The discovery that hyperactivation of the NOTCH1 pathway contributes to the majority of T-ALL cases revolutionized both scientific and clinical perspectives in this field, and has spurred a search for NOTCH1-silencing therapeutic agents. At the core of this search is the need for a more complete understanding of NOTCH1 protein stability regulation, as the majority of the mutations affect proteasome-mediated degradation. We demonstrate that FBW7 targets NOTCH1 for ubiquitination and map in detail the amino acid degron sequence required for this interaction. Furthermore, we identify inactivating FBW7 mutations in ~40% of human T-ALL lines and 16% of primary leukemias. These mutations abolish the ability of FBW7 to interact with its targets, including NOTCH1, c-Myc, and cyclin E.
Initially, using a series of amino acid point mutants, we have mapped the N1-IC–FBW7 interaction to a degron centered around T2512
. We further analyzed the degron, demonstrating that a T+4 acidic glutamate is necessary for interaction with FBW7. The reason for this requirement is not clear. Possibly, a negative charge at position 2516 either facilitates the phosphorylation of T2512
or directly promotes interaction with FBW7. Current evidence suggests that phosphorylation of N1-IC by CDK8 plays a key role in recognition by FBW7. Work by Fryer et al. showed that MAML1 recruits CDK8 to the N1-IC transcriptional activation complex, and this kinase phosphorylates N1-IC to promote its degradation (31
). Two of the three CDK8 phosphorylation sites reported in that study are within the T2512
degron we identified. Although mutation of either S2514
by itself allowed at least some interaction with FBW7 (albeit compromised in the case of S2514
), a N1-IC with both residues mutated lost all ability to bind FBW7. Together with the work of Fryer et al., our observations suggest that CDK8 must phosphorylate N1-IC (minimally at S2514
) for it to be efficiently recognized by FBW7, either by priming the site for a different kinase that phosphorylates T2512
, or by directly facilitating physical interaction with FBW7. We do not know the identity of the kinase that phosphorylates T2512
; CDK8 might be responsible for this, too, but that possibility has not been directly tested. Although the N1-IC degron identified here is both similar to and different from degrons in other FBW7 substrates, it appears to occupy the canonical FBW7-binding pocket. Crystal structures of Cdc4 bound to a cyclin E peptide show that the phospho-T in the degron makes physical contact with three arginine residues, R465
, and R505
, in WD40 repeats III and IV (numbering based on FBW7α isoform) (33
). We have found that the FBW7 point mutations identified in T-ALL cell lines and primary leukemia affect these arginine residues, which is consistent with findings in solid human tumors (35
). It has been suggested that Fbw7 is a haploinsufficient tumor suppressor in p53+/−
), which supports the idea that decreased gene dosage stabilizes FBW7 targets in vivo. However, it is also possible that these mutations act as DNs because their inabilities to interact with the NOTCH1 and c-Myc degrons might create a pool of nonfunctional SCFFBW7
Although previously detected in certain solid tumors, FBW7-inactivating mutations do not appear to be a general oncogenic trigger in leukemia. Indeed, a recent sequencing of a large number of acute myeloid leukemia (AML) patient samples failed to detect any such mutations (38
). Thus, it was a surprise to detect FBW7-inactivating mutations in both T-ALL lines and primary T-ALL samples. These findings suggested that FBW7 mutations could be frequent in T-ALL because of the ability of FBW7 to recognize, bind to, and degrade important oncogenes, including N1-IC and its transcriptional target c-Myc. Interestingly, it seems that FBW7 deficiency could either activate the NOTCH pathway in the absence of NOTCH1 mutations or amplify its signaling strength by cooperating with already existing NOTCH1-HD mutations. If loss of FBW7 is, indeed, a NOTCH1 signal amplifier that facilitates NOTCH1-induced transformation, one would predict that selective pressure to acquire an FBW7 mutation preferentially affects lymphocytes in which the NOTCH1 PEST domain remains intact, as no increase in NOTCH1 signaling capacity could be gained by eliminating FBW7 function in the context of an existing PEST deletion. In agreement with this hypothesis, none of the 13 T-ALL cell lines or 96 primary samples showed any coexisting FBW7 and NOTCH1 PEST mutations. On the other hand, 60% of FBW7mut
samples had mutations in the NOTCH1-HD domain. These results suggest that, in T-ALL, the selective pressure to increase NOTCH1 stability and signaling strength is a primary force that drives oncogenesis. However, we do not want to imply that FBW7-mediated transformation depends solely on NOTCH1 activation. Both c-Myc and cyclin E could also be the mediators of this process. However, c-Myc is also a direct target of NOTCH signaling, so it is possible that FBW7 could impinge on both signaling pathways. The specific contribution and the importance of each FBW7 target in the induction of T-ALL remains to be analyzed using in vivo genetic approaches.
Moreover, we found that FBW7 mutations were more frequent in relapse samples and that more than half of the patients with FBW7 mutations at relapse did not carry the mutation at diagnosis, suggesting that FBW7 mutation might be an event that confers resistance to treatment. Interestingly, all T-ALL lines carrying FBW7 mutations were resistant to treatment with GSI. FBW7 is a very attractive treatment resistance candidate because of its ubiquitin ligase function and its ability to target c-Myc protein stability. c-Myc is a direct tran scriptional target of NOTCH1, and overexpression of c-Myc can rescue most T-ALL lines treated with GSI (16
). As c-Myc is a posttranslational target of FBW7 (40
), but—at the same time—a transcriptional target of NOTCH activity (16
), the transforming ability of FBW7 inactivation could be twofold; by losing FBW7 function, a cell should not only stabilize c-Myc but also produce more of it because of increased NOTCH1 signaling. In agreement with this hypothesis, we have shown that T-ALL FBW7 mutants lose their ability to recognize c-Myc. Also, our experiments demonstrate that c-Myc protein remains stable upon GSI treatment of lines carrying FBW7 inhibitors. Although these experiments strongly argue for a role for FBW7, further experimentation, preferably in vivo, is required to prove the role of FBW7 deficiency in GSI resistance. Moreover, it is possible that there are multiple resistance mechanisms, as we have found that a T-ALL line (PF382) was FBW7WT
, yet resistant to GSI treatment.
We report the identification of FBW7 mutations in human T-ALL and their direct connection to the NOTCH1–c-Myc oncogenic pathway caused by the ability of FBW7 to recognize, bind, and ubiquitinate these target proteins. These mutations target conserved arginines that form the binding pocket of FBW7 and abolish its binding to its targets. We believe that the identification of FBW7 mutations is of unique importance for the design of future molecular therapies for T-ALL. Indeed, our data suggest that these treatments should be multifaceted, covering both NOTCH1 activation and potential escape mechanisms, such as FBW7 mutations, and their effects on the stability of downstream target proteins.