BAK Autoactivation in Hematological Malignancies

摘要 线粒体凋亡途径在对各种细胞应激的反应中起着关键作用，包括 靶向抗癌治疗。该途径受BCL 2不同成员之间的相互作用调节。 蛋白质家族特别是，BAX和巴克通过透化细胞膜在该途径中发挥不可或缺的作用。 线粒体外膜（mitochondrial outer membrane，ORM）。虽然BAX在上皮组织中起主要作用，特别是在 出生后，巴克在正常白色血细胞、白血病细胞系和临床白血病中特别丰富 标本我们先前的研究已经证明巴克激活由两个不同的过程启动： i）响应于某些刺激（例如，短暂结合 NOXA，其响应于NEDD 8活化酶抑制剂pevonedistat而上调），和 ii）浓度依赖性巴克自激活，我们最初描述的过程。一旦激活，巴克形成 多聚体，其使细胞透化。我们最近的研究表明，这种渗透作用涉及 巴克激活时外化并与BAK相互作用的C-末端脂质结合结构域的作用 心磷脂然而，巴克可以被抗-CD 4+细胞结合和中和，从而抵消这种促凋亡作用。 淋巴造血细胞系和原发性急性髓系白血病（AML）中的凋亡BCL 2旁系同源物 标本重要的是，这些细胞对BH 3模拟物的反应，BH 3模拟物是促凋亡小分子， 选择性结合和中和BCL 2、BCL XL和/或MCL 1，反映了抗凋亡BCL 2家族中 成员对巴克具有宪法约束力。总的来说，这些观察结果导致了这样的假设， 较高的巴克水平将包含更多的组成型活化巴克，并且将对BH 3特别敏感 模拟物以及激活仅BH 3蛋白的靶向疗法。我们现在提出三个目标， 将测试这一假设，并提供更多的了解巴克在AML抗白血病治疗中的作用。 首先，我们将评估巴克在一些AML中高表达的机制，而不是其他AML， 较高巴克表达有助于巴克自激活。其次，我们将确定生物化学基础， 巴克自激活和随后的抑制抗凋亡BCL 2家族成员，因为这部分- 活化然后抑制的巴克是在暴露于BH 3模拟物时准备杀死白血病细胞的种类 以及上调BH 3-only蛋白的靶向疗法。第三，我们将评估高 巴克表达、各种抗凋亡BCL 2家族成员对巴克的抑制以及临床AML对 正在进行早期临床试验的新型含pevonedistat联合用药，从而评估 构成性巴克激活在临床环境中的潜在重要性。这些研究建立在我们最近的 在分子水平上理解巴克作用的进展，共同设计以增强电流 了解BCL 2家族生物学，同时提供新的见解，一个潜在的重要 AML敏感性的决定因素。

ABSTRACT The mitochondrial apoptotic pathway plays a critical role in the response to various cellular stresses, including targeted anticancer therapies. This pathway is regulated by interactions between various members of the BCL2 family of proteins. In particular, BAX and BAK play an indispensible role in this pathway by permeabilizing the mitochondrial outer membrane (MOM). While BAX plays a predominant role in epithelial tissues, especially in postnatal life, BAK is particularly abundant in normal white blood cells, leukemia cell lines, and clinical leukemia specimens. Our previous studies have demonstrated that BAK activation is initiated by two distinct processes: i) Transient binding of BH3-only members of the BCL2 family in response to certain stimuli (e.g., transient binding of NOXA, which is upregulated in response to the NEDD8 activating enzyme inhibitor pevonedistat), and ii) concentration-dependent BAK autoactivation, a process we initially described. Once activated, BAK forms multimers that permeabilize the MOM. Our recent studies indicate that this MOM permeabilization involves the action of a C-terminal lipid binding domain that is externalized upon BAK activation and interacts with the MOM lipid cardiolipin. Counterbalancing this pro-apoptotic effect, however, BAK can be bound and neutralized by anti- apoptotic BCL2 paralogs in lymphohematopoietic cell lines and primary acute myeloid leukemia (AML) specimens. Importantly, the response of these cells to BH3 mimetics, proapoptotic small molecules that selectively bind and neutralize BCL2, BCLXL and/or MCL1, reflects which of the anti-apoptotic BCL2 family member(s) constitutively bind BAK. Collectively, these observations lead to the hypothesis that AMLs with higher BAK levels will harbor more constitutively activated BAK and will be particularly sensitive to BH3 mimetics as well as targeted therapies that activate BH3-only proteins. We now propose three aims that will test this hypothesis and provide additional insight into the action of BAK in AML during anti-leukemic therapy. First, we will assess the mechanisms responsible for high BAK expression in some AMLs but not others because high BAK expression contributes to BAK autoactivation. Second, we will determine the biochemical basis for BAK autoactivation and subsequent restraint by anti-apoptotic BCL2 family members because this partially- activated-and-then-restrained BAK is the species poised to kill leukemia cells upon exposure to BH3 mimetics and targeted therapies that upregulate BH3-only proteins. Third, we will assess the relationship between high BAK expression, BAK restraint by various anti-apoptotic BCL2 family members, and response of clinical AML to a novel pevonedistat-containing combination undergoing early phase clinical testing, thereby assessing the potential importance of constitutive BAK activation in the clinical setting. These studies, which build on our recent advances in understanding the action of BAK at the molecular level, are collectively designed to enhance current understanding of BCL2 family biology and simultaneously provide new insight into a potentially important determinant of AML sensitivity in the clinic.