Modeling Oncogenic KIT Signaling and Drug Resistance in the Mouse
Dissertation zur Erlangung des
akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von Benedikt Bosbach
an der
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie
Konstanz, 2012
Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-204678
Modeling Oncogenic KIT Signaling and Drug Resistance in the Mouse
Dissertation zur Erlangung des
akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)
vorgelegt von Benedikt Bosbach
an der
Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie
Tag der mündlichen Prüfung: 12. September 2012 1. Referent: Prof. Dr. Thomas Brunner
2. Referent: Prof. Dr. Peter Besmer, MSKCC, New York, NY, USA 3. Referent: PD Dr. Edward Málaga-Trillo
Table of Contents
Table of Contents ... 3
List of Figures ... 5
List of Tables ... 7
Summary ... 8
Deutsche Zusammenfassung ... 11
Acknowledgements ... 14
1. General Introduction ... 15
1.1 Protein kinases ... 16
1.2 The receptor tyrosine kinase KIT ... 17
1.3 Interstitial Cells of Cajal ... 21
1.4 Gastrointestinal stromal tumor (GIST) ... 23
1.5 Tyrosine kinase inhibitors with KIT selectivity ... 26
2. Imatinib resistance and microcytic erythrocytosis in a KitV558Δ;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor... 28
2.1 Abstract ... 29
2.2 Introduction ... 29
2.3 Results ... 31
2.4 Discussion ... 43
2.5 Materials and Methods ... 45
2.6 Acknowledgments ... 48
2.7 Supplementary Information ... 48
2.8 Author Summary ... 53
3. In vivo expansion of hematopoietic stem and progenitor cells in fetal and adult Kit-mutant mice with microcytic erythrocytosis ... 56
3.1 Abstract ... 57
3.2 Introduction ... 58
3.3 Results ... 59
3.4 Discussion ... 71
3.5 Materials and Methods ... 72
3.6 Supplementary information ... 75
4. Dissecting oncogenic KIT signaling: mouse models revealing a crucial role of the KIT/PI3K-axis for the initiation and maintenance of gastrointestinal stromal tumor and recapitulating human chromosomal deletions during tumor progression ... 77
4.1 Abstract ... 78
4.2 Introduction ... 79
4.3 Results ... 81
4.4 Discussion ... 95
4.5 Materials and Methods ... 97
Declaration of Contributions ... 101
Concluding Remarks ... 102
Abbreviations ... 108
References ... 110
List of Figures
Fig. 1: Derivation and phenotypic characterization of Kit-gatekeeper mice. ... 32 Fig. 2: Cecal GIST and pronounced gastric and colonic ICC hyperplasia in
KitV558Δ;T669I/+ mice. ... 33 Fig. 3: The KitV558Δ;T669I double mutation confers resistance to imatinib in vivo.
Sunitinib overcomes resistance. ... 35 Fig. 4: Resistance to dasatinib and sensitivity to sorafenib treatment in
KitV558Δ;T669I/+ mice. ... 36 Fig. 5: Increased mast cell and red blood cell numbers in KitV558Δ;T669I/+ mice... 39 Fig. 6: KitV558Δ;T669I/+
erythroid progenitor growth is hypersensitive to KitL and is susceptible to sunitinib inhibition. ... 42 Fig. S7: Targeting strategy for the knock-in of V558Δ into exon 11 of the 129/Sv
Kit locus. ... 48 Fig. S8: Drug response after long-term treatment. (A) The KitV558Δ;T669I double
mutation confers resistance to imatinib in vivo. Sunitinib overcomes resistance. ... 49 Fig. S9: Histological and immunohistochemical comparison of interstitial cell of
Cajal (ICC) hyperplasia in stomach and colon of KitV558Δ/+ and KitV558Δ;T669I/+ mice. ... 50 Fig. S10:ICC hyperplasia in KitV558Δ;T669I/+
mice is resistant to imatinib treatment and susceptible to sunitinib treatment. ... 51 Fig. S11:KitV558Δ;T669I/+ mice exhibit microcytic erythrocytosis and elevated mean
platelet volume. ... 52 Fig. 12: Expansion of erythroid progenitors in the KitV558Δ;T669I/+ BM and spleen. ... 61 Fig. 13: Expansion of the myeloid lineage in the adult BM and spleen of
KitV558Δ;T669I/+ mice... 63 Fig. 14: Reduced frequency of lymphopoiesis in KitV558Δ;T669I/+ mice. ... 64 Fig. 15: Analysis of the stem cell compartment in wild-type and KitV558Δ;T669I/+
mice. ... 65 Fig. 16: Analysis of fetal livers from wild-type and KitV558Δ;T669I/+
mice. ... 66 Fig. 17: In vivo functionality of HSC. ... 68 Fig. 18: Stem cell cycling in wild-type and KitV558Δ;T669I/+ mice... 70 Fig. 19: Response of wild-type and KitV558Δ;T669I/+
mice to 5-FU. ... 71 Fig. S20:Cytospin of wild-type and KitV558Δ;T669I/+ spleen. ... 75 Fig. S21:CFU-S assay of wild-type and KitV558Δ;T669I/+ mice. ... 75
Fig. 22: Targeting strategy for the simultaneous knock-in of V558Δ and Y567F (A) into exon 11 of the 129/Sv Kit locus. An analogous strategy was employed for the KitV558Δ;Y719F allele (scheme B). ... 81 Fig. 23: Kaplan-Meier survival plot showing increased survival of
KitV558Δ;Y567F/Y567F mice in comparison to fully signaling-competent KitV558Δ/+ mice. Survival of KitV558Δ;Y719F/Y719F mice is like wild-type. ... 82 Fig. 24: KitV558Δ;Y567F/Y567F mice develop gastric and focal colonic ICC hyperplasia
and cecal GIST. ... 83 Fig. 25: Attenuation of GIST growth in KitV558Δ;Y567F/Y567F mice lacking the SRC
binding site on KIT. ... 84 Fig. 26: Reduced MAPK activation but unaffected KIT/PI3K signal transduction in
GIST of KitV558Δ;Y567F/Y567F mice. ... 85 Fig. 27: Loss of chromosome 14p regions is an early, highly recurrent genomic
event in human GIST. ... 86 Fig. 28: Tumor progression and heterogeneity in KitV558Δ;Y567F/Y567F mice older than
1 year. ... 87 Fig. 29: Histologic heterogeneity in polynodular GIST of old KitV558Δ;Y567F/Y567F
mice. ... 88 Fig. 30: Genomic heterogeneity and hemizygous whole chromosomal deletions in
GIST nodules of old KitV558Δ;Y567F/Y567F mice. ... 89 Fig. 31: Absence of tumorigenesis in KitV558Δ;Y719F/Y719F mice. ... 90 Fig. 32: Preservation of ICC development, but absence of ICC hyperplasia in
KitV558Δ;Y719F/Y719F mice. ... 91 Fig. 33: Impaired spermatogenesis in KitV558Δ;Y719F/Y719F
mice. ... 92 Fig. 34: XL147 pharmacologically inhibits the PI-3K axis in GISTs of KitV558Δ/+
mice. ... 93 Fig. 35: Long-term treatment of KitV558Δ/+ mice with XL147 diminishes GIST cell
proliferation. ... 94
List of Tables
Tab. 1: KIT Fact Box. ... 19 Tab. 2: Overview of “gatekeeper” mutations conferring resistance to kinase
inhibitors, as detected clinically and with special emphasis on GIST. ... 25 Tab. 3: Histological response of GIST lesions in KitV558Δ/+ and KitV558Δ;T669I/+ mice
to 7-d treatment with imatinib, dasatinib, sunitinib, or sorafenib. ... 37 Tab. 4: Increased erythroid progenitors in spleen of KitV558Δ;T669I/+ mice. ... 41 Tab. 5: Overview of phenotypes in mice with Kit mutations and their sensitivity to
targeted kinase inhibition. ... 55 Tab. 6: CFU-GM and CFU-GEMM assays KitV558Δ;T669I/+
mice. ... 64 Tab. S7: List of Antibodies. ... 76 Tab. 8: Impact of 7-day treatments on body mass of Kit-mutant mice. ... 95
Summary
KIT is a receptor tyrosine kinase critical for gametogenesis, hematopoiesis, melanogenesis, and the pacemaker system of the gastrointestinal tract, the interstitial cells of Cajal (ICC). The majority of gastrointestinal stromal tumors (GIST) harbor an activating mutation in the KIT gene. Introduction of such gain-of-function mutations into the endogenous Kit locus of the mouse, e.g., the knock-in of KitV558Δ, has been shown previously to result in interstitial cell of Cajal (ICC) hyperplasia throughout the gastrointestinal tract and development of cecal GIST lesions with full penetrance. This experimentally demonstrated the central role mutant KIT plays in the pathogenesis of GIST. Aberrant signal transduction in GIST lesions of KitV558Δ/+ mice includes sustained activation of the PI3K/AKT/S6, MAPK, and STAT3 pathways, which is amenable to inhibition with the tyrosine kinase inhibitor (TKI) imatinib. Imatinib is the first-line treatment for advanced GIST and can be given adjuvantly after surgery; it improves re- currence-free survival but is not curative. This necessitates chronic imatinib treatment, eventually resulting in relapse of patients. At this point, second-site mutations in Kit can be detected in the majority of the imatinib-resistant tumor lesions. A prominent second- site mutation abrogating the inhibitory effect of most TKIs is the so-called gatekeeper mutation, which affects a critical, conserved threonine residue in the kinase pocket of multiple oncogenic RTKs. The homologous mutations in human and murine KIT are KITT670I and KitT669I, respectively.
