ALBERT

Description: Abstract 5042 Background In acute myeloid leukemia (AML) a recurrent chromosome abnormality t(12;15)(p13;q25) fuses ETV6 with NTRK3. This rearrangement uniquely occurs in both solid tumors – including secretory breast cancer where it has been recently shown to target WNT signalling (Li et al., Cancer Cell 2007, 12: 542) - and leukemia, but has yet to be characterized in the hematologic setting. Tyrosine receptor kinases (TRK) play key roles in leukemogenesis and already serve as therapeutic targets. We set out to characterize potential downstream targets of ETV6-NTKR3 in AML cells. Methods and Cells By applying molecular cytogenetics, rapid amplification of c-DNA ends, microarray transcriptional profiling, reverse transcriptase quantitative-PCR, sequencing technology, and pathway analysis we defined and characterized the transcriptosome of a t(12;15) cell line (AP-1060) recently established from a patient with acute promyelocytic leukemia. We also investigated the transcriptional responses of AP-1060 cells to TRKi(nhibitor). For comparison we used, firstly a panel of 12 AML cell lines lacking ETV6-NTRK3 or PML-RARA, followed by NB-4 cells with solo PML-RARA. Results FISH confirmed ETV6 rearrangement, while 3′-RACE and RT-PCR identified and confirmed ETV6-NTRK3 fusion transcripts. Sequencing revealed both ETV6 exon-4 / NTKR3 exon-14, and ETV6 exon-2 / exon-18 of NTKR3 (hematopoietic) transcripts - the former dominating. Comparative transcriptional profiling of AP-1060 and control AML cells with or without PML-RARA showed upregulation of RAS-MAPK and PI3K-AKT related genes, highlighting the involvement of both TRK physiological signaling pathways via ETV6-NTRK3. Top genes upregulated in AP-1060 confer signatures both for AML - CCNA1, CD96, DSU, EVI1, HGF, IL32, LGALS3, MDS1, TLE1, TSPAN2; and lymphocyte development - BSPRY, BST1, CCR6, EMP1, GIMAP1, GZMA, PLEKHG1. Several primitive hematopoietic or stem cell mRNAs were also overexpressed, including PRSS2, CD96, SIPA1L2, and PYHIN1. Prominent downregulated genes also included: ADD3, CD36, HOXA-9/10, LGALS9, MALAT1, PGDS, PLA2G4A (AML signature); HOXB4, KIAA1949, NR2F6, TEAD4 (stem cell); and LY6E, TRIM44 (lymphocyte signature). Growth and proliferation of ETV6-NTKR3 cells was exquisitely sensitive to TRKi treatments which spared control AML and to which NB-4 cells were highly resistant. Accordingly we used pharmacologic modulation of conspicuously expressed genes by small molecule TRKi treatment to highlight likely kinase signaling targets among conspicuously expressed genes. Several candidate target genes thus emerged, notably AWNT1, IL32, and the MDS-EVI1 fusion transcript. Salient pharmacologically unmodulated genes were preferentially stem cell in character highlighting this setting for t(12;15) formation in AP-1060 cells. Bioinformatic pathway analysis (http://david.abcc.ncifcrf.gov/) of both up- and down- conspicuously regulated genes identified “Alternative Splicing” as top category, with respectively 743 and 373 alternate spliceform genes up- and down-regulated. These included several genes whose spliceforms may be differentially expressed in oncogenesis, including MDS1-EVI1/EVI1, MALAT1, and WT1/AWT1. Interestingly, a key pre-mRNA splicing gene, MBNL2 was conspicuously downregulated, while another spliceosomal component THOC5 (C22orf19), recently identified as a leukemic kinase signalling target (Pierce et al., Br J Haematol 2008;141:641), is upregulated. Conclusions We present a human leukemia model and resource for ETV6-NTRK3. Taken together, our findings support spliceosomal targeting by ETV6-NTRK3 and suggest a possible underlying mechanistic framework. Additional targets, e.g. WNT signaling, seem to be shared with solid tumors bearing the same oncogene fusion. Perspectives: Future work includes transcriptosomal analysis of AP-1060 cells after knockdown of ETV6-NTRK3 and key splicesomal genes, such as THOC5, by short-hairpin RNAs, and novel, highly selective 4-aminopyrazolylpyrimidine TRKi (Thress et al., Mol Cancer Therapy 2009;8:1818). Disclosures No relevant conflicts of interest to declare.

