Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair

Abstract

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O(6)-methylguanine due to elevated expression of the repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ-induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. On the basis of our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of nicotinamide adenine dinucleotide (NAD(+)), we have hypothesized that combined BER and NAD(+) biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD(+) biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD(+) biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM.

Figure 1. Expression of MPG and PARP1 modulate BER dependent PAR generation and cell death after alkylation damage

A, Cell viability of LN428 or LN428/MPG cells after 1 h MMS treatment, as measured by an MTS assay 48 hours after exposure. Plots show the % viable cells as compared to untreated (control) cells. Means are calculated from quadruplicate values in each experiment. Results indicate the mean ± S.E. of three independent experiments. B, PAR generation in LN428 or LN428/MPG cells before or after 15 minute MMS exposure (15 and 30 min.; 1.5 mM), as measured by quantitative ELISA (see Materials & Methods). C, Cell viability of LN428/MPG cells or LN428/MPG cells with stable knockdown of either PARP1 or PARP2 after 1 h MMS treatment, as measured by an MTS assay 48 hours after exposure. Plots show the % viable cells as compared to untreated (control) cells. Means are calculated from quadruplicate values in each experiment. Results indicate the mean ± S.E. of three independent experiments. D, Cells were depleted of PARP1 or PARP2 via shRNA. PAR generation in LN428/MPG cells LN428/MPG/PARP1KD and LN428/MPG/PARP2KD is shown before or after MMS exposure (15 and 30 min.; 1.5 mM), as measured by quantitative ELISA (see Materials & Methods). All values are normalized to LN428/MPG signal at 15 min. exposure (comparable to Fig. 1B) and reported as fold change in PAR level.

Figure 2. FK866 transiently decreases the level of NAD⁺ and enhances alkylation-induced cell death via a BER dependent mechanism

A, Cellular NAD⁺ levels in LN428 and LN428/MPG cells before exposure, after exposure to FK866 (10nM; 24 hrs) and following an additional 24 hours of recovery from FK866 treatment. The bars are defined in the legend B, Cytotoxicity profile of FK866. Viability of LN428 and LN428/MPG cells was measured by an MTS assay 48 hours after FK866 (24 hrs) treatment. Viable cells were determined as in Fig. 1A and reported as percentage relative to vehicle control treated cells (% Control). C, Cell viability after FK866-mediated NAD⁺ depletion immediately followed by alkylation damage. LN428 and LN428/MPG cells were treated with FK866 (24 hours) at varying doses followed by 1 hour MMS treatment at 0.5 mM. Cells were allowed to recover in normal media for 48 hours prior to analysis for cell viability by the MTS assay. Viable cells were determined as in Fig. 1A.

Figure 3. Chemical inhibition of BER and NAD⁺ biosynthesis potentiates alkylation-induced cell death

A, Cell viability of LN428 cells measured by an MTS assay after treatment with MMS alone, MMS in combination with 30 mM MX, MMS in combination with 10nM FK866, or with the 3-drug combination of MX, FK866 and MMS. Drug treatments were carried out as described in the Materials and Methods section. Viable cells were determined as in Fig. 1A and reported as percentage relative to vehicle control treated cells (% Control). B, Cell viability measured by an MTS assay in the LN428/MPG cells after treatment with MMS alone, MMS in combination with 30 mM MX, MMS in combination with 10nM FK866, or with the 3-drug combination of MX, FK866 and MMS. Drug treatments were carried out as described in the Materials and Methods section. Viable cells were determined as in Fig. 1A and reported as percentage relative to vehicle control treated cells (% Control). SER was calculated as IC₅₀ TMZ/ IC₅₀ TMZ + sensitizer.

Figure 4. Enhanced ATP depletion following MMS, MX and FK866 combined treatment

A, Cellular ATP content two hours after 0.5mM MMS treatment (or media control) as measured by the ATP-lite luminescent kit. Results are reported as percent of untreated control. Cells were pre-treated for 24 hours with either 10nM FK866 or media control. B, Cellular ATP content two hours after 0.5mM MMS treatment (or media control) as measured by the ATP-lite luminescent kit. Results are reported as a percentage of untreated control. Cells were pre-treated for 30 minutes with MX or media control followed by co-treatment with MMS or media and post treatment as described in the Materials and Methods section. *Represents statistical significance with a p < 0.05.

Figure 5. Combining NAD⁺ biosynthesis inhibition with BER inhibition sensitizes chemotherapy resistant glioma cells to TMZ

A, Cell survival determined by a long-term survival assay in cells modified to over-express MGMT. LN428/MPG/MGMT cells were treated with either TMZ alone for 12 days, 24 hours of 10nM FK866 followed by 12 days of TMZ, a 30 minute pre-treatment of 10mM MX followed by MX (5mM) and TMZ co-treatment for 12 days, or the three-drug combination (a 24 hour pre-treatment of FK866, followed by a 30 minute pre-treatment with MX and a 12 day co-treatment with MX and TMZ. TMZ was used at a 75 μm dose. B–D, Cell survival determined by a long-term survival assay in cells stably expressing shRNA to MLH1 (B), MSH2 (C) or MSH6 (D). LN428/MPG MMR knockdown cells were treated with either TMZ alone for 12 days, 24 hours of 10nM FK866 followed by 12 days of TMZ, a 30 minute pre-treatment of 10mM MX followed by MX (5mM) and TMZ co-treatment for 12 days, or the three-drug combination (a 24 hour pre-treatment of FK866, followed by a 30 minute pre-treatment with MX and a 12 day co-treatment with MX and TMZ. TMZ was used at a 75 μm dose. *Represents statistical significance with a p < 0.05.

Figure 6. Combining NAD⁺ biosynthesis inhibition with BER inhibition sensitizes chemotherapy resistant T98G cells to TMZ

A, Cell survival determined by a long-term survival assay in the T98G cells after a 24 hour pre-treatment with varying doses of FK866 followed by a 6 hour exposure to 500 μM TMZ. B, Cell survival determined by a long-term survival assay in T98G cells after treatment with either TMZ alone for 6 hours, a 30 minute pre-treatment with 10mM MX followed by 5mM MX and TMZ co-treatment for 6 hours, or the three-drug combination with varying doses of FK866 as a 24 hour pre-treatment followed by a 30 minute pre-treatment with 10mM MX and a 6 hour co-treatment with 5mM MX and TMZ. A dose of 500μM TMZ was used in each treatment condition. *Represents statistical significance with a p < 0.05 as compared to TMZ + MX.