Targeting DNA binding proteins for cancer therapy

2.1 Cancer-associated TFs in leukemia

Leukemia is classified into acute myeloid leukemia (AML), acute lymphocytic leukemia, Chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia. Chromosomal rearrangements at 11q23 are observed in both pediatric and adult leukemias, which led to the identification of the mixed-lineage leukemia gene (MLL).⁶

Approximately 10% of acute leukemia patients harbor MLL translocations.⁷ MLL belongs to a family of histone methyltransferases and specifically methylates histone H3 on lysine 4. MLL frequently fuses with a component of the AEP coactivator complex in acute leukemia, resulting in immortalization of hematopoietic progenitors through the aberrant activation of genes associated the hematopoietic stem cell program, such as HOXA9 and MEIS1.^{8, 9} Okuda et al¹⁰ reported that AEP and MLL-AEP fusion proteins induce transcriptional activation through physical interaction with selectivity factor 1, which is a core component of the RNA polymerase I preinitiation complex. Acute myeloid leukemia 1 (AML1/RUNX1) is a TF and forms a fusion protein with the ETO gene, which is involved in transcriptional repression.¹¹ The AML1-ETO fusion protein is associated with development of acute leukemia by upregulating a number of genes such as SOX4, IL-17BR, CD200, and γ-catenin.^{12, 13} A recent study reported that AML1-ETO cooperates with AP-1 to drive cyclin D2 expression, resulting in G₁ cell cycle progression and leukemic propagation.⁴

Chronic myelogenous leukemia is (CML) caused by a genetic abnormality that leads to generation of BCR-ABL, a constitutively active tyrosine kinase. Therefore, the tyrosine kinase inhibitor imatinib was a breakthrough CML therapy. However, Naka et al¹⁴ reported that initiating cells in CML are resistant to imatinib. In a mouse model used in this study, treatment of leukemia-initiating cells with transforming growth factor-β induced nuclear localization of Foxo3a through Akt activation, leading to development of imatinib resistance.

2.2 Cancer-associated TFs in solid tumors

Recent studies reported that TF are also dysregulated in solid cancers, which results in acquisition of tumor malignancies such as cell proliferation, drug resistance, invasiveness, immune evasion, and metastasis (Table 1). c-Myc is an important cancer-associated TF and promotes tumor cell growth and proliferation by regulating numerous target genes such as Cdc25A, hTERT, and glutamine synthetase.^15-17 Santoro et al¹⁸ reported that p53 loss induces c-Myc activation in breast tumors. Although c-Myc is important for symmetric cell division of mammary stem cells and induces reprogramming of progenitor cells to stem cells in a mouse model, concomitant p53 loss and c-Myc activation upregulates expression of 189 mitosis-associated genes, resulting in the expansion of cancer stem cells in human breast cancer cells.

Sry-related high-mobility box 2 is a pluripotency-associated TF and is essential for the maintenance of stemness. Therefore, SOX2 plays important roles in regulating developmental processes.¹ Aberrant SOX2 expression is present in many cancers and promotes tumor seeding ability and drug resistance. For example, in breast cancer cells, SOX2 suppression induces sensitization to tamoxifen by activating Wnt-signaling pathway-related genes such as DKK1 and AXIN2.^{19, 20}

Immune checkpoint blockade against T-cell inhibitory receptors such as CTL-associated protein-4 (CTLA-4) and PD-1 is considered to be one of the most effective approaches across diverse cancers.²¹ However, it is difficult to predict patient response to these approaches during cancer treatment. Therefore, it is critical to identify genes that modulate antigen presentation and tumor-immune interaction. Chew et al²² reported that the early embryonic TF DUX4, which is silenced in somatic tissues, is reexpressed in diverse solid cancers, resulting in suppression of MHC class I. Therefore, DUX4 inhibits T cell recognition of cancer cells. Consistent with these findings, low DUX4 expression is correlated with progression-free and overall survival in response to anti-CTLA-4 therapy in metastatic melanoma patients.

Nuclear receptors are also important TFs and known to function as a critical regulator in cancer biology.²³ Estrogen receptor (ER) is one of the nuclear receptors and associated with luminal type breast cancer.²⁴ Therefore, ER is important to determine the subtypes of breast cancer and therapeutic approaches. As the expression level of ER is transcriptionally regulated by ER factor-1,²⁵ ER factor-1 is also considered to regulate the gene expression that is characteristic of the luminal type breast cancer phenotype.

