Targeting the C481S Ibrutinib-Resistance Mutation in Bruton’s Tyrosine Kinase Using PROTAC-Mediated Degradation

Inhibition of Bruton’s
tyrosine kinase (BTK) with the irreversible
inhibitor ibrutinib has emerged as a transformative treatment option
for patients with chronic lymphocytic leukemia (CLL) and other B-cell
malignancies, yet >80% of CLL patients develop resistance due to
a
cysteine to serine mutation at the site covalently bound by ibrutinib
(C481S). Currently, an effective treatment option for C481S patients
exhibiting relapse to ibrutinib does not exist, and these patients
have poor outcomes. To address this, we have developed a PROteolysis
TArgeting Chimera (PROTAC) that induces degradation of both wild-type
and C481S mutant BTK. We selected a lead PROTAC, MT-802, from several
candidates on the basis of its potency to induce BTK knockdown. MT-802
recruits BTK to the cereblon E3 ubiquitin ligase complex to trigger
BTK ubiquitination and degradation via the proteasome. MT-802 binds
fewer off-target kinases than ibrutinib does and retains an equivalent
potency (>99% degradation at nanomolar concentrations) against
wild-type
and C481S BTK. In cells isolated from CLL patients with the C481S
mutation, MT-802 is able to reduce the pool of active, phosphorylated
BTK whereas ibrutinib cannot. Collectively, these data provide a basis
for further preclinical study of BTK PROTACs as a novel strategy for
treatment of C481S mutant CLL.

布鲁顿酪氨酸激酶（Bruton’s tyrosine kinase, BTK）的不可逆抑制剂依鲁替尼（ibrutinib），已成为慢性淋巴细胞白血病（chronic lymphocytic leukemia, CLL）及其他B细胞恶性肿瘤患者的突破性治疗选择。然而，超过80%的CLL患者会因依鲁替尼共价结合位点发生半胱氨酸向丝氨酸的C481S突变而产生耐药性。目前，针对依鲁替尼耐药的C481S突变患者尚无有效的治疗方案，此类患者预后极差。为解决这一临床未满足需求，我们开发了一款蛋白水解靶向嵌合体（PROteolysis TArgeting Chimera, PROTAC），可同时诱导野生型及C481S突变型BTK的降解。我们基于诱导BTK敲降的活性，从多个候选化合物中筛选出先导PROTAC MT-802。MT-802可将BTK招募至cereblon E3泛素连接酶复合物，通过蛋白酶体途径触发BTK的泛素化与降解。相较于依鲁替尼，MT-802的脱靶激酶结合更少，且对野生型及C481S突变型BTK均保持同等强效活性（纳摩尔浓度下可实现99%以上的降解率）。在从携带C481S突变的CLL患者体内分离的细胞中，MT-802能够降低活性磷酸化BTK的水平，而依鲁替尼则无法做到这一点。综上，本研究数据为BTK靶向PROTAC作为治疗C481S突变型CLL的新型策略开展进一步临床前研究提供了依据。