The early successes of molecularly targeted therapy, such as imatinib mesylate directed against the BCR-ABL fusion protein in chronic myelogenous leukemia, stimulated enormous interest in subverting abnormal signaling pathways in cancer cells. The NOTCH signaling cascade influences normal development by regulating differentiation, proliferation, and apoptosis (52
). Activation of the NOTCH signaling pathway is firmly established in T-ALL and is likely involved in the genesis of many other tumor types (53
). Thus, it is not surprising that efforts to block NOTCH signaling as a novel therapeutic strategy are under way in T-ALL as well as in solid tumors. One of the most promising approaches has been to inhibit NOTCH receptor signaling using GSIs. This strategy suppresses the generation of NICD and thus, in principle, should inhibit the downstream transcriptional events normally induced by this key signaling component after it traverses to the nucleus. Although effective against some T-ALL cell lines, GSIs do not uniformly eliminate leukemic cells with activated NOTCH signaling. Understanding the mechanisms of GSI resistance may lead to better treatments for T-ALL.
We found missense FBW7 mutations or homozygous FBW7 deletion in GSI-resistant T-ALL cell lines and in primary T-ALL samples. Moreover, we have defined the NOTCH phosphodegron and demonstrated that the mutant forms of FBW7 found in T-ALL cannot bind to the NICD. Each T-ALL cell line with constitutive NICD expression harbored either NOTCH1 PEST domain or FBW7 mutations, suggesting that these two classes of mutations provide a mutually exclusive means of prolonging the NICD half-life. We also show that the expression of NOTCH target genes including DELTEX1 and MYC are not affected by GSI treatment in five resistant T-ALL cell lines with mutations in FBW7 (CEM, BE13, PEER, DU528, and HSB2), demonstrating that the mechanism of resistance in these leukemias lies upstream of MYC and DELTEX1 transcription. It appears that in these T-ALL lines, one consequence of FBW7 mutation is stabilization of the NICD resulting in sustained NOTCH signaling, and thus promoting resistance to GSIs. However, other FBW7 substrates, such as MYC, may also account for selection for FBW7 mutations and for GSI resistance in T-ALL, as suggested by our finding that ALL-associated FBW7 mutations can dominantly inhibit MYC degradation.
An important question is why FBW7 mutation confers GSI resistance, whereas NOTCH PEST domain truncations that remove the FBW7 interaction domain do not. In each cell line with PEST mutations, only one allele is affected by these heterozygous mutations, leaving the remaining normal allele, which encodes an NICD that is stabilized in the presence of mutant FBW7. Thus, although the amount of increased NOTCH activity resulting from the single allele PEST mutations may be sufficient to underlie the primary selection for these mutations, disruption of FBW7 function may be more active in sustaining NOTCH signaling and also may prolong the half-life of MYC (as well as other substrates), thus promoting GSI drug resistance.
The association between FBW7
mutations and resistance to GSIs has implications for clinical testing of these agents in patients whose cancers show deregulation of the NOTCH pathway. Molecular analysis of the FBW7
gene, as well as genes encoding the relevant NOTCH receptors and other key components of the NOTCH signaling pathway, such as NUMB, may contribute to identification of patients likely to be most responsive to GSI therapy. Because Myc has recently been shown to be an important target of Notch in mammary tumorigenesis as well as in T-ALL (5
), it might also be possible to overcome GSI resistance by combining GSIs with other drugs that block the MYC pathway to synergistically reduce MYC levels and block tumor cell growth. One attractive candidate is TMPyP4, a cationic porphyrin that binds to and stabilizes guanine quadruplexes in DNA. MYC
contains a sequence in its promoter that forms a guanine quadruplex, and TMPyP4 has been shown to inhibit MYC
transcription and the growth of tumor cells in vivo (54
). Thus, TMPyP4 or other agents that inhibit MYC
transcription may be useful in combination with GSIs to overcome resistance in patients harboring FBW7
mutations. Pharmacologic or genetic strategies that restore the normal function of FBW7 in tumor cells could also be therapeutically useful.