Targeting the MAPK Pathway in KRAS-Driven Tumors.

KRAS mutations occur in a quarter of all of human cancers, yet no selective drug has been approved to treat these tumors. Despite the recent development of drugs that block KRASG12C, the majority of KRAS oncoproteins remain undruggable. Here, we review recent efforts to validate individual components of the mitogen-activated protein kinase (MAPK) pathway as targets to treat KRAS-mutant cancers by comparing genetic information derived from experimental mouse models of KRAS-driven lung and pancreatic tumors with the outcome of selective MAPK inhibitors in clinical trials. We also review the potential of RAF1 as a key target to block KRAS-mutant cancers.This work was supported by grants from the European Research Council (ERC-AG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00 and RTC2017-6576-1), the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM), and the Asociacion Espanola contra el Cancer (GC166173694BARB). M.B. is a recipient of an Endowed Chair from the AXA Research Fund.S

Targeting KRAS mutant lung cancer: light at the end of the tunnel.

For decades, KRAS mutant lung adenocarcinomas (LUAD) have been refractory to therapeutic strategies based on personalized medicine owing to the complexity of designing inhibitors to selectively target KRAS and downstream targets with acceptable toxicities. The recent development of selective KRASG12C inhibitors represents a landmark after 40 years of intense research efforts since the identification of KRAS as a human oncogene. Here, we discuss the mechanisms responsible for the rapid development of resistance to these inhibitors, as well as potential strategies to overcome this limitation. Other therapeutic strategies aimed at inhibiting KRAS oncogenic signaling by targeting either upstream activators or downstream effectors are also reviewed. Finally, we discuss the effect of targeting the mitogen-activated protein kinase (MAPK) pathway, both based on the failure of MEK and ERK inhibitors in clinical trials, as well as on the recent identification of RAF1 as a potential target due to its MAPK-independent activity. These new developments, taken together, are likely to open new avenues to effectively treat KRAS mutant LUAD.This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6576, RTI2018-094664-BI00) the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM) and the CRIS Cancer Foundation (to MB) as well as the Spanish Ministry of Science and Innovation (PID2020-116705RB-100) (to MD). MB is a recipient of an Endowed Chair from the AXA Research Fund.S

Gentherapeutische Intervention beim medullären Schilddrüsenkarzinom durch adenoviral vermittelte Expression dominant-negativer RET Proto-Onkogen Mutanten