Description: The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-cell acute lymphoblastic leukemia (T-ALL). To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 fusion signals were observed, confirming the low frequency of this rearrangement in T-ALL, but we did observe marked amplification (〉 10 signals per nucleus) ABL1 of in 5 of 90 (5.5 %) T-ALL patients. Amplification of ABL1 occurred on small extrachromosomal elements that were not detectable by conventional cytogenetics. and hence are referred to as episomes. FISH, and array-CGH analyses delineated the amplicon as a 500 kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (CAN). Molecular analysis led to the identification of a NUP214-ABL1 fusion gene, which is generated as result of the circularization of the genomic region between ABL1 and NUP214 to form the episomes. This is the first example of an oncogenic fusion gene generated by extrachromosomal amplification. The NUP214-ABL1 transcript was detected in 5 patients with ABL1 amplification, in 5 of 85 (5.8 %) additional T-ALL patients, and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib mesylate (STI-571). The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased expression TLX1 of (HOX11) or TLX3 (HOX11L2), and with deletion of CDKN2A (p16), consistent with a multi-step pathogenesis of T-ALL. Our results identify a novel mechanism for the generation of a fusion gene on extrachromosomal elements, and indicate the importance of activated tyrosine kinase signaling in the pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of T-ALL patients that could benefit from imatinib treatment.

Description: Three NK-like (NKL) homeobox genes, TLX1/HOX11, TLX3/HOX11L2 and NKX2- 5/CSX, have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Here we screened further NKL genes in 24 T-ALL cell lines by RT-PCR and identified common expression of MSX2, highlighting this homeobox gene as a potential physiological family member in T-cells. Subsequent quantification of MSX2 confirmed expression in primary hematopoietic cells demonstrating higher levels in CD34+ stem cells when compared to peripheral blood cells or mature CD3+ T-cells. Analysis of core thymic factors in T-ALL cell lines, including IL7, BMP4, TGFbeta, NOTCH and T-cell receptor signaling, suggests their involvement in MSX2 regulation during T-cell differentiation. Chromosomal and genomic analysis of the MSX2 locus (at 5q35) uncovered deletion in t(5;14)(q35;q32) positive T-ALL cell lines associated with low expression levels of MSX2 and ectopic activation of TLX3 or NKX2-5, respectively. For functional analysis we lentivirally transduced T-ALL cells for overexpression of either MSX2 or oncogenic TLX1 and NKX2-5. These cells displayed transcriptional activation of NOTCH3-signaling, as indicated by expression array profiling and real-time PCR analysis of NOTCH3, HES1 and HEY1. The sensitivities to gamma-secretase inhibitor analyzed by MTT-assay of cells overexpressing MSX2, TLX1 or NKX2-5, respectively, were consistently decreased. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with repressor proteins of the NOTCH-pathway, SPEN/MINT and TLE1/GRG1, as shown by co-immunoprecipitation, probably representing one mechanism of (de)regulation. Elevated expression of NOTCH3 and HEY1 mRNA was detected in TLX1/3 positive T-ALL patients, confirming data obtained from cell lines. In conclusion, we have defined expression patterns, regulation and targets of MSX2 in hematopoietic cells, to reveal a novel modulatory activity in T-cell differentiation operating via NOTCH-signaling, and in leukemogenesis when replaced or supplemented by oncogenic NKL homeodomain proteins.

