MYC Expression in Concert with BCL2 and BCL6 Expression Predicts Outcome in Chinese Patients with Diffuse Large B-Cell Lymphoma,Not Otherwise Specified

Recent studies provide convincing evidence that a combined immunohistochemical or fluorescence in situ hybridization (FISH) score of MYC, BCL2, BCL6 proteins and MYC translocations predicted outcome in diffuse large B-cell lymphoma (DLBCL) patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). However, by far, all these researches are based on Western populations. Therefore, we investigate the prognostic relevance of MYC-, BCL2- and BCL6-rearrangements and protein expression by immunohistochemistry and FISH from 336 de novo DLBCL, NOS treated with CHOP or R-CHOP. Breaks in MYC and BCL6, and fusion in IGH/BCL2 were detected in 9.7%, 20.0%, and 11.1% of the cases, respectively, and were not significantly associated with clinical outcomes. Protein overexpression of MYC (≥40%), BCL2 (≥70%) and BCL6 (≥50%) was encountered in 51%, 51% and 36% of the tumors, respectively. On the basis of MYC, BCL2 and BCL6 expression, double-hit scores (DHSs) and triple-hit score (THS) were assigned to all patients with DLBCL. Patients with high MYC/BCL2 DHS, high MYC/BCL6 DHS and high THS had multiple adverse prognostic factors including high LDH level, poor performance status, advanced clinical stage, high International Prognostic Index (IPI) score, and non-germinal center B-cell. In univariate analysis, high MYC/BCL2 DHS, high MYC/BCL6 DHS and high THS were associated with inferior OS and PFS in both CHOP and R-CHOP cohorts (P<0.05). The highly significant correlations with OS and PFS were maintained in multivariate models that controlled for IPI (P<0.05). DLBCLs with high DHSs and high THS share the clinical features and poor prognosis of double-hit lymphoma (P>0.05). These data together suggest that the immunohistochemical DHSs and THS defined a large subset of DLBCLs with double-hit biology and was strongly associated with poor outcome in patients treated with R-CHOP or CHOP.     

Ancient Mitochondrial DNA Analyses of Ascaris Eggs Discovered in Coprolites from Joseon Tomb

Analysis of ancient DNA (aDNA) extracted from Ascaris is very important for understanding the phylogenetic lineage of the parasite species. When aDNAs obtained from a Joseon tomb (SN2-19-1) coprolite in which Ascaris eggs were identified were amplified with primers for cytochrome b (cyt b) and 18S small subunit ribosomal RNA (18S rRNA) gene, the outcome exhibited Ascaris specific amplicon bands. By cloning, sequencing, and analysis of the amplified DNA, we obtained information valuable for comprehending genetic lineage of Ascaris prevalent among pre-modern Joseon peoples.     

Flow sorting of C-genome chromosomes from wild relatives of wheat Aegilops markgrafii,Ae. triuncialis and Ae. cylindrica,and their molecular organization

Background and Aims Aegilops markgrafii (CC) and its natural hybrids Ae. triuncialis (U^tU^tC^tC^t) and Ae. cylindrica (D^cD^cC^cC^c) represent a rich reservoir of useful genes for improvement of bread wheat (Triticum aestivum), but the limited information available on their genome structure and the shortage of molecular (cyto-) genetic tools hamper the utilization of the extant genetic diversity. This study provides the complete karyotypes in the three species obtained after fluorescent in situ hybridization (FISH) with repetitive DNA probes, and evaluates the potential of flow cytometric chromosome sorting.Methods The flow karyotypes obtained after the analysis of 4'',6-diamidino-2-phenylindole (DAPI)-stained chromosomes were characterized and the chromosome content of the peaks on the flow karyotypes was determined by FISH. Twenty-nine conserved orthologous set (COS) markers covering all seven wheat homoeologous chromosome groups were used for PCR with DNA amplified from flow-sorted chromosomes and genomic DNA.Key Results FISH with repetitive DNA probes revealed that chromosomes 4C, 5C, 7C^t, T6U^tS.6U^tL-5C^tL, 1C^c and 5D^c could be sorted with purities ranging from 66 to 91 %, while the remaining chromosomes could be sorted in groups of 2–5. This identified a partial wheat–C-genome homology for group 4 and 5 chromosomes. In addition, 1C chromosomes were homologous with group 1 of wheat; a small segment from group 2 indicated 1C–2C rearrangement. An extensively rearranged structure of chromosome 7C relative to wheat was also detected.Conclusions The possibility of purifying Aegilops chromosomes provides an attractive opportunity to investigate the structure and evolution of the Aegilops C genome and to develop molecular tools to facilitate the identification of alien chromatin and support alien introgression breeding in bread wheat.     