As a proof-of-concept and, more importantly, to provide a preclinical model for the development of salvage therapies, the first aim of this study was the generation of a mouse model of imatinib-resistant GIST. By targeted mutagenesis the activating muta- tion KitV558Δ and the gatekeeper mutation KitT669I were introduced simultaneously into the Kit locus. The derived KitV558Δ;T669I/+ mice are viable and developed ICC hyperplasia and cecal GIST lesions. While GIST lesions were smaller in KitV558Δ;T669I/+ mice in comparison with KitV558Δ/+ mice, ICC hyperplasia was more pronounced, with no appar- ent changes in oncogenic signal transduction in both tissues/mouse models. Importantly, KitV558Δ;T669I/+ GIST were resistant to intervention with imatinib and dasatinib at the bi- ochemical as well as at the histological/cellular level. This is further remarkable as both these TKIs are known to inhibit wild-type KIT (Kit+), which is present in the tumors of
these heterozygous mice and likely in the majority of tumors of patients. The resistance mediated by the gatekeeper mutation was overcome by treatments with sunitinib and sorafenib, providing a rationale for their utilization in gatekeeper-mutant GIST cases.
Unexpectedly, KitV558Δ;T669I/+ mice uniformly and within six weeks after birth devel- oped a polycythemia vera-like microcytic erythrocytosis. Macrocytic anemia is one of the hallmark phenotypes of mice with Kit loss-of-function dominant white-spotting (W) mutations. Together, this indicates that the KitV558Δ;T669I mutation is a strong gain-of- function mutation. Concordantly, KitV558Δ;T669I/+ mice had increased mast cell hyperplas- ia in addition to the observed microcytic erythrocytosis and the increased ICC hyper- plasia. Besides the hyperplastic ICC and GIST lesions, also burst-forming units erythroid (BFU-Es) obtained from bone marrow and spleen of KitV558Δ;T669I/+ mice were resistant to imatinib and susceptible to sunitinib. This indicates that the observed erythrocytosis is largely dependent on aberrant kinase activity. It remains to be studied if the addition of the KitT669I mutation to the KitV558Δ mutation introduces qualitative changes in KIT, understanding of which might guide the design of improved inhibitors for gatekeeper-mutant kinases.
The second aim of this study was to molecularly abrogate specific phosphorylation sites of the KitV558Δ kinase to elucidate the contribution of the respective downstream signaling cascades to GIST development. Therefore, in the context of the KitV558Δ mutation two well-characterized phosphorylation sites of KIT, the Src family binding site pY567 and the PI3K binding site pY719, were substituted individually with phenyl- alanine by targeting the corresponding codons in the mouse genome. KitV558Δ;Y567F/Y567F
mice developed attenuated GIST and ICC hyperplasia, associated with diminished acti- vation of the MAPK pathway. Remarkably, GIST of KitV558Δ;Y567F/Y567F mice showed tumor progression along similar cytogenetic pathways as human GIST, with recurrent hemizygous deletions of mouse chr12, the chromosome which is largely syntenic to human chr14, the most frequently lost chromosome in GIST.
No tumor development was observed in the KitV558Δ;Y719F/Y719F mice up to the oldest animals analyzed at 23 months and median survival was wild type-like. Of note, ICC and accordingly their progenitors, which are the presumed cells-of-origin of GIST, were unaffected in KitV558Δ;Y719F/Y719F mice. While other KIT-dependent processes, namely hematopoiesis and melanogenesis, also did not show any apparent defects in
KitV558Δ;Y719F/Y719F
mice, the known male sterility phenotype of KitY719F/Y719F
mice was not rescued in KitV558Δ;Y719F/Y719F mice. This indicates that cell-specific and absolute pathway requirements can exist in RTK signaling, even in the context of a potent acti- vating mutation.
Translation of these genetic findings by treating established GIST of KitV558Δ/+ mice with a pharmacologic inhibitor of pan-class I PI3Ks, XL147, resulted in pathway- specific inhibition of ribosomal protein S6 and reduction of tumor cell proliferation. As responses to XL147 in comparison with results obtained in previous studies with imatinib were not complete, combination therapy with a MAPK pathway inhibitor might be of benefit, as indicated by the attenuated tumor growth of KitV558Δ;Y567F/Y567F
mice. Approaches like this, to elucidate and target multiple proximal downstream medi- ators of oncogenic signaling, in the future might offer treatment options to circumvent the emergence of resistance to targeted cancer therapy and to potentiate it altogether.
Deutsche Zusammenfassung
Die Rezeptor-Tyrosin-Kinase KIT ist notwendig für die Gametogenese, die Hämato- poese, die Melanogenese und das Schrittmachersystem des Gastrointestinaltrakts, die Interstitiellen Zellen von Cajal (ICC). Die meisten gastrointestinalen Stromatumore (GIST) haben aktivierende Mutationen im KIT-Gen. Die Insertion einer solchen Zugewinnmutation (KitV558Δ) in den eigentlichen Kit-Genlokus der Maus verursacht, wie in Vorarbeiten gezeigt, ICC-Hyperplasie entlang des gesamten Magen-Darmtrakts und die Entwicklung zäkaler GIST-Läsionen in den entsprechenden KitV558Δ/+-Mäusen mit kompletter Penetranz. Dies demonstrierte experimentell KITs entscheidende Rolle in der GIST-Pathogenese. Die aberrante Signaltransduktion in GIST-Läsionen umfasst die permanente Aktivierung der PI3K-AKT-S6-, MAPK- und STAT3-Signalwege und ist umkehrbar durch die Gabe des Tyrosin-Kinase-Inhibitors (TKI) Imatinib. Imatinib ist die Standardtherapie für maligne und metastasierende GIST und ist zudem zugelassen als Zusatztherapie nach GIST-Resektion. Imatinib verbessert das rückfallfreie Überlebensrisiko, aber ist nicht kurativ. Dies macht eine chronische Therapie notwendig, was schließlich in ein Rezidiv von Patienten resultiert. In solchen Fällen können in der überwiegenden Zahl der Imatinib-resistenten Tumorläsionen Zweitmutationen in KIT detektiert werden. Eine bedeutende Zweitmutation, die die Wirkung der meisten TKIs aufhebt, ist die sogenannte gatekeeper-Mutation. Sie mutiert eine entscheidende, hochkonservierte Aminosäure im aktiven Zentrum mehrerer onkogener Rezeptor-Tyrosin-Kinasen. Die entsprechende homologe Mutation im menschlichen KIT-Gen ist KitT670I und in der Maus KitT669I.
