Profiling the specificity of SPI1 and SPIB activity for drug discovery

PROJECT SUMMARY/ABSTRACT: Profiling the specificity of SPI1 and SPIB activity for drug discovery Diseases involving cells of hematopoietic origin are extensively involved in rare diseases, defined as affecting 200,000 or fewer people in the U.S. SPI1 (PU.1) and SPIB (Spi-B) are two master transcription factors in the hematopoietic system that are deregulated in a broad spectrum of rare diseases, including: leukemias, lymphomas, myeloma, histiocytic sarcomas, rheumatic diseases, systemic fibroses, inborn errors of immunity, and osteopetrosis. The broad relevance of PU.1 and Spi-B to rare diseases makes them particularly attractive therapeutic targets. As members of the Class III ETS superfamily of transcription factors, PU.1 and Spi-B are structurally homologous proteins despite their functional and pathophysiologic differentiation. As progress by our group and others is being made in asserting pharmacologic control over PU.1, it is apt to consider strategies by which structurally conserved transcription factors may be distinguished in drug discovery. To this end, we have discovered that low-MW ligands can modulate PU.1 binding efficiently outside the orthosteric protein/DNA interface. Such allosteric modulation opens the possibility that a transcription factor may be selected with a ligand at a non-orthosteric site that is critical for cooperative DNA-binding with or recruitment of a distinct protein binding partner. In this novel hypothesis, the targeted unit is a more precisely specified combinatorial complex, rather than a single constituent that engages DNA in a conserved fashion. From the literature, partnerships with Oct-2 (POU2F2) and interferon regulatory factor IRF4/8 (IRF4/IRF8) are known from ChIP-Seq studies to attend PU.1- and SpiB-specific occupancy in the genome. Functionally, PU.1/Oct-2 and Spi-B/IRF partnerships are associated with rare diseases such as the auto-immune disorders mediated by microRNA-155 (e.g., idiopathic arthritis) and Williams syndrome (a rare congenital multi-system disorder), respectively. The molecular specification of these disease-relevant complexes by the two structurally homologous ETS factors are currently not understood. Moreover, we lack experimental tools capable of distinguishing PU.1- and SpiB-selective binding in cells. This pilot project is aimed at addressing these gaps in order to test the novel hypothesis that PU.1 and Spi-B can be selectively targeted as the PU.1/Oct-2 and Spi- B/IRF4(8) complexes respectively. We propose to characterize the structure, affinity, and cooperative properties of these complexes, and develop a reporter platform capable of detecting selective PU.1 and Spi-B activity in cells. The results will support future proposals aimed at discovering new compounds that target PU.1 and Spi-B with specificity, including library screening for ligands to serve as probes or therapeutic leads in rare diseases. The prospect of effectively targeting PU.1 and Spi-B would impact a broad spectrum of rare diseases and presents a strong value proposition over single-indication targets for this RFA.