Combination therapy for KIT-mutant mast cells: targeting constitutive NFAT and KIT activity.

Resistant KIT mutations have hindered the development of KIT kinase inhibitors for treatment of patients with systemic mastocytosis. The goal of this research was to characterize the synergistic effects of a novel combination therapy involving inhibition of KIT and calcineurin phosphatase, a nuclear factor of activated T cells (NFAT) regulator, using a panel of KIT-mutant mast cell lines. The effects of monotherapy or combination therapy on the cellular viability/survival of KIT-mutant mast cells were evaluated. In addition, NFAT-dependent transcriptional activity was monitored in a representative cell line to evaluate the mechanisms responsible for the efficacy of combination therapy. Finally, shRNA was used to stably knockdown calcineurin expression to confirm the role of calcineurin in the observed synergy. The combination of a KIT inhibitor and a calcineurin phosphatase inhibitor (CNPI) synergized to reduce cell viability and induce apoptosis in six distinct KIT-mutant mast cell lines. Both KIT inhibitors and CNPIs were found to decrease NFAT-dependent transcriptional activity. NFAT-specific inhibitors induced similar synergistic apoptosis induction as CNPIs when combined with a KIT inhibitor. Notably, NFAT was constitutively active in each KIT-mutant cell line tested. Knockdown of calcineurin subunit PPP3R1 sensitized cells to KIT inhibition and increased NFAT phosphorylation and cytoplasmic localization. Constitutive activation of NFAT appears to represent a novel and targetable characteristic of KIT-mutant mast cell disease. Our studies suggest that combining KIT inhibition with NFAT inhibition might represent a new treatment strategy for mast cell disease.

Sorafenib inhibits many kinase mutations associated with drug-resistant gastrointestinal stromal tumors.

Sorafenib has substantial clinical activity as third- or fourth-line treatment of imatinib- and sunitinib-resistant gastrointestinal stromal tumors (GIST). Because sorafenib targets both angiogenesis-related kinases (VEGFR) and the pathogenetic kinases found in GIST (KIT or PDGFRA), the molecular basis for sorafenib efficacy in this setting remains unknown. We sought to determine the spectrum of activity of sorafenib against different mutant kinases associated with drug-sensitive and drug-resistant GIST. We compared the activity of imatinib and sorafenib against transiently expressed mutant forms of KIT and PDGFRA, including various secondary mutations that have been identified in imatinib-resistant or sunitinib-resistant GISTs. We also examined these drugs against four GIST cell lines, three of which are imatinib resistant. In our in vitro studies, we determined that sorafenib inhibited imatinib-resistant mutations in exons encoding the ATP/drug-binding pocket and in exons encoding the activation loop, with the exception of substitutions at KIT codon D816 and PDGFRA codon 842. Notably our data indicate that sorafenib is more effective than imatinib or sunitinib for inhibiting the kinase activity of drug-resistant KIT mutants (as assessed by biochemical IC(50)). We hypothesize that a major determinant of the efficacy of sorafenib for treatment of advanced GIST is the activity of this agent against KIT or PDGFRA-mutant kinases. These results have implications for the further development of treatments for drug-resistant GIST.

PDGFRA activating mutations in gastrointestinal stromal tumors.

Most gastrointestinal stromal tumors (GISTs) have activating mutations in the KIT receptor tyrosine kinase, and most patients with GISTs respond well to Gleevec, which inhibits KIT kinase activity. Here we show that approximately 35% (14 of 40) of GISTs lacking KIT mutations have intragenic activation mutations in the related receptor tyrosine kinase, platelet-derived growth factor receptor alpha (PDGFRA). Tumors expressing KIT or PDGFRA oncoproteins were indistinguishable with respect to activation of downstream signaling intermediates and cytogenetic changes associated with tumor progression. Thus, KIT and PDGFRA mutations appear to be alternative and mutually exclusive oncogenic mechanisms in GISTs.

SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase.

