ALBERT

Description: Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression of, or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) patients, and is a diagnostic marker which predicts a poor outcome. Recently, microRNA deregulation was identified as a major contributor to cancer initiation and progression. Moreover, miRNA genes were shown to be directly regulated by activated proto-oncogenes. In this study, we investigated which miRNA genes are implicated in the transcriptional pathways governed by the EVI1 oncogene. A total of 384 miRNAs were profiled through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR (Applied Biosystems). In a first step, differential miRNA expression was determined in 5 EVI1 rearranged/EVI1 overexpressing samples, 5 non-rearranged/EVI1 overexpressing samples and 5 non-rearranged/non-EVI1 overexpressing bone marrow samples. Next, up and down regulated miRNAs were analyzed in the leukemic EVI1 overexpressing cell lines Kasumi-3 and UCSD-AML1 following transient EVI1 RNAi knockdown. We will present the detailed results of these profiling studies as well as the results of data mining for identification of putative EVI1 regulated miRNAs. Further functional studies will be performed in order to select validated miRNA target genes and to asses their contribution to the leukemic phenotype. We anticipate that the discovery of such miRNAs may provide new targets for therapeutic intervention.

Description: Erythrocyte sickling on deoxygenation in vitro occurs in transgenic SAD mice, hemizygous for a modified human sickle hemoglobin, HbSAD [alpha 2 beta 2S(beta 6val)Antilles (beta 23 lle)D- Punjab (beta 121Gln)] (SAD- 1, 19% HbSAD; beta-thal/SAD-1, 26% HbSAD). The present study examines the cellular defects in vivo and pathologic changes observed in SAD-1 mice at atmospheric oxygenation as well as the effect of acute hypoxia. The transgenic mice showed generalized congestion and microvascular occlusions, occasionally with thrombosis and infarctions of lung, kidneys, penis, and myocardium. The most prevalent chronic organ lesions were congestive splenomegaly (83% of animals) and renal glomerulopathy, which affected 75% of animals by 10 months of age. Further, SAD mice have a mean lifespan that was reduced by 40% when compared with nontransgenic littermates. Premature death of SAD mice was associated with acute vasoocclusive events or severe renal disease. SAD mice developed lethal vasoocclusive processes when exposed to reduced pO2 conditions, whereas control mice survived normally. The sensitivity to hypoxia appears to depend on the cellular level of HbSAD, because death occurred at pO2 of 42 mmHg for SAD mice and 49 mmHg for beta-thal/SAD. Administration of an antisickling agent that increases oxygen affinity (BW12C79) protected SAD and beta-thal/SAD mice from the lethal hypoxic stress. In conclusion, the transgenic SAD and beta-thal/SAD mice developed a pathophysiology that strongly resembles human sickle cell disease. Moreover, this animal model allows studies on the effect of antisickling agents.

Description: Chromosomal translocations involving the IGH locus on 14q32 are a hallmark of B-cell malignancies. These translocations are particularly frequent in non-hyperdiploid (NHD) multiple myeloma (MM), representing approximately 50% of myeloma cases. MM-associated primary t(14q32) target at least 7 partner genes including cyclins D (CCND1, −D3), MAF transcription factors (CMAF, MAF B and A) and MMSET/FGFR3. Some of the translocations are predictive of clinical outcome. Recently, we identified a novel interstitial del(14q) involving IGH and recurrent in chronic lymphocytic leukaemia and MM (Pospisilova et al, Leukemia, 2007). In spite of extensive studies, the mechanism(s) and molecular consequences of del(14q) remain unknown. We report here 34 cases of plasma cell (PC) malignancies with del(14q) involving IGH, as proven by FISH. Cases were collected in UK and Belgium. The estimated incidence of these aberrations in PC malignancies was 1.4%. There were 13 female and 21 male patients ranging in age from 49 to 86 years (average 68). Twenty seven patients had MM, one had SMM and 6 had MGUS. In almost all cases, the del(14q) was detected at diagnosis. Clinical data of the reported cases have been collected. The del(14q) were roughly mapped by FISH and grouped into 3 categories according to the proximal breakpoint: del(14)(q24.1q32.33) involving the ZFP36L1 region (11 cases); deletions proximal to ZFP36L1 (14 cases) and deletions distal to ZFP36L1 (7 cases). The size of del(14q) was not determined in 2 cases. Biallelic deletion of TRAF3/14q32.33 recurrently occurring in MM, was detected by FISH in 1 out of 9 analyzed cases. Additional FISH analysis showed that the del(14q) was predominantly associated with NHD tumors (62% vs 38% with hyperdiploid karyotypes) and frequently (60%) accompanied by del(13q), regarded as a poor prognostic factor. All reported cases were negative for t(4;14) and t(11;14); they also showed a normal status of CCND3, CMAF, MAFB and CMYC, when examined. A gain of 1q/CKS1B was found in 57% (8/14) of analyzed cases. The expression pattern of cyclin D1−D3 has been examined.

