TP53 gene alterations remain one of the major determinants of poor prognosis in multiple myeloma (MM). However, p53 dysfunction can also arise through non-genetic mechanisms, like post-translational modifications (PTMs). Indeed, phosphorylation and acetylation play essential roles in modulating p53 stability and activity, but their functional relevance in primary MM samples has not been systematically explored. Here, we quantified the expression of four key p53 PTMs, phosphorylation at S15, S20, and T55, and acetylation at K382, using capillary nanoimmunoassay, in 127 newly diagnosed MM patients. We also evaluated the DNA damage marker γ-H2AX, the kinase Chk2, and the phosphatases PP1A and PP2A-C. We found that p53 PTM expression was highly heterogeneous among patients. p-p53 T55 and Ac-p53 K382 were the most frequently expressed, detected in more than 70% of samples. Patients with double-hit TP53 showed significantly lower p-p53 S20 and p-p53 T55 levels compared with those with normal TP53. Low p53 phosphorylation at S20, S15 and T55 was correlated with higher progression risk (HR = 2.39, p = 0.02; HR = 2.14, p = 0.04; and HR = 2.13, p = 0.005, respectively). We also identified deregulated expression of Chk2 and phosphatases, suggesting upstream regulatory imbalances. This is the first study to quantify p53 PTMs in a large set of MM patients, revealing that post-translational dysregulation contributes to p53 impairment independently of TP53 gene alterations. Characterizing p53 PTMs sheds light on the mechanisms underlying its dysfunction, particularly in patients with an intact TP53 gene, but dysfunctional p53 protein regulation.

Cyclins D could be a unifying event in multiple myeloma (MM), even though MM is not typically considered a proliferative disease. In this study, we hypothesized that cyclins D might have additional roles in the pathogenesis of MM beyond cell cycle control. We showed that overexpression of CCND1 and CCND2 in MM cell lines lacking these proteins revealed a mutually exclusive expression pattern, with both cyclins D localized in the cytoplasm and no impact on proliferation. To investigate non‐canonical roles of cyclin D1, we performed transcriptome analysis and multidimensional flow cytometry. Cyclin D1 overexpression led to upregulation of several key cell adhesion pathway proteins, including STAT1 and ZO‐1, along with alterations in the actin cytoskeleton and decreased adhesion to certain matrices. Immunophenotypic analysis showed a significant reduction in CD56 expression following cyclin D1 overexpression, validated in a cohort of 85 MM patients, in which 73% with high cyclin D1 were CD56‐negative. High cyclin D1 was also associated with increased circulating tumor cells (CTCs) ( P < 0.001). Overall, we revealed novel functions of cyclin D1 in MM pathogenesis, particularly in cell adhesion and dissemination.

Antiapoptotic Bcl-2 family proteins are involved in myeloma cell survival. To date, their expression in multiple myeloma (MM) patients has mostly been analyzed at the RNA level. In the present study, we quantified for the first time the protein expression of the Bcl2-family members using a capillary electrophoresis immunoassay in 120 newly diagnosed MM patients, aged ≤65 years, treated in the context of the PETHEMA/GEM2012 study. We found that the pattern of expression of Bcl-2 family proteins was highly heterogeneous among patients. Although cases with t(11;14) had significantly higher levels of Bcl-2/Bcl-xL and Bcl-2+Bim+Bax/Bcl-xL ratios than those without t(11;14), the presence of this translocation was not synonymous with such high levels of expression. Conversely, some patients with other genetic alterations also showed higher levels of those ratios. Survival analysis revealed that the high expression of Bad and Puma proteins was associated with significantly longer overall survival (p = 0.001 and p < 0.001, respectively). Bcl-2 protein ratios predicting sensitivity to venetoclax in vitro were also able to distinguish patients with shorter time to progression after triplet-based induction therapy and ASCT. This is the first study to assess the expression of the most important Bcl-2 family proteins by a quantitative method in a large set of MM patients according to their cytogenetic abnormalities. We shed light on the impact of these proteins on MM prognosis, which could help to consider the levels of proteins involved in apoptosis in the development of new therapeutic strategies.

