Modeling Oncogenic KIT Signaling and Drug Resistance in the Mouse

Bosbach, Benedikt

Modeling Oncogenic KIT Signaling and Drug Resistance in the Mouse

Dateien

Dissertation Benedikt Bosbach OA.pdfGröße: 13.54 MBDownloads: 942

Datum

2012

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Biologie

Publikationstyp

Dissertation

Publikationsstatus

Published

Zusammenfassung

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 Kit^V558Δ, 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 Kit^V558Δ/+ 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 recurrence-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 KIT^T670I/+ and Kit^T669I, 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 mutation Kit^V558Δ and the gatekeeper mutation Kit^T669I were introduced simultaneously into the Kit locus. The derived Kit^{V558Δ;T669I/+} mice are viable and developed ICC hyperplasia and cecal GIST lesions. While GIST lesions were smaller in Kit^{V558Δ;T669I/+} mice in comparison with Kit^V558Δ/+ mice, ICC hyperplasia was more pronounced, with no apparent changes in oncogenic signal transduction in both tissues/mouse models. Importantly, Kit^{V558Δ;T669I/+} GIST were resistant to intervention with imatinib and dasatinib at the biochemical 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, Kit^{V558Δ;T669I/+} mice uniformly and within six weeks after birth developed 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 Kit^V558Δ;T669I mutation is a strong gain-of-function mutation. Concordantly, Kit^{V558Δ;T669I/+} mice had increased mast cell hyperplasia in addition to the observed microcytic erythrocytosis and the increased ICC hyperplasia. Besides the hyperplastic ICC and GIST lesions, also burst-forming units erythroid (BFU-Es) obtained from bone marrow and spleen of Kit^{V558Δ;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 Kit^T669I mutation to the Kit^V558Δ 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 Kit^V558Δ kinase to elucidate the contribution of the respective downstream signaling cascades to GIST development. Therefore, in the context of the Kit^V558Δ mutation two well-characterized phosphorylation sites of KIT, the Src family binding site pY567 and the PI3K binding site pY719, were substituted individually with phenylalanine by targeting the corresponding codons in the mouse genome. Kit^{V558Δ;Y567F/Y567F} mice developed attenuated GIST and ICC hyperplasia, associated with diminished activation of the MAPK pathway. Remarkably, GIST of Kit^{V558Δ;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 Kit^{V558Δ;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 Kit^{V558Δ;Y719F/Y719F} mice. While other KIT-dependent processes, namely hematopoiesis and melanogenesis, also did not show any apparent defects in Kit^{V558Δ;Y719F/Y719F} mice, the known male sterility phenotype of Kit^Y719F/Y719F mice was not rescued in Kit^{V558Δ;Y719F/Y719F} mice. This indicates that cell-specific and absolute pathway requirements can exist in RTK signaling, even in the context of a potent activating mutation.

Translation of these genetic findings by treating established GIST of Kit^V558Δ/+ 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 Kit^{V558Δ;Y567F/Y567F} mice. Approaches like this, to elucidate and target multiple proximal downstream mediators 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.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

gametogenesis, spermatogenesis, melanogenesis, hematopoiesis, hematopoietic stem cell, HSC, hematopoietic progenitor cell, BFU-E, CFU-E, erythropoiesis, macrocytic anemia, microcytic erythrocytosis, polycythemia vera, EPO, JAK2, splenectomy, mast cell, receptor tyrosine kinase, KIT, interstitial cell of Cajal, ICC, hyperplasia, soft-tissue sarcoma, Gastrointestinaler Stromatumor, gastrointestinal stromal tumor, familial GIST, gain-of-function, loss-of-function, site-directed mutagenesis, conditional knock-in mouse model, loxP, signal transduction, PI3K, aneuploidy, chr12, chr14, tyrosine kinase inhibitor, sorafenib tosylate, dasatinib salt, sunitinib malate, drug resistance, gatekeeper

Zitieren

ISO 690

BOSBACH, Benedikt, 2012. Modeling Oncogenic KIT Signaling and Drug Resistance in the Mouse [Dissertation]. Konstanz: University of Konstanz

BibTex

@phdthesis{Bosbach2012Model-20467,
  year={2012},
  title={Modeling Oncogenic KIT Signaling and Drug Resistance in the Mouse},
  author={Bosbach, Benedikt},
  note={Teilw. ersch. in: Proceedings of the National Academy of Sciences of the United States of America : PNAS ; 109 (2012), 34. - E2276-E2283. - doi: 10.1073/pnas.1115240109},
  address={Konstanz},
  school={Universität Konstanz}
}

