Band Q22 Research Articles

Lost in HELLS: Disentangling the mystery of SALNR existence in senescence cellular models.

Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular senescence by transcriptionally and post-transcriptionally modulating the expression of many important genes involved in senescence-associated pathways and processes. Among the different lncRNAs associated to senescence, Senescence Associated Long Non-coding RNA (SALNR) was found to be down-regulated in different cellular models of senescence. Since its release in 2015, SALNR has not been annotated in any database or public repository, and no other experimental data have been published. The SALNR sequence is located on the long arm of chromosome 10, at band 10q23.33, and it overlaps the 3' end of the HELLS gene. This investigation helped to unravel the mystery of the existence of SALNR by analyzing publicly available short- and long-read RNA sequencing data sets and RT-PCR analysis in human tissues and cell lines. Additionally, the expression of HELLS has been studied in cellular models of replicative senescence, both in silico and in vitro. Our findings, while not supporting the actual existence of SALNR as an independent transcript in the analyzed experimental models, demonstrate the expression of a predicted HELLS isoform entirely covering the SALNR genomic region. Furthermore, we observed a strong down-regulation of HELLS in senescent cells versus proliferating cells, supporting its role in the senescence and aging process.

Fusion Gene-Based Classification of Variant Cytogenetic Rearrangements in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) represents a heterogeneous disease entity that is continuously moving to a more genetically defined classification. The classification of AML with recurrent chromosomal translocations, including those involving core binding factor subunits, plays a critical role in diagnosis, prognosis, treatment stratification, and residual disease evaluation. Accurate classification of variant cytogenetic rearrangements in AML contributes to effective clinical management. We report here the identification of four variant t(8;V;21) translocations in newly diagnosed AML patients. Two patients showed a t(8;14) and a t(8;10) variation, respectively, with a morphologically normal-appearing chromosome 21 in each initial karyotype. Subsequent fluorescence in situ hybridization (FISH) on metaphase cells revealed cryptic three-way translocations t(8;14;21) and t(8;10;21). Each resulted in RUNX1::RUNX1T1 fusion. The other two patients showed karyotypically visible three-way translocations t(8;16;21) and t(8;20;21), respectively. Each resulted in RUNX1::RUNX1T1 fusion. Our findings demonstrate the importance of recognizing variant forms of t(8;21) translocations and emphasize the value of applying RUNX1::RUNX1T1 FISH for the detection of cryptic and complex rearrangements when abnormalities involving chromosome band 8q22 are observed in patients with AML.

A cryptic EWSR1::DDIT3 fusion in myxoid liposarcoma: Potential pitfalls with FISH and cytogenetics.

Myxoid liposarcoma (MLS) is molecularly characterized by fusions involving the DDIT3 gene in chromosome band 12q13; the fusion partner is FUS in band 16p11 in 90-95% of the cases and EWSR1 in band 22q12 in the remaining 5-10%. Hence, molecular studies, often fluorescence in situ hybridization (FISH) for DDIT3 rearrangement, are useful for establishing a correct diagnosis. Although all MLS tumors should have DDIT3 fusions, it is important to be aware of reasons for potential false-negative results. We here present a case of MLS that was negative for FISH for DDIT3, that showed an unexpected t(11;22) at G-banding, but that displayed a characteristic EWSR1::DDIT3 fusion at RNA-sequencing. The results suggest that neoplasia-associated fusions that, due to the transcriptional orientations of the two genes involved, cannot arise through only two double-strand breaks are more likely to be associated with negative FISH-findings and unexpected karyotypes.

Tifab Deficiency Protects Against Stress-Induced Bone Marrow Failure at the Expense of Pre-Leukemic Selection

