Abstract LB-46: Chemo-sensitization by modulation of Bcl-2 expression: The first example of an i-motif interactive small molecule that modulates gene expression

For the first time we provide evidence that not only illustrates the i-motif DNA secondary structure has potential to serve as a molecular target, but that within the Bcl-2 promoter this targeting can result in the modulation of gene expression and increase in chemo-sensitivity. The Bcl-2 oncoprotein is a key regulator and inhibitor of the intrinsic apoptotic pathway. Evasion of apoptosis is a well-known hallmark of cancer and is also recognized to play an important role in chemo-resistance in a variety of cancers particularly of lymphocytic origin. The significant over-expression of Bcl-2 common in low-grade lymphomas and lymphocytic leukemias has been associated with prolonged cell survival despite apoptotic stimuli and therefore is an attractive target for chemo-sensitization. Transcriptional regulation of Bcl-2 involves two promoter sites, P1 and P2, with P1 exerting the majority of control. Directly upstream of the P1 promoter is a GC-rich element capable of transitioning to DNA secondary structures. Previously we have shown the G-rich promoter element forms a mixed parallel/antiparallel G-quadruplex that has potential to adopt three different G-quadruplex structures. In recently published studies we have demonstrated that the complementary C-rich sequence has potential to form a stable i-motif structure. We now show that Bcl-2 expression can be modulated at the transcriptional level through small molecule targeting and destabilization of the i-motif. A high-throughput fluorescence resonance energy transfer (FRET) screening assay yielded a steroid derivative (compound 59) from the NCI diversity set that increased the i-motif FRET probe fluorescence by 2.5 fold indicative of substantial destabilization of the i-motif structure. The destabilization of the Bcl-2 i-motif was then confirmed with CD spectral and thermal stability analyses. As proof of principle we demonstrated that following treatment with compound 59, Bcl-2 gene expression is significantly decreased as detected by Real-Time RT-PCR in MCF-7, MCF-7 tamoxifen resistant breast cancer cell lines and the GRANTA-519 mantle cell lymphoma cell line. In addition, the GRANTA-519 cell line displays resistance to etoposide-induced apoptosis; however, in the presence of compound 59 the cells exhibited an increase in capase activity in comparison to cells that were treated with etoposide alone. These studies have been expanded to include a chemo-sensitization lymphoma cell line model in which two cell lines differ in Bcl-2 expression, low versus high, and sensitivity to etoposide-induced apoptosis, sensitive versus resistant, respectively. We anticipate from preliminary data that in response to compound 59 treatment the etoposide-resistant cell line that over-expresses Bcl-2 will have an increased apoptotic response similar to its etoposide sensitive counter-part. A Bcl-2 i-motif non-interactive compound is used as a negative control. Small molecules that target the Bcl-2 i-motif and result in the down-regulation of Bcl-2 expression have the potential to act as chemo-sensitizing agents to improve current chemotherapy in refractory patients by an alternative mechanism to ABT-737, which is in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-46.