PRDM16 Research Articles

Analysis of the genomic landscape of multiple myeloma highlights novel prognostic markers and disease subgroups

In multiple myeloma, next-generation sequencing (NGS) has expanded our knowledge of genomic lesions, and highlighted a dynamic and heterogeneous composition of the tumor. Here we used NGS to characterize the genomic landscape of 418 multiple myeloma cases at diagnosis and correlate this with prognosis and classification. Translocations and copy number abnormalities (CNAs) had a preponderant contribution over gene mutations in defining the genotype and prognosis of each case. Known and novel independent prognostic markers were identified in our cohort of proteasome inhibitor and immunomodulatory drug-treated patients with long follow-up, including events with context-specific prognostic value, such as deletions of the PRDM1 gene. Taking advantage of the comprehensive genomic annotation of each case, we used innovative statistical approaches to identify potential novel myeloma subgroups. We observed clusters of patients stratified based on the overall number of mutations and number/type of CNAs, with distinct effects on survival, suggesting that extended genotype of multiple myeloma at diagnosis may lead to improved disease classification and prognostication.

B cell-intrinsic epigenetic modulation of local and systemic antibody response by gut microbiota through catabolic short-chain fatty acids

Abstract Immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM), as initiated by AID, and plasma cell differentiation, as mediated by Blimp-1, are critical for the maturation of the antibody response. This, as we have suggested, can be modulated by epigenetic modifications arising from environmental factors, such as the gut microbiota. Here we show that the gut microbiota regulates host immune responses through the production of metabolites such as short chain fatty acids (SCFAs). Butyrate and propionate are two of the major metabolic products generated by gut microbiota through processing of dietary fibers. They effectively dampened local and systemic IgG, IgA and IgE responses through B cell-intrinsic modulation of Aicda and Prdm1 gene expression, and, therefore, CSR/SHM and plasma cell differentiation. This SCFA-mediated reduction of class-switched B cells and plasma cells in PP, MLNs and the spleen curtailed the antibody response to T-dependent and T-independent antigens. It also virtually abrogated the allergic IgE responses to house dust mite and peanut as well as the lupus IgG autoantibody response. Mice fed fiber-free diets showed reduced levels of gut SCFAs and increased titers of serum IgG, IgA, and IgE, which were reversed by the administration of exogenous butyrate and propionate. SCFA-mediated modulation of B cell was not due to their G-protein coupled receptor signaling or their use as an energy source. Rather, it resulted from their HDAC inhibitory activity, which upregulated select miRNAs silencing Aicda and Prdm1 mRNAs. Thus, the gut microbiota modulates B cell differentiation and antibody responses through B cell-intrinsic epigenetic regulation, by fiber processing metabolic products.

Role of LncRNA ROR in Ad36-induced browning of human adipose-derived stem cells

Objective To investigate the role of LncRNA ROR in Ad36-induced browning of human adipose-derived stem cells(hADSC). Methods After hADSC was induced by cocktail and Ad36 for 2, 4, 6, and 8 days, Oil red O staining was performed for observing the adipogenic status. The mRNA expressions of LncRNA ROR, uncoupling protein 1(UCP1), and PRDM16 were detected by real- time PCR and the protein expressions of UCP1 and PRDM16 were detected by Western-blot. After LncRNA ROR was knocked down by siRNA, UCP1 and PRDM16 mRNA and protein expression levels in the process of Ad36-induced adipocyte differentiation were detected by real- time PCR and Western-blot. Results Oil red O staining showed that fat droplets in the cocktail-induced group were larger than those in the Ad36-induced group. Compared with the cocktail group, the mRNA expressions of LncRNA ROR, UCP1 and PRDM16, and the protein expressions of UCP1 and PRDM16 in Ad36 group were significantly increased(P<0.05). The mRNA and protein expressions of UCP1 and PRDM16 in LncRNA ROR knockdown group were significantly lower than those in control group(P<0.05). Conclusion In the process of Ad36-induced hADSC differentiation, the up-regulation of LncRNA ROR may stimulate UCP1 and PRDM16 expression, and thus promote the browning of hADSC. (Chin J Endocrinol Metab, 2018, 34: 318-324) Key words: Human adipose-derived stem cells; Browning; Cocktail; Ad36; Long noncoding RNA

Prdm16 and Mecom mutants exhibit cleft secondary palate as a result of perturbations that affect different stages of palatogenesis

