Analysis of Molecular-Cellular Responses to Proteasome Inhibitors in Multiple Myeloma Patients; A Translational Approach of Proteasome Inhibitors In Vivo Effects from the Drosophila Experimental Model to Humans

Abstract Organisms require efficient surveillance of proteome functionality to prevent disruption of proteostasis. Central to the proteostasis ensuring network is the proteasome, which degrades both normal short-lived ubiquitinated proteins and damaged or mutated proteins. Over-activation of the proteasome seems to represent a hallmark of advanced tumors and thus, its selective inhibition provides a strategy for the development of novel anti-tumor therapies. This approach is applied in multiple myeloma (MM) that represents the second most common hematological malignancy. Specifically, proteasome inhibitors have demonstrated clinical efficacy in the treatment of MM and mantle cell lymphoma and are evaluated for the treatment of other malignancies. Nevertheless, the impact of proteasome dysfunction in normal human tissues (which relates to side effects in the clinic) remains poorly understood. By using the fruit fly Drosophila melanogaster as an in vivo experimental platform to study proteasome physiology we found that proteasome functionality is sex-, tissue- and age-dependent. Oral administration of proteasome inhibitors (e.g. Bortezomib or Carfilzomib) in young flies suppressed proteasome activities in the somatic tissues; reduced motor function (recapitulating peripheral neuropathy of Bortezomib treatment in the clinic) and caused premature aging. It also increased oxidative stress and activated an Nrf2-dependent feedback regulatory circuit that upregulated proteasome genes in order to restore normal proteasome functionality. Moreover, in line with observations in the clinic, Carfilzomib was found to cause milder (as compared to Bortezomib) neuromusculatory toxicity and reduction of flies' lifespan. To address the question whether these findings can be translated in humans we started characterizing proteasome physiology in both healthy donors, as well as in MM patients treated with therapeutic proteasome inhibitors. For our studies we used isolated red blood cells (RBCs; represent an anucleate relatively "long-lived" proteome) and peripheral blood mononucleated cells (PBMCs; represent cell lineages with active genomic responses). Our analyses in healthy donors of different ages revealed significant variability of basal proteasome peptidase activities in both cell types. PBMCs expressed (as compared to RBCs) higher basal proteasome activities and RBCs from females had higher chymotrypsin-like activity as compared to RBCs from males of similar age. Furthermore, as in the flies' somatic tissues, proteasome activities were found (independently of sex and cell type) to decline during aging. Studies in RBCs and PBMCs isolated from MM patients treated with Bortezomib revealed donor-, cell type- and drug-specific readouts. In most (but not all) cases proteasome activities were suppressed in both cell types at 24-hrs post-drug administration. RBCs were particularly sensitive to the inhibitor and their proteasome activities remained low during the entire course of treatment. On the contrary, PBMCs were characterized by phases of rebound proteasome activities during the periods of no drug administration; these phases correlated with upregulation of proteasome genes expression, indicating that the feedback regulatory circuit which functions to restore proteasome activities in flies is also operational in humans. Additional gene expression analyses in PBMCs showed that proteasome inhibition also triggers the induction of genes involved in chaperon, autophagy, unfolded protein- and antioxidant-responses pathways; while, as in the fly model, the intensity of genes induction seems to decline during aging. Interestingly, in those patients who (despite treatment) showed no reduction of proteasome activities we found marginal gene expression alterations, suggesting that the observed gene induction largely depends on proteasome loss of function. Importantly, at the clinical level we observed a positive correlation between the degree of proteasome inhibition (in PBMCs or RBCS) and the depth of disease responses. The similarities between the Drosophila pharmacological model and the MM patients indicate that the molecular responses to proteasome malfunction are largely conserved in higher metazoans. We foresee that our ongoing studies will support a more personalized clinical therapeutic approach in hematological malignancies. Disclosures Terpos: Amgen: Honoraria, Other: Travel expenses, Research Funding; Takeda: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Novartis: Honoraria; Celgene: Honoraria, Other: Travel expenses. Dimopoulos:Celgene: Honoraria; Onyx: Honoraria; Novartis: Honoraria; Genesis: Honoraria; Janssen-Cilag: Honoraria; Janssen: Honoraria; Amgen: Honoraria.

