Antitumor Effects of Blocking Protein Neddylation in T315I-BCR-ABL Leukemia Cells and Leukemia Stem Cells

Abstract

Imatinib revolutionized the treatment of chronic myeloid leukemia (CML), but drug resistance and disease recurrence remain a challenge. In this study, we suggest a novel strategy based on blocking protein neddylation to address BCR-ABL point mutations and leukemia stem cells (LSC) that lie at the root of imatinib-resistant recurrences. On the basis of the finding that the NEDD8-activating enzyme subunit NAE1 is overexpressed in CML cells, we hypothesized that the function of certain neddylation-dependent protein substrates might be targeted to therapeutic ends in imatinib-resistant CML cells and LSCs. In support of this hypothesis, we demonstrated that the NAE1 inhibitor MLN4924 induced G-M-phase arrest and apoptosis in bulk CML cells with wild-type p53, regardless of their T315I mutation status in BCR-ABL. Moreover, MLN4924 inhibited the survival and self-renewal of primary human CML CD34 cells and LSCs in CML-bearing mice via accumulation of p27 in the nucleus. Notably, silencing attenuated the suppressive effect of MLN4924 on the maintenance of LSCs in CML-bearing mice. Taken together, our findings offer a preclinical proof of concept for targeting protein neddylation as a novel therapeutic strategy to override mutational and LSC-derived imatinib resistance in CML. These findings highlight a mediator of protein neddylation, a type of protein turnover mechanism, as a viable therapeutic target against imatinib-resistant forms of chronic myelogenous leukemia. .

The NEDD8-activating enzyme inhibitor MLN4924 induces DNA damage in Ph+ leukemia and sensitizes for ABL kinase inhibitors

“…We show that the CHK1 kinase inhibitor AZD7762 had an almost perfect additive effect in combination with MLN4924 ( Fig S3), suggesting that CHK1 and Nedd8-conjugation act in the same pathway that prevents replication stress in Ph+ leukemia cells. These findings are in good agreement with a recent study in which it was shown that MLN4924 induces DNA damage as reflected by accumulation of CDT1, γH2AX and phospho-Chk2 [47].…”

“…We demonstrate here that the first-in-class Nedd8activating enzyme inhibitor MLN4924 very efficiently induced cell death of Ph+ leukemia cells, surpassing the effect in MM cells, for which MLN4924 is currently under investigation in a phase I/II clinical trial [39]. In line with previous data [47] we showed that MLN4924 induced caspase-dependent apoptosis and was equally active in the imatinib-resistant KBM5-T315I subline as in the parental KBM5 cell line, indicating that MLN4924 may be of use for treatment of imatinib-resistant leukemias. Furthermore, we show that MLN4924 sensitized Ph+ cell lines that are characterized by a limited TKI sensitivity for both imatinib and the third generation ABL TKI ponatinib.…”

“…We show that MLN4924 had no appreciable effect on NF-κB signaling in BV173 and SUPB15 cells. The nuclear levels of the NF-κB p65 subunit were slightly decreased by MLN4924 treatment, as well as the levels of ser32-phosphorylated IκBα ( Fig S3A), which in contrast to our results, were previously shown to accumulate in MLN4924-treated DLBCL cells and also in CML cells harboring wild type BCR-ABL or T315I-BCR-ABL [33,47]. STAT5 tyr694-phosphorylation was not affected by MLN4924, whereas it was abrogated by imatinib treatment, as expected ( Fig S3B), indicating that MLN4924 did not impede BCR-ABL signaling.…”

“…Our results indicate that MLN4924-and imatinib-induced DNA damage and replication stress triggered apoptosis by altering the MCL1/NOXA balance. In other cancer cell types it was also found that MLN4924 increased NOXA protein and mRNA expression [35,47]. Of note, the induction of NOXA was also observed in p53 mutant cell lines (KCL22 and K562, data not shown), indicating its p53 independence.…”

“…Interestingly, our results show that MLN4924 significantly improved the efficacy of HU treatment, suggesting that this combination might be useful for leukemia debulking regimens. Moreover, it was shown recently that MLN4924 is specifically effective in CML LICs since these have increased levels of NAE1 and single treatment with MLN4924 inhibited survival and self-renewal of these cells [47]. Therefore, we speculate that MLN4924 can be a valuable addition to the current treatment protocols for Ph+ leukemia patients and may significantly aid in not only achieving early and deep MR in these patients, but also eradicating the LICs and thereby reducing the risk of TKI resistance.…”