MicroRNA-223 is a novel negative regulator of HSP90B1 in CLL

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: MicroRNAs are known to inhibit gene expression by binding to the 3'UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients. [Methods]: By applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3'UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status, HSP90B1 expression and clinico-biological data were assessed. [Results]: HSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome. [Conclusions]: HSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target.This work was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543 and PI12/00281, Proyectos de Investigación del SACYL 355/A/09, COST Action EuGESMA (BM0801), Fundación Manuel Solórzano, Obra Social Banca Cívica (Caja Burgos), Fundación Española de Hematología y Hemoterapia (FEHH) and by a grant (RD12/0036/0069) from the Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa” (Innocampus). The research leading to these results has received funding from the European Union Seventh Framework Programme [FP7/2007-2013] under Grant Agreement n°306242-NGS-PTL. MHS is fully supported by an Ayuda predoctoral de la Junta de Castilla y Leon by the Fondo Social Europeo. ME Sarasquete is supported by Contrato Miguel Servet (CP13/00080).Peer Reviewe

Stroma-Mediated Resistance to S63845 and Venetoclax through MCL-1 and BCL-2 Expression Changes Induced by miR-193b-3p and miR-21-5p Dysregulation in Multiple Myeloma.

BH3-mimetics targeting anti-apoptotic proteins such as MCL-1 (S63845) or BCL-2 (venetoclax) are currently being evaluated as effective therapies for the treatment of multiple myeloma (MM). Interleukin 6, produced by mesenchymal stromal cells (MSCs), has been shown to modify the expression of anti-apoptotic proteins and their interaction with the pro-apoptotic BIM protein in MM cells. In this study, we assess the efficacy of S63845 and venetoclax in MM cells in direct co-culture with MSCs derived from MM patients (pMSCs) to identify additional mechanisms involved in the stroma-induced resistance to these agents. MicroRNAs miR-193b-3p and miR-21-5p emerged among the top deregulated miRNAs in myeloma cells when directly co-cultured with pMSCs, and we show their contribution to changes in MCL-1 and BCL-2 protein expression and in the activity of S63845 and venetoclax. Additionally, direct contact with pMSCs under S63845 and/or venetoclax treatment modifies myeloma cell dependence on different BCL-2 family anti-apoptotic proteins in relation to BIM, making myeloma cells more dependent on the non-targeted anti-apoptotic protein or BCL-XL. Finally, we show a potent effect of the combination of S63845 and venetoclax even in the presence of pMSCs, which supports this combinatorial approach for the treatment of MM

Expression of p53 protein isoforms predicts survival in patients with multiple myeloma

Loss and/or mutation of the TP53 gene are associated with short survival in multiple myeloma, but the p53 landscape goes far beyond. At least 12 p53 protein isoforms have been identified as a result of a combination of alternative splicing, alternative promoters and/or alternative transcription site starts, which are grouped as α, β, γ, from transactivation domain (TA), long, and short isoforms. Nowadays, there are no studies evaluating the expression of p53 isoforms and its clinical relevance in multiple myeloma (MM). We used capillary nanoimmunoassay to quantify the expression of p53 protein isoforms in CD138‐purified samples from 156 patients with newly diagnosed MM who were treated as part of the PETHEMA/GEM2012 clinical trial and investigated their prognostic impact. Quantitative real‐time polymerase chain reaction was used to corroborate the results at RNA levels. Low and high levels of expression of short and TAp53β/γ isoforms, respectively, were associated with adverse prognosis in MM patients. Multivariate Cox models identified high levels of TAp53β/γ (hazard ratio [HR], 4.49; p < .001) and high‐risk cytogenetics (HR, 2.69; p < .001) as independent prognostic factors associated with shorter time to progression. The current cytogenetic‐risk classification was notably improved when expression levels of p53 protein isoforms were incorporated, whereby high‐risk MM expressing high levels of short isoforms had significantly longer survival than high‐risk patients with low levels of these isoforms. This is the first study that demonstrates the prognostic value of p53 isoforms in MM patients, providing new insights on the role of p53 protein dysregulation in MM biology