Als Ursache für das Auftreten sowohl sporadischer als auch hereditärer Formen des medullären Schilddrüsenkarzinoms (MTC) gelten aktivierende Mutationen im RET Proto-Onkogen, die in über 95% aller untersuchten Karzinome gefunden wurden. Das RET Proto-Onkogen kodiert für eine Rezeptor-Tyrosinkinase, die im Normalfall an Entwicklungsprozessen beteiligt ist. Durch onkogene Mutationen kommt es dabei zur konstitutiven Aktivierung der Tyrosinkinase, was hier eine entscheidende Bedeutung für die Tumorentstehung hat. Ziel der Arbeit war es daher, durch Gentransfer dominant-negativer Mutanten des RET Rezeptors, die onkogen veränderten RET Proteine in einer MTC-Zelllinie (TT) zu inhibieren und die daraus entstehenden Konsequenzen in Bezug auf eine potenzielle therapeutische Anwendung hin zu untersuchen. Zum Gentransfer dieser Mutanten wurden demzufolge adenovirale Vektoren eingesetzt, wobei ein synthetischer Calcitonin-Promotor (TSE2.CP1) zur Gewährleistung einer Zielzell-selektiven Expression verwendet wurde. Durch Reportergenanalysen konnte hier verdeutlicht werden, dass der TSE2.CP1-Promotor, auch im Kontext adenoviraler Vektoren, die Selektivität für C-Zellen, wie hier am Beispiel der TT-Zelllinie gezeigt, behält. Zwei zuvor beschriebene, dominant-negative RET Mutanten (RET HSCR32 und RET flag) wurden so unter der Kontrolle des TSE2.CP1-Promotors mit Hilfe adenoviraler Vektoren in der TT-Zelllinie, die eine häufige MTC-assoziierte RET Mutation trägt, exprimiert. Durch Mutationen in der extrazytoplasmatischen Region werden diese Mutanten nicht auf der Zelloberfläche exprimiert und stattdessen im endoplasmatischen Retikulum (ER) zurückgehalten. Der dominant-negative Effekt der Mutanten beruht dabei auf der intrazellulären Dimerisierung mit onkogenen RET Mutanten. Wie hier gezeigt wurde, wird endogenes onkogenes RET in TT-Zellen nach Gentransfer der dominant-negativen Mutanten effizient im ER zurückgehalten. Darüber hinaus war eine ausgeprägte Zytotoxizität bei Expression von RET HSCR32 und RET flag zu beobachten, die auf Apoptose-Induktion beruhte. Ein weiterer Schwerpunkt der vorliegenden Untersuchungen lag in der in vivo-Analyse des Antitumor-Potenzials der dominant-negativen Mutante RET flag, exemplarisch für beide extrazytoplasmatischen Mutanten, im Xenotransplantatmodell.. Die erzielten Ergebnisse verdeutlichten dabei, dass eine signifikante Lebensverlängerung der so behandelten Mäuse zu beobachten war. In einem einzigen Fall (10 %) konnte darüber hinaus eine vollständige Heilung erzielt werden. Da der pro-apoptotische Effekt von RET HSCR32 und RET flag jedoch, wie anschließend gezeigt werden konnte, unabhängig von ihren dominant-negativen Eigenschaften war und stattdessen auf Apoptose-Mechanismen, die zu Stressreaktionen im endoplasmatischen Retikulum führten, zurückzuführen war, wurde weiterhin das Antitumor-Potenzial einer Tyrosinkinase-deletierten RET Mutante (RET DTK) untersucht, die keine extrazytoplasmatischen Mutationen aufwies. Diese Mutante dimerisiert mit onkogenem RET an der Zelloberfläche und inhibiert dadurch die RET trans-Autophosphorylierung. Der adenovirale Gentransfer von RET DTK unter der Kontrolle des CMV-Promotors in TT-Zellen bestätigt diesen Sachverhalt. Nach Inhibition der endogenen trans-Autophosphorylierung onkogener RET Mutanten war eine signifikante Wachstumsreduktion von TT-Zellen zu beobachten. Diese war selektiv auf die Inhibition der RET trans-Autophosphorylierung zurückzuführen, da RET-negative Zellen unbeeinflusst blieben. Als Ursache konnte sowohl eine gesteigerte Apoptoserate wie auch eine reduzierte Proliferationsrate identifiziert werden. Die in dieser Arbeit erzielten Resultate liefern also einen ersten Hinweis darauf, dass die Inhibition von onkogen aktiviertem RET durch dominant-negative Mutanten in der Zukunft eine alternative Strategie zur Behandlung von MTCs darstellen könnte

Clasp2 ensures mitotic fidelity and prevents differentiation of epidermal keratinocytes

Epidermal homeostasis is tightly controlled by a balancing act of self-renewal or terminal differentiation of proliferating basal keratinocytes. An increase in DNA content as a consequence of a mitotic block is a recognized mechanism underlying keratinocyte differentiation, but the molecular mechanisms involved in this process are not yet fully understood. Using cultured primary keratinocytes, here we report that the expression of the mammalian microtubule and kinetochore-associated protein Clasp2 is intimately associated with the basal proliferative makeup of keratinocytes, and its deficiency leads to premature differentiation. Clasp2-deficient keratinocytes exhibit increased centrosomal numbers and numerous mitotic alterations, including multipolar spindles and chromosomal misalignments that overall result in mitotic stress and a high DNA content. Such mitotic block prompts premature keratinocyte differentiation in a p53-dependent manner in the absence of cell death. Our findings reveal a new role for Clasp2 in governing keratinocyte undifferentiated features and highlight the presence of surveillance mechanisms that prevent cell cycle entry in cells that have alterations in the DNA content.This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (MINECO) [BFU2012-33910 and BFU2015-71376-R (MINECO/ European Regional Development Fund (ERDF), European Union) to M.P.-M.]. Deposited in PMC for immediate release.S

