Background: The KIT‐inhibitor imatinib (IM) has greatly improved the palliative treatment of metastatic gastrointestinal stromal tumors (GIST). IM exhibits strong antiproliferative effects but fails to induce sufficient levels of apoptosis resulting in low pCR rates and a high rate of secondary progression. Upregulation of p53 by MDM2 inhibitors has been shown to induce apoptosis in p53 wildtyp tumors. Given the rare incidence of inactivating mutations of p53 in GIST we sought to evaluate the therapeutic potential of MDM2/p53 modulation in GIST.
Methods: MDM2‐inhibitors nutlin‐3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) were evaluated in IM‐sensitive (GIST T1 [Ex11‐mutation], GIST882 [Ex13]) and IM‐resistant (GIST430 [Ex11/13], GIST48 [Ex11/17], GIST48B [KIT‐neg]) GIST cell lines. Biological consequences of MDM2/p53 modulation alone and in combination with different KIT‐inhibitors (IM, sunitinib, nilotinib, dasatinib, sorafenib, 17‐AAG) were determined by immunoblotting for p53‐ (p53, MDM2, p21, hnRNP K) and KIT‐related signalling pathways, immunoprecipitation, cell viability assays, effects on cell cycle and apoptosis as measured by flow cytometry and luminescence‐based caspase activation assays.
Results: Nutlin‐3 (5µM) exhibited moderate antiproliferative effects in the IM‐resistant GIST cell lines (20–30% after 3 days, 30–60% after 6 days). Notably RITA (1µM) exhibited strong antiproliferative effects in two cell lines (GIST T1: IC50 200nM, GIST48B: 300nM) but showed only minor effects the other cell lines (IC50 >5µM). Inhibition of MDM2 did not affect KIT‐ or KIT‐related signalling pathways. Nutlin‐3 resulted in strong induction of MDM2 (10‐fold), p53 (3‐fold) and p21 (4–10‐fold) in the IM‐resistant cell lines. In contrast, induction of p53 by RITA was not accompanied by induction of MDM2 and p21. Cell cycle and apoptosis assays showed moderate induction of apoptosis in the IM‐resistant GIST cells by nutlin‐3. RITA strongly induced apoptosis in GIST T1 and GIST48B. In cell viability assays combinational treatments showed no antagonistic effects in any combination. Mild additive effects (5–20%) with various inhibitors were observed both with nutlin‐3 (IM‐resistant cell lines) and RITA (GIST T1, GIST48B). Pronounced additive proapoptotic effects as measured by caspase activation were seen in combination of nutlin‐3 with the HSP90 inhibitor 17‐AAG. Binding of MDM2 and hnRNP K decreased following nutlin‐3 but remained unchanged following RITA treatment.
Conclusion: Nutlin‐3 and RITA may induce p53 in IM‐resistant GIST and show differential antiproliferative and proapoptotic effects in GIST cell lines. MDM2‐inhibition may enhance the proapoptotic effect of some KIT‐inhibitors at doses that are therapeutically achievable. No antagonistic effects with any KIT‐inhibitory drug were found. Notably, GIST‐T1 and GIST48B exhibited a striking sensitivity towards RITA but not nutlin‐3. Our data provide first evidence that modulation of the MDM2/p53 pathway may be therapeutically useful in GIST.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C154.