The paralogous Prdm16 and Mecom transcription factor genes play widespread and important roles during mouse embryogenesis and postnatal development. Each gene is required for the maintenance of various stem cell populations (i.e. hematopoietic and neuronal stem cells). Our studies have focused on increasing understanding of Prdm16 function in developing orofacial structures, specifically mandible, tongue and palate. Prdm16 loss‐of‐function mutations result in cleft secondary palate (CP) due to failed palate shelf elevation secondary to micrognathia. This mechanism of clefting models that observed in human Pierre Robin Sequence CP.We are examining the requirement for Prdm16 during palate formation through neural crest‐, mandible‐ and palate‐specific ablation or rescue of Prdm16 using an invertible conditional gene trap null allele of Prdm16. These experiments largely support a palate‐extrinsic mechanism of clefting in Prdm16 mutants, since null and mandible‐specific Prdm16 mutant embryos exhibit micrognathia and CP and palate‐specific loss results in CP with reduced penetrance. To characterize the underlying cellular defects, we are assessing cell proliferation and apoptosis in the palate shelves and Meckel's cartilage. We also continue to screen craniofacial markers by in situ hybridization and have carried out RNA‐seq expression profiling in the developing mandible of wt and Prdm16 mutant embryos to identify and validate differential gene expression as a consequence of Prdm16 loss.We also hypothesized that Mecom is also required during orofacial development. Mecom or Mds1‐Evi1 complex locus includes the genes Mds1 and Evi1 and the product of intergenic splicing, Mds1‐Evi1. This study characterizes two targeted Mecom alleles, Evi1ko1 (ko1) and Evi1ko3 (ko3), which knockout Evi1 alone or both Evi1 and Mds1‐Evi1, respectively. To maintain a consistent strain background as our Prdm16 mutants, we initiated a series of successive outcrosses matings between Mecom heterozygotes and FVB/NJ mice. We observed incompletely penetrant CP in ko1 homozygotes examined at birth after the 4th generation, although this penetrance did not increase beyond 25% by the 10th generation (F10). In contrast, ko3 homozygous mutants exhibit CP with very high, but incomplete penetrance by F10. Histologic assessment of coronal head sections showed that mutant palate shelves had elevated but failed to fuse at the midline, in contrast to Prdm16 mutants that exhibit failed palate shelf elevation. To explore a potential genetic interaction between Prdm16 and Mecom, we examined Prdm16; Mecom (ko3) double heterozygous newborn pups and identified CP with high penetrance. Interestingly, these pups show both pre‐ and post‐elevation CP. These studies demonstrate a requirement for both Prdm16 and Mecom during normal palate formation, albeit via alternate mechanisms, and also evidence for the influence of Prdm16‐Mecom genetic interactions.Support or Funding InformationNIH/NIDCR R15 DE023982 and Intramural funding from the Midwestern University Office of Research and Sponsored Programs and College of Health Sciences Biomedical Sciences Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Induction of Short NFATc1/αA Isoform Interferes with Peripheral B Cell Differentiation.

In lymphocytes, immune receptor signals induce the rapid nuclear translocation of preformed cytosolic NFAT proteins. Along with co-stimulatory signals, persistent immune receptor signals lead to high levels of NFATc1/αA, a short NFATc1 isoform, in effector lymphocytes. Whereas NFATc1 is not expressed in plasma cells, in germinal centers numerous centrocytic B cells express nuclear NFATc1/αA. When overexpressed in chicken DT40 B cells or murine WEHI 231 B cells, NFATc1/αA suppressed their cell death induced by B cell receptor signals and affected the expression of genes controlling the germinal center reaction and plasma cell formation. Among those is the Prdm1 gene encoding Blimp-1, a key factor of plasma cell formation. By binding to a regulatory DNA element within exon 1 of the Prdm1 gene, NFATc1/αA suppresses Blimp-1 expression. Since expression of a constitutive active version of NFATc1/αA interfered with Prdm1 RNA expression, LPS-mediated differentiation of splenic B cells to plasmablasts in vitro and reduced immunoglobulin production in vivo, one may conclude that NFATc1/αA plays an important role in controlling plasmablast/plasma cell formation.

Impact of PRDM1 gene inactivation on C-MYC regulation in diffuse large B-cell lymphoma

