RNR inhibitors have shown high clinical activity and favorable toxicity profiles when co-administered with cytotoxic anticancer therapies, such as cisplatin and radiation [7, 8]. Use of RNR inhibitors to improve cytotoxic anticancer agent response is not new [7, 8, 24–30]. However, the optimal way to integrate RNR inhibitor therapy into cytotoxic anticancer regimens involving cancer cell DNA damage remains unknown. Several clinical trials have shown lower than anticipated anticancer responses when sequencing RNR inhibitors before cytotoxic therapy [27–29]. Other clinical trials have shown substantial gains in therapeutic efficacy when RNR inhibitors are sequenced after cytotoxic therapy, perhaps most conspicuous when RNR inhibitors are administered after irradiation [7, 8, 24–26]. Administration of 3-AP after a DNA damaging agent has emerged, over time of its clinical development, as the more clinically relevant cytotoxic sequence [7,10-11].
Here, we interrogated whether sequencing 3-AP prior to cisplatin better restored platinum-sensitivity in platinum-resistant ovarian cancer. Our findings that cisplatin plus 3-AP led to substantial DNA damage (i.e. increased number of γH2AX foci), led to impaired RNR activity when dNTPs were most demanded, and led to significant cytoreduction in platinum-resistant ovarian cancer cells are clinically relevant. This is especially noteworthy considering the modest clinical activity seen among the six women with platinum-resistant ovarian cancer treated by an overlapping four-day 3-AP then cisplatin sequence. Sequencing 3-AP, and therefore targeted inhibition of RNR after cisplatin treatment, not only increases cisplatin-mediated DNA damage in “platinum-resistant” ovarian cancer cells, but also blocks de novo dNTP supply when needed most for cisplatin-DNA adduct repair. Such data mimics radiochemotherapy sensitizing properties of 3-AP in cervix cancer cells [10, 11]. Our study would be strengthened by a more rigorous molecular interrogation of RNR inactivated by 3-AP, subsequent recovery of RNR activity, and high RNR activity facilitated cisplatin-induced DNA damage repair in “platinum-resistant” cancer cells.
The finding of relatively high levels of RNR M2 in non-responders is of interest. RNR M2 is a short-lived protein as a consequence of sequences promoting proteosome-dependent breakdown in late mitosis . It is reasonable to speculate that “platinum-resistant” ovarian cancers with high RNR M2 levels may have a large S-phase population, escaping cisplatin-mediated cytotoxicity through enhanced repair of stalled forks formed at cisplatin-DNA adducts during S-phase DNA replication . Alternatively, IHC-detected elevated levels of intracellular RNR M2 may reflect elevated RNR activity which would facilitate cisplatin-DNA adduct repair through timely on-demand supply of de novo dNTPs . Current research is exploring each intriguing possibility more closely.
Lastly, dose-limiting methemoglobinemia was observed in two women after 3-AP intravenous infusion, halting GOG protocol #126O clinical trial accrual. The mechanism of RNR inhibition by 3-AP is via inactivation of the tyrosyl free radical within the M2 or M2b (p53R2) small subunits [23, 33, 34]. Basically, this is a molecular interaction of a Fe2+-3-AP chelate and of oxygen generating local reactive oxygen species capable of annihilating the nearby tyrosyl free radical. In a similar manner, a Fe2+-3-AP chelate interferes with methemoglobin-hemoglobin cycling. Oxygenated Fe2+ hemoglobin oxidizes to Fe3+ methemoglobin and superoxide at a rate of 3% per day. Methemoglobin is normally reduced to hemoglobin by cytochrome b5 reductase, accounting for 94% of recycling methemoglobin to hemoglobin . Methemoglobin is thus maintained at a level of 1% of total hemoglobin. Symptomatic dyspnea occurs in fit adults when methemoglobin blood levels reach 25%, but symptoms could occur at much lower levels of methemoglobin when co-morbid conditions exist. Dose-limiting methemoglobinemia was encountered twice in six women, but among a total of 100 individual 3-AP infusions. Mechanistically, 3-AP methemoglobinemia is expected to be independent of 3-AP mediated augmentation of DNA damaging agent effects. Gains in best sequenced and timed RNR inhibitor and DNA damaging agent therapy should translate into overall clinical anticancer benefit without undue methemoglobin toxicity. For example, in patients with cervical cancer where 3-AP is administered immediately after irradiation for maximal radiosensitizing effect and on a different day from cisplatin to lessen “off-target” toxicity from a cisplatin-3-AP effect, symptomatic methemoglobinemia is not encountered .