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. TRAF-interacting protein with forkhead-associated domain B (TIFAB) resides within the proximal commonly-deleted region on band 5q31.1, and belongs to a family of forkhead-associated domain proteins. TIFAB is deleted in nearly all reported cases of del(5q) MDS and AML. As expected, TIFAB expression is significantly lower in CD34+ and BM mononuclear cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q. Recently we have shown that hematopoietic-specific deletion of Tifab in mice results in progressive BM and blood defects, including aberrant HSPC proportions, altered myeloid differentiation, and progressive cytopenia (Varney and Niederkorn et al., JEM 2015). Gene expression analysis of Tifab KO lineage-Sca1+cKit+ (LSK) cells identified dysregulation of immune-related signatures, hypersensitivity to Toll-like receptor stimulation, and enrichment of a p53 regulatory gene-set.Importantly, p53 is implicated in the pathogenesis of del(5q) MDS: 1) BM cells from murine models and BM from del(5q) patients exhibit increased p53 activity, which is thought to contribute to ineffective hematopoiesis and anemia; and 2) del(5q) MDS patients often acquire concurrent TP53 mutations that result in rapid transformation to AML and poor treatment response. To examine the effects of TIFAB on p53 function, we examined p53 target genes in TIFAB-overexpressing and -deficient cells. Gene expression profiling and qRT-PCR analysis of Tifab KOHSPCs revealed significant upregulation of p53 regulatory genes. In contrast, overexpression of TIFAB in a p53-competent cell line reduced the expression of p53 target gene, p21 . We also observed increased p53 protein in Tifab KO total BM, lineage- BM, and spleen. At steady-state and after in vitro treatment with 2 Gy irradiation, Tifab KO lineage- BM cells exhibit increased p53 protein compared to WT cells.To test whether this increased p53 stability renders Tifab -deficient HSPCs more sensitive to stress, we treated Tifab KO HSPCs both in vivo and ex vivo using irradiation. When treated with low dose irradiation ex vivo, Tifab KO HSPCs initially exhibit significantly reduced colony forming ability; however, upon secondary re-plating, Tifab KO HSPCs strikingly form more colonies than WT HSPC, resembling a clonal selection event. To model this effect in vivo, we transplanted WT or Tifab KO BM into lethally-irradiated recipient mice. After 4 weeks of hematopoietic reconstitution, recipient mice were treated with 6.5 Gy sublethal irradiation. We find that Tifab KO recipient mice are less likely to succumb to IR-induced bone marrow failure as compared to mice transplanted with WT BM cells. Instead, after 3+ months, the remaining Tifab KO recipient mice have an increased tendency to develop hematologic malignancies, evident by myeloid blasts. Collectively, these data suggest that TIFAB-deficiency protects against cellular stress by eliminating “less fit” HSPCs, at the expense of selecting for pre-leukemic populations, paralleling observations in patients with transformed MDS.To investigate the mechanism of this phenomenon, we performed a tandem-affinity tag purification and mass-spectrometry analysis of TIFAB complexes in a del(5q) AML cell line (HL60), and identified unique TIFAB-interacting proteins. The top interacting candidate was an ubiquitin-specific peptidase (USP), USP15. USPs play a major role in ubiquitin-dependent processes including DNA damage response signaling, protein degradation, and kinase activation. Specifically, USP15 has been shown to promote p53 degradation via deubiquitination and stabilization of its major negative regulator, MDM2. Through biochemical assays and a series of deletion mutants, we confirmed that TIFAB interacts with USP15 and enhances the deubiquitination of biological USP15 substrates, including MDM2. Thus, our data implicate TIFAB as an important negative regulator of p53 and modulator of USP15 DUB activity.Altogether, our findings identify a novel role for TIFAB as a modifier of p53 function, potentially via a USP15-MDM2 axis. These findings have important implications in the potential role of TIFAB and p53 signaling in the pathogenesis of del(5q) MDS and transformation to AML. DisclosuresNo relevant conflicts of interest to declare.

Involvement of the AML1 Gene in the t(3;21) in Therapy-Related Leukemia and in Chronic Myeloid Leukemia in Blast Crisis

AbstractA nonrandom translocation between chromosomes 3 and 21, t(3;21)(q26.2;q22) has been detected in patients with a myelodysplastic syndrome or acute myeloid leukemia after treatment (t-MDS/t-AML) for a primary malignant disease and in chronic myelogenous leukemia in blast crisis (CML-BC). In these patients, the breakpoint on chromosome 21 is at band 21q22. This band is also involved in the t(8;21)(q22;q22) detected in 40% of the patients with acute myeloid leukemia subtype M2 (AML-M2) de novo who have an abnormal karyotype. In the t(8;21), the AML1 gene is the site of the breakpoint on chromosome 21. The AML1 gene is transcribed from telomere to centromere, and in the t(8;21) the 5′ part of AML1 is fused to the ETO gene on chromosome 8 to produce the chimeric AML1/ETO on the der(8) chromosome. We found that AML1 is also rearranged in two t-AML patients and in one CML-BC patient with the t(3;21), but the breakpoints are approximately 40 to 60 kb downstream to those of AML-M2 patients. This region contains at least one additional exon of AML1, as determined by using an AML1 cDNA as a probe in Southern blot analysis. The t(3;21) breakpoints for the remaining patients could not be determined because, by fluorescence in situ hybridization analysis, the breaks are outside of the region covered by the available probes.

A case report of familial 4q13.3 microdeletion in three individuals with syndromic intellectual disability

BackgroundInterstitial 4q deletions are rare chromosomal alterations. Most of the previously reported deletions involving the 4q13.3 region are large chromosomal alterations with a common loss of band 4q21 resulting in marked growth restriction, severe intellectual disability, and absent or severely delayed speech. A microdeletion of 4q13.3 hasn’t been previously reported. We discuss the involvement of genes and the observed phenotype, comparing it with that of previously reported patients.Case presentationWe report on a 4q13.3 microdeletion detected in three affected individuals of a Lithuanian family. The clinical features of two affected children and their affected mother are very similar and include short stature, congenital heart defect, skeletal anomalies, minor facial anomalies, delayed puberty, and intellectual disability. Whole genome SNP microarray analysis of one child revealed an interstitial 4q13.3 microdeletion, 1.56 Mb in size. FISH analysis confirmed the deletion in the proband and identified the same deletion in her affected sib and mother, while it was not detected in a healthy sib. Deletion includes ADAMTS3, ANKRD17, COX18, GC, and NPFFR2 protein-coding genes.ConclusionsOur findings suggest that 4q13.3 microdeletion is a cause of a recognizable phenotype of three affected individuals. The detected microdeletion is the smallest interstitial deletion in 4q13. We highlight ADAMTS3, ANKRD17 and RNU4ATAC9P as candidate genes for intellectual disability, growth retardation and congenital heart defect.

