2753 Poster Board II-729 Activating mutations of the KIT class III receptor tyrosine kinases are associated with core binding factor leukemias (CBF AML), systemic mastocytosis (SM), gastrointestinal stromal tumors (GIST), melanomas, seminoma/dysgerminoma and sinonasal natural killer/T-cell non-Hodgkin lymphoma. Despite the encouraging therapeutic potential of KIT-tyrosine kinase inhibitors (TKI), resistance leading to disease progression occurs in many patients, specifically after TKI monotherapy. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways which override TKI inhibition. To explore the downstream signaling pathways of class III receptor tyrosine kinases, we performed unbiased phoshoproteomic analyses of mutant FLT3 or KIT leukemia and mastocytosis cell lines before and after TKI treatment. Tantalizingly, immunoaffinity purification of phosphopeptides followed by tandem mass spectrometry following KIT-inhibition with Imatinib at IC90 (100nM) revealed a significant upregulation of phosphorylation levels of peptides identified as members of the heat shock protein (HSP) family. Of interest, mRNA GeneChip® Array analysis of hematopoietic Ba/F3 cells transfected with either a mutant KIT isoform (D816V) or a mutant FLT3 isoform (ITD) and treated with TKI revealed significant downregulation of HSP family members in the FLT3 model – but stable mRNA levels in the KIT model. Taken together, our phosphoproteome and mRNA data suggest a protective function of HSP in mutant-KIT tumors treated with TKI. Next we studied the antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504, a 17-AGG-derivative, in mutant-KIT cell models. IPI-504 potently inhibited proliferation and induced apoptosis with an IC50 of 0.5 up to 5μM depending on the KIT isoform. Importantly, combination of IPI-504 with TKIs resulted in potentiation of the antiproliferative and proapoptotic effects achieved by either drug alone. Antitumor efficacy in combination therapy was observed even at HSP90 inhibitor concentrations that did not display antitumor activity if administered alone. In conclusion, our model suggests that inhibition of KIT affects heat shock protein activity serving to stabilize the functionality of targeted autoactivated receptor tyrosine kinases, which provides a potential mechanism for resistance to TKI therapy. Importantly, we provide a rationale to combine TKI with (low-dose) HSP-inhibitors such as IPI-504 to optimize TKI therapy. Disclosures: Normant: Infinity: Employment.
3982 Activating mutations of FLT3 and KIT class III receptor tyrosine kinases (TK) are associated with acute leukemias and systemic mastocytosis. Despite the encouraging therapeutic potential of TK inhibitors (TKI), monotherapy often is not sufficient to obtain lasting responses. Here we show that TKI therapy may lead to cellular protection mechanisms including stabilization/repair of DNA, as well as protection of the function of heat shock proteins. We further demonstrate that degradation of heat shock protein 90 (HSP90) client proteins, FLT3 and KIT, by the HSP90 inhibitor IPI-504, leads to antitumor effects in acute leukemia and mastocytosis models; including additive to synergistic antiproliferative and proapoptotic activity after TKI treatment. To explore the immediate downstream signaling effects of mutant FLT3 and KIT inhibition in acute leukemia and mastocytosis models, we performed unbiased phoshoproteomic analyses of cells before and after TKI treatment. Phosphopeptides that displayed a significant difference in phosphorylation before and after FLT3/KIT inhibition with Imatinib or Sunitinib at IC90 were immunoaffinity purified, and identified by tandem mass spectrometry. Degradation of FLT3 and KIT after HSP90 inhibition was studied by immunoblotting. Antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504 alone, or in combination with TKI treatment, were studied in different leukemia and mastocytosis cell models, and in an isogenic BaF/3 model harboring different clinically relevant KIT and FLT3 mutations. Phoshoproteomics revealed a significant upregulation in phosphorylation of DNA and protein function stabilizing proteins, including HSP members in mutant KIT and FLT3 cell lines. Both FLT3 and KIT turned out to be client proteins of HSP90, and IPI-504 was able to degrade FLT3 and KIT protein, resulting in a strong potentiation of the antiproliferative and proapoptotic effects achieved by TK inhibitors alone. Of interest, IPI-504 displayed antitumor activity even as monotherapy in some leukemia and mastocytosis cell line models. Our data suggest that TK inhibition of FLT3 or KIT causes initiation of cell protective mechanisms, including activation of HSPs, which can therapeutically be targeted by HSP90 inhibitors such as IPI-504. These results provide a rationale to combine TKI with HSP inhibitors to optimize TKI therapy in patients with hematopoietic malignancies. Disclosures: Normant: Infinity pharmaceuticals: Employment.
Introduction Activating mutations of the KIT class III receptor tyrosine kinase (TK) are associated with the pathophysiology of acute leukemia, especially core binding factor leukemia (CBFL), and systemic mastocytosis (SM). Despite considerable antiproliferative and proapoptotic activity of several KIT TK inhibitors in vitro, clinical efficacy in AML and SM is generally moderate. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways. Previously we reported that KIT TK inhibition results in significantly increased phosphorylation of heat shock protein (HSP) family members and that KIT is a client protein of phosphorylated (p)HSP90 putatively stabilizing KIT protein function in the presence of KIT TK-inhibitors (Kampa-Schittenhelm et al., ASH annual meeting 2010). This prompted us to further test the HSP inhibitor IPI-504 in KIT dependent CBFL, including the leukemic stem cell fraction. Methods Protein expression levels of (p)HSPs in leukemic blasts of high-risk and CBFL patients were studied by flow cytometry focusing on the CD34+/CD38- leukemia stem/progenitor cell fraction. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays. Results (p)HSP90/60 levels were preferentially upregulated in CBFL associating with KIT dysregulation. Consequently, HSP90 inhibition with IPI-504 potently degraded KIT expression causing a direct antiproliferative and antiapoptotic effect in CBFL in in vitro and ex vivo models. Efficacy of IPI-504 was potentiated when combined with TK inhibitors. Importantly, high expression of (p)HSP90 and HSP60 was particularly observed in the CD34+/CD38- putative leukemia stem cell fraction arguing for a function as protection mechanism to conserve (leukemic) progenitor cell function upon cell stress such as antileukemic treatment. Conclusion HSPs are upregulated in CBFL, and IPI-504 induces antiproliferative and proapoptotic effects in primary leukemia samples. Importantly, in particular the putative malignant progenitor cell pool in KIT-associated acute leukemia expresses high levels of (p)HSP90, identifying HSP90 inhibition as an attractive novel strategy to overcome the therapy-refractory behavior of malignant stem/progenitor cells. Our results provide a rationale for the evaluation of HSP90 inhibitors such as IPI-504 in CBFL. Disclosures: No relevant conflicts of interest to declare.
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