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. 2024 Feb;38(2):424-429.
doi: 10.1038/s41375-024-02147-4. Epub 2024 Jan 23.

Analysis of autophagy in DLBCL reveals subtype-specific differences and the preferential targeting of ULK1 inhibition in GCB-DLBCL provides a rationale as a new therapeutic approach

Harpreet K Mandhair  1   2   3 , Ramin Radpour  4   5 , Mira Westerhuis  6 , Yara Banz  6 , Magali Humbert  6 , Miroslav Arambasic  4   5 , Jörn Dengjel  7 , Andrew Davies  8 , Mario P Tschan  9   6 , Urban Novak  10   11
Affiliations

Analysis of autophagy in DLBCL reveals subtype-specific differences and the preferential targeting of ULK1 inhibition in GCB-DLBCL provides a rationale as a new therapeutic approach

Harpreet K Mandhair et al. Leukemia. 2024 Feb.
No abstract available

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Conflict of interest statement

Andrew Davies: Celgene (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. Urban Novak: reports consulting fees and advisory board participation to/through the institution from and with Janssen-Cilag, Celgene (BMS), Takeda, AstraZeneca, Roche, Novartis, Incyte, Beigene, Kyowa Kirin, Gilead, Pierre Fabre and Miltenyi, payment of honoraria to the institution form Celgene (BMS), Novartis, Takeda, and Gilead, and meeting and/or travel support to the institution from Janssen, Roche, Gilead, and Takeda. The remaining authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1. Characterisation of autophagy dependency in primary lymphomas and its preferential targeting in GCB-DLBCL cell lines.
A Representative images of immunohistochemistry (IHC) staining of excised lymph nodes from aggressive diffuse large B-cell lymphoma (DLBCL, NOS) (top) and follicular lymphoma (FL) (bottom) (10X). Upper and lower lymphoma panel view of autophagy markers: LC3B and p62, and haematoxylin and eosin (H&E) staining. Left panel view of haematoxylin and eosin (H&E) staining, middle and right panels are stained for LC3B and p62. Intense cytoplasmic staining for LC3B and p62 (brown colouration) was defined in DLBCL, NOS compared to FL. B 318 primary lymphoma cases were grouped according to their clinical classifications being indolent (n = 149) and aggressive (n = 169) lymphomas (Supplementary Fig. 1B defines the classification of lymphoma entities). Bar graph summarising the LC3B staining positivity (%) of indolent and aggressive lymphomas. Unpaired student t test two-tailed test was used for statistical analysis. C Representative blot of two independent experiments for GCB Oci-Ly1 and SUDHL-6 cells treated with vehicle, TORIN1 (0.1 and 0.15 µM) and ibrutinib (0.5 and 1 µM) for 4 h. D Cell viability assessment of GCB Oci-Ly1 was subjected to vehicle, ibrutinib (as indicated), ascending MRT68921 (0.5, 1, 2.5, 3 and 5 µM) concentrations in combination for 24 h. P values were calculated using one-way ANOVA and Dunnett’s multiple comparisons test, and student t test between variables. E GCB Oci-Ly1 and SUDHL-6 was treated with vehicle, ibrutinib, MRT68921 and in combination for 24 h in the presence and absence of bafilomycin A1 (Baf A1, 0.2 µM) in the last 2 h. Statistical significance was determined using Mann-Whitney U test. F Representative image of Oci-Ly1 treated with vehicle, ibrutinib (1 µM), MRT68921 (2.5 µM) and combination in presence and absence of pan-caspase inhibitor Q-VD-OPh (10 and 20 µM) for 8 h (two independent experiments). Activation of caspase 8 was determined through the engagement of cleavage p43, p41 and active p18. Caspase 9 was activated through cleavage p37 and p35. In the presence of Q-VD-OPh caspase cleavages were abolished. *Non-specific protein. Representative error bars of mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 2
Fig. 2. Characterisation of ULK1 inhibition in GCB cell lines and provides a rationale for its targeting in GCB-DLBCL patients.
A Gene Ontology (GO) analysis of downregulated genes in Oci-Ly1 (MRT68921-treated with 2.5 µM vs. vehicle). B GSEA representing negative NES values of genes downregulated associated to regulation of gene expression, c-MYC transcriptional activation and c-MYC pathway. C GCB Oci-Ly1 and Oci-Ly18 cell lines treated with MRT68921 for 0.5, 1 and 2 h and phosphorylation of c-MYC Ser62 was measured. This blot represents three independent experiments. The relative protein expression was normalised to the total protein and a fold was generated. One-way ANOVA with post hoc Dunnett’s multiple comparisons test. Representative error bars of mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. D Endoplasmic reticulum stress is induced in response to autophagy inhibition. GCB Oci-Ly1 and Oci-Ly18 cells were treated with 2.5 µM MRT68921 in a time-dependent manner. Activation of the PERK pathway was confirmed by phosphorylation of eIF2a and activating transcription factor ATF-4. Immunoblotting was used for detection. E GSEA compared autophagy transcripts between gene expression profiling (GEP) of GCB (n = 240) and ABC (n = 121) patients assigned to R-CHOP treatment arm. Venn diagram illustrating the co-expression of differentially expressed genes from the ULK1 and VPS34 complexes in GCB patients. Increased gene expression of the ULK1 complex is associated with poor overall survival upon R-CHOP treatment. Transformed p values from (Supplementary Fig. 9A) were plotted, data presents mean ± SD and student t test unpaired two tailed, *P < 0.05.
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