A Novel Screen for Compounds that Outflank BCR-ABL Drug-Resistance

描述（由申请人提供）：突变激活的ABL酪氨酸激酶是许多白血病的病因。ABL激酶抑制剂在治疗BCR-ABL1融合的慢性髓性白血病（CML）中很有用，证明了癌蛋白导向治疗的价值。然而，一些ABL活化的白血病对这些抑制剂没有反应，一些有反应的CML患者随后产生耐药性。耐药和疾病复发最常见的原因是BCR-ABL1激酶结构域突变维持了激酶活性，但阻止了抑制剂的结合。为了了解白血病发生的机制，并探索激酶抑制剂抵抗白血病的潜在治疗靶点，我们研究了ABL激酶的生理激活剂。RIN1直接结合ABL1并通过去抑制自抑制酶刺激激酶活性。在体外和体内，RIN1都能强烈刺激ABL激酶。此外，RIN1结合并增强BCR-ABL1的催化、转化和致白血病特性。这表明BCR-ABL1的酪氨酸激酶活性虽然相对于ABL1有所升高，但仍然对RIN1的刺激有反应。缺失RIN1阻断BCR-ABL1对骨髓细胞的转化。通过重新引入RIN1挽救了转化，表明这是一种细胞自主表型。白血病细胞中RIN1的沉默降低了细胞磷酸酪氨酸水平，并使细胞对ABL抑制剂伊马替尼敏感。BCR- ABL1T315I是一种在CML患者中发现的耐药突变体，也依赖于RIN1进行转化。通过RIN1激活ABL激酶提示了激酶抑制的另一种方法：破坏与正调节因子的相互作用。由于BCR-ABL1依赖于RIN1进行完全转化，这代表了一个独特的脆弱性点，可以用于治疗激酶抑制剂耐药白血病。将抑制RIN1激活BCR-ABL1的药物与标准ABL激酶抑制剂联合使用，可以提供更有效、更不易产生耐药性和疾病复发的治疗方法。我们创建了一种定量分析RIN1和ABL1协同作用的方法。该分析结合了时间分辨荧光共振能量转移（TR-FRET）的新应用，并在具有稳健特性（Z' > 0.5）的高通量格式中进行了验证。在筛选抑制RIN1和ABL1相互作用的化合物时，将包括一个异常多样化的化学文库。二级筛选将用于消除假阳性，并优先考虑可以阻断BCR-ABL1转化的抑制剂。该建议解决了信号转导中的一个基本问题（非受体酪氨酸激酶是如何调节的？），具有明确的疾病相关性（致白血病ABL激酶如何转化细胞？）和直接的临床意义（如何预防或规避对标准激酶抑制剂的耐药性？）。在此筛选中鉴定的化合物将成为了解正常和致癌ABL蛋白生物化学的宝贵工具，也可能作为新治疗方法的先导。公共卫生相关性：拟议的研究使用一种新的测定方法来鉴定在白血病发生中起直接作用的ABL酪氨酸激酶调节因子的抑制剂。该检测包括对荧光技术的创新使用，将用于独立于标准抑制剂阻断白血病相关激酶的化合物的高通量筛选。这类化合物应该是了解这种疾病的生物化学基础的宝贵工具。这些抑制剂的衍生物可能最终发展成为对抗越来越多的耐药白血病的有效疗法，并可能与标准激酶抑制剂协同作用，以减少这些和其他酪氨酸激酶依赖性癌症的耐药发生率。

DESCRIPTION (provided by applicant): Mutationally activated ABL tyrosine kinases are causative in many leukemias. ABL kinase inhibitors have been useful in treating chronic myeloid leukemia (CML) with a BCR-ABL1 fusion, demonstrating the value of oncoprotein-directed therapeutics. However, some leukemias with activated ABL do not respond to these inhibitors and some CML patients that respond, subsequently become resistant. Resistance, and disease relapse, most often results from BCR-ABL1 kinase domain mutations that maintain kinase activity but prevent inhibitor binding. To understand the mechanism of leukemogenesis, and to explore potential therapeutic targets in kinase inhibitor-resistant leukemias, we examined a physiological activator of ABL kinases. RIN1 directly binds ABL1 and stimulates kinase activity through de-repression of the autoinhibited enzyme. RIN1 strongly stimulates ABL kinases in vitro and in vivo. In addition, RIN1 binds to and enhances the catalytic, transforming and leukemogenic properties of BCR-ABL1. This demonstrates that the tyrosine kinase activity of BCR-ABL1, while elevated and constitutive relative to ABL1, is still responsive to stimulation by RIN1. Deletion of RIN1 blocks transformation of bone marrow cells by BCR-ABL1. Transformation is rescued by re-introduction of RIN1, indicating that this is a cell autonomous phenotype. Silencing of RIN1 in leukemia cells reduced cellular phospho-tyrosine levels and sensitized cells to the ABL inhibitor imatinib. BCR- ABL1T315I, a drug resistant mutant found in CML patients, was also dependent on RIN1 for transformation. The activation of ABL kinases by RIN1 suggests an alternative approach to kinase inhibition: disrupting the interaction with a positive regulator. Because BCR-ABL1 is dependent on RIN1 for full transformation, this represents a unique point of vulnerability that could be exploited to treat kinase inhibitor-resistant leukemias. Combining drugs that inhibit BCR-ABL1 activation by RIN1 with standard ABL kinase inhibitors could provide therapy that is more efficacious and less prone to the development of resistance and disease relapse. We have created an assay that quantifies the collaboration of RIN1 and ABL1. The assay incorporates a novel application of time-resolved fluorescence resonance energy transfer (TR-FRET) and was validated in a high throughput format with robust characteristics (Z' > 0.5). An exceptionally diverse chemical library will be included in a screen for compounds that inhibit the interaction of RIN1 and ABL1. Secondary screens will be used to eliminate false positives and to prioritize inhibitors that can block transformation by BCR-ABL1. This proposal addresses a fundamental question in signal transduction (how are non-receptor tyrosine kinases regulated?), with clear disease relevance (how do leukemogenic ABL kinases transform cells?) and immediate clinical implications (how can resistance to standard kinase inhibitors be prevented or circumvented?). The compounds identified in this screen will be invaluable tools for understanding the biochemistry of normal and oncogenic ABL proteins, and may also serve as leads to new therapeutics. PUBLIC HEALTH RELEVANCE: The proposed research uses a novel assay to identify inhibitors of an ABL tyrosine kinase regulator with a direct role in leukemogenesis. The assay, which includes an innovative use of fluorescence technologies, will be used in a high throughput screen for compounds that block leukemia-associated kinases independently of standard inhibitors. Compounds in this category should be invaluable tools for understanding the biochemistry underlying this disease. Derivatives of such inhibitors may eventually be developed into effective therapeutics against the increasing number of drug resistant leukemias and might synergize with standard kinase inhibitors to reduce the incidence of drug resistance in these and other tyrosine kinase-dependent cancers.