PAX5 Research Articles

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options.

TCF3-HLF-fusion positive acute lymphoblastic leukemia (ALL) is currently incurable. Employing an integrated approach, we uncovered distinct mutation, gene expression, and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. Recurrent intragenic deletions of PAX5 or VPREB1 were identified in constellation with TCF3-HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin towards a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics, but sensitivity towards glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease.

Ebf1 heterozygosity results in increased DNA damage in pro-B cells and their synergistic transformation by Pax5 haploinsufficiency

AbstractEarly B-cell factor 1 (Ebf1) is a transcription factor with documented dose-dependent functions in normal and malignant B-lymphocyte development. To understand more about the roles of Ebf1 in malignant transformation, we investigated the impact of reduced functional Ebf1 dosage on mouse B-cell progenitors. Gene expression analysis suggested that Ebf1 was involved in the regulation of genes important for DNA repair and cell survival. Investigation of the DNA damage in steady state, as well as after induction of DNA damage by UV light, confirmed that pro-B cells lacking 1 functional allele of Ebf1 display signs of increased DNA damage. This correlated to reduced expression of DNA repair genes including Rad51, and chromatin immunoprecipitation data suggested that Rad51 is a direct target for Ebf1. Although reduced dosage of Ebf1 did not significantly increase tumor formation in mice, a dramatic increase in the frequency of pro-B cell leukemia was observed in mice with combined heterozygous mutations in the Ebf1 and Pax5 genes, revealing a synergistic effect of combined dose reduction of these proteins. Our data suggest that Ebf1 controls DNA repair in a dose-dependent manner providing a possible explanation to the frequent involvement of EBF1 gene loss in human leukemia.

Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors.

To investigate how transcription factor levels impact B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro. The alteration in lineage fate depended on a Notch1-mediated conversion process, whereas no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5(+/-)Ebf1(+/-) pro-B cells were reflected in the transcriptional response. Both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, but only the Pax5(+/-)Ebf1(+/-) pro-B cells down-regulated genes central for the preservation of stable B cell identity. This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling. This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.

PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia

AbstractAlterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL.

MTOR is necessary for proper satellite cell activity and skeletal muscle regeneration

The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7CreER and Mtorflox/flox mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes.

Bone marrow transcriptome and epigenome profiles of equine common variable immunodeficiency patients unveil block of B lymphocyte differentiation

Common variable immunodeficiency (CVID) is a late-onset humoral deficiency characterized by B lymphocyte dysfunction or loss, decreased immunoglobulin production, and recurrent bacterial infections. CVID is the most frequent human primary immunodeficiency but still presents challenges in the understanding of its etiology and treatment. CVID in equine patients manifests with a natural impairment of B lymphocyte differentiation, and is a unique model to identify genetic and epigenetic mechanisms of disease. Bone marrow transcriptome analyses revealed decreased expression of genes indicative of the pro-B cell differentiation stage, importantly PAX5 (p≤0.023). We hypothesized that aberrant epigenetic regulation caused PAX5 gene silencing, resulting in the late-onset and non-familial manifestation of CVID. A significant increase in PAX5 enhancer region methylation was identified in equine CVID patients by genome-wide reduced-representation bisulfite sequencing and bisulfite PCR sequencing (p=0.000). Thus, we demonstrate that integrating transcriptomics and epigenetics in CVID enlightens potential mechanisms of dysfunctional B lymphopoiesis or function.

Abstract P4-07-10: MiRNAs are important regulators of Pax-5 expression and function during breast cancer progression