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Molecular Analyses Of The Effects Induced By Orally Administered Bortezomib In Drosophila Flies: A Novel In Vivo Experimental Platform To Screen For The Tissue- and Age-Dependent Effects Of Proteasome Inhibitors

Blood ◽

10.1182/blood.v122.21.2910.2910 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2910-2910 ◽

Cited By ~ 1

Author(s):

Eleni N. Tsakiri ◽

Evangelos Terpos ◽

Gerasimos P. Sykiotis ◽

Issidora S. Papassideri ◽

Vassilis G. Gorgoulis ◽

...

Keyword(s):

Signaling Pathway ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Molecular Analyses ◽

Regulatory Circuit ◽

Somatic Tissues ◽

Age Dependent ◽

The Impact ◽

Dose Dependent

Abstract Organisms require efficient surveillance of proteome quality to prevent disruption of proteostasis (homeostasis of the proteome). Central to the proteostasis ensuring mechanisms is the proteasome, which is involved in the degradation of both normal short-lived ubiquitinated proteins and mutated or damaged proteins. Proteome quality control also depends on the activity of the Nrf2/Keap1 signaling pathway which upon increased oxidative stress stimulates the expression of phase II and antioxidant enzymes. Recent findings indicate that over-activation of the proteostasis ensuring mechanisms (e.g. the proteasome) represents a hallmark of advanced tumors, and thus their inhibition provides a strategy for the development of novel anti-tumor therapies. This approach is effectively applied in multiple myeloma (MM) that represents the second most common hematological malignancy. Bortezomib is the first-in-class proteasome inhibitor that is used in the clinic for the treatment of MM, both as a single agent and as part of combination regimens. Nevertheless, the impact of the in vivo impaired proteasome functionality in tissues of higher metazoans (which maybe related to adverse effects in the clinic) remains poorly understood. To address this issue we harnessed the power of Drosophila genetics and developed a novel in vivo model of specific dose-dependent pharmacological inhibition of proteasome in adult flies. Drosophila is well-suited to this line of investigation, due to its powerful genetics and its similarities in key metabolic and aging pathways with mammals; the fact that its proteasome resemble those from mammals and finally, because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cell lineages. We have found that feeding of bortezomib to young flies causes dose-dependent decrease of proteasome activities in the hemolymph and the somatic tissues, disruption of proteostasis, reduced motor function (a phenotype that recapitulates peripheral neuropathy of bortezomib treatment in the clinic) and a marked reduction of flies’ lifespan. Further molecular analyses showed that proteasome dysfunction is signaled to the proteostasis network of the young (but not the aged) somatic tissues by reactive oxygen species that originated from damaged mitochondria and downstream activated the Nrf2/Keap1 signaling pathway. Nrf2 activation was essential for stimulation of the genomic antioxidant response elements and the upregulation of the proteasome subunits in order to restore normal proteasome proteolysis rates. Interestingly, the reproductive tissues of the flies were more resistant than somatic tissues to proteasome inhibition triggering (in an age-independent manner) a more intense upregulation of proteasome components after bortezomib-mediated proteasome dysfunction. Additional observations indicated that the toxicity of the bortezomib may also relates to the type of diet and that aged flies are extremely sensitive (compared to young organisms) to proteasome inhibition, while even short term exposures of young flies to bortezomib still affected their overall longevity. Finally, our studies showed that the lower threshold of proteasome activities that can support life is ∼30-40% of the physiological basal activities. Taken together, our findings establish that impaired proteasome function triggers the activation of a tissue- and age-dependent regulatory circuit aiming to adjust the actual cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis regulatory circuit has significant detrimental effects and accelerates aging. These studies at the in vivo setting of fruit flies add new knowledge on the proteasome inhibitors effects in higher metazoans. Also, as research in this area of high biomedical interest has been developing fast they will, most likely, be of interest to a broader scientific community from distinct disciplines and they have the potential to enter the important, yet challenging, arena of translational medicine. To this end we have started translating findings from our Drosophila model in the clinical setting in order to demonstrate that our Drosophila pharmacological model fit in the spectrum of bench to bedside research. Disclosures: No relevant conflicts of interest to declare.

