The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance. However, it has proven difficult to develop small molecule inhibitors of MYC. One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small molecule-mediated stabilization of the G-quadruplex (G4) present in its promoter. Although molecules that bind globally to quadruplex DNA and influence gene expression are well-known, the identification of new chemical scaffolds that selectively modulate G4-driven genes remains a challenge. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs). We use the SMM screening platform to identify a novel G4-binding small molecule that inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes. Surface plasmon resonance (SPR) and thermal melt assays demonstrated that this molecule binds reversibly to the MYC G4 with single digit micromolar affinity, and with weaker or no measurable binding to other G4s. Biochemical and cell-based assays demonstrated that the compound effectively silenced MYC transcription and translation via a G4-dependent mechanism of action. The compound induced G1 arrest and was selectively toxic to MYC-driven cancer cell lines containing the G4 in the promoter but had minimal effects in peripheral blood mononucleocytes or a cell line lacking the G4 in its MYC promoter. As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G4-driven genes was not affected. In addition to providing a novel chemical scaffold that modulates MYC expression through G4 binding, this work suggests that the SMM screening approach may be broadly useful as an approach for the identification of new G4-binding small molecules.

G-quadruplexes (G4s) are noncanonical DNA structures that frequently occur in the promoter regions of oncogenes, such as MYC, and regulate gene expression. Although G4s are attractive therapeutic targets, ligands capable of discriminating between different G4 structures are rare. Here, we describe DC-34, a small molecule that potently downregulates MYC transcription in cancer cells by a G4-dependent mechanism. Inhibition by DC-34 is significantly greater for MYC than other G4-driven genes. We use chemical, biophysical, biological, and structural studies to demonstrate a molecular rationale for the recognition of the MYC G4. We solve the structure of the MYC G4 in complex with DC-34 by NMR spectroscopy and illustrate specific contacts responsible for affinity and selectivity. Modification of DC-34 reveals features required for G4 affinity, biological activity, and validates the derived NMR structure. This work advances the design of quadruplex-interacting small molecules to control gene expression in therapeutic areas such as cancer.

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared to single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared to single agents. Mice, bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared to single agents. A set of 37 genes cooperatively affected (34 down-regulated; 3 up-regulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes down-regulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared to normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo.

Seeds of Phaseolus vulgaris L. cv. White Kidney were germinated and grown either in a nitrogen‐free or in an ammonia‐supplied medium. The changes in the soluble protein concentration and in the levels of glutamine synthetase (GS, EC 6.3.1.2), NADH–glutamate synthase (NADH‐GOGAT, EC 1.4.1.14), ferredoxin‐glutamate synthase (Fd‐GOGAT, EC 1.4.7.1) and glutamate dehydrogenase (GDH, EC 1.4.1.2), both NADH‐ and NAD+‐dependent, were examined in cotyledons and roots during the first 10 days after sowing. Soluble protein declined rapidly in the cotyledons and increased slightly in the roots. GS activity was initially high both in cotyledons and roots but subsequently decreased during seedling growth. Exogenous ammonia hardly affected GS activity. High levels of NADH‐GOGAT were present both in cotyledons and roots during the first days of germination. The activity then gradually declined in both organs. In contrast, Fd‐GOGAT in cotyledons was initially low and progressively increased with seedling development. In roots, the levels of Fd‐GOGAT were higher in young than in old seedlings. Supply of ammonia to the seedlings increased the levels of NADH‐GOGAT and Fd‐GOGAT both in cotyledons and roots. NADH‐GDH (aminating) activity gradually increased during germination. In contrast, the levels of NAD+‐GDH (deaminating) activity were highest during the first days of germination. Exogenous ammonia did not significantly affect the activities of GDH.

MYC is deregulated in many malignancies and its aberrant expression is associated with tumorigenesis and tumor maintenance. We employed a therapeutic strategy to target both MYC transcription, and protein stability. Previously, we developed a small molecule microarray and screened it for MYC G4-binding compounds. The stabilization of G-quadraplex (G4) structures by nucleic acid binding small molecules in the NHE III region of the MYC promoter slows MYC transcription. We identified a benzofuran containing small molecule that could selectively and reversibly stabilize MYC-G4 DNA and reduce MYC expression. This compound demonstrated a G4-dependent mechanism of action showing toxicity in multiple myeloma cell lines containing a G4 in their MYC promoter whereas minimal effects were seen in a Burkitt’s lymphoma line lacking the MYC G4 sequence. Gene expression analysis validated that this compound selectively inhibited MYC and other MYC effectors, but did little to limit the expression of other G4 containing genes. The synthesis of more than 15 analogs identified a new benzofuran-containing analog with enhanced inhibitory activity in a diverse panel of human cancer cell lines (NCI-60), including a number of myeloma cell lines. EDGE-seq data confirmed that the new analog is similar to the original compound in having higher affinity for the G4 in MYC compared to the G4 in BCL2, KRAS, VEGFA and HIF1A. In addition, the analog was synergistic with entinostat, an HDAC inhibitor that can affect MYC protein stability, in limiting myeloma cells, but not PBMCs. The more potent growth inhibitory activity of the new analog allowed us to generate sufficient quantities to evaluate its pharmacokinetics and toxicity in mice. The compound was well tolerated in a dose-escalation toxicity study; no adverse effects were observed and preliminary pharmacokinetic studies showed promising serum bioavailability and exposure properties when administered either intravenously, intraperitoneally or orally. In an assessment of short term in vivo activity, MYC protein expression was inhibited in multiple myeloma xenografts. Longer term studies to evaluate the anti-tumor activity of the compound are currently in progress. Thus, our data provide evidence that small molecule stabilization of the MYC G4 can drive transcriptional silencing of oncogenic MYC both in vitro and in vivo. Furthermore, synergistic tumor cell killing was achieved when the MYC G4 inhibitor was coupled with HDAC inhibition. Citation Format: Snehal M. Gaikwad, David R. Calabrese, Elena C. Leon, John K. Simmons, Shuling Zhang, Aleksandra Michalowski, Sayeh Gorjifard, Zaw Phyo, Daniel Connors, John S. Schneekloth, Beverly A. Mock. Effective targeting of MYC expression with a novel nucleic acid binding (G4-quadruplex) small molecule coupled with HDAC inhibition synergizes to limit myeloma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 194. doi:10.1158/1538-7445.AM2017-194