Description: Hodgkin lymphoma (HL) is a rare malignancy arising from pre-apoptotic germinal center B-cells which mainly affects young adults. Classical HL (cHL), the most common form, is unusual in that only a small proportion of the tumour consists of the typical malignant Hodgkin/Reed-Sternberg (HRS) cells, whereas the bulk of the remaining cells consisting of infiltrating reactive cells. The scarcity of tumour cells in lymphoma biopsies and low proliferative index has greatly hampered genetic analyses. The isolation of individual HRS cells through micromanipulation offers a possibility to overcome these problems. Given the important role that recently emerged for small non-coding RNAs (miRNAs) in cancer development and the development of a platform which allows reliable miRNA profiling starting from as little as 10 ng total RNA, we decided to determine the miRNA signature for 360 miRNAs in a series of 9 classical HL and four common HL cell lines as well as CD77+ germinal center B-cells as normal counter part.First, we profiled a total of 360 miRNAs through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR in four commonly used cHL cell lines and compared these data to the miRNA signatures obtained from 3 different subsets of CD77+ germinal center B-cells. These analyses revealed 35 up-regulated and 16 down-regulated miRNAs in the cHL cell lines. Next, we wondered whether the miRNA signature obtained from the cHL cell lines would correspond with miRNA expression levels in primary Hodgkin lymphoma patients. Therefore, we determined the miRNA expression signature of microdissected HRS cells from 9 cHL patients and compared these data to the miRNA profiles from CD77+ B-cells. These analyses revealed 30 up-regulated and 73 down-regulated miRNAs in the microdissected HRS cells. Comparing the data from the cHL cell lines and HRS cells, a signature of 17 miRNAs was shown to be consistently up-regulated in both cHL cell lines and HRS cells, whereas a signature of 15 miRNAs was consistently down-regulated. For a number of the most highly under- and over-expressed miRNAs, the differential expression between cHL cell lines and CD77+ B-cells was re-confirmed using monoplex qPCR analysis. Combined knockdown of different over-expressed miRNAs in HL cell lines induced significant inhibition of cell viability, further confirming a potential role of these miRNAs in cHL pathogenesis. In conclusion, this is the first report describing profiling of a large set of 360 miRNAs in pure microdissected populations of HRS cells obtained from frozen biopsies from cHL patients. These miRNA profiles were compared to those from HL cell lines and CD77+ germinal center B-cells yielding a distinct subset of 17 over- and 15 under-expressed miRNAs. This study paves the way for further investigations directed at the role of these miRNAs in the pathogenesis of cHL.