Objective: To investigate the role of PRDM1 gene inactivaion in the regulation of C-MYC in diffuse large B-cell lymphoma (DLBCL), and to explore the correlation of its immunophenotype and prognosis. Methods: 100 cases paraffin-embedded DLBCL tissues were collected from January 2009 to December 2015 at the First Affiliated Hospital of Xinjiang Medical University along with 20 cases of reactive proliferative lymph nodes as control. Immunohistochemical methods were used to detect the expression of CD20, CD10, MUM1, Ki-67, bcl-6, PRDM1/Blimp1, C-MYC and PAX5 protein. The tumors were classified into two subtypes according to Hans classification.The expression of PRDM1 and C-MYC gene in tumor group and control group was detected by reverse transcription PCR (RT-PCR) and the relationship between PRDM1 and C-MYC gene was analyzed.OCI-LY1 (GCB subtype) and OCI-LY3 (non-GCB subtype) cell lines were transfected with small interfering RNA by cationic liposome reagent transfection, and the expression of C-MYC in the transfected cell lines was detected by RT-PCR and Western blot. The Kaplan-Meier method was used to analyze the prognostic significance of PRDM1/Blimp1 and C-MYC at protein and mRNA levels. Results: There were 27 cases of GCB subtype and 73 cases of non-GCB subtype according to Hans classification. The positive expression of Blimp1 in DLBCL group and proliferative lymph nodes in control group was seen in 26(26.0%) and 20 cases(100%), respectively. There were 58 cases with high expression of PRDM1 at mRNA level, including 22 cases of GCB subtype and 36 cases non-GCB subtype, and the difference was statistically significant (P=0.004). There were differences in PRDM1 gene expression between the two immunological subtypes, serum lactate dehydrogenase (serum LDH) level, presence of B symptoms, tumor primary sites and other clinical pathological parameters, while C-MYC expression was different in gender, IPI score, and serum LDH levels. Upon PRDM1/Blimp1 gene silencing in the two cell lines, C-MYC protein and gene expression were up-regulated in the transfection group, compared with the blank control group and negative control group by reverse transcription PCR and Western blot analyses. Moreover, PRDM1 expression was significantly associated with C-MYC(χ(2)=7.648, P=0.006) at mRNA level. Conclusion: The up-regulation of C-MYC gene expression induced by PRDM1 inactivation in DLBCL may play an important role for the development of DLBCL.PRDM1 protein and mRNA are associated with immunophenotyping and PRDM1 mRNA is a marker of poor prognosis.

PRDI-BF1 and PRDI-BF1P isoform expressions correlate with disease status in multiple myeloma patients.

Human positive regulatory domain I binding factor 1 (PRDI-BF1 or BLIMP-1) is a transcription factor that acts as a master regulator and has crucial roles in the control of differentiation and in maintaining survival of plasma cells (PC). The PRDM1 gene, which codifies for PRDI-BF1, contains an alternative promoter capable of generating a PRDI-BF1 deleted protein (called PRDI-BF1β), which lacks 101 amino acids comprising most of the regulatory domain. PRDI-BF1β has been detected in relevant quantities especially in multiple myeloma cell lines (U266 and NCI- H929). The first aim of the study was to compare, using real time polymerase chain reaction (RT-PCR), the levels of PRDI-BF1 and PRDI-BF1β in myeloma patients and in normal human bone marrow. The second step was the examination of the expression of PRDI-BF1 and PRDI-BF1β isoform depending on disease status and treatment response. We demonstrate the correlation of PRDI-BF1 and the shorter PRDI-BF1β isoform protein levels with the clinical evolution and the management of myeloma patients.

Analysis of the genomic landscape of multiple myeloma highlights novel prognostic markers and disease subgroups

In multiple myeloma, next generation sequencing (NGS) has expanded our knowledge of genomic lesions, and highlighted a dynamic and heterogeneous composition of the tumor. Here, we used NGS to characterize the genomic landscape of 418 multiple myeloma cases at diagnosis and correlate this with prognosis and classification. Translocations and copy number changes (CNAs) had a preponderant contribution over gene mutations in defining the genotype and prognosis of each case. Known and novel independent prognostic markers were identified in our cohort of proteasome inhibitor and IMiD-treated patients with long follow-up, including events with context-specific prognostic value, such as deletions of the PRDM1 gene. Taking advantage of the comprehensive genomic annotation of each case, we used innovative statistical approaches to identify potential novel myeloma subgroups. We observed clusters of patients stratified based on the overall number of mutations and number/type of CNAs, with distinct effects on survival, suggesting that extended genotype of multiple myeloma at diagnosis may lead to improved disease classification and prognostication.Leukemia accepted article preview online, 06 December 2017. doi:10.1038/leu.2017.344.

Relationship between four SNPs of PRDM16 gene and dyslipidemia and their interaction

Objective The aim of this study was to evaluate whether PRDM16 gene polymorphisms were associated with dyslipidemia. Methods The polymorphisms of rs2651899, rs2236518, rs870171, and rs2282198 in PRDM16 gene in 528 participants were genotyped by the method of snapshot or ligase detection reaction. The genotype differences and the allele differences between the case group and the control group were analyzed. Linkage disequilibrium analysis was performed with SHE-sis online software. The interaction between rs2651899, rs2236518, rs870171, rs2282198 and gender, age, BMI were analyzed by MDR software. Results The frequency of allele A in rs2651899 locus was significantly higher in low HDL-C group compared with that in control group[OR(95%CI)=1.32(1.02-1.71), P=0.033]. The frequency of A/C genotype in rs870171 was significantly different between LDL-C abnormal group and control group[OR(95%CI)=1.97(1.01-3.86), P=0.037]. There may be interaction between rs2236518 and sex, which is a risk factor for low HDL-C[Model Ⅱ: OR(95%CI)=1.958(1.366-2.809), P<0.01]. There may be interactions among rs2651899, rs2236518, rs870171, and rs2282198, which seemed to be risk factors for lower HDL-C[Model Ⅳ: OR(95%CI)=3.991(2.707-5.884), P<0.01]. rs870171, rs2282198 may have interaction with age, which is a risk factor for high LDL-C [Model Ⅶ: OR(95%CI)=3.991(2.707-5.884), P<0.01]. Conclusion Allele A of rs2651899 may be a risk factor to low HDL-C. Under the codominant inheritance patterns, genotype A/C of rs870171 may be a risk factor to high LDL-C. In addition, there may be interaction between SNPs with gender and age. (Chin J Endocrinol Metab, 2017, 33: 651-655) Key words: PRDM16; Single nucleotide polymorphism; Interaction; Dyslipidemia