Zum Nachweis des Wirkprinzips und vor allem um ein präklinisches Modell zur Entwicklung von Folgetherapien zur Verfügung zu stellen, war das erste Ziel dieser Arbeit die Generierung eines Mausmodells für Imatinib-resistenten GIST. Mittels gezielter Mutagenese wurden die aktivierende Mutation KitV558Δ und die Resistenzmutation KitT669I gleichzeitig in den Kit-Genlokus inseriert. Die dermaßen generierten KitV558Δ;T669I/+ Mäuse waren überlebensfähig und entwickelten ICC- Hyperplasie und zäkalen GIST. Während die Tumore in KitV558Δ;T669I/+ Mäusen im Vergleich zu KitV558Δ/+ Mäusen kleiner waren, war die ICC-Hyperplasie ausgeprägter, wobei in beiden Geweben beider Mausmodelle keine Unterschiede in der onkogenen
Signaltransduktion festgestellt werden konnten. Ein Hauptergebnis war, dass die KitV558Δ;T669I/+-mutierten GIST-Läsionen Imatinib- sowie Dasatinib-Resistenz aufwiesen, sowohl auf biochemischer, wie auch zellulärer/histologischer Ebene. Dies ist weiterhin bemerkenswert, da sowohl Imatinib als auch Dasatinib Wildtyp-KIT inhibieren, welches in den Tumoren der heterozygoten KitV558Δ;T669I/+ Mäuse exprimiert wird und wahrscheinlich auch in den GIST-Läsionen der meisten Patienten. Die durch die gatekeeper-Mutation vermittelte Resistenz konnte durch Behandlungen mit Sunitinib und Sorafenib unterdrückt werden. Dies liefert eine wissenschaftliche Begründung für den Einsatz dieser TKIs in gatekeeper-mutierten GIST-Fällen.
Unerwarteterweise entwickelten KitV558Δ;T669I/+-Mäuse innerhalb von sechs Wochen nach der Geburt und uniform eine Polycythaemia vera-ähnliche sogenannte mikrozytische Erythrozytose. Makrozytische Anämie hingegen ist ein Hauptmerkmal von Mäusen mit Funktionsverlustmutationen im Kit-Gen. Zusammengenommen weist dies darauf hin, dass die KitV558Δ;T669I-Mutation eine starke Funktionsgewinnmutation ist. Damit übereinstimmend wiesen die KitV558Δ;T669I/+-Mäuse, zusätzlich zur beobachteten mikrozytischen Erythrozytose und der ausgeprägteren ICC-Hyperplasie, eine erhöhte Mastzellen-Hyperplasie auf. Neben der ICC-Hyperplasie und den GIST- Läsionen waren auch burst-forming units erythroid (BFU-Es) sowohl aus dem Knochenmark als auch aus der Milz der KitV558Δ;T669I/+
-Mäuse resistent gegen Imatinib und sensibel für Sunitinib. Dies weist darauf hin, dass die beobachtete Erythrozytose hauptsächlich auf aberranter Kinaseaktivität beruht. Künftige Studien könnten klären, ob die Addition der KitT669I-Mutation zur KitV558Δ-Mutation auch qualitative Unterschiede in KIT verursacht. Dieses Wissen könnte richtungsweisend sein für die Entwicklung verbesserter Inhibitoren für gatekeeper-mutierte Kinasen.
Das zweite Hauptziel dieser Studie war die molekulare Abrogation spezifischer Phosphorylierungsstellen der KitV558Δ-Kinase, um den Beitrag der jeweils abhängigen Signaltransduktionswege zur GIST-Entwicklung zu untersuchen. Hierfür wurden zwei bereits gut untersuchte Kit-Phosphorylierungsstellen – die Src-Protein-Familien- Bindestelle pY567 und die PI3K-Bindestelle pY719 – jeweils mit Phenylalanin substituiert über gezielte Mutagenese der entsprechenden Codons im Erbgut der Maus;
gleichzeitig wurde die oncogene KitV558Δ-Mutation inseriert. Die KitV558Δ;Y567F/Y567F- Mäuse entwickelten abgeschwächten GIST, assoziiert mit verminderter Aktivierung des
MAPK-Signalwegs. Bemerkenswerterweise wiesen GIST von KitV558Δ;Y567F/Y567F
- Mäusen eine Tumor-Progression entlang ähnlicher cytogenetischer Wege auf wie humane GIST, mit wiederholter hemizygoter Deletion von Maus-Chromosom 12.
Chromosom 12 in der Maus ist größtenteils syntenisch zum humanen Chromosom 14, welches das am häufigsten deletierte Chromosom in GIST ist.
Keine Tumorentwicklung wurde in den KitV558Δ;Y719F/Y719F-Mäusen festgestellt, bis hin zu den ältesten untersuchten Tieren im Alter von 23 Monaten. Die mediane Überlebenswahrscheinlichkeit von KitV558Δ;Y719F/Y719F-Mäusen glich derjenigen von Wildtypmäusen. Ein entscheidende Feststellung war, dass ICC und dementsprechend ihre Vorläuferzellen, die die mutmaßlichen Ursprungszellen von GIST sind, keine Veränderung in KitV558Δ;Y719F/Y719F-Mäusen aufwiesen. Während andere KIT-abhängige Prozesse, namentlich die Hämatopoese und die Melanogenese, ebenfalls keine offensichtlichen Defekte in KitV558Δ;Y719F/Y719F-Mäusen hatten, wurde der in Vorarbeiten beschriebene Sterilitätsphänotyp von KitY719F/Y719F
-Mäusen in den KitV558Δ;Y719F/Y719F- Mäusen nicht gerettet (no genetic rescue). Dies ist ein starker Hinweis, dass zellspezifische und absolute Signalwegabhängigkeiten in der Signaltransduktion von Rezeptor-Tyrosin-Protein-Kinasen existieren können, selbst im Kontext stark aktivier- ender Mutationen.
Die Übersetzung dieser genetischen Erkenntnisse durch die Behandlung von etablierten GIST in KitV558Δ/+-Mäusen mit einem pharmakologischen Pan-Klasse-I-PI3- Kinasen-Inhibitor, XL147, hatte die signaltransduktionsweg-spezifische Inhibition des ribosomalen-Proteins-S6 und eine Reduktion der Tumorzellproliferation zur Folge. Da die Wirkung von XL147, d. h. die Inhibition nur des PI3K-Signaltransduktionswegs, jedoch im Vergleich mit Ergebnissen vorangegangener Experimente mit dem KIT- Inhibitor Imatinib verringert war, könnte eine Kombinationstherapie von XL147 zusammen mit einem Inhibitor des MAPK-Signalwegs effektiver sein, wie durch das abgeschwächte Tumorwachstum in KitV558Δ;Y567F/Y567F-Mäusen mit der genetischen Reduktion des MAPK-Signalweg impliziert. Ansätze zur Entdeckung und Inhibierung mehrerer essentieller Mediatoren onkogener Signaltransduktion, wie sie in dieser Arbeit demonstriert wurden, könnten in Zukunft Therapiemöglichkeiten eröffnen, die das Entstehen von Resistenzen gegen zielgerichtete Therapien umgehen und ihre Wirksamkeit insgesamt erhöhen.
Acknowledgements
First and foremost I would like to thank my thesis mentor and Doktorvater Prof. Dr. Pe- ter Besmer, who enabled this study and taught me science. His modesty in combination with passion for science and his continuous support will always be a role model for me.
I am indebted to Prof. Dr. Thomas Brunner for cordially accepting me as his external graduate student, the entire extra work this entails and the superb hospitality of his groups in Bern and Konstanz.
I want to thank Prof. Dr. Claudia A.O. Stürmer for her willingness to chair my thesis committee. She conveyed her enthusiasm for science and teaching to me right from her first lecture in my undergraduate studies.
Frau Edelgard Matzner, Leiterin des Zentralen Prüfungsamts der Universität Konstanz, möchte ich für ihre jederzeitige Hilfsbereitschaft und Fachkenntnis im Umgang mit den bürokratischen Hürden einer externen Promotion herzlich danken.
Dem Fachbereich Biologie der Universität Konstanz möchte ich meinen Dank und meine Verbundenheit aussprechen, insbesondere dem Fachbereichssprecher Herrn Prof.
Dr. Marcus Groettrup und dem Fachbereichsreferenten Herrn Dr. Roland Kissmehl für ihre unbürokratische Hilfe. Frau Regine Winter gilt mein besonderer Dank für ihre liebe Unterstützung beim Druck und bei der Abgabe meiner Dissertation.
My labmates Dr. Shayu Deshpande, Dr. Ferdinand Rossi and Yasemin Yozgat I would like to thank for being with me in the lab for countless hours and challenges.
Dr. Markus Hafner from the Rockefeller University as well as Jeff Smith and John Maciejowski from the Gerstner Sloan-Kettering Graduate School I would like to thank for vectors, cell lines and stimulating discussions on bike rides and elsewhere.
