BTK is a major kinase in BCR signaling pathway, which is highlighted by the clinical effectiveness of irreversible small-molecule BTKi, Ibrutinib, Acalabrutinib and Zanubrutinib. In this study, we made a parallel analysis of Ibrutinib, Acalabrutinib and Zanubrutinib to uncover their potential similarities and differences in anti-survival effects in MCL, since they have differential binding selectivity against their common target, BTK, but have similar biologic effects and comparable clinical responses. Our MCL-cell viability data showed that Ibrutinib demonstrated a high cytotoxic activity in JeKo-1 cells, but not in Mino cells, which was consistent with previous findings that JeKo-1was classified as an Ibrutinib-sensitive cell line, while Mino was classified as an Ibrutinib-resistant cell line [
28]. Also, the sensitivities of both JeKo-1 and Mino cells to Acalabrutinib and Zanubrutinib were low. In order to explain the mechanism underlying such effects, RNA-seq analysis followed by KEGG analysis were performed to identify the critical upregulated pro-apoptotic genes and downregulated anti-apoptotic genes controlled by each BTKi, and the data showed that Ibrutinib was more powerful to upregulate pro-apoptotic genes,
HRK,
GADD45A and
ATM in JeKo-1 cells than in Mino cells. In addition, Acalabrutinib and Zanubrutinib had low capacities to modulate the expression of such three apoptotic genes in both JeKo-1 and Mino cells. Of note, these three apoptosis-related genes identified in this study were well-known in apoptosis signaling. GADD45A is associated with DNA damage and is proapoptotic [
29], and ATM, as a tumor suppressor gene, plays a role in the initiation and/or progression of MCL [
30]. As a member of the pro-apoptotic subgroup of BCL-2 family, HRK is an essential initiators of apoptosis that can function as tumor suppressors [
31]. All of these findings could support the idea that these genes play roles in mediating MCL-cell apoptosis induced by BTK inhibitors, even though overall apoptotic effects induced by BTKi were moderate in MCL.
Both Ibrutinib and Acalabrutinib have been shown to decrease levels of CCL3 and CCL4, two critical chemokines inducing migration or homing of leukemia cells, in CLL-cell cultures and their separate clinical trials [
14‐
16,
32]. Zanubrutinib could inhibit homing of CLL cells through downregulating CXCR5, a homing receptor mediating migration or homing and BCR signaling activation [
18,
33]. However, regulatory impact of these three BTKi on chemotaxis and chemotaxis-related genes in MCL still need to be analyzed. Our study showed that conditioned medium from HS-5 human marrow stromal cells induced migration of MCL cells, which was inhibited by Ibrutinib, Acalabrutinib and Zanubrutinib without significant differences between drug treatments. Consistent with these functional data, our results indicated that Ibrutinib, Acalabrutinib and Zanubrutinib similarly reduced the expression of
CCL3,
CCL4 and
CXCR5 via RNA-seq followed by KEGG analysis, and the production of CCL3 and CCL4 was validated by ELISA quantification, which did not show significant differences between drug treatments as well. Except these known chemotaxis-related target genes of BTKi, we also found that Ibrutinib, Acalabrutinib and Zanubrutinib could similarly reduce the expression of
CCL3L1,
CCL4L2 and
CXCL16.
Since elevated LDs could enhance MCL-cell survival [
34], we detected whether these three BTKi could inhibit pro-survival LD accumulation in MCL-cell via SRS imaging analysis, a label-free live-cell imaging technique for testing intracellular components accumulation, including LDs [
35]. As expected, quantitative analysis of lipogenesis at single-cell level via SRS imaging revealed that treatment with BTKi significantly reduced the accumulation of LDs in MCL. Based on the KEGG classification of RNA-seq data, we found that BTKi treatment dramatically reduced the expression of several pivotal lipogenic genes,
DGAT2,
ENPP2,
SCD and
ACACA (
ACC1). DGAT2 catalyzes the final step in synthesis triglyceride, which is a major component of LDs [
36], and genetic deletion of DGAT2 was lethal with knockout mice presenting severe and systemic reductions in triglyceride [
37]. SCD is a principal enzyme responsible for fatty acid desaturation, which is critical for growth, survival and tumorigenesis [
25,
38]. ENPP2, also known as Autotaxin (ATX), is always overexpressed in many malignancies [
39], including MCL [
40]. Interestingly, ATX catalyzes the extracellular biosynthesis of lysophosphatidic acid (LPA), and LPA is responsible for cancer cells growth and anti-cancer therapy resistance of many cancer cells [
27]. Combined with our findings showing BTKi-associated ENPP2 downregulation, the downregulation of SCD might be caused by the reduction of LPA levels at least partially, since LPA could stimulate SCD expression and therefore accelerate the formation of lipid droplets [
41]. Besides, ACC1, also known as ACACA, controls de novo lipogenesis, whose chemical inhibition suppresses lipogenesis and induces apoptosis in cancer cells [
26]. Previous study showed that Zanubrutinib could downregulate the expression of ACACA in MCL [
19], and our study demonstrated that both Ibrutinib and Acalabrutinib downmodulated ACACA expression in MCL as well. Importantly, inhibition of fatty acid synthesis, a crucial step of LDs accumulation, triggers significant apoptosis in MCL [
17]. Accordingly, our data suggested that BTKi-induced downregulation of
DGAT2,
ENPP2,
SCD and
ACACA might result in LD accumulation inhibition, which trigger modest MCL-cell death at least partially, and such findings provide a new evidence that targeting the lipid metabolism pathway might be a strategy to treat MCL, or other B-cell malignancies, which deserves further studies.