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Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T cells associated with chronic infection by human T-cell lymphotropic virus type-1 (HTLV-1). ATLL patients with aggressive subtypes have dismal outcomes. We demonstrate that ATLL cells co-opt an early checkpoint within the tumor necrosis factor receptor 1 (TNFR1) pathway, resulting in survival advantage. This early checkpoint revolves around an interaction between the deubiquitinase CYLD and its target RIPK1. The status of RIPK1 K63-ubiquitination determines cell fate by creating either a prosurvival signal (ubiquitinated RIPK1) or a death signal (deubiquitinated RIPK1). In primary ATLL samples and in cell line models, an increased baseline level of CYLD phosphorylation was observed. We therefore tested the hypothesis that this modification of CYLD, which has been reported to inhibit its deubiquitinating function, leads to increased RIPK1 ubiquitination and thus provides a prosurvival signal to ATLL cells. CYLD phosphorylation can be pharmacologically reversed by IKK inhibitors, specifically by TBK1/IKKε and IKKβ inhibitors (MRT67307 and TPCA). Both of the IKK subfamilies can phosphorylate CYLD, and the combination of MRT67307 and TPCA have a marked effect in reducing CYLD phosphorylation and triggering cell death. ATLL cells overexpressing a kinase-inactive TBK1 (TBK1-K38A) demonstrate lower CYLD phosphorylation and subsequently reduced proliferation. IKK blockade reactivates CYLD, as evidenced by the reduction in RIPK1 ubiquitination, which leads to the association of RIPK1 with the death-inducing signaling complex (DISC) to trigger cell death. In the absence of CYLD, RIPK1 ubiquitination remains elevated following IKK blockade and it does not associate with the DISC. SMAC mimetics can similarly disrupt CYLD phosphorylation and lead to ATLL cell death through reduction of RIPK1 ubiquitination, which is CYLD dependent. These results identify CYLD as a crucial regulator of ATLL survival and point to its role as a potential novel target for pharmacologic modification in this disease.

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Summary We previously identified the N -quinoline-benzenesulfonamide (NQBS) scaffold as a potent inhibitor of nuclear factor-κB (NF-κB) translocation. Now, we report the structure-activity relationship of compounds with the NQBS scaffold in models of diffuse large B-cell lymphoma (DLBCL). We identified CU-O42, CU-O47, and CU-O75 as NQBS analogs with the most potent cytotoxic activity in DLBCL lines. Their anti-lymphoma effect was mediated by NF-κB sequestration to the cytoplasm of DLBCL cells. Internal Coordinates Mechanics analysis suggested direct binding between CU-O75 and IκBα/p50/p65 which leads to the stabilization of the NF-κB trimer. A whole cellular thermal shift assay confirmed direct binding of the NQBS to IκBα, an inhibitory component of the IκBα/p50/p65 trimer. Lymphoma cell line sequencing revealed CU-O75 induced downregulation of NF-κB-dependent genes and DeMAND analysis identified IκBα as one of the top protein targets for CU-O75. CU-O42 was potent in inhibiting tumor growth in two mouse models of aggressive lymphomas.

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy of mature T lymphocytes associated with a chronic infection by human T-cell lymphotropic virus type-1 (HTLV-1). Multistep oncogenic process induced by HTLV-1 relies on the viral transactivator protein Tax interaction with pathways important in cell survival. Prognosis of the more aggressive subtypes of ATLL is abysmal and new treatment options are urgently needed. The TNFR1 pathway is one of the best characterized pathways determining cellular fate. TNF binding to its receptor induces a survival response in a vast majority of cells. This signal can however also trigger a death response if one of its cell death checkpoints is disrupted. Since these checkpoints act by antagonizing the cell death machinery, it is presumed that they can be used by malignant cells to avoid cell death. An early checkpoint within the TNFR1 pathway is centered around RIPK1, which has a somewhat bipolar role by inducing either cell survival or cell death. These diametrically opposite endpoints are determined by the state of lysine 63 (K63) poly-ubiquitination of the RIPK1. When RIPK1 is K63-ubiquitinated, a pro-survival signal is generated, while deubiquitination leads to death signal. CYLD, an enzyme that removes K63-linked ubiquitin chains from RIPK1, is crucial regulator as it converts RIPK1 into a death-signaling molecule. CYLD was initially identified as a tumor suppressor in rare hereditary tumors such as cylindromatosis. Its role in hematologic malignancies is less clear. Our data shows that CYLD tumor suppressor activity can instead be inactivated by phosphorylation, which inhibits its catalytic activity. We found CYLD to be constitutively phosphorylated in ATLL cell line models (MT4 and C1866) as well as primary samples from patients with ATLL. This was accompanied by an increase in the phosphorylation of TBK1/IKKɛ and IKKɑ/β, upstream kinases for CYLD. Additionally, we demonstrated that Tax by itself is sufficient to induce CYLD phosphorylation by transfection of a Tax-encoding plasmid into HEK293 cells. We subsequently examined the effect of IKK inhibitors MRT67307 and TPCA on CYLD phosphorylation. Both inhibitors were potent in reducing CYLD phosphorylation individually and more potently in combination. This effect was followed by induction of cell death, mediated by both apoptosis (blocked by zVAD-FMK) and necroptosis (blocked by necrostatin-1). Next, we transfected MT4 cells with a kinase-inactive TBK1-K38A mutant. MT4 cells stably transfected with TBK1-K38A exhibited reduced CYLD phosphorylation and a significantly slower growth rate compared to cells transfected with a control gene. Knockdown of CYLD in MT4 cells reduced the level of cell death induced by IKK inhibitors. Furthermore, both apoptosis and necroptosis were diminished in CYLD-deficient cells treated with the IKK inhibitors. Association of RIPK1 with the FADD-containing death-inducing signaling complex (DISC), which is a biochemical signature of the RIPK1 switch to become a death-inducing molecule, was enhanced by IKK inhibitors and was dependent on CYLD. Finally, we show that IKK inhibitors reduced RIPK1 ubiquitination in control knockdown MT4 cells. More strikingly, RIPK1 ubiquitination was basally elevated in CYLD-deficient MT4 cells where it remained elevated upon IKK blockade. We conclude that in ATLL cells, TAX induces phosphorylation of CYLD to keep it inactive in order to prevent RIPK1 from inducing cell death. This process of tumor suppressor inactivation could be targeted as a therapeutic option in ATLL. Disclosures No relevant conflicts of interest to declare.