Pirtobrutinib (LOXO-305), a reversible inhibitor of Bruton’s tyrosine kinase (BTK), was designed as an alternative strategy to treat ibrutinib-resistant disease that develops due to C481 kinase domain mutations. The clinical activity of pirtobrutinib has been demonstrated in CLL, but the mechanism of action has not been investigated. We evaluated pirtobrutinib in 4 model systems: first, MEC-1, a CLL cell line overexpressing BTKWT, BTKC481S, or BTKC481R; second, murine models driven by MEC-1 overexpressing BTKWT or BTKC481S; third, in vitro incubations of primary CLL cells; and finally, CLL patients during pirtobrutinib therapy (NCT03740529, ClinicalTrials.gov). Pirtobrutinib inhibited BTK activation as well as downstream signaling in MEC-1 isogenic cells overexpressing BTKWT, BTKC481S, or BTKC481R. In mice, overall survival was short due to aggressive disease. Pirtobrutinib treatment for 2 weeks led to reduction of spleen and liver weight in BTKWT and BTKC481S cells, respectively. In vitro incubations of CLL cells harboring wild-type or mutant BTK had inhibition of the BCR pathway with either ibrutinib or pirtobrutinib treatment. Pirtobrutinib therapy resulted in inhibition of BTK phosphorylation and downstream signaling initially in all cases irrespective of their BTK profile, but these effects started to revert in cases with other BCR pathway mutations such as PLCG2 or PLEKHG5. Levels of CCL3 and CCL4 in plasma were marginally higher in patients with mutated BTK; however, there was a bimodal distribution. Both chemokines were decreased at early time points and mimicked BCR pathway protein changes. Collectively, these results demonstrate that pirtobrutinib is an effective BTK inhibitor for CLL harboring wild-type or mutant BTK as observed by changes in CCL3 and CCL4 biomarkers and suggest that alterations in BCR pathway signaling are the mechanism for its clinical effects. Long-term evaluation is needed for BTK gatekeeper residue variation along with pathologic kinase substitution or mutations in other proteins in the BCR pathway.
吡托布鲁替尼（LOXO-305）作为一种可逆性布鲁顿酪氨酸激酶（BTK）抑制剂，其设计旨在针对因C481激酶结构域突变而产生的伊布替尼耐药性疾病提供替代治疗策略。该药物在慢性淋巴细胞白血病（CLL）中已显示临床活性，但其作用机制尚未明确。我们通过四种模型系统评估吡托布鲁替尼：首先采用过表达BTK野生型、BTKC481S或BTKC481R的CLL细胞系MEC-1；其次建立由过表达BTK野生型或BTKC481S的MEC-1细胞驱动的鼠类模型；第三进行原代CLL细胞的体外培养实验；最后通过吡托布鲁替尼治疗的CLL患者临床试验（NCT03740529，ClinicalTrials.gov）进行验证。
在过表达BTK野生型、BTKC481S或BTKC481R的MEC-1同源细胞中，吡托布鲁替尼能有效抑制BTK活化及下游信号传导。动物实验中因疾病进展迅猛导致总体生存期较短，但两周的吡托布鲁替尼治疗分别减轻了BTK野生型和BTKC481S模型小鼠的脾脏与肝脏重量。体外培养携带野生型或突变型BTK的CLL细胞显示，伊布替尼或吡托布鲁替尼处理均能抑制BCR通路。
临床治疗中，吡托布鲁替尼在所有病例初期均能抑制BTK磷酸化及下游信号传导（与BTK状态无关），但在存在其他BCR通路突变（如PLCG2或PLEKHG5）的病例中，这些抑制效应逐渐减弱。血浆CCL3和CCL4水平在BTK突变患者中略高，但呈双峰分布。两种趋化因子在治疗早期均出现下降，其变化趋势与BCR通路蛋白改变相一致。
综合研究表明，通过CCL3和CCL4生物标志物的变化可观察到，吡托布鲁替尼对携带野生型或突变型BTK的CLL均具有抑制作用，同时提示其对BCR通路信号传导的调节是实现临床疗效的作用机制。未来需对BTK守门残基变异、病理激酶替换及BCR通路其他蛋白突变进行长期评估。
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