br Introduction Core binding factor CBF is a heterodimeric

Introduction Core binding factor (CBF) is a heterodimeric transcription factor composed of a DNA-binding RUNX subunit (encoded by one of three genes: RUNX1, RUNX2, or RUNX3) and a non-DNA-binding CBFβ subunit which increases the affinity of RUNX proteins for DNA. All three RUNX proteins as well as CBFβ have been shown to be critical regulators of specific developmental pathways. RUNX1 and CBFβ are essential for definitive hematopoiesis, where they regulate expression of genes associated with proliferation, differentiation, and survival of stem and progenitor menin (Friedman, 2009; de Bruijn and Speck, 2004; Wang et al., 2010; Link et al., 2010). RUNX2 is essential for normal bone formation by way of transcriptional regulation of genes critical for bone development (Komori et al., 1997; Otto et al., 1997). Both RUNX1 and RUNX3 play key roles in neuronal development. Perhaps not surprisingly, based on their critical roles in normal development, RUNX proteins and CBFβ are targets of genetic alteration in a variety of cancers. Both RUNX1 and CBFB undergo chromosomal translocations in a subset of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) patients where the corresponding fusion proteins have clearly been shown to be drivers of disease (Blyth et al., 2005). For the fusion proteins AML1-ETO and TEL-AML1, the binding of the fusion proteins to CBFβ has been shown to be essential for transformation (Roudaia et al., 2009). RUNX1 is also mutated in a subset of AML and myelodysplastic syndrome (MDS) patients. In addition, RUNX1 has recently been implicated in a number of epithelial cancers (Scheitz et al., 2012; Scheitz and Tumbar, 2013). Altered expression of RUNX2 has been implicated in breast and prostate cancers (Blyth et al., 2005). Silencing of RUNX3 by DNA methylation has been linked to intestinal and lung cancers (Lee et al., 2013). Due to the importance of these proteins for normal development as well as in a variety of cancers, small molecules which can modulate their activity are useful tools to address function and test new therapeutic approaches. Small molecule inhibitors of protein-protein interactions, particularly in the context of transcription factors, is still a relatively nascent field, in part due to the long and widely held belief that this class of interactions is “undruggable”. With an increasing number of success stories of small molecule inhibitors modulating protein-protein interactions (Arkin et al., 2014a; Laraia et al., 2015; Arkin and Whitty, 2009), including transcription factors, this paradigm is clearly changing. Along this vein, we have developed tool compounds which bind to CBFβ and inhibit CBFβ binding to RUNX proteins as a probe for the role of this important protein-protein interaction in function as well as its potential therapeutic applications. The most potent compounds we have developed inhibit this protein-protein interaction at low micromolar concentrations, use an allosteric mechanism to achieve inhibition, displace CBFβ from RUNX1 in cells, change occupancy of RUNX1 on target genes, alter expression of RUNX1 target genes, and show clear effects on leukemia and basal-like breast cancer cells consistent with on-target activity on RUNX protein activity.
Materials and Methods
Results
Discussion Transcription factors represent an important as well as challenging class of proteins for the development of small molecule inhibitors. Because of their function as crucial regulators of cell fate decisions in a normal and malignant context and as mediators of a wide swath of signaling pathways they are particularly attractive targets for cancer therapy. Moreover, it is now abundantly clear that tumorigenesis in multiple tissues involves the deregulation of gene expression and a subversion of the normal cell differentiation processes (Bonifer and Cockerill, 2011). However, transcription factors do not act alone, but function in the context of large multi-protein assemblies, which requires the targeting of intracellular protein-protein or protein-DNA interactions. This has proved to be more challenging than targeting enzymatic activity due to the nature of the interaction surfaces and the complexity of the assemblies, provoking the notion that this class of proteins is “undruggable”. Nevertheless, efforts in a number of labs have now demonstrated successful targeting of this class of proteins, opening up numerous avenues for novel modes of action to approach the treatment of cancer. Herein, we describe the development of a small molecule inhibitor which targets the heterodimeric core binding factor (CBF) transcription factor, specifically the protein-protein interaction between CBFβ and RUNX, the two subunits of CBF. We have optimized the activity as well as the ADMET properties based on an initial hit to generate tool compounds which we have shown bind to CBFβ and inhibit binding to RUNX1, resulting in decreased binding of RUNX1 to target sites in the genome and concomitant changes in the level of expression of RUNX1 target genes. While we have only experimentally demonstrated disruption of binding with RUNX1, all three RUNX proteins are known to bind CBFβ. In addition, the Runt domain of the RUNX proteins, to which CBFβ binds, is almost identical in sequence among the family members and no differences are found on the surface of the Runt domain which interacts with CBFβ. Based on this, it is our expectation that similar effects will be seen for these inhibitors on CBFβ-RUNX2 and CBFβ-RUNX3 binding. As such, the biological effects observed likely reflect activity against all the RUNX proteins present in a particular cell type. These compounds meet the criteria outlined by Frye for a high quality chemical probe (Frye, 2010), including a clear molecular profile of activity, mechanism of action, identity of active species, and proven utility.