Description: Abstract 1281 Poster Board I-303 Many oncogenes code for transcription factors involved in regulation of developmental pathways. The activity of these pathways is tissue specific and restricted to certain developmental stages. Here, we searched for T-cell acute lymphoblastic leukemia (T-ALL) oncogenes which physiologically regulate differentiation of natural killer (NK) cells. NK- and T-cells are closely related lymphocytes, sharing the same early progenitors which can differentiate into either lineage. We compared expression profiles of malignant NK- and T-cell lines to identify aberrantly expressed genes in T-ALL. This analysis revealed high expression of HOXA9, HOXA10 and ID2 in NK-cell lines and in one T-ALL line, LOUCY, suggesting leukemic deregulation therein. Subsequently, we analyzed mechanisms underlying their regulation. Overexpression and chromatin immuno-precipitation experiments demonstrated that HOXA9 and HOXA10 directly activate ID2 expression. Analysis of other ALL and acute myeloid leukemia cell lines with and without mixed lineage leukemia (MLL) gene translocations demonstrated a correlated expression of HOXA9/10 and ID2, highlighting ID2 as an indirect target of MLL fusion proteins which deregulate HOXA genes. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including the components EZH2 and HOP of polycomb repressor complex 2 (PRC2), showed downregulation of EZH2 in LOUCY and limited expression of HOP to NK-cell lines. Subsequent treatment of T-ALL cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively, confirming repressive activity of EZH2 in T-cells. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression. Taken together, our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 and may contribute to T-cell leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Description: The summarizes the salient characteristics of 560 cell lines: precursor B (85), mature B (155), plasma cell leukemia/myeloma (66), immature T (55), mature T (21), natural killer (10), Hodgkin lymphoma (10), anaplastic large cell lymphoma (16), myelocytic (62), monocytic (33) and erythrocytic-megakaryocytic cell lines (45). Along with other improvements, the advent of continuous human leukemia-lymphoma (LL including myeloma) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt lymphoma-derived lines, were established in 1963. Since then more than 1500 cell lines have been described, some 500 of them in detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro with preservation of most cellular features, and specific genetic alterations characteristic of their tumor of origin. Recent transcriptional profiling studies have confirmed that LL cell lines stably retain the aberrant profiles typical of their tumors of origin. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines should be discerned from Epstein-Barr virus-immortalized normal cells. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be defined. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 20%) or are cross-contaminated with other cell lines (14% in our DNA fingerprinting analysis on 622 samples covering 560 LL cell lines). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection and proper, regular authentication of cell lines. The underlying cause appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high quality LL cell lines to which every scientist has access. These are offered by public cell line banks like the DSMZ which holds 169 LL cell lines which all have undergone a strict quality, identity and characterization program (immunoprofile, karyotype, DNA fingerprint, virus/mycoplasma check). An example of the practical utility of LL cell lines are the recent advances in studies of classical and molecular cytogenetics which in large part were made possible by cell lines. We propose a list of the most useful, robust and publicly available reference cell lines which may be used for a variety of experimental purposes. The is available on CD; inquire at .

Description: In T-cell acute lymphoblastic leukaemia (T-ALL) alternative t(5;14)(q35;q32.2) forms effect leukemic dysregulation of either TLX3 or NKX2-5 homeobox genes at 5q35 by juxtaposition with 3′-BCL11B at 14q32.2. Putative regulatory sequences underlying ectopic homeobox gene activation in t(5;14), and their mode of action have remained poorly understood mainly because breakpoints at 14q32.2 are widely scattered over the ~1 Mbp genomic desert region. We pooled cytogenetic data from t(5;14) cell lines together with published clinical data to refine the BCL11B downstream breakpoint cluster region (bcr). Ectopic homeobox gene dysregulation was investigated by DNA-i(nhibitory-treatments) with 26-mer double-stranded DNA oligo(nucleotide)s directed against putative enhancers using NKX2-5 expression as endpoints. Enhancer targets were provisionally identified from orphan T-cell DNase-I hypersensive sites (DNaseI-HS) located between 3′-BCL11B and VRK1. NKX2-5 downregulation in t(5;14) PEER cells was almost entirely restricted to DNAi targeting enhancers within the distal bcr and was dose- and sequence-dependent. Interestingly, enhancers near 3′-BCL11B regulated that gene only. These data imply that enhancer-promoter distances and/or locations are important for long-range gene regulation. Chromatin immunoprecipation assays showed that the four most effectual NKX2-5 ectopic enhancers were hyperacetylated. These enhancers clustered ~1 Mbp downstream of BCL11B, within a region displaying multiple regulatory stigmata, including a TCRA-enhancer motif, and abyssal sequence-conservation (“5-Way Regulatory Potential”). Paradoxically, although TLX3/NKX2-5 promoter/exon-1 regions were hypo-acetylated, their expression decreased after TSA treatment, implying extrinsic regulation by factor(s) subject to acetylation-control. PU.1 is known to get transcriptionally repressed by TSA and potentially binds TLX3/NKX2-5 upstream promoter regions. Knockdown of PU.1 effected downregulation of both homeobox genes. Moreover, genomic analysis showed preferential enrichment near validated ectopic enhancers of binding sites for the PU.1-cofactor HMGA1, knockdown of which also inhibited NKX2-5 in PEER cells. Analysis of nuclear matrix attachment (NMA) in PEER cells showed enhanced attachment near to the most effectual enhancer cluster which was alleviated by TSA-treatment. Interestingly, the juxtapositional genomic regions of “active” ins(14;5) rearrangements driving NKX2-5 expression exhibited tight NMA, forming structures reminiscent of “active chromatin hubs”. These findings lead us to propose that HMGA1 and PU.1 co-regulate ectopic homeobox gene expression in t(5;14) T-ALL by interactions mediated at the nuclear matrix, possibly mediated by SATB1 binding. Our data document homeobox gene dysregulation by a novel regulatory region at 3′-BCL11B responsive to HDAC-inhibition and highlight a novel class of potential therapeutic target amid “junk” DNA.

