Targeting proximal bcr signaling pathway in diffuse large b-cell lymphoma

Introduction: Stimulation and activation of the B cell receptor (BCR) signaling propagates multiple proliferative and survival pathways in normal and malignant B cells. Thus, targeting oncogenic BCR pathways via pharmacological inhibitions holds promise and has fundamentally changed the treatment paradigm for B cell lymphoma patients. Despite the rapid development of BCR pathway inhibitors and success in treating patients, much work remains to be done to achieve more effective and long-lasting therapies. Methods: In an effort to identify the new molecular targets in BCR signaling, a genome wide CRISPR screening in BCR-dependent diffuse large B cell lymphoma (DLBCL) was performed. The signaling networks and interactome of the novel BCR regulator were assessed by multiplatform -omic analysis (quantitative proteomics, phospho-proteomics, ubiquitinomics, transcriptomics) and were validated by flow cytometry and biochemical methods. Furthermore, CRISPR-Cas9 drug modifier screening and high-throughput drug screening were used to identify synergistic synthetic lethality and define potential therapeutic drug combinations. Results: We found that C-terminal SRC Kinase (CSK) is an essential gene for BCR-dependent DLBCL survival. Deletion of CSK using an inducible CRISPR/Cas9 system decreased cell viability over time. Expression of CSK (WT) was able to rescue the toxic effects of CSK deletion while kinase-dead CSK (K222R) mutant isoform could not, suggesting that the survival of lymphoma cells depends on phosphorylation targets of CSK. CSK inhibition by a small molecule inhibitor (CSKi) globally changed the dynamics of ubiquitylation and phosphorylation of BCR regulators with the increased BCR signaling output in a short period of time. However, it ultimately led to the proteasomal degradation of active and lymphoma specific Src family of protein tyrosine kinases (SFKs), direct targets of CSK, and diminished proximal BCR signaling. Combinatorial deletion of these SFKs resulted in a decrease in cell proliferation and survival. Consistently, transcriptomics data revealed that CSK-inhibited cells show a reduced downstream survival gene expression when compared to untreated cells. Lastly, CSKi synergized with BCL2 family inhibitors, potentiating the toxicity of CSKi for lymphoma cell line models. Conclusion: In summary, we have uncovered that CSK functions as a positive BCR signaling regulator that sustains active SFKs in the context of oncogenic BCR signaling. CSK inhibition, by blocking proximal BCR signaling, represents a novel therapeutic strategy for aggressive lymphomas that is non-redundant with existing agents now used to treat these cancers. The research was funded by: Intramural Research Program of the National Institutes of Health, National Cancer Institute, and Center for Cancer Research Keywords: Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies, Aggressive B-cell non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.