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Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs.

Cell cycle stimulation is a major transforming mechanism of Myc oncoprotein. This is achieved through at least three concomitant mechanisms: upregulation of cyclins and Cdks, downregulation of Cdk inhibitors p15 and p21 and the degradation of p27. The Myc-p27 antagonism has been shown to be relevant in human cancer. To be degraded, p27 must be phosphorylated at Thr-187 to be recognized by Skp2, a component of the ubiquitination complex. We previously described that Myc induces Skp2 expression. Here we show that not only Cdk2 but Cdk1 phosphorylates p27 at the Thr187, which was previously unreported. Moreover, Myc induced p27 degradation in murine fibroblasts through Cdk1 activation, which was achieved by Myc-dependent cyclin A and B induction. In the absence of Cdk2, p27 phosphorylation at Thr-187 was mainly carried out by cyclin A2-Cdk1 and cyclin B1-Cdk1. We also show that Cdk1 inhibition was sufficient for the synthetic lethal interaction with Myc. This result is relevant because Cdk1 is the only Cdk strictly required for cell cycle and the reported synthetic lethal interaction between Cdk1 and Myc.

KRAS oncogenes are responsible for one fourth of all human tumors, including lung and pancreatic adenocarcinomas, two tumor types with some of the worse prognoses. Unfortunately, development of suitable therapies to treat these tumors has remained elusive for the last 35 years. Although KRAS remains an undruggable target (with the possible exception of the KRASG12C isoform), most of its downstream effectors are druggable kinases. Indeed, pharma and biotech companies have already developed dozens of selective inhibitors against these kinases. Yet none of them has been approved by the FDA, mostly due to unacceptable toxicities at antitumor effective doses. Hence, patients are still treated with old chemotherapy drugs. To address this important health issue, we have used a new generation of genetically engineered mouse (GEM) tumor models of KRas/Tp53 driven lung and pancreatic tumors to deconstruct oncogenic KRas signaling to identify those effectors whose systemic ablation/inactivation in tumor-bearing mice will result in therapeutic responses without inducing unacceptable toxicities. We have used this experimental strategy to interrogate the effect of ablating/inactivating each of the members of the MAPK cascade, including the Raf, Mek, and Erk kinases, as well as key effectors of the PI3K pathway, including PI3K p110alpha and mTOR. We have also evaluated additional downstream signaling elements such as the cell cycle Cdks. This systematic approach has revealed that most of the KRas signaling effectors are not suitable therapeutic targets due to either lack of therapeutic activity, such as Cdk2, Cdk6 A-Raf, or B-Raf, or to the induction of unacceptable toxicities such as the Mek1/2 and Erk1/2 kinases, PI3K p110alpha, mTOR, and Cdk1. Only Raf1 and Cdk4 turned out to be suitable therapeutic targets, based not only on their antitumor properties but also on the well-tolerated toxicities. Surprisingly, EGFR, an upstream effect of KRas, also has suitable therapeutic activity in pancreatic but not in lung tumors. We are now combining these targets to define more efficacious therapeutic strategies that could be eventually translated to the clinic. Moreover, we are using these experimental GEM tumor models to identify potential resistance mechanisms to these therapies. Citation Format: Mariano Barbacid. The use of GEM tumor models to identify effective therapies against KRAS mutant tumors [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA11.

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

MEK inhibition in combination with a glycogen synthase kinase-3β (GSK3β) inhibitor, referred as the 2i condition, favors pluripotency in embryonic stem cells (ESCs). However, the mechanisms by which the 2i condition limits ESC differentiation and whether RAS proteins are involved in this phenomenon remain poorly understood. Here we show that RAS nullyzygosity reduces the growth of mouse ESCs (mESCs) and prohibits their differentiation. Upon RAS deficiency or MEK inhibition, ERF (E twenty-six 2 [Ets2]-repressive factor), a transcriptional repressor from the ETS domain family, translocates to the nucleus, where it binds to the enhancers of pluripotency factors and key RAS targets. Remarkably, deletion of Erf rescues the proliferative defects of RAS-devoid mESCs and restores their capacity to differentiate. Furthermore, we show that Erf loss enables the development of RAS nullyzygous teratomas. In summary, this work reveals an essential role for RAS proteins in pluripotency and identifies ERF as a key mediator of the response to RAS/MEK/ERK inhibition in mESCs. We thank Cian Lynch, Jorge Monsech, and Diego Megias for their help with microarray, ChIP-seq, and high-throughput microscopy analyses. We also thank Dr. Manuel Serrano and Dr. André Nussenzweig for their input on the manuscript, and Dr. Diego Sanz for his support throughout the project. C.M.-R. was funded by a PhD fellowship from La Caixa Foundation, T.O. was funded by a PhD fellowship from the Boehringer Ingelheim Fonds, and S.R. was funded by a Ramon y Cajal contract (RYC-2011-09242). Research was funded by Fundación Botín and Banco Santander through its Santander Universities Global Division; grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-23753 and SAF2014- 57791-REDC; these projects were cofinanced with European Fonds Européen de Développement Économique et Régional [FEDER] funds), Fundació La Marato de TV3, Howard Hughes Medical Institute, and the European Research Council (ERC- 617840) to O.F.-C.; and grants from the Spanish Ministryof Economy and Competitiveness (SAF2013-49147-P and SAF2016-80874-P; these projects were cofinanced with European FEDER funds) to S.R. Author contributions: C.M.-R. and S.R. conducted most of the experiments; T.O. helped with the characterization of RASlox/lox mESCs and with ERF localization studies; E.L. helped with ChIP-seq experiments; M.D., S.O., and M.B. contributed to the work on RAS-deficient cells; M.V.-S. provided technical help; O.D. helped with genomics experiments and bioinformatics analysis; and S.R. and O.F.-C. coordinated the study and wrote the manuscript. Sí

On the basis of clinical trials using first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), it became a doctrine that V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-RAS) mutations drive resistance to EGFR inhibition in non-small cell lung cancer (NSCLC). Conversely, we provide evidence that EGFR signaling is engaged in K-RAS-driven lung tumorigenesis in humans and in mice. Specifically, genetic mouse models revealed that deletion of Egfr quenches mutant K-RAS activity and transiently reduces tumor growth. However, EGFR inhibition initiates a rapid resistance mechanism involving non-EGFR ERBB family members. This tumor escape mechanism clarifies the disappointing outcome of first-generation TKIs and suggests high therapeutic potential of pan-ERBB inhibitors. On the basis of various experimental models including genetically engineered mouse models, patient-derived and cell line-derived xenografts, and in vitro experiments, we demonstrate that the U.S. Food and Drug Administration-approved pan-ERBB inhibitor afatinib effectively impairs K-RAS-driven lung tumorigenesis. Our data support reconsidering the use of pan-ERBB inhibition in clinical trials to treat K-RAS-mutated NSCLC.