Thank you, Jason, for being the best neighbor in the world.
Meinen Eltern und meiner Frau gilt mein allergrößter Dank für die nicht in Worte zu fassende Unterstützung auf meinem Lebensweg. Bernadette, Andreas und Eva – diese Arbeit ist Euch gewidmet.
1. General Introduction
The quest for a long, healthy life is a motor of mankind. Despite tremendous scientific success over the last 150 years in the cure of infectious diseases, progress in treating the seemingly myriad ways our own cells transform to a state of uncontrolled proliferation, often simply summarized as “cancer”, has been limited (Bailar and Smith 1986;
Eheman et al. 2012). With the advent of molecular biology in the 1970s and 1980s, though, first individual proto-oncogenes like KIT could be discovered as mammalian cellular counterparts of viral oncogenes like v-kit and shortly thereafter their mutant ver- sions – human oncogenes – as underlying entities of distinct cancer subsets (Stehelin et al. 1976; Tabin et al. 1982; Taparowsky et al. 1982; Besmer et al. 1986). This target identification brought about efforts to develop targeted therapeutics, finally resulting over the last decade in improved clinical outcome in specific cancer types like chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST) (Gambacorti- Passerini et al. 2011; Joensuu and DeMatteo 2012).
The knowledge gained during this long chain of incremental progress justifies further attempts to rationally dissect “cancer”. Most CML and GIST cases show features of ad- diction to one oncogene, BCR-ABL and mutant KIT, respectively. The majority of these and other cancer types, though, has more than one genetic lesion. Interindividual and intratumoral heterogeneity further complicates optimal treatment decisions that should be made for and with each patient. While the prospect of individualized medicine raises the issue of financing it, eventually, diagnosing the complete inventory of genetic le- sions on a routine basis is likely to be possible with the rapid decrease in the cost of methods like whole genome sequencing and high density array comparative genomic hybridization (aCGH). The validation of molecular drivers of cellular transformation is more challenging. Ideally, analogous to the interplay of theoretical and experimental physics, model systems can be developed to define (targetable) patterns in the broad spectrum of singular (genetic) events within the studied system (of cancerous cells).
These models are likely to be expansive, incorporating previous achievements. In this study we built on the understanding gained from modeling oncogenic KIT signaling in the organism of the mouse.
1.1 Protein kinases
The posttranslational modification of proteins by phosphorylation is a central biological mechanism to regulate the activity, location and/or structure of proteins. Interestingly, a high rate of turnover of phosphorus was initially observed in a (phospho-)protein frac- tion that had been isolated from tumor tissues (Burnett and Kennedy 1954). Phosphory- lation is catalyzed by kinases which transfer the γ-phosphate group of adenosine-5’- triphosphate (ATP) onto hydroxyl-groups of substrate proteins. While the substrate specificity of individual kinases is confined by their own as well as their substrate’s polypeptide structure, two main classes of kinases exist: Serine/threonine kinases (STKs) phosphorylate the hydroxyl group of a serine and/or threonine residue in their target recognition sequence whereas tyrosine kinases (TKs) catalyze the phosphoryla- tion of tyrosine residues. Besides phosphorylating other proteins a number of STKs and TKs can also target themselves via (trans-)autophosphorylation, oftentimes resulting in activation and/or recruitment of downstream mediator proteins.
Cytoplasmic tyrosine kinases are being utilized for intracellular signaling in metazo- ans as well as closely related premetazoans (King et al. 2008). These tyrosine kinases have been found to share conserved domains in premetazoans as well as metazoans, im- plying their first occurrence in a common unicellular ancestor (Suga et al. 2012). The evolution of receptor tyrosine kinases (RTKs) has been linked to the necessity of cell- cell signaling with the development of multicellular organisms, but one might argue that outside-in signal transduction as mediated by RTKs could be beneficial for unicellular organisms as well in terms of nutrient, stress and quorum sensing . Recently, RTKs were detected in premetazoans, but as they lack homology with RTKs in multicellular organisms it has been proposed that they developed independently from TKs after the premetazoan/metazoan divergence (Suga et al. 2012).
The long evolution of TKs notwithstanding, it took until the late 1970s until tyrosine phosphorylation was discovered and the search for tyrosine kinases began (Eckhart et al. 1979; Hunter and Sefton 1980). Three decades later, 58 RTKs in human have been identified and classified into 20 subfamilies based on the composition of their extracel- lular domains and the presence or absence of a peptide stretch called the kinase insert domain (KID) in-between the characteristic N-terminal β-sheets and C-terminal α-
helices of the canonical kinase structure (Lemmon and Schlessinger 2010). Members of the subfamily with 5 immunoglobulin (Ig)-like extracellular domains and KID are the receptor tyrosine kinases v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene hom- olog (KIT), platelet-derived growth factor receptor, alpha and beta (PDGFRA/B), colo- ny stimulating factor 1 receptor (CSF1R) and fms-related tyrosine kinase 3 (FLT3) . The phosphorylated tyrosines of proteins act as docking sites for interactions with pro- teins containing “read-out” domains, namely Src homology region 2 (SH2) domains which can bind a pYXXX consensus motif (strictly dependent on the presence of phos- phorylated tyrosine) or phosphotyrosine-binding (PTB) domains which show specificity towards NPXpY peptide epitopes (most of which bind to this target also when not phosphorylated) (Nolte et al. 1996; Schlessinger and Lemmon 2003).
1.2 The receptor tyrosine kinase KIT
KIT was originally discovered as a viral oncogene in the sarcoma of a cat (Besmer et al.
1986). While consequently its name v-kit was derived from the word “kitten“, its cellu- lar wild-type counterpart – initially designated c-KIT, now KIT – was found to be con- served in all vertebrates and encoding an essential receptor tyrosine kinase (RTK) (Yarden et al. 1987; Qiu et al. 1988). In the mouse Kit turned out to be encoded in the dominant white-spotting (W) locus on chromosome 5 (Chabot et al. 1988; Geissler et al.
1988). Now, phenotypes known for decades and linked to the W locus like depigmenta- tion (with the exception of the eye), infertility, macrocytic anemia and lack of mast cells (Cuénot 1905; Little 1915; Kitamura et al. 1978; Russell 1979) could be quite readily explained by loss-of-function mutations impairing the expression and/or function of KIT (Nocka et al. 1989). As indicated by the W mice, Kit is expressed in dedicated and diverse cell lineages. As such, it is functionally critical to sustain hematopoiesis, game- togenesis, melanogenesis and the development of pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICCs) (overview of these and following features of KIT, cf. Tab. 1).
Structurally, KIT is a member of the RTK subfamily for which five extracellular Ig- like domains (D1-D5) and a cytoplasmic split kinase/KID are characteristic. The distal
extracellular domains D2 and D3 mediate binding of a homodimer, the only known lig- and for KIT, KIT ligand (Huang et al. 1990; Yuzawa et al. 2007). Murine Kit ligand is encoded in the Steel (sl) locus, loss-of-function mutations of which result in similar phenotypes like the observed in W-mutant mice (Huang et al. 1990), reviewed in (Besmer 1997). Contrary to most kinases, not the canonical activation loop but an inhib- itory constraint imposed by the juxtamembrane domain has to be relieved to activate KIT kinase activity (Chan et al. 2003; Mol et al. 2004; DiNitto et al. 2010). This likely is achieved by the Kit ligand mediated rotation of two facing KIT proteins around their perpendicular axis, thus overcoming repulsive interactions of their D5 domains which become aligned more closely, facilitating intracellularly the trans-autophosphorylation and full activation of the kinase domains by flipping out the juxtamembrane and activa- tion loop domains (Mol et al. 2003; Mol et al. 2004; Yuzawa et al. 2007).
KIT has 22 intracellular tyrosine phosphorylation sites, all of which – at least in vitro – can be phosphorylated, in a sequence of phosphorylation events in which the Y567/Y569 bimotif of the juxtamembrane domain is an early target (DiNitto et al.
2010), possibly facilitating the release of its inhibitory constraint on kinase activity. The phospho-Y567-site on KIT, depending on their presence in the cellular context, can in- teract with a variety of positive or negative regulators and signal transducers, namely, SRC family kinases (SFKs), e.g., SRC itself (Lennartsson et al. 1999) and LYN (Timokhina et al. 1998), but also with the Cbl adaptor protein APS (Wollberg et al.
