ULK1 Blockade Preferentially Targets Germinal Centre B-Cell - Diffuse Large B-Cell Lymphoma Subtype By Attenuating Autophagy, c-MYC and Inflammation

Diffuse Large B-cell Lymphoma (DLBCL) is a heterogeneous lymphoid malignancy. Gene Expression profiling (GEP) has characterized two distinct subsets: Germinal Centre B-cell (GCB) and Activated B-cell (ABC) DLBCL. Oncogenic PI3K-AKT signaling has been identified in primary DLBCL subtypes[1]. PI3K-AKT is activated in response to the loss of PTEN in GCB-DLBCL. In ABC-DLBCL the activation is associated with CD79B mutations and concomitant MYD88 alterations. The Bruton Tyrosine Kinase (BTK) functions as an intermediary kinase. Ibrutinib (BTK inhibitor) has demonstrated promising capability in ABC-DLBCL unlike GCB tumors, warranting further investigations[2]. PI3K-AKT-mTOR signaling is also a known regulator of macro-autophagy (herein referred to as autophagy). ULK1 complex is critical for the initiation of autophagy. Aberrant signaling in this pathway promotes nutritional adaptation to malignant cells and contributes to drug resistance. We hypothesize autophagy facilitates cytoprotection and contributes to drug resistance including R-CHOP and Ibrutinib in GCB-DLBCL. Dual targeting of the BTK with Ibrutinib and autophagy initiating complex(es) would be a strategic method to impair these oncogenic functions.

To determine the presence of increased autophagy related genes (ATGs) and its association to R-CHOP treatment outcome, we analyzed the GEP of patients from the REMODL-B trial (R-CHOP: GCB n=240 and ABC n=121)[3]. Gene set enrichment analysis (GSEA) showed no difference between the DLBCL patients indicating mutual autophagy dependency. Genes associated to worst overall survival (OS) in autophagy initiating complex I/ ULK1 complex (ULK1, RB1CC1/FIP200 and ATG13) and complex II (AMBRA1, PIK3C3/VPS34, PIK3R4/VPS15 and USO1/p115) were identified using Kaplan-Meier curves and overlapped according to their respective complexes. Statistical analysis confirmed ULK1 complex was significantly dominate in response to R-CHOP treatment leading to poor OS (p=0.0403). These findings provide a rational to inhibit autophagy via the ULK1 complex in GCB patients. Alike, multiple GCB (n=7) and ABC (n=5) cell lines showed substantial autophagy dependency using LC3B autophagy marker expression. Ibrutinib treatment enhanced autophagic flux in GCB cell lines. Addition of ULK1 complex inhibitor (MRT68921) augmented the anti-tumor activity of Ibrutinib in HT, Oci-Ly1, Oci-Ly18 and SUDHL-6 GCB cell lines and activated caspase dependent apoptosis. Interestingly, ULK1 complex inhibitors (MRT68921/ ULK1-101) induced acute NF-ҡB activation in multiple GCB and ABC lymphoma lines irrespective of their NF-ҡB mutational status. RNA sequencing of GCB and ABC cell lines treated with MRT68921, or vehicle reported diverse ULK1 inhibitory signaling pathways. Oci-Ly1 (GCB, c-MYC amplification) treated cells significantly downregulated genes associated to PI3K-mTOR activity/ signaling, autophagy, metabolism, and IL-6. GSEA reported a substantial decrease in c-MYC transcriptional activation/ pathway and TNF signaling. Immunoblotting data confirmed ULK1 inhibition dephosphorylated c-myc(ser62) in a time dependent manner in Oci-Ly1 and Oci-Ly18. In contrast, Oci-Ly3 (ABC, c-MYC amplification) treated cells upregulated genes involved in JAK-STAT pathway, IL-6, and apoptosis. C-MYC remained unaffected.

In summary, GCB-DLBCL patients treated with R-CHOP harbor increased autophagy signatures that potentially dictate treatment outcome. Our in vitro data demonstrates ULK1 inhibition sensitizes GCB cell lines to Ibrutinib due to diverse role in regulating NF-ҡB, c-MYC and inflammation. Unlike, ABC, where ULK1 blockade promotes immunogenic-dependent cell death.

References:

1. Erdmann, T., et al., Sensitivity to PI3K and AKT inhibitors is mediated by divergent molecular mechanisms in subtypes of DLBCL. Blood, 2017. 130(3): p. 310-322.

2. Younes, A., et al., Randomized Phase III Trial of Ibrutinib and Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone in Non-Germinal Center B-Cell Diffuse Large B-Cell Lymphoma. J Clin Oncol, 2019. 37(15): p. 1285-1295.

3. Davies, A., et al., Gene-expression profiling of bortezomib added to standard chemoimmunotherapy for diffuse large B-cell lymphoma (REMoDL-B): an open-label, randomised, phase 3 trial. Lancet Oncol, 2019. 20(5): p. 649-662.

Davies:Celegne (a Bristol Myers Squibb company): Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel to scientific conferences, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research support. Travel to scientific conferences, Research Funding; Kite, a Gilead company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research support; GSK: Other: Research support; Ascerta Pharma/Astra Zeneca: Honoraria, Other: Research support; Incyte: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research support. Novak:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Ideogen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pierre Fabre: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; H & O Communication: Honoraria.