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Translating Findings of Proteasome Inhibitors Effects from the in VivoDrosophila Experimental Model to Humans: The Paradigm of the Molecular-Cellular Responses to Bortezomib and Carfilzomib

Blood ◽

10.1182/blood.v124.21.4814.4814 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4814-4814

Author(s):

Ioannis P. Trougakos ◽

Eleni-Dimitra Papanagnou ◽

Tina Bagratuni ◽

Efstathios Kastritis ◽

Issidora S. Papassideri ◽

...

Keyword(s):

Multiple Myeloma ◽

Experimental Model ◽

Drug Administration ◽

Proteasome Inhibitors ◽

Cellular Responses ◽

Antioxidant Responses ◽

Healthy Donors ◽

Genes Expression ◽

Somatic Tissues

Abstract Cellular proteostasis (homeostasis of the proteome) is ensured by the efficient maintenance of proteome quality and functionality. Central to the proteostasis process is the proteasome, which is involved in the degradation of both normal short-lived ubiquitinated proteins and mutated or damaged proteins. Recent findings indicate that the over-activation of the proteostasis ensuring mechanisms represents a hallmark of advanced tumors, and thus their inhibition provides a strategy for the development of novel anti-tumor therapies. In line with this concept, proteasome inhibitors (i.e. bortezomib and carfilzomib) have demonstrated clinical efficacy in the treatment of multiple myeloma (MM) and mantle cell lymphoma and are under evaluation for the treatment of other malignancies. By using the fruit fly Drosophila melanogaster as an in vivo platform for screening the effects of proteasome inhibitors at the whole organism level we recently reported that proteasome functionality in flies’ somatic tissues is sex- and tissue- dependent and that it declines with aging. Administration of bortezomib to young flies caused dose-dependent decrease of proteasome activities in the somatic tissues; induction of the proteasome genes expression, disruption of proteostasis, reduced motor function (a phenotype that recapitulates peripheral neuropathy of bortezomib treatment in the clinic) and a marked reduction of flies’ lifespan. Our in vivo data also showed that carfilzomib was less toxic compared to bortezomib, including neuromusculatory toxicity and effects on flies’ longevity. To address the question whether these findings can be translated to humans we started characterizing proteasome regulation and functionality in both healthy donors, as well as in MM patients treated with either bortezomib or carfilzomib. Initially, we screened isolated red blood cells (RBCs) and peripheral blood mononucleated cells (PBMCs) from male and female healthy donors of different ages; these two cell types represent either an anucleate relatively “long-lived” proteome (RBCs) or cell lineages with the capacity to mobilize genome responses after proteasome inhibition (PBMCs). Our analyses revealed significant variability of basal proteasome peptidase activities among different donors in both RBCs and PBMCs. PBMCs expressed (independently of sex) higher basal proteasome activities as compared to RBCs. Moreover, RBCs isolated from female donors had elevated (as compared to males’ RBCs) basal chymotrypsin-like activity; whereas, males’ PBMCs exhibited higher trypsin-like and caspase-like enzymatic activities as compared to PBMCs from females of similar age. In line with our observations in flies’ somatic tissues, we also found that the proteasome peptidase activities decrease significantly during aging (in a sex-independent manner) in both RBCs and PBMCs. Our studies in isolated RBCs and PBMCs from MM patients treated with either bortezomib or carfilzomib revealed drug-, donor- and cell type-specific readouts. Specifically, in most cases proteasome activities were suppressed in both RBCs and PBMCs after drug administration. Also, we noted that RBCs were particularly sensitive to both inhibitors and their proteasome activities remained low during the entire course of treatment. On the other hand, PBMCs were characterized by phases of relapsed proteasome activities during the periods of no drug administration. Finally, as in the case of the in vivo Drosophila experimental model, proteasome dysfunction in PBMCs triggered in most patients a significant upregulation of the proteasome 20S and 19S genes expression. Moreover, we noted an induction of genes involved in cellular antioxidant responses; this finding is in line with our observations in flies showing that administration of proteasome inhibitors results in increased cellular oxidative stress that mobilizes genomic antioxidant responses. Data on the clinical outcomes of the treated patients in correlation with the recorded molecular responses will be presented at the meeting. Overall, our findings indicate that the molecular-cellular responses to proteasome inhibitors observed at the in vivo Drosophila Experimental model are largely translatable to humans. Moreover, we anticipate that our employed methodologies will set the basis towards a more personalized clinical therapeutic approach for multiple myeloma patients. Disclosures No relevant conflicts of interest to declare.

