PARPi/cisplatin or PARPi/carboplatin combinations as compared to single drug treatments increase the survival of BRCA-deficient xenograft models (16
). However, it is unclear whether a PARPi combined with cisplatin or carboplatin kills the tumor more efficiently, and only a limited number of studies have used isogenic models and examined the drug mechanisms. Here we report that ABT-888/carboplatin rather than ABT-888/cisplatin is generally associated with a more favorable outcome: additive or synergistic killing or growth inhibition of Brca/BRCA cells.
Our examination of combination treatments in human and mouse cells dependent on Brca/BRCA status demonstrated that ABT-888/carboplatin was more effective overall than ABT-888/cisplatin in enhancing cell killing/growth inhibition of Brca/BRCA cells. In Brca/BRCA cells, ABT-888/cisplatin combination treatment did not enhance lethality or reduce proliferation compared to ABT-888 or cisplatin treatments alone. In contrast, either synergistic or additive responses were observed in Brca/BRCA cells following ABT-888/carboplatin treatment. Several studies have already established that cisplatin has better activity than carboplatin in BC (independent of BRCA status) (37
). Therefore, it is likely that cisplatin potency was too high in our in models to be enhanced in the combination studies. In addition, the two platinum drugs have differential kinetics for adduct formation (carboplatin is ~100 fold slower than cisplatin) (39
). It would be interesting to determine whether ABT-888 followed by cisplatin treatment (sequential treatment) would mimic the results obtained with simultaneous ABT-888/carboplatin treatment. The differences in cell sensitivity/synergism observed between cell lines are influenced by types and position of BRCA mutation, underlying genetic factors other than BRCA status, as well as their TNBC status (i.e.
, HCC1937 line). Currently, no specific targeted therapy exists for TNBC but PARP targeting may be possible, even when tumors are not BRCA-linked (18
Because ABT-888/carboplatin improved Brca/BRCA-deficient cell killing versus the drugs used singly in vitro, we determined if tumor inhibition/response was also observed in vivo. So far, xenograft studies using the ABT-888/carboplatin combination have been carried out using only Brca/BRCA-deficient cells without any comparison to an isogenic BRCA complemented line. We showed that the ABT-888/carboplatin combination not only delayed tumor growth in Brca2-deficient xenografts, but also in Brca2-complemented xenografts. However, an actual response to carboplatin and ABT-888/carboplatin treatment was observed only in the Brca2-deficient xenografts. Moreover, no toxicity was observed in either model. These in vitro and in vivo studies strongly suggest that, although PARPi and carboplatin are not part of the first line of treatment for BRCA-associated BC therapy, their use in combination should be considered. Hence, our interest in evaluating the mechanism of ABT-888 and carboplatin combined versus single agent treatment in order to understand the factors contributing to their efficacy.
ABT-888, carboplatin, or their combination generated DNA damage in all cell lines examined, but only the BRCA-complemented cells used HRR. To assess DNA damage, we evaluated γH2AX foci (40
) in cells exposed to the drugs. A complexity in studying γH2AX response to ABT-888/carboplatin is that ABT-888 rapidly inhibits PARP activity, whereas carboplatin continues to form DNA adducts for up to 2 days in vitro
. As expected, the number of cells positive for γH2AX foci following PARPi single agent treatment was similar between HRR proficient and BRCA cells (3
). However, we have demonstrated that γH2AX foci produced after PARPi, carboplatin, or PARPi/carboplatin treatments in the tested cell lines are similar. RAD51 focus formation is dependent on both BRCA1 and BRCA2 and occurs following γH2AX focus formation (44
). Both BRCA proteins are required for RAD51 foci formation, and only cells that are specifically in S/G2 phase after drug treatment will show increased numbers of RAD51 foci (45
). Upon treatment, increased formation of RAD51 foci occurred only in BRCA-complemented cells, attesting to the normal repair response of those cells upon complementation.
Although single drug treatments with carboplatin and cisplatin are known to trigger apoptosis, BRCA cell response to combinations of these agents with PARPi has not been extensively studied. All BRCA lines exhibited apoptosis post-treatment, indicating that this pathway is not disabled. However, an apoptotic response consistent with synergistic drug action was observed only for HCC1937 cells exposed to high ABT-888 concentrations (200μM) and was most apparent from caspase 3 cleavage. A similar apoptotic response was not detected in EUFA423F cells at the concentrations and times tested, but those cells are an FA line. Cell death mechanisms in FA cells are less well-studied, and some cells constitutively produce caspase 3 and show PARP cleavage (46
). Although we show evidence that the apoptotic response remains, this response might be different for BRCA lines that are not FA.
PARP activity correlated with chemoresponse to PARPi, in non-isogenic human BRCA2 cell lines (BXPC3 and CAPAN1) and isogenic VC8 CHO cell lines (32
). Here, we show that all the isogenic human and mESC BRCA1 and BRCA2 lines had higher PARP activity than their respective BRCA/Brca-proficient cell lines, indicating that PARP activity in cells with either BRCA deficiency could serve as a biomarker. PAR formation depletes NADPH. Similarly, increased PARP activity in BRCA cell extracts was associated with a decrease in NADPH. PARP activity could be greater in BRCA cells for several reasons, including changes in the HRR-deficient cells to a more open chromatin structure or increased reliance on BER in an HRR-deficient background. The lines most resistant to ABT-888 alone or in combination with carboplatin (Brca2 and BRCA1) had PARP activity more similar to their respective BRCA-proficient cell lines. Upregulation of PARP1 transcripts has recently been noted in TNBC (47
), supporting the use of PARP1 RNA levels as a possible biomarker to guide treatment. Thus, PARP activity or NADPH levels could indicate patient response to PARPi. Platinum drug lesions are generally repaired by NER and HRR (48
). ERCC1, an NER protein, has been used to track the response of cells to platinum drugs. The high ERCC1 levels in BRCA1-complemented cells were associated with a ~3 or 4-fold higher carboplatin IC50
value as compared to the BRCA2-complemented cells. Additionally, the BRCA1- or BRCA2-deficient lines had ~2-fold lower carboplatin IC50
values than their respective complemented cells. Thus, ERCC1 expression, which most likely correlates with cellular ability to remove adducts/crosslinks, is a good indication of BRCA cell response to platinum drugs.
In conclusion, these data support the use of ABT-888/carboplatin combinations to treat BRCA tumors. Moreover, ABT-888/carboplatin appears to be a more effective combination than ABT-888/cisplatin, because after various doses and exposure times to BRCA cells, ABT-888/carboplatin was associated with a more additive or synergistic response than was ABT-888/cisplatin. Endogenous PARP activity and ERCC1 expression could serve as a guide for predicting treatment outcomes. However, further clinical and preclinical studies are needed to explore whether the efficiency of ABT-888/carboplatin is significantly enhanced in patient tumor cells that have high PARP activity and low ERCC1 levels. Overall, our data suggest that the ABT-888/carboplatin combination will likely be more successful than monotherapy in treating BRCA-associated cancers, and a phase II clinical trial (NCI#8264) is underway to test this hypothesis.