Multiple selective events at the PRDM16 functional pathway shaped adaptation of western European populations to different climate conditions.

Several studies highlighted the role of climate in shaping many human evolutionary processes. This occurred even in relatively recent times, having affected various human phenotypic traits, among which metabolic processes that orchestrate absorption and accumulation of substances to maintain energy homeostasis, that is critical for the survival of individuals in high energy-expenditure environments. To date, most researches have focalized on detection of climatic influence on SNPs' frequency in populations exposed to extreme environmental conditions or by comparing variation patterns between populations from different continents. In this study, we instead explored the genetic background of distinct western European human groups at loci involved in nutritional and thermoregulation processes, to test whether patterns of differential local adaptation to environmental conditions could be appreciated also at a lower geographical scale. Taking advantage from the 1000 Genomes Project data, genetic information for 21 genes involved in nutritional and thermoregulation processes was analysed for three western European populations. The applied Anthropological Genetics methods pointed to appreciable differentiation between the examined groups especially for the PRDM16 gene. Moreover, several neutrality tests suggested that balancing selection has acted on different regions of the gene in people from Great Britain, as well as that more recent positive selection could have also targeted some PRDM16 SNPs in Finn and Italian populations. These series of adaptive footprints are plausibly related to climate variability in both ancient and relatively recent times. Since this locus is involved in thermoregulation mechanisms and adipogenesis, local adaptations mediated by a pathway related to the brown adipose tissue activity could have evolved in response to changing cold temperature exposures of such populations.

Characterization and functional analysis of the Paralichthys olivaceus prdm1 gene promoter

PR domain containing protein 1 (Prdm1) is a transcriptional repressor identified in various species and plays multiple important roles in immune response and embryonic development. However, little is known about the transcriptional regulation of the prdm1 gene. This study aims to characterize the promoter of Paralichthys olivaceus prdm1 (Po-prdm1) gene and determine the regulatory mechanism of Po-prdm1 expression. A 2000bp-long 5'-flanking region (translation initiation site designated as +1) of the Po-prdm1 gene was isolated and characterized. The regulatory elements in this fragment were then investigated and many putative transcription factor (TF) binding sites involved in immunity and multiple tissue development were identified. A 5'-deletion analysis was then conducted, and the ability of the deletion mutants to promote luciferase and green fluorescent protein (GFP) expression in a flounder gill cell line was examined. The results revealed that the minimal promoter is located in the region between -446 and -13bp, and the region between -1415 and -13bp enhanced the promoter activity. Site-directed mutation analysis was subsequently performed on the putative regulatory elements sites, and the results indicated that FOXP1, MSX and BCL6 binding sites play negative functional roles in the regulation of the Po-prdm1 expression in FG cells. In vivo analysis demonstrated that a GFP reporter gene containing 1.4kb-long promoter fragment (-1415/-13) was expressed in the head and trunk muscle fibres of transient transgenic zebrafish embryos. Our study provided the basic information for the exploration of Po-prdm1 regulation and expression.

Molecular genetics of aggressive B-cell lymphoma.

Molecular genetics of aggressive B-cell lymphoma.

Multilocus analysis reveals three candidate genes for Chinese migraine susceptibility.

Several genome-wide association studies (GWASs) in Caucasian populations have identified 12 loci that are significantly associated with migraine. More evidence suggests that serotonin receptors are also involved in migraine pathophysiology. In the present study, a case-control study was conducted in a cohort of 581 migraine cases and 533 ethnically matched controls among a Chinese population. Eighteen polymorphisms from serotonin receptors and GWASs were selected, and genotyping was performed using a Sequenom MALDI-TOF mass spectrometry iPLEX platform. The genotypic and allelic distributions of MEF2D rs2274316 and ASTN2 rs6478241 were significantly different between migraine patients and controls. Univariate and multivariate analysis revealed significant associations of polymorphisms in the MEF2D and ASTN2 genes with migraine susceptibility. MEF2D, PRDM16 and ASTN2 were also found to be associated with migraine without aura (MO) and migraine with family history. And, MEF2D and ASTN2 also served as genetic risk factors for the migraine without family history. The generalized multifactor dimensionality reduction analysis identified that MEF2D and HTR2E constituted the two-factor interaction model. Our study suggests that the MEF2D, PRDM16 and ASTN2 genes from GWAS are associated with migraine susceptibility, especially MO, among Chinese patients. It appears that there is no association with serotonin receptor related genes.