Numerical and Structural Genomic Aberrations Are Reliably Detectable in Tissue Microarrays of Formalin-Fixed Paraffin-Embedded Tumor Samples by Fluorescence In-Situ Hybridization

Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches.     

Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t_1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G₀ are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t_1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1⁻ GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.     

GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner

GNA13, encoding one of the G protein alpha subunits of heterotrimeric G proteins that transduce signals of G protein-coupled receptors (GPCR), is frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) with poor prognostic outcomes. Due to the “undruggable” nature of GNA13, targeted therapy for these patients is not available. In this study, we found that palmitoylation of GNA13 not only regulates its plasma membrane localization, but also regulates GNA13’s stability. It is essential for the tumor suppressor function of GNA13 in GCB-DLBCL cells. Interestingly, GNA13 negatively regulates BCL2 expression in GCB-DLBCL cells in a palmitoylation-dependent manner. Consistently, BCL2 inhibitors were found to be effective in killing GNA13-deficient GCB-DLBCL cells in a cell-based chemical screen. Furthermore, we demonstrate that inactivating GNA13 by targeting its palmitoylation enhanced the sensitivity of GCB-DLBCL to the BCL2 inhibitor. These studies indicate that the loss-of-function mutation of GNA13 is a biomarker for BCL2 inhibitor therapy of GCB-DLBCL and that GNA13 palmitoylation is a potential target for combination therapy with BCL2 inhibitors to treat GCB-DLBCL with wild-type GNA13.Subject terms: B-cell lymphoma, B-cell lymphoma, Translational research     

Biomarkers of response to ibrutinib plus nivolumab in relapsed diffuse large B-cell lymphoma,follicular lymphoma,or Richter's transformation

We analyzed potential biomarkers of response to ibrutinib plus nivolumab in biopsies from patients with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and Richter''s transformation (RT) from the LYM1002 phase I/IIa study, using programmed death ligand 1 (PD-L1) immunohistochemistry, whole exome sequencing (WES), and gene expression profiling (GEP). In DLBCL, PD-L1 elevation was more frequent in responders versus nonresponders (5/8 [62.5%] vs. 3/16 [18.8%]; p = 0.065; complete response 37.5% vs. 0%; p = 0.028). Overall response rates for patients with WES and GEP data, respectively, were: DLBCL (38.5% and 29.6%); FL (46.2% and 43.5%); RT (76.5% and 81.3%). In DLBCL, WES analyses demonstrated that mutations in RNF213 (40.0% vs. 6.2%; p = 0.055), KLHL14 (30.0% vs. 0%; p = 0.046), and LRP1B (30.0% vs. 6.2%; p = 0.264) were more frequent in responders. No responders had mutations in EBF1, ADAMTS20, AKAP9, TP53, MYD88, or TNFRSF14, while the frequency of these mutations in nonresponders ranged from 12.5% to 18.8%. In FL and RT, genes with different mutation frequencies in responders versus nonresponders were: BCL2 (75.0% vs. 28.6%; p = 0.047) and ROS1 (0% vs. 50.0%; p = 0.044), respectively. Per GEP, the most upregulated genes in responders were LEF1 and BTLA (overall), and CRTAM (germinal center B-cell–like DLBCL). Enriched pathways were related to immune activation in responders and resistance-associated proliferation/replication in nonresponders. This preliminary work may help to generate hypotheses regarding genetically defined subsets of DLBCL, FL, and RT patients most likely to benefit from ibrutinib plus nivolumab.     

Differential expression of genes linked to the leukemia inhibitor factor signaling pathway during the estrus cycle and early pregnancy in the porcine endometrium

The objective of this study was to determine the expression profiles of leukemia inhibitory factor (LIF) and its receptor (LIFR), interleukin 6 receptor (IL6R), tumor protein p53 (TP53) and B-cell CLL/lymphoma 2 (BCL2) in the porcine endometrium on selected days of the estrous cycle and pregnancy. Time- and reproductive status (estrous cycle/pregnancy)-specific patterns of expression were identified for all investigated genes. The most pronounced changes were seen on Days 12 and 14 of pregnancy when maternal recognition of pregnancy and implantation, respectively, occurs in pigs.     