Internal tandem duplication (ITD) in the juxtamembrane portion of Fms-like tyrosine kinase 3 (FLT3), a type III receptor tyrosine kinase (RTK), is the most common molecular defect associated with acute myeloid leukemia (AML). The high prevalence of this activating mutation makes it a potential target for molecularly based therapy. Indolinone tyrosine kinase inhibitors have known activity against KIT, another member of the type III RTK family. Given the conserved homology between members of this family, we postulated that the activity of some KIT inhibitors would extend to FLT3. We used various leukemic cell lines (BaF3, MV 4-11, RS 4;11) to test the activity of indolinone compounds against the FLT3 kinase activity of both wild-type (WT) and ITD isoforms. Both SU5416 and SU5614 were capable of inhibiting autophosphorylation of ITD and WT FLT3 (SU5416 concentration that inhibits 50% [IC(50)], 100 nM; and SU5614 IC(50) 10 nM). FLT3-dependent activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) was also inhibited by treatment in the same concentration ranges. FLT3 inhibition by SU5416 and SU5614 resulted in reduced proliferation (IC(50), 250 nM and 100 nM, respectively) and induction of apoptosis of FLT3 ITD-positive leukemic cell lines. Treatment of these cells with an alternative growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF]) restored MAPK signaling and cellular proliferation, demonstrating specificity of the observed inhibitory effects. We conclude that SU5416 and SU5614 are potent inhibitors of FLT3. Our finding that inhibition of FLT3 induces apoptosis of leukemic cells supports the feasibility of targeting FLT3 as a novel treatment strategy for AML.

Kaposi's sarcoma-associated herpesvirus-induced upregulation of the c-kit proto-oncogene, as identified by gene expression profiling, is essential for the transformation of endothelial cells.

Kaposi's sarcoma (KS), the most frequent malignancy afflicting AIDS patients, is characterized by spindle cell formation and vascularization. Infection with KS-associated herpesvirus (KSHV) is consistently observed in all forms of KS. Spindle cell formation can be replicated in vitro by infection of dermal microvascular endothelial cells (DMVEC) with KSHV. To study the molecular mechanism of this transformation, we compared RNA expression profiles of KSHV-infected and mock-infected DMVEC. Induction of several proto-oncogenes was observed, particularly the receptor tyrosine kinase c-kit. Consistent with increased c-Kit expression, KHSV-infected DMVEC displayed enhanced proliferation in response to the c-Kit ligand, stem cell factor (SCF). Inhibition of c-Kit activity with either a pharmacological inhibitor of c-Kit (STI 571) or a dominant-negative c-Kit protein reversed SCF-dependent proliferation. Importantly, inhibition of c-Kit signal transduction reversed the KSHV-induced morphological transformation of DMVEC. Furthermore, overexpression studies showed that c-Kit was sufficient to induce spindle cell formation. Together, these data demonstrate an essential role for c-Kit in KS tumorigenesis and reveal a target for pharmacological intervention.

A functional genomics approach to Kaposi's sarcoma.

Kaposi's sarcoma (KS) is the most frequent malignancy afflicting acquired immune-deficiency syndrome (AIDS) patients. Tumor lesions are characterized by spindle cells of vascular origin and vascularization. Kaposi's sarcoma-associated herpes virus (KSHV) is consistently found in all forms of KS. Infection of dermal microvascular endothelial cells (DMVEC) with KSHV recapitulates spindle cell formation in vitro. We studied this transformation process by DNA microarray analysis comparing the RNA expression profiles of KSHV-infected and mock-infected DMVEC. Genes involved in tumorigenesis, angiogenesis, host defense, cell growth and differentiation, transcription, and metabolism were observed to change significantly upon infection with KSHV. One of the most consistently KSHV-induced genes was the receptor tyrosine kinase and proto-oncogene c-Kit. Inhibition of c-Kit activity with the pharmacological inhibitor of c-Kit signaling STI571 reversed the KSHV-induced morphological transformation of DMVEC. Moreover, overexpression studies showed that c-Kit was sufficient to induce spindle cell formation (Moses et al. J. Virol. 76(16): 8383-8399). These data demonstrate that microarrays are useful for the identification of pharmacological targets essential for KS tumorigenesis.