Coordinated Implementation of Chikungunya Virus Reverse Transcription–PCR

A preformulated chikungunya virus real-time reverse transcription–PCR, quality-confirmed oligonucleotides, and noninfectious virus controls were distributed by the European Network for the Diagnosis of Imported Viral Diseases. An international proficiency study with 31 participants demonstrated that ad hoc implementation of molecular diagnostics was feasible and successful

Results of the 1st external quality assurance for SARS new coronavirus diagnostic PCR and serology : talk

Background The detection of the new Coranavirus (CoV) causing agent of the severe acute respiratory syndrome (SARS) for diagnostic purposes is still a critical step in prevention of secondary hospital infections. In this respect the PCR for SARS diagnostic is the fastest and most sensitive method and was published very early after the description of the new pathogen by different groups. To evaluate the quality and sensitivity of the SARS PCR performed in diagnostic laboratories all over the world an external quality assurance (EQA) for SARS PCR was initiated by the WHO, the European Network for Diagnostics of "Imported" Viral Diseases (ENIVD) and the Robert Koch-Institut. Methods Therefore 10 samples of inactivated SARS CoV strains isolated in Frankfurt and Hong Kong in different dilutions and negative controls were prepared. The freeze dried samples were send by mail to 62 different laboratories, in 37 countries in Europe and Israel (35), Asia (11), The Americas (11), Australia and New Zealand (4) and Africa (1). The results were returned by email or fax 1 week (13), 2 weeks (14), 3 weeks (6) and later (29) after receiving the material which does not mimic at all the possible speed of this fast method. But this was not considered in the evaluation of these first SARS EQA. Results 44 laboratories showed good or excellent results (26 = 100%, 18 = 90%) and even the 14 laboratories which archived only 80% (10) or 70% (4) correct results are mostly lacking sensitivity. The results of the other 4 laboratories show basic problems in regard to sensitivity, specificity and consistency of results and must be overcome as soon as possible. 4 laboratories seem to have problems with the specificity finding a positive signal in negative samples. The different methods used for preparation of the SARS CoV genome and diagnostic PCR test procedure used by the participating laboratories will be discussed in more detail in the presentation. Conclusion However, in contrast to previous EQAs for Ebola, Lassa and Orthopoxviruses the quality of PCR results was rather good which might be caused by the early publication and distribution of well developed PCR methods. An EQA for evaluation of SARS specific serology is still ongoing, first results will be available beginning of April 2004

Requirement for epithelial p38α in KRAS-driven lung tumor progression

Malignant transformation entails important changes in the control of cell proliferation through the rewiring of selected signaling pathways. Cancer cells then become very dependent on the proper function of those pathways, and their inhibition offers therapeutic opportunities. Here we identify the stress kinase p38α as a nononcogenic signaling molecule that enables the progression of KrasG12V-driven lung cancer. We demonstrate in vivo that, despite acting as a tumor suppressor in healthy alveolar progenitor cells, p38α contributes to the proliferation and malignization of lung cancer epithelial cells. We show that high expression levels of p38α correlate with poor survival in lung adenocarcinoma patients, and that genetic or chemical inhibition of p38α halts tumor growth in lung cancer mouse models. Moreover, we reveal a lung cancer epithelial cell-autonomous function for p38α promoting the expression of TIMP-1, which in turn stimulates cell proliferation in an autocrine manner. Altogether, our results suggest that epithelial p38α promotes KrasG12V-driven lung cancer progression via maintenance of cellular self-growth stimulatory signals

