Engineering tyrosine kinase-activated caspases for selective cancer cell killing

描述（由申请人提供）：大多数化疗药物的疗效依赖于有效诱导凋亡细胞死亡。对于表达活化酪氨酸激酶的白血病（例如，表达Bcr-Abl的慢性髓系白血病、表达Tel-PDGFR2的慢性髓系白血病和表达活化FLT3的急性髓系白血病），由于活化酪氨酸激酶是凋亡细胞死亡的有效抑制剂，传统化疗的效用受到限制。此外，目前的靶向激酶抑制剂受到获得性耐药性问题的困扰，即使有效，它们也不能完全消除白血病细胞库，因此患者必须无限期地接受治疗。这将是非常有利的治疗方法，可以选择性地和强制诱导白血病细胞表达活化酪氨酸激酶死亡，而保留正常细胞。许多细胞信号通路影响细胞的凋亡决定。然而，在各种不同的情况下，细胞死亡程序的执行是由半胱氨酸蛋白酶家族（称为半胱天冬酶）执行的。尽管caspase（如caspase 8）可以通过细胞外“死亡配体”（如Fas和TNF）的受体结合以相当直接的方式被激活，但caspase在响应损伤剂（辐射，化疗）时通常通过细胞的线粒体进行激活。凋亡刺激诱导呼吸链酶细胞色素c从线粒体膜间隙转运到细胞质，其机制尚不完全清楚。细胞色素c与细胞质蛋白Apaf-1结合，形成一种称为凋亡小体的结构，在这种结构中，caspase 9被募集到Apaf1/细胞色素c中并被其激活。在先前的报道中，Bcr-Abl已被证明可以抑制线粒体细胞色素c的释放，这一功能使细胞对化疗诱导的细胞死亡具有显著的抵抗力。我们最近的研究表明，Tel-PDGFR2和FLT3/D835Y具有类似的耐药性。此外，即使存在胞浆细胞色素c，这些活化的酪氨酸激酶也能阻止凋亡细胞的活化。我们假设需要新的策略来诱导白血病细胞的选择性死亡，因为这些活化的酪氨酸激酶的抗凋亡活性可能会限制大多数化疗药物的效用。为此，我们制定了一种创新的策略，通过一种独特的方法在表达活化酪氨酸激酶的细胞中促进选择性半胱天冬酶的激活。

DESCRIPTION (provided by applicant): The efficacy of most chemotherapeutic agents relies upon the efficient induction of apoptotic cell death. In the case of leukemias expressing activated tyrosine kinases (e.g., chronic myelogenous leukemias expressing Bcr-Abl, chronic myelomoncytic leukemias expressing Tel-PDGFR2, and acute myeloid leukemias expressing activated FLT3), the utility of conventional chemotherapeutics is limited by the fact that activated tyrosine kinases are potent inhibitors of apoptotic cell death. In addition, current targeted kinase inhibitors are plagued by problems of acquired resistance and, even when efficacious, they don't entirely eliminate the reservoir of leukemic cells and thus patients must remain on therapy indefinitely. It would be highly advantageous to develop treatments that could selectively and forcibly induce the death of leukemic cells expressing activated tyrosine kinases, while sparing normal cells. Many cellular signaling pathways impinge upon the cellular decision to die by apoptosis. However, under a variety of different circumstances, execution of the cell death program is carried out by a family of cysteine proteases known as caspases. Although caspases (such as caspase 8) can be activated in a fairly direct manner by receptor binding of extracellular "death ligands" such as Fas and TNF, caspase activation in response to damaging agents (radiation, chemotherapeutics) typically proceeds through the cell's mitochondria. By a mechanism that is still not entirely clear, apoptotic stimuli induce transit of the respiratory chain enzyme cytochrome c from the intermembrane space of the mitochondria to the cytoplasm. Engagement of a cytosolic protein, Apaf-1, by cytochrome c then nucleates the formation of a structure known as the apoptosome, in which caspase 9 is recruited to and activated by Apaf1/cytochrome c. In previous reports, Bcr-Abl has been shown to inhibit mitochondrial cytochrome c release, a function that renders cells remarkably resistant to chemotherapy-induced cell death. We have recently shown that Tel-PDGFR2 and FLT3/D835Y are similarly resistant. Moreover, even in the presence of cytosolic cytochrome c, these activated tyrosine kinases prevent activation of the apoptosome. We hypothesize that novel strategies are required to induce selective death of leukemic cells as the anti-apoptotic activity of these activated tyrosine kinases are likely to limit the utility of most chemotherapeutic agents. Towards this end, we have formulated an innovative strategy to promote selective caspase activation by a unique means in cells expressing activated tyrosine kinases. PUBLIC HEALTH RELEVANCE: We propose to engineer and test novel activators of cell death-promoting enzymes (caspases) that are activated by oncogenic tyrosine kinases with the goal of eliminating leukemic cells.