Upregulation of a cyclin D gene determined by expression microarrays is an almost universal event in multiple myeloma (MM), but this finding has not been properly confirmed at the protein level. For this reason, we carried out a quantitative analysis of cyclin D proteins using a capillary electrophoresis nanoimmunoassay in newly diagnosed MM patients. Exclusive expression of cyclin D1 and D2 proteins was detected in 54 of 165 (33%) and 30 of 165 (18%) of the MM patients, respectively. Of note, cyclin D1 or D2 proteins were undetectable in 41% of the samples. High levels of cyclin D1 protein were strongly associated with the presence of t(11;14) or 11q gains. Cyclin D2 protein was detected in all the cases bearing t(14;16), but in only 24% of patients with t(4;14). The presence of cyclin D2 was associated with shorter overall survival (hazard ratio =2.14; P=0.017), although patients expressing cyclin D2 protein, but without 1q gains, had a favorable prognosis. In conclusion, although one of the cyclins D is overexpressed at the mRNA level in almost all MM patients, in approximately half of the patients this does not translate into detectable protein. This suggests that cyclins D could not play an oncogenic role in a proportion of patients with MM (clinicaltrials gov. identifier: NCT01916252).

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: Evasion of apoptotic mechanisms due to the deregulation of Bcl-2 family proteins may play an important role in the resistance of multiple myeloma (MM) to different therapeutic schemes. Therapies targeting these proteins, such as venetoclax, which inhibits the Bcl-2 protein, have recently been developed. To date, the expression of Bcl2 family proteins in MM patients have mostly been analyzed at RNA level, even though transcript levels by themselves may not be appropriate to predict protein levels. Although, some studies analyze the expression of apoptotic proteins in MM by immunohistochemistry techniques or Western blot, the low protein concentration obtained after CD138 isolation have largely limited the quantification of proteins so far. Aims: - To investigate the expression of the Bcl-2 family proteins in newly diagnosed MM (NDMM) patients, and its association with clinical features and cytogenetic abnormalities. - To analyze the prognostic impact of Bcl-2 family proteins in NDMM patients who underwent autologous stem cell transplantation. - To explore the Bcl-2/Bcl-xl, Bcl-2/Mcl-1 and Bcl-2+Bim+Bax/Bcl-xl protein ratios, which have been associated with response to venetoclax when analyzed at RNA level, depending on the genetic alterations. Methods: Bone marrow samples from 120 NDMM patients enrolled in the Spanish Myeloma Group clinical trial GEM2012MENOS65 were included in this study. The expression of the antiapoptotic (Bcl-2, Bcl-xl, Mcl-1) and proapoptotic (Bax, Bak, Bim, Puma, and Bad) proteins were analyzed by the capillary electrophoresis nanoimmunoassay methodology (ProteinSimple, WES™ system). Results: All the Bcl-2 family proteins analyzed were expressed in more than 80% of MM patients, except Bax which was found in less than 30%. Bak, Bim and Mcl-1 were the proteins with the highest level of expression. Patients with low expression levels of Bim protein had a significant higher incidence of plasmacytomas (p = 0.019). We found that Bak protein expression was significantly higher in patients with t(14;16) (p = 0.028), while Bcl-xl and Puma expression was lower in patients with t(11;14) and 1p deletion (p = 0.014, p = 0.046), respectively. High levels of Bad and Puma proteins were associated with longer OS (p = 0.017 and p = 0.001). Conversely, high levels of Bcl-2 had a negative impact on PFS (p = 0.018).When we analyzed the values of three ratios that have demonstrated to be predictors of the response to venetoclax, we found that patients with t(11;14) had higher levels of Bcl-2/Bcl-xl and Bcl-2+Bim+Bax/Bcl-xl ratios than patients without t(11;14) [14/18 (79%) vs. 41/91 (45%), and 14/18 (79%) vs. 42/91 (46%), respectively, p = 0.01], as has been published at RNA level. Strikingly, the high Bcl-2+Bim+Bax/Bcl-xl protein expression ratio was also enriched in patients harbouring TP53 mutations compared to patients without these mutations [7/8 (87%) vs. 40/82 (49%), p = 0.036]. Summary/Conclusion: - The expression of anti and proapoptotic proteins was well-balanced in MM. - High levels of the proapoptotic proteins Bad and Puma were associated with favorable prognosis, while the overexpression of the antiapoptotic protein Bcl-2 had a negative impact on survival. - High Bcl-2/Bcl-xl and Bcl-2+Bim+Bax/Bcl-xl protein ratios were significantly enriched in patients with t(11;14). Patients harboring TP53 mutations were also significantly associated with high Bcl-2+Bim+Bax/Bcl-xl protein ratio. FUNDING: This study has been funded by ISCIII (PI19/00674) (co-funded by FEDER), Castilla y Leon (GRS 2058/A/19, GRS 2331/A/21) and AECC (PROYE20047GUTI). CDR, EAR and IJCB were supported by AECC (CLJUN18010DERA), “Consejería Educación Castilla y León” and ISCIII (FI20/00226), respectively. Keywords: Protein Expression, BCL2, Apoptosis, Multiple myeloma

Regional Health Management of Castilla y León (GRS 2142/A/2020);); Carlos III Health Institute European Union (PI19/00674); AECC (PROYE20047GUTI).

