Mapping lung cancers Achilles heels through functional genomics

Lungenkrebs hat weiterhin eine schlechte Prognose, vor allem, weil er in fortgeschrittenem Stadium diagnostiziert wird. Wenn sich Metastasen bereits auf ferne Organe ausgebreitet haben, ist Chirurgie keine praktikable Option mehr. Trotz der Fortschritte bei Krebstherapeutika hat sich im Fall von Lungenkrebs wenig verbessert, da die Langzeitüberlebensraten in den letzten Jahrzehnten nahezu unverändert geblieben sind. Lungentumore zeichnen sich durch einen hohen Grad an Mutationen aus, was auch zu einer erheblichen Tumorheterogenität zwischen Patienten führt. Patienten mit einem ähnlichen Tumor-Subtyp zeigen gewöhnlich ein dramatisch divergentes klinisches Ergebnis auf die gleiche Behandlung. In der Ära der personalisierten Medizin bleibt es eine Herausforderung, jedem Patienten die Behandlung zuzuweisen, die am ehesten einen maximalen Nutzen bietet. Zu diesem Zweck lieferten Deep-Sequencing-Technologien detaillierte Informationen über genetische Veränderungen von Lungentumoren. Gleichzeitig wurde eine breite Palette von kleinen Molekülen zielgerichtet auf Onkoproteine entwickelt, die derzeit präklinisch untersucht werden. Tumorveränderungen einem dieser neuartigen Medikamente zuordnen zu können, gehört zu den Prioritäten der klinischen Krebsforschung. In diesem Dissertationsprojekt wird ein maßgeschneidertes in vitro Modell des Lungenadenokarzinoms vorgestellt. Wir haben die häufigsten Genveränderungen, die in der Literatur beschrieben sind, in eine immortalisierte epitheliale Lungenzelllinie eines gesunden Spenders eingeführt. Auf diese Weise entwickelten wir eine Gruppe von isogenen Zelllinien, die alle aus der ursprünglichen nicht-transformierten Zelllinie stammen und sich nur in einer bestimmten Veränderung voneinander unterscheiden. Unser isogenes Modell erlaubt eine einfache Interpretation der mit dem Arzneimittel assoziierten Änderung der Überlebensfähigkeit spezifisch für jeden genetischen Hintergrund. Solch eine leistungsstarke Methodik erlaubte es uns, den ganzen einzigartigen Satz von Zelllinien gegen eine kuratierte Auswahl von State-of- the-Art-Kinase-Inhibitoren und Chemotherapeutika zu testen. Im Einzelnen umfaßte der hier beschriebene pharmakogenetische Screen über 100 Arzneimittel und ermöglichte es uns, Hunderte von einzigartigen Gen- Arzneimittel-Wechselwirkungen zu studieren. Unser High-Throughput-Ansatz konnte erwartete Abhängigkeiten erfassen, die zuvor bei Lungenadenokarzinom beschrieben wurden, was das Potenzial unserer Technologie stärkte. Darüber hinaus beschreiben wir neue zielgerichtete Zusammenhänge mit Arzneimitteln, die derzeit in klinischen Studien getestet werden oder bereits für die Behandlung von Lungenkrebs zugelassen sind. Darunter beschreiben wir eine neuartige Rolle von ATM, die Zelllebensfähigkeit als Antwort auf MEK-Inhibierung zu modulieren. Insbesondere fanden wir, dass Krebs-assoziierte ATM-Mutationen sowie ATM-Verlust Lungenkrebszellen überempfindlich gegen Moleküle, die auf MEK1 / 2-Kinasen zielen, macht. Die ATM (Ataxia telangiectasia mutated) -Kinase fungiert als der Hauptsensor von DNA-Doppelstrangbrüchen (DSB) und ist verantwortlich für die Verstärkung der Signalisierung, die die DNA-damage response Maschinerie rekrutiert. ATM gehört zu den am häufigsten mutierten Tumorsuppressorgenen bei Krebs. Darüber hinaus werden auch große Deletionen, die den ATM-Locus betreffen, beobachtet, insbesondere bei hämatologischen Malignitäten. Fehlende ATM- Funktion in diesen Zellen trägt zu einem höheren Grad an genomischer Instabilität bei, ein Markenzeichen von Krebs. MEK1/2-Kinasen (mitogenaktivierte Proteinkinase-Kinase 1/2) sind nachgeschaltete Glieder des RAS / RAF-mitogenen Signalwegs. Obwohl MEK selten mutiert ist, ist es zu einem relevanten Ziel in der Arzneimittelentwicklung geworden, um die Signaltransduktion aus hyperaktivierten mutierten RAS- oder RAF-Onkogenen abzuschalten. Es gibt derzeit mehrere MEK-Inhibitoren in der klinischen Entwicklung zur Behandlung einer breiten Palette von malignen Erkrankungen. Vor kurzem wurde der MEK-Inhibitor Trametinib von der FDA zugelassen, der erste seiner Klasse, der die klinische Entwicklung abschliessen konnte. Bisher werden jedoch Patienten, die einen MEK-Inhibitor erhalten, nicht routinemßig auf Basis einer definierten Tumoraberration stratifiziert. Auf der Grundlage unserer Ergebnisse schlagen wir den ATM-Status als Biomarker für die Patientenstratifizierung vor, um eine MEK-Inhibitor-Behandlung zu erhalten. Da Lungenkrebs weltweit zu den häufigsten Tumoren gehört, stellt dies eine signifikante Anzahl an Patienten