Yoon, Hyejin

Abstract Waldenströms Macroglobulinemia (WM) is a unique form of lymphoplasmacytic lymphoma, in which the tumor compartment is comprised of B-lymphocytes, plasmacytoid lymphocytes and plasma cells. The majority of WM cases are immunophenotypically characterized by expression of surface antigens that are present on both B-cells (CD19/20) and plasma cells (CD38/138). However, in advanced stage disease, which has been treated with various chemoimmunotherapeutics the molecular profile of WM cells can drastically shift resulting in a majority of the WM tumor compartment exhibiting loss of CD20 and concurrent upregulation of atypical surface antigens (Barakat et al, Am J Clin Pathol 2011; Morice et al Mod Pathol 2009). These external immunophenotypic changes are associated with underlying epigenetic and genomic alterations, whose integrated study may provide further insight into the biology of advanced stage WM. Moreover, such an analysis holds clinical potential by uncovering of therapeutic vulnerabilities, which can direct the selection of appropriate therapeutics for patients with CD20- WM. With this in mind, we conducted comprehensive molecular testing coupled with functional studies to understand if underlying genomic/epigenetic changes correlated with drug sensitivity using appropriate preclinical models of CD20+ WM (BCWM.1 cell line) and CD20- WM (RPCI-WM1 cell line). Importantly, the index patient from whom the CD20- cells were developed had received a total of 6 lines of treatment, two of which contained rituximab and was documented as being refractory to rituximab. Immunophenotyping of CD20- cells showed differential downregulation of 19 and upregulation of 7 CD antigens relative to CD20+ WM cells (cutoff at 1.5 fold; >20% gated expression). Upregulated antigens were associated with a phenotype reminiscent of a memory B-cell with plasmacytoid features. Next, we conducted RNA-Seq analysis (Illumina HiSeq), which revealed downregulation of 6,673 and upregulation of 4,594 genes (>1.5 fold cutoff) in CD20- WM cells compared with CD20+ cells. Gene pathway enrichment analysis (NextBio, Illumina Inc.) of mRNA differentially expressed in CD20- cells showed significant enrichment of upregulated genes corresponding to Protein Transport (p<1.8E-98) and Response to Unfolded Protein Gene Ontology groups (p<5.9E-20). Interestingly, pathway analysis of downregulated genes in CD20- WM cells showed enrichment within the B-cell receptor-signaling (BCR) pathway (Broad MSigDB) (p<6.1E-14) relative to CD20+ WM cells. BCR signaling has been linked to dysregulated expression of several micro-RNAs (miRs). Consistent with pathway analysis results, we observed decreased levels of numerous miRs (Exiqon qPCR) in CD20- vs. CD20+ WM cells, which target and regulate components involved in BCR activation. To examine further epigenetic changes that may account for decreased BCR signaling, we examined the global methylation profile (Infinium HumanMethylation 450K array) of CD20- and CD20+ WM cells and noted hypermethylation of the promoter sites (200 base pairs upstream) for a number of transcription factors involved in the BCR pathway. We therefore examined sensitivity of CD20- WM cells to the BTK inhibitor ibrutinib, which abrogates BCR signaling. As anticipated, CD20- WM cells retained >90% viability and demonstrated significantly less apoptosis in presence of ibrutinib (4uM) compared to CD20+ cells, suggesting decreased reliance on canonical BCR signaling for survival. In parallel and guided by pathway analysis results, we exposed both CD20- and CD20+ WM cells to a novel 19S proteasome inhibitor (VLX1570), which caused profound proteotoxicity and ER stress at concentrations ranging between 250 – 500nM. We noted CD20- cells were significantly more sensitive (p<0.01) to 19S proteasome inhibition than CD20+ WM cells indicating the formers critical dependence on tightly regulated protein homeostasis. In summary we describe for the first time, a comprehensive molecular and immunophenotypic investigation of CD20- WM cells compared to CD20+ WM cells, using validated preclinical models of the two disease phenotypes. Overall our data suggests significant architectural differences between CD20+ and CD20- WM, which can potentially impact the behavior of the cells and their sensitivity towards novel therapeutics being developed for treatment of advanced stage WM. Disclosures Martin: Janssen: Honoraria.

Purpose of review APOE4 genotype is the strongest genetic risk factor for Alzheimer's disease. Prevailing evidence suggests that amyloid β plays a critical role in Alzheimer's disease. The objective of this article is to review the recent findings about the metabolism of apolipoprotein E (ApoE) and amyloid β and other possible mechanisms by which ApoE contributes to the pathogenesis of Alzheimer's disease. Recent findings ApoE isoforms have differential effects on amyloid β metabolism. Recent studies demonstrated that ApoE-interacting proteins, such as ATP-binding cassette A1 (ABCA1) and LDL receptor, may be promising therapeutic targets for Alzheimer's disease treatment. Activation of liver X receptor and retinoid X receptor pathway induces ABCA1 and other genes, leading to amyloid β clearance. Inhibition of the negative regulators of ABCA1, such as microRNA-33, also induces ABCA1 and decreases the levels of ApoE and amyloid β. In addition, genetic inactivation of an E3 ubiquitin ligase, myosin regulatory light chain interacting protein, increases LDL receptor levels and inhibits amyloid accumulation. Although amyloid β-dependent pathways have been extensively investigated, there have been several recent studies linking ApoE with vascular function, neuroinflammation, metabolism, synaptic plasticity, and transcriptional regulation. For example, ApoE was identified as a ligand for a microglial receptor, TREM2, and studies suggested that ApoE may affect the TREM2-mediated microglial phagocytosis. Summary Emerging data suggest that ApoE affects several amyloid β-independent pathways. These underexplored pathways may provide new insights into Alzheimer's disease pathogenesis. However, it will be important to determine to what extent each mechanism contributes to the pathogenesis of Alzheimer's disease.

AbstractAccumulation of amyloid β (Aβ) in the brain is a key pathological hallmark of Alzheimer's disease (AD). Because aging is the most prominent risk factor for AD, understanding the molecular changes during aging is likely to provide critical insights into AD pathogenesis. However, studies on the role of miRNAs in aging and AD pathogenesis have only recently been initiated. Identifying miRNAs dysregulated by the aging process in the brain may lead to novel understanding of molecular mechanisms of AD pathogenesis. Here, we identified that miR‐186 levels are gradually decreased in cortices of mouse brains during aging. In addition, we demonstrated that miR‐186 suppresses β‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) expression by directly targeting the 3′UTR of Bace1 mRNA in neuronal cells. In contrast, inhibition of endogenous miR‐186 significantly increased BACE1 levels in neuronal cells. Importantly, miR‐186 over‐expression significantly decreased Aβ level by suppressing BACE1 expression in cells expressing human pathogenic mutant amyloid precursor protein. Taken together, our data demonstrate that miR‐186 is a potent negative regulator of BACE1 in neuronal cells and it may be one of the molecular links between brain aging and the increased risk for AD during aging. image We identified that miR‐186 levels are gradually decreased in mouse cortices during aging. Furthermore, we demonstrated that miR‐186 is a novel negative regulator of beta‐site amyloid precursor protein‐cleaving enzyme 1 (BACE1) expression in neuronal cells. Therefore, we proposed that reduction in miR‐186 levels during aging may lead to the up‐regulation of BACE1 in the brain, thereby increasing a risk for Alzheimer's disease in aged individuals. Read the Editorial Highlight for this article on page 308.