The p53 Slavic Gene Mutation Nijmegen Breakage Syndrome

Nijmegen breakage syndrome is a rare autosomal congenital disorder. It originates from mutations in the NBS1 gene located in band 8q21, which encodes for the DNA double strand repair protein Nibrin. Immunodeficiency with increased susceptibility to infection. It is a condition characterized by short stature, an unusually small head size (microcephaly), distinctive facial features, recurrent respiratory tract infections, an increased risk of cancer, intellectual disability, and other health problems. People with this condition typically grow slowly during infancy and early childhood. After this period of slow growth, affected individuals grow at a normal rate but remain shorter than their peers. Microcephaly is apparent from birth in the majority of affected individuals. The head does not grow at the same rate as the rest of the body, so it appears that the head is getting smaller as the body grows (progressive microcephaly).Individuals with Nijmegen breakage syndrome have distinctive facial features that include a sloping forehead, a prominent nose, large ears, a small jaw, and outside corners of the eyes that point upward (up slanting palpebral fissures). These facial features typically become apparent by age 3. Keywords: Nijmegen breakage syndrome; Chromosomal instability; Immunodeficiency; Microcephaly

Deep sequencing of myxoinflammatory fibroblastic sarcoma.

Myxoinflammatory fibroblastic sarcoma (MIFS) has recurrent genetic features in the form of a translocation t(1;10)(p22-31;q24-25), BRAF gene fusions, and/or an amplicon in 3p11-12 including the VGLL3 gene. The breakpoints on chromosomes 1 and 10 in the t(1;10) cluster in or near the TGFBR3 and OGA genes, respectively. We here used a combination of deep sequencing of the genome (WGS), captured sequences (Cap-seq), and transcriptome (RNA-seq) and genomic arrays to investigate the molecular outcome of the t(1;10) and the VGLL3 amplicon, as well as to assess the spectrum of other recurrent genomic features in MIFS. Apart from a ROBO1-BRAF chimera in a t(1;10)-negative MIFS-like tumor, no fusion gene was found at RNA-seq. This was in line with WGS and Cap-seq results, revealing variable breakpoints in chromosomes 1 and 10 and genomic breakpoints that should not yield functional fusion transcripts. The most common genomic rearrangements were breakpoints in or around the OGA, NPM3, and FGF8 genes in chromosome band 10q24, and loss of 1p11-p21 and 10q26-qter (all simultaneously present in 6/7 MIFS); a breakpoint in or near TGFBR3 in chromosome 1 was found in four of these tumors. Amplification and overexpression of VGLL3 was a consistent feature in MIFS and MIFS-like tumors with amplicons in 3p11-12. The significant molecular genetic outcome of the recurrent t(1;10) could be loss of genetic material from 1p and 10q. Other recurrent genomic imbalances in MIFS, such as homozygous loss of CDKN2A and 3p- and 13q-deletions, are shared with other sarcomas, suggesting overlapping pathogenetic pathways.

Role of Genetic Evolution and Germline Mutations in SAMD9 and SAMD9L Genes

Acquired deletions on chromosome 7 (monosomy 7/del7q) are common in myeloid neoplasms, especially pediatric MDS and AML. Although these tumors have historically been reported to occur within families, suggesting a genetic predisposition, the genetic lesion(s) that initiate these diseases has remained elusive until the last few years. Following a series of publications in which germline mutations in SAMD9 and SAMD9L were reported in a MIRAGE syndrome and Ataxia Pancytopenia syndrome, respectively, our group and others described similar heterozygous missense germline mutations in pediatric MDS, especially non-syndromic familial MDS with monosomy 7. Mutations in SAMD9 and SAMD9L have now also been reported in transient monosomy 7, inherited bone marrow failure and AML. Collectively, it is estimated that germline mutations in these genes are present in nearly 20% of children with MDS, with a strong enrichment in those with monosomy 7. Surprisingly, SAMD9 and SAMD9L are paralogous genes adjacently located on human chromosome 7 at band 7q21, and the monosomy 7 clone that expands in children universally lacks the pathologic germline variant. Expression of the mutant proteins in cells results in profound growth suppression, suggesting that there is strong selective pressure for hematopoietic cells to not express the mutant alleles. In addition to chromosome loss, additional methods that suppress expression of the pathologic allele have been described. These include copy neutral loss of heterozygosity (CN-LOH) with duplication of the wild-type allele or the somatic acquisition of additional mutations in cis with the germline mutation that counteract the growth suppressive effect of the germline mutation. The clinical phenotype is largely dictated by the revertant mutation in the dominant hematopoietic clone within the patient's bone marrow. Those with an expansion of a CN-LOH clone are more commonly asymptomatic, in contrast to those patients with a dominant monosomy 7 clone. Progression to higher grade MDS or AML is associated with the acquisition of additional somatic mutations including mutations in SETBP1, KRAS and RUNX1. The recognition of these germline mutations has had an immediate impact on the clinical management of children with MDS, including their family members, and ongoing clinical work in the pediatric MDS community is aimed at establishing guidelines for the pathologic diagnosis, clinical monitoring and treatment for these patients. In addition to these ongoing clinical pursuits, there is significant research interest in these genes, the function of their proteins in hematopoietic cells and how the germline mutations alter the function of the wild-type protein. The SAMD9 and SAMD9L proteins are largely uncharacterized and have been shown to be important in endocytosis, growth factor signaling and to have antiviral properties. Intriguingly, SAMD9 and SAMD9L are both induced by inflammatory signals, including interferons, suggesting a link between inflammatory stress and the disease phenotype. Ongoing studies are aimed at developing models, including in vitro and in vivo models, to understand the mechanisms by which these germline mutations can ultimately lead to the development of pediatric MDS and related disorders. Disclosures No relevant conflicts of interest to declare.

Generation of four induced pluripotent stem cell lines, GZWWTi001-A, GZWTZi001-A, GZWXYi001-A, and GZWXDi001-A, derived from peripheral blood mononuclear cells from a family with asparagine synthetase deficiency.

