Exceptional Response to BTK Inhibitors in Aggressive Lymphomas Linked to Chronic Selective Autophagy

• Published in: Blood 2023-11, Vol.142 (Supplement 1), p.850-850
• Main Authors: Phelan, James D, Scheich, Sebastian, Choi, Jaewoo, Wright, George, Häupl, Björn, Young, Ryan M., Rieke, Sara, Pape, Martine, Ji, Yanlong, Urlaub, Henning, Doebele, Carmen, Zindel, Alena, Wotapek, Tanja, Kasbekar, Monica, Huang, Da Wei, Coulibaly, Zana, Morris, Vivian M, Yu, Xin, Xu, Weihong, Yang, Yandan, Zhao, Hong, Shoemaker, Christopher J., Wang, Zhuo, Shaffer, Arthur, Staudt, Louis M., Oellerich, Thomas
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-190851

Diffuse large B cell lymphoma (DLBCL) is an aggressive cancer that is profoundly heterogeneous, both molecularly and phenotypically, presenting a challenge for precision medicine. Inhibitors of Bruton tyrosine kinase (BTK) block B cell receptor (BCR)-dependent NF-κB signaling and are particularly effective in DLBCL with mutations in the BCR subunit CD79B and MYD88 (MCD DLBCL). MCD tumors are enriched for a multiprotein supercomplex, termed the My-T-BCR, that is nucleated by MYD88 L265P, TLR9 and the BCR, and serves as a central hub of NF-κB signaling. The integrity of the My-T-BCR complex is rapidly compromised following BTK inhibition, but the molecular mechanisms responsible for the dissolution of this molecular machine have not been elucidated. To investigate the underlying mechanisms regulating the My-T-BCR, we used genome wide CRISPR-Cas9 screens in MCD DLBCL cell line models treated with the BTK inhibitors (BTKi) and compared the results to screens with inhibitors of SYK, IKK, mTOR, and BRD4. We identified several drug resistance genes encoding known negative regulators of BCR, NF-κB, and PI3 kinase signaling that were recurrently inactivated by mutation and/or deletion in DLBCL biopsies. Unexpectedly, we identified multiple autophagy-related genes involved autophagosome formation ( ATG9A, ATG101, ATG13, RB1CC1 and ATG14) and autophagosome membrane expansion ( ATG2A, WIPI2, WDR45) that strongly counteracted the toxicity of BTKi (≥3 SD). Notably, these autophagy genes did not promote resistance to the mTOR inhibitors, which induces classical autophagy. To gain further insight, we generated BTKi-resistant cell lines deficient in ATG9A or ATG101 (ATG KO) and performed genome-wide CRISPR screens and RNA-seq with or without BTKi. In doing so, we observed 11 ATG genes that displayed epistatic interactions, no longer conferring BTKi resistance in ATG KO MCD cells (p≤0.05). We also observed the buffering of many NF-κB negative regulators (p≤0.001) and the increased sensitivity to loss of NF-κB positive regulators (p≤0.05). Furthermore, gene expression studies showed a marked decrease of BCR, MYD88 and NF-κB signatures in control BTKi-treated cells, whereas ATG KOs demonstrated a rescue of these same gene signatures and displayed higher levels of nuclear NF-κB localization upon BTKi treatment. MCD patients display the highest levels of NF-κB gene expression compared to other genetic subtypes. Interestingly, MCD patients also displayed the lowest gene expressi