Differential expression of ribonucleotide reductase regulatory subunit M2 in triple negative breast cancer.

Women diagnosed with triple negative breast cancer can benefit neither from endocrine therapy nor from HER2-targeted therapies (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding ribonucleotide reductase regulatory subunit M2, RRM2, when comparing the tumor cells of patients with triple negative breast cancer to normal mammary ductal cells (2). RRM2 was also differentially expressed in bulk tumor in human breast cancer (3). RRM2 mRNA was present at significantly increased quantities in TNBC tumor cells relative to normal mammary ductal cells. Analysis of human survival data revealed that expression of RRM2 in primary tumors of the breast was correlated with recurrence-free survival in patients with luminal A type cancer, while within triple negative breast cancer, primary tumor expression of RRM2 was correlated with distant metastasis-free survival in patients with luminal androgen receptor subtype disease. RRM2 may be of relevance to initiation, maintenance or progression of triple negative breast cancers.

Ribonucleotide Reductase Regulatory Subunit M2 as a Driver of Glioblastoma TMZ-Resistance through Modulation of dNTP Production

Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies have examined primary and recurrent GBM tumors, but it is difficult to study tumor evolution during therapy where resistance develops. To investigate this, we performed an in vivo single-cell RNA sequencing screen in a patient-derived xenograft (PDX) model. Primary GBM was modeled by mice treated with DMSO control, recurrent GBM was modeled by mice treated with temozolomide (TMZ), and during therapy GBM was modeled by mice euthanized after two of five TMZ treatments. Our analysis revealed the cellular population present during therapy to be distinct from primary and recurrent GBM. We found the Ribonucleotide Reductase gene family to exhibit a unique signature in our data due to an observed subunit switch to favor RRM2 during therapy. GBM cells were shown to rely on RRM2 during therapy causing RRM2-knockdown (KD) cells to be TMZ-sensitive. Using targeted metabolomics, we found RRM2-KDs to produce less dGTP and dCTP than control cells in response to TMZ (p<0.0001). Supplementing RRM2-KDs with deoxycytidine and deoxyguanosine rescued TMZ-sensitivity, suggesting an RRM2-driven mechanism of chemoresistance, established by regulating the production of these nucleotides. In vivo, tumor-bearing mice treated with the RRM2-inhibitor, Triapine, in combination with TMZ, survived longer than mice treated with TMZ alone (p<0.01), indicating promising clinical opportunities in targeting RRM2. Our data present a novel understanding of RRM2 activity, and its alteration during therapeutic stress as response to TMZ-induced DNA damage.

CHK1 inhibition exacerbates replication stress induced by IGF blockade

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

A Study of Ribonucleotide Reductase mRNA Expression and Its Prognostic Role in Patients with Chronic Lymphocytic Leukemia

Background Ribonucleotide Reductase (RNR) is responsible for converting ribonucleotides to deoxyribonucleotides required for DNA replication and repair. RNR consists of two subunits, termed subunit 1 (RRM1) and 2 (RRM2). Imbalance in the regulation of RNR activity and control of dNTPs' pool leads to genomic instability and increases mutation rate. RNR expression has been associated with prognosis in pancreatic, non-small-cell lung, breast, and biliary tract cancer. However, RNR expression in chronic lymphocytic leukemia (CLL) and its possible prognostic role have not been investigated yet. Aim In this study we evaluate the possible prognostic role of RNR expression in CLL. Method The study comprised patients with immunophenotypically confirmed disease at the time of sample collection. Peripheral whole blood samples were collected from 84, 27, 15, and 9 patients before treatment, after one, two, and three lines of treatment respectively. RNA extraction and reverse transcription were carried out using standard protocols. A Taqman based real-time PCR was performed on a CFX96 RT-PCR system (Bio-Rad Laboratories, Hercules, CA, USA). For both the housekeeping and target genes, a Taqman primer/probe mix was used according to the manufacturer's instructions (Applied Biosystems, Foster City, CA, USA). RRM1 and RRM2 mRNA levels were expressed as an RRM1-2/GAPDH ratio. Western blot analysis was performed to quantify the RRM1 protein levels in a random sample of 41 patients. Antibodies used were: RRM1 #3388, β-actin #4967 and anti-rabbit IgG HRP-conjugated #7074 (Cell Signaling Technology, Danvers, MA, USA). Detection was done using the ECL western blotting reagents. Statistical analysis was conducted to study the possible correlations between the variables. All reported p values are two-tailed. Statistical significance was set at p&lt;0.05 and analyses were conducted using SPSS statistical software (version 22.0). Results From 135 CLL patients included in the study 56.3% were female and the median age at diagnosis was 64 years. Peripheral blood was collected in 84 treatment-naïve patients (62.2%). Median follow up was 6.66 years (3.47 ─ 11.13) and median time from diagnosis until 1st line treatment was 23.1 months (IQR: 5.8 - 56.5 months). Out of 135 patients, 69 (51,1%) received 1 st line treatment and 35 patients (25,9%) 2 nd line treatment with median time between the two treatment lines being 26.5 months (IQR: 7.8 - 40.8 months). Furthermore, 48.5%, 33.8%, 12.3%, 3.1% and 2.3% of the patients had Rai score 0, I, II, III, IV respectively. The median mRNA expression of RRM1 was 0.04 (IQR: 0 - 0.09) and of RRM2 was 0.01 (IQR: 0 - 0.1). RRM1 mRNA expression was significantly higher in patients without anemia (p=.025) and without lymphadenopathy (p=.002). Higher values of ESR (r=-.30; p=.028), LDH (r=-.20; p=.026) and Rai score (r=-.18; p=.037) were associated with lower expression of RRM1 mRNA. In addition, TP53 gene deletion detected by FISH was associated with higher RRM1 mRNA expression (p=.036). Significantly higher RRM2 mRNA expression was reported in patients without lymphadenopathy (p=.021) and Rai score 0 (p=.003). Moreover, higher was the expression of RRM2 mRNA in cases with Trisomy 12 (p=.050). In samples collected before treatment, higher values of RRM1 mRNA expression were statistically significantly associated with lower RAI score (r=-.30; p=.005) and longer time periods between the first two lines of treatment (r=.95; p=.050). Western blot analysis confirmed detection of RRM1 protein but statistical correlation was not carried out due to lack of material from the whole group of patients. Conclusion For the first time, mRNA expression of RRM1 and RRM2 is studied in patients with CLL. These results show RNR involvement in the pathophysiology of CLL. RRM1 and RRM2 mRNA higher expression found in 17p deletion and trisomy 12 cases respectively may be consistent with the existence of a methylation-depended mechanism proposed by other studies. Therefore, these results demonstrate RNR's potential role as a prognostic factor, and make it a probable therapeutic target. A study including a larger number of cases could further confirm our results. Figure 1 Figure 1. Disclosures Kyrtsonis: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Genesis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees. Panagiotidis: Abbvie: Research Funding; Pfizer: Research Funding; Janssen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Sandoz: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Research Funding; Astellas: Research Funding. Viniou: Sandoz: Research Funding; Takeda: Research Funding; Novartis: Honoraria, Research Funding; Sanofi: Research Funding; Janssen: Honoraria, Research Funding; Pfizer: Research Funding; Abbvie: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Roche: Research Funding; Astellas: Research Funding; Celgene: Research Funding.