Description: Abstract 140 Molecular mechanisms underlying the pathogenesis of classical Hodgkin lymphoma (cHL) are poorly understood. Although no characteristic chromosomal translocation has been identified in cHL, gain and amplification of the 9p24 region harbouring JAK2 has been observed in up to 50% of cHLs. JAK2 encodes a protein tyrosine kinase (PTK) that plays a key role in the JAK/STAT signalling pathway. Chromosomal translocations and gain-of-function mutations involving JAK2 occur in several haematological malignancies. The aim of this study was to characterize a novel t(4;9)(q21;p24) found in a case of nodular sclerosis HL (NSHL), and to determine the in vitro and in vivo consequences of the fusion associated with this translocation. FISH with BAC clones flanking JAK2/9p24 was used to identify the 9p breakpoint and demonstrated involvement of JAK2. A BAC- and fosmid-walking interphase FISH strategy was further applied to identify the 4q21 breakpoint which was eventually mapped in the region of SEC31A. SEC31A is ubiquitously expressed in human cells and is known to play a role in ER-to-Golgi vesicular transport. Further molecular studies led to the identification of a SEC31A-JAK2 in-frame fusion transcript in which exon 24 of SEC31A is fused to exon 17 of JAK2. Of note, our recent studies showed involvement of SEC31A as a partner of ALK in ALK+ LBCL (Van Roosbroeck et al., Haematologica 2009, in press). To determine the in vitro oncogenic potential of SEC31A-JAK2, a chimeric expression construct was designed and introduced into mouse haematopoietic IL3-dependent Ba/F3 cells. SEC31A-JAK2 was found to transform Ba/F3 cells to IL3-independent growth, demonstrating its implication in oncogenic transformation. The fusion protein is likely to function as a constitutively activated tyrosine kinase, due to SEC31A-mediated oligomerization of JAK2. Attempts to identify the SEC31A domain responsible for the constitutive JAK2 activation are ongoing. Initial experiments with deletion mutants containing or lacking the WD40-like repeats of SEC31A exclude these repeats to be the driving force of JAK2 activation. An in vivo role of the fusion was assessed with a murine bone marrow transplant model. All six recipients of SEC31A-JAK2 transduced bone marrow cells developed a fatal disease after 107 – 174 days, showing involvement of the blood, bone marrow and spleen, and in a subset of mice also of lymph nodes and thymus. FACS and histopathological examination of the involved tissues in 3 mice revealed the development of a T-lymphoblastic lymphoma. Analysis of the remaining mice is still ongoing. In addition, we showed that the T-lymphoblastic disease is transplantable to secondary recipients. Downstream of the SEC31A-JAK2 fusion we could demonstrate constitutive activation of the ERK pathway in Ba/F3 cells bearing the SEC31A-JAK2 construct as well as in the reconstituted mouse tissues. To determine the incidence of JAK2 rearrangements in cHL, we screened 60 unselected cHL cases, including 25 with NSHL, by FISH and cDNA-based nested PCR. Using this approach, we identified one additional case with a SEC31A-JAK2 fusion showing 4q21 and 9p24 breakpoints identical to these in the index case. Moreover, we found a third case with a JAK2 rearrangement and two extra copies of the 3'JAK2. As SEC31A is not involved in the latter aberration, further studies aiming at the identification of the JAK2 partner in this case of cHL are ongoing. The vast majority (80%) of the remaining cHL cases analyzed by FISH revealed recurrent gains/amplifications of JAK2. In summary, we proved that JAK2 is recurrently targeted by chromosomal translocations in cHL. We identified and molecularly characterized the novel t(4;9)(q21;p24) resulting in a SEC31A-JAK2 fusion found in two NSHL cases and identified another not yet characterized JAK2 rearrangement in the third cHL case. We demonstrated the oncogenic potential of the SEC31A-JAK2 fusion both in vitro in the mouse haematopoietic IL3-dependent Ba/F3 cell line and in vivo in a murine bone marrow transplant model. Of note, this is the first report of a recurrent translocation associated with cHL. Although aberrant expression of various PTKs including JAK2 has already been documented in cHL, our results indicate that at least in some cHL cases, this aberration can be driven by a chromosomal translocation. Disclosures: No relevant conflicts of interest to declare.

Description: Erythrocyte sickling on deoxygenation in vitro occurs in transgenic SAD mice, hemizygous for a modified human sickle hemoglobin, HbSAD [alpha 2 beta 2S(beta 6val)Antilles (beta 23 lle)D- Punjab (beta 121Gln)] (SAD- 1, 19% HbSAD; beta-thal/SAD-1, 26% HbSAD). The present study examines the cellular defects in vivo and pathologic changes observed in SAD-1 mice at atmospheric oxygenation as well as the effect of acute hypoxia. The transgenic mice showed generalized congestion and microvascular occlusions, occasionally with thrombosis and infarctions of lung, kidneys, penis, and myocardium. The most prevalent chronic organ lesions were congestive splenomegaly (83% of animals) and renal glomerulopathy, which affected 75% of animals by 10 months of age. Further, SAD mice have a mean lifespan that was reduced by 40% when compared with nontransgenic littermates. Premature death of SAD mice was associated with acute vasoocclusive events or severe renal disease. SAD mice developed lethal vasoocclusive processes when exposed to reduced pO2 conditions, whereas control mice survived normally. The sensitivity to hypoxia appears to depend on the cellular level of HbSAD, because death occurred at pO2 of 42 mmHg for SAD mice and 49 mmHg for beta-thal/SAD. Administration of an antisickling agent that increases oxygen affinity (BW12C79) protected SAD and beta-thal/SAD mice from the lethal hypoxic stress. In conclusion, the transgenic SAD and beta-thal/SAD mice developed a pathophysiology that strongly resembles human sickle cell disease. Moreover, this animal model allows studies on the effect of antisickling agents.

Description: Abstract 361 Chromosomal rearrangements involving the EVI1 gene are a recurrent finding in malignant myeloid disorders. These translocations or inversions contribute to ectopic expression or to the formation of fusion genes involving the EVI1 gene. EVI1 transcriptional activation has been reported in up to 10% of acute myeloid leukemia (AML) and is a prognostic marker of poor outcome. MicroRNA (miRNA) deregulation was recently identified as a major contributor to cancer initiation and progression. As miRNA genes were shown to be directly regulated by activated proto-oncogenes, we aimed to identify miRNAs under direct or indirect control of EVI1. To this purpose, we analyzed the expression of 366 miRNAs in 38 EVI1 rearranged/overexpressing patient samples, 6 normal bone marrow controls and 2 EVI1 knockdown model systems (siRNA mediated EVI1 knockdown in the EVI1 rearranged/overexpressing cell lines Kasumi-3 and UCSD-AML1). In total, 24 upregulated and 25 downregulated miRNAs (p