Genome-wide methylation analysis identifies novel CpG loci for perimembranous ventricular septal defects in human.

Congenital heart diseases are the most common birth defects worldwide and leading cause of infant mortality. The perimembranous ventricular septal defect is most prevalent. Epigenetics may provide an underlying mechanism of the gene-environment interactions involved. We examined epigenome-wide DNA methylation using the Illumina HumanMethylation450 BeadChip in 84 case children and 196 control children. We identified differential methylation of a CpG locus (cg17001566) within the PRDM16 gene after Bonferroni correction (p = 9.17 × 10-8). This was validated by bisulfite pyrosequencing. PRDM16 functions as a repressor of TGF-β signaling controlling tissue morphogenesis crucial during cardiogenesis. At 15% false-discovery rate, we identified seven additional CpG loci. These findings provide novel insights in the pathogenesis of perimembranous ventricular septal defect, which is of interest for future prediction and prevention.

Current diagnosis and treatment for pediatric acute myeloid leukemia.

Acute myeloid leukemia (AML) is a complex disease caused by chromosomal aberrations, mutations, epigenetic modifications, and the deregulated expression of genes, leading to increased myeloid cell proliferation and decreased hematopoietic progenitor cell differentiation. Although most of these aberrations are correlated with prognosis, accurate risk stratification remains a challenge even after incorporating these molecular markers. Currently, some genetic mutations that allow risk stratification have been identified in adult AML, including DNMT3A and IDH1/2. However, these mutations are rare in pediatric AML cases, indicating that a different pathogenesis may exist between adult and pediatric AML. To reveal further details of pediatric AML pathogenesis, the authors performed whole-exome sequencing and whole-transcriptome analysis using massively parallel sequencing technologies in addition to gene expression array. We found that PRDM16 and EVI1-overexpressing patients had significantly worse overall survival and event-free survival, and these overexpressed genes were useful for stratifying patients with FLT3-ITD positive and/or normal karyotype. In order to further this work and establish more appropriate risk classification and molecular target drug development, target validation clinical studies are needed and expected.

Potential mechanism and prognostic value of promoter methylation of PRDM1 gene in diffuse large B cell lymphoma

Objective: To investigate PRDM1 gene methylation status, immune classification and their prognostic significance in diffuse large B cell lymphoma (DLBCL). Methods: Immunohistochemical (IHC) staining for CD20, CD10, bcl-2, bcl-6, PRDM1/Blimp-1 and MUM1 was carried out in 100 cases of DLBCL specimens and 20 reactive lymphoid proliferation samples. All patients were classified into germinal center B cell-like (GCB) and activated B cell-like (ABC) subtype according to Hans' algorithmin. PRDM1 gene methylation was detected by methylation-specific PCR (MSP) and its relationship with clinicopathologic parameters was analyzed. OCI-Ly1 (GCB type) and OCI-Ly3 (ABC type) cell lines were transfected by Small interfering RNA(siRNA) with cationic lipid reagent transfection mediated, and the PRDM1/Blimp-1 expression in before and after transfected cell lines were detected with reverse transcription-PCR and Western blot methods. The relationship between PRDM1 gene methylation, clinicopathologic parameter and survival was analyzed using one-way analysis of variance. Results: One hundred patients were classified into 73 (73%) cases of GCB subtypes and 27 (27%) cases of ABC. PRDM1/Blimp-1 was expressed in 21 DLBCL and highly expressed in 20 reactive lymphoid proliferation. PRDM1 gene methylation was detected in 23% (23/100) of DLBCL, while no methylation was detected in all 20 reactive lymphoid proliferation. The difference of the PRDM1 methylation status between DLBCL and the control samples was statistically significant (P=0.004). However, there was no significant correlation between the PRDM1 gene methylation and clinicopathologic parameters (P>0.05). Reverse transcription-PCR and Western blot showed that PRDM1 gene expression was reduced in siRNA-induced group compared with blank control group and negative control group. One-way analysis of variance revealed that aged ≥60 years, performance status score above 3, and the presence of general symptoms were associated with significantly lower overall survival rate. Conclusions: PRDM1 gene silencing with aberrant CpG methylation is probably one of the critical events in the oncogenesis of DLBCL. This may have important implications as a candidate marker for diagnosis and targeted gene therapy. Meanwhile in vitro siRNA transfected OCI-Ly1 and OCI-Ly3 cell lines confirm that PRDM1 gene is a suppressor gene in DLBCL and may represent a novel therapeutic target.

