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 BTK^WT, BTK^C481S, or BTK^C481R; second, murine models driven by MEC-1 overexpressing BTK^WT or BTK^C481S; 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 BTK^WT, BTK^C481S, or BTK^C481R. In mice, overall survival was short due to aggressive disease. Pirtobrutinib treatment for 2 weeks led to reduction of spleen and liver weight in BTK^WT and BTK^C481S 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.

Pirtobrutinib (LOXO-305) 是布鲁顿酪氨酸激酶 (BTK) 的可逆抑制剂，被设计为治疗因 C481 激酶结构域突变而发展的耐依鲁替尼疾病的替代策略。pirtobrutinib 的临床活性已在 CLL 中得到证实，但尚未研究其作用机制。^{我们在 4 个模型系统中评估了 pirtobrutinib：首先，MEC-1，一种过表达 BTK WT}、BTK ^C481S或 BTK ^C481R的 CLL 细胞系；第二，由过表达 BTK ^WT或 BTK ^{C481S的 MEC-1 驱动的小鼠模型}; 第三，原代CLL细胞的体外培养；最后是 pirtobrutinib 治疗期间的 CLL 患者（NCT03740529，ClinicalTrials.gov）。^{Pirtobrutinib 在过表达 BTK WT}、BTK ^C481S或 BTK ^C481R的 MEC-1 同基因细胞中抑制 BTK 活化以及下游信号传导。在小鼠中，由于侵袭性疾病，总生存期很短。Pirtobrutinib 治疗 2 周导致 BTK ^WT和 BTK ^{C481S的脾脏和肝脏重量减少}细胞，分别。含有野生型或突变型 BTK 的 CLL 细胞的体外培养对使用依鲁替尼或 pirtobrutinib 治疗的 BCR 途径具有抑制作用。Pirtobrutinib 治疗最初在所有病例中导致 BTK 磷酸化和下游信号传导的抑制，无论其 BTK 谱如何，但在具有其他 BCR 途径突变（如 PLCG2 或 PLEKHG5）的病例中，这些效果开始恢复。BTK 突变患者血浆中 CCL3 和 CCL4 水平略高；但是，存在双峰分布。两种趋化因子在早期时间点均减少并模拟 BCR 通路蛋白变化。集体，这些结果表明，通过 CCL3 和 CCL4 生物标志物的变化观察到，pirtobrutinib 是一种有效的 BTK 抑制剂，可用于含有野生型或突变 BTK 的 CLL，并表明 BCR 通路信号传导的改变是其临床效果的机制。需要对 BTK 看门人残基变异以及 BCR 通路中其他蛋白质的病理性激酶替代或突变进行长期评估。