MCL-1 is a prognostic indicator and drug target in breast cancer

Analysis of publicly available genomic and gene expression data demonstrates that MCL1 expression is frequently elevated in breast cancer. Distinct from other pro-survival Bcl-2 family members, the short half-life of MCL-1 protein led us to investigate MCL-1 protein expression in a breast cancer tissue microarray and correlate this with clinical data. Here, we report associations between high MCL-1 and poor prognosis in specific subtypes of breast cancer including triple-negative breast cancer, an aggressive form that lacks targeted treatment options. Deletion of MCL-1 in the mammary epithelium of genetically engineered mice revealed an absolute requirement for MCL-1 in breast tumorigenesis. The clinical applicability of these findings was tested through a combination of approaches including knock-down or inhibition of MCL-1 to show triple-negative breast cancer cell line dependence on MCL-1 in vitro and in vivo. Our data demonstrate that high MCL-1 protein expression is associated with poor outcome in breast cancer and support the therapeutic targeting of MCL-1 in this disease

Dysregulation of Hepatitis B Virus Nucleocapsid Assembly in vitro by RNA-binding Small Ligands

RNA sequences/motifs dispersed across the genome of Hepatitis B Virus regulate formation of nucleocapsid-like particles (NCPs) by core protein (Cp) in vitro, in an epsilon/polymerase-independent fashion. These multiple RNA Packaging Signals (PSs) can each form stem-loops encompassing a Cp-recognition motif, -RGAG-, in their loops. Drug-like molecules that bind the most important of these PS sites for NCP assembly regulation with nanomolar affinities, were identified by screening an immobilized ligand library with a fluorescently-labelled, RNA oligonucleotide encompassing this sequence. Sixty-six of these "hits", with affinities ranging from low nanomolar to high micromolar, were purchased as non-immobilized versions. Their affinities for PSs and effects on NCP assembly were determined in vitro by Surface Plasmon Resonance. High-affinity ligand binding is dependent on the presence of an -RGAG-motif within the loop of the PS, consistent with ligand cross-binding between PS sites. Simple structure−activity relationships show that it is also dependent on the presence of specific functional groups in these ligands. Some compounds are potent inhibitors of in vitro NCP assembly at nanomolar concentrations. Despite appropriate logP values, these ligands do not inhibit HBV replication in cell culture. However, modelling confirms the potential of using PS-binding ligands to target NCP assembly as a novel antiviral strategy. This also allows for computational exploration of potential synergic effects between antiviral ligands directed at distinct molecular targets in vivo. HBV PS-regulated assembly can be dysregulated by novel small molecule RNA-binding ligands opening a novel target for developing directly-acting anti-virals against this major pathogen

Selective Small-Molecule Targeting of a Triple Helix Encoded by the Long Noncoding RNA, MALAT1

Metastasis-associated lung adenocarcinoma transcript 1 (Malat1/MALAT1, mouse/human), a highly conserved long noncoding (lnc) RNA, has been linked with several physiological processes, including the alternative splicing, nuclear organization, and epigenetic modulation of gene expression. MALAT1 has also been implicated in metastasis and tumor proliferation in multiple cancer types. The 3′ terminal stability element for nuclear expression (ENE) assumes a triple-helical configuration that promotes its nuclear accumulation and persistent function. Utilizing a novel small molecule microarray strategy, we identified multiple Malat1 ENE triplex-binding chemotypes, among which compounds 5 and 16 reduced Malat1 RNA levels and branching morphogenesis in a mammary tumor organoid model. Computational modeling and Förster resonance energy transfer experiments demonstrate distinct binding modes for each chemotype, conferring opposing structural changes to the triplex. Compound 5 modulates Malat1 downstream genes without affecting Neat1, a nuclear lncRNA encoded in the same chromosomal region as Malat1 with a structurally similar ENE triplex. Supporting this observation, the specificity of compound 5 for Malat1 over Neat1 and a virus-coded ENE was demonstrated by nuclear magnetic resonance spectroscopy. Small molecules specifically targeting the MALAT1 ENE triplex lay the foundation for new classes of anticancer therapeutics and molecular probes for the treatment and investigation of MALAT1-driven cancers. © 2019 American Chemical Society

Development of Small Molecules with a Noncanonical Binding Mode to HIV‑1 Trans Activation Response (TAR) RNA

Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemistry of targeting RNA. Here we report the synthesis, biochemical, and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue, <b>15</b>, retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC₅₀ of 40 μM. NMR characterization of a soluble analogue, <b>2</b>, revealed a noncanonical binding mode for this class of compounds. Finally, evaluation of <b>2</b> and <b>15</b> by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5′-untranslated region (UTR). Thus, these compounds exhibit a novel and specific mode of interaction with TAR, providing important suggestions for RNA ligand design

MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies

Overexpression of anti-apoptotic BCL-2 family members is a hallmark of many lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) that can be targeted with small molecule inhibitors. ABT-199 is a rationally designed BCL-2 homology (BH)-3 mimetic that specifically binds to BCL-2, but not to MCL-1 and BCL-xL. Although the thrombocytopenia that occurs with navitoclax treatment has not been a problem with ABT-199, clinical trials in CLL could benefit by lowering the ABT-199 concentration through targeting other survival pathways. In this study, we investigated the mechanisms of resistance that develops to ABT-199 therapy by generating ABT-199-resistant (ABT199-R) cell lines via chronic exposure of NHL cell lines to ABT-199. Acquired resistance resulted in substantial AKT activation and upregulation of MCL-1 and BCL-xL levels that sequestered BIM. ABT199-R cells exhibited increased MCL-1 stability and failed to activate BAX in response to ABT-199. The ABT-199 acquired and inherent resistant cells were sensitized to treatment with ABT-199 by inhibitors of the PI3K, AKT, and mTOR pathways, NVP-BEZ235 and GS-1101. NVP-BEZ235, a dual inhibitor of p-AKT and mTOR, reduced MCL-1 levels causing BIM release from MCL-1 and BCL-xL, thus leading to cell death by BAX activation. The PI3Kδ inhibitor GS-1101 (idelalisib) downregulated MCL-1 and sensitized ABT199-R cells through AKT-mediated BAX activation. A genetic approach, through siRNA-mediated down-regulation of AKT, MCL-1, and BCL-xL, significantly decreased cell survival, demonstrating the importance of these cell survival factors for ABT-199 resistance. Our findings suggest a novel mechanism that modulates the expression and activity of pro-survival proteins to confer treatment resistance that could be exploited by a rational combination therapeutic regimen that could be effective for treating lymphoid malignancies