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.
No relevant conflicts of interest to declare.
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