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The Novel Proteasome Inhibitors Carfilzomib and Oprozomib Induce Milder Degenerative Effects Compared To Bortezomib When Administered Via Oral Feeding In An In Vivo Drosophila Experimental Model: A Biological Platform To Evaluate Safety/Efficacy Of Proteasome Inhibitors

Blood ◽

10.1182/blood.v122.21.1930.1930 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1930-1930

Author(s):

Evangelos Terpos ◽

Eleni N. Tsakiri ◽

Efstathios Kastritis ◽

Tina Bagratuni ◽

Vassilis G. Gorgoulis ◽

...

Keyword(s):

Oral Administration ◽

Proteasome Inhibitor ◽

Germ Line ◽

Conflicts Of Interest ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Somatic Tissue ◽

Oral Feeding ◽

Somatic Tissues

Abstract The proteasome is involved in the degradation of both normal, short-lived ubiquitinated proteins and mutated or damaged proteins. Carfilzomib is a tetrapeptide epoxyketone–based proteasome inhibitor and oprozomib is an orally bioavailable tripeptide epoxyketone-based proteasome inhibitor. The primary target for both agents is the chymotrypsin-like β5 subunit of the constitutive proteasome and immunoproteasome. Oprozomib is 5-fold less potent than carfilzomib, but displays similar cytotoxic potential with longer exposure times due to its time-dependent proteasome inhibition. In contrast, bortezomib is a slowly reversible proteasome inhibitor with potency of proteasome inhibition similar to carfilzomib. We propose the fruit fly Drosophila melanogaster as an in vivo platform for screening and characterizing proteasome inhibitors at the whole organism level. Drosophilais well-suited to this line of investigation, due to its powerful genetics, its similarities in key metabolic and aging pathways with humans, the fact that it expresses proteasomes that structurally resemble those from mammals, and also because it comprises a soma-germ line demarcation composed of both post-mitotic and mitotic cells. Moreover, flies live for few months and thus, drug screening on large cohorts can be completed in a reasonable time. We validate our model by investigating the effects of orally administered carfilzomib and oprozomib vs. bortezomib. In isolated Drosophila proteasome in vitroassays, carfilzomib showed a pattern of inhibitory activity similar to bortezomib, whereas oprozomib was less effective. After continuous oral administration of the inhibitors (∼50 μM of carfilzomib and ∼300-400 μM of oprozomib) to young flies (by adding the inhibitor in the flies’ culture medium) a proteasome inhibitory effect in somatic tissues roughly similar to 1 μM bortezomib was induced. Similar findings were noted when we analyzed distinct somatic tissue parts (i.e., head, thorax and abdomen), indicating that orally administered proteasome inhibitors are equally distributed to different body parts. As in the case of bortezomib, the effects of the inhibitors were less pronounced in the reproductive tissues. At the molecular level, carfilzomib (as compared to bortezomib) induced a milder disruption of fly somatic tissue proteostasis, lower rates of somatic tissue oxidative stress and less intense activation of genomic antioxidant response elements that correlated with reduced intensities of proteasome genes and protein subunit upregulation. Proteasome subunit induction was found to depend on the activity of the transcription factor Nrf2, a master regulator of cellular anti-oxidant responses. Furthermore, carfilzomib promoted the induction of lysosomal enzymes (e.g. cathepsins) and autophagy-related genes but less intensively compared to bortezomib. At concentrations that induced rates of proteasome inhibition that were similar to bortezomib, there were no significant toxic effects of either carfilzomib or oprozomib to oogenesis or to embryogenesis. Compared to bortezomib, both inhibitors exerted a significantly milder impact on the neuromusculatory system (locomotor performance) of the flies. Finally, we found that sustained oral administration of either carfilzomib or oprozomib exerted significantly milder effects (as compared to bortezomib) on flies’ mortality rate, healthspan and overall longevity. Our in vivo data support that carfilzomib is significantly less toxic compared to bortezomib, including neuromusculatory toxicity. Oprozomib was also less toxic but it is worth noting that it showed reduced activity against fly proteasomes. In support, our preliminary analyses indicated that in comparison to bortezomib and carfilzomib, oprozomib was less potent when tested in human osteosarcoma cancer cell lines. The validity of our in vivo pharmacological model is exemplified by the observed similarities with the reported clinical adverse effects, while the ratio of the different doses used to achieve similar rates of proteasome inhibition in Drosophila somatic tissues (i.e. ∼1 μM bortezomib, ∼50 μM carfilzomib) is reminiscent of the doses used in the clinic (i.e. ∼1.3 mg/m2 bortezomib and ∼25-56 mg/m2 carfilzomib). We conclude that fruit flies represent a valid biological platform for evaluating the efficacy and toxicity of proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

