Abstract 3913: Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy
Authors: Choi Jaewoo , Ceribelli Michele , Phelan James D. , Häupl Björn , Huang Da Wei , Wright George W. , Hsiao Tony , Morris Vivian , Ciccarese Francesco , Wang Boya , Corcoran Sean , Scheich Sebastian , Yu Xin , Xu Weihong , Yang Yandan , Zhao Hong , Zhou Joyce , Zhang Grace , Muppidi Jagan , Inghirami Giorgio Ga , Oellerich Thomas , Thomas Craig J. , Wilson Wyndham H. , Staudt Louis M. 
Published: March 2024

Abstract
Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas. However, the mechanisms by which GCs kill malignant lymphoma cells still largely remains elusive due to the pleiotropic consequences of GC binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. This prompted us to search for critical direct targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To determine effector genes that increase glucocorticoid toxicity, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone. Screens in cell line models of Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor (BCR) signaling pathway (≥2 SD). To identify direct transcriptional GR targets and binding sites genome-wide, we performed the cleavage under targets and release using nuclease (CUT&RUN) assay and integrated this dataset with RNA-seq upon GC treatment. The pairwise comparison analysis (p&lt;0.05) for GR-bound genes across BL and DLBCL cell lines revealed a significant enrichment of negative regulators of BCR/PI3K signaling and BCR-related tumor suppressors (LAPTM5, KLHL14, ARID5B, DDIT4, DAPP1, INPP5D, and CSK). Interestingly, expression of CSK, an inhibitor of all Src-family kinases (SFKs), was repressed while that of other BCR negative regulators were directly activated by binding of GR. Combinatorial deletion of activated genes counteracted the toxicity of GC up to 83.9%. In contrast, depletion of CSK paradoxically increased the cytotoxic effects of GC in BCR-dependent aggressive lymphoma with decreased proximal BCR signaling. To gain further insights on CSK function in aggressive lymphomas, we performed phospho-proteome and ubiquitinome profiling by treating lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi). The CSKi treatment initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-κB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs. Furthermore, small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers.
Citation Format: Jaewoo Choi, Michele Ceribelli, James D. Phelan, Björn Häupl, Da Wei Huang, George W. Wright, Tony Hsiao, Vivian Morris, Francesco Ciccarese, Boya Wang, Sean Corcoran, Sebastian Scheich, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Joyce Zhou, Grace Zhang, Jagan Muppidi, Giorgio Ga Inghirami, Thomas Oellerich, Craig J. Thomas, Wyndham H. Wilson, Louis M. Staudt. Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3913.