Dual DNA Damage Repair Inhibition Synergizes with Alkylator Chemotherapy for Myeloma and Acute Leukemia

Alkylator chemotherapy remains important in pretransplant conditioning for multiple hematologic malignancies. Resistance to alkylators such as busulfan and melphalan results in part from the ability to repair DNA damage. DNA repair inhibition has therefore become an attractive treatment target. Here we tested the hypothesis that inhibitors of DNA repair overcome resistance to alkylator therapy.

We initially treated REH, K562, HL60, NB4, RPMI8226 and U266 cells with the PARP inhibitor ABT-888. We noted widely varying GI50 values from 8.7 - 11x10⁴ μM. Acute leukemia cells were overall insensitive to ABT-888 alone. However myeloma cells appeared relatively sensitive to ABT-888 with lower GI50 values (8.7μM for RPMI8226 cells, 49μM for U266 cells) and increased γH2AX foci at 24 hours after treatment. Synergy between ABT-888 and alkylator chemotherapy was observed in proliferative assays in all cell lines when treated with a fixed dose of ABT-888 (4μM) and increasing doses of either melphalan or busulfan. Combination index for RPMI8226 and HL60 cells was 0.5 at 50% proliferation. These findings were confirmed in a clonogenic assay. Increased cytotoxicity did not appear to be a result of apoptotic cell death as shown by Annexin V/PI co-staining. Instead combination treated RPMI8226 cells underwent accelerated senescence compared to melphalan treated cells at 24 hours as shown by βGal staining (27% vs. 51%, p=0.02). This was confirmed by upregulation of senescence related genes p16 (1.6 fold increase) and p21 (1.5 fold increase). Combination treated RPMI8226 cells were also noted to arrest in G2/M phase (43% vs. 50%). We hypothesized that cells may acquire the senescence associated secretory phenotype. We noted a 1.5-fold upregulation in IL6 gene expression in combination treatments compared to melphalan alone. CD86 gene expression increased by 1.6-fold and mean fluorescence intensity also increase by a factor of 1.3 compared to melphalan treatment. In mixed lymphocyte culture of treated RPMI8226 cells and CD3+ T cells, the stimulation index was 1.2 for melphalan treated cells and 3.2 for combination treated cells (p=0.003) showing that potentially RPMI8226 cells were able to directly present antigen to T-cells following combination treatment.

As combination treatment resulted in upregulation of double strand repair genes such as BRCA1, BRCA2, FANCD2 and DNA-PK in HL60 cells we hypothesized that inhibitors of double strand repair may be synergistic with the alkylator chemotherapy and ABT-888. Treatment with NU7026 at fixed concentrations (10μM) to inhibit DNA-PK in addition to ABT-888 (4μM) resulted in 46% reduction in proliferation in RPMI8226 cells and 52% in U266 cells. No effect was seen in HL60 cells. However when used in combination with alkylator chemotherapy we were able to show marked synergy with alkylator and PARP inhibition in HL60 and RPMI8226 cells. Combination index in melphalan, ABT888 and NU7026 treated RPMI 8226 cells was 0.6.

Finally we hypothesized that efficacy of DNA repair inhibition combination treatments was reliant on abnormal DNA repair in malignant cells in comparison to normal cells. To illustrate this we tested the impact of treatment on CD34+ cell hematopoietic colony formation. We show that NU7026, ABT-888 or a combination of both drugs with alkylator chemotherapy did not decrease colony formation over alkylator chemotherapy alone.

Here we show that PARP and DNA-PK inhibition synergizes with alkylator chemotherapy in AML, ALL and myeloma cells lines, providing a rationale for the addition of these agents to conditioning chemotherapy. In addition we show the activity of these agents in myeloma without the use of alkylating agents.