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性细胞死亡。在表达活化酪氨酸激酶的白血病（例如，表达Bcr-Abl的慢性骨髓性白血病、表达Tel-PDGFR 2的慢性骨髓单核细胞白血病和表达活化的FLT 3的急性骨髓性白血病），常规化疗剂的效用受到活化的酪氨酸激酶是凋亡性细胞死亡的有效抑制剂这一事实的限制。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的储存库，因此患者必须无限期地接受治疗。 这将是非常有利的开发治疗，可以选择性地和强制性地诱导表达活化酪氨酸激酶的白血病细胞的死亡，同时保留正常细胞。许多细胞信号传导途径影响细胞通过凋亡而死亡的决定。然而，在各种不同的情况下，细胞死亡程序的执行是由称为半胱氨酸蛋白酶的半胱氨酸蛋白酶家族进行的。虽然半胱天冬酶（例如半胱天冬酶8）可以通过细胞外“死亡配体”（例如Fas和TNF）的受体结合以相当直接的方式被激活，但是响应于损伤剂（辐射、化疗剂）的半胱天冬酶激活通常通过细胞的线粒体进行。通过一种尚不完全清楚的机制，凋亡刺激诱导呼吸链酶细胞色素c从线粒体的膜间隙转运到细胞质。细胞溶质蛋白Apaf-1与细胞色素c的结合使一种被称为线粒体的结构的形成成核，其中半胱天冬酶9被Apaf 1/细胞色素c募集并激活。在以前的报道中，Bcr-Abl已被证明可以抑制线粒体细胞色素c的释放，这是一种使细胞对化疗诱导的细胞死亡具有显著抗性的功能。我们最近发现Tel-PDGFR 2和FLT 3/D835 Y具有相似的耐药性。此外，即使在胞质细胞色素c的存在下，这些活化的酪氨酸激酶也阻止了线粒体的活化。我们假设，需要新的策略来诱导白血病细胞的选择性死亡，因为这些激活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，我们制定了一个创新的策略，以促进选择性caspase激活的细胞表达激活酪氨酸激酶的独特手段。 公共卫生相关性：我们建议设计和测试新的细胞死亡促进酶（半胱天冬酶）的激活剂，这些酶被致癌酪氨酸激酶激活，目的是消除白血病细胞。

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.