The interest around the graphene family of materials is constantly growing due to their potential application in biomedical fields. The effect of graphene and its derivatives on cells varies amongst studies depending on the cell and tissue type. Since the toxicity against non-adherent cell lines has barely been studied, we investigated the effect of graphene and two different graphene oxides against four multiple myeloma cell lines, namely KMS-12-BM, H929, U226, and MM.1S, as well as two non-Hodgkin lymphoma cells lines, namely KARPAS299 and DOHH-2. We performed two types of viability assays, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion) and ATP (adenosine triphosphate detection), flow cytometry analysis of apoptosis induction and cell cycle, cell morphology, and direct interaction analysis using two approaches-visualization of living cells by two different systems, and visualization of fixed and dyed cells. Our results revealed that graphene and graphene oxides exhibit low to moderate cytotoxicity against cells, despite visible interaction between the cells and graphene oxide. This creates possibilities for the application of the selected graphene materials for drug delivery systems or theragnostics in hematological malignancies; however, further detailed studies are necessary to explain the nature of interactions between the cells and the materials.

The search for biomarkers based on the mechanism of drug action has not been thoroughly addressed in the therapeutic approaches to multiple myeloma (MM), mainly because of the difficulty in analyzing proteins obtained from purified plasma cells. Here, we investigated the prognostic impact of the expression of 12 proteins involved in the mechanism of action of bortezomib, lenalidomide, and dexamethasone (VRD), quantified by capillary nanoimmunoassay, in CD138-purified samples from 174 patients with newly diagnosed MM treated according to the PETHEMA/GEM2012 study. A high level of expression of 3 out of 5 proteasome components tested (PSMD1, PSMD4, and PSMD10) negatively influenced survival. The 5 analyzed proteins involved in lenalidomide's mode of action were associated with time to progression (TTP); low levels of cereblon and IRF4 protein and high levels of Ikaros, AGO2, and Aiolos were significantly associated with shorter TTP. Although the glucocorticoid receptor (GCR) level by itself had no significant impact on MM prognosis, a high XPO1 (exportin 1)/GCR ratio was associated with shorter TTP and progression-free survival (PFS). The multivariate Cox model identified high levels of PSMD10 (hazard ratio [HR] TTP, 3.49; P = .036; HR PFS, 5.33; P = .004) and Ikaros (HR TTP, 3.01, P = .014; HR PFS, 2.57; P = .028), and low levels of IRF4 protein expression (HR TTP, 0.33; P = .004; HR PFS, 0.35; P = .004) along with high-risk cytogenetics (HR TTP, 3.13; P < .001; HR PFS, 2.69; P = .002), as independently associated with shorter TTP and PFS. These results highlight the value of assessing proteins related to the mechanism of action of drugs used in MM for predicting treatment outcome.

Protein analysis in bone marrow samples from patients with multiple myeloma (MM) has been limited by the low concentration of proteins obtained after CD138(+) cell selection. A novel approach based on capillary nano-immunoassay could make it possible to quantify dozens of proteins from each CD138(+) purified MM sample in an automated manner. Up to now, the knowledge of protein level in those cells was limited because a relatively small quantity of sample is available after the diagnostic procedure. Moreover, the sample often is required for nucleic acids analysis. We have developed the procedure for obtaining proteins from bone marrow samples preserved in RLT+ buffer, and we have successfully applied this approach for the quantification of proteins in the setting of patients with MM. Proteins are extracted from RLT+ buffer, the content is quantified by total protein assay with WES machine and finally, the particular protein expression level is evaluated using specific antibodies by capillary nano-immunoassay with WES machine. The present protocol enables us to quantify many proteins from a limited amount of sample, without losing the opportunity to obtain nucleic acids at the same time. Proteins are quantified automatically in an assay with a low probability of human errors, which makes it a useful tool for biomarkers development.

