Irene M. Pedersen

A unified model for apical caspase activation.

Apoptosis is orchestrated by the concerted action of caspases, activated in a minimal two-step proteolytic cascade. Existing data suggests that apical caspases are activated by adaptor-mediated clustering of inactive zymogens. However, the mechanism by which apical caspases achieve catalytic competence in their recruitment/activation complexes remains unresolved. We explain that proximity-induced activation of apical caspases is attributable to dimerization. Internal proteolysis does not activate these apical caspases but is a secondary event resulting in partial stabilization of activated dimers. Activation of caspases-8 and -9 occurs by dimerization that is fully recapitulated in vitro by kosmotropes, salts with the ability to stabilize the structure of proteins. Further, single amino acid substitutions at the dimer interface abrogate the activity of caspases-8 and -9 introduced into recipient mammalian cells. We propose a unified caspase activation hypothesis whereby apical caspases are activated by dimerization of monomeric zymogens.

Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity

Apoptosis resistance commonly occurs in cancers, preventing activation of Caspase family cell death proteases. XIAP is an endogenous inhibitor of Caspases overexpressed in many cancers. We developed an enzyme derepression assay, based on overcoming XIAP-mediated suppression of Caspase-3, and screened mixture-based combinatorial chemical libraries for compounds that reversed XIAP-mediated inhibition of Caspase-3, identifying a class of polyphenylureas with XIAP-inhibitory activity. These compounds, but not inactive structural analogs, stimulated increases in Caspase activity, directly induced apoptosis of many types of tumor cell lines in culture, and sensitized cancer cells to chemotherapeutic drugs. Active compounds also suppressed growth of established tumors in xenograft models in mice, while displaying little toxicity to normal tissues. These findings validate IAPs as targets for cancer drug discovery.

Src homology 2 domain–containing inositol-5-phosphatase and CCAAT enhancer-binding protein β are targeted by miR-155 in B cells of Eμ-MiR-155 transgenic mice

We showed that Emicro-MiR-155 transgenic mice develop acute lymphoblastic leukemia/high-grade lymphoma. Most of these leukemias start at approximately 9 months irrespective of the mouse strain. They are preceded by a polyclonal pre-B-cell proliferation, have variable clinical presentation, are transplantable, and develop oligo/monoclonal expansion. In this study, we show that in these transgenic mice the B-cell precursors have the highest MiR-155 transgene expression and are at the origin of the leukemias. We determine that Src homology 2 domain-containing inositol-5-phosphatase (SHIP) and CCAAT enhancer-binding protein beta (C/EBPbeta), 2 important regulators of the interleukin-6 signaling pathway, are direct targets of MiR-155 and become gradually more down-regulated in the leukemic than in the preleukemic mice. We hypothesize that miR-155, by down-modulating Ship and C/EBPbeta, initiates a chain of events that leads to the accumulation of large pre-B cells and acute lymphoblastic leukemia/high-grade lymphoma.

Onco-miR-155 targets SHIP1 to promote TNFα-dependent growth of B cell lymphomas

Non‐coding microRNAs (miRs) are a vital component of post‐transcriptional modulation of protein expression and, like coding mRNAs harbour oncogenic properties. However, the mechanisms governing miR expression and the identity of the affected transcripts remain poorly understood. Here we identify the inositol phosphatase SHIP1 as a bonafide target of the oncogenic miR‐155. We demonstrate that in diffuse large B cell lymphoma (DLBCL) elevated levels of miR‐155, and consequent diminished SHIP1 expression are the result of autocrine stimulation by the pro‐inflammatory cytokine tumour necrosis factor α (TNFα). Anti‐TNFα regimen such as eternacept or infliximab were sufficient to reduce miR‐155 levels and restored SHIP1 expression in DLBCL cells with an accompanying reduction in cell proliferation. Furthermore, we observed a substantial decrease in tumour burden in DLBCL xenografts in response to eternacept. These findings strongly support the concept that cytokine‐regulated miRs can function as a crucial link between inflammation and cancer, and illustrate the feasibility of anti‐TNFα therapy as a novel and immediately accessible (co)treatment for DLBCL.