et al., The Cip/Kip family, namely, p21Cip1, p27Kip1, and p57Kip2, are stoichiometric cyclin-dependent kinase inhibitors (CKIs). Paradoxically, they have been proposed to also act as positive regulators of Cdk4/6-cyclin D by stabilizing these heterodimers. Loss of p21Cip1 and p27Kip1 reduces Cdk4/6-cyclin D complexes, although with limited phenotypic consequences compared to the embryonic lethality of Cdk4/6 or triple cyclin D deficiency. This milder phenotype was attributed to Cdk2 compensatory mechanisms. To address this controversy using a genetic approach, we generated Cdk2-/- p21-/- p27-/- mice. Triple-knockout mouse embryonic fibroblasts (MEFs) displayed minimal levels of D-type cyclins and Cdk4/6-cyclin D complexes. p57Kip2 downregulation in the absence of p21Cip1 and p27Kip1 aggravated this phenotype, yet MEFs lacking all Cip/Kip proteins exhibited increased retinoblastoma phosphorylation, together with enhanced proliferation and transformation capacity. In vivo, Cdk2 ablation induced partial perinatal lethality in p21-/- p27-/- mice, suggesting partial Cdk2-dependent compensation. However, Cdk2-/- p21-/- p27-/- survivors displayed all phenotypes described for p27-/- mice, including organomegalia and pituitary tumors. Thus, Cip/Kip deficiency does not impair interphasic Cdk activity even in the absence of Cdk2, suggesting that their Cdk-cyclin assembly function is dispensable for homeostatic control in most cell types., This work was supported by grants from the European Research Council (ERC-AG/250297-RAS AHEAD), EU-Framework Programme (LSHG-CT-2007-037665/CHEMORES, HEALTH-F2-2010-259770/LUNGTARGET, and HEALTH-2010-260791/EUROCANPLAT-FORM), Spanish Ministry of Economy and Competitiveness (SAF2011-30173), and Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE) to M.B. C.E.S. is supported by a grant from the La Caixa/CNIO International Ph.D. Fellowship Program sponsored by the La Caixa Foundation (Spain).

The liver has a strong regenerative capacity. After injury, quiescent hepatocytes can reenter the mitotic cell cycle to restore tissue homeostasis. This G0/G1-S cell-cycle transition of primed hepatocytes is regulated by complexes of cyclin-dependent kinase 2 (Cdk2) with E-type cyclins (CcnE1 or CcnE2). However, single genetic ablation of either E-cyclin or Cdk2 does not affect overall liver regeneration. Here, we systematically investigated the contribution of CcnE1, CcnE2, and Cdk2 for liver regeneration after partial hepatectomy (PH) by generating corresponding double- and triple-knockout (KO) mouse mutants. We demonstrate that conditional deletion of Cdk2 alone in hepatocytes resulted in accelerated induction of CcnE1, but otherwise normal initiation of S phase in vivo and in vitro. Excessive CcnE1 did not contribute to a noncanonical kinase activity, but was located at chromatin together with components of the pre-replication complex (pre-RC), such as the minichromosome maintenance (MCM) helicase. Concomitant ablation of Cdk2 and CcnE1 in hepatocytes caused a defect in pre-RC formation and further led to dramatically impaired S-phase progression by down-regulation of cyclin A2 and cell death in vitro and substantially reduced hepatocyte proliferation and liver regeneration after PH in vivo. Similarly, combined loss of CcnE1 and CcnE2, but also the Cdk2/CcnE1/CcnE2 triple KO in liver, significantly inhibited S-phase initiation and liver mass reconstitution after PH, whereas concomitant ablation of CcnE2 and Cdk2 had no effect. Conclusion: In the absence of Cdk2, CcnE1 performs crucial kinase-independent functions in hepatocytes, which are capable of driving MCM loading on chromatin, cyclin A2 expression, and S-phase progression. Thus, combined inactivation of Cdk2 and CcnE1 is the minimal requirement for blocking S-phase machinery in vivo. (Hepatology 2014;59:651–660)

Hypoactive BRAF mutants bind more tightly than wild type to the upstream regulator RAS, thus amplifying to amplify ERK signalling; tumours expressing these mutants require coexistent mechanisms for RAS activation to grow and are sensitive to their inhibition. Mutant alleles of the kinase BRAF have been identified in human tumours, some of which have activated catalytic functions, whereas others have impaired or absent kinase activity. Here Neal Rosen and colleagues have characterized the less-understood hypoactive BRAF mutants. They find that these mutants bind more tightly to the upstream regulator RAS in order to amplify signalling and require coexistent mechanisms for RAS activation in the tumour in order to function. The mechanistic action of these hypoactive BRAF mutants is different to that of the activated mutants and dictates their sensitivity to therapeutic inhibitors of the pathway. Elsewhere in this issue, David Santamaria and colleagues show that kinase-inactive BRAF mutants can initiate the development of lung adenocarcinoma in mice. Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2)1. Here we characterize a third class of BRAF mutants—those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS–GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

Cyclin-dependent kinase (Cdk)7, the catalytic subunit of the Cdk-activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)-mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7-expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death.

The presence of neurogenic precursors in the adult mammalian brain is now widely accepted, but the mechanisms coupling their proliferation with the onset of neuronal differentiation remain unknown. Here, we unravel the major contribution of the G1 regulator cyclin-dependent kinase 6 (Cdk6) to adult neurogenesis. We found that Cdk6 was essential for cell proliferation within the dentate gyrus of the hippocampus and the subventricular zone of the lateral ventricles. Specifically, Cdk6 deficiency prevents the expansion of neuronally committed precursors by lengthening G1 phase duration, reducing concomitantly the production of newborn neurons. Altogether, our data support G1 length as an essential regulator of the switch between proliferation and neuronal differentiation in the adult brain and Cdk6 as one intrinsic key molecular regulator of this process.

