Abstract
Background
The most aggressive diffuse large B-cell lymphomas (DLBCL) display concurrent over-expression of the B-cell lymphoma gene-2 (BCL2) and cellular-myelocytomatosis (MYC) oncogenes. DLBCL with these features are nearly uniformly fatal due to the lethal combination of resistance to apoptosis and high proliferation. Currently, there are no effective strategies to treat these patients. We propose directly targeting BCL2/MYC transcription through DNA secondary structures to induce chemosensitization in BCL2/MYC overexpressing DLBCL cell lines. DNA secondary structures are proposed to act as molecular switches, turning gene expression on or off. In support of this, genome-wide analyses have detected DNA secondary structure-forming sequences within close proximity of transcriptional start sites in 43% of promoter regions, which were mostly oncogene promoters including BCL2 and MYC (Huppert et al., Nucleic Acids Res 2007). Directly upstream of the BCL2 and MYC promoters are guanine and cytosine (GC)-rich elements capable of transitioning to G-quadruplex and i-motif secondary structures, respectively.
Previous high-throughput fluorescence resonance energy transfer (FRET) screening assays yielded a steroid derivative (NSC59276) and an ellipticine analog (NSC338258) from the NCI diversity library of small molecules. Compound NSC59276 targets the BCL2 i-motif and subsequently decreases mRNA and protein levels that leads to increased sensitivity in etoposide-resistant Burkitt’s and mantle cell lymphoma cell lines in vitro and in vivo (Kendrick et al., Nature Chemistry, under revision 2013). Similarly for the MYC G-quadruplex, NSC338258 down-regulates MYC expression and induces cytotoxicity in Burkitt’s lymphoma cell lines (Brown et al., J Biol Chem. 2011). Here, we assess the efficacy of the NSC59276 and NSC338258 as proof of principle for the targeting of BCL2/MYC concurrently in DLBCL, which has clinical implications for improving treatment for the most aggressive DLBCL.
Methods
We evaluated the cytotoxic and chemosensitizing effects of the BCL2 and MYC inhibitors using the MTS cytotoxcity assay in a DLBCL cell line model that consists of varied BCL2/MYC expression levels and mechanisms of regulation (Table 1). The cytotoxicity assays were conducted with either NSC59276 (BCL2i) or NSC338258 (MYCi) alone or concurrently in combination with standard chemotherapy agents for DLBCL (cyclophosamide and doxorubicin).
Results
The simultaneous targeting of BCL2/MYC with BCL2i and MYCi in combination with cyclophosphamide treatment had a potent effect on decreasing cell viability by 23–64% compared to cyclophosphamide alone. This effect was observed in DLBCL cell lines that over-express BCL2/MYC regardless of mechanism of deregulation and was negligible in the HT cells that do not express detectable levels. Interestingly, the most significant decrease in cell viability was seen in cell lines with BCL2/MYC gene amplification. Furthermore, BCL2i and MYCi had a synergistic effect on lowering cyclophosphamide IC50 concentration by 30- to a 100-fold in cell lines, particularly of the ABC subtype. Cell viability and cyclosphosphamide IC50 were also decreased when cells were treated with BCL2i or MYCi; however, BCL2i alone had a more significant effect than MYCi. A similar, but less pronounced effect was observed with doxorubicin combination experiments.
Conclusion
These data support our hypothesis that modulation of BCL2 and MYC with transcriptional repressors in combination with standard chemotherapy agents will improve the efficacy of chemotherapy alone. Further investigations that are ongoing will determine if the combination treated DLBCL cells are more significantly undergoing apoptosis compared to cyclophosphamide or doxorubicin alone. This is a novel, alternative approach of using small molecules that target BCL2 and MYC DNA secondary structures to down-regulate expression and act as chemosensitizing agents to improve current chemotherapy in refractory patients.
Disclosures:
Hurley: TetraGene: Consultancy, Equity Ownership.
DNA secondary structures: stability and function of G-quadruplex structures