Dysregulation of apoptosis genes in hematopoietic malignancies

Ever since the discovery of Bcl-2 and the elucidation of its role in apoptosis, tremendous interest has arisen in prospects for triggering suicide of malignant cells by exploiting knowledge emerging from apoptosis research. In this review, we summarize information about the multiple genetic lesions which have been identified in apoptosis-regulatory genes of hematopoietic and lymphoid neoplasms. Emerging data about the structural and biochemical details of apoptosis proteins and their upstream regulators have reveal novel strategies for therapeutic intervention, some of which are under interrogation in clinical trials currently.

Microenvironmental interactions and survival of CLL B-cells.

Chronic Lymphocytic Leukemia (CLL) B cells display characteristics consistent with a defect in programmed cell death (apoptosis) and exhibit prolonged survival in vivo. When recovered from peripheral blood or lymphoid tissues from the patient and cultured in vitro, these malignant cells rapidly undergo spontaneous apoptosis. This observation suggests that the selective survival advantage enjoyed by CLL B-cells is not entirely autonomous, raising the possibility of manipulating CLL B-cell survival by iatrogenic means. The extended survival of the neoplastic B-cells creates a permissive soil on which oncogene activation, genetic instability and accumulation of gene mutations favoring disease progression can occur. In addition, such survival-promoting microenvironments can rescue leukemia cells from cytotoxic therapy, giving way to disease relapse. Survival of CLL B-cells is influenced by interactions with non-leukemia cells in the microenvironment of lymph nodes, marrow and other tissues. CLL B-cells have developed many different ways to escape undergoing apoptosis. These include: (a) expression of survival receptor as well as their ligands, giving rise to autocrine survival pathways which are leukemia cell specific; (b) defects in plasma membrane receptor cell signaling, triggered by death receptors such as Fas- and TRAIL; and (c) constitutively active survival signaling pathways such as NFκB and PI3K/Akt. Here we discuss some of the molecular mechanisms by which interaction with other cells and factors in the microenvironment provides survival advantages to CLL B-cells in specific in vivo niches, and we suggest some strategies for overcoming these anti-apoptotic mechanisms for improving treatment of CLL.

MicroRNAs in the Immune Response

MicroRNAs (miRs) were only discovered little more than a decade ago, yet it has become rapidly clear that they are crucial posttranscriptional regulators of gene expression by decreasing the abundance or translational efficiency of mRNAs [Maroney PA, Yu Y, Nilsen TW. MicroRNAs, mRNAs, and translation. Cold Spring Harb Symp Quant Biol 2006;71: 531-5; Nilsen TW. Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet 2007;23: 243-9], [1,2]. While the role of miRs in cell fate decisions linked to proliferation, differentiation and apoptosis was recognized early on, the importance of these noncoding small RNAs on immune system development and response has only recently become evident. In addition to facilitating cell fate decisions of immune cells (e.g. miR-181a and miR-223), miRs also regulate central elements of the adaptive immune response such as antigen presentation (e.g. miR-155) and T cell receptor signaling (mir-181a). Furthermore, miRs are involved in innate immunity through regulation of Toll-like receptor signaling and cytokine responses (e.g. miR-146). Intriguingly, cellular miRs not only alter immune cell development and function, but are also able to directly affect viral replication. Conversely, virus-encoded miRs shape the host-virus interactions and regulate the viral life cycle. Here, we provide a brief overview on the role of cellular and viral miRs in the development and function of the immune system.