Activated oncogenes induce compensatory tumour-suppressive responses, such as cellular senescence or apoptosis, but the signals determining the main outcome remain to be fully understood. Here, we uncover a role for Cdk2 (cyclin-dependent kinase 2) in suppressing Myc-induced senescence. Short-term activation of Myc promoted cell-cycle progression in either wild-type or Cdk2 knockout mouse embryo fibroblasts (MEFs). In the knockout MEFs, however, the initial hyper-proliferative response was followed by cellular senescence. Loss of Cdk2 also caused sensitization to Myc-induced senescence in pancreatic beta-cells or splenic B-cells in vivo, correlating with delayed lymphoma onset in the latter. Cdk2-/- MEFs also senesced upon ectopic Wnt signalling or, without an oncogene, upon oxygen-induced culture shock. Myc also causes senescence in cells lacking the DNA repair protein Wrn. However, unlike loss of Wrn, loss of Cdk2 did not enhance Myc-induced replication stress, implying that these proteins suppress senescence through different routes. In MEFs, Myc-induced senescence was genetically dependent on the ARF-p53-p21Cip1 and p16INK4a-pRb pathways, p21Cip1 and p16INK4a being selectively induced in Cdk2-/- cells. Thus, although redundant for cell-cycle progression and development, Cdk2 has a unique role in suppressing oncogene- and/or stress-induced senescence. Pharmacological inhibition of Cdk2 induced Myc-dependent senescence in various cell types, including a p53-null human cancer cell line. Our data warrant re-assessment of Cdk2 as a therapeutic target in Myc- or Wnt-driven tumours.

It's the degree of activation, not a specific cyclin-dependent kinase, that is important for genome maintenance in postreplicative cells., In response to DNA damage, cells activate a phosphorylation-based signaling cascade known as the DNA damage response (DDR). One of the main outcomes of DDR activation is inhibition of cyclin-dependent kinase (Cdk) activity to restrain cell cycle progression until lesions are healed. Recent studies have revealed a reverse connection by which Cdk activity modulates processing of DNA break ends and DDR activation. However, the specific contribution of individual Cdks to this process remains poorly understood. To address this issue, we have examined the DDR in murine cells carrying a defined set of Cdks. Our results reveal that genome maintenance programs of postreplicative cells, including DDR, are regulated by the overall level of Cdk activity and not by specific Cdks.

Unicellular organisms such as yeasts require a single cyclin-dependent kinase, Cdk1, to drive cell division. In contrast, mammalian cells are thought to require the sequential activation of at least four different cyclin-dependent kinases, Cdk2, Cdk3, Cdk4 and Cdk6, to drive cells through interphase, as well as Cdk1 to proceed through mitosis. This model has been challenged by recent genetic evidence that mice survive in the absence of individual interphase Cdks. Moreover, most mouse cell types proliferate in the absence of two or even three interphase Cdks. Similar results have been obtained on ablation of some of the activating subunits of Cdks, such as the D-type and E-type cyclins. Here we show that mouse embryos lacking all interphase Cdks (Cdk2, Cdk3, Cdk4 and Cdk6) undergo organogenesis and develop to midgestation. In these embryos, Cdk1 binds to all cyclins, resulting in the phosphorylation of the retinoblastoma protein pRb and the expression of genes that are regulated by E2F transcription factors. Mouse embryonic fibroblasts derived from these embryos proliferate in vitro, albeit with an extended cell cycle due to inefficient inactivation of Rb proteins. However, they become immortal on continuous passage. We also report that embryos fail to develop to the morula and blastocyst stages in the absence of Cdk1. These results indicate that Cdk1 is the only essential cell cycle Cdk. Moreover, they show that in the absence of interphase Cdks, Cdk1 can execute all the events that are required to drive cell division.

Cyclin-dependent kinases (Cdks) are the catalytic subunits of a family of mammalian heterodimeric serine/threonine kinases that have been implicated in the control of cell-cycle progression, transcription and neuronal function. Recent genetic evidence obtained with gene-targeted mice has shown that Cdk4 and Cdk6 are not needed for entry into the cell cycle after mitogenic stimuli and organogenesis; however, they are essential for the proliferation of some endocrine and hematopoietic cells. Cdk2 is also dispensable for the mitotic cell cycle. Indeed, mice without Cdk2 are normal except for their complete sterility: unexpectedly, Cdk2 is crucial for the first meiotic division of male and female germ cells. These findings have important implications both for our current understanding of the role of Cdks in regulating the mammalian cell cycle and for their potential use as therapeutic targets in cancer.

Most human tumors harbor mutations that misregulate the early phases of the cell cycle. Here, we summarize genetic evidence, mostly obtained in our laboratory using strains of gene-targeted mice, that provides direct experimental support for a role of Cdk4 in tumor development. Moreover, these genetic studies challenge some well-established concepts regarding the role of Cdks during the early phases of the cell cycle. For instance, they have illustrated that Cdk4 and Cdk6 are not essential for cell division during embryonic development except in the hematopoietic system. More surprisingly, mice lacking Cdk2 survive for over 2 years without detectable abnormalities except in their germ cells, indicating that Cdk2 is essential for meiosis but dispensable for the normal mitotic cell cycle. Cdk2 is also dispensable for cell cycle inhibition and tumor suppression by the Cip/Kip inhibitors, p21(Cip1) and p27(Kip1). These observations have important implications not only to understand cell cycle regulation, but also to validate Cdks as potential targets for the development of therapeutic strategies to block proliferation of tumor cells.

We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2-/- mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.

