PROJECT SUMMARY/ABSTRACT Histone deacetylases (HAT) are zinc-dependent enzymes that catalyze the removal of acetyl groups from the epsilon amine of lysine side chains. These proteins have been widely pursued as anti-cancer drug targets, but therapeutic development has been largely unsuccessful given the essential nature of many HDAC proteins. Recently, we discovered a synthetic lethal relationship between HDAC1 and HDAC2, which is caused by recurrent chromosomal deletions that result in hemizygous deletion of HDAC1 in neuroblastoma and HDAC2 in multiple myeloma. As a result of HDAC1 deletion, neuroblastoma cells are hypersensitive to disruption of HDAC2, and vice versa in multiple myeloma. Using dTAG-mediated degradation or CRISPR/Cas9-based gene disruption, we discovered that targeting HDAC1/2 synthetic lethality (e.g. degrading HDAC2 in neuroblastoma cells with a hemizygous HDAC1 deletion) results in dissociation of the NuRD chromatin remodeler complex, of which HDAC1/2 are members. Dissociation of the complex results in degradation of NuRD subunits that are selectively required for neuroblastoma and multiple myeloma survival, suggesting that HDAC1/2 synthetic lethality can be leveraged to target subunit-specific NuRD vulnerabilities in cancer. We hypothesize that HDAC1 deletions cause the NuRD subunits, HDAC2 / MBD3 / MTA3, to be essential for neuroblastoma, whereas HDAC2 deletions cause vulnerabilities to loss of their paralogs, HDAC1 / MBD2 / MTA2, in multiple myeloma. Here, we will address this hypothesis and explore the translational potential of these vulnerabilities by developing small- molecule modulators that target NuRD structure and/or function. In Aim 1, we will (i) Determine whether MBD and MTA vulnerabilities are caused by HDAC1/2 deletions using CRISPR/Cas9, inducible RNAi, and dTAG- based approaches in vitro and in vivo, (ii) Reveal whether the loss of NuRD subunits required for cancer cell survival leads to dissociation and/or degradation of the NuRD complex using unbiased proteomics approaches, and (iii) Establish if HDAC1/MBD2/MTA2 and HDAC2/MBD3/MTA3 form distinct NuRD sub-complexes as a result of HDAC2 and HDAC1 deletions, respectively. These experiments will determine if subunit-specific NuRD vulnerabilities are caused by HDAC1/2 deletions or simply exploited by HDAC1/2 synthetic lethality. In Aim 2, we will develop small molecules targeting the NuRD complex to exploit NuRD vulnerabilities in genetically defined cancer sub-types. Specifically, we will: (i) develop paralog-selective PROTACs that distinguish between HDAC1 and HDAC2, (ii) determine the potential for covalent ligands of MTA3-Cys532 to disrupt NuRD structure and/or function in MTA3-dependent cancers, and (iii) develop MTA3-targeted PROTACs based on ligands that covalently engage MTA3-C532. Altogether, successful completion of these aims will determine the mechanisms underlying NuRD vulnerabilities in cancer and advance novel chemical tools to drug and study the...