SCFFBW 7 regulates cellular apoptosis by targeting MCL 1 for ubiquitylation and destruction

The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia (T-ALL). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL, validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun, Myc, cyclin E and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth, overexpression of Jun, Myc or notch 1 can also induce programmed cell death. Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCF (a SKP1–cullin-1–F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL. MCL1 is frequently overexpressed in various leukaemias through mechanisms that are not fully understood. MCL1 is distinct from other BCL2 family members in its extremely unstable nature, which provides a mechanism for cells to switch to either survival or apoptotic mode in response to various stresses. Phosphorylation of MCL1 by glycogen synthase kinase 3 (GSK3) regulates the stability of MCL1 (ref. 13), but little is known about the identity of the E3 ubiquitin ligase that targets phosphorylated MCL1 for destruction. On examination of the GSK3-mediated phosphorylation sites in MCL1, we surmised that they resemble a degron sequence that can be recognized by FBW7 (also known as FBXW7) (Fig. 1a), prompting us to test the possibility that GSK3-mediated phosphorylation of MCL1 triggers the degradation of MCL1 by FBW7. Depletion of FBW7 (Fig. 1b) or the SCF components cullin 1 (CUL1), RBX1 and SKP1 (Fig. 1c), but not other F-box proteins that we examined (Fig. 1b), resulted in a significant increase in the amount of MCL1 protein. T-cell-lineage-specific depletion of FBW7 in Fbw7 conditional knockout (Lck-Cre/Fbw7)mice3 resulted in increased MCL1 levels in the thymuses of these mice (Fig. 1d), as well as thymic lymphoma (Supplementary Fig. 1a) and the presence of acute leukaemia cells in the thymuses (Supplementary Fig. 1b). Consistent with a recent study, FBW7 human DLD1 cells (Fig. 1e) and HeLa cells treated with short interfering RNA (siRNA) directed against FBW7 (Supplementary Fig. 1c) have elevated MCL1 expression mainly in the mitosis (M) and early G1 phases of the cell cycle. The clinical relevance of this finding is further demonstrated by the finding that human T-ALL cell lines harbouring FBW7 mutations and/ or deletions have a significant increase in MCL1 (Fig. 1f). Additionally, depletion of FBW7 in DND41 cells or Loucy cells (both of which have wild-type FBW7) leads to increased MCL1 expression (Fig. 1g), whereas reintroduction of wild-type FBW7 dramatically reduced MCL1 expression in FBW7-deficient T-ALL cells (Fig. 1h), supporting a causal relationship between loss of FBW7 activity and elevated MCL1 expression in the T-ALL cells examined. More importantly, elevated MCL1 expression is also observed in both primary human and mouse T-ALL samples with deficient FBW7 activity (Fig. 1i, j and Supplementary Fig. 1a, b), and depletion of MCL1 impaired T-ALL disease progression in vivo (Fig. 1k–m). Consistent with a post-translational mode of regulation, no changes in MCL1 mRNA levels were observed after depletion of FBW7 in DLD1 cells (Supplementary Fig. 2d), and no positive relationship was observed between MCL1 mRNA levels and loss of FBW7 in T-ALL cells (Supplementary Fig. 2e). The half-life of MCL1 was significantly extended in the thymuses of Fbw7 mice and FBW7-deficient human T-ALL cells (Supplementary Fig. 3a–c), and experimental manipulation of FBW7 levels changed MCL1 stability accordingly (Supplementary Fig. 3d, e). Together, these results suggest that MCL1 is a downstream ubiquitylation target of SCF. As the proper substrate phosphorylation events are required for FBW7 to recognize and target its substrates for ubiquitylation, we next investigated which phosphorylation events trigger MCL1 destruction by FBW7. Mass spectrometry analysis showed that MCL1 is phosphorylated at multiple sites in vivo (Fig. 2a and Supplementary Fig. 5a–c). In addition to serine at position 159 (S159) and threonine at position 163 (T163), S64 and S121 were also phosphorylated in vivo. Consistent with previous reports, MCL1 destruction is promoted by GSK3 (Fig. 2b) but not by the protein kinases ERK1 (also