Approximately two hundred mutant BRAF alleles have been identified in human tumors. Physiologic activation of RAF isoforms requires RAS-dependent induction of their dimerization. Activating BRAF mutants cause ERK dependent feedback inhibition of RAS.GTP and are RAS independent. As we have shown previously, they signal either as active monomers or RAS-independent constitutively active dimers. Here, we characterized a third class of BRAF mutants—those that have impaired kinase activity or are kinase dead. These class 3 BRAF mutants are sensitive to ERK-mediated feedback and they function in a RAS-dependent manner. In tumors, they bind more tightly to active RAS, thus enhancing their heterodimerization with CRAF. This is associated with the amplification of RAS-RAF-MEK-ERK signaling. Since these mutants are sensitive to ERK-dependent feedback inhibition of RAS, their enhancement of ERK signaling in tumors requires concurrent dysregulation of RAS activation. Thus, melanomas with Class 3 mutations usually harbor coexistent RAS mutation or NF1 mutants/deletion, whereas receptor tyrosine kinase signaling is activated in lung and colorectal cancers with these mutants. Our model suggests that these tumors will be sensitive to the inhibition of RAS activation. Currently, no direct inhibitors of RAS activation are available. However, in support of this idea, inhibitors of activated RTK signaling in carcinomas with Class 3 BRAF mutants and wild type RAS is sufficient to markedly inhibit ERK signaling and their growth in in vivo murine models and in patients. We have thus defined a third subset of BRAF mutants, which is RAS-dependent. Tumors harboring such mutants are sensitive to tyrosine kinase inhibitors in tumors expressing wild type RAS and NF1. Citation Format: Zhan Yao, Rona Yaeger, Vanessa S. Rodrik-Outmezguine, Anthony Tao, Neilawattie M. Torres, Matthew T. Chang, Matthias Drosten, Huiyong Zhao, Fabiola Cecchi, Todd Hembrough, Judith Michels, Hervé Baumert, Linde Miles, Naomi M. Campbell, Elisa de Stanchina, David B. Solit, Mariano Barbacid, Barry S. Taylor, Neal Rosen. Tumors with class 3 BRAF mutants are sensitive to the inhibition of activated RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2017-4078

Entry of quiescent cells into the cell cycle is driven by the cyclin D‐dependent kinases Cdk4 and Cdk6. These kinases are negatively regulated by the INK4 cell cycle inhibitors. We report the generation of mice defective in P15 INK4b and P18 INK4c . Ablation of these genes, either alone or in combination, does not abrogate cell contact inhibition or senescence of mouse embryo fibroblasts in culture. However, loss of P15 INK4b , but not of P18 INK4c , confers proliferative advantage to these cells and makes them more sensitive to transformation by H‐ ras oncogenes. In vivo , ablation of P15 INK4b and P18 INK4c genes results in lymphoproliferative disorders and tumor formation. Mice lacking P18 INK4c have deregulated epithelial cell growth leading to the formation of cysts, mostly in the cortical region of the kidneys and the mammary epithelium. Loss of both P15 INK4b and P18 INK4c does not result in significantly distinct phenotypic manifestations except for the appearance of cysts in additional tissues. These results indicate that P15 INK4b and P18 IKN4c are tumor suppressor proteins that act in different cellular lineages and/or pathways with limited compensatory roles.

Recent studies have suggested a role for neurotrophins in the growth and refinement of neural connections, in dendritic growth, and in activity-dependent adult plasticity. To unravel the role of endogenous neurotrophins in the development of neural connections in the CNS, we studied the ontogeny of hippocampal afferents intrkB (−/−) andtrkC (−/−) mice. Injections of lipophilic tracers in the entorhinal cortex and hippocampus of newborn mutant mice showed that the ingrowth of entorhinal and commissural/associational afferents to the hippocampus was not affected by these mutations. Similarly, injections of biocytin in postnatal mutant mice (P10–P16) did not reveal major differences in the topographic patterns of hippocampal connections.In contrast, quantification of biocytin-filled axons showed that commissural and entorhinal afferents have a reduced number of axon collaterals (21–49%) and decreased densities of axonal varicosities (8–17%) in bothtrkB (−/−) andtrkC (−/−) mice. In addition, electron microscopic analyses showed thattrkB (−/−) andtrkC (−/−) mice have lower densities of synaptic contacts and important structural alterations of presynaptic boutons, such as decreased density of synaptic vesicles. Finally, immunocytochemical studies revealed a reduced expression of the synaptic-associated proteins responsible for synaptic vesicle exocytosis and neurotransmitter release (v-SNAREs and t-SNAREs), especially intrkB (−/−) mice. We conclude that neithertrkB nortrkC genes are essential for the ingrowth or layer-specific targeting of hippocampal connections, although the lack of these receptors results in reduced axonal arborization and synaptic density, which indicates a role for TrkB and TrkC receptors in the developmental regulation of synaptic inputs in the CNSin vivo. The data also suggest that the genes encoding for synaptic proteins may be targets of TrkB and TrkC signaling pathways.

The impact of the nerve growth factor (NGF) family of neurotrophins and their receptors was examined on the cutaneous innervation in the mystacial pads of mice. Ten sets of unmyelinated and thinly myelinated sensory and autonomic innervation were evaluated that terminated in the epidermis, upper dermis, and upper part of the intervibrissal hair follicles. Mystacial pads were analyzed from newborn to 4-week-old mice that had homozygous functional deletions of the genes for NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), tyrosine kinase (trk) A, trkB, trkC, or p75. Mystacial pads were also analyzed in adult transgenic mice that had overproduction of NGF, BDNF, or NT-3 driven by a keratin promoter gene. The innervation was revealed by using immunofluorescence and immunocytochemistry with antibodies for protein gene product (PGP) 9.5, calcitonin gene-related product (CGRP), substance P (SP), galanin (GAL), neuropeptide Y (NPY), tyrosine hydroxylase (TH), and a neurofilament protein. The cumulative results indicated that NGF/trkA signaling plays a major role in the outgrowth and proliferation of sensory axons, whereas NT-3/ trkA signaling plays a major role in the formation of sensory endings. TrkC is also essential for the development of three sets of trkA-dependent sensory innervation that coexpress CGRP, SP, and GAL. Another set of sensory innervation that only coexpressed CGRP and SP was solely dependent upon NGF and trkA. Surprisingly, most sets of trkA-dependent sensory innervation are suppressed by trkB perhaps interacting with p75. BDNF and NT-4 appear to mediate this suppressing effect in the upper dermis and NT-4 in the epidermis. In contrast to sensory innervation, sympathetic innervation to the necks of intervibrissal hair follicles depends upon NGF/trkA signaling interacting with p75 for both the axon outgrowth and ending formation. Although NT-3/trkA signaling is essential for the full complement of sympathetic neurons, NT-3 is detrimental to the formation of sympathetic terminations to the necks of hair follicles. TrkB signaling mediated by BDNF but not NT-4 also suppresses these sympathetic terminations. One sparse set of innervation, perhaps parasympathetic, terminating at the necks of hair follicles is dependent solely upon NT-3 and trkC. Taken together, our results indicate that the innervation of the epidermis, upper dermis, and the upper portion of hair follicles is regulated by a competitive balance between promoting and suppressing effects of the various neurotrophins.

