Therapeutic resistance in leukemic cells: targeting BCL-2 family and autophagy

 描述（由申请方提供）：慢性淋巴细胞白血病（CLL）在临床上具有异质性，反映了多个途径的功能改变，包括凋亡和B细胞信号传导。处于后期开发阶段的新型靶向药物包括PI 3 K和促凋亡B细胞白血病/淋巴瘤2（BCL-2）同源性3（BH 3）模拟物。我们长期以来一直有兴趣了解对BH 3模拟物和活性剂（如氟达拉滨）的反应和抗性的基本机制。我们已经确定了几个具有生物学和临床意义的决定因素，在细胞凋亡反应和这些治疗的阻力，BCL-2家族蛋白作为细胞存活的关键调节因子。我们将解决两个关键的治疗耐药机制：受损的细胞凋亡和自噬，抗凋亡BCL-2家族是至关重要的过程中，支持我们发表的和初步的数据。我们已经开发了一种基于BCL-2家族基因表达的凋亡指数，该指数定义了CLL对ABT-737的敏感性，并产生和表征了ABT- 737和ABT-199耐药细胞系。耐药细胞中MCL-1表达的增加对于防止细胞死亡至关重要，过量的MCL-1与BH 3-only蛋白、BIM或关键的自噬调节因子Beclin 1和Beclin 2相关，Beclin 1和Beclin 2也含有BH 3结构域。ABT-199耐药细胞还产生了高BCL-xL表达，其通过miR 377表观遗传学调节。miR 377和BCL-xL表达均预测CLL的临床结果，因此可以提供新的干预靶点以及治疗生物标志物。耐药细胞依赖于Beclin 2而不是Beclin 1和AMPK的高基础自噬，并且令人惊讶的是，也具有高mTORC 1活性。我们的中心假设是，对CLL治疗的反应性和抗性由调节细胞凋亡和自噬的BCL-2家族蛋白决定。我们将用ABT-199临床治疗后的白血病细胞系和原代CLL患者细胞进行实验，在悬浮液中或基质细胞上离体培养，以模拟它们与体内微环境的相互作用。我们的具体目标是：1）确定BCL-xL和MCL-1在对BH 3模拟物的抗性和致敏中的作用，2）将自噬定义为抗性机制，以及3）通过整合从细胞系获得的基因表达数据与来自临床或离体治疗的患者的基因表达数据，为优化BCL-2靶向剂的使用奠定基础。PI在BCL-2家族生物学、细胞凋亡和自噬方面的专业知识增强了我们成功的可能性。史密斯，B。Hill（Co-I）在慢性淋巴细胞白血病和淋巴系统恶性肿瘤中的应用; Hsi（Co-I）和A. Ting（顾问）在甲基化和下一代测序方面。根据不同的细胞敏感性，了解关键的BCL-2家族蛋白质的关联将允许开发有效靶向的分子和药理学方法。通过表征淋巴恶性肿瘤中的凋亡和自噬途径，我们寻求最终使用它们来预测反应，从而个性化靶向治疗药物的选择。

 DESCRIPTION (provided by applicant): Chronic lymphocytic leukemia (CLL) is heterogeneous clinically, reflecting functional alterations in multiple pathways, including apoptotic and B cell signaling. Novel targeted agents in late-stage development include PI3K and pro-apoptotic B cell leukemia/lymphoma 2 (BCL-2) homology 3 (BH3) mimetics. We have a long- standing interest in understanding the fundamental mechanisms for the response and resistance to BH3 mimetics and active agents, such as fludarabine. We have identified several determinants with biologic and clinical significance in the apoptotic response and resistance to these therapeutics, with the BCL-2 family proteins emerging as key regulators of cellular survival. We will address two critical therapy resistance mechanisms: impaired apoptosis and autophagy, processes in which the anti-apoptotic BCL-2 family is crucial, as supported by our published and preliminary data. We have developed an apoptotic index based on BCL-2 family gene expression that defines sensitivity to ABT-737 in CLL and have generated and characterized ABT- 737 and ABT-199-resistant cell lines. Increased MCL-1 expression in resistant cells was critical to preventing cell death, with the excess MCL-1 being associated with the BH3-only protein, BIM, or key autophagy regulators Beclin 1 and Beclin 2, which also contain a BH3 domain. ABT-199-resistant cells also developed high BCL-xL expression that is regulated epigenetically through miR377. Both miR377 and BCL-xL expression predicted clinical outcome in CLL and thus can provide new targets for intervention as well as biomarkers for therapy. Therapy-resistant cells were addicted to high basal autophagy that depended on Beclin 2, instead of Beclin 1, and AMPK, and surprisingly, also had high mTORC1 activity. Our central hypothesis is that responsiveness and resistance to CLL therapeutics are governed by the BCL-2 family proteins that regulate apoptosis and autophagy. We will perform experiments with leukemia cell lines and primary CLL patient cells after clinical therapy with ABT-199, cultured ex vivo in suspension or on stromal cells to mimic their interaction with their in vivo microenvironment. Our specific aims are: 1) To define the role of BCL-xL and MCL-1 in resistance to and sensitization to BH3 mimetics, 2) To define autophagy as a mechanism of resistance, and 3) To lay the foundations for optimizing the use of BCL-2 targeting agents by integrating gene expression data obtained from cell lines with that from patients treated in the clinic or ex vivo. Our likelihood of success is enhanced by the expertise of the PI in the BCL-2 family biology, apoptosis, and autophagy, M. Smith, B. Hill (Co-I) in CLL and lymphoid malignancies, E. Hsi (Co-I) in pathology, and A. Ting (Consultant) in methylation and next-generation sequencing. Understanding the critical BCL-2 family protein associations according to differing cell sensitivities will allow development of molecular and pharmacologic approaches for their effective targeting. By characterizing apoptotic and autophagy pathways in lymphoid malignancies, we seek to ultimately use them to predict response and thus personalize the selection of targeted therapeutic agents.