MicroRNA-223 is a novel negative regulator of HSP90B1 in CLL

Background MicroRNAs are known to inhibit gene expression by binding to the 3′UTR of the target transcript. Downregulation of miR-223 has been recently reported to have prognostic significance in CLL. However, there is no evidence of the pathogenetic mechanism of this miRNA in CLL patients. Methods By applying next-generation sequencing techniques we have detected a common polymorphism (rs2307842), in 24% of CLL patients, which disrupts the binding site for miR-223 in HSP90B1 3′UTR. We investigated whether miR-223 directly targets HSP90B1 through luciferase assays and ectopic expression of miR-223. Quantitative real-time polymerase chain reaction and western blot were used to determine HSP90B1 expression in CLL patients. The relationship between rs2307842 status,HSP90B1 expression and clinico-biological data were assessed. Results HSP90B1 is a direct target for miR-223 by interaction with the putative miR-223 binding site. The analysis in paired samples (CD19+ fraction cell and non-CD19+ fraction cell) showed that the presence of rs2307842 and IGHV unmutated genes determined HSP90B1 overexpression in B lymphocytes from CLL patients. These results were confirmed at the protein level by western blot. Of note, HSP90B1 overexpression was independently predictive of shorter time to the first therapy in CLL patients. By contrast, the presence of rs2307842 was not related to the outcome. Conclusions HSP90B1 is a direct target gene of miR-223. Our results provide a plausible explanation of why CLL patients harboring miR-223 downregulation are associated with a poor outcome, pointing out HSP90B1 as a new pathogenic mechanism in CLL and a promising therapeutic target. Keywords Chronic lymphocytic leukemia MicroRNAs Next-generation sequencingEuropean Commision (EC). Funding FP7/SP1/HEALTH. Project Code: 30624

Nuevos conocimientos sobre la diferenciación de las células plasmáticas y la respuesta a las proteínas mal plegadas en el mieloma múltiple: papel de DEPTOR e IRE1

[ES]La transición de células B (CB) a células plasmáticas (CP) implica profundas modificaciones transcripcionales que dan como resultado el fenotipo de CP, caracterizado por una capacidad de supervivencia a largo plazo, y la producción y secreción de anticuerpos. Los defectos en el desarrollo de las CP pueden provocar enfermedades graves. De hecho, la interrupción del proceso de diferenciación de la CB, como consecuencia de mutaciones en factores clave en su maduración, se ha asociado con la transformación maligna de las CB. Aunque el conocimiento de las bases moleculares que regulan la formación de la CP ha avanzado en los últimos años, muchos de los mecanismos implicados en el mantenimiento de las CP de larga duración, que son las células que se acumulan anormalmente en la medula ósea de los pacientes con MM, son aún desconocidos. Profundizar en el conocimiento de los factores implicados en la maduración de las CP podría ayudarnos a comprender la patogenia del MM y, por tanto, brindar nuevas perspectivas para el desarrollo de estrategias terapéuticas más efectivas que ayuden a bloquear la actividad tumoral de las CP en el MM. En un análisis previo de expresión génica mediante microarrays, habíamos observado que el ARNm que codifica DEPTOR e IRE1 estaba sobreexpresado en las CP normales y en las células del mieloma múltiple (MM) en comparación con los linfocitos B normales. Por este motivo, planteamos la hipótesis de que la expresión variable de las proteínas DEPTOR e IRE1 podría contribuir a inducir diferentes estados de maduración de las células del MM. DEPTOR es un inhibidor de mTOR que está sobreexpresado en los MM con translocaciones de CCND1 o CCND3, o MAF o MAFB. En estas células, se observó que se necesitaba una alta expresión de DEPTOR para mantener la activación de PI3K y AKT, y que la reducción de los niveles de DEPTOR conducía a la apoptosis. Sin embargo, no se ha descrito el posible papel de DEPTOR en la maduración de la CP. Se ha demostrado que el sensor de estrés en el retículo endoplásmico, IRE1, es necesario durante la diferenciación temprana y tardía de las CB. Por un lado, es fundamental en la etapa de células pro-B para la recombinación de los segmentos V(D)J de los genes de las inmunoglobulinas, y por otro lado, se ha demostrado que IRE1 es indispensable para la diferenciación terminal de la CB en CP a través de la activación de XBP1. En los últimos años, se ha descubierto que numerosos microRNAs participan en la maduración de las CB. Este hecho nos llevó a pensar que la expresión tanto de DEPTOR como de IRE1 también podría estar regulada a nivel postranscripcional por microRNAs. Además de participar en la maduración de la CP, también se ha demostrado que IRE1 es capaz de degradar algunos ARNm de una manera diferente a como lo hace con XBP1. Este mecanismo conocido como “regulated IRE1-dependent decay (RIDD)” puede depender de la naturaleza de los estímulos de estrés y del tejido donde se produzca. Diferentes investigaciones sugieren que aún quedan muchas dianas de RIDD por identificar. En las células de mieloma, la única diana de RIDD que se ha identificado es el ARNm de BLOC1S1. Nuestra hipótesis era que podrían existir muchas otras dianas RIDD en el MM y que algunas de ellas podrían regular la supervivencia o proliferación de las células del MM

FAM46C controls antibody production by the polyadenylation of immunoglobulin mRNAs and inhibits cell migration in multiple myeloma