The Eph family of tyrosine protein kinase receptors and their cognate ligands, a family of membrane-bound molecules known as ephrins, have been implicated in diverse biological processes, such as axon guidance, the formation of topographic neural projections, axon fasciculation, the regionalization of the central nervous system, and cell migration. The recent generation of mutant mice lacking some of these receptors has made possible the dissection of the role that these molecules play in the development of the mammalian nervous system.

The impact of null mutations of the genes for the NGF family of neurotrophins and their receptors was examined among the wide variety of medium to large caliber myelinated mechanoreceptors which have a highly specific predictable organization in the mystacial pad of mice. Immunofluorescence with anti-protein gene product 9.5, anti-200-kDa neurofilament protein (RT97), and anti-calcitonin gene-related product was used to label innervation in mystacial pads from mice with homozygous null mutations for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), the three tyrosine kinase receptors (trkA, trkB, trkC), and the low-affinity nerve growth factor receptor p75. Specimens were sacrificed at birth and at 1, 2, and 4 weeks for each type of mutation as well as at 11 weeks and 1 year for p75 and trkC mutations, respectively. Our results demonstrate several major concepts about the role of neurotrophins in the development of cutaneous mechanoreceptors that are supplied by medium to large caliber myelinated afferents. First, each of the high-affinity tyrosine kinase receptors, trkA, trkB, and trkC, as well as the low-affinity p75 receptor has an impact on at least one type of mechanoreceptor. Second, consistent with the various affinities for particular trk receptors, the elimination of NGF, BDNF, and NT-3 has an impact comparable to or more complex than the absence of their most specific high-affinity receptors: trkA, trkB, and trkC, respectively. These complexities include potential NT-3 signaling through trkA and trkB to support some neuronal survival. Third, most types of afferents are dependent on a different combination of neurotrophins and receptors for their survival: reticular and transverse lanceolate afferents are dependent upon NT-3, NGF, and trkA; Ruffini afferents upon BDNF and trkB; longitudinal lanceolate afferents upon NGF, trkA, BDNF, and trkB; and Merkel afferents on NGF, trkA, NT-3, trkC, and p75. NT-4 has no obvious detrimental impact on the mechanoreceptor development in the presence of BDNF. Fourth, NT-4 and BDNF signaling through trkB may suppress Merkel innervation and NT-3 signaling through trkC may suppress Ruffini innervation. Finally, regardless of the neurotrophin/receptor dependency for afferent survival and neurite outgrowth, NT-3 has an impact on the formation of all the sensory endings. In the context of these findings, indications of competitive and suppressive interactions that appear to regulate the balance of innervation density among the various sets of innervation were evident.

Analysis of mice carrying targeted mutations in genes encoding neurotrophins and their signalling Trk receptors has provided critical information regarding the role that these molecules play in the mammalian nervous system. In this study we generated mice defective in both TrkB and TrkC tyrosine kinase receptors to determine the biological effects of these receptors in the absence of compensatory mechanisms. trkB(-/-);trkC(-/-) double-mutant mice were born at the expected frequency, indicating that TrkB and TrkC signalling are not required for embryonic survival. However, these double-mutant mice had a significantly shorter lifespan and displayed more severe sensory defects than their single-mutant trkB(-/-) and trkC(-/-) littermates. The most dramatic sensory deficit observed in trkB(-/-);trkC(-/-) mutant mice was the absence of vestibular and cochlear ganglia. Interestingly, these mice developed inner ear sensory epithelia in spite of the complete absence of sensory innervation. Analysis of the CNS in trkB(-/-);trkC(-/-) mutant mice revealed a well formed hippocampus, cortex and thalamus. Moreover, the pattern of expression of several neuronal markers appeared normal in these animals. These observations suggest that neurotrophin signalling through TrkB and TrkC receptors is essential for the development of sensory ganglia; however, it does not play a major role in the differentiation and survival of CNS neurons during embryonic development.

The alleged ability of taste afferents to induce taste buds in developing animals is investigated using a mouse model with a targeted deletion of the tyrosine kinase receptor trk B for the neurotrophin BDNF. This neurotrophin was recently shown to be expressed in developing taste buds and the receptor trk B has been shown to be expressed in the developing ganglion cells that innervate the taste buds. Our data show a reduction of geniculate ganglion cells to about 5% of control animals in neonates. Degeneration of ganglion cells starts when processes reach the central target (solitary tract[ but before they reach the peripheral target (taste buds). Degeneration of ganglion cells is almost completed in trk B knockout mice before taste afferents reach in control animals the developing fungiform papillae. Four days later the first taste buds can be identified in fungiform papillae of both control and trk B knockout mice in about equal number and density. Many taste buds undergo a normal maturation compared to control animals. However, the more lateral and caudal fungiform papillae grow less in size and become less conspicuous in older trk B knockout mice. No intragemmal innervation can be found in trk B knockout taste buds but a few extragemmal fibers enter the apex and end between taste bud cells without forming specialized synapses. Taste buds of trk B knockout mice appear less well organized than those of control mice, but some cells show similar vesicle accumulations as control taste bud cells in their base but no synaptic contact to an afferent. These data strongly suggest that the initial development of many fungiform papillae and taste buds is independent of the specific taste innervation. It remains to be shown why others appear to be more dependent on proper innervation.

Determinations of dorsal root ganglion (DRG) neuron loss in nerve growth factor (NGF) and neurotrophin-3 (NT-3) null mutant mice have supported the concept that neurons can switch neurotrophin dependence by revealing that many neurons must require both of these factors acting either sequentially or simultaneously during development. The situation is complex, however, in thatNT-3(−/−)mutant mice show far greater neuron loss than mice deficient in the NT-3 receptor TrkC, suggesting that NT-3 may support many DRG neurons via actions on the NGF receptor TrkA. To assess the possibility of ligand-receptor cross-talk as a developmental mechanism, we have compared the onset of survival dependence of lumbar DRG neurons on NT-3, TrkC, NGF, and TrkA signaling in mice deficient in these molecules as a result of gene targeting. At embryonic day 11.5 (E11.5), virtually all lumbar DRG cells express TrkC mRNA and many require NT-3 and TrkC signaling for survival. In contrast, although many lumbar DRG cells also express TrkA at E11.5, there is little survival dependence on TrkA signaling. By E13.5, most lumbar DRG cells have downregulated TrkC mRNA. The onset of survival dependence on NGF and TrkA-signaling is concurrent and of equal magnitude at E13.5, demonstrating that NT-3 alone does not support DRG neurons via TrkA, nor can NT-3 compensate for the loss of NGF. We conclude that many murine DRG cells require NT-3 for survival before exhibiting NGF dependence and that NT-3 activation of TrkA is unimportant to these early NT-3 survival-promoting actions. We suggest that the discrepancy in cell loss betweenNT-3(−/−)andtrkC(−/−)mutants is attributable to the ability of NT-3 to support DRG neurons via TrkA in the artificial situation where TrkC is absent.