Abstract Recent studies have enabled the identification of important factors regulating cancer progression, one of these being the Pax-5 gene. Pax-5, an essential developmental factor of B cells, is aberrantly expressed in various B cell cancer lesions and solid tumors such as breast carcinoma. Although Pax-5 downstream activity is relatively well characterized, the regulation of aberrant Pax-5 expression in a cancer specific context is poorly understood. To investigate the regulation of Pax-5 expression, we turned our attention to micro-RNAs (miRNAs). MiRNAs are highly conserved, small non-coding RNA molecules that regulate key biological processes. Extensive studies also show their deregulation in multiple cancer lesions. In this study, we aim to elucidate a causal link between differentially expressed miRNAs in cancer cells and their putative targeting of Pax-5-dependent cancer processes. With the help of biobank data and bioinformatics analyses, we observe that miRNAs 484 and 210 are aberrantly expressed in breast cancer cells and cross-reference with their predicted capacity to target the Pax-5 mRNA 3’ untranslated region (3’UTR). Using anti- or pre-miRNAs transfected into Pax-5 expressing breast cancer cell lines (MCF-7 and MB231), we demonstrate that miRNAs 484 and 210 are capable of regulating Pax-5 expression. In addition, miRNA-regulated Pax-5 expression resulted in a concomitant alteration in Pax-5-mediated phenotype and cancer processes. This is the first study demonstrating the regulation of Pax-5 expression and function by non-coding RNAs in cancer cells. We believe that the aberrant expression of Pax-5 in cancer cells is in part due to deregulated miRNA expression profiles. This study will bring insight in regards to cancer regulating processes associated with miRNA and Pax-5 deregulations and help us better understand aberrant Pax-5 expression levels within cancerous states. This study can therefore provide the eventual possibility of earlier, more efficient diagnostics as well as more targeted treatments for cancer patients. Citation Format: Jason M Harquail, Gilles A Robichaud. MiRNAs are important regulators of Pax-5 expression and function during breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-10.

Defects in subventricular zone pigmented epithelium-derived factor niche signaling in the senescence-accelerated mouse prone-8.

We studied potential changes in the subventricular zone (SVZ) stem cell niche of the senescence-accelerated mouse prone-8 (SAM-P8) aging model. Bromodeoxyuridine (BrdU) assays with longtime survival revealed a lower number of label-retaining stem cells in the SAM-P8 SVZ compared with the SAM-Resistant 1 (SAM-R1) control strain. We also found that in SAM-P8 niche signaling is attenuated and the stem cell pool is less responsive to the self-renewal niche factor pigmented epithelium-derived factor (PEDF). Protein analysis demonstrated stable amounts of the PEDF ligand in the SAM-P8 SVZ niche; however, SAM-P8 stem cells present a significant expression decrease of patatin-like phospholipase domain containing 2, a receptor for PEDF (PNPLA2-PEDF) receptor, but not of laminin receptor (LR), a receptor for PEDF (LR-PEDF) receptor. We observed changes in self-renewal related genes (hairy and enhancer of split 1 (Hes1), hairy and enhancer of split 1 (Hes5), Sox2] and report that although these genes are down-regulated in SAM-P8, differentiation genes (Pax6) are up-regulated and neurogenesis is increased. Finally, sheltering mammalian telomere complexes might be also involved given a down-regulation of telomeric repeat binding factor 1 (Terf1) expression was observed in SAM-P8 at young age periods. Differences between these 2 models, SAM-P8 and SAM-R1 controls, have been previously detected at more advanced ages. We now describe alterations in the PEDF signaling pathway and stem cell self-renewal at a very young age, which could be involved in the premature senescence observed in the SAM-P8 model.

A critical role of the Thy28-MYH9 axis in B cell-specific expression of the Pax5 gene in chicken B cells.

Accumulating evidence suggests that Pax5 plays essential roles in B cell lineage commitment. However, molecular mechanisms of B cell-specific expression of Pax5 are not fully understood. Here, we applied insertional chromatin immunoprecipitation (iChIP) combined with stable isotope labeling using amino acids in cell culture (SILAC) (iChIP-SILAC) to direct identification of proteins interacting with the promoter region of the endogenous single-copy chicken Pax5 gene. By comparing B cells with macrophage-like cells trans-differentiated by ectopic expression of C/EBPβ, iChIP-SILAC detected B cell-specific interaction of a nuclear protein, Thy28/Thyn1, with the Pax5 1A promoter. Trans-differentiation of B cells into macrophage-like cells caused down-regulation of Thy28 expression. Loss-of-function of Thy28 induced decrease in Pax5 expression and recruitment of myosin-9 (MYH9), one of Thy28-interacting proteins, to the Pax5 1A promoter. Loss-of-function of MYH9 also induced decrease in Pax5 expression. Thus, our analysis revealed that Thy28 is functionally required for B cell-specific expression of Pax5 via recruitment of MYH9 to the Pax5 locus in chicken B cells.

