBD development. Further reduction of Notch2 gene dosage in Notch2 conditional/mutant (N2F/LacZAlbCre) animals further enhanced IHBD abnormalities and concomitant liver pathology. Conclusion: Notch2 is required for proper IHBD development and morphogenesis. (HEPATOLOGY 2008;48:607–616.)

Notch2 is required for progression of pancreatic intraepithelial neoplasia and development of pancreatic ductal adenocarcinoma

Pancreatic cancer is one of the most fatal malignancies lacking effective therapies. Notch signaling is a key regulator of cell fate specification and pancreatic cancer development; however, the role of individual Notch receptors and downstream signaling is largely unknown. Here, we show that Notch2 is predominantly expressed in ductal cells and pancreatic intraepithelial neoplasia (PanIN) lesions. Using genetically engineered mice, we demonstrate the effect of conditional Notch receptor ablation in KrasG12D-driven pancreatic carcinogenesis. Deficiency of Notch2 but not Notch1 stops PanIN progression, prolongs survival, and leads to a phenotypical switch toward anaplastic pancreatic cancer with epithelial–mesenchymal transition. By expression profiling, we identified increased Myc signaling regulated by Notch2 during tumor development, placing Notch2 as a central regulator of PanIN progression and malignant transformation. Our study supports the concept of distinctive roles of individual Notch receptors in cancer development.

Smyd3 links lysine methylation of map3k2 to ras-driven cancer

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

Notch Signaling Is Required for Exocrine Regeneration After Acute Pancreatitis

BACKGROUND & AIMS The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. METHODS Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. RESULTS Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. CONCLUSIONS Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors.

Upregulation of the ERK1 and ERK2 (ERK1/2) MAP kinase (MAPK) cascade occurs in >30% of cancers, often through mutational activation of receptor tyrosine kinases or other upstream genes, including KRAS and BRAF. Efforts to target endogenous MAPKs are challenged by the fact that these kinases are required for viability in mammals. Additionally, the effectiveness of new inhibitors of mutant BRAF has been diminished by acquired tumor resistance through selection for BRAF-independent mechanisms of ERK1/2 induction. Furthermore, recently identified ERK1/2-inducing mutations in MEK1 and MEK2 (MEK1/2) MAPK genes in melanoma confer resistance to emerging therapeutic MEK inhibitors, underscoring the challenges facing direct kinase inhibition in cancer. MAPK scaffolds, such as IQ motif–containing GTPase activating protein 1 (IQGAP1), assemble pathway kinases to affect signal transmission, and disrupting scaffold function therefore offers an orthogonal approach to MAPK cascade inhibition. Consistent with this, we found a requirement for IQGAP1 in RAS-driven tumorigenesis in mouse and human tissue. In addition, the ERK1/2-binding IQGAP1 WW domain peptide disrupted IQGAP1-ERK1/2 interactions, inhibited RAS- and RAF-driven tumorigenesis, bypassed acquired resistance to the BRAF inhibitor vemurafenib (PLX-4032) and acted as a systemically deliverable therapeutic to significantly increase the lifespan of tumor-bearing mice. Scaffold-kinase interaction blockade acts by a mechanism distinct from direct kinase inhibition and may be a strategy to target overactive oncogenic kinase cascades in cancer.

Early Requirement of Rac1 in a Mouse Model of Pancreatic Cancer

BACKGROUND & AIMS Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease without effective chemopreventive or therapeutic approaches. Although the role of oncogenic Kras in initiating development of PDAC is well established, downstream targets of aberrant Ras signaling are poorly understood. Acinar-ductal metaplasia (ADM) appears to be an important prerequisite for development of pancreatic intraepithelial neoplasia (PanIN), a common precursor to PDAC. RAS-related C3 botulinum substrate 1 (Rac1), which controls actin reorganization, can be activated by Ras, is up-regulated in several human cancers, and is required for cerulein-induced morphologic changes in acini. We investigated effects of loss of Rac1 in Kras-induced pancreatic carcinogenesis in mice. METHODS Using a Cre/lox approach, we deleted Rac1 from pancreatic progenitor cells in different mouse models of PDAC and in mice with cerulein-induced acute pancreatitis. Acinar epithelial explants of mutant mice were used to investigate the role of Rac1 in vitro. RESULTS Rac1 expression increased in mouse and human pancreatic tumors, particularly in the stroma. Deletion of Rac1 in Kras(G12D)-induced PDAC in mice reduced formation of ADM, PanIN, and tumors and significantly prolonged survival. Pancreatic epithelial metaplasia was accompanied by apical-basolateral redistribution of F-actin, along with basal expression of Rac1. Acinar epithelial explants that lacked Rac1 or that were incubated with inhibitors of actin polymerization had a reduced ability to undergo ADM in 3-dimensional cultures. CONCLUSIONS In mice, Rac1 is required for early metaplastic changes and neoplasia-associated actin rearrangements in development of pancreatic cancer. Rac1 might be developed as a diagnostic marker or therapeutic target for PDAC.

Origin of pancreatic ductal adenocarcinoma from atypical flat lesions: a comparative study in transgenic mice and human tissues†

Pancreatic ductal adenocarcinoma (PDAC) and its precursor lesions, pancreatic intraepithelial neoplasia (PanIN), display a ductal phenotype. However, there is evidence in genetically defined mouse models for PDAC harbouring a mutated kras under the control of a pancreas‐specific promoter that ductal cancer might arise in the centroacinar‐acinar region, possibly through a process of acinar‐ductal metaplasia (ADM). In order to further elucidate this model of PDAC development, an extensive expression analysis and molecular characterization of the putative and already established (PanIN) precursor lesions were performed in the Kras

Genetically engineered mouse models of pancreatic cancer: unravelling tumour biology and progressing translational oncology

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Conditional ablation of Notch signaling in pancreatic development

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