Exceptional Response to BTK Inhibitors in Aggressive Lymphomas Linked to Chronic Selective Autophagy
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 
Published: December 2023

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 expression profile for a subset of the ATG genes (p≤2.7x10 -6), suggesting that this pathway may be counter-selected during MCD pathogenesis.

As autophagy promotes the lysosomal degradation of proteins, and disruption of autophagy promoted NF-κB signaling in MCD cell lines, we assessed global protein levels and localization of proteins to the My-T-BCR in ATG KO cells using mass spectrometry. Selective autophagy receptors TAX1BP1, NBR1 and p62 were significantly upregulated in ATG KOs and displayed significant enrichment of proximity to MYD88 L265Pin ATG KOs (p≤0.01), suggesting MYD88 L265P is targeted for degradation by selective autophagy. To test this, we engineered MCD cell lines with dual fluorescent autophagy reporters (GFP-RFP fusions) for TAX1BP1, NBR1, p62, or MYD88 L265P. Each reporter displayed an accumulation of GFP relative to RFP in ATG KOs or upon treatment with the autophagy blocking v-ATPase inhibitor bafilomycin.

To identify genes that modulate selective autophagy of MYD88 L265P, we performed a CRISPR screen in MCD cells stably expressing a MYD88 L265P-RFP-GFP reporter. We observed deletion of the same epistatic interactors of ATG9A in BTKi survival screens also blocked MYD88 L265P autophagic degradation. Conversely, deletion of BTK, mTORC1-related genes and IRF4 were all among the top-ranked genes that promoted MYD88 L265P autophagic degradation. We validated these findings using chemical inhibitors of each gene alone or in combination and observed synergy for the promotion of MYD88 L265P autophagic degradation.

Collectively, we identified a non-canonical form of selective autophagy that chronically degrades MYD88 L265P, is counter-selected in MCD tumors, and is promoted by BTK inhibitors. Our findings help to elucidate the exceptional benefit of BTK-targeted therapies in the MCD DLBCL subtype and offer a rationally designed combination therapy regimen to specifically degrade this mutant allele of MYD88.