SQSTM1: A NEW PARTNER FOR NUP214 IN ADULT T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Background. NUP214, a FXFG nucleoporin located on the cytoplasmic side of the nuclear pore complex (NPC), plays a critical role in cell cycle progression and import/export trafficking. In T-cell acute lymphoblastic leukemia (T-ALL) NUP214 fusions appear to predict a poor prognosis. So far two NUP214 fusions were identified in T-ALL: when cryptic del(9q) is present SET fuses with NUP214, while episomal amplification leads NUP214 to rearrange with ABL1. Supervised gene expression profiling (GEP) analysis in adult T-ALL identified SET-NUP214 positive cases with high expression of HOXA cluster genes, MEIS1 and NUP214, and low expression of SET, FNBP1. C9orf78 and USP20 genes.1 Among 69 cases of T-ALL, an additional case emerged at GEP analysis with over-expression of HOXA, MEIS1, NUP214, and also of SET. This 20-year old man with a chemo-resistant pre-T ALL showed 46,XY, del(6p) karyotype at diagnosis. Aims. To investigate last case as an hypothetical new rearrangement involving NUP214 in T-ALL. Methods. We set up FISH assays with RP1-112N13 for the 9q sub-telomere, RP11-143H20, flanking the 3’NUP214, RP11-544A12, spanning the gene, and two overlapping fosmids, G248P89801E11 (exons 25-31) and G248P8659A12 (exons 31-36). Additional FISH experiments were done with 5q35-qter probes: RP11-117L6, RP11-286C20, RP11-549A4, RP11-319K2, RP11-718N2, RP1-240G13. NESTED RT-PCR was performed using primers SQSTM1_ex3_528F (5’-TGCCCAGACTACGACTTGTG-3’) / NUP214_ex36_6543R (5’-AGTAATCATGCGCCTTGTGAGTT-3’), for the first amplification round, and SQSTM1_ex4/5_763F (5’-AATCAGCTTCTGGTCCATCG-3’)/NUP214_ex33/34_6337R (5’-CAAAGCTGAACCCTCCTGTG -3’) for the second. PCR products were cloned into pGEM-T easy vector system and sequenced. Results. FISH probe RP11-143H20 gave two signals on normal chromosomes 9 while RP11-544A12 and RP1-112N13 gave 3 signals on chromosomes 9 and on der(5). The NUP214 breakpoint fell between G248P89801E11, which was retained on chromosome 9, and G248P8659A12, translocated to der(5). The 5q35 breakpoint was telomeric to RP11-718N2, a ~1.7Mb region containing 15 candidate genes, with an appropriate centromere-telomere orientation. GEP indicated down-regulation of genes telomeric to SQSTM1, which was thus selected as first candidate gene. RT-PCR detected an amplification product of 852bp. Molecular cloning and sequencing identified the in-frame fusion between nucleotide 849 (exon 5) of SQSTM1 and nucleotide 6014 (exon 33) of NUP214. Nested-PCR and molecular screening of 60 T-ALL adults did not identify any additional case. Conclusions. SQSTM1 point mutation and over-expression were described in congenital Paget’s Bone Disease and solid tumours, respectively. No translocations involving SQSTM1 have been reported, yet. In this adult with T-ALL NUP214/9q34 rearranged with SQSTM1 as a result of a cryptic unbalanced translocation with an apparently normal chromosome 5, i.e, ish der(5)t(5;9)(q35;q34). As in other NUP214 leukemic fusions, the FG repeats were maintained, suggesting similarities with leukemic recombinations of other nucleoporins such as NUP98. However, together with the NUP214 FG repeats, the SQSTM1 N-terminal structural and regulatory motifs, such as the Zinc-finger domain may contribute to the leukemogenic process. To elucidate the incidence and the clinical impact of NUP214 rearrangements in T-ALL we strongly recommend NUP214 FISH screening in large prospective studies. DDG and PG shared co-authorship. Aknowledgements. PRIN 20078C9NRT_003.