Deferasirox Induces Apoptosis in Mantle Cell Lymphoma in a Mechanism Involving Cyclin-D1, p53 and the Ribonucleotide Reductase

Mantle cell lymphoma (MCL) is a difficult to treat B-cell malignancy characterized by a rapid progressive course and poor prognosis. Despite advances in conventional and novel agents such as bortezomib, lenalidomide and ibrutinib, many MCL patients are still subject to rapid relapses of the disease, responsible for the relatively short overall survival. MCL is characterized by the t(11,14) translocation, resulting in cyclin-D1 (CD1) overexpression and dysregulation of the cell cycle. Recently, it has been demonstrated that depletion of CD1 results in cell cycle arrest, apoptosis and cell death of MCL cells thus labeling CD1 as a therapeutic target of this aggressive malignancy. Accumulating findings indicate that not only overexpressed CD1 but also deregulated signaling pathways such as PI3K/AKT/mTOR and Wnt are accountable for the tumorigenesis of MCL and for the maintenance of its proliferative and survival capacity. We thus believe that MCL treatment should target multiple deregulated signaling pathways and key molecules. Iron chelators have emerged as a novel strategy to treat tumors due to their capacity to target a multitude of key molecules, signaling pathways and cellular processes. Yet, the application of common iron chelators is limited due to their low bioavailability and high toxicities. Deferasirox (DFX), a clinically approved iron chelator applied for the treatment of iron overload, in contrast to the majority of iron chelators, is highly bioavailable and exhibits little to no side effects. To date, DFX is reported as being an orally effective antitumor agent against solid tumors by various mechanisms. In our project, we examined the in vitro impact of DFX on three MCL cell lines; Jeko-1, REC-1 and Z138; aiming thereby to resolve its molecular mode of action. DFX inhibited the growth of the MCL cell lines with 50% inhibitory concentrations (IC₅₀) of 8.07±1.08µM, 1.8±1.03µM and 1.52±1.54µM for Jeko-1, REC-1 and Z138 cell lines, respectively. Moreover, we demonstrated that DFX treatment trigged a rapid and intense (~2-fold) production of reactive oxygen species (ROS), induced severe DNA damage and arrested cells at the G1 phase within 6 hrs. Ultimately, a 72h treatment of 10µM DFX led to apoptosis in 60% to 86% of the MCL cells. Most importantly, we found DFX to substantially reduce CD1 protein levels in a dose- and time-dependent manner. The depletion of CD1 levels was accompanied with its phosphorylation at Thr-286 (pCD1), a phosphorylation known to sentence CD1 to proteasomal degradation. In agreement with previous studies that have shown a correlation between ROS induction, CD1-phosphorylation, CD1 depletion and DNA damage, we found CD1 levels to negatively correlate with increasing DNA damage levels following DFX treatment.Interestingly, the efficacy of DFX treatment was dependent on the p53-status of the respective cell line. The wild-type p53 expressing lines, REC-1 and Z138, were found to be ~5-fold more sensitive to DFX than the p53 negative Jeko-1 cell line. In an attempt to understand this intriguing finding, we found DFX treatment to post-translationally elevate and stabilize p53 levels, most likely by depleting the intracellular heme pool which serves as a negative p53-regulator. Furthermore, we showed that DFX plays an important role in the attenuation of the iron dependent ribonucleotide reductase enzyme (RR); the rate limiting enzyme in dNTP biosynthesis and thus the key enzyme in DNA synthesis, cell cycle regulation and proliferation. DFX inhibited the enzymatic activity of RR with an IC₅₀ of ~25 µM; which is ~20-fold more effective than other established inhibitors such as deferoxamine.

In conclusion and pending further analysis, we demonstrated a potent impact of DFX on MCL cells. The vigorous anti-tumoral activity of DFX is most likely mediated via its ability to induce oxidative stress, to inhibit the RR enzyme and to eliminate the main oncogene in MCL pathogenesis; CD1. The depleting of intracellular iron/heme pools and the modulation of p53 only intensify the effect of DFX. The ability to inhibit the RR activity qualifies DFX as a sensitizer to existing DNA damaging radio- and chemotherapy options of MCL. DFX possesses the unique capacity to affect a multitude of cellular mechanisms in an auto-potentiating manner that propose this clinically established molecule as a potential therapeutic option for mantle cell lymphoma.