Inactivation of Capicua in adult mice causes T-cell lymphoblastic lymphoma

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4 Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy.We are grateful to Carol MacKintosh (University of Dundee, UK) for the pcDNA5/FRT/TO-GFP-CIC plasmid, and Huda Zoghbi (Baylor College of Medicine, Houston, TX) and Yoontae Lee (University of Pohang, Korea) for Cic antisera. We thank Scott Brown and Robert Holt (University of Vancouver, Canada) for their help with TCR abundance calculations. We also thank Carmen G. Lechuga, Marta San Roman, Raquel Villar, Beatriz Jimenez, and Nuria Cabrera for excellent technical assistance. We value the support of Sagrario Ortega (Transgenic Mice Core Unit, CNIO) for help in generating the Cic mutant mice, Orlando Dominguez (Genomics Core Unit, CNIO) for the RNA-seq analysis, and the Histopathology Core Unit. This work was supported by grants from the Fundacio La Marato de TV3 (20131730/1) to G.J. and M.B., and the European Research Council (ERC-AG/250297-RAS AHEAD), the EU-Framework Programme (HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010-260791/EUROCANPLATFORM), the Spanish Ministry of Economy and Competitiveness (SAF2014-59864-R), the Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE), and the Asociacion Espanola contra el Cancer (AECC) (GC16173694BARB) to M.B. M.B. is the recipient of an Endowed Chair from the AXA Research Fund. L.S.-C. was supported by a fellowship from the Programa de Formacion de Personal Investigator (FPI) of the Spanish Ministry of Economy and Competitiveness. M.D. and M.B. conceived and designed the study. L.S.-C., O.G., G.J., M.D., and M.B. developed the methodology. L.S.-C., O.G., M.S., and M.D. acquired the data. L.S.-C., O.G., M.S., H.K.C.J., G.J., M.D., and M.B. analyzed and interpreted the data. L.S-C., O.G., G.J., M.D., and M.B. wrote, reviewed, and/or revised the manuscript. G.J. provided material support. A. G. analyzed the T-ALL sequencing. M.D. and M.B. supervised the study.S

Coronavirus Antibodies in African Bat Species

Asian bats have been identified as potential reservoir hosts of coronaviruses associated with severe acute respiratory syndrome (SARS-CoV). We detected antibody reactive with SARS-CoV antigen in 47 (6.7%) of 705 bat serum specimens comprising 26 species collected in Africa; thus, African bats may harbor agents related to putative group 4 CoV

KSR induces RAS-independent MAPK pathway activation and modulates the efficacy of KRAS inhibitors.

The kinase suppressor of rat sarcoma (RAS) proteins (KSR1 and KSR2) have long been considered as scaffolding proteins required for optimal mitogen-activated protein kinase (MAPK) pathway signalling. However, recent evidence suggests that they play a more complex role within this pathway. Here, we demonstrate that ectopic expression of KSR1 or KSR2 is sufficient to activate the MAPK pathway and to induce cell proliferation in the absence of RAS proteins. In contrast, the ectopic expression of KSR proteins is not sufficient to induce cell proliferation in the absence of either rapidly accelerated fibrosarcoma (RAF) or MAPK-ERK kinase proteins, indicating that they act upstream of RAF. Indeed, KSR1 requires dimerization with at least one member of the RAF family to stimulate proliferation, an event that results in the translocation of the heterodimerized RAF protein to the cell membrane. Mutations in the conserved aspartic acid-phenylalanine-glycine motif of KSR1 that affect ATP binding impair the induction of cell proliferation. We also show that increased expression levels of KSR1 decrease the responsiveness to the KRASG12C inhibitor sotorasib in human cancer cell lines, thus suggesting that increased levels of expression of KSR may make tumour cells less dependent on KRAS oncogenic signalling.We thank M. San Roman and R. Villar for technical assistance. This work was supported by grants from the European Research Council (ERC-AG/695566, THERACAN), the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00 and RTC2017-6576-1), the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM), the CRIS Cancer Foundation and the Asociacion Espanola contra el Cancer (AECC) (GC166173694BARB). MB is a recipient of an Endowed Chair from the AXA Research Fund. GP has been supported by a fellowship from the Programa de Atraccion de Talento of the Autonomous Community of Madrid. SGA is a recipient of a postdoctoral fellowship from the AECC. OB is a recipient of a fellowship from the Formacion de Personal Investigador (FPI) program of the Spanish Ministry of Science, Innovation and Universities.S