Sebastien Didier

Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation.

Chronic lymphocytic leukemia (CLL) represents the outgrowth of a CD5+ B cell. Its etiology is unknown. The structure of membrane Ig on CLL cells of unrelated patients can be remarkably similar. Therefore, antigen binding and stimulation could contribute to clonal selection and expansion as well as disease promotion. Initial studies suggest that CLL mAbs bind autoantigens. Since apoptosis can make autoantigens accessible for recognition by antibodies, and also create neo-epitopes by chemical modifications occurring naturally during this process, we sought to determine if CLL mAbs recognize autoantigens associated with apoptosis. In general, ~60% of CLL mAbs bound the surfaces of apoptotic cells, were polyreactive, and expressed unmutated IGHV. mAbs recognized two types of antigens: native molecules located within healthy cells, which relocated to the external cell surface during apoptosis; and/or neoantigens, generated by oxidation during the apoptotic process. Some of the latter epitopes are similar to those on bacteria and other microbes. Although most of the reactive mAbs were not mutated, the use of unmutated IGHV did not bestow autoreactivity automatically, since several such mAbs were not reactive. Particular IGHV and IGHV/D/J rearrangements contributed to autoantigen binding, although the presence and degree of reactivity varied based on specific structural elements. Thus, clonal expansion in CLL may be stimulated by autoantigens occurring naturally during apoptosis. These data suggest that CLL may derive from normal B cells whose function is to remove cellular debris, and also to provide a first line of defense against pathogens.

Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin

Many B-cell chronic lymphocytic leukemia (CLL) monoclonal antibodies (mAbs) can be grouped into subsets based on nearly identical stereotyped sequences. Subset 6 CLL mAbs recognize nonmuscle myosin heavy chain IIA (MYHIIA). Herein, we report that during apoptosis, MYHIIA becomes exposed on the cell surface of a subgroup of apoptotic cells, allowing subset 6 CLL mAbs to bind with it. Because other non-subset 6 CLL mAbs interact with apoptotic cells, 26 CLL mAbs, including 24 not belonging to subset 6, were tested for reactivity with MYHIIA-exposed apoptotic cells (MEACs). More than 60% of CLL mAbs bound MEACs well; most of these mAbs expressed unmutated IGHV (15 of 16) and belonged to a stereotyped subset (14 of 16). Binding to MEACs inversely correlated with the degree of IGHV mutation. Interestingly, high binding to MEACs significantly correlated with poor patient survival, suggesting that the basis of IGHV mutation status as a CLL prognostic factor reflects antigen binding. Finally, natural antibodies from human serum also reacted with MEACs. Taken together, our data indicate that a large proportion of CLL clones emerge from natural antibody-producing cells expressing immunoglobulins that recognize MEACs, and that this reactivity is associated with poor clinical outcome.

The small GTPase RhoG mediates glioblastoma cell invasion.

BackgroundThe invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.ResultsWe found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.ConclusionsOur functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.

Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors

Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with β-hemoglobinopathies.

AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo.

Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer’s disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology.

Torque Teno Virus 10 Isolated by Genome Amplification Techniques from a Patient with Concomitant Chronic Lymphocytic Leukemia and Polycythemia Vera

An infectious etiology has been proposed for many human cancers, but rarely have specific agents been identified. One difficulty has been the need to propagate cancer cells in vitro to produce the infectious agent in detectable quantity. We hypothesized that genome amplification from small numbers of cells could be adapted to circumvent this difficulty. A patient with concomitant chronic lymphocytic leukemia (CLL) and polycythemia vera (PV) requiring therapeutic phlebotomy donated a large amount of phlebotomized blood to test this possibility. Using genome amplification methods, we identified a new isolate (BIS8-17) of torque teno virus (TTV) 10. The presence of blood isolate sequence 8–17 (BIS8-17) in the original plasma was confirmed by polymerase chain reaction (PCR), validating the approach, since TTV is a known plasma virus. Subsequent PCR testing of plasmas from additional patients showed that BIS8-17 had a similar incidence (~20%) in CLL (n = 48) or PV (n = 10) compared with healthy controls (n = 52). CLL cells do not harbor BIS8-17; PCR did not detect it in CLL peripheral blood genomic deoxyribonucleic acid (DNA) (n = 20). CLL patient clinical outcome or prognostic markers (immunoglobulin heavy chain variable region [IGHV] mutation, CD38 or zeta-chain associated protein kinase 70kDa [ZAP-70]) did not correlate with BIS8-17 infection. Although not causative to our knowledge, this is the first reported isolation and detection of TTV in either CLL or PV. TTV could serve as a covirus with another infectious agent or TTV variant with rearranged genetic components that contribute to disease pathogenesis. These results prove that this method identifies infectious agents and provides an experimental methodology to test correlation with disease.

Semapimod Sensitizes Glioblastoma Tumors to Ionizing Radiation by Targeting Microglia

Glioblastoma is the most malignant and lethal form of astrocytoma, with patients having a median survival time of approximately 15 months with current therapeutic modalities. It is therefore important to identify novel therapeutics. There is mounting evidence that microglia (specialized brain-resident macrophages) play a significant role in the development and progression of glioblastoma tumors. In this paper we show that microglia, in addition to stimulating glioblastoma cell invasion, also promote glioblastoma cell proliferation and resistance to ionizing radiation in vitro. We found that semapimod, a drug that selectively interferes with the function of macrophages and microglia, potently inhibits microglia-stimulated GL261 invasion, without affecting serum-stimulated glioblastoma cell invasion. Semapimod also inhibits microglia-stimulated resistance of glioblastoma cells to radiation, but has no significant effect on microglia-stimulated glioblastoma cell proliferation. We also found that intracranially administered semapimod strongly increases the survival of GL261 tumor-bearing animals in combination with radiation, but has no significant benefit in the absence of radiation. In conclusion, our observations indicate that semapimod sensitizes glioblastoma tumors to ionizing radiation by targeting microglia and/or infiltrating macrophages.