Asparagine synthetase (ASNS) deficiency (ASNSD; MIM #615574) is a rare neurodevelopmental disorder caused by mutations in the ASNS gene. The ASNS gene maps to cytogenetic band 7q21.3 and is 35kb long. ASNSD is characterised by congenital microcephaly, severely delayed psychomotor development, seizures, and hyperekplexic activity. Here, we reported a family with compound heterozygous mutations in ASNS (NM_001178076:c.551C>T; c. 944A>C) and established induced pluripotent stem cells (iPSCs) from blood samples. To date, limited functional data have been reported to explain the underlying pathophysiology of ASNSD; therefore, iPSCs from these patients may be powerful tools for studying disease mechanisms.

PS1330 VARIABILITY IN THE EXTENT OF DEL(5Q) AND ITS CLINICAL IMPLICATION IN MYELODYSPLASTIC SYNDROMES (MDS)

Background:The interstitial deletion of the long arm of chromosome 5—del(5q)—is the most common cytogenetic abnormality in MDS occurring either as a sole aberration or as a part of complex karyotypes (CK). MDS with isolated del(5q) are associated with a favorable outcome while MDS with CK relate to poor prognosis. Extent of deleted segment is highly variable. However, it remains unclear whether its size matters for the different MDS phenotypes.Aims:The aim was to compare the extent of deletion in 264 MDS patients [116 with isolated del(5q) and 148 with del(5q) involved in CK] and to assess the relationship of del(5q) extent and TP53 gene mutation.Methods:Extent of del(5q) was analyzed using I‐FISH (Abbott, MetaSystems), mBAND (MetaSystems) and array CGH/SNP (CytoChip Cancer SNP 180K, Illumina or SurePrint G3 Cancer CGH+SNP 4x180K, Agilent). CK were analyzed with mFISH (MetaSystems). Sequence analysis of TP53 gene was performed in 84 cases using amplicon deep sequencing (exons 4–11) on a 454 GS Junior system (Roche) or with TruSight Myeloid Panel Kit on MiSeq sequencing instruments (Illumina).Results:In the group with isolated del(5q), the most frequently deleted segment ranged between the bands 5q14 and 5q33.3, with the smallest deletion encompassing the 5q31.1–5q31.3 region (18.527 Mb). The TP53 mutation was proved in 19.4% cases. The size of the deletion did not significantly differ in patients with and without TP53 mutation. In the group with CK, the deletion often involved entire long arm including the telomeric region. In some cases, a part of short arm was also deleted, but we never observed the loss of the entire chromosome 5. The commonly conserved region (CCR) was localized in bands 5p12 to 5p14.1. In this group, the mutation of TP53 and/or LOH17p was detected in 49% of patients. The commonly deleted regions (CDR) were in the band 5q31.1 (5.522 Mb) in cases with isolated del(5q) and between the bands 5q31.1 and 5q31.3 (18.527 Mb) in patients with CK.Summary/Conclusion:Patients with isolated del(5q) had a smaller size of the deleted segment. More extensive 5q deletion was associated with higher karyotype complexity, increased frequency of TP53 aberrations and worse prognosis. Accurate analysis of breakpoints and range of del(5q) points out to the correlation of deletion size with increasing genomic instability in MDS and contributes to a better understanding of the MDS pathogenesis.Supported by RVO‐VFN64165, GACR P302/12/G157, ProgresQ28/LF1, MHCR 00023736.

Deletions of Chromosome 7q Affect Nuclear Organization and HLXB9Gene Expression in Hematological Disorders.

The radial spatial positioning of individual gene loci within interphase nuclei has been associated with up- and downregulation of their expression. In cancer, the genome organization may become disturbed due to chromosomal abnormalities, such as translocations or deletions, resulting in the repositioning of genes and alteration of gene expression with oncogenic consequences. In this study, we analyzed the nuclear repositioning of HLXB9 (also called MNX1), mapping at 7q36.3, in patients with hematological disorders carrying interstitial deletions of 7q of various extents, with a distal breakpoint in 7q36. We observed that HLXB9 remains at the nuclear periphery, or is repositioned towards the nuclear interior, depending upon the compositional properties of the chromosomal regions involved in the rearrangement. For instance, a proximal breakpoint leading the guanine-cytosine (GC)-poor band 7q21 near 7q36 would bring HLXB9 to the nuclear periphery, whereas breakpoints that join the GC-rich band 7q22 to 7q36 would bring HLXB9 to the nuclear interior. This nuclear repositioning is associated with transcriptional changes, with HLXB9 in the nuclear interior becoming upregulated. Here we report an in cis rearrangement, involving one single chromosome altering gene behavior. Furthermore, we propose a mechanistic model for chromatin reorganization that affects gene expression via the influences of new chromatin neighborhoods.

Abstract 3885: 11q13 amplification selects for sensitivity to the ERK inhibitor KO-947 in squamous cell carcinomas

