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: 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 1567 Fusions of the tyrosine kinase domain of JAK2 with multiple partners occur in leukemia/lymphoma where they are believed to promote JAK2-oligomerization and autonomous signalling although the underlying mechanisms remain unclear. Affected entities are candidates for therapy with JAK2 signalling inhibitors. Among 200 peripheral T-cell lymphomas surveyed, we identified only two with JAK2 amplification and none with JAK2 translocations, confirming their rarity in T-cell neoplasias. Here we describe the genomic, transcriptional and signalling characteristics of PCM1-JAK2 formed by t(8;9)(p22;p24) in cell lines established at indolent and aggressive phases of a cutaneous T-cell lymphoma. To investigate signalling, PCM1-JAK2 was subjected to lentiviral knockdown which inhibited 7 genes most upregulated in t(8;9) cells, notably SOCS2/3. SOCS3, but not SOCS2, was also upregulated in a chronic eosinophilic leukemia bearing PCM1-JAK2. Conversely, expression of GATA3, a key T-cell developmental gene silenced in aggressive phase cells, was partially restored by PCM1-JAK2 knockdown. Activation of the tumor suppressor SOCS3 by PCM1-JAK2 may follow structural alteration of JAK2 affecting the ternary complex it forms with SOCS3 and receptor proteins. Treatment with JAK2 inhibitor (TG101348) to which MAC-1/2A/2B cells were conspicuously sensitive mimicked knockdown results, highlighting JAK2 as the active moiety. PCM1-JAK2 signalling required pSTAT5 as reported for JAK2V617F or ETV6/TEL-JAK2, thus extending the paradigm of STAT5 activation by JAK2 alterations in hematopoietic malignancies. MAC-1/2A/2B are the first JAK2– translocation cell line models. Our data support further investigation of SOCS2/3 as signalling effectors, prognostic indicators and potential therapeutic targets in cancers with JAK2 rearrangements. Disclosures: No relevant conflicts of interest to declare.

Description: MLL translocations in adult B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) are largely restricted to the immature CD10− immunophenotypes. MLL-AF4 is known to be the most frequent fusion transcript, but the exact frequencies of MLL aberrations in CD10− adult BCP-ALL are unknown. We present a genetic characterization of 184 BCR-ABL− CD10− adult ALL cases (156 cyIg−, 28 cyIg+) diagnosed between 2001 and 2007 at the central diagnostic laboratory of the GMALL study group. Patient samples were investigated by RT-PCR for MLL-AF4, MLL-ENL, and MLL-AF9 and by long-distance inverse polymerase chain reaction, thus also allowing the identification of unknown MLL fusion partners at the genomic level. MLL-AF4 was detected in 101 (54.9%) and MLL-ENL in 11 (6.0%) cases. In addition, rare MLL fusion genes were found: 2 MLL-TET1 cases, not previously reported in ALL, 1 MLL-AF9, 1 MLL-PTD, a novel MLL-ACTN4, and an MLL-11q23 fusion. Chromosomal breakpoints were determined in all 118 positive cases, revealing 2 major breakpoint cluster regions in the MLL gene. Characteristic features of MLL+ patients were significantly lower CD10 expression, expression of the NG2 antigen, a higher white blood count at diagnosis, and female sex. Proposals are made for diagnostic assessment. The clinical studies are registered at http://www.clinicaltrials.gov as NCT00199056 and NCT00198991.