Latent sensitivity to Fas-mediated apoptosis after CD40 ligation may explain activity of CD154 gene therapy in chronic lymphocytic leukemia

Patients with chronic lymphocytic leukemia (CLL) treated with adenovirus (Ad)-CD154 (CD40L) gene therapy experience reductions in leukemia cell counts and lymph node size associated with induction of the death receptor Fas (CD95). CD4 T cell lines can induce apoptosis of CD40-activated CLL cells via a CD95 ligand (CD95-L)-dependent mechanism. To examine whether CD95-L was sufficient to induce cytolysis of CD40-activated CLL cells, we used Chinese hamster ovary cells transfected with CD95-L as cytotoxic effector cells. CD40-activated CLL cells were initially resistant to CD95-mediated apoptosis despite high-level expression of CD95. However, after 72 h, CLL cells from seven of seven patients became increasingly sensitive to CD95-mediated apoptosis. This sensitivity correlated with a progressive decline in Flice-inhibitory protein (FLIP), which was induced within 24 h of CD40 ligation. Down-regulation of FLIP with an antisense oligonucleotide or a pharmacologic agent, however, was not sufficient to render CLL cells sensitive to CD95-mediated apoptosis in the 24–72 h after CD40 activation. Although the levels of pro-Caspase-8 appeared sufficient, inadequate levels of Fas-associated death domain protein (FADD) and DAP3 may preclude assembly of the death-inducing signaling complex. Seventy-two hours after CD40 ligation, sensitivity to CD95 and a progressive increase in FADD and DAP3 were associated with the acquired ability of FADD and FLIP to coimmunoprecipitate with the death-inducing signaling complex after CD95 ligation. Collectively, these studies reveal that CD40 ligation on CLL B cells induces a programmed series of events in which the cells initially are protected and then sensitized to CD95-mediated apoptosis through shifts in the balance of the anti- and proapoptotic proteins FLIP and FADD.

MiR-128 represses L1 retrotransposition by binding directly to L1 RNA

Long interspersed element 1 (LINE-1 or L1) retrotransposons compose 17% of the human genome. Active L1 elements are capable of replicative transposition (mobilization) and can act as drivers of genetic diversity. However, this mobilization is mutagenic and may be detrimental to the host, and therefore it is under strict control. Somatic cells usually silence L1 activity by DNA methylation of the L1 promoter. In hypomethylated cells, such as cancer cells and induced pluripotent stem cells (iPSCs), a window of opportunity for L1 reactivation emerges, and with it comes an increased risk of genomic instability and tumorigenesis. Here we show that miR-128 represses new retrotransposition events in human cancer cells and iPSCs by binding directly to L1 RNA. Thus, we have identified and characterized a new function of microRNAs: mediating genomic stability by suppressing the mobility of endogenous retrotransposons.

Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.

SAMHD1 is a phosphohydrolase maintaining cellular dNTP homeostasis but also acts as a critical regulator in innate immune responses due to its antiviral activity and association with autoimmune disease, leading to aberrant activation of interferon. SAMHD1 expression is differentially regulated by interferon in certain primary cells, but the underlying mechanism is not understood. Here, we report a detailed characterization of the promotor region, the 5′- and 3′-untranslated region (UTR) of SAMHD1, and the mechanism responsible for the cell type-dependent up-regulation of SAMHD1 protein by interferon. We demonstrate that induction of SAMHD1 by type I and II interferons depends on 3′-UTR post-transcriptional regulation, whereas the promoter drives basal expression levels. We reveal novel functional target sites for the microRNAs miR-181a, miR-30a, and miR-155 in the SAMHD1 3′-UTR. Furthermore, we demonstrate that down-regulation of endogenous miR-181a and miR-30a levels inversely correlates with SAMHD1 protein up-regulation upon type I and II interferon stimulation in primary human monocytes. These miRNAs are not modulated by interferon in macrophages or dendritic cells, and consequently protein levels of SAMHD1 remain unchanged. These results suggest that SAMHD1 is a non-classical interferon-stimulated gene regulated through cell type-dependent down-regulation of miR-181a and miR-30a in innate sentinel cells.