Protein prenylation is increasingly recognized as an important mechanism by which functional association of proteins to membranes is mediated. Ras proteins, regulators of cell proliferation and differentiation, are among the proteins that undergo farnesylation, one of the two prenylation modifications known. Since ras proteins are activated into hyperactive oncogenic versions in a wide variety of human cancers, agents that down modulate ras activity could be antineoplastic. Therefore, inhibitors of farnesyltransferase have the potential to be of therapeutic value as anticancer agents due to their ability to block ras processing and hence its function. We describe the identification of two farnesyl pyrophosphate (FPP) analogs that are potent and selective inhibitors of farnesyltransferase. While showing no toxicity to untransformed cells, a pivaloyloxymethyl ester of one of these inhibitors blocked ras mediated transformation of NIH 3T3 cells. In addition, both the ester and its parent acid inhibited ras farnesylation as measured by incorporation of labeled mevalonate into ras proteins in whole cells. Thus, this is the first report of an FPP analog to show biological activity by inhibiting ras processing in whole cells. © 1995 Wiley-Liss, Inc.

Several new members of the nerve growth factor family of neurotrophins, which comprises nerve growth factor itself, brain-derived neurotrophic factor and neurotrophins-3, -4 (also known as neurotrophin-5) and -6, have been isolated in recent years. Their signaling receptors have been identified as the Trk family of tyrosine protein kinases, thus facilitating the dissection of the signaling pathways responsible for mediating their trophic properties. More recently, the advent of gene targeting has made it possible to generate strains of mice lacking neurotrophins and their receptors. Analysis of the phenotypes of these mutant animals has provided detailed information on the role that neurotrophins and their receptors play in the ontogeny of the mammalian nervous system.

Accumulating evidence indicates that the Trk family of tyrosine protein kinase receptors, Trk (also known as TrkA), TrkB, and TrkC, are responsible for mediating the trophic effects of the NGF family of neurotrophins. Nerve growth factor (NGF) specifically recognizes Trk, a receptor identified in all major NGF targets, including sympathetic, trigeminal, and dorsal root ganglia as well as in cholinergic neurons of the basal forebrain and the striatum. Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) specifically activate the TrkB tyrosine kinase receptor. trkB transcripts encoding this receptor are found throughout multiple structures of the central and peripheral nervous system. Neurotrophin-3 (NT-3) primarily activates the TrkC tyrosine protein kinases, four related isoforms encoded by alternative splicing of trkC, a gene also widely expressed throughout the mammalian nervous system. Unlike the other neurotrophins, NT-3 appears to be somewhat promiscuous since it can activate Trk and TrkB kinase receptors, at least in certain cell systems. The trkB and trkC genes also encode noncatalytic neurotrophin receptor isoforms of an as yet, unknown function. Recently, strains of mice lacking each of these tyrosine kinase receptors have been generated. Preliminary characterization of these mutant mice has provided significant information regarding the role of these receptors in the ontogeny of the mammalian nervous system. For instance, mice deficient for Trk receptors lack most sympathetic neurons and do not display nociceptive and temperature sensations, two defects likely to result from severe neuronal cell loss in their trigeminal and dorsal root ganglia. Mice lacking TrkB tyrosine kinase receptors die postnatally due to their inability to intake food. Neuron cell loss in their trigeminal, nodose and petrosal sensory ganglia as well as in the facial motor nucleus are likely to contribute to this phenotype. Finally, TrkC-deficient mice display strikingly abnormal movements consistent with loss of proprioception, a defect likely to be a consequence of the complete loss of Ia muscle afferents observed in this mutant mice.

K-RAS oncogenes have been implicated in about one fifth of all human cancers including lung adenocarcinoma and pancreatic ductal adenocarcinoma, two tumor types with some of the worse prognosis. In order to identify effective therapeutic strategies to treat these tumors, we have developed genetically engineered mouse (GEM) models that closely recapitulate their natural history. We are using these mice to validate targets of potential therapeutic value with the ultimate goal to translate these findings to the clinic. In previous studies, we have crossed these GEM models with mice that carried either germ line or lox-Cre conditional knock out loci encoding each of the downstream kinases of the Raf/Mek/Erk pathway as well as the cell cycle interphase Cdks to interrogate their role in the development of K-Ras driven lung adenocarcinomas. These studies have led us to validate the c-Raf and Cdk4 kinases as essential targets for these tumors (Puyol et al., Cancer Cell 2010; Blasco et al., Cancer Cell 2011). Following a similar strategy, we have demonstrated that the EGF Receptor and c-Raf were also absolutely essential for the development of pancreatic ductal adenocarcinomas (Navas et al., Cancer Cell 2012). Importantly, systemic ablation of either c-Raf or Cdk4 has no significant effect on normal homeostasis. Now, we have decided to interrogate the role of these targets in advanced tumors. To this end, we have generated new GEM models in which expression of the resident K-Ras oncogene, as well as ablation of the p53 tumor suppressor is mediated by the frt-FLp(o) targeting system, a strategy that allows temporal separation of tumor development from target ablation. In addition, we have generated lox-Cre based conditional knock-in strains that upon recombination direct the expression of kinase dead isoforms instead of inducing protein ablation. We feel that these strains should serve as better models to mimic drug intervention in the clinic. Finally, we are now combining ablation (or inactivation) of these targets in order to identify combinations that may eventually eradicate advanced tumors. We hope that these studies will serve to guide the design of future clinical trails to treat patients carrying K-RAS mutant tumors. Citation Format: Mariano Barbacid. Targeting K-RAS driven cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL03-01.