LCK over-expression drives STAT5 oncogenic signaling in PAX5 translocated BCP-ALL patients.

The PAX5 gene is altered in 30% of BCP-ALL patients and PAX5 chromosomal translocations account for 2-3% of cases. Although PAX5 fusion genes significantly affect the transcription of PAX5 target genes, their role in sustaining leukemia cell survival is poorly understood. In an in vitro model of PAX5/ETV6 leukemia, we demonstrated that Lck hyper-activation, and down-regulation of its negative regulator Csk, lead to STAT5 hyper-activation and consequently to the up-regulation of the downstream effectors, cMyc and Ccnd2. More important, cells from PAX5 translocated patients show LCK up-regulation and over-activation, as well as STAT5 hyper-phosphorylation, compared to PAX5 wt and PAX5 deleted cases. As in BCR/ABL1 positive ALL, the hyper-activation of STAT5 pathway can represent a survival signal in PAX5 translocated cells, alternative to the pre-BCR, which is down-regulated. The LCK inhibitor BIBF1120 selectively reverts this phenomenon both in the murine model and in leukemic primary cells. LCK inhibitor could therefore represent a suitable candidate drug to target this subgroup of ALL patients.

One missense mutation in exon 2 of the PAX5 gene in Iran.

The PAX5 gene, which encodes the B-cell specific activator protein, is one of the most important factors in determination of B-cell development. This gene is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). For example, point mutations, deletions, as well as other gene rearrangements may lead to several forms of B-cell malignancy. In this study, we obtained 50 blood samples from patients diagnosed with ALL, and screened for PAX5 mutations using sequencing in exons 1, 2 and 3. We found a heterozygous germline variant, c.113G>A (p.Arg38His), which affects the paired domain of PAX5. It seems that this mutation is pathogenic, but is recessive. Our findings suggest that this mutation in a single allele of the PAX5 gene is not sufficient to cause disease, and it is possible that other alleles are also involved in the onset of B-ALL.

Molecular characterization of mouse lens epithelial cell lines and their suitability to study RNA granules and cataract associated genes

The discovery of cytosolic RNA granule (RG) component proteins associated with human cataract has initiated investigations on post-transcriptional mechanisms of gene expression control in the lens. Application of established mouse lens epithelial cell lines (LECs) can provide rapid insights on RG function in lens cells, especially because mouse mutants in several RG components are not available. However, although these LECs represent potential reagents for such analyses, they are uncharacterized for lens gene expression or RG formation. Therefore, a detailed molecular and cellular characterization of three permanent mouse LECs 17EM15, 21EM15 and αTN4 is performed in this study. Comparative analysis between microarray gene expression datasets on LEC 21EM15 and iSyTE lens tissue demonstrates that 30% of top 200 iSyTE identified lens-enriched genes are expressed in these cells. Majority of these candidates are independently validated to either have lens expression, function or linkage to cataract. Moreover, analysis of microarray data with genes described in Cat-Map, an online database of cataract associated genes and loci, demonstrates that 131 genes linked to cataract loci are expressed in 21EM15 cells. Furthermore, gene expression in LECs is compared to isolated lens epithelium or fiber cells by qRT-PCR and by comparative analyses with publically available epithelium or fiber-specific microarray and RNA-seq (sequencing) datasets. Expression of select candidate genes was validated by regular and real-time quantitative RT-PCR. Expression of lens epithelium-enriched genes Foxe3, Pax6, Anxa4 and Mcm4 is up-regulated in LEC lines, compared to isolated lens fiber cells. Moreover, similar to isolated lens epithelium, all three LECs exhibit down-regulation of fiber cell-expressed genes Crybb1, Mip and Prox1 when compared to fiber cells. These data indicate that the LEC lines exhibit greater similarity to lens epithelium than to fiber cells. Compared to non-lens cell line NIH3T3, LECs exhibit significantly enriched expression of transcription factors with important function in the lens, namely Pax6, Foxe3 and Prox1. In addition to these genes, all three LECs also express key lens- and cataract-associated genes, namely Dkk3, Epha2, Hsf4, Jag1, Mab21l1, Meis1, Pknox1, Pou2f1, Sfrp1, Sparc, Tdrd7 and Trpm3. Additionally, 21EM15 microarrays indicate expression of Chmp4b, Cryab and Tcfap2a among others important genes. Immunostaining with makers for Processing bodies (P-bodies) and Stress granules (SGs) demonstrates that these classes of RGs are robustly expressed in all three LECs. Moreover, under conditions of stress, 17EM15 and αTN4 exhibit significantly higher numbers of P-bodies and SGs compared to NIH3T3 cells. In sum, these data indicate that mouse LECs 21EM15, 17EM15 and αTN4 express key lens or cataract genes, are similar to lens epithelium than fiber cells, and exhibit high levels of P-bodies and SGs, indicating their suitability for investigating gene expression control and RG function in lens-derived cells.

Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

Hidden DNA Copy Number Alterations and Mutations in IKZF1, TP53, CRLF2 and JAK2 Genes Are Associated with a Poor Prognosis in B-Progenitor Acute Lymphoblastic Leukemia

Background: In B-progenitor acute lymphoblastic leukemia (B-ALL) the identification of additional genetic alterations associated with treatment failure is still a challenge.Aims: 1.To identify genomic gains and losses in B-ALL at the time of diagnosis and to correlate these abnormalities with the genetic characteristics and the patients outcome. 2.To assess the prevalence and prognostic impact of genetic lesions in IKZF1, TP53, CRLF2, IL7R, PAX5, JAK2 and LEF1 genes in B-ALL.Methods: A total of 215 B-ALL patients were eligible for this study. 115 (53.5%) had less than <18 years. In all cases oligonucleotide microarrays (aCGH) were carried out. For genetic gain and losses analysis, the NimbleGen CGH 12x135K array (Roche) was used. The data analysis was performed with GISTIC and aCGHweb software. In 118 of these cases (65 children and 53 adults), an integrative analysis of aCGH and NGS was performed to identify genetic lesions in genes associated with B-ALL: JAK2 (Exon 12-Exon 16), PAX5 (E2-E3), LEF1 (E2-E3), CRLF2 (E6), IL7R (E5), IKZF1 (E2-E8) and TP53 (E4-E11). The 454 GS Junior system (Roche) was used. The variant analysis was performed using Amplicon Variant Analyzer (AVA-Roche 454) and Sequence Pilot (JSI Medical Systems) software.Results: DNA copy number aberrations (CNAs) were observed in 96.5% of cases. Gains on chromosomes 4 (10%), 6 (10%), 10 (12%), 14 (13%), 21 (31%) and X (18%) were more common in children, whereas losses in 7p (13%) and 9p21 (24%) were frequent in adults. In children, gains on chromosomes 6, 10, 14q, 17, 18, 21 and X were associated with longer overall survival (OS) while loss on 7p and 17p were associated with shorter OS in adults (Table 1).The integrative analysis of aCGH and NGS showed that 60% of patients carried at least one alteration (deletion and/or mutation) in the seven genes analyzed. Focal deletions were common in IKZF1, CRLF2, PAX5, and LEF1 genes while broad deletions were more frequent in TP53, JAK2 and IL7R. Forty-one percent of patients harbored IKZF1 alterations (IKZF1+), 22.9% in PAX5, 18.6% in JAK2 and 11% had TP53 or CRLF2 alterations. LEF1 and IL7R genomic lesions were only present in 4.2% and 2.5% of the B-ALL, respectively. JAK2 and CRLF2 mutations were associated (p=0.01). Moreover abnormalities in IKZF1+ were associated with alterations in JAK2 (p=0.004), TP53 (p=0.04) and PAX5 (p=0.03).The presence of alterations was most frequent in high-risk (HR) B-ALL (38% vs 18%, p=0.02), although 8% low-risk (LR) childhood patients showed genetic alterations. Fourteen B-ALL patients carried mutations in TP53, CRLF2 and/or JAK2 genes: all of them were Ph-, and 11 were classified as HR. Of note, 3 cases were children. In the HR group the presence of mutation in TP53, CRLF2 and/or JAK2 was related to a shorter OS (3-year OS: 56% vs. 36%; P=0.034) and event-free survival (3-year EFS: 56% vs. 11%; P<0.002) (Figure 1), due to an increased cumulative incidence of relapse (3-year CIR: 38% vs. 88%; P<0.004).The presence of IKZF1 alterations stratified HR-Ph- B-ALL cases. Thus HR-Ph- patients with normal IKZF1 were associated with longer EFS compared to HR-Ph- B-ALL IKZF1+ (3-year EFS: 60% vs. 33%; P=0.005) (Figure 2). The presence of IKZF1 deletion was associated with shorter EFS (89% vs. 67%, P= 0.04) and increased CIR (3-year CIR: 12% vs. 33%; P=0.05) in children.Summary/Conclusions: CNAs are frequent in B-ALL and are associated with genetic subtype, age and overall survival. The integrative analysis by aCGH and NGS techniques demonstrated that alterations in IKZF1 gene and mutations in TP53, CRLF2 and/or JAK2 genes could stratify high-risk Ph- B-ALL patients.Subvention:FP7/2007-13, Nº306242-NGS-PTL; HUS272U13, JCyL,Consejería de Educación; BIO/SA31/13 Gerencia Regional de Salud, SACYL, SpainTable 1DNA copy number aberrations (CNAs) associated with overall survival in 215 B-ALL patientsAssociationCNAnMedian (months)P valueShort overall survival7p-ChildrenAdults1961396150.0517p-ChildrenAdults12576940.029+19ChildrenAdults351421Not reached100.00122q+ChildrenAdults261214Not reached7<0.0001Longoverall survival+6ChildrenAdults26197Not reached7<0.0001+10ChildrenAdults25196Not reached70.001+14qChildrenAdults26188Not reached4<0.0001+17ChildrenAdults24177Not reached600.012+18ChildrenAdults25187Not reachedNot reached<0.000121q+ChildrenAdults473116Not reached8<0.0001XChildrenAdults522725Not reached9<0.0001 [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genetic Abnormalities and Prognosis in Pediatric B–precursor Acute Lymphoblastic Leukemia Treated on Tokyo Children′s Cancer Study Group Protocol