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S-2209, a Novel, Non-Boronic Proteasome Inhibitor, Induces Apoptosis and Cell Growth Arrest in Multiple Myeloma Cells.

Blood ◽

10.1182/blood.v110.11.1513.1513 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1513-1513

Author(s):

Philipp Baumann ◽

Karin Mueller ◽

Sonja Mandl-Weber ◽

Helmut Ostermann ◽

Ralf Schmidmaier ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Wistar Rats ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Inhibition Assay ◽

Induction Of Apoptosis

Abstract Purpose: Multiple Myeloma (MM) is still an incurable disease. Patients become resistant to cytotoxic drugs and die of disease progression. Bortezomib is the first approved member of a new class of antineoplastic agents, the proteasome inhibitors. It has synergistic effects with genotoxic drugs and steroids in vitro and in vivo. However, single agent activity in humans is only moderate and specific toxicity (e.g. neurotoxicity) often limits its clinical use. Further proteasome inhibitors need to be developed to optimize this promising treatment option. Methods: The new proteasome inhibitor S-2209 was characterized by several assays. Inhibition of the chymotryptic activity of the human 20S proteasome was determined with the in-vivo protease inhibition assay. Additionally, proteasome inhibition was determined in isolated PBMCs from S2209-pretreated wistar rats. Inhibition of NFκB activity was determined using a NFκB reporter gene assay. Cell growth rates of MM cells (OPM-2, U266, RPMI-8226 and NCI-H929) were measured with the WST-1 assay. Induction of apoptosis was shown by flow cytometry after staining with annexin-V-FITC and propidium iodide. Intracellular signal modulation was demonstrated by western blotting. Toxicity of the substance was tested in male wistar rats. Results: The proteasome inhibition assay revealed an IC50 at ∼220nM. The NFκB inhibition assay using an A549-NFκB-SEAP transfected cell line showed an EC50 of 0.9μM. Upon incubation with S-2209, cell growth as well as cell proliferation in MM cell lines was significantly inhibited (IC50 100nM – 600nM). Furthermore, the incubation with S-2209 resulted in strong induction of apoptosis in all four MM cell lines even at nanomolar concentrations (IC50 at ∼300nm) as well as primary cells. Western blotting revealed caspase-3 cleavage and upregulon of p53 and increased phosphorylation of IκB. No induction of apoptosis was detected in PBMCs from healthy humans. Despite the administration of 5, 10 or 15mg/kg/day in wistar rats, no toxicity with respect to body weight, hepatic enzymes (ALAT ASAT, ALP), creatinin or hemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals than in control animals treated with 0.1mg/kg/d bortezomib (89% vs. 70% respectively). Conclusions: The proteasome inhibitor S-2209 inhibitis MM cell growth and induces apoptosis. This is accompanied by a strong inhibition of proteasome and of the NFκB activity. Because S-2209 shows a favourable toxicity profile in vivo, further clinical development of this promising drug is urgently needed.