dar, die von einer Behandlung mit MEK-Inhibitoren profitieren können. Daher hat der Beitrag der hier vorgestellten Ergebnisse das Potenzial, eine bemerkenswerte klinische Wirkung zu erzielen.Lung cancer continues having a poor prognosis mainly because it is diagnosed at advanced stages. At this point, where metastatic disease has spread to distant organs, surgery is not a viable option anymore. Despite the improvements on cancer therapeutics, little has improved in the case of lung cancer, as long-term survival rates have remained almost unchanged during the last decades. Lung tumours are characterized by a high degree of mutational burden, which also results in substantial interpatient tumour heterogeneity. Consequently, patients with similar tumour subtype are usually displaying a dramatically divergent clinical outcome to the same treatment. In the era of personalized medicine, it remains a challenge to assign each patient to the treatment that is likely to provide a maximal benefit. To this aim, deep-sequencing technologies have provided detailed information regarding lung tumour genetic alterations. At the same time, a wide range of small molecules targeting oncoproteins have been developed and they are currently under pre-clinical investigation. To complete the puzzle by matching a tumour alteration to one of these novel drugs is among the priorities in clinical cancer research. In this doctoral thesis project, a tailored lung adenocarcinoma in vitro model is presented. Employing an immortalized epithelial lung cell line from a healthy donor, we have introduced the most common gene alterations described in the literature. This way, we have engineered a panel of isogenic cell lines, all deriving from the original non-transformed cell line and only differing from each other in one particular alteration. Our isogenic model permits a straightforward interpretation of drug-associated changes in viability specifically for each genetic background. Such a powerful methodology has allowed us to test the whole unique set of cell lines against a curated selection of state-of-the-art kinase inhibitors and chemotherapeutics. In detail, the pharmacogenetic screen described here included over 100 drugs and enabled us to study hundreds of unique gene-drug interactions. Notably, our high-throughput approach captured expected dependencies previously described in lung adenocarcinoma, strengthening the potential of our technology. Furthermore, we describe new targetable relationships with drugs currently in clinical trials or already approved for the management of lung cancer. Among them, we describe here a novel role of ATM modulating cell viability in response to MEK inhibition. Specifically, we found that cancer- associated ATM mutations as well as ATM loss renders lung cancer cells hypersensitive to small molecules targeting MEK1/2 kinases. The ATM (ataxia telangiectasia mutated) kinase acts as the major sensor of DNA double strand breaks (DSB) and it is responsible for amplifying the signalling recruiting the DNA damage response machinery. ATM is among the most frequently mutated tumour suppressor genes in cancer. In addition, large deletions affecting the ATM locus are also observed, particularly in hematologic malignancies. Lack of ATM function in these cells contributes to a higher level of genomic instability, a hallmark of cancer. MEK1/2 kinases (mitogen-activated protein kinase kinase 1/2) are downstream members of the RAS/RAF mitogenic signalling pathway. Although MEK is rarely mutated, it has become a relevant target in drug development in order to shut down signal transduction coming from hyperactivated mutant RAS or RAF oncogenes. There are currently several MEK inhibitors in clinical development for the treatment of a wide range of malignancies. Recently, the MEK inhibitor trametinib has been granted FDA approval, being the first of its class to complete clinical development. To date, however, patients receiving a MEK inhibitor are not routinely stratified based on a defined tumour aberration. Based on our finding, we propose ATM status as a biomarker for patient stratification to receive a MEK-inhibitor treatment. As lung cancer is among the most prevalent tumours worldwide, this constitutes a significant number of patients who could benefit from a MEK-inhibitor based treatment. Therefore, the contribution of the findings presented here has the potential to achieve a remarkable clinical impact.submitted by Ferran Fece de la Cruz, MScZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2017OeB

Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer.