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

Description: Micro-RNA (miR) genes posttranscriptionally modulate target gene expression via imperfect 3′-UTR matching sequences and play key roles in development, homeostasis and cancer. Little is known how miR genes are themselves regulated, or deregulated in cancer. Chief paradigm for neoplastic miR deregulation concerns miR-17/92 cluster members subject to genomic amplification in B-cell lymphoma. While the repeated occurrence of oncogenic miR genes at or near chromosomal breakpoints in cancer links chromosome fragility to oncogenic miR deregulation, direct evidence of a causal connection remains tenuous. We found that t(3;7)(q27;q32) in a B-cell lymphoma cell line joins 5′-BCL6 to a noncoding region of chromosome 7 inside a common chromosomal fragile site (FRA7H). In these cells hybrid mRNA was absent, unlike canonical BCL6 translocations which involve promoter exchange yielding hybrid mRNA. Affected cells instead showed downregulation of miR-29b-1, the only gene located within FRA7H - a recurrent transcriptional feature of B-cell lymphoma subsets. In another BCL6 translocation, t(3;13)(q27;q31)t(13;12)(q31;p11), which 5′-RACE also showed to be non-fusogenic, long distance inverse (LDI)-PCR revealed junction of 5′-BCL6 to chromosome 13 sequences inside the miR-17/92 host gene MIRH1 (alias c13orf25). FISH using a sensitive tyramide amplification protocol with c13orf25 clones confirmed the presence of a cryptic BCL6-MIRH1 rearrangement. Surprisingly, reverse transcriptase quantitative (q) PCR assay revealed weak MIRH1 expression using 3′-primers. In contrast, repeating the assay using more central primers covering the miR-17/92 coding region showed massive upregulation. 3′-RACE confirmed a novel high level MIRH1 transcript truncated by 3.1 kbp. Quantitative genomic PCR and FISH excluded miR-17/92 genomic copy number alteration, while LDI-PCR analysis showed that formation of truncated MIRH1 involved multiple DNA cuts at 3q27 (x1), 12p11 (x1), and 13q31 (x5) – the last including a complex excision/inversion/insertion rearrangement. Stress induced DNA duplex destabilization (SIDD) analysis revealed that 6 of 7 breaks precisely coincided with fragility peaks. Taken together, these data suggest a novel role for BCL6 translocations in the deregulation of miR genes near sites of chromosome or DNA instability. BCL6 has been shown to suppress p53 in germinal center B-cells thus protecting B-cells from apoptosis induced by DNA damage, offering a possible explanation for chromosome rearrangements associated with genomic fragility therein. Chromosomal MIRH1 dysregulation is not limited to BCL6 expressing lymphomas, however: cytogenetic investigations performed on diverse leukemia-lymphoma cell lines, including those derived from multiple myeloma and plasma cell leukemia, showed 11/50 with cytogenetic rearrangements at or near MIRH1. In sister cell lines sequentially established at diagnosis and relapse of multiple myeloma, only the latter showed miR-17/92 chromosomal rearrangement and upregulation. Interestingly miR overexpression was limited to miR-92, while miR-17/18 were barely expressed. FISH analysis and qPCR showed that discrepant expression was associated with rearrangement upstream of MIRH1. In brief, our data show that like other cancer genes, oncogenic miRs are subject to dysregulation mediated by structural chromosome rearrangements.

Description: Genomic amplifications of the 11q23 region occur in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) where MLL and a few neighboring genes, notably DDX6, are deemed salient targets. However, the extent to which amp(lified)-MLL and translocated MLL share effector targets remains to be established. Even less is known about the target(s) of deletions affecting the long arm of chromosome 5 (5q-) which reportedly partner amp-MLL. We analyzed three AML/MDS cell lines by cytogenetics (conventional and FISH) in parallel with real time q(uantitative)-PCR at both 11q23 and 5q2 to measure copy number and expression of salient target genes together with putative downstream targets. The cell lines comprised: MOLM-17 (transforming-MDS), SAML-2 (therapy-related AML), and UOC-M1 (AML-M1). All three cell lines exhibited approximately four-fold genomic amplification of 11q23 including MLL and DDX6, while the amplicon extended telomerically to include FLI1 (11q24) and HNT (11q25) in MOLM-17 and UOC-M1 only. Expression, quantified relative to AML/MDS cell lines without MLL rearrangement, revealed that of the genomically amplified genes only MLL was generally overexpressed, namely by 9.5x (MOLM-17), 5.1x (UOC-M1), and 4.6x (SAML-2). In addition to the highest MLL expression, in MOLM-17 FLI1 (3.8x) and DDX6 (2.8x) were significantly upregulated. Expression was also quantified among reputed MLL target genes, and showed that in the three cell lines MEIS1 was upregulated in MOLM-17 only (by 6x), and CDKN2C in all cell lines (by about 2x), while HOXA9 and CDKN1B showed near-normal levels of expression. All three cell lines carried 5q- with a common deleted region at 5q31 extending from 134.2–137.5 Mbp. Of a panel of genes recently identified as 5q- deletion targets (centromere-TIGA1, CAMLG, C5orf15, C5orf14, BRD8, HARS, KIAA0141, CSNK1A1, RBM22-telomere), only C5orf15 (function unknown) and BRD8 (a component of the nua4 histone acetyltransferase complex involved in transcriptional activation) were generally downregulated - to about 0.25x, and about 0.4x normalized expression levels, respectively. Both genes lie within the common deleted region. In summary, we have characterized amp-MLL and 5q- in MOLM-17, the first MDS cell line to be described with these rearrangements, together with two AML cell lines with similar cytogenetic profiles. Our data suggest that MLL is the only clear object of 11q23 amplification hitherto identified and CDKN2C its sole unequivocal target in AML/MDS cell lines. It is possible that MEIS1 is also targeted for activation in specific cell types or disease phases in MDS. These findings also highlight C5orf15 and/or BRD8 as possible leukemogenic accomplices targeted for downregulation in accompanying 5q-. These findings may point to differences in signalling pathways targeted by amp-MLL in AML and MDS.