Abstract The MAPK pathway is a major driver of malignant progression, particularly in cancers arising from mutations in pathway components, and BRAF and MEK inhibitors have been approved for treatment of BRAF-mutant melanoma. ERK1/2 kinases are the final node in the MAPK signaling pathway and offer the possibility of clinical benefit in settings where earlier drugs are ineffective. We have previously reported that KO-947, a potent and selective inhibitor of ERK1/2 with extended target residence time and favorable pharmaceutic properties, displays robust single-agent antitumor activity in PDX models of adenocarcinomas with RAS/RAF mutations, and in squamous cell carcinomas (SCCs) that lack mutations in MAPK pathway components. Here we report the discovery of a novel biomarker that is associated with sensitivity to KO-947 in head and neck SCC and esophageal SCC PDX models. Recurring amplification of chromosome 11 between bands q13.3 and q13.4 is a feature of several tumor types, including ESCC and HNSCC. TCGA reports incidence rates of >50% in ESCC and ~20% in HNSCC, although higher frequencies are seen in some subtypes, such as pharyngeal and HPV-negative SCC. The 11q13 amplicon commonly contains about a dozen genes, including several potential oncogenes with functional linkage to the MAPK pathway, such as CCND1, FADD and the recently described calcium-dependent chloride channel ANO1. PDX campaigns were carried out in 24 ESCC and 18 HNSCC models, including 16 and 7 of each with 11q13 amplification, respectively. Groups of three animals were dosed with KO-947 at 300mg/kg QW, tumor growth was monitored for 3-6 weeks and responses were categorized as progressive disease, stable disease (SD, >80% TGI) or partial/complete response (PR/CR, >30% regression). In ESCC tumor-bearing animals, the overall response rates (ORR = PR+CR) were 33% in the overall population, 51% in the 11q13-amplified (11q-AMP) and 3% in 11q13-WT. The disease control rates (DCR = PR/CR+SD) were 54% overall, 77% in 11q-AMP and 21% in 11q-WT. In HNSCC tumor-bearing animals, the ORRs were 24% in the overall population, 56% in 11q-AMP and 9% in 11q-WT, with DCRs of 40%, 69% and 26% in the three subsets, respectively. 11q-AMP was significantly associated with response to KO-947 in both tumor types. ROC analysis defined the minimum effective copy number as 4 and revealed significant associations between expression levels of several 11q13 amplicon genes and response to KO-947. A key role for ANO1 in driving ERK-dependent tumor growth in 11q-AMP cases was further indicated by the observation that ANO1 expression was silenced in some 11q-AMP models and these tumors failed to respond the ERK inhibition. The results suggest that 11q13 amplification in SCCs can drive tumor growth and survival in a MAPK-dependent manner and that 11q-AMP may be a useful biomarker for predicting clinical response to ERK inhibitors. Citation Format: Francis J. Burrows, Linda Kessler, Tao Wu, Xin Gao, Jeffrey Chen, Rasmus Hansen, Shuangwei Li, Carol Thach, Shisheng Li, Ke Yu, Jeff Kucharski, Ulf Peters, Jun Feng, Yi Wang, Yvonne Yao, Ata Zarieh, Matt Janes, Jingchuan Zhang, Liansheng Li, Dana Hu-Lowe, Pingda Ren, Yi Liu. 11q13 amplification selects for sensitivity to the ERK inhibitor KO-947 in squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3885.

Tifab, a Del(5q) MDS/AML Gene, Regulates USP15 Activity and p53

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. TRAF-interacting protein with forkhead-associated domain B (TIFAB) resides within the proximal commonly-deleted region on band 5q31.1, and belongs to a family of forkhead-associated domain proteins. TIFAB is deleted in nearly all reported cases of del(5q) MDS and AML. As expected, TIFAB expression is significantly lower in CD34+ and BM mononuclear cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q. Recently we have shown that hematopoietic-specific deletion of Tifab results in progressive BM and blood defects, including aberrant HSPC proportions, altered myeloid differentiation, and progressive cytopenia (Varney and Niederkorn et al., JEM 2015). Approximately 10% of mice transplanted with Tifab KO HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. Gene expression analysis of Tifab KO lineage-Sca1+cKit+ (LSK) cells identified dysregulation of immune-related signatures, and hypersensitivity to Toll-like receptor stimulation. To investigate the molecular function of TIFAB, we performed a tandem-affinity tag purification and mass-spectrometry analysis of TIFAB complexes in a del(5q) AML cell line (HL60), and identified unique TIFAB-interacting proteins. The top interacting candidate was an ubiquitin-specific peptidase (USP), USP15. USPs play a major role in ubiquitin-dependent processes including DNA damage response signaling, protein degradation, and kinase activation. Specifically, USP15 has been shown to promote p53 degradation via deubiquitination and stabilization of its major negative regulator, MDM2. Through biochemical assays and a series of deletion mutants, we confirmed that TIFAB interacts with USP15. Moreover, we find that TIFAB enhances the deubiquitination of USP15 substrates, including MDM2 and histone 2B. To examine whether TIFAB directly regulates USP15 DUB activity, we performed in vitro deubiquitination assays in a cell-free system using fluorescent reporter di-ubiquitin substrates with purified USP15. We found that the addition of purified TIFAB increases the rate of USP15 catalytic activity on both lysine (K)48 and K63-linked di-ubiquitins in a dose-dependent manner. Collectively, these findings indicated that the USP15-TIFAB interaction leads to increased USP15 activity.USP15 stabilizes MDM2 via its DUB function, and MDM2 is known to bind and inhibit p53. Moreover, p53 is implicated in the pathogenesis of del(5q) MDS: 1) BM cells from murine models and BM from del(5q) patients exhibit increased p53 activity, which is thought to contribute to ineffective hematopoiesis and anemia; and 2) del(5q) MDS patients often acquire concurrent TP53 mutations that result in rapid transformation to AML and poor treatment response. To examine the effects of TIFAB on p53 function, we examined p53 target genes in TIFAB-overexpressing and -deficient cells. Gene expression profiling and qRT-PCR analysis of Tifab KOHSPCs revealed significant upregulation of p53 regulatory genes. In contrast, overexpression of TIFAB in a p53-competent cell line reduced the expression of p53 target gene, p21. Collectively, our findings identify a novel role for TIFAB as an activating adapter of USP15 and mediator of p53 activity. These findings have important implications in the potential role of TIFAB and p53 signaling in the pathogenesis of del(5q) MDS and transformation to AML. DisclosuresNo relevant conflicts of interest to declare.