Introduction; Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Most childhood cases of B-precursor ALL (BCP-ALL) may be subclassified by the presence of either gross or submicroscopic genetic alteration. Recent genomewide analyses have identified genetic or cytogenetic subtypes, such as Ph-like ALL, deletion of ERG, intrachromosomal amplification of chromosome 21 (iAMP21) in BCP-ALL. Deletion of ERG and iAMP21 were associated with frequent IKZF1 deletions, which were found in Ph-like ALL associated with poor prognosis. However, the frequency and prognostic implications of these genomic abnormalities have not been defined in Japan.Patients and Methods; We studied 208 children with de novo BCP-ALL enrolled on Tokyo Children Cancer Study Group (TCCSG) ALL L0416 and L0616 trials. All patients had a diagnosis of BCP-ALL without BCR-ABL1 phenotype confirmed by morphology, immunophenotyping and mRNA Real-time PCR. Alterations of ERG and iAMP21 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) kit P327-B1. To identify Ph-like ALL and BCR-ABL1 like ALL, We investigated gene expression of 235 BCP-ALL cases enrolled on TCCSG L0416 and L0616 trials by microarray GeneChip Human Genome U133 Plus 2.0 and analyzed using clustering analysis as described previously. To assess whether these genomic alterations overlap with known gene abnormalities, such as mutations of CRLF2,JAK1, JAK2 and IL7R, rearrangement of P2RY8-CRLF2, PAX5-JAK2 and PDGFRB-EBF1 genes and copy number alteration of IKZF1, CDKN2A/2B, PAX5, EBF1 and SH2B3 genes, mutational analyses of these genes were also performed using direct sequencing, RT-PCR or MLPA kit P335-A1 and P200.Results; We identified Ph-like signature in 9 (3.8%) patients and BCR-ABL1 like ALL signature in 11 (4.7%) patients. Five (55.6%) of 9 Ph-like ALL patients and 3 (27.3%) of 11 BCR-ABL1 like ALL patients have IKZF1 deletions. Five patients were classified into both Ph-like ALL and BCR-ABL1 like ALL. Among these 5 patients, one patient had CRLF2 high expression, JAK2 R683S/R683G mutation, CDKN2A/2B deletion and EBF1 deletion, and another patient have PAX5-JAK2 rearrangement. These two patients relapsed, and the latter patient died. Three of 9 Ph-like ALL patients relapsed and 2 died, while 7 of 11 BCR-ABL1 like ALL patients relapsed and 2 died. We identified iAMP21 in 5 (2.4%) patients and deletion of ERG in 8 (3.8%). One of 5 patients with iAMP21 and 3 of 8 patients with ERG deletion had IKZF1 deletion. One patient with iAMP21 had BCR-ABL1 like ALL signature, but not Ph-like ALL signature. CRLF2 high expression was identified in two of 5 patients with iAMP21, and P2RY8-CRLF2 rearrangement and SH2B3 deletion was identified in one of them. No mutations of CRLF2 and JAK2 were identified in patients with iAMP21 and ERG deletion. Among 5 iAMP21 patients, patient with BCR-ALB1 signature and IKZF1 deletion relapsed, and one patient died. No patients with ERG deletion relapsed or died.Conclusions; Using GeneChip microarray analysis, low frequency of Ph-like ALL signature and BCR-ABL1 like ALL signature was found. Using MLPA as a novel screenings technique, frequency of iAMP21 and ERG deletion in pediatric BCP-ALL was similar to that of previous studies. BCR-ABL1 like ALL and iAMP21 was associated poor prognosis, while ERG deletion was associated good prognosis in pediatric ALL. These data suggest that screening for these genetic abnormalities in pediatric patients with BCP-ALL is critical not only for outcome prediction but also for risk-adapted therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Background of BCP-ALL Cases with an Early Switch to Monocytic Lineage

