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1744 Humań Molecułar Generics. 1999. Vol. 8. No. 9

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Figurę 2. Deteciion ofihc STATSb gene rcurningemem at the genonuc le\*el by Southern biot analysis. Southern biot anaiysis was donc on DNA from parient bonę marrow cells (P> and from a norma! contro! (O with the STATSb cDNA probe. A junction fragment is detected with ligfll (11 kb). The presence of a rearranged band confirms. at the genomie levcl. the result obcained with toial RNAs after 5-RACE-PCR.

show complete reniission after differentiation therapy with ali-trans RA (ATRA) (41). Addition of ATRA mediates replacement of the repressor comple\ by an activator compIex with acetyl-transferase activit\ (42.38). The transcriptional activator comp]ex is composed of CREB binding protein (CBP)/p300 and histone acetvltran$ferase. APL-Ls with the PLZF-RARA eene fusion are

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not sensitive to ATRA because the PLZF componeni of the ehi-meric protein is also able to reeruit a repressor complex with histone deacenlase activitv but RA has not effect on the PLZF-

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repressor compiex (36-38). It is noteworthy that our case did not respond to ATRA. The STATSb protein-protem interaction domain has been shown to associate with N-Myc inieractor (Nmi), which in tum enhances association of CBP/p300 with STAT5 (29). The chimeric STAT5b-RARA protein could seąuester CBP/p300 cotactors and therefore prevent ATRA to release the repressor complex from the RA target genes. AJtema-tively. an ATRA-insensitive association of N-CoR co-repressor with the N-terminal coiled-coil domain derived from the STATSb component of the fusion protein could explain the unresponsive-ness of the present APL-L to RA.

Apart from its role in prolactin and growth hormone-induced functions. STAT5b is also invoived in differentiation and prolifer-ation of myeloid progenitors. NotabJy. G-CSF. which plays a crit-icai role in granuiopoiesis. stimulates STAT5 transcription factor activitv (23). The STATSb component of the fusion protein could therefore participatc in leukaemogenesis in tiic present APL-Land explain the atypicai phenotype as compared with classicai APL. Thus far. scvcraJ repons link aberrations in the JAK/STAT patii-ways to malignant phenotypes. In Dmsophila. u dominant mutant Jak kinase (hopTum-1) causes leukaemia-Iike abnormaiities <43;. Constitulivc STAT5 activation has becn found in T celi leukacmia/lymphoma (44.45; ;md in malignant T lymphocytcs dcrived from culaneous anapiastic large T ccii lymphoma and Sezary syndromc (46The STA TS transcription factors were also found consiitutively activalcd mainly in aculc lymphohlastic Icu-kaemias (ALL; (47.4K; and in hacmutopoiclic celi lines trans* formed by BCR/ABL tyrosine kinase (49.50). It was also Jound conslitutive!v aciivated in acute mvdoid leukaemias

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(47.48,51.52) and in familial erytlirocytosis (53). Before this report. TEL-JAK2 fusions in a T ccii childhood acute lympho-blastic leukaemia (.54) and in myeloid leukaemias (55) were the unique gene rearrangements in the JAK/STAT pathways described in human cancers. Ali TEL-JAK2 variants stronclv

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activate STAT5 (56). 'lite finding of constitutively activated STAT5 transcription factors in numerous malignant haematopoi-etic disorders strongly supports the idea of STATS-dependent oncogene activation. Verv recently, Nieborowska-Skorska et al. (57) have demonstrated a causal involvement of STAT5 activa-tion in BCR/ABL-mediated leukaemogenesis both in vitro and in vivo.

The present APL-L with a gene fusion between STATSb and

RARA is the first malignancy that harbours a rearranged member of

the ST AT gene family. The STATSb-RARA chimeric protein con-

tains 636 amino acids fromjthe N-terminus of STATSb: the fusion

sile occurs within the C terminal SH2 domain of STATSb. before

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Tyt699. Tlie SH2 domain eoordinates interaction of SiatSb with the

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phosphotyrosine docking site on the cytoplasmic pan of the cytokine receptor. The phosphotyrosine docking site reeruits the STATSb protein by interacting with a criticaJ arginine residue (ArgólS) in the highly conserved core of SH2. When reemited. Tyr699 on STATSb i$ phosphoryiated ITyr(P)]. the activated fonn of STATSb is then able to dimerize by reciprocal SH2-Tyr(P) inter-actions. The hishlv conserved core of tlie SH2 domain with Arc618 is present in the ST AT5 b- RARA protein: it could bind to the cytokine receptor. The cytoplasmic truncated STATSb protein could therefore act in a dominant negative inanner by competing with the nomial STATSb transcription factor for tlie interaction with the receptor docking site. Studies on homo- and heterodimeri-zation between the STAT1 and STAT2 proteins showed that an unphosphoiy lated STAT can dimerize w ith a TynP)-STAT: a single TyrtPP-SH2 interaction therefore seems sufficient fordimeriza-tion (58.59). II' the chimeric STATSb-RARA protein could 'dimerize' by a single Tvr(P)-SH2 interaction with the wild-type STATSa and STATSb proteins. it w ill seąuester nomial STAT proteins and perturbate tlie JAK/STAT5 pathways. Moreover. tlie fusion protein is mainly nuclear and conuiins a functionaJ DNA-binding region. One couid anticipate that stablc homo- or hei-erodimers with nomial STAT5a/b proteins are generated and bind to GAS elements. STAT5b-RARA-STAT5a/b heterodimers could either constimtivclv activate gene transcription or act in a dominant negative manner on activated wild-type STATSa/b transcription factors. If the STAT5b-RARA gene fusion leads to a constitutively activated STATSb transcription factor. cytokine independent growth may participate in leukaemogenesis in this APL-L. Lastly. and as mentionned above. the STAT5 transcription lactors ha\e been shown to internet wńh other proteins such as CBP/p300 to Jbnn enhanceosomes (29). In the present APL-L case. abnomial STATSb proteins could seąuester co-aclivators of other signalling pathways. including the retinoid pathway.

As palient materia! is no longcr availahle. nngoing iransfection studies willi the chimeric cDNA in the Ba/F3 celi linę. which is dependent on IL-3 for growth. w ill allow lesting of the effect of the fusion protein on the STAT5 pathways. Morcovcr. co-trans-lection experiments with an RA-imlucible reporter plasmid will also pennit the study ol the effect ol STATSb-RARA protein on RA-reeulaicd uenes.

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