ZNF-Mediated Resistance to Imatinib Mesylate in Gastrointestinal Stromal Tumor

描述（由申请人提供）：甲磺酸伊马替尼（IM）彻底改变了胃肠道间质瘤（GIST）患者的治疗;然而，尽管观察到了初步疗效，但IM的临床耐药性已成为现实。此外，治疗开始时难治性患者的选择非常有限（约20%）。使用临床前 在一项前瞻性新辅助治疗II期试验（RTOG 0132）的治疗活检样本中，我确定了一个32基因签名，其中包括KRAB-ZNF 91亚家族成员，可以预测对IM的反应。我随后证明，这些基因中的许多不仅预测IM反应，而且介导药物的活性。为了从机制上确定这些ZNF可能如何调节对IM的应答，使用RNAi方法敲低GIST细胞中预测标签（包括10个ZNF）内的基因，并使用外显子1.0 ST阵列进行表达谱分析。这导致发现骨膜蛋白、NEDD 9和TGF 23在siRNA介导的敲低后普遍下调。这是令人感兴趣的，因为TGF 23是骨膜蛋白和NEDD 9的已知诱导剂，这两者都被报道为在各种人类癌症中频繁过表达，并且表明从这些临床研究中发现的这些ZNF可能是这些途径的“主控制器”。有趣的是，缺氧已显示上调NEDD 9和骨膜蛋白的表达（通过PI 3 K），导致肿瘤细胞的侵袭性增加和存活增强。因此，有可能骨膜蛋白和NEDD 9两者都以类似的机制由TGF 2和缺氧调节，由ZNF控制，以调节GIST细胞中的IM反应。还显示NEDD 9在IM抗性GIST细胞系中过表达并且SRC超活化，其中NEDD 9的敲低恢复IM敏感性。我推测GIST中的IM抵抗可能与缺氧相关，缺氧导致ZNF反式激活TGF 23，导致随后分别诱导骨膜蛋白和/或NEDD 9以及PI 3-K或SRC通路的激活。这些潜在的“拯救途径”的激活独立于KIT/PDGFRA信号传导发生，从而克服了IM对促存活途径的抑制活性。我的初步数据表明，这些ZNF负责调节这一途径，并且这些基因的过表达与GIST患者对IM治疗的临床耐药性有关。 为了检验我的假设，我在目的1中提出确定原发性GIST样品中TGF 23、NEDD 9和/或骨膜蛋白的表达是否与IM反应（短期和长期）和ZNF表达相关。在目标2中，我将研究这些ZNF如何影响TGF 23，NEDD 9和骨膜蛋白表达，并最终对IM的反应。目的3将确定体内靶向低氧诱导的TGF 23->骨膜蛋白-> PI-3 K/AKT和/或低氧诱导的TGF 23-> NEDD 9-> SRC拯救途径是否将消除对IM的抗性。这一建议的中心原则是，靶向药物在同时阻断多种致癌途径的组合中使用时将变得有效，从而防止绕过可以作为这些肿瘤细胞的“拯救途径”的平行途径。 这项研究将在宾夕法尼亚州费城的Fox Chase癌症中心（FCCC）完成。FCCC是由国家癌症研究所指定的综合性癌症中心，因其在医疗，放射和外科肿瘤学方面的领导地位而获得国家认可。FCCC领导层致力于为我成功完成拟议的学习提供必要的基础设施和支持。我将由Margaret von Mehren博士（教授兼主任，肉瘤项目），Andrew K. Godwin（堪萨斯大学医学院（KUMC）分子肿瘤学教授兼主任）和埃里卡Golemis（FCCC教授兼副首席科学官），他们是FCCC和KUMC备受尊敬的教员，也是转化研究和医学肿瘤学领域国际公认的专家。我将有机会在工作组的定期会议上互动和讨论我的研究，包括肉瘤计划，转化研究论坛，发展治疗学和由冯·梅伦博士和戈德温博士参加的双月实验室会议（通过视频会议）。我的主要目标是在癌症研究中获得独立的学术生涯。为了帮助实现这一目标，我组建了一个咨询委员会，由我的导师和其他FCCC教职员工（博士后培训计划主任Maureen Murphy博士和Edna Cukierman）组成，以审查我的研究进展并提供反馈。在资助的头两年，我将继续与我的导师密切合作， 实现目标1和2中提出的研究目标。在此期间，我将过渡到独立寻找终身教职的位置。在过去三年的资助中，我将继续进行目标2和3中提出的研究，同时建立我的独立研究计划。作为我未来研究目标的一部分，我不仅对新型生物制剂（如IM）的耐药机制感兴趣，而且还对建立模型和挑战现有范式以合理开发联合疗法以减轻癌症负担感兴趣。 在我短暂的职业生涯中，我已经取得了重大的科学成就，并使自己成为GIST研究界的重要成员。我写了10篇研究文章和学术评论。我获得了许多奖项和培训奖学金，并被选中在2009年的AACR和ASCO会议上口头介绍我的工作，这是本提案的基础。

DESCRIPTION (provided by applicant): Imatinib mesylate (IM) has revolutionized the treatment of patients with gastrointestinal stromal tumors (GISTs); however, clinical resistance to IM has become a reality, despite the initial efficacy observed. Furthermore, very limited options exist for patients (~20%) that are refractory at the start of treatment. Using clinical pre treatment biopsy samples from a prospective neoadjuvant phase II trial (RTOG 0132), I identified a 32-gene signature that includes KRAB-ZNF 91 subfamily members that can predict response to IM. I subsequently demonstrated that many of these genes were not only predictive of IM response but mediated the drug's activity. In order to determine mechanistically how these ZNFs might be modulating response to IM, RNAi approaches were used to knockdown genes within the predictive signature (including 10 ZNFs) in GIST cells and expression profiling was performed using exon 1.0 ST arrays. This led to the finding that periostin, NEDD9 and TGF23, are universally downregulated following siRNA-mediated knockdown. This is intriguing because TGF23 is a known inducer of periostin and NEDD9, both of which have been reported as frequently overexpressed in a variety of human cancers and indicates that these ZNFs uncovered from these clinical studies may be a "master controller" over these pathways. Interestingly, hypoxia has been shown to upregulate expression of both NEDD9 and periostin (through the PI3K) leading to increased aggressiveness and enhanced survival of tumor cells. Therefore, it is possible that both periostin and NEDD9 are regulated in a similar mechanism by TGF2 and hypoxia, controlled by ZNFs, to modulate IM response in GIST cells. NEDD9 was also shown to be overexpressed and SRC hyper-activated in an IM-resistant GIST cell line, with knockdown of NEDD9 restoring IM sensitivity. I hypothesize that IM resistance in GISTs may