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A human cell atlas of fetal chromatin accessibility

Science ◽

10.1126/science.aba7612 ◽

2020 ◽

Vol 370 (6518) ◽

pp. eaba7612 ◽

Cited By ~ 1

Author(s):

Silvia Domcke ◽

Andrew J. Hill ◽

Riza M. Daza ◽

Junyue Cao ◽

Diana R. O’Day ◽

...

Keyword(s):

Gene Expression ◽

Human Cell ◽

Single Cells ◽

Complex Trait ◽

Cell Types ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Cell Type ◽

Cell Type Specific

The chromatin landscape underlying the specification of human cell types is of fundamental interest. We generated human cell atlases of chromatin accessibility and gene expression in fetal tissues. For chromatin accessibility, we devised a three-level combinatorial indexing assay and applied it to 53 samples representing 15 organs, profiling ~800,000 single cells. We leveraged cell types defined by gene expression to annotate these data and cataloged hundreds of thousands of candidate regulatory elements that exhibit cell type–specific chromatin accessibility. We investigated the properties of lineage-specific transcription factors (such as POU2F1 in neurons), organ-specific specializations of broadly distributed cell types (such as blood and endothelial), and cell type–specific enrichments of complex trait heritability. These data represent a rich resource for the exploration of in vivo human gene regulation in diverse tissues and cell types.

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Expression of XIAP in Multiple Myeloma Patients

Blood ◽

10.1182/blood.v112.11.5118.5118 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5118-5118 ◽

Cited By ~ 1

Author(s):

Tatyana Gaponova ◽

Andrey Misurin ◽

Larisa Mendeleeva ◽

Elena Varlamova ◽

Elena Parovichnikova ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Expression Level ◽