2003), the E3 ubiquitin-protein ligase Casitas B-lineage lymphoma (Cbl) itself directly (Masson et al. 2006), the megakaryocyte-associated tyrosine kinase (MATK; formerly Csk-homologous kinase, CHK) (Price et al. 1997) and the protein tyrosine phosphatase SHP-2/PTPN11 (Kozlowski et al. 1998). The presence of PTPN11 has been shown in mast cells to be required for the ability of the scaffolding adapter Gab2 (Grb2- associated-binding protein 2) to activate the Rac/JNK pathway after Gab2 itself had been tyrosine-phosphorylated via P-Y567-KIT/SFKs (Yu et al. 2006). Docking of these proteins onto KIT does not necessarily entail their activation, e.g., different crystal structures of Cbl bound to ZAP-70 peptide showed that its E3 ubiquitin-protein ligase is by default autoinhibited, requiring phosphorylation of its Y371 for full E3 activity (Dou et al. 2012). In KIT regulation Cbl has been proposed to be phosphorylated by SFKs leading to monoubiquitination of KIT and targeting for lysosomal degradation (Masson
et al. 2006). The KID phosphorylation site pY719 is the only site on KIT for the re- cruitment of PI-3K family members (Serve et al. 1994; Mali et al. 2012). Unfortunately, the KID is not stably structured, so crystal structures could not be obtained from this domain yet (Mol et al. 2003; Gajiwala et al. 2009).
Tab. 1: KIT Fact Box.
Chromosome location Mouse: chr5:75,971,012-76,052,747 (5 42.0 cM, MGSCv37 C57BL/6J).
Human: chr4:55,524,095-55,606,881 (4q11-q12, GRCh37.p5).
Gene size Mouse: 81,735 bp.
Human: 82,786 bp.
Exon/Intron structure 21 exons (3’-end of exon 9 alternatively spliced to GNNK+/- isoforms).
20 introns.
mRNA size Mouse: 5,205 bp (NCBI Reference Sequence: NM_001122733.1) 5´-UTR: 1-65; ORF: 66-3,005; 3´-UTR: 3,006-5,205.
Human: 5,190 bp (NCBI Reference Sequence: NM_000222.2) 5´-UTR: 1-87; ORF: 88-3,018; 3´-UTR: 3,019-5,190.
Amino acids Mouse: 979 aa (long exon 9 GNNK+ isoform).
Human: 976 aa (long exon 9 GNNK+ isoform).
Molecular weight 120-150 kDa (depending on tissue/amount of glycosylation).
Post-translational modifications
Glycosylation, phosphorylation, ubiquitination.
Domains 5 Ig-like extracellular domains (exons 2-9) Transmembrane domain (exon 10)
Kinase domain (exons 11-20) with the subdomains:
Juxtamembrane domain (exon 11)
Kinase domain, N-terminal lobe (exons 11-14) Kinase insert domain (KID; exons 14/15) Kinase domain, C-terminal lobe (exons 16-20) with non-canonical activation loop (exons 17-18).
Ligand Kit ligand (mmu: Kitl; hsa: KITLG), formerly also: SCF (stem cell fac- tor) / MGF (mast cell growth factor).
Dimerization partners Homodimerization only.
Pathways activated ERK/MAPK, PI3K, STAT, PLCγ.
Tissues expressed Hematopoietic stem and progenitor cells (HSCs), mast cells,
melanoblasts, melanocytes, interstitial cells of Cajal (ICC), Leydig cells, primordial germ cells (PGCs), spermatogonia, oocytes, cerebellum, sub- populations in dorsal root ganglia.
Human diseases with gain-of-function mutation
Gastrointestinal stromal tumor (GIST), mastocytosis, subsets of acute myeloid leukemia (AML) and testicular germ cell tumor (TGCT).
Human diseases with loss-of-function mutation
Piebaldism.
Knock-out mouse phenotype Perinatal lethal.
References are given in text; mmu, Mus musculus; hsa, Homo sapiens.
Even though all 22 intracellular tyrosine phosphorylation sites, the juxtamembrane do- main critical for kinase autoinhibition and all important kinase active side residues are conserved between human and mouse KIT, differences in the amino acid sequence and consequently numbering are present (e.g. murine KitT669 corresponds to human KITT670; murine KitY719F to human KITY721F). The main known functional consequence of these subtle differences is that murine KIT ligand can stimulate both human and mouse KIT, whereas human KIT ligand only weakly stimulates mouse KIT (Mitsui et al. 1993).
Taking this fact into consideration could facilitate the interpretation of KIT overexpres- sion/phosphorylation experiments: When murine KIT is expressed in a human cell line like HEK293, false-positive stimulation by potentially host-expressed (human) KitL is unlikely. Still, for host-expressed KIT should be controlled. Also, in the specific context of dexamethasone induced stress erythropoiesis assays in vitro, differences in the sensi- tivity of human erythroid precursors to stimulation with soluble murine and human KitL and their ability to upregulate the glucocorticoid receptor alpha have been reported re- cently (Varricchio et al. 2012).
Tissues that express KIT are mast cells (Nocka et al. 1989), melanoblasts and mela- nocytes (Nocka et al. 1989), interstitial cells of Cajal (ICC) (Maeda et al. 1992), Leydig cells (Manova et al. 1990), primordial germ cells (PGCs) and spermatogonia (Manova et al. 1990; Yoshinaga et al. 1991), oocytes (Manova et al. 1990), cerebellum (Manova et al. 1992), and subpopulations of neurons in embryonic dorsal root ganglia (Hirata et al. 1993). KIT is an important marker for murine hematopoietic stem cells (HSCs) and progenitor cells of the hematopoietic lineage (Ikuta and Weissman 1992). Human long- term repopulating (LT)-HSCs can be enriched by sorting for CD34+KITlow, but not KIT- cells (Gunji et al. 1993; Kawashima et al. 1996).
Human diseases with gain-of-function mutations or overexpression of KIT are pre- dominantly mastocytosis (Furitsu et al. 1993; Nagata et al. 1995; Longley et al. 1996) and gastrointestinal stromal tumor (GIST) (Hirota et al. 1998) and subsets of acute mye- loid leukemia (AML) (Gari et al. 1999), testicular germ cell tumors (Tian et al. 1999;
Looijenga et al. 2003) and melanoma (Curtin et al. 2006; Antonescu et al. 2007). Inter- estingly, a monoclonal antibody specific for a cell surface antigen expressed by a sub- group of human myeloid leukemias had been identified even before Kit, the antibody later turned out to bind a KIT epitope (Gadd and Ashman 1985; Lerner et al. 1991). Re-
cently, increased KIT expression as a consequence of a SNP in the 3’ UTR leading to diminished micro-RNA 221 binding has been implicated to account for a more than fourfold increased risk of acral melanoma (Godshalk et al. 2011). A human autosomal dominant disorder with KIT loss-of-function mutations is piebaldism, characterized by deafness, constipation and local depigmentation (e.g., a white forelock of hair); mecha- nistically characterized best are mutations in the extracellular Ig-like domain D2 which are proposed to diminished binding of Kit Ligand, but deletions and intracellular do- main mutations are frequent, too (Giebel and Spritz 1991; Ezoe et al. 1995; Fleischman et al. 1996; Yuzawa et al. 2007).
Besides the long history of mice and rats with W (“white”) alleles, which turned out to be Kit loss-of-functions mutations of different severity, the utility of rodent models to study KIT biology is substantiated by the fact that gain-of-function mutations homolo- gous to the KITD816V mutation prevalent in human mastocytosis have been detected in murine (KitD814V) and rat (KitD817Y) mast cell lines (Tsujimura et al. 1994; Tsujimura et al. 1995). Furthermore, the conditional expression of KitD814V by means of BAC- transgenesis results in severe mastocytosis in mice (Gerbaulet et al. 2011). Mice homo- zygous for strong loss-of-function W alleles that survive beyond birth (accompanied with severe macrocytic anemia) presumable retain low levels of KIT activity because mice homozygous for a Kit knock-out mutation in exon 1 (insertion of LacZ) are perina- tal lethal (Bernex et al. 1996; Waskow et al. 2004).
