BIM MEDIATES IMATINIB-INDUCED APOPTOSIS OF GASTROINTESTINAL STROMAL TUMORS: TRANSLATIONAL IMPLICATIONS

Gastrointestinal stromal tumors (GISTs) are oncogene-addicted cancers driven by activating mutations in the genes encoding receptor tyrosine kinases KIT and PDGFR-α. Imatinib mesylate, a specific inhibitor of KIT and PDGFR-α signaling, delays progression of GIST, but is incapable of achieving cure. Thus, most patients who initially respond to imatinib therapy eventually experience tumor progression, and have limited therapeutic options thereafter. To address imatinib-resistance and tumor progression, these studies sought to understand the molecular mechanisms that regulate apoptosis in GIST, and evaluate combination therapies that kill GISTs cells via complementary, but independent, mechanisms. BIM (Bcl-2 interacting mediator of apoptosis), a pro-apoptotic member of the Bcl-2 family, effects apoptosis in oncogene-addicted malignancies treated with targeted therapies, and was recently shown to mediate imatinib-induced apoptosis in GIST. This dissertation examined the molecular mechanism of BIM upregulation and its cytotoxic effect in GIST cells harboring clinically-representative KIT mutations. Additionally, imatinib-induced alterations in BIM and pro-survival Bcl-2 proteins were studied in specimens from patients with GIST, and correlated to apoptosis, FDG-PET response, and survival. Further, the intrinsic pathway of apoptosis was targeted therapeutically in GIST cells with the Bcl-2 inhibitor ABT-737. These studies show that BIM is upregulated in GIST cells and patient tumors after imatinib exposure, and correlates with induction of apoptosis, response by FDG-PET, and disease-free survival. These studies contribute to the mechanistic understanding of imatinib-induced apoptosis in clinically-relevant models of GIST, and may facilitate prediction of resistance and disease progression in patients. Further, combining inhibition of KIT and Bcl-2 induces apoptosis synergistically and overcomes imatinib-resistance in GIST cells. Given that imatinib-resistance and GIST progression may reflect inadequate BIM-mediated inhibition of pro-survival Bcl-2 proteins, the preclinical evidence presented here suggests that direct engagement of apoptosis may be an effective approach to enhance the cytotoxicity of imatinib and overcome resistance.

Targeting Histone deacetylases (HDAC) for the treatment of soft tissue sarcoma

Targeting Histone deacetylases (HDAC) for the treatment of genetically complex soft tissue sarcoma Histone deactylase inhibitors (HDACi) are a new class of anticancer therapeutics; however, little is known about HDACi or the individual contribution of HDAC isoform activity in soft tissue sarcoma (STS). We investigated the potential efficacy of HDACi as monotherapy and in combination with chemotherapy in a panel of genetically complex STS. We found that HDACi combined with chemotherapy significantly induced anti-STS effects in vitro and in vivo. We then focused our study of HDACi in malignant peripheral nerve sheath tumor (MPNST), a subtype of highly aggressive, therapeutically resistant, and commonly fatal malignancies that occur in patients with neurofibromatosis type-1 (NF1) or sporadically. The therapeutic efficacy of HDACi was investigated in a panel of NF1-associated and sporadic MPNST cell lines. Our results demonstrate the NF1-assocaited cohort to be highly sensitive to HDACi while sporadic cell lines exhibited resistance. HDACi-induced productive autophagy was found to be a mode of resistance and inhibiting HDACi-induced autophagy significantly induced pro-apoptotic effects of HDACi in vitro and in vivo. HDACs are not a single enzyme consisting of 11 currently known isoforms. HDACis used in these studies inhibit a variety of these isoforms, namely class I HDACs which include HDAC1, 2, 3, and 8. Recently, HDAC8-specific inhibitors (HDAC8i) have been created and tested in various cancer cell lines. Lastly, the potential therapeutic efficacy of HDAC8i was investigated in human (NF1-associated and sporadic) and NF1-associated murine-derived MPNST. HDAC8i abrogated cell growth in human and murine-derived MPNST cells. Similar to the pattern noticed with pan-HDACis NF1-associated cells, especially murine-derived, were more sensitive to HDAC8i compared to human sporadic MPNST cell lines. S-phase arrest was observed in human and murine MPNST cells, independent of p53 mutational and NF1 status. HDAC8i induced apoptosis is all cell lines tested, with a more pronounced effects in human and murine-derived NF1-associated cells. Most importantly, HDAC8i abrogated murine-derived MPNST xenograft growth in vivo. Taken together, these findings support the evaluation of pan-HDACi and isoform-specific inhibitors as a novel therapy to treat MPNST, including in combination with autophagy blocking combination regimens in particular for patients with sporadic MPNST.

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^