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    <dcterms:abstract xml:lang="eng">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 Kit&lt;sup&gt;V558Δ&lt;/sup&gt;, 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 Kit&lt;sup&gt;V558Δ/+&lt;/sup&gt; 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 recurrence-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 KIT&lt;sup&gt;T670I/+&lt;/sup&gt; and Kit&lt;sup&gt;T669I&lt;/sup&gt;, respectively.&lt;br /&gt;&lt;br /&gt; 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 mutation Kit&lt;sup&gt;V558Δ&lt;/sup&gt; and the gatekeeper mutation Kit&lt;sup&gt;T669I&lt;/sup&gt; were introduced simultaneously into the Kit locus. The derived Kit&lt;sup&gt;V558Δ;T669I/+&lt;/sup&gt; mice are viable and developed ICC hyperplasia and cecal GIST lesions. While GIST lesions were smaller in Kit&lt;sup&gt;V558Δ;T669I/+&lt;/sup&gt; mice in comparison with Kit&lt;sup&gt;V558Δ/+&lt;/sup&gt; mice, ICC hyperplasia was more pronounced, with no apparent changes in oncogenic signal transduction in both tissues/mouse models. Importantly, Kit&lt;sup&gt;V558Δ;T669I/+&lt;/sup&gt; GIST were resistant to intervention with imatinib and dasatinib at the biochemical as well as at the histological/cellular level. This is further remarkable as both these TKIs are known to inhibit wild-type KIT (Kit&lt;sup&gt;+&lt;/sup&gt;), 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.&lt;br /&gt;&lt;br /&gt; Unexpectedly, Kit&lt;sup&gt;V558Δ;T669I/+&lt;/sup&gt; mice uniformly and within six weeks after birth developed 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 Kit&lt;sup&gt;V558Δ;T669I&lt;/sup&gt; mutation is a strong gain-of-function mutation. Concordantly, Kit&lt;sup&gt;V558Δ;T669I/+&lt;/sup&gt; mice had increased mast cell hyperplasia in addition to the observed microcytic erythrocytosis and the increased ICC hyperplasia. Besides the hyperplastic ICC and GIST lesions, also burst-forming units erythroid (BFU-Es) obtained from bone marrow and spleen of Kit&lt;sup&gt;V558Δ;T669I/+&lt;/sup&gt; 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 Kit&lt;sup&gt;T669I&lt;/sup&gt; mutation to the Kit&lt;sup&gt;V558Δ&lt;/sup&gt; mutation introduces qualitative changes in KIT, understanding of which might guide the design of improved inhibitors for gatekeeper-mutant kinases.&lt;br /&gt;&lt;br /&gt; The second aim of this study was to molecularly abrogate specific phosphorylation sites of the Kit&lt;sup&gt;V558Δ&lt;/sup&gt; kinase to elucidate the contribution of the respective downstream signaling cascades to GIST development. Therefore, in the context of the Kit&lt;sup&gt;V558Δ&lt;/sup&gt; mutation two well-characterized phosphorylation sites of KIT, the Src family binding site pY567 and the PI3K binding site pY719, were substituted individually with phenylalanine by targeting the corresponding codons in the mouse genome. Kit&lt;sup&gt;V558Δ;Y567F/Y567F&lt;/sup&gt; mice developed attenuated GIST and ICC hyperplasia, associated with diminished activation of the MAPK pathway. Remarkably, GIST of Kit&lt;sup&gt;V558Δ;Y567F/Y567F&lt;/sup&gt; 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.&lt;br /&gt;&lt;br /&gt; No tumor development was observed in the Kit&lt;sup&gt;V558Δ;Y719F/Y719F&lt;/sup&gt; 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 Kit&lt;sup&gt;V558Δ;Y719F/Y719F&lt;/sup&gt; mice. While other KIT-dependent processes, namely hematopoiesis and melanogenesis, also did not show any apparent defects in Kit&lt;sup&gt;V558Δ;Y719F/Y719F&lt;/sup&gt; mice, the known male sterility phenotype of Kit&lt;sup&gt;Y719F/Y719F&lt;/sup&gt; mice was not rescued in Kit&lt;sup&gt;V558Δ;Y719F/Y719F&lt;/sup&gt; mice. This indicates that cell-specific and absolute pathway requirements can exist in RTK signaling, even in the context of a potent activating mutation.&lt;br /&gt;&lt;br /&gt; Translation of these genetic findings by treating established GIST of Kit&lt;sup&gt;V558Δ/+&lt;/sup&gt; 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 Kit&lt;sup&gt;V558Δ;Y567F/Y567F&lt;/sup&gt; mice. Approaches like this, to elucidate and target multiple proximal downstream mediators 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.</dcterms:abstract>
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Prüfungsdatum der Dissertation

September 12, 2012

Kommentar zur Publikation

Teilw. ersch. in: Proceedings of the National Academy of Sciences of the United States of America : PNAS ; 109 (2012), 34. - E2276-E2283. - doi: 10.1073/pnas.1115240109