1.3 Interstitial Cells of Cajal
While the crucial role of the KIT protein for hematopoiesis, melanogenesis and gameto- genesis was established soon after its discovery quite readily based on the well studied phenotypes of macrocytic anemia, depigmentation and infertility in W mice, the in- volvement of KIT in the biology of a peculiar cell type of the gastrointestinal tract was somewhat hidden deep in the muscle layers of the gut.
Interstitial cells of Cajal, named after their discoverer Santiago Ramón y Cajal, form a loose network of single cells throughout the gastrointestinal tract. As first proposed by Lars Thuneberg in the early 1980s, ICC are required for intestinal pacemaker activity,
which could be experimentally proven by treating postnatal wild-type mice with an an- tagonistic KIT antibody that elicited severe anomalous gut movement and in W mice that lacked ICC, concomitantly demonstrating a KIT-dependence of ICCs (Thuneberg 1982; Maeda et al. 1992; Ward et al. 1994; Huizinga et al. 1995). Subsequently, ICC in the mouse have been classified, without a strict consensus nomenclature, into different subgroups: The majority of ICC, mainly called ICC-MY, is localized in the plane of the myenteric plexus around enteric neurons, in-between the circular and longitudinal smooth muscle layers (Beckett et al. 2007). Intramuscular ICC (ICC-IM) reside dis- persed within the smooth muscle of the stomach, cecum and colon, whereas in the small intestine they cluster as ICC-DMP within the deep muscular plexus near the submucosal surface of the circular muscle layer (Chen et al. 2007a). ICC at the submucosal/submuscular interface of the colon are designated ICC-SM or ICC-SMP and in a study of unclear significance have been reported to undergo ultrastructural changes in patients with Crohn’s disease of the colon (Rumessen et al. 2011).
In contrast to enteric neurons, ICCs are not neural crest-derived but are of mesoder- mal origin according to chicken/quail chimera and mouse experiments. (Lecoin et al.
1996; Gershon 1999; Wu et al. 2000). During mouse embryogenesis KIT- immunoreactive ICCs appear between E12 in the foregut and E14 in the terminal hind- gut and develop independently of enteric neurons (Wu et al. 2000). ICC-like cells do have been reported to develop embryonically in the absence of full-length KIT using mice homozygous for a knock-in of LacZ into the first intron of Kit and these mice die perinatally (Bernex et al. 1996). Intervention experiments with the KIT-blocking anti- body ACK2 and the TKI imatinib late in gestation (E17 to P0) came to a different con- clusion, as these interventions caused loss of functionally developed ICC-MY networks, which – as the authors observed – might be reconciled by the plasticity of ICC and its marker KIT: Upon removal of KIT-inhibition, ICC-MY and pacemaker activity recov- ered within nine days (Beckett et al. 2007). The analysis of ICC is limited by the fact that ICCs cannot be cultured in vitro and thus have to be studied either in vivo, in fixed tissue, in organ explants or by the technically challenging isolation by microdissection and cell sorting (Chen et al. 2007b). Besides KIT, markers for ICC are Nestin, Ano1, and Etv1, the latter being involved in the biogenesis of ICC-MY and ICC-IM, but not detected in ICC-DMP and ICC-SMP (Tsujimura et al. 2001; Gomez-Pinilla et al. 2009;
Chi et al. 2010). All these markers are not restricted to ICC, though, and thus a combi- nation of histological assessment and labeling techniques such as IHC, IF and tissue specific stains has to be employed for their detection (Faussone-Pellegrini and Thuneberg 1999).
In 1998, the involvement of ICC in the pathogenesis of a rare stromal tumor of the gastrointestinal tract (GIST) was proposed, based on the shared expression of KIT and the presence of activating point mutations in KIT (Hirota et al. 1998).
1.4 Gastrointestinal stromal tumor (GIST)
KIT is a sensitive diagnostic marker for GIST (Sarlomo-Rikala et al. 1998) and in the majority of GISTs activating mutations in KIT can be detected. In various national and international studies KIT mutation rates of 69-89% percent in GIST have been reported (Antonescu et al. 2003; Heinrich et al. 2003a; Wozniak et al. 2012). A subgroup of about 5%-13% GIST cases has mutations in the closely related PDGF-receptor alpha (PDGFRA), mutually exclusive with mutations in KIT (Heinrich et al. 2003b; Corless et al. 2005; Wozniak et al. 2012). The remaining fraction of cases often is classified as
“wild-type GIST” based on the absence of KIT and PDGFRA mutations and containing the majority of pediatric GIST.
Within the WT GIST class, KIT is usually still expressed (Antonescu et al. 2003;
Agaram et al. 2008a) and a few candidate driver mutations have been detected. The au- tosomal dominant disorder neurofibromatosis type 1 (NF-1; von Recklinghausen's dis- ease) had occasionally been associated with large, solid stromal tumors of the gastrointestinal tract (Schaldenbrand and Appelman 1984; Fuller and Williams 1991).
In a subset of cases with germline NF1 mutations, GIST development without KIT and PDGFRA mutations has been reported (Kinoshita et al. 2004; Miettinen et al. 2006). As NF1 is a negative regulator of the RAS/MAPK pathway, mutations in this pathway might contribute to GIST tumorigenesis. Indeed, BRAFV660E mutations have been identi- fied in some cases of WT GIST (Agaram et al. 2008b; Hostein et al. 2010; Daniels et al.
2011). Also, amplification of insulin-like growth factor 1 receptor (IGF1R) has been implicated in WT/pediatric GIST (Prakash et al. 2005; Agaram et al. 2008a). Recently,
germline and spontaneous inactivating mutations in the succinate dehydrogenase subu- nit genes SDHB and SDHC have been found in patients with WT/KIT-expression posi- tive GIST (Janeway et al. 2011; Miettinen et al. 2011). Notably, this diverse spectrum of mutations apparently gives rise to the same clinical tumor entity, GIST, implying common oncogenic pathways beneath the dominating roles the expression and activat- ing mutations of KIT play in GIST.
GIST are refractory to radiation therapy as well as standard chemotherapy and up to today only surgery can be curative in about half the cases presenting with localized dis- ease (Silberhumer et al. 2009; Joensuu and DeMatteo 2012). Metastatic and/or surgical- ly unresectable GIST could not be treated effectively until 2000. The first targeted ther- apy using a small molecule kinase inhibitor, imatinib (formerly CGP 57148B and STI571), had produced promising results in chronic myelogenous leukemia (CML).
(Druker et al. 2001). This stimulated the initiation of clinical trials in ad- vanced/metastatic GIST, as imatinib was know to inhibit the KIT and PDGF receptor besides the fusion protein BCR-ABL underlying and defining CML (Carroll et al. 1997;
Tefferi and Vardiman 2008). Imatinib proved to be efficacious also in advanced solid stromal tumors of the gastrointestinal tract and as adjuvant treatment after surgery pro- longing recurrence-free survival (Joensuu et al. 2001; Demetri et al. 2002; Dematteo et al. 2009). As imatinib is not curative, continuous treatment is necessary and patients over time relapse, most often with second-site mutations in KIT (Antonescu et al. 2005).
The second-site mutations in acquired imatinib-resistant GIST tend to be single ami- no acid substitutions in KIT, located on the allele with the primary mutation (Tamborini et al. 2004; Antonescu et al. 2005; Nishida et al. 2008). Though not the most frequent, one specific type of second-site mutation gained fame as the “gatekeeper” mutation, as it confers resistance to most known TKIs and occurs in the same conserved residue of the ATP-binding pocket of different targeted kinases and across various malignancies (Tab. 2). The gatekeeper mutation in KIT is a T670I conversion, caused by a missense mutation of ACA to ATA in the codon for this amino acid residue. As of July 2012, the database Catalogue of somatic mutations in cancer (Cosmic) was listing 21 tumors of 14 individual cases of GIST with mutation of the KIT gatekeeper residue, 13 cases had the T670I mutation and 1 case of the T670E mutation (e.g., the database lists 3 tumors from
Nishida et al. 2008, but all are from the same case; Wardelmann et al. cite the T670E case in two publications, which is listed as 4 tumors in the database)1.
Tab. 2: Overview of “gatekeeper” mutations conferring resistance to kinase inhibitors, as detected clinically and with special emphasis on GIST.