Transcription Factors and Checkpoint Inhibitor Expression with Age: Markers of Immunosenescence?

Background: Aging is characterized by a progressive decline in immune surveillance that favors tumor development in older patients. One mechanism used by pre-malignant and/or malignant cells to escape immune surveillance is the upregulation of inhibitory immune checkpoint molecules, such as PD-1 and PD-L1. Another process associated with aging is genetic or epigenetic modifications of tumor suppressor genes (TSGs). We have reported a correlation between 6q deletion and progression into a T cell lymphoproliferative disease, identifying the BACH2 gene as a candidate TSG(1). We thus examined - on a molecular level - the expression of specific transcription factors (BACH2 and PRDM1) and checkpoint molecules (PD-1 and PD-L1) in the major lymphocytes subsets for their potential as predictive markers of immunosenescence.Methods: Lymphocyte subpopulations (CD3+/CD4+; CD3+/CD8+ and CD19+) were isolated for subsequent molecular analyses using the MACS technology (Miltenyi), with the purity of each lymphocyte subpopulation between 95%-99%. PD-1 (PDCD1), PD-L1 (CD274), IL4, IFNG, BACH2 and PRDM1 (Blimp1) mRNA transcripts were quantified in the purified subpopulations using qRT-PCR. BACH2 and PRDM1 protein expression were examined by Western blotting. Peripheral blood mononuclear cells were treated by Etoposide (VP16) to induce intracellular oxidative stress. Apoptotic function was assessed by flow cytometry (Annexin-V and Propidium Iodide).Results: Peripheral blood samples were obtained from 40 healthy donors (HDs) between the ages of 20 to 90 yrs, subdivided into <50 yrs (n=21; median: 36yo) and ³50 yrs (n=19; median: 61yo). And 40 blood samples from untreated patients with chronic lymphocytic leukemia (CLL; median: 67yo) were comparatively analyzed.BACH2-deficient mice have been shown to have increased numbers of IL4-producing CD4+ T cells after activation(2), with BACH2 known to repress PRDM1 expression in B andT cells(2,3). We, therefore, examined BACH2 gene expression in the HD groups finding it was significantly downregulated in CD4+, CD8+ T cells and CD19+ B cells from the older HD group (p=0.001; 0.004 and 0.03, respectively). BACH2 expression was further reduced in CD4+, CD8+ T cells and CD19+ B cells from CLL patients compared to HD of similar age (p=0.001; <0.001 and 0.004). In contrast, PRDM1 was significantly upregulated in CD4+ and CD8+ T cells (p=0.003; 0.001) from CLL patients but not in their leukemic B cells. As expected, BACH2 expression was inversely correlated with PRDM1 in CD4+, CD8+ T cells and CD19+ B cells (r=0.57; 0.71 and 0.62, respectively). Western blot analysis demonstrated that BACH2 and Blimp1 (PRDM1) protein expressions in the T and B cell subpopulations were significantly correlated with transcript expression. We also observed that BACH2 downregulation is correlated with increased IL-4 gene expression (r=0.61) but not IFNγ in CD4+ T cells. These observations suggest that BACH2 downregulation in CD4+ T cells could enhance expressed Th2-related genes, such as IL-4 and PRDM1.PD-1 gene expression was significantly upregulated in CD4+, CD8+ T cells (p=0.015 and 0.02) in the older HD group. PD-1 was also significantly upregulated in the same T cells subpopulations isolated from untreated CLL patients (p=0.001 and 0.002) when compared to a similar HD age group. High PD-L1 gene expression was correlated with increased age in HD B cells (p=0.04) with a further increase detected in leukemic B cells compared with the older HD group (p=0.001). We also observed an inverse correlation between BACH2 and PD-1 in CD4+, CD8+ T cells (r=0.59 and 0.68); and between BACH2 and PD-L1 in CD19+ B cells (r=0.66).We further examined VP16-induced apoptosis in isolated lymphocytes subpopulations. As recently reported in BACH2-deficient mice(4), our observation showed that BACH2 downregulation was strongly correlated with resistance to apoptosis in CD4+, CD8+ T cells and leukemic B cells (r=0.61; 0.75 and 0.69, respectively).Conclusion: These data suggest that BACH2, PRDM1, PD-1 and PD-L1 gene expression is correlated with aging and age-related immunosuppression. These effects are even more pronounced in leukemic cells from CLL patients.1. C. Sibille, et al.,(personal data) ; 2. S. Tsukumo, et al. Proc. Natl Acad Sci USA. 2013 ; 3. H. Tanaka et al. J Biol Chem. 2016 ; 4. Muto, A., et al. J Biol Chem. 2002; DisclosuresNo relevant conflicts of interest to declare.

