Reversal of CYLD phosphorylation as a novel therapeutic approach for adult T-cell leukemia/lymphoma (ATLL)

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T cells driven by chronic infection with human T-cell lymphotropic virus type 1 (HTLV-1). Patients with aggressive ATLL subtypes face particularly poor prognoses. Our findings reveal that ATLL cells exploit an early regulatory checkpoint within the tumor necrosis factor receptor 1 (TNFR1) signaling pathway to gain a survival advantage. This checkpoint centers on the interaction between the deubiquitinase CYLD and its substrate RIPK1. The ubiquitination status of RIPK1 at lysine 63 (K63) dictates cell fate: K63-ubiquitinated RIPK1 promotes survival, while deubiquitinated RIPK1 favors cell death.
In both primary ATLL samples and cell line models, we observed elevated baseline phosphorylation of CYLD. Given that CYLD phosphorylation is known to inhibit its deubiquitinating activity, we hypothesized that this modification increases RIPK1 ubiquitination, thereby enhancing prosurvival signaling in ATLL cells. Pharmacologic inhibition of CYLD phosphorylation using IKK inhibitors—specifically, TBK1/IKKε inhibitor MRT67307 and IKKβ inhibitor TPCA—reduces CYLD phosphorylation, restores its activity, and induces cell death. Both IKK subfamilies contribute to CYLD phosphorylation, and their combined inhibition produces a potent effect on promoting ATLL cell death.
ATLL cells expressing a kinase-inactive TBK1 mutant (TBK1-K38A) exhibit reduced CYLD phosphorylation and decreased proliferation. IKK inhibition reactivates CYLD, leading to decreased RIPK1 ubiquitination and facilitating RIPK1′s recruitment to the death-inducing signaling complex (DISC), thereby triggering apoptosis. In contrast, in CYLD-deficient cells, RIPK1 remains ubiquitinated even after IKK blockade and fails to associate with the DISC.
SMAC mimetics also reduce CYLD phosphorylation in a CYLD-dependent manner and induce ATLL cell death by decreasing RIPK1 ubiquitination. Collectively, these findings identify CYLD as a critical regulator of ATLL cell survival and highlight its potential as a novel therapeutic target in this disease.