Cancer/syndrome Kinase inhibitor Gatekeeper mutation Reference CML Imatinib/dasatinib BCR-ABL(T315I) (Gorre et al. 2001)
HES Imatinib FIP1L1-PDGFRA(T674I) (Cools et al. 2003)
(von Bubnoff et al. 2005) NSCLC Gefitinib/erlotinib EGFR(x;T790M) (Kobayashi et al. 2005)
(Pao et al. 2005) EGFR(T790M) (Bell et al. 2005) GIST Imatinib/dasatinib KIT(x;T670I) (Tamborini et al. 2004)
(Debiec-Rychter et al. 2005) (Antonescu et al. 2005) (Wardelmann et al. 2006) (Nishida et al. 2008) (Liegl et al. 2008) (Cameron et al. 2010) (Bauer et al. 2010)
Abbreviations: CML, chronic myelogenous leukemia; HES, hypereosinophilic syndrome; NSCLC, non small-cell lung cancer; x, primary mutation.
The central role of KIT activating mutations in GIST was experimentally proven by the introduction of KIT mutations found in familial cases of GIST into the germline of mice by targeted mutagenesis of the Kit locus. Three such mouse models have been reported, all of which developed ICC hyperplasia and cecal GIST lesions (Sommer et al. 2003;
Rubin et al. 2005; Nakai et al. 2008). Advantages of knock-in models like the KitV558Δ/+
mouse in contrast to transgenic mice are that the targeted gene is expressed from its en- dogenous locus (including all possible tissue specific regulators/enhancers); as the tar- geted chromosome is known, mutations on other chromosomes can be combined by in- tercrossing; phenotypes/tumors develop orthotopic in an immunocompetent environ- ment; and unphysiologic alteration/silencing of the mutant allele is not expected in germline transmissions over the generations.
1URL:http://www.sanger.ac.uk/perl/genetics/CGP/cosmic?action=bygene&ln=KIT&start=670&end=670
&coords=AA%3AAA (July 30, 2012).
1.5 Tyrosine kinase inhibitors with KIT selectivity
Imatinib mesylate (formerly CGP 57148B and STI571) emerged from various attempts to inhibit protein kinases and as the most promising compound for clinical development to target the abnormal protein product BCR-ABL caused by the fusion of the breakpoint cluster region gene (BCR; chr22) and the non-receptor tyrosine kinase c-abl oncogene 1 (ABL1; chr9) in the Philadelphia chromosome (Deininger et al. 2005). Given the struc- tural and amino acid conservation in the core of most kinases (e.g., the “gatekeeper”
residue, Tab. 2), imatinib’s fairly specific inhibition of ABL in vitro was a landmark success for the field (Buchdunger et al. 1996). As it turned out, its initial “off-targets”
KIT and PDGFRA/B are key targets in GIST. Based on imatinib’s clinical and financial success, a plethora of kinase inhibitors has been and is being developed, partly motivat- ed by the search for second and higher generation TKIs to overcome acquired re- sistance.
The inhibitor sunitinib malate is approved as second-line treatment for imatinib- refractory or primary resistant GIST and has been reported to inhibit KITT670I (Carter et al. 2005; Prenen et al. 2006). Sunitinib has been tested preclinically only in a non- orthotopic immunocompromised xenograft mouse model without the gatekeeper muta- tion (Revheim et al. 2010). Sorafenib tosylate is the second TKI that has been shown in vitro to inhibit T670I-mutant KIT (Guida et al. 2007) (Guo et al. 2007)and in vivo only tested in an immunocompromised xenograft mouse model of GIST without second-site mutations/with exon 11 mutations only (Huynh et al. 2009). Dasatinib has been tested by our group in the single-mutant KitV558Δ/+ mouse model (Rossi et al. 2010). Dasatinib does not inhibit the gatekeeper mutant versions of RTKs as shown for BCR- ABL(T315I) (Shah et al. 2004). Of the four kinase inhibitors with KIT specificity uti- lized in this study, only dasatinib is predicted to bind the activated conformation of KIT based on DS/ABL interaction data (type I inhibition), whereas imatinib, sorafenib and sunitinib bind and are thought to stabilize the closed/autoinhibited conformation of KIT (type II inhibition) (Mol et al. 2004; Manley et al. 2006; Gajiwala et al. 2009; Davis et al. 2011)2.
2 In Suppl. Tab. 5 of Davis et al. 2011 sunitinib is classified as a type I inhibitor based on affinity data with phosphorylated versus nonphosporylated wt ABL1, but according to data in the respective Suppl.
Tab. 1 sunitinib has low affinity for phos./nonphos. ABL1 [150 nm/250 nM] in comparison with its pri-
All these compounds have the potential to inhibit other kinases than KIT and it is not clear which contribution the inhibition of KIT alone has on the observed inhibition of proteins supposed to be downstream of KIT, which might be actually downstream of off-targets. Additionally, these TKIs all inhibit mutant as well as wild-type KIT (Davis et al. 2011) and the role of the wild-type KIT protein in GIST signal transduction as well as the importance (if any) of its inhibition for GIST treatment outcome is not known.
mary target KIT [0.21 nM] and sunitinib has been shown directly to target the autoinhibited and not the active conformation of KIT by Gajiwala et al. 2009.
2. Imatinib resistance and microcytic erythrocytosis in a Kit
V558Δ;T669I/+gatekeeper-mutant mouse model of gastrointestinal stromal tumor
Benedikt Bosbach1, Shayu Deshpande1, Ferdinand Rossi1, Jae-Hung Shieh3, Gunhild Sommer5, Elisa de Stanchina2, Darren R. Veach2, Joseph M. Scandura6, Katia Manova- Todorova1, Malcolm A.S. Moore3, Cristina R. Antonescu4, Peter Besmer1#
1Developmental Biology, 2Molecular Pharmacology & Chemistry, and 3Cell Biology Programs, Sloan-Kettering Institute, and 4Department of Pathology, Memorial Sloan- Kettering Cancer Center, 10065 New York, New York, 5Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 29425 Charleston, South Carolina and 6Department of Medicine, Weill Cornell Medical College, 10065 New York, NY.
Proc Natl Acad Sci U S A. 2012 May 31. [Epub ahead of print]
Classification: BIOLOGICAL SCIENCES: Medical Sciences PMID: 22652566
Corresponding author#: Peter Besmer
Developmental Biology Program Sloan-Kettering Institute
1275 York Avenue New York, NY 10065.
Phone: 212-639-8188;
Fax: 646-422-2355.
E-mail: p-besmer@ski.mskcc.org
2.1 Abstract
Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the KIT receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequent- ly develop imatinib resistance as a result of second-site KIT mutations. To investigate the consequences of second-site KIT mutations on GIST development and imatinib sen- sitivity, we engineered a mouse model carrying in the endogenous Kit locus both the KitV558Δ mutation found in a familial case of GIST and the KitT669I (human KITT670I)
“gatekeeper” mutation found in imatinib-resistant GIST patients. Similar to KitV558Δ/+
mice, KitV558Δ;T669I/+ mice developed gastric and colonic interstitial cell of Cajal hyper- plasia as well as cecal GIST. In contrast to the single-mutant KitV558Δ/+ control mice, treatment of the KitV558Δ;T669I/+ mice with either imatinib or dasatinib failed to inhibit oncogenic KIT signaling and GIST growth. However, this resistance could be overcome by treatment of KitV558Δ;T669I/+ mice with sunitinib or sorafenib. Although tumor lesions were smaller in KitV558Δ;T669I/+ mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in KitV558Δ;T669I/+ mice.
Strikingly, the KitV558Δ;T669I/+ mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.
Key words:
soft tissue sarcoma | hematopoiesis | erythropoiesis | drug resistance
2.2 Introduction
Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract. GISTs express receptor tyrosine kinase KIT and are thought to derive from a KIT+ or KITlow interstitial cell of Cajal (ICC) progenitor or from ICCs themselves (Kwon et al. 2009). The principal genetic events responsible for the patho-
genesis of GIST are thought to be gain-of-function mutations in the KIT gene or in a small subset in the PDGFR-alpha gene (Hirota et al. 1998; Heinrich et al. 2003b). KIT- activating mutations in GIST are found predominantly in the juxtamembrane domain of the KIT receptor (exon 11) (Antonescu et al. 2003), but mutations in the extracellular (exon 9) and kinase domains of KIT have been described as well (Lasota et al. 2000;
Rubin et al. 2001). The KIT juxtamembrane domain has an autoinhibitory role and sta- bilizes an inactive conformation of the KIT kinase; mutation of this domain disrupts the conformational integrity and thus diminishes autoinhibition (Mol et al. 2004). KIT acti- vation-loop mutations found in acute myeloid leukemias, mast cell neoplasms, and sem- inomas stabilize an active conformation of the KIT kinase.