We identified a subset of BCP-ALL with switch towards the monocytic lineage within the first month of treatment (swALL)[Slámová et al Leukemia 2014]. During the switch cells gradually lose CD19 and CD34 expression and acquire CD33 and CD14 positivity. We proved clonal relatedness of switched monocytic blasts with the diagnostic leukemic cells based on identical Ig-TCR rearrangements. SwALL cases are not associated with MLL or BCR/ABL1 aberrancies and lack any known genetic markers of lineage ambiguity (detected by FISH or MLPA).We analyzed transcriptomes of swALL samples at diagnosis (n=4) and at d8 (n=4) where the immunophenotypic switching was already apparent as well as control BCP-ALL (n=4). RNA was isolated form either FACS sorted cells or whole BM when blasts constituted >80% of cells. For RNA-Seq we used Illumina HiSeq 2000 paired-end or single end sequencing. Raw sequencing data were analyzed using adapted protocol from Anders at al [Anders et al Nature Protocols 2013] and custom scripts. For methylome analysis we used Enhanced Reduced Representation Bisulfite Sequencing (ERRBS)[Akalin et al PLoS Genetics 2012]. ERRBS quantitatively measures DNA methylation at ~3M CpGs genome-wide. Samples from swALL at diagnosis (n=7) and at d8 (n=4) and control BCP-ALL (n=4) were processed. Analysis was performed according to [Akalin et al Genome Biology 2012] and followed with custom analysis in R statistical language.Comparison (generalized exact binomial test) of transcriptomes of B-lineage blasts from diagnosis between swALLs and control BCP-ALLs revealed a number of differentially expressed genes. Among 300 most significantly differentially expressed were KLF4, CEBPD, CLEC12A and CLEC12B (upregulated in swALL) and ANXA5, VPREB1, CD9 and IGHG3 (downregulated in swALL). Hierarchical clustering separated not only swALL and control BCP-ALL, but also swALL cells before and during the monocytic switch. Changes in gene expression during lineage switch included downregulation of ITGA6, Id2, EBF1, CD19, CD34, FLT3, MYB, CD79a, BCR, PAX5, GATA3 and TCF3 genes and upregulation of S100A10, AIF1, CD14, CD33, LGALS1, RNF130 and MNDA.When comparing all three cell types (swALL B cell and monocytic blasts and control BCP-ALL blasts) we concentrated on 1) immunophenotype switch markers and 2) lineage related transcription factors (TF):1) Both markers typical for B cell blasts (CD19, CD34) decreased during the switch. However while CD19 was expressed in swALL at diagnosis at same levels as in control BCP-ALL, CD34 was overexpressed in swALL compared to BCP-ALL at diagnosis. Both monocytic markers (CD33, CD14) increased their expression during the switch. CD14 showed no difference between swALL and control BCP-ALL at diagnosis. However CD33 was interestingly upregulated in swALL already at diagnosis and continued to rise during the switch. SwALL had therefore deregulated expression of lineage commitment markers already at diagnosis favoring stemness marker CD34 and myeloid marker CD33.2) B lineage commitment related TFs (EBF1, TCF3, PAX5) were expressed in B lineage blasts in both swALL and control BCP-ALL. However they were all downregulated during the switch. On the other hand myeloid lineage related transcription factor CEBPA is overexpressed in diagnostic B lineage blasts in swALL compared to control BCP-ALL cases. Similarly CEBPD is overexpressed in swALL and its expression further rises during the switch. Other hematopoietic TFs upregulated in swALL cases include KLF4, NANOG and GATA3.To confirm some of the epigenetic markers of swALL cases (demethylation of CEBPA promoter) and to widen epigenetic screening we used ERRBS. While some of the upregulated genes had expectedly hypomethylated promoters in swALL (CEBPA, GATA3) other genes (TCF3, PAX5) had demethylated promoters in all cases. While the whole DNA methylation picture is still a challenge to draw both omics method could clearly separate swALL cases from control BCP-ALL using principal component analysis.In summary we show that immunophenotypic shift is associated with gene expression changes of surface markers, lineage specific transcription factors and other genes. Some of the genes have altered expression already at diagnosis. Expression of some key lineage genes is differentially regulated by DNA methylation.Supported by: GAUK 914613, GAČR P301/10/1877, UNCE 204012, IGA NT13462-4 DisclosuresNo relevant conflicts of interest to declare.

