RRM2B: An oxygen-requiring protein with a role in hypoxia

ABSTRACT

How tumor cells adapt and survive under hypoxia significantly impacts patient prognosis. We recently demonstrated that the oxygen-requiring ribonucleotide reductase (RNR) enzyme, which provides cells with deoxyribonucleotides, responds to limited oxygen availability by switching small subunits from RRM2 to RRM2B. This property of RNR is essential for hypoxic cell viability and therefore contributes to the most aggressive and therapy-resistant fraction of tumors.

Most human solid tumors contain regions of inadequate oxygen supply (hypoxia), which occur as a result of high metabolic demand and an abnormal/inefficient tumor vasculature. The degree of tumor hypoxia correlates with poor patient prognosis, in part due to resistance to chemo- and radio-therapy as well as increased genomic instability, invasion and metastatic potential.¹ Severe levels of hypoxia, which correlate with radiation resistance, induce DNA replication stress characterized by significantly reduced replication rates, accumulation of single stranded DNA (ssDNA) and ATR (Ataxia-Telangiectasia and RAD3-related)-mediated signaling. Hypoxia-induced replication stress triggers the activation of the DNA damage response (DDR) pathway. Notably, hypoxia-induced replication stress occurs in the absence of detectable DNA damage.² The activation of the DDR is associated with the early stages of cancer development, and hypoxia is likely one of the principle factors responsible, as hypoxic regions have been observed in preneoplastic lesions.³

Ribonucleotide Reductase (RNR) is the only enzyme that catalyzes the de novo conversion of all 4 ribonucleotides diphosphates (NDPs) to deoxyribonucleotides diphosphates (dNDPs), and therefore plays a pivotal role in maintaining deoxyribonucleotide (dNTP) pools at optimum levels.⁴ Maintenance of optimal concentrations of dNTPs is critically important for the faithful replication of the genome, as imbalanced nucleotide pools lead to replication stress and promote tumourigenesis.⁵ There are 3 main classes of RNR enzymes and all of them share a common reaction mechanism whereby free radicals generate the electron used to reduce the NDPs to dNDPs. Most eukaryotic organisms contain class I RNR enzymes, consisting of 2 homodimeric subunits, which associate to form the holoenzyme.⁴ The α subunit (a RRM1 dimer) contains the catalytic site whereas the β subunit (a RRM2 or RRM2B dimer), contains the essential tyrosyl radical, which is formed during the reaction between oxygen and a di-iron center.^4,6 Oxygen is therefore essential for mammalian RNRs (RRM1/RRM2 or RRM1/RRM2B), leading to the logical assumption that RNR is inactive in severely hypoxic/anoxic conditions.⁴

Using the DNA fiber technique we demonstrated that DNA replication continued in hypoxic conditions, albeit at a greatly reduced rate.^7,8 This finding, combined with the lack of DNA damage and gradual resolution of ssDNA observed in hypoxia led us to hypothesize that there must be nucleotide availability in hypoxia. In support of this, we found that the levels of all 4 nucleotides were indeed reduced in hypoxia but low levels were detectable.

To determine the source of nucleotides in hypoxia we began by investigating the levels of the 3 RNR subunits, and found that RRM2B was significantly induced in several cell lines in hypoxia, whereas the levels of the other β subunit (RRM2) and RRM1 decreased over time. Notably, the hypoxic induction of RRM2B occurred in a TP53-dependent manner at the transcriptional level and through an alternative mechanism at the translational level. Depletion of RRM2B in hypoxic conditions, using both siRNA and CRISPR technology, led to further disruption of the dNTP pools, persistent ssDNA and DNA damage, which indicated an inability to continue DNA replication. Most importantly, xenograft studies with RRM2B null cells showed delayed tumor growth, increased apoptosis in the hypoxic fraction and increased radiosensitivity. These results suggested that the basal levels of dNTPs provided by RRM1/RRM2B (as opposed to RRM1/RRM2) in hypoxia are sufficient for on-going replication and preventing the collapse of replication forks, which would otherwise ultimately lead to double strand breaks and further loss of viability (Fig. 1).

Figure 1.

RRM2B induced by hypoxia drives continued DNA replication. Severe levels of hypoxia lead to replication stress, which through the activation of the DNA damage response pathway, leads to accumulation of RRM2B. Ribonucleotide reductase (RNR) favors the RRM2B subunit in hypoxia, because it retains activity in these conditions providing the cell with a basal level of deoxyribonucleotides (dNTPs). As a result, replication fork integrity is preserved, DNA replication continues albeit at a compromised rate and the cell avoids apoptosis and eventually loss of viability. The fact that RRM2B is unable to provide the appropriate amount of dNTPs for replication to continue at the same rates as in normoxia, results in the hypoxic-specific replication stress observed in cells that survive in these conditions. NDPs (ribonucleotide diphosphates), dNDPs (deoxyribonucleotide diphosphates), RPA (replication protein A), ssDNA (single-stranded DNA).

The RRM2 and RRM2B proteins share 83% sequence homology and both bind RRM1 to form an active RNR holoenzyme.⁶ RRM2 and RRM2B have the same mechanism of tyrosyl radical formation, but although they generally share several functional domains, they also have key differences. The 2 proteins show distinct expression patterns, with RRM2 being specifically expressed during the S phase of the cell cycle, whereas RRM2B is constitutively expressed.⁶ Using molecular dynamics, enzyme kinetic assays and direct measurement of the tyrosyl radical by EPR, we asked if RRM2B is better able to function in hypoxia compared with RRM2. We found that the RRM1/RRM2B form of RNR enzyme is indeed capable of retaining activity in hypoxia, whereas the RRM1/RRM2 activity was rapidly compromised in hypoxia. The ability of RRM1/RRM2B to retain activity in hypoxia was attributed to several factors including, increased oxygen entering frequency and increased stability of the tyrosyl radical. The specific residues within RRM2B responsible for continued activity in hypoxia were identified (Y164 and K37/K151) and are not conserved in RRM2.

In the absence of DNA damage, basal levels of RRM2B are observed in quiescent and non-proliferating cells, highlighting a critical role in mitochondrial DNA synthesis.⁶ The first stress-responsive role attributed to RRM2B was to provide dNTPs during DNA repair in response to DNA damage, caused by agents such as ultraviolet light and irradiation.⁹ Our study suggests that one of the principal functions of RRM2B is to act as the hypoxic specific RNR subunit, and that RRM2B remains active in hypoxic conditions to preserve genomic stability. This conclusion is supported by reports describing variants of RRM2B (ccRRM2Bii) as responsible for continued cell division in anoxia tolerant vertebrates.¹⁰

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

ORCID

0000–0002–2335–3146