Imatinib mesylate, an inhibitor of the KIT, PDGFR, and BCR-ABL tyrosine kinases, is the first-line therapy in patients with chronic myelogenous leukemia (CML) and met- astatic GIST. Imatinib is most effective in GISTs with KIT-activating mutations in the juxtamembrane domain, some kinase domain mutations, or extracellular domain muta- tions. However, KIT mutations that destabilize the inactive form of the kinase are re- sistant to inhibition by imatinib. Imatinib binds to the inactive conformation of the ABL and KIT kinases and not to the active conformation and inhibits juxtamembrane domain KIT mutants but not activation-loop KIT mutants (Mol et al. 2004).
That oncogenic KIT mutations have a critical role in the development of human neo- plasias was strengthened by the observation of familial GIST and familial mastocytosis (Nishida et al. 1998). Patients with familial GIST also may have cutaneous mastocytosis and hyperpigmentation. The observation of germ-line KIT gain-of-function mutations provided us with a rationale for developing a mouse model for familial GIST. The KIT- V558 deletion mutation found in the first familial GIST case was introduced into the mouse genome using a knock-in strategy (Sommer et al. 2003). The mutant animals de- veloped ICC hyperplasia and neoplastic lesions in the cecum indistinguishable from human GIST with complete penetrance (Rossi et al. 2006; Rossi et al. 2010).
Long-term imatinib treatment of patients with GIST or CML is associated with the development of drug resistance. In GIST most cases of resistance appear to derive from second-site mutations in the kinase domain of the KIT receptor (Antonescu et al. 2005;
Debiec-Rychter et al. 2005). In patients who have CML, second-site mutations in BCR- ABL are the predominant mechanism of drug resistance (Gorre et al. 2001; Shah et al.
2002). The second-site mutations in acquired imatinib-resistant GIST tend to be single amino acid substitutions in KIT, located on the allele with the primary mutation (Tamborini et al. 2004; Antonescu et al. 2005). Second-site mutations in GIST occur in catalytic domain II of KIT, exons 17 and 18, as well as in the N-terminal kinase domain, exon 13 (V654A) and exon 14 (T670I) (Antonescu et al. 2005). In the gatekeeper T670I mutation, the isoleucine methyl group protrudes into the imatinib binding site and dis- rupts an important hydrogen bond formation between imatinib and the kinase, preclu- ding proper binding of imatinib (Mol et al. 2004). Second-site mutations in the activa- tion loop within the kinase domain stabilize the active conformation of KIT and main- tain it constitutively activated at a high level, thereby preventing imatinib binding. Cur- rently, several drugs, including sunitinib, dasatinib, and sorafenib, are being evaluated for efficacy in the treatment of imatinib-resistant GIST. Previous in vitro studies indica- ted that both sunitinib and sorafenib inhibit the T670I gatekeeper mutation, but imati- nib, dasatinib, and nilotinib failed to do so (Carter et al. 2005; Guo et al. 2007).
Because of the clinical importance of imatinib resistance, the development of new strategies for the treatment of GIST is highly relevant. Such strategies may be based on the development of KIT kinase inhibitors that show efficacy with the resistant forms of KIT; targeting of downstream signaling components critical for oncogenic KIT function could provide a second approach. However, models to examine these approaches and their possible side effects in vivo have not been reported. Here we describe the deriva- tion of a mouse model for imatinib-resistant GIST that includes both the juxtamembrane domain KitV558Δ and KitT669I gatekeeper mutations as a tool to develop therapeutic strat- egies for imatinib-resistant GIST and to investigate the consequences of KIT oncogenic signaling in other KIT-dependent cell lineages in particular in hematopoiesis.
2.3 Results
Derivation and Phenotypic Characterization of KitV558Δ;T669I/+ Gatekeeper Mice.
To investigate the consequences of second-site KIT mutations on imatinib susceptibility and GIST development in vivo, we generated a mouse model introducing both the KitV558Δ and the KitT669I gatekeeper mutation, corresponding to human KITT670I and
found in cases of imatinib-resistant GIST, into the endogenous Kit locus. To facilitate simultaneous introduction of the two point mutations into the mouse Kit gene, the tar- geting vector included a floxed neomycin-resistance gene (NEO) cassette in Kit intron 11 for positive selection of recombinant ES cells containing both the V558Δ (exon 11) and T669I (exon 14) mutations (Fig. 1A). After successful integration and germ-line transmission of the KitV558Δ;T669I-NEO allele, the intronic NEO cassette was removed by crossing to Tg(EIIa-cre) mice (19). The resulting KitV558Δ;T669I allele retains a single loxP site in intron 11 (Fig. 1A3). A KitV558Δ allele with a loxP site in intron 11 was gen- erated as a control for the KitV558Δ;T669I allele (Fig. S7A).
Fig. 1: Derivation and phenotypic characterization of Kit-gatekeeper mice.
(A) Targeting strategy for the simultaneous knock-in of V558Δ (exon 11) and T669I (exon 14) into the 129/Sv Kit locus. Blue and red bars denote the exons with the respective point mutations. A similar tar- geting vector with a shorter 3′ homology arm was used to generate the single-mutant KitV558Δ/+ mice (Fig.
S7A). Triangles (not drawn to scale) indicate loxP sites; white gaps indicate BamHI restriction sites; bar indicates 518-bp Southern blot probe. DTA, diphtheria toxin A gene; NEO, neomycin resistance gene. (B) Kaplan-Meier survival plot showing increased survival of gatekeeper-mutant KitV558Δ;T669I/+ mice in com- parison with KitV558Δ/+ mice (n ≥ 43 each; ticks indicate censored subjects). (C) Photographs of ileocecal junctions showing reduced length and diameter of tumor alongside the cecum in KitV558Δ;T669I/+ mice (Middle; red bracket indicates straight cecal GIST) in comparison with KitV558Δ/+ mice (Bottom; blue bracket indicates twisted cecal GIST). Of note, the cecum is significantly shorter in KitV558Δ;T669I/+ mice than in wild-type mice (Top; black arrow) and KitV558Δ/+ mice. Representative pictures of 3-mo-old ani- mals are shown with colon facing down left and ileum facing down right (n ≥ 59 each). (Scale bar, 1 cm.)
Double-mutant KitV558Δ;T669I/+ mice are viable and fertile but, in contrast to KitV558Δ/+
mice, were born at sub-Mendelian ratios when crossed to wild-type mice (35% instead of 50% heterozygous offspring). In comparison with single-mutant KitV558Δ/+ mice, dou- ble-mutant KitV558Δ;T669I/+
mice had a prolonged lifespan with a median survival of 14 mo (n > 43 each, P < 0.0001) (Fig. 1B).
Invariably, KitV558Δ;T669I/+
mice developed cecal tumors. These tumors were smaller than in KitV558Δ/+ mice, perhaps explaining the improved survival by a decreased chance
of intestinal obstruction (Fig. 1C). The average tumor diameter in 3-mo-old animals was fivefold smaller in KitV558Δ;T669I/+ than in KitV558Δ/+ mice (1.4 ± 0.1 mm vs. 7.0 ± 0.3 mm, P < 0.001). Interestingly, not only were the cecal tumors smaller, but the length of the cecum was significantly shorter in KitV558Δ;T669I/+ mice compared with KitV558Δ/+ and wild-type mice (13 ± 2 mm vs. 24 ± 2 mm, P = 0.003) (Fig. 1C).
Fig. 2: Cecal GIST and pronounced gastric and colonic ICC hyperplasia in KitV558Δ;T669I/+ mice.
Cross-sections of stomach (A–C; higher magnification is shown in A*–C*), cecum (D–F), and colon (G–
I) of 3- to 4-mo-old wild-type, KitV558Δ/+ and KitV558Δ;T669I/+ mice. Arrows indicate normal thin layer of myenteric ICC in wild-type samples. ICC hyperplasia in stomach and colon samples is indicated by black bars. Note the extensive hyperplasia involving the circular muscle layer (dotted lines) in the stomach of KitV558Δ;T669I/+ mice. Photographs show representative H&E staining; n ≥ 3 each. (Scale bars: 50 μm in A*–C*; 100 μm in A–I.)