Small Cell Variant of T-Cell Prolymphocytic Leukemia with Trisomy of the TCL1A Locus Via a Novel Translocation

Small-cell variant of T-cell prolymphocyticleukaemia (T-PLL) is a rare entity, with only 32 cases described1. A 64 year old asymptomatic female presented with lymphocytosis (15.6 x 10^9/L), already present 9 years earlier. Clinical examination was normal. Flow cytometry demonstrated 94% of lymphocytes were CD3+/CD4+/CD8- T cells with normal expression of CD2/5/7. V-beta T cell receptor repertoire and T cell gene rearrangement confirmedmonoclonality. Peripheral blood morphology showed small mature lymphocytes without cytoplasmic granulation and only a rare form showing indistinct nucleoli [arrowhead, figure 1]. Lymphocyte count initially increased slowly (to 23.7 x 10^9/L 10 months after diagnosis), then rapidly (47.2 x 10^9/L, 90.8 x 10^9/L, 151.6 x 10^9/L, 201.6 x 10^9/L at 16 months, 22 months, 28 months and 29 months after diagnosis respectively). By 29 months thrombocytopenia (platelet count 24 x 10^9/L), elevated LDH and spontaneous bruising developed. CT scan did not showadenopathyor splenomegaly. Bone marrow biopsy showed extensive infiltration by small, mature lymphocytes with an identicalimmunophenotypeto that seen at diagnosis. Treatment with CHEOP for 2 cycles did not achieve any response and causedmyelosupression, GI upset and fatigue. Progressive disease was seen during 2 cycles ofpanobinostatwith development ofadenopathy, splenomegaly and rising lymphocyte count.Alemtuzumabproduced rapid disease response with normalisation of lymphocyte count and resolution of theadenopathyand splenomegaly.Bone marrow biopsy after 28 doses ofalemtuzumabshowed no morphological orimmunophenotypicevidence of residual lymphoma.The patient declined to undertake consolidative autologous peripheral blood stem cell transplant at best response. Unfortunately the patient relapsed 4 months later with progressive lymphocytosis, splenomegaly and high LDH. Gemcitabine was given without response and the patient passed away 45 months after diagnosis. Comprehensive genetic analysis was undertaken using G-banded karyotyping, M-FISH, allele-specific FISH for TCL1A and SNP array. Multiple complex rearrangements were detected with abnormalities involving chromosomes 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, 17, 18, 20 and 22, but lacking the inv(14)(q11.2q32) commonly seen in typical T-PLL. In total, 58 copy number changes were identified, but the sole region of copy number gain was a 21Mb duplication of chromosome 14 distal to band 14q31.3. It was present in approximately 90% cells, consistent with the presence of a der(21)t(14;21)(q31;p11.2) observed by metaphase cytogeneticsand resulting in trisomy of the TCL1A locus seen on allele-specific FISH for TCL1A [arrowheads, figure 2]. Some changes detected by SNP array were present in virtually all cells and were thus likely to be have occurred early during tumour evolution. Other subclonalchanges were seen in approximately 90%, 75% or 60% cells respectively, indicating the presence of four distinct cell lines in total. Deletion of the distal portion of 12p was present in virtually all cells, in keeping with the presence of either the dicentric12;13or the dicentric12;14 chromosome in all malignant cells. Of note, a cytogenetically cryptic 1.5Mb deletion at 2p23.2 resulting in deletion of DNMT3A was also an early event, as was a heterozygous deletion of DNMT3B at 20q11.2. The deletion of DNMT3B was seen in association withchromothripsis-like changes on chromosome 20. Despite demonstrated rearrangement of both copies of chromosome 20 by metaphasecytogenetics, no regions of homozygous deletion on chromosome 20 were identified. Typically cases of T-PLL, small cell variant, possess rearrangements of 14q11, often theparacentricinversion typically seen in standard T-PLL, but with other translocations involving the 14q11 locus also described. Thet(14;21) seen in this case demonstrates a novel mechanism of TCL1A dysregulation, not previously described in T-PLL. 1Rashidi, A. and Fisher, S. I. (2015), T-cell chronic lymphocyticleukemia or small-cell variant of T-cellprolymphocyticleukemia: a historical perspective and search for consensus.Eur JHaematol, 95: 199-210.doi:10.1111/ejh.12560 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13)

A basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. Here, we constructed engineered Chinese hamster ovary cell lines (CHO-S) with a pCHO-hVR1 vector that carried an extracellular domain of a VEGF receptor (VR) fusion gene. Forty-five clones with high hVR1 expression were selected for karyotype analysis. Using fluorescence in situ hybridization (FISH) and G-banding, we found that pCHO-hVR1 was integrated into three chromosomes, including chromosomes 1, Z3 and Z4. Four clones were selected to evaluate their productivity under non-fed, non-optimized shake flask conditions. The results showed that clones 1 and 2 with integration sites on chromosome 1 revealed high levels of hVR1 products (shake flask of approximately 800 mg/L), whereas clones 3 and 4 with integration sites on chromosomes Z3 or Z4 had lower levels of hVR1 products. Furthermore, clones 1 and 2 maintained their productivity stabilities over a continuous period of 80 generations, and clones 3 and 4 showed significant declines in their productivities in the presence of selection pressure. Finally, pCHO-hVR1 localized to the same region at chromosome 1q13, the telomere region of normal chromosome 1. In this study, these results demonstrate that the integration of exogenous hVR1 gene on chromosome 1, band q13, may create a high protein-producing CHO-S cell line, suggesting that chromosome 1q13 may contain a useful target site for the high expression of exogenous protein. This study shows that the integration into the target site of chromosome 1q13 may avoid the problems of random integration that cause gene silencing or also overcome position effects, facilitating exogenous gene expression in CHO-S cells.

Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of ATG5

ABSTRACTMelanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.

Cortactin is a prognostic marker for oral squamous cell carcinoma and its overexpression is involved in oral carcinogenesis.

EMS1 (chromosome eleven, band q13, mammary tumor and squamous cell carcinoma-associated gene 1) gene amplification and the concomitant cortactin overexpression have been reported to associate with poor prognosis and tumor metastasis. In this study, we examined cortactin expression by immunohistochemistry in human oral tumors and murine tongue tumors which were induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO). The immunostaining results show over- to moderate expression of cortactin in 85% (104/122) of oral squamous cell carcinoma (OSCC) tissues and in all 15 leukoplakia tissues examined. Further, statistical analysis indicates that cortactin overexpression appears to be a predictor for shorter survival and poorer prognosis in OSCC patients. In an animal model, cortactin is shown to upregulate in infiltrating squamous cell carcinoma, papilloma, and epithelia with squamous hyperplasia, indicating that cortactin induction is an early event during oral carcinogenesis. It is suggested that cortactin expression is mediated in the progression of pre-malignancy to papilloma, based on earlier cortactin induction in pre-malignancy preceding cyclin D1 in papilloma. In conclusion, cortactin overexpression is frequently observed in human OSCC and mouse tongue tumors. Thus, cortactin may have an important role in the development of oral tumors in human and mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 799-812, 2017.

B-cell Acute Lymphoblastic Leukemia With t(9;22)(q34;q11) Translocation and Clonal Divergence Through ider(22) Chromosome and t(13;17)(q14;q25) Translocation

Dear Editor, The Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (MDBGFC) has more than 100 cases for the t(9;22)(q34;q11.2) with divergent patterns of clonal evolution [1]. Here, we present a patient with B-cell ALL who showed t(9;22)(q34;q11.2) and a clonal divergence. Hematological studies from a 54-yr-old woman revealed white blood cell count of 97.2×109/L, platelets 35×109/L, and hemoglobin 6.9 g/dL. Bone marrow blasts displayed L2 morphology and expressed cell-surface differentiation antigens defining a B-cell phenotype, namely, CD10 (80%), CD19 (78%), CD5 (11%), CD3 (0%), and CD13 (0%). The patient received vincristine, prednisone, and daunorubicin-based chemotherapy. However, four months later, the patient was in the terminal phase and died after infiltrations were detected both to the retro-ocular and central nervous systems. We analyzed metaphasic cells from non-stimulated peripheral blood samples obtained eight days before the patient's death. Her karyotype included several sub-clones, which were described according to the International System for Human Cytogenetic Nomenclature (ISCN) (2013) [2], as: 46,XX,t(9;22)(q34;q11.2)[3]/46,idem,ider(22)(22pter→22q11.2::9q34→9q?tel::9q?tel→9q34::22q11.2→22pter)[14]/46,idem,t(13;17)(q14;q25)[3]/46,idem,+1,dic(1;1)(?;?),t(13;17)(q14;q25)[8]/46,XX[1] (Fig. 1A-E). C-banding by barium hydroxide and Giemsa revealed two heterochromatic regions (1q12 band) on the dic(1;1) chromosome (Fig. 1F). Fig. 1 Chromosomes banded with standard techniques. (A) Metaphasic cell with the ider(22). (B) This metaphasic cell contains both t(9;22)(q34;q11.2) and t(13;17)(q14;q25). (C) In addition to the two translocations observed in the above image, a dic(1;1) chromosome ... FISH studies with the dual-color, single-fusion BCR/ABL1 probe (Vysis, Downers Grove, IL, USA) revealed two fusion signals on the ider(22) and only one fusion signal on the standard Philadelphia chromosome (data not shown). FISH study with the RB1 probe (Vysis) disclosed two signals on both normal 13 chromosomes in the clone containing ider(22) (Fig. 2A). Cells with t(13;17) were positive only on the normal chromosome 13 and did not display any signal on either der(13) or der(17) (Fig. 2B and C), thus demonstrating the loss of RB1 by translocation. Fig. 2 FISH analysis with the RB1 probe (red signals) in cells counterstained with DAPI (4',6-diamidino-2-phenylindole; blue color). (A) Metaphase from the clone with ider(22) showing two RB1 signals on both normal 13 chromosomes. (B) Cell with t(13;17) displaying ... Yamamoto et al. [3] reported the presence of first and only ider(22) chromosome similar to the one observed by us in another ALL patient who had a poor prognosis too. These ider(22) chromosomes could result from an U-type sister chromatid exchange. The ider(22) chromosome equals a second copy of der(22) chromosome, which is well known to be one of the main secondary chromosomal changes related to the clonal evolution of cells with t(9;22) [1]. The co-occurrence of t(9;22)(q34;q11) and t(13q14) is rare. There are only six cases registered in the MDBGFC [1], which were previously reported [4,5,6,7]. When both translocations were present, the patients had CML in blast crisis or relapse. Out of four patients tested for chromosome 13 deletions by using FISH [5,6], three tested negative, and only in the fourth patient, there was inconclusive evidence of such a deletion [6]. The t(13;17)(q14;q25), which caused the loss of RB1 sequences in the present patient has not been previously described in ALL [1]. This translocation has only been detected in an AML-M4 patient who did not show deletion of the RB1 gene [8], and in a case of Sezary syndrome in which the status of RB1 gene was not tested [9]. Although we did not know the extent of present 13q deletion, some features of the patient such as genome instability, poor prognosis, and shortened survival suggest that she had a large deletion inclusive of the RB1 gene. Moreover, the chromosomal band 17q25 has been implicated in several translocations also associated with poor prognosis and shorter survival [1]. Imbalances in chromosome 1 are frequent in human neoplasia [1]. The apparently different sizes of two heterochromatic regions observed on the dic (1;1) chromosome (Fig. 1F and ​and2C)2C) suggest a translocation between homologous chromosomes as the forming mechanism of such a dicentric chromosome. Concomitant with the clonal divergence found in peripheral blood, this patient had central nervous system and retro-ocular infiltrations that could not be further studied. Nowicki et al. [10] observed that the circulating CML-blast crisis cells showed major changes than the CML-cells from bone marrow. This observation suggests that leukemic bloodstream cells differ from those in their original niche in survival, growth, invasiveness, and metastatic potential. In our patient, we could not determine whether the divergent sub-clones found in peripheral blood were also present in the infiltrations, or whether each infiltration was derived from a unique and independent sub-clone. This case calls for research on chromosomal and genetic changes that enable the leukemic cells to infiltrate other tissues.

