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

摘要：费城染色体阳性(Ph+)白血病是由bcr-abl1基因引起的。 融合蛋白激酶。针对bcr-abl1三磷酸腺苷位点的酪氨酸激酶抑制剂(TKI)对治疗 慢性期慢性粒细胞白血病(CP-CML)对急性期CML和Ph+的微效 急性淋巴细胞性白血病。自第一个TKI被批准以来的20年里，伊马替尼，TKI 极大地提高了慢性粒细胞白血病患者的存活率，导致慢性粒细胞白血病的预计增加 流行率从2010年的7万美国人增加到2050年的18万人。尽管取得了这些进展，但对TKI耐药的慢性粒细胞白血病仍然存在 这是一个挑战，美国每年有1000人死亡。至少50%的TKI治疗失败是通过 Bcr-abl1基因突变。对FDA批准的五种BCR-ABL1 TKIs的实验室研究已经确定了它们的 在患者中观察到针对&gt；30突变的突变特征。总体而言，这些TKI涵盖临床 Bcr-abl1单点突变的谱带。Ponatinib是临床上唯一对T315I有效的TKI 守门人变种。然而，bcr-abl1复合突变体，定义为2突变在相同的bcr-abl1 包括具有任何第二个突变的T315I的等位基因对所有批准的TKI都具有抗药性，包括波纳替尼，离开 这些患者没有进一步的治疗选择。阿西米尼是临床开发中第一个结合 BCR-ABL1肉豆蔻基部位，远离ATP部位的变构部位，以强制自身抑制的、不活跃的 构象。我们证实，阿斯米尼和波纳替尼一样，对T315I包含物无效。 突变体，但将波纳替尼(但不是尼洛替尼或达沙替尼)与阿司米尼联合使用非常有效地抑制 多种含T315I的bcr-abl1复合突变形式。这一发现为一部小说提供了基础 治疗战略，以解决完全未得到满足的医疗需求，并是这项建议的基础。在目标1中， 我们将使用计算、生物物理和结晶学的方法来破译波纳替尼是如何重新敏感的。 复合突变体bcr-abl1为阿司米尼。我们将在相关的小鼠模型和初级实验中测试该组合 白血病样本。在目标2A中，我们将开发一种临床耐药bcr-abl1化合物的治疗策略 不受波纳替尼和阿司米尼联合抑制的突变体。取而代之的是，我们将针对这些突变体 对于蛋白酶体的降解，使用阿司米尼蛋白水解靶向嵌合体(PROTAC)策略。与TKI不同， PROTAC即使在瞬间或弱结合时也是有效的。我们将检验波纳替尼诱导的假说 肉豆蔻基位置的稳定是允许随后与阿司米尼-PROTAC结合的启动事件 和复合突变体bcr-abl1的蛋白酶体降解。在目标2B中，我们将开发一种Ponatinib-PROTAC 携带肉豆蔻碱基抗性突变的复合突变体的策略。我们的工作将提供一个理论基础 帕纳替尼联合阿司米尼治疗目前无法治愈的bcr-abl1的临床评价 复合突变型白血病。复合突变也是急性髓系耐药的主要原因 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.