BAK Autoactivation in Hematological Malignancies

摘要 线粒体的凋亡通路在对各种细胞应激的反应中起着关键作用，包括 有针对性的抗癌治疗。该途径由BCL2的不同成员之间的相互作用调节 蛋白质家族。特别是，BAX和BAK在这一途径中发挥着不可或缺的作用，它们通过通透性 线粒体外膜(MOM)。而Bax在上皮组织中起主导作用，特别是在 出生后，BAK在正常白细胞、白血病细胞系和临床白血病中尤其丰富 标本。我们之前的研究已经证明，BAK的激活由两个不同的过程启动： I)BCL2家族中仅含BH3的成员对某些刺激的瞬时结合(例如，瞬时结合 NOXA，它是对NEDD8激活酶抑制剂pevoneDistat的反应而上调的)，以及 Ii)依赖浓度的BAK自激活，这是我们最初描述的一个过程。一旦激活，BAK形式 能渗透母亲体内的多聚体。我们最近的研究表明，这种母体通透性涉及 在BAK激活时外化的C端脂结合域的作用并与MOM相互作用 脂类心磷脂。然而，BAK可以被抗凋亡蛋白结合并中和，以抵消这种促凋亡作用 淋巴造血细胞系和原发急性髓系白血病(AML)中的bcl2类似凋亡基因 标本。重要的是，这些细胞对BH3模拟物的反应，促凋亡小分子 选择性地结合和中和BCL2、BCLXL和/或MCL1，反映抗凋亡的BCL2家族中的哪一个 成员(S)对北卡罗来纳州具有宪法约束力。总的来说，这些观察结果导致了这样的假设，即AML与 较高的BAK水平将包含更多结构性激活的BAK，并将对BH3特别敏感 模拟以及激活BH3-Only蛋白质的靶向治疗。我们现在提出三个目标， 将检验这一假说，并为BAK在抗白血病治疗期间在AML中的作用提供更多的见解。 首先，我们将评估在一些AML中高表达BAK的机制，而不是其他AML，因为 BAK的高表达有助于BAK的自我激活。第二，我们将确定生物化学基础 抗凋亡的BCL2家族成员对BAK的自激活和随后的抑制，因为这部分- 先激活后抑制的BAK是一种在暴露于BH3模拟物后准备杀死白血病细胞的物种 以及上调仅BH3蛋白的靶向治疗。第三，我们将评估高与高的关系 BAK的表达、不同抗凋亡BCL2家族成员对BAK的抑制以及临床AML对 一种新的含有PEVONEDART的组合物正在进行早期临床测试，从而评估 结构性BAK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基础上 在分子水平上理解BAK作用的进展，共同旨在增强电流 对BCL2家族生物学的理解，同时为潜在的重要 急性髓系白血病临床敏感性的决定因素。

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.