Identification of Candidate Oncogenes and Chromosomal Breakpoint Sequencing By Targeted Locus Amplification in T-Cell Acute Lymphoblastic Leukemia

Background: T-cell acute lymphoblastic leukemia is characterized by clonal and mutual exclusive chromosomal rearrangements that recurrently activate TAL1, LMO2, TLX1, NKX2-1, TLX3, HOXA or MEF2C oncogenes. Most of these translocations or chromosomal rearrangements occur as erroneous D-J or V-DJ rearrangement attempts of T-cell receptor beta (TCRB) or TCR alpha/delta (TCRAD) genes, mostly positioning oncogenes under the transcriptional control of TCR enhancer elements. Alternatively, oncogenes can also be activated as consequence of BCL11B chromosomal rearrangements. Although many oncogenes are known in T-ALL, the driving oncogenic lesion in particular T-ALL cases remains unknown.Aims: In this study, we aimed to clone reciprocal breakpoint sequences to elucidate cellular mechanisms that lead to recurrent BCL11B -TLX3 chromosomal translocations. Moreover, we want to identify oncogene candidates in various T-ALL patient samples with BCL11B-, TCRB- or TCRAD-translocations for which the candidate oncogene so far has not been identified.Methods: We used Targeted Locus Amplification procedure, a recently developed method that relies on the crosslinking of DNA in live cells, DNA digestion and re-ligation to allow formation of circular DNA ligation fragments and inverted polymerase chain reaction amplification from specific view-point loci. Amplified DNA fragments are sequenced by next generation sequencing, allowing sequence identification in a region covering 2MB around selected regions of interest, including TLX3, TLX1, TAL1, LMO2, BCL11B, TCRAD (TRAJ61), TCRB (TCRBC2)Results: TLA was successfully performed on 10 T-ALL patients having FISH validated TAL1 translocations (2 patients), LMO2 translocations (3 patients), TLX3 translocations (3 patients), TLX1 translocations (2 patients) or an inversion targeting NKX2.1 (1 patient). Analysis of both TAL1 translocated cases revealed a TAL1-TCRAD genomic fusion due to a classical t(1;14)(q32;q11) in 1 patient, but surprisingly reveal a TAL1-TCF7 genomic fusion due to a t(1;5)(q32;q31.1) chromosomal translocation in the second patient. For the LMO2 translocated cases, two patients showed classical LMO2-TCRAD (t(11;14)(p13;q11)) or LMO2-TCRB (t(7;11)(q35;p13) translocations, whereas the third patient presented with an unusual LMO2-BCL11B genomic fusion due to a t(11;14)(p13;q32). Two out of 3 TLX3-translocated patients had classical t(5;14)(q35;q32) translocations, whereas the TLX3 gene in the third patient was rearranged to the calcyphosine-like gene (CAPSL, which flanks the IL7Ra gene) on chromosomal 5p13.2 due to a t(5;5)( p13.2;q35) or an inv(5). One patient had an inversion on chromosome 14, i.e. inv(14)(q11;q13), that brings the NKX2.1 oncogene under the transcriptional control of the TCRAD enhancer. Finally, one TLX1-rearranged patient had a classical TLX1-TCRAD translocation, whereas the other presented with a chromosomal inversion involving the chromosomal band 10q24 (which included TLX1), but also revealed a novel translocation involving the centromere protein P gene (CENPP) on chromosome 9q22.31 with the TCRAD locus.Summary/Conclusions: Targeted Locus Amplification identification of chromosomal rearrangements and genomic breakpoint sequences reveals novel complex translocations in 3 out of 10 T-ALL cases analyzed thus far, indicating higher complexity of chromosomal translocations of known T-ALL oncogenes as thus far anticipated. It further proved a useful tool to identify novel translocation partners from various loci such as the TCR or BCL11B genes that are recurrently involved in these chromosomal rearrangements in T-ALL. Cloning of molecular translocation breakpoints of diagnostic T-ALL patient samples may further provide excellent minimal residual disease markers for disease monitoring during the course of treatment. DisclosuresSplinter:Cergentis BV: Employment. van Min:Cergentis BV: Employment.