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3265 The Deubiquitinase CYLD Acts As an Oncogene in a Cellular Model of Chronic Lymphocytic Leukemia

Program: Oral and Poster Abstracts
Session: 641. Chronic Lymphocytic Leukemias: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, Translational Research, CLL, bioinformatics, Diseases, immune mechanism, Lymphoid Malignancies, metabolism, Biological Processes, Technology and Procedures, gene editing, Study Population, Human, omics technologies
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Marina Gerousi1*, Georgios Gavriilidis1*, Sofoklis Keisaris1*, Artemis Kourouni1*, Aspasia Orfanou1*, Anastasia Iatrou1*, Athanasios Pseftogkas2,3*, George Mosialos4*, Eleni Theodosiou1*, Anastasia Chatzidimitriou1*, Fotis Psomopoulos1*, Paolo Ghia, MD, PhD2,3, Kostas Stamatopoulos1 and Konstantinos Xanthopoulos1,5*

1Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thessaloniki, Greece
2Vita-Salute San Raffaele University, Milan, Italy
3Division of Experimental Oncology, B cell neoplasia, IRCCS Ospedale San Raffaele, Milan, Italy
4School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece
5School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece

The cylindromatosis protein (CYLD) is a functional deubiquitinase that regulates critical signaling pathways, e.g. NF-κB and Wnt, thus modulating several cellular functions. CYLD acts as a tumor suppressor gene in solid tumors, and is also involved in the pathogenesis of hematological malignancies, including B cell lymphomas, in as yet unclear ways. In chronic lymphocytic leukemia (CLL), preliminary evidence suggests that reduced expression of CYLD correlates with a worse clinical prognosis, which appears to be in line with its postulated role as a tumor suppressor. Here we sought to gain insight into the function of CYLD in CLL through genetic engineering, molecular characterization and bioenergetic profiling. To this end, we used CRISPR/Cas9 technology and a CYLD-targeting or an unrelated gRNA to generate stable CYLD-knockout (CYLDko) and control (CYLDwt) MEC1 cells. Phenotypic characterization of CYLDko versus CYLDwt MEC1 cells by flow cytometry showed (i) significantly reduced viability, assessed by Annexin V (fold difference, FD=1.2, p<0.05); (ii) lower cell proliferation rate, assessed by Ki67 expression (FD=1.4, p<0.05); (iii) increased apoptosis, determined by measuring active caspase 3 expression levels (FD=6.1, p<0.01); and, (iv) reduced expression of CD86 (FD=4.3, p<0.001) and CD40 (FD=4.1, p=0.05). Western blotting analysis of CYLDko versus CYLDwt MEC1 cells revealed down-regulation of the NF-κB pathway evidenced by diminished expression of ΙΚΚβ (FD=2.4, p<0.01), phospho-ΙκΒα (FD=3.4, p<0.01) and phospho-p105 (FD=2.5, p=0.2); and, the Wnt pathway evidenced by reduced β-catenin levels (FD=2, p<0.05). Transcriptome profiling by RNA-seq gave concordant results, in documenting increased apoptosis and decreased NF-κΒ signaling in CYLDko versus CYLDwt MEC1 cells. The former also showed downregulation of calcium, BcR and PI3K/AKT/mTOR signaling pathways and, in contrast, upregulation of the glutathione and KEAP1-NFE2L2 pathways, which contribute to antioxidant defense and nutrient metabolism, as well as, regulation of redox balance and cellular metabolism, respectively. To evaluate the impact of CYLD deletion on CLL bioenergetics, we assessed ATP production rate, as a marker of active cellular metabolism, using the Seahorse XF Real-Time ATP Rate assay. CYLDko MEC1 cells exhibited impaired ATP production reflected in decreased OCR (oxygen consumption rate) and ECAR (extracellular acidification rate), that are proportional to OXPHOS (oxidative phosphorylation) and glycolysis, respectively. Regarding the metabolic phenotype, CYLDwt MEC1 cells displayed a shift towards mitochondrial OXPHOS, whereas, in CYLDko MEC1 cells ATP production was mostly based on glycolysis. High-performance liquid chromatography was performed using culture supernatants obtained at sequential time points to quantify glucose uptake and lactate secretion rates. The analysis revealed that CYLDwt MEC1 cells primarily directed glucose carbon toward biomass biosynthetic pathways, as reflected in both higher cell number and proliferation rate achieved, while CYLD deletion redirected carbon flux enhancing lactate formation. In both cases, once glucose was consumed (day 4), the secreted lactate was re-used, yet this was more pronounced in CYLDko MEC1 cells. Finally, we explored whether CYLD knockout might impact MEC1 sensitivity to targeted agents i.e. the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax. We found that CYLDko MEC1 cells presented increased apoptosis compared to their CYLDwt counterparts when cultured in the presence of either drug. Moreover, treatment with ibrutinib or venetoclax led to reduced ATP production rates in both CYLDko and CYLDwt MEC1 cells, albeit the reduction was more pronounced in the former (FD=4.5 for ibrutinib; FD=10 for venetoclax compared to the respective CYLDwt MEC1 treated cells). Taken together, we demonstrate for the first time that CYLD can also act as an oncogene, at least in the context of CLL and in particular in the MEC1 cell line model of CLL, since its elimination leads to (i) lower proliferation and increased apoptosis rates coupled with diminished signaling capacity; (ii) metabolic rewiring toward enhanced lactate formation; and, (iii) augmented sensitivity to CLL therapeutic agents. It remains to be elucidated under which condition this could also occur in the patients with CLL or other B lymphoproliferative disorders.

Disclosures: Chatzidimitriou: Novartis: Other; Jansenn: Other. Ghia: Roche: Consultancy, Honoraria, Research Funding; MSD: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Lilly/Loxo Oncology: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding.

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