2.3 Telomeric DNA binding protein

In addition to cancer-associated TFs, telomere binding proteins are considered to be viable therapeutic targets, as these proteins are associated with tumor malignancies such as tumorigenesis and proliferation.²⁶ Mammalian telomeres are comprised of a double-stranded TTAGGG-repeat tract terminating in a 3′ single-stranded overhang that forms a T-loop with a specialized protein complex known as the shelterin complex. The shelterin complex is comprised of six proteins, including TRF1, TRF2, POT1, RAP1, TIN2, and TPP1. Among them, TRF1, TRF2, and POT1 are telomeric DNA binding proteins. The telomere-repeat binding factors TRF1 and TRF2 bind duplex TTAGGG repeats to stabilize telomeric DNA.

Bejarano et al²⁶ reported that TRF1 is highly expressed in mouse and human glioblastoma multiforme (GBM). In GBM mouse models, brain-specific Trf1 genetic deletion efficiently inhibits GBM initiation and progression, improving survival rate. Importantly, TRF1 small molecule inhibitors have similar effects in human GBM cell lines and xenografts generated from patient-derived primary glioblastoma CSCs .

Because TRF2 expression is elevated in several types of cancers, including breast, liver, lung, and colon cancer, TRF2 is considered to be associated with tumorigenesis.^27-30 Blanco et al³¹ used telomerase-deficient mice that also expressed TRF2 under the keratin 5 promoter to demonstrate that telomerase deficiency promotes TRF2-mediated epithelial carcinogenesis. These findings suggest that TRF2 inhibition could be an effective therapeutic approach for treatment of telomerase-deficient cancers.

The 3′ overhang of human telomeres forms tetra-stranded DNA structures known as G-quadruplexes, which are important for elongation of telomeric DNA by telomerase.³² Because stabilization of G-quadruplexes has the potential to inhibit telomere replication by interfering with the telomerase-mediated elongation step, small molecules that stabilize G-quadruplexes are promising agents for cancer therapy. Consistent with these findings, the G-quadruplex stabilizer telomestatin induces tumor suppression by promoting dissociation of TRF2 from the telomeres.³³

4.1 Microfluidic-based EMSA (QPID) assay

Electrophoretic mobility-shift assay is considered a gold standard method to analyze and measure the binding affinity between a DNA binding protein and its target sequence.⁵⁴ Combining the EMSA assay with microfluidics technology facilitated more rapid and quantitative performance of the EMSA. Glick et al⁵¹ succeeded in developing a high-throughput microfluidic platform termed quantitative protein interaction with DNA (QPID). This is an integrated microfluidic-based assay with a DNA microarray that enables analysis of 4096 samples and calculation of the binding affinity of DNA binding proteins to their target sequences in a single run (Figure 1). The interaction of cyclic AMP-dependent transcription factor 1 (ATF1) and ATF3 with 128 genomic cAMP response elements was examined using this platform. The study revealed that the difference in DNA binding affinity between ATF1 and ATF3 was due to a minor groove width of genomic DNA.

4.2 DNA-protein interaction-ELISA

The ELISA is a well-known and useful method for detection of target proteins such as antigens and Abs, quantifying relative amounts of the target protein based on an enzymatic reaction. Brand et al⁴⁸ developed a novel method based on the ELISA termed DPI-ELISA, in which biotin-labelled oligonucleotides are conjugated to streptavidin-coated microplates. The DPI-ELISA enables analysis of DNA-protein interactions in multiple samples in a single run (Figure 1). Using the DPI-ELISA method, Alonso et al⁴⁹ identified netropsin as a specific inhibitor of mammalian high-mobility group protein AT-hook 2 (HMGA2), which is associated with metastasis in several types of cancers.^55-57

4.3 DNA strand exchange-fluorescence resonance energy transfer

To investigate the interactions between DNA binding proteins and their target sequences more simply and rapidly, Miyagi et al⁵⁰ developed a novel method based on 2 phenomena: DNA binding protein-dependent inhibition of spontaneous DSE between partially double-stranded DNA probes, and FRET (Figure 1). For the DSE-FRET assay, 2 types of oligonucleotides are used. One is a quencher-conjugated oligo, and the other is a fluorescence-labelled oligo. During the interaction of the DNA binding protein with these 2 oligonucleotides, strand exchange is inhibited. Therefore, after inhibition of the interaction of the DNA binding protein with the oligonucleotides, fluorescence is detected.

4.4 Amplified luminescent proximity homogeneous assay

An amplified luminescent proximity homogeneous assay is based on a luminescent oxygen-channeling chemistry.⁵⁸ In this assay, an analyte is sandwiched by a biotinylated Ab conjugated to streptavidin-coated donor beads and a second Ab conjugated to acceptor beads. Donor beads are located in proximity to acceptor beads through the binding of these Abs to the analyte. After the excitation of donor beads at 680 nm, singlet oxygen is transferred from donor beads to acceptor beads, which results in the chemiluminescent emission at 615 nm. Using this system, Nomura et al⁵³ established a high-throughput screening system and identified pyrrothiogatain as a novel inhibitor of GATA3 DNA-binding activity, which is important for T helper 2 cell differentiation.