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Kitlow stem cells cause resistance to Kit/platelet-derived growth factor alpha inhibitors in murine gastrointestinal stromal tumors.

ABSTRACT: Gastrointestinal stromal tumors (GIST) are related to interstitial cells of Cajal (ICC) and often contain activating stem cell factor receptor (Kit) or platelet-derived growth factor receptor alpha (Pdgfra) mutations. Kit/Pdgfra inhibitors such as imatinib mesylate have increased progression-free survival in metastatic GIST but are not curative. In mouse models we investigated whether Kit(low) ICC progenitors could represent an inherently Kit/Pdgfra inhibitor-resistant reservoir for GIST.Isolated Kit(low)Cd44(+)Cd34(+) cells were characterized after serial cloning. The tumorigenic potential of spontaneously transformed cells was investigated in nude mice. The Kit(low)Cd44(+)Cd34(+) cells' responsiveness to Kit activation and blockade was studied by enumerating them in Kit(K641E) mice (a GIST model), in mice with defective Kit signaling, and pharmacologically.Single isolated Kit(low)Cd44(+)Cd34(+) cells were clonogenic and capable of self-renewal and differentiation into ICC. In nude mice, spontaneously transformed cells formed malignant tumors expressing GIST markers. The Kit(low)Cd44(+)Cd34(+) cells were resistant to in vitro Kit blockade, including by imatinib, and occurred in normal numbers in mice with reduced Kit signaling. In Kit(K641E) mice, the mutant ICC stem cells were grossly hyperplastic but remained imatinib-resistant. In contrast, the cancer stem, cell-targeting drug salinomycin blocked the proliferation of Kit(low)Cd44(+)Cd34(+) cells and increased their sensitivity to imatinib.Kit(low)Cd44(+)Cd34(+) progenitors are true stem cells for normal and hyperplastic ICC and give rise to GIST. Resistance to Kit/Pdgfra inhibitors is inherent in GIST and is caused by the native ICC stem cells' lack of dependence on Kit for survival, which is maintained after the acquisition of oncogenic Kit mutation. Cancer stem cell drugs may target these cells.

SUBMITTER: Bardsley MR

PROVIDER: S-EPMC2933938 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Kitlow stem cells cause resistance to Kit/platelet-derived growth factor alpha inhibitors in murine gastrointestinal stromal tumors.

Bardsley Michael R MR Horváth Viktor J VJ Asuzu David T DT Lorincz Andrea A Redelman Doug D Hayashi Yujiro Y Popko Laura N LN Young David L DL Lomberk Gwen A GA Urrutia Raul A RA Farrugia Gianrico G Rubin Brian P BP Ordog Tamas T

Gastroenterology 20100604 3

<h4>Background & aims</h4>Gastrointestinal stromal tumors (GIST) are related to interstitial cells of Cajal (ICC) and often contain activating stem cell factor receptor (Kit) or platelet-derived growth factor receptor alpha (Pdgfra) mutations. Kit/Pdgfra inhibitors such as imatinib mesylate have increased progression-free survival in metastatic GIST but are not curative. In mouse models we investigated whether Kit(low) ICC progenitors could represent an inherently Kit/Pdgfra inhibitor-resistant ...[more]

PMID: 20621681

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Project description:Background & aimsIn gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1.MethodsWe studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation.ResultsKIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA.ConclusionsIn KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.

Project description:Gastrointestinal stromal tumors (GISTs) are the most common Mesenchymal Neoplasm of the gastrointestinal tract. The tumorigenesis of GISTs has been associated with the gain-of-function mutation and abnormal activation of the stem cell factor receptor (c-KIT) and platelet-derived growth factor receptor alpha (PDGFR?) kinases. Hence, inhibitors that target c-KIT and PDGFR? could be a therapeutic option for the treatment of GISTs. The available approved c-KIT/PDGFR? inhibitors possessed low efficacy with off-target effects, which necessitated the development of potent inhibitors. We performed computational studies of 48 pyrazolopyridine derivatives that showed inhibitory activity against c-KIT and PDGFR? to study the structural properties important for inhibition of both the kinases. The derivative of phenylurea, which has high activities for both c-KIT (pIC50 = 8.6) and PDGFR? (pIC50 = 8.1), was used as the representative compound for the dataset. Molecular docking and molecular dynamics simulation (100 ns) of compound 14 was performed. Compound 14 showed the formation of hydrogen bonding with Cys673, Glu640, and Asp810 in c-KIT, and Cys677, Glu644, and Asp836 in PDGFR?. The results also suggested that Thr670/T674 substitution in c-KIT/PDGFR? induced conformational changes at the binding site of the receptors. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed based on the inhibitors. Contour map analysis showed that electropositive and bulky substituents at the para-position and the meta-position of the benzyl ring of compound 14 was favorable and may increase the inhibitory activity against both c-KIT and PDGFR?. Analysis of the results suggested that having bulky and hydrophobic substituents that extend into the hydrophobic pocket of the binding site increases the activity for both c-KIT and PDGFR?. Based on the contour map analysis, 50 compounds were designed, and the activities were predicted. An evaluation of binding free energy showed that eight of the designed compounds have potential binding affinity with c-KIT/PDGFR?. Absorption, distribution, metabolism, excretion and toxicity (ADMET) and synthetic feasibility tests showed that the designed compounds have reasonable pharmaceutical properties and synthetic feasibility. Further experimental study of the designed compounds is recommended. The structural information from this study could provide useful insight into the future development of c-KIT and PDGFR? inhibitors.

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Kitlow stem cells cause resistance to Kit/platelet-derived growth factor alpha inhibitors in murine gastrointestinal stromal tumors.

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Kitlow stem cells cause resistance to Kit/platelet-derived growth factor alpha inhibitors in murine gastrointestinal stromal tumors.

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