Protein analysis in bone marrow samples from patients with multiple myeloma (MM) has been limited by the low protein concentration obtained after CD138+ selection. A novel approach based on capillary nano-immunoelectrophoresis (CNIA) could make it possible to automatically quantify tens of proteins from each myeloma sample. We here present the results of a pilot study in MM using this platform. Our aims were, (i) to simultaneously quantify the protein levels of 13 proteins involved in MM biology and correlate them with the corresponding gene expression (mRNA) levels; (ii) to compare the impact of protein quantification with the value of mRNA assessment on predicting MM survival. Bone marrow aspirates from 63 newly diagnosed MM patients, treated with bortezomib and lenalidomide containing regimens were analyzed. Myeloma cells were purified by anti-CD138 magnetic microbeads (AutoMACs) and stored in RLT+ buffer at -80 C and simultaneous extraction of DNA, RNA and proteins from the same sample was performed in all cases. Proteins were analyzed using the CNIA methodology (ProteinSimple, WES™) and mRNA expression by q-RT-PCR using TaqMan™ assays. After the analysis of the total protein content, 60 MM samples of 63 (95.2%) fulfilled the quantity and quality requirements for the quantification of the 13 selected proteins in each sample. The highest correlation between protein and mRNA level was observed for Cyclin D1 (ρ= 0.687, p<0.001) and Cyclin D2 (ρ=0.654, p<0.001), whereas no correlation was found for proteins such as PSME1 (Proteasome Activator Subunit 1), cereblon, c-myc, ikaros, HSP90, RIPK1 (Receptor interacting protein kinase 1) and XAF1 (XIAP Associated Factor 1). We observed modest degree of correlation for aiolos, dicer, calnexin (protein of the endoplasmic reticulum) and DDX21 (RNA helicase). High levels of cereblon and PSME1 proteins were associated with longer time to progression (TTP) compared to low levels. Thus, patients with high levels of cereblon and PSME1 had a median TTP of 25.8 and 43.8 months compared to 10.8 and 17.3 months, respectively (p<0.01 and FDR<0.05 for both comparisons). On the other hand, no significant impact on prognosis was observed when TTP was estimated using the mRNA levels. To conclude, the CNIA platform provides the novel opportunity to automatically quantify, for the first time, the expression of more than 10 proteins in CD138+ primary MM samples. We observed a weak correlation between the quantification of proteins and mRNAs encoded by several relevant genes in MM biology. Interestingly, protein measurement of cereblon and PSME1, involved in mechanism of action of IMIDs and proteasome inhibitors respectively, discriminated prognosis of patients with MM better than the corresponding mRNA levels. This work is funded by a grant from the International Myeloma Foundation's Black Swan Research Initiative®, and WES™ by INNOCAMPUS Program (CEI10-1-0010).

Jesus Ma Hernandez Rivas (Med. Adjunto Hematologia HUS, Prof. Titular, Uvd. Salamanca) Norma Gutierrez Gutierrez (Med. Adjunto H matologia) Juan Luis Garcia Hernandez (IESCYL) Invt. Posdoctoral Ma Rocio Benito Sanchez (FICUS) Invt. Posdoctoral Irena Misiewicz-Krzeminska (HUS) Invt. Posdoctoral Isabel M. Isidro (Fundacion Pethema) Eva Lumbreras Garcia (FICUS) Invt. Predoctora Cristina Robledo Montero (FICUS) Invt. Predoctora Ana E. Rodriguez Vicente (Beca FIS) Invt. Predoctora Monica del Rey Gonzalez (Beca FEHH) Invt. Predoctora Maria Abaig r Alvarado (Beca CSIC) Invt. Predoctora Andreia Aires (Beca F Ciencia y Tecnl) Ana Diez Martin (FICUS) Tecnico Ana Simon Munoz (FICUS) Tecnico

In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.

In this study, we used comparative genomic hybridization to provide an overview of chromosomal imbalances in a series of 20 adult and 8 childhood ependymomas. All tumors displayed multiple genomic imbalances. Loss of genetic material was observed in chromosomes 22q (71%), 16 (57%), 17 (46%), 6 (39%), 19q (32%), 20q (32%), and 1p (29%), with the overlapped deletion regions determined at 16p13.1–13.3, 16q22–q24, 19q13.1–13.4, 20q13.1–13.2 and 1p36.1–36.3. Gain of DNA was commonly detected on chromosomes 5q (46%), 12q (39%), 7q (36%), 9q (36%), and 4q (32%), with overlapped regions of gain mapped to 5q21–22, 12q15–24.1, 7q11.2–31.2, 9q12–32, and 4q23–28, respectively. These findings suggest a greater degree of genomic imbalance in ependymomas than has been recognized previously and highlight chromosomal loci likely to contain oncogenes or tumor suppressor genes that may contribute to the molecular pathogenesis of this tumor. Our study also confirmed previous findings on frequent losses of 17 and 22q in ependymomas and further identified chromosome 16 loss as a common recurrent genetic aberration in ependymomas.