Strategies to target BCR-ABL1 compound mutants in CML and Ph+ ALL

翻译后摘要：费城染色体阳性（Ph+）白血病是由BCR-ABL 1，一个组成性活性 融合激酶靶向BCR-ABL 1 ATP位点的酪氨酸激酶抑制剂（TKI）可有效治疗 慢性期慢性粒细胞白血病（CP-CML），但对治疗急变期CML和Ph+有效性极低 急性淋巴细胞白血病自第一个TKI获批以来的20年中，伊马替尼，TKI 大大提高了CP-CML患者的生存率，导致CML的预计增加 患病率从2010年的7万人上升到2050年的18万人。尽管取得了这一进展，但TKI耐药CML仍然存在 这是一个挑战，在美国每年有超过1,000例死亡，至少50%的TKI治疗失败是由于 BCR-ABL 1突变。对FDA批准的5种BCR-ABL 1 TKI的实验室研究已确定其 针对患者中观察到的>30个突变的突变谱。总体而言，这些TKI涵盖了临床 BCR-ABL 1单点突变体谱。Ponatinib是唯一对T315 I具有临床有效性的TKI 看门人突变体然而，BCR-ABL 1复合突变体，定义为在相同的BCR-ABL 1基因中的突变位点2， 等位基因，包括具有任何第二突变的T315 I，对所有获批的TKI（包括泊那替尼）具有耐药性， 这些患者没有进一步的治疗选择。Asciminib是临床开发中的第一种抑制剂， BCR-ABL 1肉豆蔻酰位点，一个远离ATP位点的变构位点， 构象我们确定，与泊那替尼一样，阿昔替尼对含T315 I的化合物无效 突变体，但联合泊那替尼（但不是尼洛替尼或达沙替尼）与阿昔替尼在抑制 BCR-ABL 1的许多含T315 I的化合物突变形式。这一发现为一部小说提供了基础。 治疗策略，以解决一个完全未满足的医疗需求，是这个建议的基础。在目标1中， 我们将使用计算，生物物理和晶体学方法来破译泊那替尼如何重新增敏 复合突变体BCR-ABL 1与阿昔替尼。我们将在相关小鼠模型和原发性小鼠模型中测试该组合。 白血病样本在目标2A中，我们将开发临床耐药BCR-ABL 1化合物的治疗策略 不被泊那替尼与阿昔替尼的组合抑制的突变体。相反，我们会把目标对准这些变种人 使用asciminib蛋白水解靶向嵌合体（PROTAC）策略进行蛋白酶体降解。与TKI不同， PROTAC即使在瞬时或弱结合时也是有效的。我们将检验泊那替尼诱导的 肉豆蔻酰位点的稳定化是允许随后的阿昔替尼-PROTAC结合的起始事件 和复合突变体BCR-ABL 1的蛋白酶体降解。在目标2B中，我们将开发ponatinib-PROTAC 携带肉豆蔻酰位点抗性突变复合突变体的策略。我们的工作将提供一个理论基础 用于临床评价泊那替尼联合阿昔替尼治疗目前无法治疗的BCR-ABL 1 复合突变型白血病复合突变也是急性髓系白血病耐药的主要原因。 白血病、黑色素瘤和肺癌，我们的研究将为治疗这些恶性肿瘤提供蓝图。

Abstract: Philadelphia chromosome-positive (Ph+) leukemia is caused by BCR-ABL1, a constitutively active fusion kinase. Tyrosine kinase inhibitors (TKIs) targeting the ATP site of BCR-ABL1 are effective in treating chronic-phase chronic myeloid leukemia (CP-CML) yet minimally effective at treating blast-phase CML and Ph+ acute lymphoblastic leukemia. In the 20 years since the approval of the first TKI in all of medicine, imatinib, TKIs have dramatically improved survival of patients with CP-CML, resulting in a projected increase of CML prevalence from 70,000 Americans in 2010 to 180,000 in 2050. Despite this progress, TKI-resistant CML remains a challenge, with >1,000 deaths annually in the U.S. At least 50% of TKI treatment failure arises through mutations in BCR-ABL1. Laboratory studies on the five FDA-approved BCR-ABL1 TKIs have established their mutational profiles against the >30 mutations observed in patients. In aggregate, these TKIs cover the clinical spectrum of BCR-ABL1 single point mutants. Ponatinib is the only TKI that is clinically effective against the T315I gatekeeper mutant. However, BCR-ABL1 compound mutants, defined as 2 mutations in the same BCR-ABL1 allele, that include T315I with any second mutation are resistant to all approved TKIs, including ponatinib, leaving these patients with no further treatment options. Asciminib is the first inhibitor in clinical development that binds the BCR-ABL1 myristoyl site, an allosteric site distant from the ATP site, to enforce an autoinhibited, inactive conformation. We established that asciminib, like ponatinib, is not effective against T315I-inclusive compound mutants, yet combining ponatinib (but not nilotinib or dasatinib) with asciminib is extremely effective at inhibiting many T315I-inclusive compound mutant forms of BCR-ABL1. This discovery provides the basis for a novel therapeutic strategy to address an entirely unmet medical need and is the foundation of this proposal. In Aim 1, we will use computational, biophysical and crystallographic methods to decipher how ponatinib re-sensitizes compound mutant BCR-ABL1 to asciminib. We will test the combination in relevant mouse models and in primary leukemia samples. In Aim 2A, we will develop a therapeutic strategy for clinically resistant BCR-ABL1 compound mutants that are not inhibited by the combination of ponatinib with asciminib. Instead, we will target these mutants for proteasomal degradation using an asciminib proteolysis targeting chimera (PROTAC) strategy. Unlike TKIs, PROTACs are effective even upon transient or weak binding. We will test the hypothesis that ponatinib-induced stabilization of the myristoyl site is the initiating event that allows subsequent binding of an asciminib-PROTAC and proteasomal degradation of compound mutant BCR-ABL1. In Aim 2B, we will develop a ponatinib-PROTAC strategy for compound mutants carrying a myristoyl site resistance mutation. Our work will provide a rationale for clinical evaluation of ponatinib combined with asciminib as a therapy for currently untreatable BCR-ABL1 compound mutant leukemia. Compound mutations are also a major cause of resistance in acute myeloid leukemia, melanoma, and lung cancer, and our study will provide a blueprint for treating these malignancies.