Description: Activating mutations and deletions affecting specific NOTCH1 protein domains have been recently shown to occur widely in T-cell neoplasia, e.g. in T-acute lymphoblastic leukemia (T-ALL). However, knowledge of NOTCH1 chromosomal alterations is largely based on a single cell line model (SUP-T1) with t(7;9)(q35;q34) in which NOTCH1 truncated at exon 24 is juxtaposed with TCRB. We describe the characterization of a novel rearrangement, t(9;14)(q34.3;q11) in two T-cell lymphoma cell lines, HD-MAR and HT-1. FISH analysis using fosmid clones and sequencing of fragments identified by long distance inverse PCR showed that in both cases t(9;14) effected tail-to-tail juxtaposition of intron 27 of NOTCH1 with TCRA genes, namely 5′-TRAV40 in HD-MAR, and intron 2 of TRAV5 in HT-1. Thus, in both cell lines t(9;14) places NOTCH1, truncated immediately 3′ of the HD-domain, under transcriptional control of TCRA. The 14q11.2 breakpoints in HD-MAR and HT-1 lie, respectively, near the proximal E-delta enhancer and amid a cryptic enhancer region represented by a cluster of T-cell specific DNase-I hypersensitive sites. Western blotting revealed prominent expression of truncated activated NOTCH1 polypeptides, ranging in size from 100 to 115 kDa in both cell lines. Antibodies recognizing ANK and TAD domains, believed essential for inducing T-ALL, detected the aberrant polypeptides. Moreover, treatment with gamma-secretase inhibitor (GSI) altered expression patterns of NOTCH1 polypeptides and induced growth inhibition due to G0/G1 cell cycle arrest in both t(9;14) cell lines, in stark contrast to GSI-resistant SUP-T1 cells wherein truncation occurs before the heterodimerization (HD) domain. (Another recently described t(7;9) cell line (CUTLL1) which is GSI-sensitive also carries a NOTCH1 breakpoint at intron 27.) The same protein species were not detectable by antibodies recognizing the transmembrane domain of NOTCH1 which requires GS for exposure suggesting nuclear access requires GS-cleavage. Immunostaining confirmed extranuclear blocking of NOTCH1 in response to GSI in HD-MAR/HT-1 but not in SUP-T1. In contrast, repression of HES1 occurred in response to GSI irrespective of NOTCH1 breakpoint location, suggesting its non-involvement in growth signaling. In addition to providing cell line models for a new NOTCH1 disease translocation, these data suggest that the sensitivities of T-cell neoplasias bearing NOTCH1 translocations may critically depend on whether 9q34 breakpoints lie upstream or downstream of the HD domain.

Description: Abstract 2641 Poster Board II-617 Background: In T-cell acute lymphoblastic leukemia (T-ALL) the LMO2 transcription factor locus is juxtaposed with T-cell receptor (TCR) genes by a recurrent chromosome translocation, t(11;14)(p13;q11). Recent molecular cytogenetic data indicate that unlike classical TCR rearrangements, t(11;14) operates synonymously with submicroscopic del(11)(p13p13) by removing a negative upstream LMO2 regulator (Dik et al., Blood 2007;110:388). The combined incidence of both LMO2 rearrangements is ∼10-15% (Van Vlierberghe and Huret, Atlas Genet Cytogenet Oncol Haematol, November 2007). However, aberrant LMO2 expression occurs in nearly half of all T-ALL cases, a discrepancy which may indicate a significant contribution by cryptic chromosome alterations. We attempted the extended characterization of the LMO2 genomic region in T-ALL cell lines to look for such rearrangements. Cells and Methods: We investigated a panel of 26 well characterized and authenticated T-ALL cell lines using parallel fluorescence in situ hybridization (FISH) with a tilepath BAC/fosmid contig and both conventional and quantitative reverse transcriptase (Rq)-PCR. Global gene expression was additionally measured in some cell lines by Affymetrix array profiling. Results: LMO2 rearrangements were detected in 5/26 (19.2%) cell lines including both established rearrangements, t(11;14) and del(11)(p13p13) in one cell line apiece (3.8%). Interestingly, we found two novel LMO2 translocations: t(X;11)(q25;p13) in 2/26 (7.7%), and t(3;11)(q25;p13) in 1/26 (3.8%) cell lines, respectively. Comparing transcription levels in cell lines with and without genomic rearrangements showed that LMO2 expression was significantly higher in T-ALL cell lines carrying LMO2 rearrangements (P