Retrospective Evaluation of Correlations Between Genetic Backgrounds and Stem Cell Transplantation for De Novo Pediatric Acute Myeloid Leukemia: A Study from the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 Clinical Trial

IntroductionPediatric acute myeloid leukemia (AML) comprises less than 20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology. Approximately 40% of patients still have a relapse after first-line therapies, and the expected long-term survival rate decreases following relapse. Stem cell transplantation (SCT) was a conclusive strategy for de novo AML patients with a high risk and relapsed or refractory patients with standard and intermediate risk in a recent clinical trial. AML is a molecularly and clinically heterogeneous disease caused by various genetic alterations. Thus, it is difficult to accurately evaluate risk stratification even if known representative molecular markers, including KIT, FLT3-ITD, t(8;21)/RUNX1-RUNX1T1, and KMT2A (also known as MLL)-rearrangements, are used.MethodsWe investigated differences in the genetic background between SCT and non-SCT groups in participants of the Japan Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. Among 369 patients with de novo AML, 175 patients received SCT. A standardized form was used to record clinical variables, including patient demographic information. The clinical data of patients in each risk group were followed for 3 years after the date of final registration. JPLSG performed a central review of morphologic classification and karyotyping based on the World Health Organization Classification, French-American-British classification, and cytogenetic analysis using conventional G-banding. Molecular characterization included mutational analyses of KIT (exons 8 and 17), N- and K-RAS (exons 1 and 2), NPM1 (exon 12), CEBPA (exon 12), FLT3-ITD, NUP98-NSD1,and CBFA2T3-GLIS2 gene rearrangement, as well as KMT2A- partial tandem duplication (MLPA methods). We also evaluated the gene expression of MECOM (also known as EVI1) and PRDM16 (also known as MEL1) because their high expressions are known poor prognostic markers. Overall survival (OS) was defined as the time from AML diagnosis to death or censorship at the last follow-up. Event-free survival (EFS) was defined as the time from AML diagnosis to treatment failure, relapse, death, or last follow-up.ResultsThe 3-year OS among SCT patients (n = 175) was approximately 50%. It was significantly worse than that of non-SCT patients (n = 194, 90%; P < 0.001). Among 137 CBF-AML patients, 44 patients (32%) received SCT and their 3-year OS was 80%. On the other hand, among 232 non-CBF-AML patients, 131 patients (57%) received SCT, and their 3-year OS was only 35%. This result indicated that SCT is beneficial for relapsed CBF-AML, whereas most non-CBF patients who received SCT did not obtain the clinical benefit. In terms of the molecular characteristics, among 58 patients with high EVI1 expression, 39 patients (67%) received SCT, and their 3-year OS was approximately 35%. EVI1 expression was especially useful when using with MLL rearrangement because prognosis of patients with both MLL rearrangement andhigh EVI1 expression were extremely poor. On the other hand, among 84 patients with high PRDM16 expression, 63 patients (75%) received SCT, and their 3-year OS was approximately 20%. Although all AML patients with FLT3-ITD were assigned to receive SCT in the AML-05 trial, FLT3-ITD(+) patients withlow PRDM16 expression had a better outcome than FLT3-ITD(+) patients with high PRDM16 expression (3-year OS: 70% vs. 21%; P < 0.001). This result indicated that FLT3-ITD itself might not necessarily be associated with poor prognosis. PRDM16 gene expression is a useful marker to select patients needing SCT, particularly for patients with FLT3-ITD.ConclusionSCT was beneficial for patients with CBF-AML, whereas SCT was insufficient to rescue patients with non-CBF AML who relapsed, particularly patients with both FLT3-ITD(+) and high PRDM16 expression. New strategies, such as gemtuzumab ozogamicin or haploidentical SCT, are urged to rescue high risk/refractory patients. DisclosuresNo relevant conflicts of interest to declare.