Tyrosine protein kinases are likely to play an important role in the maintenance and/or development of the nervous system. In this study we have used the PCR cloning technique to isolate sequences derived from tyrosine kinase genes expressed in cultured hippocampal neurons obtained from 17.5-day-old rat embryos. Nucleotide sequence analysis of 209 independent clones revealed sequences derived from 25 tyrosine kinases, of which two corresponded to previously unreported genes. One of the PCR clones, ptk-2, belongs to the Jak family of cytoplasmic tyrosine kinases. The second clone, ptk-3, was derived from a gene encoding an additional class of tyrosine kinase receptors whose extracellular domains contain regions of homology with coagulation factors V and VIII and complement component C1. Transcripts encoding the Ptk-3 receptor are present in a variety of embryonic and adult tissues with highest levels observed in brain. During development, ptk-3 transcripts are most abundant in the proliferative neuroepithelial cells of the ventricular zone, raising the possibility that this receptor may play an important role in the generation of the mammalian nervous system.

Interleukin-2 (IL-2) is a growth factor involved in the clonal expansion of antigen-activated T lymphocytes. Interaction of IL-2 with its receptor triggers tyrosine phosphorylation of a series of proteins and results in the activation of p21ras. We report here that Shc, an SH2-containing adaptor protein, is tyrosine-phosphorylated following IL-2 stimulation. IL-2-induced tyrosine phosphorylation of Shc was detectable within seconds following IL-2 addition, reaching its highest level by 15 min. Tyrosine phosphorylation of Shc was induced in multiple IL-2-dependent T cell lines and was found to correlate with IL-2-dependent cell proliferation. Tyrosine-phosphorylated Shc was found to be capable of associating with the SH2 domain of Grb2 following IL-2 stimulation. These results indicate that tyrosine phosphorylation of Shc and its association with Grb2 may be important events in IL-2-initiated signal transduction events in T cells.

Toll-like receptors (TLRs) modulate the expression of multiple microRNAs (miRNAs). Here, we report the down-regulation of miR-107 by TLR4 in multiple cell types. The miR-107 sequence occurs in an intron within the sequence encoding the gene for pantothenate kinase 1α (PanK1α), which is regulated by the transcription factor peroxisome proliferator-activating receptor α (PPAR-α). PanK1α is also decreased in response to lipopolysaccharide (LPS). The effect on both miR-107 and PanK1α is consistent with a decrease in PPAR-α expression. We have found that the putative miR-107 target cyclin-dependent kinase 6 (CDK6) expression is increased by TLR4 as a result of the decrease in miR-107. This effect is required for increased adhesion of macrophages in response to LPS, and CDK6-deficient mice are resistant to the lethal effect of LPS. We have therefore identified a mechanism for LPS signaling which involves a decrease in miR-107 leading to an increase in CDK6.

K-Ras mutations are frequent in colorectal cancer (CRC), albeit K-Ras is the only Ras isoform that can elicit apoptosis. Here, we show that mutant K-Ras directly binds to the tumor suppressor RASSF1A to activate the apoptotic MST2-LATS1 pathway. In this pathway LATS1 binds to and sequesters the ubiquitin ligase Mdm2 causing stabilization of the tumor suppressor p53 and apoptosis. However, mutant Ras also stimulates autocrine activation of the EGF receptor (EGFR) which counteracts mutant K-Ras-induced apoptosis. Interestingly, this protection requires the wild-type K-Ras allele, which inhibits the MST2 pathway in part via AKT activation. Confirming the pathophysiological relevance of the molecular findings, we find a negative correlation between K-Ras mutation and MST2 expression in human CRC patients and CRC mouse models. The small number of tumors with co-expression of mutant K-Ras and MST2 has elevated apoptosis rates. Thus, in CRC, mutant K-Ras transformation is supported by the wild-type allele., This work was supported by Cancer Research UK, the European Union FP6 STREP Growthstop (LSHC-CT-2006-037731), Science Foundation Ireland under Grant No. 06/CE/B1129, and a Kuwait Foundation for the Advancement of Sciences grant (2006-1302-07) and Research Core Facility grant GM01/05.

Summary We have generated mice carrying a germline mutation in the tyrosine kinase catalytic domain of the trkB gene. This mutation eliminates expression of gp145 trkB , a protein-tyrosine kinase that serves as the signaling receptor for two members of the nerve growth factor family of neurotrophins, brain-derived neurotrophic factor and neurotrophin-4. Mice homozygous for this mutation, trkB TK (−l−), develop to birth. However, these animals do not display feeding activity, and most die by P1. Neuroanatomical examination of trkB TK (−l−) mice revealed neuronal deficiencies in the central (facial motor nucleus and spinal cord) and peripheral (trigeminal and dorsal root ganglia) nervous systems. These findings illustrate the role of the gp 145 trkB protein-tyrosine kinase receptor in the ontogeny of the mammalian nervous system.

We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein.

Trk-immunoreactivity was observed in basal forebrain and striatal cholinergic neurons, whereas low-affinity NGF receptor immunoreactivity was observed in basal forebrain but not striatal cholinergic neurons. Since NGF exerts trophic actions on both basal forebrain and striatal cholinergic populations, the presence of Trk in these neurons lends strong support for an essential role of Trk in NGF-responsive neurons, but suggests that the low affinity receptor is not necessary for NGF actions in the striatum.

We have exploited a battery of approaches to address several controversies that have accompanied the expansion of the nerve growth factor (NGF) family of neurotrophic factors and the identification of the Trk tyrosine kinases as receptors for these factors. For example, we find that a recently cloned mammalian neurotrophin, known as either neurotrophin-4 or neurotrophin-5 and assigned widely differing receptor specificities, represents the functional counterpart of Xenopus neurotrophin-4 and is a “preferred” ligand for TrkB. However, its interactions with TrkB can be distinguished from those of brain-derived neurotrophic factor (BDNF) with TrkB. We also find that all of the Trks display similar dose responses to their “preferred” ligands in neuronal as compared with nonneuronal cells (i.e., NGF for TrkA, BDNF and NT-4/5 for TrkB, and NT-3 for TrkC), providing evidence against a role for accessory molecules expressed in neurons in generating receptors that would allow for responses to lower concentrations of the neurotrophins. However, we find that a neuronal environment does restrict the Trks in their ability to respond to their “nonpreferred” neurotrophin ligands.