be associated with hypoxia leading to the transactivation of TGF23 by ZNFs causing subsequent induction of periostin and/or NEDD9 and activation of the PI3-K or SRC pathways, respectively. The activation of these potential "rescue routes" occurs independent of KIT/PDGFRA signaling, thereby overcoming inhibiting activities of IM on pro- survival pathways. My preliminary data suggest that these ZNFs are responsible for regulating this pathway and overexpression of these genes is associated with clinical resistance of GIST patients to IM-based therapy. To test my hypothesis, I propose in Aim 1 to determine if expression of TGF23, NEDD9 and/or periostin in primary GIST samples correlate to IM response (both short and long-term) and ZNF expression. In Aim 2 I will investigate how these ZNFs affect TGF23, NEDD9 and periostin expression and ultimately response to IM. Aim 3 will establish whether targeting the hypoxia-induced TGF23-> periostin-> PI-3K/AKT and/or hypoxia- induced TGF23-> NEDD9-> SRC rescue pathway in vivo will abrogate resistance to IM. The central tenet of this proposal is that targeted agents will become potent when used in combinations that simultaneously block multiple oncogenic pathways, preventing the circumvention of parallel pathways that can act as "rescue routes" for these tumor cells. The proposed research will be accomplished at Fox Chase Cancer Center (FCCC) in Philadelphia, PA. FCCC is a comprehensive cancer center as designated by the National Cancer Institute, nationally recognized for its leadership in medical, radiation and surgical oncology. FCCC leadership is committed to providing the necessary infrastructure and support for me to successfully complete my proposed studies. I will be mentored by Drs. Margaret von Mehren (Professor & Director, Sarcoma Program), Andrew K. Godwin (Professor & Director of Molecular Oncology at the University of Kansas Medical School (KUMC), and Erica Golemis (Professor & Deputy Chief Scientific Officer at FCCC), esteemed faculty members at FCCC and KUMC and internationally recognized experts in the field of translational research and medical oncology. I will have the opportunity to interact and discuss my research in regular meetings of working groups including the Sarcoma Program, Translational Research Forum, Developmental Therapeutics and bi-monthly lab meetings attended by both Drs. von Mehren and Godwin (by videoconference). My primary goal is to obtain an independent academic career in cancer research. To help accomplish this goal, I have assembled an advisory committee consisting of my mentors and other FCCC faculty members (Drs. Maureen Murphy, Director of Postdoctoral Training Program and Edna Cukierman) in order to review my research progress and provide feedback. During the first two years of funding, I will continue to work closely with my mentors to address the research goals proposed in Aims 1 and 2. During this time I will transition into independence seeking out a tenure-track faculty position. In the last three years of funding, I wil continue the studies proposed in Aims 2 and 3 while establishing my independent research program. As part of my future research objectives, I am interested not only in mechanisms of resistance to novel biologics, such as IM, but in establishing models and challenging existing paradigms to rationally develop combination therapies in order to reduce the burden of cancer. I have already made significant scientific accomplishments in my short career and have established myself to be an important member of the GIST research community. I have authored 10 research articles and scholarly reviews. I have received a number of awards and training fellowships and was selected to orally present my work, the basis of this proposal, at both the AACR and ASCO meetings in 2009.