Control Group ◽

Mitochondrial Activity ◽

High Dose ◽

Xiap Expression ◽

Group I

Abstract Introduction of novel drugs, in particular, proteasome inhibitors, for the treatment of Multiple Myeloma (MM) patients has significantly improved treatment response and overall survival. One of the effects of proteasome inhibition is down-regulation of the transcription factor NF kB that stimulates the expression of apoptosis inhibitors (IAPs). The expression of IAPs protects cells from the death due to temporary apoptotic stimuli. The overexpression of IAPs is one of the characteristics of malignant cells. A crucial gene of the IAPs family is XIAP which encodes a protein which contains not only the caspase 3 and 7 blocking domain BIR2, but also a unique caspase 9 inhibiting domain BIR3. Therefore, XIAP is able to block both apoptosis pathways: one that depends on external signals and the other that depends on mitochondrial activity. In addition, the RING domain of XIAP has an E3 ubiquitin ligase activity. The aim of our study was to investigate the XIAP expression in MM patients at diagnosis and during chemotherapy, especially with proteosome inhibitors. Our study included 25 primary MM patients; all of them have given informed consent. The median age was 48 years (range 31–62). IgG MM was diagnosed in 22 cases, IgA MM in 1 and Light Chain MM in 2. Initial treatment consisted of 3 cycles of VAD. If CR or PR were not achieved, the treatment was changed to bortezomib 1.3 mg/m2 on days 1,4,8 and 11 and dexamethasone (dex) 40 mg daily on days 1–4 days (4–6 cycles). If CR or PR was attained, Stem Cell mobilization was performed with Cyclophosphamide 6 mg/m2 +G-CSF. Melphalan at 200 mg/m2 was given before auto-SCT. The XIAP expression level was analyzed before therapy (n=25), after VAD (n=12), after bortezomib (n=6) and after auto-SCT (n=4). XIAP expression was evaluated quantitatively by means of RQ-PCR using ABL gene expression for normalization. In primary MM patients the XIAP expression was found in 100%. The meaning of XIAP/ABL*100% varied in the range of 5 to 5382% (median 22%). 24% of MM patients demonstrated XIAP hyperexpression (XIAP/ABL*100%>40%). In the control group of healthy donors the XIAP expression level was not more 13%. We subdivided MM patient into two groups according to XIAP/ABL*100% meaning: I<40%, II>40%. The comparison of M-protein, beta-microglobulin and albumin levels did not reveal any difference between these two groups. However, in group II (with primary XIAP hyperexpression) we observed the decrease of XIAP expression paralled tumor reduction (from more then 40% to 5–20%). On the contrast, in group I the XIAP gene expression increased right after chemotherapy initiation to extremely high levels of 2425%. But, after high dose melphalan and auto-SCT, the XIAP level significantly decreased (22–157%) along with the attainment of CR or very good PR. The level of the XIAP gene expression was also evaluated after the bortezomib treatment. After 4–6 courses of bortesomib + dex in all 6 MM patients CR + PR were achieved, that correlated with XIAP reduction to 8–25%. Conclusion: In MM patients at diagnosis, the level of the XIAP expression is high. The decrease of the XIAP expression correlates with the chemotherapy and proteasome inhibitor treatment efficicacy. XIAP expression comes to the normal values at the time of CR and PR achievement.

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An intein-split transactivator for intersectional neural imaging and optogenetic manipulation

10.1101/2021.04.05.438407 ◽

2021 ◽

Author(s):

Hao-Shan Chen ◽

Xiao-Long Zhang ◽

Rong-Rong Yang ◽

Guang-Ling Wang ◽

Xin-Yue Zhu ◽

...

Keyword(s):

Gene Expression ◽

Cell Types ◽

Specific Cell ◽

Control Gene ◽

Cell Type ◽

Neural Imaging ◽

Control Gene Expression ◽

Numerous Cell ◽

Marker Location

The complexity of brain circuitry is manifested by numerous cell types based on genetic marker, location and neural connectivity. Cell-type specific recording and manipulation is essential to disentangle causal neural mechanisms in physiology and behavior; however, many current approaches are largely limited by number of intersectional features, incompatibility of common effectors and insufficient gene expression. To tackle these limitations, we devise an intein-based intersectional synthesis of transactivator (IBIST) to selectively control gene expression of common effectors in specific cell types defined by a combination of multiple features. We validate the specificity and sufficiency of IBIST to control common effectors including fluorophores, optogenetic opsins and Ca2+ indicators in various intersectional conditions in vivo. Using IBIST-based Ca2+ imaging, we show that the IBIST can intersect up to five features, and that hippocampal cells tune differently to distinct emotional valences depending on the pattern of projection targets. Collectively, the IBIST multiplexes the capability to intersect cell-type features and is compatible with common effectors to effectively control gene expression, monitor and manipulate neural activities.

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Behavioral state coding by molecularly defined paraventricular hypothalamic cell type ensembles

Science ◽

10.1126/science.abb2494 ◽

2020 ◽

Vol 370 (6514) ◽

pp. eabb2494 ◽

Cited By ~ 4

Author(s):

Shengjin Xu ◽

Hui Yang ◽

Vilas Menon ◽

Andrew L. Lemire ◽

Lihua Wang ◽

...