Invader or resident? Ancient-DNA reveals rapid species turnover in New Zealand little penguins

The expansion of humans into previously unoccupied parts of the globe is thought to have driven the decline and extinction of numerous vertebrate species. In New Zealand, human settlement in the late thirteenth century AD led to the rapid demise of a distinctive vertebrate fauna, and also a number of ''turnover'' events where extinct lineages were subsequently replaced by closely related taxa. The recent genetic detection of an Australian little penguin (Eudyptula novaehollandiae) in southeastern New Zealand may potentially represent an additional ‘cryptic’ invasion. Here we use ancient-DNA (aDNA) analysis and radiocarbon dating of pre-human, archaeological and historical Eudyptula remains to reveal that the arrival of E. novaehollandiae in New Zealand probably occurred between AD 1500 and 1900, following the anthropogenic decline of its sister taxon, the endemic Eudyptula minor. This rapid turnover event, revealed by aDNA, suggests that native species decline can be masked by invasive taxa, and highlights the potential for human-mediated biodiversity shifts.     

Commentary on the WHO classification of tumors of lymphoid tissues (2008): ��Gray zone�� lymphomas overlapping with Burkitt lymphoma or classical Hodgkin lymphoma

The 2008 WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues has introduced two new categories of high-grade B-cell lymphomas: entities in which features of diffuse large B-cell lymphoma (DLBCL) overlap with Burkitt lymphoma (DLBCL/BL) or classical Hodgkin lymphoma (DLBCL/HL). The DLBCL/BL category encompasses cases that resemble Burkitt lymphoma morphologically, but have one or more immunophenotypic or molecular genetic deviations that would exclude it from the BL category; conversely, some cases have immunophenotypic and/or genetic features of BL, but display cytologic variability unacceptable for BL. Many of the cases in the DLBCL/BL category contain a translocation of MYC as well as either BCL2 or BCL6 (so-called double-hit lymphomas) and have a very aggressive clinical behavior. The DLBCL/HL category encompasses lymphomas that exhibit the morphology of classical Hodgkin lymphoma but the immunophenotype of DLBCL, or vice versa. Most DLBCL/HL cases described present as mediastinal masses, but this category is not limited to mediastinal lymphomas. These new categories acknowledge the increasing recognition of cases that display mixed features of two well-established diseases. Whether the existence of such cases reflects shortcomings of our current diagnostic armamentarium or a true disease continuum in which such hybrid or intermediate neoplasms actually exist remains to be determined.     

Combining FISH and model-based predictions to understand chromosome evolution in Typhonium (Araceae)

Background and Aims

Since the advent of molecular phylogenetics, numerous attempts have been made to infer the evolutionary trajectories of chromosome numbers on DNA phylogenies. Ideally, such inferences should be evaluated against cytogenetic data. Towards this goal, we carried out phylogenetic modelling of chromosome number change and fluorescence in situ hybridization (FISH) in a medium sized genus of Araceae to elucidate if data from chromosomal markers would support maximum likelihood-inferred changes in chromosome numbers among close relatives. Typhonium, the focal genus, includes species with 2n = 65 and 2n = 8, the lowest known count in the family.

Methods

A phylogeny from nuclear and plastid sequences (96 taxa, 4252 nucleotides) and counts for all included species (15 of them first reported here) were used to model chromosome number evolution, assuming discrete events, such as polyploidization and descending or ascending dysploidy, occurring at different rates. FISH with three probes (5S rDNA, 45S rDNA and Arabidopsis-like telomeres) was performed on ten species with 2n = 8 to 2n = 24.

Key Results

The best-fitting models assume numerous past chromosome number reductions. Of the species analysed with FISH, the two with the lowest chromosome numbers contained interstitial telomeric signals (Its), which together with the phylogeny and modelling indicates decreasing dysploidy as an explanation for the low numbers. A model-inferred polyploidization in another species is matched by an increase in rDNA sites.

Conclusions

The combination of a densely sampled phylogeny, ancestral state modelling and FISH revealed that the species with n = 4 is highly derived, with the FISH data pointing to a Robertsonian fusion-like chromosome rearrangement in the ancestor of this species.     