TP53 Alterations Are Frequent in Patients over 60 Years with B-Precursor Acute Lymphoblastic Leukemia (ALL) and Low Hypodiploid/Near Triploid (HoTr) Karyotype; They Correlate with RB1 Deletion and Leukemic Telomere Gain

IntroductionTP53 is the most frequently altered gene in cancer. Until recently data on its frequency and prognostic impact in ALL has been scant, particularly in the elderly. TP53 alteration in the context of telomere shortening (the hallmark of aging) results in telomere attrition and genomic instability with resultant overexpression of telomerase to overcome the genomic crisis.ObjectiveTo determine TP53 alteration frequency and its association with cytogenetic subgroups, key B cell differentiation/cell cycle genes abnormalities and induction therapy response in ALL patients aged ≥60 and its impact on leukemic telomere stateMethodsPatients enrolled in the UK NCRI UKALL14 and UKALL60+ trials aged ≥60 years were assessed at diagnosis for 17p deletions by cytogenetics, TP53 alterations by FISH and/or direct sequencing of hot spots exons 5-8. Data were correlated with IKZF1, CDKN2A/B, PAR1, BTG, RB1, ETV6, EBF and PAX5 gene copy number status, assessed by MLPA (P335-B1). MYC-rearrangement was an exclusion criterion. Minimal residual disease (MRD) was assessed by quantitative PCR (qPCR) for Ig/TCR rearrangements or BCR-ABL1, where applicable. Leukemic DNA telomere length relative to that of remission DNA was assessed by monochrome multiplex (MM) qPCR and expressed as a log2 ratio. Using expectation maximization mixture model a log2 ratio >3.18 defined gain (reflecting telomerase overexpression) and values below this were defined as loss.ResultsThe cohort included 63 patients; characteristics are presented in table 1. Median age was 63 years (range 60–83). TP53 state by cytogenetics and/or sequencing was available in all. Eleven had TP53 alteration (17%); TP53 mutation only, n=3; TP53 deletion only, n=6; TP53 deletion and mutation, n=2. Cytogenetics was available in 60 patients. Nine patients had HoTr (14%). Seven of 9 patients with HoTr had TP53 alteration (78%) versus 4/51 without HoTr (4%); p<0.001. MLPA was done in 57 patients. Those with TP53 alteration had more copy number alterations (CNA)(median 4 versus 1; p<0.01). Twelve (21%) had RB1 deletion; 3 were due to cytogenetically visible 13q abnormalities rather than ‘true’ intragenic aberration. Four of 9 patients with TP53 alteration had RB1 deletion versus 8/51 without TP53 alteration, p=0.07. All other microaberrations were not significantly associated with TP53 alteration. Sixty-two achieved complete remission after 2 cycles of induction. MRD state (n=32) was not impacted by TP53 (2/5 with TP53 alteration and 13/27 without were MRD positive after 2 cycles of induction; p=0.563). Fourteen of 59 (24%) patients relapsed; median of four months from diagnosis (range 2–21), three had TP53 alteration versus 11 without TP53 alteration; p=0.65. Relapse occurred significantly earlier in patients with TP53 alteration (median two months versus eight months; p=0.03). Leukemic telomere state was assessed in 28 patients. Three of 4 patients with telomere gain had TP53 alteration versus 3/24 without; p=0.02ConclusionsThis is one of the largest reports of TP53 frequency in older patients with non-MYC-rearranged ALL. TP53 was deleted and/or mutated in 17%, with highest frequency in those with HoTr (7/9). RB1 deletion was the commonest