In this study, we used a panel of leukemia cell lines to assess the relative effects of MGMT activity, MMR status, and PARP activity on the ability of ABT-888 to potentiate the antitumor effects of TMZ. Although our data indicate that ABT-888 was most effective in potentiating TMZ in MMR-deficient cell lines with low MGMT activity, ABT-888 also potentiated TMZ in MMR-proficient cell lines. In primary acute leukemia cells, ABT-888 potentiation of TMZ seemed to correlate only with leukemia subtype, providing >3-fold potentiation in two of four primary AML samples.
Although Tentori et al. (41
) had also noted that leukemia cell lines could be sensitized to TMZ using the PARPi benzamine, this is the first report to examine the effect of MGMT activity on the ability of PARPis to potentiate TMZ in leukemia MMR-proficient and MMR-deficient cell lines. This is also the first reported examination of ABT-888 potentiation of TMZ in primary acute leukemia. Similar to previous reports in leukemia cell lines, colon cancer, and glioma, PARPi potentiation of TMZ was more pronounced in cells with MMR deficiencies (11
Several PARPis are under investigation in oncology clinical trials. ABT-888 is undergoing phase 1 testing in adults with melanoma. Other PARPis under clinical study include AZD2281 (AstraZeneca), AG014699 (Pfizer) in combination with TMZ for adults with solid tumors and metastatic malignant melanoma (42
), INO-1001 (Inotek/Genentech) in combination with TMZ for metastatic melanoma and glioblastoma multiforme (43
), KU59436 (KuDOS/AstraZeneca) in advanced solid tumors (including patients with BRCA1/BRCA2 mutations; ref. 44
), and BS201 (BiPar) for refractory solid tumors and lymphomas (12
). A better understanding of the factors that govern PARPi potentiation of TMZ may allow for the prospective determination of which tumors most likely to respond to this combination.
Prior clinical studies in both adults and pediatrics have suggested that TMZ may be effective in AML patients with low/absent MGMT tumor activity but is less effective in the presence of MMR deficiency or elevated MGMT activity (3
). Our data suggest that PARPi may be effective at potentiating TMZ in AML patients without MMR deficiencies and that this potentiation may be independent of MGMT activity. Although common in adults with treatment-related AML (45
), MMR deficiencies are uncommon in pediatric patients with newly diagnosed AML. However, pediatric patients with relapsed leukemia frequently have elevated MGMT (4
) and this group of patients would benefit from potentiating agents that could overcome both MMR deficiencies and elevated MGMT activity.
In MMR-deficient cell lines, we found an inverse correlation between MGMT activity and potentiation of TMZ by ABT-888. MGMT activity seemed less important in determining ABT-888 potentiation of TMZ than MMR status in these leukemia cell lines. First, we observed 3-fold ABT-888 potentiation of TMZ in MMR-proficient leukemia cell lines and 10-fold ABT-888 potentiation of TMZ in MMR-deficient leukemia cells with elevated MGMT activity (). Second, overexpression of MGMT in both MMR-deficient and MMR-proficient cell lines was unable to completely block ABT-888 potentiation of TMZ (). PARPi potentiation of TMZ in the presence of MGMT was also observed using the PARPi 3AB in colon cancer cell lines (46
). These data suggest that neither increased MGMT activity nor MMR proficiency precludes the ability of PARPi to potentiate TMZ.
In some cases, the effects of ABT-888 seemed to depend on a threshold level of PARP activity because ABT-888 did not potentiate TMZ in either the U937 AML cell line or primary leukemia cells with very low PARP activity ( and ). However, ABT-888 did not result in TMZ potentiation in many primary ALL samples with high PARP activity, indicating that the presence of robust PARP activity is not sufficient for ABT-888 potentiation. There was also one AML cell line (KG1) with very low PARP activity, which showed 3-fold ABT-888 potentiation of TMZ, suggesting that either ABT-888 potentiation of TMZ may be dependent on nonribosylation functions of PARP. TMZ potentiation effects that are independent of PARP inhibition (i.e., off-target effects) cannot be excluded. Previous data showed that the PARPi GPI-15427 increased the antitumor activity of TMZ and irinotecan in colon cancer xenografts irrespective of PARP activity (16
). Although the range of PARP activity in most primary tumors is unknown, Plummer et al. (47
) examined PARP activity in adult melanoma specimens and noted that PARP activity was quite variable, ranging from 1.7 to 3,600 pmol PARP/mg protein. Hence, it is likely that the effectiveness of PARPi will vary, reflecting variability in both baseline PARP activity and the efficiency of PARP inhibition.
Although best known for its role in BER, PARP also participates in DSB repair (5
). Evidence suggests that PARP competes with Ku for DSBs and PARP can either preserve DSBs for homologous recombination (48
) or (in the absence of Ku) initiate the use of a backup NHEJ repair pathway involving DNA ligase III (40
). The effects of PARP inhibition could be influenced by the intactness of the DSB repair pathways; for instance, in the absence of Ku or BRCA1/2, tumor cells may be very sensitive to PARP inhibition (49
). We have shown here that NHEJ status did not affect the ability of ABT-888 to potentiate TMZ in leukemia cell lines. Further study is needed to determine whether the status of the backup NHEJ repair pathway or homologous recombination influences ABT-888 potentiation of TMZ.
The influence of MGMT activity, PARP activity, and MSI on the ability of ABT-888 to potentiate TMZ is derived from a limited number of leukemia cell lines and therefore should be interpreted with caution. PARPi potentiation of TMZ in MMR-proficient cell lines may also be limited clinically because ABT-888 potentiation is seen only at high TMZ concentrations (>100 µmol/L). PARPi potentiation of TMZ could also be influenced by factors that were not examined, such as ABT-888 transport or metabolism.
In limited phase 1 studies of TMZ in patients with leukemia, where patients may have either ALL or AML, clinical responses have been observed exclusively in patients with AML. In a phase 1 study of TMZ in pediatric leukemia (4
), two of eight patients with AML had objective responses. These patients had absent or low MGMT activity and no MSI. Similarly, in an adult phase 1 study of TMZ in acute leukemia, the four patients with an objective response to TMZ (complete remission or complete remission with incomplete platelet recovery) had AML (3
). In this study, in vitro
ABT-888 potentiation of TMZ was seen in some primary samples from patients with AML (). Although the reason for this is unclear, it may be that more complete PARP inhibition can be obtained in patients with AML.
In summary, we have shown that the PARPi ABT-888 can effectively enhance the activity of TMZ, potentiating TMZ growth inhibition 3- to 21-fold in leukemia cell lines. ABT-888 potentiation of TMZ was most effective in cells with MMR deficiencies and low MGMT activity. Unexpectedly, PARP inhibition also potentiated TMZ activity in MMR-proficient leukemia cell lines, providing a 3- to 7-fold enhancement of TMZ growth inhibition independent of MGMT activity. Our data suggest that ABT-888 may function as a useful potentiator of TMZ; further studies, particularly in AML, are warranted.