PurposeAlthough patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood.Experimental designTargeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC.ResultsProminent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib.ConclusionsOur study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes

TAS-120 Overcomes Resistance to ATP-Competitive FGFR Inhibitors in Patients with FGFR2 Fusion–Positive Intrahepatic Cholangiocarcinoma

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating FGFR2 gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary FGFR2 kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with FGFR2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with FGFR2 fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in FGFR2-altered ICC; however, acquired FGFR2 kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models.This article is highlighted in the In This Issue feature, p. 983

Supplementary Figures S1 to S11 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Supplementary Figure S1: PI3K pathway activity in selected cases with acquired PTEN alteration. Supplementary Figure S2. Validation of AKT constructs expression in T47D cells. Supplementary Figure S3. AKT activating mutations confer resistance to PI3Ka inhibitors. Supplementary Figure S4: Free energy calculations predict resistance to orthosteric PI3K inhibitors due to specific double PIK3CA mutants. Supplementary Figure S5: Free energy perturbation predicts reduced binding of orthosteric PI3K inhibitors to double mutants. Supplementary Figure S6. Expression of PIK3CA mutations in T47D cells. Supplementary Figure S7. MCF7 cells expressing W780R or Q859H double mutants show differential response to PIK3CA orthosteric inhibitors. Supplementary Figure S8: Chemical structure of RLY-2608. Supplementary Figure S9: Surface plasmon resonance (SPR) binding assay. Supplementary Figure S10. Alpelisib shows reduced potency of downstream signaling inhibition in the presence of W780R or Q859H/K. Supplementary Figure S11. T47D cells expressing I817F or E726K double mutants do not show a differential response to inavolisib (A) or RLY-2608 (B).</p

Supplementary Tables S1 to S5 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Table S1: Eligibility Criteria. Table S2: Genomic alterations within the PIK3CA pathway and other documented alterations for this study. Table S3: VAF of EOT alterations. Table S4: Comparison of Acquired PIK3CA mutations based on baseline activating mutation. Table S5: Comparison of Acquired PIK3CA mutations based on number of baseline activating PIK3CA mutations.</p

Supplementary Tables S1 to S5 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Table S1: Eligibility Criteria. Table S2: Genomic alterations within the PIK3CA pathway and other documented alterations for this study. Table S3: VAF of EOT alterations. Table S4: Comparison of Acquired PIK3CA mutations based on baseline activating mutation. Table S5: Comparison of Acquired PIK3CA mutations based on number of baseline activating PIK3CA mutations.</p

Supplementary Figures S1 to S11 from Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary <i>PIK3CA</i> Mutations

Supplementary Figure S1: PI3K pathway activity in selected cases with acquired PTEN alteration. Supplementary Figure S2. Validation of AKT constructs expression in T47D cells. Supplementary Figure S3. AKT activating mutations confer resistance to PI3Ka inhibitors. Supplementary Figure S4: Free energy calculations predict resistance to orthosteric PI3K inhibitors due to specific double PIK3CA mutants. Supplementary Figure S5: Free energy perturbation predicts reduced binding of orthosteric PI3K inhibitors to double mutants. Supplementary Figure S6. Expression of PIK3CA mutations in T47D cells. Supplementary Figure S7. MCF7 cells expressing W780R or Q859H double mutants show differential response to PIK3CA orthosteric inhibitors. Supplementary Figure S8: Chemical structure of RLY-2608. Supplementary Figure S9: Surface plasmon resonance (SPR) binding assay. Supplementary Figure S10. Alpelisib shows reduced potency of downstream signaling inhibition in the presence of W780R or Q859H/K. Supplementary Figure S11. T47D cells expressing I817F or E726K double mutants do not show a differential response to inavolisib (A) or RLY-2608 (B).</p