Ran GTPase-Activating Protein 1 Is a Therapeutic Target in Diffuse Large B-Cell Lymphoma

Lymphoma-specific biomarkers contribute to therapeutic strategies and the study of tumorigenesis. Diffuse large B-cell lymphoma (DLBCL) is the most common type of malignant lymphoma. However, only 50% of patients experience long-term survival after current treatment; therefore, developing novel therapeutic strategies is warranted. Comparative proteomic analysis of two DLBCL lines with a B-lymphoblastoid cell line (LCL) showed differential expression of Ran GTPase-activating protein 1 (RanGAP1) between them, which was confirmed using immunoblotting. Immunostaining showed that the majority of DLBCLs (92%, 46/50) were RanGAP1⁺, while reactive lymphoid hyperplasia (n = 12) was RanGAP1⁺ predominantly in germinal centers. RanGAP1 was also highly expressed in other B-cell lymphomas (BCL, n = 180) with brisk mitotic activity (B-lymphoblastic lymphoma/leukemia: 93%, and Burkitt lymphoma: 95%) or cell-cycle dysregulation (mantle cell lymphoma: 83%, and Hodgkin’s lymphoma 91%). Interestingly, serum RanGAP1 level was higher in patients with high-grade BCL (1.71 ± 2.28 ng/mL, n = 62) than in low-grade BCL (0.75 ± 2.12 ng/mL, n = 52) and healthy controls (0.55 ± 1.58 ng/mL, n = 75) (high-grade BCL vs. low-grade BCL, p = 0.002; high-grade BCL vs. control, p < 0.001, Mann-Whitney U test). In vitro, RNA interference of RanGAP1 showed no effect on LCL but enhanced DLBCL cell death (41% vs. 60%; p = 0.035) and cell-cycle arrest (G0/G1: 39% vs. 49%, G2/M: 19.0% vs. 7.5%; p = 0.030) along with decreased expression of TPX2 and Aurora kinases, the central regulators of mitotic cell division. Furthermore, ON 01910.Na (Estybon), a multikinase inhibitor induced cell death, mitotic cell arrest, and hyperphosphorylation of RanGAP1 in DLBCL cell lines but no effects in normal B and T cells. Therefore, RanGAP1 is a promising marker and therapeutic target for aggressive B-cell lymphoma, especially DLBCL.     

Therapeutic implication of concomitant chromosomal aberrations in patients with aggressive B-cell lymphomas

A subset of diffuse large B-cell lymphomas (DLBCL) harbors concomitant rearrangements of MYC, BCL2 and BCL6 and is characterized by clinical aggressiveness and intrinsic refractoriness to standard chemo-immunotherapy. Commonly identified as “double or triple hit” lymphomas, these diseases represent a therapeutic challenge to chemotherapy-based regimens and likely require a more targeted approach. Herein we summarize the unique biological behavior of double and triple hit lymphomas focusing on the coordinated network of pathways that enable cancer cells to tolerate the oncogenic stress imposed by the co-expression of MYC, BCL2 and BCL6. We discuss how these enabling pathways contribute to the chemo-refractoriness of these tumors. We propose to exploit lymphoma cells' addiction to these oncogenic networks to design combinatorial treatments for this aggressive disease based on the modulation of epigenetically-silenced pathways and decreasing expression and activity of these oncogenic drivers.     

Targeting the G-quadruplex-forming region near the P1 promoter in the human BCL-2 gene with the cationic porphyrin TMPyP4 and with the complementary C-rich strand

The B-cell lymphoma-2 (bcl-2) gene contains a region that has been implicated in the regulation of bcl-2 gene expression. This region can form G-quadruplex structures in solution [J.X. Dai, T.S. Dexheimer, D. Chen, M. Carver, A. Ambrus, R.A. Jones, D.Z. Yang, An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution, J. Am. Chem. Soc. 128 (2006) 1096–1098.]. Here, we examined the acid–base and conformational equilibria of this G-quadruplex-forming region (BCL2G), as well as its interaction with both the porphyrin TMPyP4 and with the complementary C-rich strand. We used molecular absorption and circular dichroism techniques, in tandem with multivariate analysis tools. The results revealed the formation of an interaction complex BCL2G:TMPyP4 with a stoichiometry of 1:2 and an equilibrium constant equal to 5.0 (±2.3) × 10¹³ M⁻². Addition of the complementary C-rich strand to BCL2G induces the predominant formation of the Watson–Crick double-helix with an equilibrium constant equal to 10^7.7 M⁻¹ (at pH 7.1). Finally, the pH-induced formation of quadruplex structures from the Watson–Crick double-helix is characterized.     

