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  • Treatment of all of the GYN cancer cells in

    2024-05-15

    Treatment of all of the GYN cancer nucleoside analog in this study with ATRi not only reduced phosphorylation of Chk1 at Ser345 following genotoxic stress, but also phosphorylation of ATM. Loss of phospho-ATM following ATRi treatment is not likely due to non-specific targeting of ATM by ETP-46464 [28]. While ATR kinase inhibitors have been shown to sensitize ATM-deficient cells to cisplatin [27], the seven cell lines studied here expressed ATM and this ATM was functional as it autophosphorylated on serine-1981 following IR. Though ATM becomes activated in response to platinum-based DNA damage, and although this activation is partially abrogated by ATMi, treatment of GYN cells with ATMi and cisplatin or carboplatin did not enhance the response of either drug. Moreover, the enhanced response of cisplatin or carboplatin in the presence of the combined inhibition of ATR and ATM was equivalent to that seen with ATRi alone. These findings are relevant as progressive accumulation of DNA lesions following acute platinum-treatment results in the formation of lethal DSBs and activation of apoptosis [4], [33], [34], [35]. As ATM has been shown to play a key role in DSB repair, our data suggest that progressive DSB accumulation by cisplatin or carboplatin either circumvents or may not temporally align with ATM-associated DNA repair pathways. Cisplatin-mediated phospho-Chk2 (Thr68) and phospho-ATM (Ser1981) levels were reduced by ATM inhibition, though more markedly by ATR inhibition. However, low levels of phospho-Chk2 (Thr68) persisted with ATR inhibition alone, indicating that there may be an ATR-independent activity of ATM responsible for this basal level of phosphorylation of Chk2. This notion is supported by the observed complete loss of phospho-Chk2 (Thr68) following combined inhibition of ATR and ATM without any further augmentation of response over that seen with the combination of cisplatin and ATRi alone. We find increased levels of cleaved PARP1 and caspase 3 in GYN cancer cells treated with cisplatin and ATRi demonstrating that the enhancement in the response of the cisplatin and ATRi combination occurs, at least in part, through elevated apoptotic signaling. These data clarify the roles of ATR and ATM in response to genotoxic stressors that induce replicative stress, such as platinum drugs, and clearly point to opportunities for pharmacologic targeting of ATR to increase the therapeutic potential of platinum-based drugs in GYN cancers. IR therapy is commonly used as a first- and second-line treatment in management of primary and recurrent endometrial and cervical cancer, and even in select settings in ovarian cancer. Our data show that ATM is activated in response to IR-mediated DNA damage. The phosphorylation status of ATM or Chk2 was only modestly reduced in irradiated cells in which ATR was pharmacologically inhibited. This finding recapitulates previous evidence demonstrating autoactivation of ATM, followed by ATM-dependent phosphorylation of Chk2, in response to IR-induced DNA damage [3], [4]. The results here suggest that ATM activation in response to IR-induced DNA damage results in the activation of ATR, which in turn phosphorylates Chk1. This conclusion is supported by our data showing that inhibition of ATM in IR-treated cells did not result in a pronounced induction of phospho-Chk1 (Ser345) compared to cells exposed to IR alone. This interpretation is further supported by the observed complete loss of basal phospho-Chk1 (Ser345) levels in ATR inhibited GYN cancer cells exposed to IR, a loss that is not observed in IR exposed cells in which ATM-alone is inhibited. Further, the interplay between ATR and ATM is underscored by the loss of phospho-Chk1 (Ser345) and phospho-Chk2 (Thr68) in IR exposed cells in which both ATR and ATM are inhibited. In the context of Chk1 activation, ATM-dependent activation of ATR resulting from the generation of ssDNA during DSB resection has been previously described [36]. An assessment of radioresistance profiles in cervical cancer cell lines has shown that abundant levels of the p53 family member, p73α is associated with increased radiosensitivity [37]. We show that pharmacologic inhibition of ATR or ATM augmented the response of IR in HELA and SIHA cervical cancer cell lines, both of which represent different levels of radioresistance arising from their low level of p73α expression [37], with further increases in IR sensitization by coordinate inhibition of both kinases. These data have translational potential to enhance the therapeutic response of IR (alone or with a radiation sensitizer such as cisplatin) with the combination of ATR and ATM inhibitors, particularly in the setting of newly diagnosed disease as well as persistent and recurrent disease in cervical, endometrial and ovarian cancer. These findings also support previous studies showing that ATM and ATR inhibition both induces sensitization to radiation and demonstrates further enhancement when inhibitors of ATR and ATM are combined with IR [17], [18], [19], [21].