BTK是一种非受体酪氨酸激酶，主要表达在B细胞的细胞质中。在B细胞发育过程中，受抗原刺激BCR信号被活化并激活BTK在内的多种蛋白激酶，进而激活下游多种基因的转录，调节B细胞的增殖和分化等生物学过程。外界微生物来源的抗原刺激、自身抗原刺激、原癌基因突变或抑癌基因失活导致BCR信号通路异常激活，促进B细胞淋巴瘤的发生发展。靶向BTK蛋白的抑制剂用于治疗B细胞淋巴瘤具有划时代意义，然而在用药过程中出现的BTK突变（如C481S/F/Y/R和L528W）严重限制了抑制剂的长期治疗效果。前人的研究发现BTK突变通过阻碍抑制剂和激酶结构域的结合产生耐药性，但这些耐药突变是否对BTK蛋白的生化活性和功能产生影响还不清楚。

首先，我们测试了这些突变对BTK蛋白生化活性的影响。在预测结构模型中，ATP结合在BTK激酶结构域N lobe和C lobe之间的活性口袋中，C481S突变不影响ATP的结合模式，但C481F/Y/R和L528W突变与ATP之间产生了明显的空间位阻。为了测试结构模型预测结果的准确性，我们纯化出激酶结构域重组蛋白，并通过体外激酶活性实验发现C481S突变不影响BTK激酶活性，但C481F/Y/R和L528W突变明显削弱了BTK激酶活性。体外实验结果提示我们进一步探究C481S/F/Y/R和L528W突变在恶性B淋巴细胞中对BTK激酶活性的影响。我们通过全细胞酪氨酸磷酸化定量质谱发现，在弥散性大B细胞淋巴瘤（diffuse large B-cell lymphoma，DLBCL）细胞中C481S突变对BTK激酶活性没有明显影响，但C481F/Y/R和L528W突变导致BTK激酶活性丧失。

然后，我们探究了DLBCL细胞中BTK激酶活性丧失带来的功能影响。通过检测BCR活化后下游Ca²⁺信号变化，我们发现BTK激酶活性对DLBCL细胞中BCR信号通路活化不重要，且BTK激酶活性丧失也不影响BCR信号通路下游基因的转录表达。与BTK激酶活性正常的DLBCL细胞类似，BTK激酶活性丧失的DLBCL细胞对BCR信号通路依然具有依赖性。基于以上发现，我们设想通过靶向蛋白降解技术克服BTK抑制剂用药过程中产生的耐药问题。我们开发了一款新型BTK蛋白水解靶向嵌合体（proteolysis-targeting chimera）分子BGB-15741，通过招募E3泛素连接酶CRBN和靶蛋白BTK形成CRBN-BGB-15741-BTK三元复合物，催化C481S/F/Y/R和L528W蛋白发生蛋白酶体依赖性降解，从而诱导BTK激酶活性丧失的DLBCL细胞死亡。

接下来，我们通过无偏见的全基因组CRISPR-Cas9遗传筛选分析BTK激酶活性丧失的DLBCL细胞的基因依赖性变化。全基因组遗传筛选共发现12个基因的富集程度在BTK激酶活性丧失的DLBCL细胞中显著下降，蛋白功能相关性富集分析显示，TLR9、UNC93B1和CNPY3之间存在蛋白-蛋白相互作用。与BTK激酶活性正常的DLBCL细胞相比，BTK激酶活性丧失的DLBCL细胞对TLR9信号通路产生了更强的依赖性。

综上所述，临床实践中发现的BTK第481位和528位氨基酸突变可以分成两类，一类对激酶活性没有显著影响（C481S），另一类导致激酶活性丧失（C481F/Y/R和L528W）。BTK激酶活性丧失的DLBCL细胞BCR信号通路依然完整、基因表达模式没有变化且对BCR信号通路依然具有依赖性。因此，BTK激酶活性对DLBCL细胞中BCR信号通路活化并不重要，上述工作为进一步研究DLBCL细胞中BTK非催化功能奠定了基础。