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

Background and Aims

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification.

Methods

A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling.

Key Results

Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species.

Conclusions

The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.     

Variable Responses of MYC Translocation Positive Lymphoma Cell Lines To Different Combinations of Novel Agents: Impact of BCL2 Family Protein Expression

Several newly developed drugs including JQ1 (BET inhibitor), ABT199 (BCL2 inhibitor), and bortezomib (proteasome inhibitor) may offer novel therapeutic strategies for aggressive diffuse large B-cell lymphoma (DLBCL). We tested these drugs together with doxorubicin in a series of combinations in 16 DLBCL cell lines including 4 ABC-DLBCL (OCI-Ly3, OCI-Ly10, SUDHL2, RIVA) and 12 GCB-DLBCL lines (OCI-Ly4, OCI-Ly18, BJAB, SUDHL4, SUDHL6, SUDHL10, DB, PR1, VAL, SC1, Karpas-231, Karpas-422). Among these cell lines, ABT199 and doxorubicin, and to a lesser extent JQ1 and bortezomib, showed high variations in their ED50 values. Of the six cell lines showing high ABT199 ED50 values, four (SUDHL10, OCI-Ly4, SUDHL2, and BJAB) had no or little BCL2 expression, and SUDHL6 also displayed a low BCL2 expression. There was no association between the ED50 value of doxorubicin, JQ1 and bortezomib, and TP53/MYC/BCL2 genetic abnormalities or cell of origin subtype. A synergistic effect in all or the majority of drug combinations was seen in 11 cell lines, while an antagonistic effect in a high proportion of drug combinations was observed in the remaining 5 cell lines including the 3 (SUDHL10, OCI-Ly4, and SUDHL2) with little BCL2 expression, and additionally OCI-Ly18 and RIVA. Extensive Western blot analyses revealed high MCL1 expression in SUDHL10 and OCI-Ly4 but no apparent alterations in other cell lines. The molecular mechanism underlying the antagonistic effect of drug combinations in DLBCL is heterogeneous with the altered BCL2 family protein expression (absent BCL2, but high MCL1) in some cell lines.     

Positioning the expanded akirin gene family of Atlantic salmon within the transcriptional networks of myogenesis

Vertebrate akirin genes usually form a family with one-to-three members that regulate gene expression during the innate immune response, carcinogenesis and myogenesis. We recently established that an expanded family of eight akirin genes is conserved across salmonid fish. Here, we measured mRNA levels of the akirin family of Atlantic salmon (Salmo salar L.) during the differentiation of primary myoblasts cultured from fast-skeletal muscle. Using hierarchical clustering and correlation, the data was positioned into a network of expression profiles including twenty further genes that regulate myogenesis. akirin1(2b) was not significantly regulated during the maturation of the cell culture. akirin2(1a) and 2(1b), along with IGF-II and several igfbps, were most highly expressed in mononuclear cells, then significantly and constitutively downregulated as differentiation proceeded and myotubes formed/matured. Conversely, akirin1(1a), 1(1b), 1(2a), 2(2a) and 2(2b) were expressed at lowest levels when mononuclear cells dominated the culture and highest levels when confluent layers of myotubes were evident. However, akirin1(2a) and 2(2a) were first upregulated earlier than akirin1(1a), 1(1b) and 2(2b), when rates of myoblast proliferation were highest. Interestingly, akirin1(1b), 1(2a), 2(2a) and 2(2b) formed part of a module of co-expressed genes involved in muscle differentiation, including myod1a, myog, mef2a, 14-3-3β and 14-3-3γ. All akirin paralogues were expressed ubiquitously across ten tissues, although mRNA levels were regulated between cell-types and family members. Gene expression patterns were often highly correlated between akirin paralogues, suggesting that natural selection has maintained an intricate network of co-regulation among family members. We concluded that the Atlantic salmon akirin family performs a multifaceted role during myogenesis and has physiological functions spanning many cell-types.