1002 - m6A-modified circTET2 interacting with HNRNPC regulates fatty acid oxidation to promote the proliferation of chronic lymphocytic leukemia

Background:
Chronic lymphocytic leukemia (CLL) is a hematological malignancy with high metabolic heterogeneity. The adaptation of CLL cell metabolism is more likely to be dependent upon lipid metabolism, which is one of the most prominent metabolic alterations in cancer because of the abundant lipid deposits when compared with normal B cells. Although accumulating evidence reveals that altered lipid metabolism is associated with the progression and treatment responsiveness of CLL, the extent of its heterogeneity and relationship to molecular heterogeneity have not been systematically studied.N6- methyladenosine (m6A) modification plays an important role in metabolism through regulating circular RNAs (circRNA). However, the underlying mechanism is not yet fully understood in CLL.

Methods:
To determine the significance of m6A modification in CLL, we conducted whole-transcriptome sequencing of a cohort of 53 newly diagnosed CLL patients, and 35 m6A regulators were included in the analysis. Next, we constructed a m6A scoring system and found that the m6Sig score displayed a potential prognostic value, and we also explored the differential status of 35 m6A regulators in CLL. To further construct a risk model according to m6A-related circRNAs, we screened circRNAs with a correlation coefficient greater than 0.5. The identification of circTET2 was performed using northern blotting, RNase R treatment, actinomycin D assay, nucleocytoplasmic separation and RNA-FISH. To investigate the potential RBPs involved in this process, catRAPID and RBPBD were then applied, splicing factors RBMX and YTHDC1 was found to bind to the flanking sequences of circTET2, and co-IP assay verified their interaction. We then constructed stable CLL cell lines showing circTET2 overexpression and lentiviral silencing. CCK8 analysis and seahorse assay were performed to detected the cellular viability and oxygen consumption rate (OCR) which represents fatty acid oxidation (FAO) levels of CLL cells. Furthermore, We applied five inhibitors: indisulam (targeting splicing by inducing RBM39 degradation), H3B-8800 (a modulator of the SF3b complex), and the three pre-mRNA splicing inhibitors isoginkgetin, madrasin, and CP028. And the apoptotic rate with the drug combination compared with single-drug treatment was examined by flow cytometric analysis.

Results:
Herein, we established an m6A-related circRNA prognostic signature and identified circTET2 with a higher risk score and higher level of m6A modification that is a potential prognostic biomarker for CLL. By interacting with the RNA-binding protein (RBP) heterogeneous nuclear ribonucleoprotein C (HNRNPC), circTET2 is involved in the regulation of FAO and the mTORC1 signaling pathway to fulfill the energy demands and promote the proliferation of CLL cells. The mTOR inhibitor dactolisib and FAO inhibitor perhexiline exert a synergistic effect on CLL cells. In addition, the biogenesis of circTET2 can be affected by the splicing process and RBPs. CP028, a splicing inhibitor, modulates the expression of circTET2 and shows pronounced inhibitory effects.

Conclusion:
In summary, we identified m6A-modified circTET2 as a prognostic marker in CLL. CircTET2 that was upregulated in CLL was modulated by the splicing factors RBMX and YTHDC1, and that circTET2 interacted with HNRNPC, occupying a vital role in the modulation of lipid metabolism and cell proliferation of CLL. The data generated by this study collectively provide new evidence for a role and underlying mechanism for circRNAs in CLL lipid metabolism, and this may engender novel potential targets in clinical treatment.