The Clinical Features and Prognostic Impact of PRDM16 gene Expression in Adult Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) is a complex disease caused by various genetic alterations. Some prognosis-associated cytogenetic aberrations or gene mutations such as FLT3-internal tandem duplication (ITD), t(8;21)(q22;q22)/RUNX1-RUNX1T1, and inv(16)(p13q22)/CBFB-MYH11 have been found and used to stratify the risk. Numerous gene mutations have been implicated in the pathogenesis of AML, including mutations of DNMT3A, IDH1/2, TET2 and EZH2 in addition to RAS, KIT, NPM1, CEBPA and FLT3in the recent development of massively parallel sequencing technologies. However, even after incorporating these molecular markers, the prognosis is unclear in a subset of AML patients.Recently, NUP98-NSD1 fusion gene was identified as a poor prognostic factor for AML. We have reported that all pediatric AML patients with NUP98-NSD1 fusion showed high expression of the PR domain containing 16 (PRDM16; also known as MEL1) gene, which is a zinc finger transcription factor located near the breakpoint at 1p36. PRDM16 is highly homologous to MDS1/EVI1, which is an alternatively spliced transcript of EVI1. Furthermore, PRDM16 is essential for hematopoietic stem cell maintenance and remarkable as a candidate gene to induce leukemogenesis. Recent reports revealed that high PRDM16 expression was a significant marker to predict poor prognosis in pediatric AML. However, the significance of PRDM16 expression is unclear in adult AML patients.MethodsA total of 151 adult AML patients (136 patients with de novo AML and 15 patients with relapsed AML) were analyzed. They were referred to our institution between 2004 and 2015 and our collaborating center between 1996 and 2013. The median length of follow-up for censored patients was 30.6 months. Quantitative RT-PCR analysis was performed using the 7900HT Fast Real Time PCR System with TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay. In addition to PRDM16, ABL1 was also evaluated as a control gene. We investigated the correlations between PRDM16 gene expression and other genetic alterations, such as FLT3-ITD, NPM1, and DNMT3A, and clarified the prognostic impact of PRDM16 expression in adult AML patients. Mutation analyses were performed by direct sequence analysis, Mutation Biased PCR, and the next-generation sequencer Ion PGM.ResultsPRDM16 overexpression was identified in 29% (44/151) of adult AML patients. High PRDM16 expression correlated with higher white blood cell counts in peripheral blood and higher blast ratio in bone marrow at diagnosis; higher coincidence of mutation in NPM1 (P = 0.003) and DNMT3A (P = 0.009); and lower coincidence of t(8;21) (P = 0.010), low-risk group (P = 0.008), and mutation in BCOR (P = 0.049). Conversely, there were no significant differences in age at diagnosis and sex distribution. Patients with high PRDM16 expression tended to be low frequency in M2 (P = 0.081) subtype, and the remaining subtype had no significant differences between high and low PRDM16 expression. Remarkably, PRDM16 overexpression patients were frequently observed in non-complete remission (55.8% vs. 26.3%, P = 0.001). Patients with high PRDM16 expression tended to have a cumulative incidence of FLT3-ITD (37% vs. 21%, P = 0.089) and MLL-PTD (15% vs. 5%, P = 0.121).We analyzed the prognosis of 139 patients who were traceable. The overall survival (OS) and median survival time (MST) of patients with high PRDM16 expression were significantly worse than those of patients with low expression (5-year OS, 17% vs. 32%; MST, 287 days vs. 673 days; P = 0.004). This trend was also significant among patients aged <65 years (5-year OS, 25% vs. 48%; MST, 361 days vs. 1565 days, P = 0.013). Moreover, high PRDM16 expression was a significant prognostic factor for FLT3-ITD negative patients aged < 65 years in the intermediate cytogenetic risk group (5-year OS, 29% vs. 58%; MST, 215 days vs. undefined; P = 0.032).ConclusionsWe investigated the correlations among PRDM16 expression, clinical features, and other genetic alterations to reveal clinical and prognostic significance. High PRDM16 expression was independently associated with non-CR and adverse outcomes in adult AML patients, as well as pediatric AML patients. Our finding indicated that the same pathogenesis may exist in both adult and pediatric AML patients with respect to PRDM16 expression, and measuring PRDM16 expression was a powerful tool to predict the prognosis of adult AML patients. DisclosuresInokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

Hypermethylation of PRDM1/Blimp-1 promoter in extranodal NK/T-cell lymphoma, nasal type: an evidence of predominant role in its downregulation.

The loss of PRDM1 expression is common in extranodal NK/T-cell lymphoma, nasal type (EN-NK/T-NT), but the role of promoter methylation in silencing PRDM1 expression remains unclear. Hence, we performed pyrosequencing analysis to evaluate the promoter methylation of PRDM1 gene in vivo and in vitro, to analyze the association between methylation and its expression, and to assess cellular effects of PRDM1 reexpression. The promoter hypermethylation of PRDM1 gene was detected in 11 of 25 EN-NK/T-NT cases (44.0%) and NK92 and NKL cells. The promoter hypermethylation of PRDM1 was significantly correlated with PRDM1 expression in vivo and in vitro, predominantly contributing to the loss of PRDM1 expression compared with genetic deletion and aberrant expression of miR-223 in EN-NK/T-NT. PRDM1 expression was significantly restored by demethylation treatment, which induced cell proliferation suppression, cell cycle arrest, and apoptosis increase. We also found that PRDM1 reexpression could downregulate the expression of Ets-1, T-bet, granzyme B, and c-myc. Our findings demonstrated that the promoter hypermethylation of PRDM1 harbored a predominant role in the downregulation of PRDM1 expression, significantly affecting the biological behavior of tumor cells in EN-NK/T-NT.