Keyword(s):

Gene Expression ◽

Neural Coding ◽

Cell Types ◽

Marker Genes ◽

Cell Type ◽

Systematic Classification ◽

Neuron Populations ◽

Multiple Cell ◽

Important Processing

Brains encode behaviors using neurons amenable to systematic classification by gene expression. The contribution of molecular identity to neural coding is not understood because of the challenges involved with measuring neural dynamics and molecular information from the same cells. We developed CaRMA (calcium and RNA multiplexed activity) imaging based on recording in vivo single-neuron calcium dynamics followed by gene expression analysis. We simultaneously monitored activity in hundreds of neurons in mouse paraventricular hypothalamus (PVH). Combinations of cell-type marker genes had predictive power for neuronal responses across 11 behavioral states. The PVH uses combinatorial assemblies of molecularly defined neuron populations for grouped-ensemble coding of survival behaviors. The neuropeptide receptor neuropeptide Y receptor type 1 (Npy1r) amalgamated multiple cell types with similar responses. Our results show that molecularly defined neurons are important processing units for brain function.

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Dissecting the temporal requirements for homeotic gene function

Development ◽

10.1242/dev.120.7.1983 ◽

1994 ◽

Vol 120 (7) ◽

pp. 1983-1995 ◽

Cited By ~ 16

Author(s):

J. Castelli-Gair ◽

S. Greig ◽

G. Micklem ◽

M. Akam

Keyword(s):

Gene Expression ◽

Nervous System ◽

Gene Function ◽

Cell Types ◽

Homeotic Gene ◽

Homeotic Genes ◽

Bithorax Complex ◽

Cell Type ◽

Homeotic Gene Expression

Homeotic genes confer identity to the different segments of Drosophila. These genes are expressed in many cell types over long periods of time. To determine when the homeotic genes are required for specific developmental events we have expressed the Ultrabithorax, abdominal-A and Abdominal-Bm proteins at different times during development using the GAL4 targeting technique. We find that early transient homeotic gene expression has no lasting effects on the differentiation of the larval epidermis, but it switches the fate of other cell types irreversibly (e.g. the spiracle primordia). We describe one cell type in the peripheral nervous system that makes sequential, independent responses to homeotic gene expression. We also provide evidence that supports the hypothesis of in vivo competition between the bithorax complex proteins for the regulation of their down-stream targets.

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Multiple Myeloma Cells Depend on the DDI2/NRF1-mediated Proteasome Stress Response for Survival

Blood Advances ◽

10.1182/bloodadvances.2020003820 ◽

2021 ◽

Author(s):

Tianzeng Chen ◽

Matthew Ho ◽

Jenna Briere ◽

Maria Moscvin ◽

Peter G Czarnecki ◽

...

Keyword(s):

Multiple Myeloma ◽

Stress Response ◽

Molecular Mechanisms ◽

Nuclear Translocation ◽

De Novo ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Knock Out

Multiple myeloma (MM) cells suffer from baseline proteotoxicity due to an imbalance between the load of misfolded proteins awaiting proteolysis and the capacity of the ubiquitin-proteasome system to degrade them. This intrinsic vulnerability is at the base of MM sensitivity to agents that perturb proteostasis such as proteasome inhibitors (PIs), the mainstay of modern-day myeloma therapy. De-novo and acquired PI resistance are important clinical limitations, adversely affecting prognosis. The molecular mechanisms underpinning PI resistance are only partially understood, limiting the development of drugs that can overcome it. The transcription factor NRF1 is activated by the aspartic protease DDI2 upon proteasome insufficiency and governs proteasome biogenesis. In this work, we show that MM cells exhibit baseline NRF1 activation and are dependent upon DDI2 for survival. DDI2 knock out (KO) is cytotoxic for MM cells, both in vitro and in vivo. Protein structure-function studies show that DDI2 KO blocks NRF1 cleavage and nuclear translocation, causing impaired proteasome activity recovery upon irreversible proteasome inhibition, thereby increasing sensitivity to PI. Add-back of wild-type, but not of catalytically-dead DDI2, fully rescues these phenotypes. We propose that DDI2 is an unexplored, promising molecular target in MM by disrupting the proteasome stress response and exacerbating proteotoxicity.

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