Response and Resistance to MEK Inhibition in Leukaemias Initiated by Hyperactive Ras

Department of Pediatrics, University of California, San Francisco, California 94143, USA.
Nature (Impact Factor: 42.35). 10/2009; 461(7262):411-4. DOI: 10.1038/nature08279
Source: PubMed

ABSTRACT The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.

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Available from: Kimberly Krisman, Feb 17, 2014
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    • "Resistance mechanisms vary considerably and include mutations blocking drug-target interaction, genetic alterations sustaining signaling in downstream pathways , or alternate survival pathways (Torti and Trusolino, 2011; Berns and Bernards, 2012). The pervasive disease recurrence following targeted therapy has motivated the use of inducible driver oncogene GEM models of cancers to proactively illuminate potential mechanisms of resistance employed by human cancers (Lauchle et al., 2009). Given the essential roles of oncogenic Kras in both PDAC initiation and maintenance, mutant KRAS and its signaling pathways have been a major focus for the development of disease models for human PDAC (Hingorani et al., 2003; Collisson et al., 2012; Collins et al., 2012; Ying et al., 2012; Eser et al., 2013; Guerra and Barbacid, 2013). "
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    ABSTRACT: Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.
    Cell 06/2014; 158(1). DOI:10.1016/j.cell.2014.06.003 · 33.12 Impact Factor
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    • "The notion that neurofibromin loss has cell type-specific effects on intracellular pathway signaling is underscored by several observations. While neurofibromin primarily regulates astrocyte and Schwann cell growth in a RAS/Akt/mTOR-dependent manner (Banerjee et al., 2010; Johannessen et al., 2005; Johansson et al., 2008; Sandsmark et al., 2007), RAS/MAPK appears to be the major neurofibromin growth control pathway in leukemic cells (Lauchle et al., 2009). Similarly, neuronal differentiation from neural stem cells (NSCs) is dependent on neurofibromin cAMP signaling, while astrocyte differentiation from NSCs requires RAS/ Akt signaling (Hegedus et al., 2007). "
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    ABSTRACT: Children with the neurofibromatosis-1 (NF1) cancer predisposition syndrome exhibit numerous clinical problems that reflect defective central nervous system (CNS) neuronal function, including learning disabilities, attention deficit disorder, and seizures. These clinical features result from reduced NF1 protein (neurofibromin) expression in NF1+/- (NF1 heterozygosity) brain neurons. Previous studies have shown that mouse CNS neurons are sensitive to the effects of reduced Nf1 expression and exhibit shorter neurite lengths, smaller growth cone areas, and attenuated survival, reflecting attenuated neurofibromin cAMP regulation. In striking contrast, Nf1+/- peripheral nervous system (PNS) neurons are nearly indistinguishable from their wild-type counterparts, and complete neurofibromin loss leads to increased neurite lengths and survival in a RAS/Akt-dependent fashion. To gain insights into the differential responses of CNS and PNS neurons to reduced neurofibromin function, we designed a series of experiments to define the molecular mechanism(s) underlying the unique CNS neuronal sensitivity to Nf1 heterozygosity. First, Nf1 heterozygosity decreases cAMP levels in CNS, but not in PNS, neurons. Second, CNS neurons exhibit Nf1 gene-dependent increases in RAS pathway signaling, but no further decreases in cAMP levels were observed in Nf1-/- CNS neurons relative to their Nf1+/- counterparts. Third, neurofibromin regulates CNS neurite length and growth cone areas in a cAMP/PKA/Rho/ROCK-dependent manner in vitro and in vivo. Collectively, these findings establish cAMP/PKA/Rho/ROCK signaling as the responsible axis underlying abnormal Nf1+/- CNS neuronal morphology with important implications for future preclinical and clinical studies aimed at improving cognitive and behavioral deficits in mice and children with reduced brain neuronal NF1 gene expression.
    Molecular and Cellular Neuroscience 08/2011; 49(1):13-22. DOI:10.1016/j.mcn.2011.08.008 · 3.73 Impact Factor
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    • "Transposons and retroviruses are widely used in insertional mutagenesis (IM) screens in mice to discover candidate cancer genes (Collier et al. 2005; Dupuy et al. 2005, 2009; Theodorou et al. 2007; Keng et al. 2009; Starr et al. 2009; Copeland and Jenkins 2010; Rad et al. 2010). IM screens have also been instrumental for the identification of genetic interactions between genes driving tumor evolution (Uren et al. 2008; Kool and Berns 2009; Kool et al. 2010) and for the identification of genes that confer resistance to anticancer drugs (Lauchle et al. 2009) or pathogens (Carette et al. 2009). Finally, retroviruses and transposons are used for germline mutagenesis in a range of experimental organisms (Amsterdam et al. 1999; Golling et al. 2002; Ding et al. 2005; Keng et al. 2005; de Wit et al. 2010). "
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    ABSTRACT: Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.
    Genome Research 08/2011; 21(12):2181-9. DOI:10.1101/gr.112763.110 · 13.85 Impact Factor
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