Inhibition of the ATR–CHK1 Pathway in Ewing Sarcoma Cells Causes DNA Damage and Apoptosis via the CDK2-Mediated Degradation of RRM2

Abstract

Inhibition of ribonucleotide reductase (RNR), the rate-limiting enzyme in the synthesis of deoxyribonucleotides, causes DNA replication stress and activates the ATR-CHK1 pathway. Notably, a number of different cancers, including Ewing sarcoma tumors, are sensitive to the combination of RNR and ATR-CHK1 inhibitors. However, multiple, overlapping mechanisms are reported to underlie the toxicity of ATR-CHK1 inhibitors, both as single-agents and in combination with RNR inhibitors, toward cancer cells. Here, we identified a feedback loop in Ewing sarcoma cells in which inhibition of the ATR-CHK1 pathway depletes RRM2, the small subunit of RNR, and exacerbates the DNA replication stress and DNA damage caused by RNR inhibitors. Mechanistically, we identified that the inhibition of ATR-CHK1 activates CDK2, which targets RRM2 for degradation via the proteasome. Similarly, activation of CDK2 by inhibition or knockdown of the WEE1 kinase also depletes RRM2 and causes DNA damage and apoptosis. Moreover, we show that the concurrent inhibition of ATR and WEE1 has a synergistic effect in Ewing sarcoma cells. Overall, our results provide novel insight into the response to DNA replication stress, as well as a rationale for targeting the ATR, CHK1, and WEE1 pathways, in Ewing sarcoma tumors.

RRM2 is a target for synthetic lethal interactions with replication stress checkpoint addiction in high-risk neuroblastoma

“…Deregulated expression, prognostic value as well as therapeutic exploitation of RRM2 has been reported in various types of cancer over the past decade including prostate cancer [11,12], glioblastoma [13], colorectal cancer [14], melanoma [15], Ewing sarcoma [10], cervical [16] and breast cancer [17,18]. Besides the finding of RRM2 as part of a four-gene prognostic signature in high-risk neuroblastoma and a recent report with limited data showing that siRNA mediated knockdown in SH-SY5Y cells caused a cell cycle arrest concomitant with apoptosis induction [19], the functional role of RRM2 in neuroblastoma tumorigenesis has remained unexplored thus far.…”

Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing’s sarcoma cells

“…ATR is activated by SSBs and replicative stress (RS) to maintain genomic stability [15,37,36]. ES cells, which are de cient in BRCA1mediated HR, suffer from permanent RS, rely on ATR-CHK1 pathways and are thus sensitive to ATRi [10,6,2,3]. In addition, we found that both ATRi and HSP90i share a set of common molecular targets, involved in cell vitality and proliferation, and ER stress response.…”

“…BALB-CTA tests and experiments in colon cancer cells showed the therapeutic potential of AUY-VE beyond ES cells. Since HSP90i and ATRi are already used to treat various types of cancer in clinical phase I/II trials [14,61,15,10,16,57], their combinations could further increase their effectiveness and improve the prognosis of patients, especially for BRCA-and p53de cient tumor entities. Figure S3.…”

“…However, effective therapeutic strategies are unavailable and thus actively pursued to cure recurrent and metastatic ES. Recently, inhibitors of ATR and ATR-mediated pathways, as well as of HSP90 have been shown to be effective in ES in vitro [10,6,11,12,4,13].…”

CDKs in Sarcoma: Mediators of Disease and Emerging Therapeutic Targets

“…One group showed that the WEE1 inhibitor, MK-1775, exacerbated the cytotoxic effects of a DNA replication stress drug, gemcitabine, in MPNST, UPS, and OS cell lines [211]. More recently, studies in EwS cells showed that inhibition of the ATR-CHK1 checkpoint pathway activates CDK2, which enhances DNA replication stress by targeting a subunit of the ribonucleotide reductase enzyme for proteasomal degradation [212]. Those findings led to concurrent targeting of ATR-CHK1 and WEE1 pathways, which killed EwS cells in a highly synergistic fashion.…”