associated microaberration, noted in over a third of patients. TP53 alteration was associated with earlier relapse but had no impact on induction response. TP53 alteration was notably associated with frequent CNA and telomere gain suggesting a putative potential role of TP53 in genomic instability though sample size for the latter limits assertion of this hypothesis. Mechanistic work to shed insight into this model is currently underway.Table 1: Patient characteristicsTP53 deleted and/or mutated n=11TP53 wild type n=52Age at Diagnosis, median(range)63(60-75)63(60-83)Gender; M:F06:0523:29Phenotype, n (%)· Precursor B cell11 (100)51 (98)· Precursor T cell0 (0)1 (2)Cytogenetics (risk stratification as per Moorman et al. Blood 2007; 109:3189–97), n (%)Standard Risk0 (0)23 (44)t(9;22)1 (9)21 (40)t(4;11)0 (0)2(4)Ho/Tri7 (64)2(4)Complex2 (18)2 (4)Unknown1 (9)2 (4)Morphological Remission after Cycle 2 Induction, n (%)11 (100)51(98)Induction Deaths, n (%)2 (18)15 (29)CNA by MLPA, median (range)4 (2-7)1 (0-2)RB1 deletion, n (%)· Present4 (36)8 (15)· Absent5 (45)43 (83)· Unknown2 (18)1 (2)Leukemic Telomere State· Gain3 (27)3 (6)· Loss1 (9)21 (40)· Unknown7 (64)28 (54) DisclosuresNo relevant conflicts of interest to declare.

Na, K-ATPase β2 isoform (atp1b2) expressed in the retina of Xenopus

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase β2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and pax6. Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

The role of Pax5 in leukemia: diagnosis and prognosis significance.

Pax5 transcription factor, also known as B-cell specific activator protein (BSAP), plays a dual role in the hematopoietic system. Pax5 expression is essential in B-cell precursors for normal differentiation and maturation of B-cells. On the other hand, it inhibits the differentiation and progress toward other lineages. The expression of this factor is involved in several aspects of B-cell differentiation, including commitment, immunoglobulin gene rearrangement, BCR signal transduction and B-cell survival, so that the deletion or inactivating mutations of Pax5 cause cell arrest in Pro-B-cell stage. In recent years, point mutations, deletions and various rearrangements in Pax5 gene have been reported in several types of human cancers. However, no clear relationship has been found between these aberrations and disease prognosis. Specific expression of Pax5 in B-cells can raise it as a marker for the diagnosis and differentiation of B-cell leukemias and lymphomas as well as account for remission or relapse. Extensive studies on Pax5 along with other genes and immunomarkers are necessary for decisive results in this regard.

Recurrent de novo mutations implicate novel genes underlying simplex autism risk.

Autism spectrum disorder (ASD) has a strong but complex genetic component. Here we report on the resequencing of 64 candidate neurodevelopmental disorder risk genes in 5,979 individuals: 3,486 probands and 2,493 unaffected siblings. We find a strong burden of de novo point mutations for these genes and specifically implicate nine genes. These include CHD2 and SYNGAP1, genes previously reported in related disorders, and novel genes TRIP12 and PAX5. We also show that mutation carriers generally have lower IQs and enrichment for seizures. These data begin to distinguish genetically distinct subtypes of autism important for etiological classification and future therapeutics.