Genetic interrogation of the mammalian cell cycle has revealed that the essential role of interphase Cdks is not to specifically drive the various phases of the cycle, as previously proposed in widely accepted models, but to sustain proliferation of specialized cells at various times during embryonic or postnatal development. Indeed, genetic studies have indicated that Cdk1 can drive the mammalian cell cycle in the absence of interphase Cdks. The molecular bases for the essential requirement of interphase Cdks in selected cell types are still poorly understood. However, these observations have important implications for understanding the role of Cdk misregulation in cancer. Indeed, it is likely that misregulation of Cdks may only confer proliferative advantages to selected cell types. More importantly, it is also possible that certain cells may become dependent of selective interphase Cdks only when their proliferation is driven by defined oncogenes. Recent studies have illustrated the requirement for Cdk4 in HER2-overexpressed mammary adenocarcinomas and K-Ras oncogene-driven lung adenocarcinomas but not in the corresponding normal tissues. Likewise, Cdk2 plays an important role in the development of Myc-induced lymphomas. These findings may open the door to the design of novel therapeutic strategies that may benefit cancer patients.

Several proteins implicated in the regulation of cellular responses to mitogenic stimuli contain a common non-catalytic domain, SH2 (for src-homologous domain 2), that mediates their interaction with activated tyrosine protein kinases. Here we report that p95vav, a proto-oncogene product specifically expressed in cells of the haematopoietic system, contains an SH2 domain and is a substrate for tyrosine protein kinases. Exposure of quiescent NIH3T3 cells ectopically expressing p95vav to either epidermal or platelet-derived growth factors induces the rapid phosphorylation of this protein on tyrosine residues. Activation of the receptors for these growth factors by their cognate ligand results in their association with p95vav, a process mediated by its SH2 domain. In T cells, co-activation of the T-cell receptor and the accessory CD4 cell-surface protein also results in the phosphorylation of the endogenous p95vav protein in tyrosine residues. Phosphorylation of p95vav is rapid, transient and precedes the appearance of most other phosphotyrosine-containing proteins. In addition to the SH2 domain, p95vav contains structural motifs not found in other tyrosine kinase substrates. One such motif is a helix-loop-helix/leucine zipper-like domain which shares some sequence similarity with these motifs in the Myc and Max proteins. Deletion of the helix-loop-helix-like motif causes oncogenic activation of p95vav. These results indicate that p95vav is a new type of signal transduction molecule and suggest a possible role for this protein in the transduction of tyrosine phosphorylation signalling into transcriptional events.

Synergism between the RAS and Hedgehog (HH) pathways has been suggested for carcinogenesis in the pancreas, lung and colon. We investigated the molecular cross-talk between RAS and HH signaling and found that, although mutant RAS induces or enhances SHH expression and favors paracrine HH signaling, it antagonizes autocrine HH signal transduction. Activated RAS can be found in primary cilia, the central organelle of HH signal transduction, but functions in a cilium-independent manner and interferes with Gli2 function and Gli3 processing. In addition, the cell-autonomous negative regulation of HH signal transduction involves the RAS effector molecule dual specificity tyrosine phosphorylated and regulated kinase 1B (DYRK1B). In line with a redirection of autocrine toward paracrine HH signaling by a KRAS-DYRK1B network, we find high levels of GLI1 expression restricted to the stromal compartment and not to SHH-expressing tumor cells in human pancreatic adenocarcinoma.

Tumour-associated cell cycle defects are often mediated by alterations in cyclin-dependent kinase (CDK) activity. Misregulated CDKs induce unscheduled proliferation as well as genomic and chromosomal instability. According to current models, mammalian CDKs are essential for driving each cell cycle phase, so therapeutic strategies that block CDK activity are unlikely to selectively target tumour cells. However, recent genetic evidence has revealed that, whereas CDK1 is required for the cell cycle, interphase CDKs are only essential for proliferation of specialized cells. Emerging evidence suggests that tumour cells may also require specific interphase CDKs for proliferation. Thus, selective CDK inhibition may provide therapeutic benefit against certain human neoplasias.

The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.

The identification of ras oncogenes in human and animal cancers including precancerous lesions indicates that these genes participate in the early stages of neoplastic development. Yet, these observations do not define the timing of ras oncogene activation in the multistep process of carcinogenesis. To ascertain the timing of ras oncogene activation, an animal model system was devised that involves the induction of mammary carcinomas in rats exposed at birth to the carcinogen nitrosomethylurea. High-resolution restriction fragment length polymorphism analysis of polymerase chain reaction-amplified ras sequences revealed the presence of both H-ras and K-ras oncogenes in normal mammary glands 2 weeks after carcinogen treatment and at least 2 months before the onset of neoplasia. These ras oncogenes can remain latent within the mammary gland until exposure to estrogens, demonstrating that activation of ras oncogenes can precede the onset of neoplasia and suggesting that normal physiological proliferative processes such as estrogen-induced mammary gland development may lead to neoplasia if the targeted cells harbor latent ras oncogenes.

We have cloned and characterized the mouse homolog of the human trk proto-oncogene, a member of the protein tyrosine kinase (TK) receptor gene family. Here, we present the first report of a trk-encoded mRNA species in vivo. In situ hybridization analysis in the mouse embryo reveals a striking temporal and spatial regulation of trk transcription, with expression confined to the sensory cranial (trigeminal, superior, jugular) and dorsal root ganglia (DRG) of neural crest origin. Recent reports have shown that TK receptors can play regulatory roles in embryonic development. Thus, the developmental mutations W in mouse and torso and sevenless in Drosophila represent genes that code for defective TK receptors. Our data show that trk, a gene associated with malignancy in humans, is a specific marker for a set of neural crest-derived sensory neurons, and are consistent with the hypothesis that this proto-oncogene may have an important role in the development or phenotype of the neurons where it is expressed.

The pursuit for drugs that inhibit cyclin-dependent kinases (CDKs) has been an intense area of research for more than 15 years. The first-generation inhibitors, Flavopiridol and CY-202, are in late-stage clinical trials, but so far have demonstrated only modest activity. Several second-generation inhibitors are now in clinical trials. Future approaches to determine clinical benefit need to incorporate both the lessons learned from these early compounds and information recently obtained from the genetic analysis of CDKs in preclinical models. Here we discuss key concepts that should be considered when validating the clinical utility of CDK inhibitors in cancer therapy.