AMP-activated protein kinase is essential for the maintenance of energy levels during synaptic activation

Summary Although the brain accounts for only 2% of the total body mass, it consumes the most energy. Neuronal metabolism is tightly controlled, but it remains poorly understood how neurons meet their energy demands to sustain synaptic transmission. Here we provide evidence that AMP-activated protein kinase (AMPK) is pivotal to sustain neuronal energy levels upon synaptic activation by adapting the rate of glycolysis and mitochondrial respiration. Furthermore, this metabolic plasticity is required for the expression of immediate-early genes, synaptic plasticity, and memory formation. Important in this context, in neurodegenerative disorders such as Alzheimer disease, dysregulation of AMPK impairs the metabolic response to synaptic activation and processes that are central to neuronal plasticity. Altogether, our data provide proof of concept that AMPK is an essential player in the regulation of neuroenergetic metabolic plasticity induced in response to synaptic activation and that its deregulation might lead to cognitive impairments.

Abstract LB-40: Guanine nucleotide exchange factors Dock7 and Ect2 mediate HGF/c-MET-induced glioblastoma cell invasion.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumor. It is an incurable disease with an average life expectancy of just 14 months following diagnosis. GBM morbidity is primarily due to its rapid growth, neovascularization and invasion throughout the brain. The angiogenesis inhibitor, bevacizumab, now approved for GBM treatment, has been shown to cause enhanced tumor cell invasion in part via c-MET (HGF-receptor) signaling. We have previously shown that several members of the Rho family of small GTPases, including Rac1, Rac3, RhoG and Cdc42, play central roles in GBM tumor cell invasion. Rho GTPases are activated by guanine nucleotide exchange factors (Rho GEFs). Herein we focus on identifying GEFs that act on Rac subfamily members and which are of functional importance in GBM. We found that Dock7 and Ect2, two GEFs that can activate Rac proteins, show elevated mRNA and protein expression in human GBM tissue in comparison with non-neoplastic brain. RNAi-mediated depletion of these GEFs in GBM cell lines resulted in decreased HGF- and serum-induced tumor cell invasion in 3-dimensional extracellular matrix in vitro and organotypic ex vivo brain slice invasion assays. GEF activity of Dock7 and Ect2 were shown to be increased by HGF using a pull-down with Rac1G15A, a nucleotide binding-deficient mutant of Rac1. Furthermore, Dock7 and Ect2 were shown to be required for HGF-induced Rac1 activation, lamellipodia formation and cell spreading. Dock7, but not Ect2, was shown to co-immunoprecipitate with c-MET and this interaction was HGF-dependent and required the c-MET adaptor protein GAB1. Dock7 and GAB1 were furthermore shown to co-immunoprecipitate in an HGF-dependent manner. GAB1 was shown to be required for HGF-induced Dock7 and Ect2 Rac1 GEF activities, Rac1 activation and HGF-induced GBM cell invasion. In summary, we have identified two GEFs that are of functional and clinical relevance in the development of the GBM pathologic phenotype. Dock7 and Ect 2 represent potential new drug targets for the management of GBM, especially in combination with an anti-angiogenic approach. Citation Format: David W. Murray, Sebastien Didier, Vincent M. Paulino, Amanda Chan, Nhan L. Tran, Annette T. Byrne, Marc H. Symons. Guanine nucleotide exchange factors Dock7 and Ect2 mediate HGF/c-MET-induced glioblastoma cell invasion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-40. doi:10.1158/1538-7445.AM2013-LB-40

Abstract LB-515: Guanine nucleotide exchange factor Dock7 expression is increased in human glioblastoma and mediates tumor cell invasion.

Glioblastoma multiforme (GBM), the most common and deadly adult brain malignancy, is an incurable disease with an average life expectancy of 14 months following diagnosis. GBM morbidity is primarily due to its rapid growth, neovascularization and invasion throughout the brain. Recently, anti-VEGF therapies approved in this indication have paradoxically been shown to stimulate the invasive behavior of GBM. Members of the Rho family of small GTPases have been established as important mediators of GBM cell invasion. These proteins are activated by guanine nucleotide exchange factors (GEFs). In the current study, we sought to identify GEFs that are up-regulated in high-grade glioma and which may be of functional importance in human GBM. In this study we have shown that that Dock7, a GEF that can mediate signaling of Rac1 and Cdc42 Rho GTPases is up-regulated in human GBM tissue in comparison with normal brain. We have further demonstrated that Dock7 silencing by RNA-interference inhibits invasion of U87 and SNB19 GBM cells into ex vivo brain slices. Dock7 silencing inhibits Hepatocyte Growth Factor (HGF)-induced cell invasion in a 3D in vitro assay. HGF is known to strongly stimulate GBM cell invasiveness and both HGF and its receptor c-MET have been shown to be up-regulated in high- versus low-grade gliomas. Interestingly, we have also shown that Dock7 binds to c-MET in an HGF-dependent manner and that this interaction requires the GAB1 adaptor protein. Dock7 silencing reduced HGF-induced activation of Rac1 and consistent with this, we have shown that Dock7 silencing results in the inhibition of HGF-induced lamellipodia formation in GBM cells. Finally Dock7 was shown to be activated in an HGF-dependant manner, using Rac1-G15A pull-down assay. In summary, our data suggests a role for Dock7 in GBM cell invasion thus, Dock7 may present a potential therapeutic target in the management of invasive GBM, in particular in the adjuvant setting in combination with anti-VEGF therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-515. doi:1538-7445.AM2012-LB-515