BTK is a non-receptor tyrosine kinase expressed mainly in the cytoplasm of B cells. During B cell development, antigen-mediated cross-linking of BCR activates many downstream protein kinases including BTK and then induces the activation of diverse transcriptional programs to promote B cell proliferation and differentiation. Microbial antigens, self-antigens, proto-oncogene activation, or tumor suppressor gene inactivation can co-opt BCR signaling to support the growth and/or survival of malignant B cells, resulting in B-cell lymphomas. Inhibitors targeting BTK (BTKi) have revolutionized the treatment for various B-cell malignancies but are limited by acquired resistance after prolonged treatment as a result of mutations in BTK, such as BTK C481S/F/Y/R and L528W mutations. Whereas previous studies have clarified their mechanism of resistance to BTKi by hindering the binding between BTKi and the kinase domain, whether these BTK mutations affect the biochemical activity and function of BTK in malignant B cells are not well understood.

In an attempt to explore the biochemical impact of clinically observed BTK mutations, we first docked ATP into the active pocket formed by the N lobe and C lobe of BTK kinase domain. In the resulting structural model, C481S was not expected to affect the mode of ATP binding, whereas C481F/Y/R and L528W were all predicted to generate steric clashes with ATP. To verify these predictions, we purified recombinant BTK kinase domain and found that C481S did not affect BTK’s kinase activity, whereas C481F/Y/R and L528W impaired BTK’s kinase activity by an in vitro kinase assay. These results prompted us to test whether these mutations impair BTK’s kinase activity in malignant B cells. By quantitative phospho-tyrosine proteomics, we found that C481S did not affect BTK’s kinase activity, whereas C481F/Y/R and L528W impaired BTK’s kinase activity in diffuse large B-cell lymphoma (DLBCL) cells.

Next, we examined the functional impact following loss of BTK’s kinase activity in DLBCL cells. By measuring Ca²⁺ flux after BCR cross-linking, we found that BTK’s kinase activity was not required for the activation of BCR signaling. Moreover, loss of BTK’s kinase activity did not affect gene expression downstream of BCR signaling. Similar to DLBCL cells expressing kinase-active BTK, DLBCL cells harboring BTK kinase-inactivating mutations remained addicted to BCR signaling for growth and/or survival. Based on these findings, we hypothesized that targeted BTK degradation by proteolysis-targeting chimeras (PROTACs) might be an effective strategy to overcome BTKi resistance. A BTK PROTAC (BGB-15741) was developed to recruit the E3 ubiquitin ligase CRBN to degrade BTK. BGB-15741 treatment induced the degradation of kinase-inactive BTK mutants and exhibited cytotoxicity in DLBCL cells harboring kinase-inactivating BTK mutants.

Next, we explored whether BTK kinase-inactivating mutations alter DLBCL genetic dependencies. By a whole genome-wide CRISPR-Cas9 screening, we identified 12 significantly depleted genes in DLBCL cells with kinase-inactive BTK relative to DLBCL cells with wild-type BTK. Among these 12 candidates, protein functional enrichment analysis revealed that TLR9, UNC93B1, and CNPY3 formed a protein-protein interaction network. Compared to DLBCL cells expressing kinase-active BTK, DLBCL cells harboring BTK kinase-inactivating mutations were more addicted to TLR9 signaling for their growth or survival.

In general, clinically observed BTK mutations in C481 and L528 can be divided into two categories, mutations maintaining BTK kinase activity (C481S) and mutations inactivating BTK kinase activity (C481F/Y/R and L528W). DLBCL cells harboring kinase-inactive BTK exhibit intact BCR signaling, unperturbed transcription, and addiction to BCR signaling. Thus, our study demonstrates that the kinase activity of BTK is not essential for oncogenic BCR signaling and lays the foundation for uncovering BTK’s noncatalytic function in DLBCL.