FAM46C, frequently mutated in multiple myeloma (MM), has recently been shown to encode a non‐canonical poly(A) polymerase (ncPAP). However, its target mRNAs and its role in MM pathogenesis remain mostly unknown. Using CRISPR‐Cas9 technology and gene expression analysis, we found that the inactivation of FAM46C in MM down‐regulates immunoglobulins (Igs) and several mRNAs encoding ER‐resident proteins, including some involved in unfolded protein response and others that affect glycosylation. Interestingly, we show that FAM46C expression is induced during plasma cell (PC) differentiation and that Ig mRNAs encoding heavy and light chains are substrates of the ncPAP, as revealed by poly(A) tail‐length determination assays. The absence of the ncPAP results in Ig mRNA poly(A) tail‐shortening, leading to a reduction in mRNA and protein abundance. On the other hand, loss of FAM46C up‐regulates metastasis‐associated lncRNA MALAT1 and results in a sharp increase in the migration ability. This phenotype depends mainly on the activation of PI3K/Rac1 signalling, which might have significant therapeutic implications. In conclusion, our results identify Ig mRNAs as targets of FAM46C, reveal an important function of this protein during PC maturation to increase antibody production and suggest that its role as a tumour suppressor might be related to the inhibition of myeloma cell migration.This work was supported by the Samuel Solórzano Barruso foundation (FS/25‐2015), the Spanish Association for Cancer Research (AECC, GCB120981SAN) and Institute of Health Carlos III/co‐funding by FEDER (PI16/01074).Peer reviewe

Factors Regulating microRNA Expression and Function in Multiple Myeloma

Intensive research has been undertaken during the last decade to identify the implication of microRNAs (miRNAs) in the pathogenesis of multiple myeloma (MM). The expression profiling of miRNAs in MM has provided relevant information, demonstrating different patterns of miRNA expression depending on the genetic abnormalities of MM and a key role of some miRNAs regulating critical genes associated with MM pathogenesis. However, the underlying causes of abnormal expression of miRNAs in myeloma cells remain mainly elusive. The final expression of the mature miRNAs is subject to multiple regulation mechanisms, such as copy number alterations, CpG methylation or transcription factors, together with impairment in miRNA biogenesis and differences in availability of the mRNA target sequence. In this review, we summarize the available knowledge about the factors involved in the regulation of miRNA expression and functionality in MM

Restoration of microRNA-214 expression reduces growth of myeloma cells through positive regulation of P53 and inhibition of DNA replication

[EN]MicroRNA have been demonstrated to be deregulated in multiple myeloma. We have previously reported that miR-214 is down-regulated in multiple myeloma compared to in normal plasma cells. The functional role of miR-214 in myeloma pathogenesis was explored by transfecting myeloma cell lines with synthetic microRNA followed by gene expression profiling. Putative miR-214 targets were validated by luciferase reporter assay. Ectopic expression of miR-214 reduced cell growth and induced apoptosis of myeloma cells. In order to identify the potential direct target genes of miR-214 which could be involved in the biological pathways regulated by this microRNA, gene expression profiling of the H929 myeloma cell line transfected with precursor miR-214 was carried out. Functional analysis revealed significant enrichment for DNA replication, cell cycle phase and DNA binding. miR-214 directly down-regulated the expression of PSMD10, which encodes the oncoprotein gankyrin, and ASF1B, a histone chaperone required for DNA replication, by binding to their 3'-untranslated regions. In addition, gankyrin inhibition induced an increase of P53 mRNA levels and subsequent up-regulation of CDKN1A (p21Waf1/Cip1) and BAX transcripts, which are direct transcriptional targets of p53. In conclusion, MiR-214 functions as a tumor suppressor in myeloma by positive regulation of p53 and inhibition of DNA replication

Additional file 5 of RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation [Dataset]

Additional file 5: Fig. S3. Validation of putative IRE1 substrates. Exon-usage plots of the 28 remaining putative mRNAs, showing the number of reads in mock (red) and IRE1-treated (blue) samples. The black arrows represent the site of primers used in the 5´ region of the putative IRE1-substrates. Red arrows represent the site of primers mapping the predicted cleavage site. Right panel of each exon-usage plot shows the abundance of mRNA in the corresponding target. All results are presented as the means ± SD of three experiments. (*p < 0.05, **p < 0.01, ***p < 0.001).Instituto de Salud Carlos III Gerencia Regional de Salud, Junta de Castilla y León Asociación Española Contra el Cancer (AECC) Consejería de Educación, Junta de Castilla y LeónPeer reviewe

RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation

[Background]: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells.[Methods]: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot.[Results]: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found.[Conclusion]: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.This study was partially supported by the Instituto de Salud Carlos III, co-financed by FEDER, “PI16/01074” and “PI19/00674”; the Gerencia Regional de Salud, Junta de Castilla y León grants “GRS 1833/A/18” and “GRS 2058/A/19″, and the "Asociación Española Contra el Cancer (AECC)”, PROYE20047GUTI. E.A.R. was supported by the Consejería de Educación de Castilla y León and FEDER funds. I.J.C.-B. was supported by a fellowship (contract PFIS-2020: FI20/00226) from the Instituto de Salud Carlos III