Description: Acute leukemias are frequently associated with specific chromosomal translocations of the human MLL gene. In general, MLL translocations define a distinct disease entity that needs to be diagnosed with precision to facilitate rapid clinical decisions. Here we present data about a new PCR based method that uses patient genomic DNA to identify any MLL fusion. Fourty different MLL translocations were successfully analyzed. We will present three novel MLL translocation partner genes and a new MLL deletion. The benefits of this novel technique for diagnosis and MRD analyses will be discussed. Supported by grant 2001.061.1 from the Wilhelm Sander foundation.

Description: Chromosomal rearrangements of the MLL gene are responsible for 5–10% of all acute leukemias, biphenotypic leukemias and myelodysplastic syndromes. 5–10% of these MLL aberrations are therapy-related leukemias with the frequent fusion partners ELL, MLLT3, MLLT10 and MLLT2. The large number of known and unknown MLL fusions (〉80) renders a precise diagnosis a demanding task. Even though all MLL rearrangements are associated with high risk acute leukemias, the outcome (favorable or poor) depends on the partner gene. Thus, the identification of MLL gene fusions is necessary for rapid clinical de-ci-sions resulting in specific therapy regimens. After their cytogenetic identification, only the most common MLL fusions MLLT2, MLLT3, MLLT1, MLLT4, ELL and MLLT10 (80%) are investigated by RT-PCR analysis, whereas infrequent or unknown MLL rearrangements (20%) are more or less excluded from further analysis. Therefore we established a LDI-PCR (long-distance inverse PCR) based method that uses small amounts of genomic DNA to determine any type of MLL associated chromosomal abnormality. With this diagnostic tool more than 400 prescreened und unscreened adult and pediatric samples (AML/ALL) from different European diagnostic centers have been analyzed. During this study more than 200 MLL rearrangements were characterized for their precise localization of genomic break-points. The identified MLL rearrangements consist of 25 different fusion genes including the following eight novel MLL partner genes: ACACA, ARGHEF17, BCL9L, MAML2, SELB, SMAP1, and TIRAP. The identified MLL aberrations are basically reciprocal translocations, but also deletions, inversions, insertions and interstitial duplications have been determined. Combining the data of our study and data retrieved from the literature, a total of 88 partner genes can now be annotated. 52 fusion partners (60%) have been characterized on the molecular level whereas at least 36 (40%) remain to be identified. These results demonstrate that this new diagnostic tool in combination with split-signal FISH is qualified for the rapid analysis of known as well as unknown MLL partner genes. Furthermore, the determined patient specific fusion sequences are useful for minimal residual disease (MRD) monitoring of MLL associated acute leukemias. The use of these MRD markers will contribute to improve the treatment and outcome of acute leukemia patients. A first prospective study was already initiated by the German ALL study group and verified the reliability of these genomic markers for MRD monitoring.