Combined inhibition of BTK and PI3K significantly increases DEGs and modulates immune-related biological processes in canine B-cell lymphoma in vitro model

Introduction: Canine B-cell lymphoma represents a unique and unpar-
alleled in vivo model for human diffuse large B-cell lymphoma (DLBCL).
The combined inhibition of Bruton’s tyrosine kinase (BTK) and the phos-
phoinositide 3-kinase (PI3K) has already shown promising antiprolifer-
ative effects in the canine DLBCL cell line, CLBL-1. Herein we analyzed
changes in differentially expressed genes (DEGs), biological process, and
molecular function of CLBL-1 driven by the combination of Ibrutinib
(BTK inhibitor) and AS605240 (PI3K inhibitor).
Methods: CLBL-1 was exposed to mono- or combined 1 μM Ibrutinib and
5 μM AS605240 for 24, 48, and 72h. In the control groups, the DMSO-con-
taining medium was added in the same volume as for the drug-treated
cells. RNA was isolated and prepared for whole transcriptome sequenc-
ing. Differential expression analysis was used for detecting and quantify-
ing changes in gene expression levels between different treatment groups.
Functional enrichment analysis (FEA) of DEGs was performed with the
DAVID Functional Annotation Tool.
Results: Compared to the controls and independently of the exposition
time, the combination of Ibrutinib and AS605240 resulted in a larger
number of DEGs (888 to 984) than the two inhibitors alone (6 to 161 for
AS605240 and 76 to 295 for Ibrutinib). Differences in the incubation time
had relatively minor effects on gene expression. FEA of DEGs revealed that
biological processes such as lymphocyte activation, cell activation, leuko-
cyte activation, immune response, positive regulation of immune system
process, lymphocyte aggregation, T cell activation, T cell aggregation were
only modulated by the combined inhibitors, not by the single inhibitor.
Conclusion: The combination of Ibrutinib and AS605240 significantly
increased the DEGs in CLBL-1, compared with these two inhibitors alone.
These significantly DEGs only led by the combination interfered with a
series of biological processes, affecting the activation and aggregation of
lymphocytes.