No abstract

Activation-induced cytidine deaminase (AID) initiates antibody diversification by mutating immunoglobulin loci in B lymphocytes. AID and related APOBEC3 (A3) enzymes also induce genome-wide mutations and lesions implicated in tumorigenesis and tumor progression. The most prevalent mutation signatures across diverse tumor genomes are attributable to the mistargeted mutagenic activities of AID/A3s. Thus, inhibiting AID/A3s has been suggested to be of therapeutic benefit. We previously used a computationalbiochemical approach to gain insight into the structure of AID's catalytic pocket, which resulted in the discovery of a novel type of regulatory catalytic pocket closure that regulates AID/A3s that we termed the "Schrodinger's CATalytic pocket". Our findings were subsequently confirmed by direct structural studies. Here, we describe our search for small molecules that target the catalytic pocket of AID. We identified small molecules that inhibit purified AID, AID in cell extracts, and endogenous AID of lymphoma cells. Analogue expansion yielded derivatives with improved potencies. These were found to also inhibit A3A and A3B, the two most tumorigenic siblings of AID. Two compounds exhibit low micromolar IC 50 inhibition of AID and A3A, exhibiting the strongest potency for A3A. Docking suggests key interactions between their warheads and residues lining the catalytic pockets of AID, A3A, and A3B and between the tails and DNAinteracting residues on the surface proximal to the catalytic pocket opening. Accordingly, mutants of these residues decreased inhibition potency. The chemistry and abundance of key stabilizing interactions between the small molecules and residues within and immediately outside the catalytic pockets are promising for therapeutic development.

Aberrant expression of Ecotropic Viral Integration Site 1 (EVI1) is a hallmark of acute myeloid leukemia (AML) with inv(3) or t(3;3), which is a disease subtype with especially poor outcome. In studying transcriptomes from AML patients with chromosome 3q rearrangements, we identified a significant upregulation of the Nuclear Receptor Interacting Protein 1 (NRIP1) as well as its adjacent non-coding RNA LOC101927745. Utilizing transcriptomic and epigenomic data from over 900 primary patient samples as well as genetic and transcriptional engineering approaches, we have identified several mechanisms that can lead to upregulation of NRIP1 in AML. We hypothesize that the LOC101927745 transcription start site harbors a context-dependent enhancer that is bound by EVI1, causing upregulation of NRIP1 in AML with chr3 abnormalities. Furthermore, we show that NRIP1 knockdown negatively affects the proliferation and survival of 3q-rearranged AML cells and increases their sensitivity towards ATRA, suggesting that NRIP1 is relevant for the pathogenesis of inv(3)/t(3;3) AML and could serve as a novel therapeutic target in myeloid malignancies with 3q abnormalities.