A novel NPM1-RARG-NPM1 chimeric fusion in acute myeloid leukaemia resembling acute promyelocytic leukaemia but resistant to all-trans retinoic acid and arsenic trioxide

The RARG gene is a member of the nuclear hormone receptor superfamily and shares high homology with RARA and RARB. RARA is involved in translocation with PML in acute promyelocytic leukaemia (APL). Little is known about RARB or RARG rearrangement. RARG fusions were reported in only five APL patients and the partner genes were NUP98, PML and CPSF6. Here, we report NPM1 as a new partner gene of RARG and identify a unique NPM1-RARG-NPM1 chimeric fusion for the first time in an old male with morphological and immunophenotypical features of hypergranular APL but lacking response to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O3) therapy. The structural features of the fusion transcript may account for the clinical resistance of the patient. RARG fusion is rare but recurrent in APL, further investigation in larger cohorts is expected to assess frequency, clinical characteristics and outcomes of RARG-translocation in APL.


BACKGROUND
Acute promyelocytic leukaemia (APL) is characterised by the PML-RARA fusion caused by t(15;17)(q22;q12) translocation. Rarely, APL cases carry gene fusions involving RARG, which is a member of the same retinoid acid receptor (RAR) family and shares high homology (90%) with RARA and RARB. In 2011, Such et al. reported the first APL case harbouring a rearrangement of RARG. 1 To date, NUP98-RARG, PML-RARG and CPSF6-RARG translocations have been reported in a total of five acute myeloid leukaemia (AML) patients resembling APL. [1][2][3][4] Here, we describe the first case with a novel NPM1-RARG-NPM1 chimeric fusion in an old male with morphological and immunophenotypical features of hypergranular APL but lacking response to all-trans retinoic acid (ATRA) and arsenic trioxide (As 2 O 3 ) therapy.
The patient was treated with As 2 O 3 (10 mg/d, days 1-34) and showed no response. Then he was switched to ATRA therapy (50 mg/d, days 35-70) after the confirmation of NPM1-RARG rearrangement. The NPM1-RARG transcripts remained positive and were highly expressed in both peripheral blood and BM samples of the patient during the course of treatment. He refused to receive chemotherapy and died 8 months after diagnosis.
www.nature.com/bjc Whole genome sequencing To clarify the genomic breakpoints in NPM1 and RARG, 30× whole genome sequencing (WGS) was performed on genomic DNA of BM sample using HiSeq X Ten (Illumina, Inc., San Diego, CA) after approval by the ethics committee at the 2nd Affiliated Hospital of Harbin Medical University. Raw reads in fastq were pre-processed and controlled for quality using fastp, followed by rapid genome analysis using speedseq with default parameters. Structural variants were called from speedseq with default options and the next annotation tool was AnnotSV.
Targeted next-generation sequencing and mutation analysis Mutational hotspots or whole coding regions of 86 genes that are known to be frequently mutated in haematologic malignancies were sequenced using a targeted, multiplexed amplicon-based high-throughput sequencing protocol as we previously reported. 5

RESULTS
Laboratory, morphology and immunophenotypic analysis of the patient suggested diagnosis of hypergranular APL. IDH1 R132H and SRSF2 P95_R102del mutations were identified.
WGS analysis revealed breakpoints in intron 4 of NPM1 and 5′ untranslated region (5′-UTR) of RARG. Interestingly, two more breakpoints in NPM1 intron 10 and RARG intron 9 were identified. Both NPM1 intron 4-RARG 5′-UTR and RARG intron 9-NPM1 intron 10 genomic fusions were confirmed by Sanger sequencing. Hence the genomic alterations of this patient were a deletion of 16,360 bp of NPM1 from intron 4 to intron 10 accompanied by an insertion   Fig. 1 Identification of a novel NPM1-RARG-NPM1 chimeric fusion in a APL case lacking t(15;17)(q22;q12)/PML-RARA. a WGS found that NPM1 and RARG each has two breakpoints (shown in red arrows). Sequencing chromatogram showed the two genomic junction sequences (NPM1 intron 4-RARG 5′-UTR and RARG intron 9-NPM1 intron 10). b RT-PCR and sequencing of the PCR products verified the presence of NPM1-RARG-NPM1 chimeric fusion with three kinds of fusion transcripts. c Expected protein sequences translated from the three fusion transcripts. The same oligomeric amino acid tail (VSLRK) as in C-terminal region of all mutant NPM1 that frequently occurred in AML was shown in yellow of 23,479 bp of RARG from 5′ UTR to intron 9 (Fig. 1a). Moreover, RT-PCR and Sanger sequencing verified the presence of three NPM1-RARG-NPM1 transcripts presumably derived from alternative splicing: NPM1 (exon 1-4) -RARG (partial exon 1-exon 9) -NPM1 (exon 11), NPM1 (exon 1-4) -RARG (exon 2-9) -NPM1 (exon 11) and NPM1 (exon 1-4) -RARG (exon 4-9) -NPM1 (exon 11) (Fig. 1b). The microdeletion of NPM1 and microinsertion of RARG at the genomic level were too subtle to be found by karyotype analysis. The NPM1 5′-region encoding the nucleoplasmin domain was fused to the DNA-binding domain (DBD) of RARG in all three transcripts. Deletion of RARG exon 10 led to 25 amino acids loss of the ligand-binding domain (LBD) of RARG. Notably, the three transcripts generated the same C-terminal oligomeric amino acid tail (VSLRK) as in all mutant NPM1 that frequently occurred in AML 6 due to the 3′ end fusion of NPM1 exon 11 and frameshift coding. The two critical C-terminal tryptophan (W) residues at positions 288 and 290 which are necessary for nucleolar localisation of NPM1 were also altered 6 (Fig. 1c).

DISCUSSION
The NPM1 gene encodes nucleophosmin, which is a highly conserved nucleo-cytoplasmic shuttling protein that shows restricted nucleolar localisation. Mutations or translocations involving NPM1 gene cause cytoplasmic ectopia of nucleophosmin and are associated with several haematological malignancies, especially the bio pathogenesis of AML. 6 NPM1-RARA has been reported as a very rare variant of RARA translocations in APL 7 (Fig. S2a). In this case, the NPM1-RARG-NPM1 fusion leads to both impairment of NPM1 protein and abnormal of RARG. The missing of NPM1 exon 5-9 and the mutation-like C-terminus of the NPM1-RARG-NPM1 transcripts may result in impaired function and ectopia of nucleophosmin in cytoplasm and contribute to the impaired differentiation and leukogenesis.
RARG, RARA and RARB are nuclear hormone receptors functioning as ligand-dependent transcriptional activators that interact specifically to modulate transcription of DNA elements, and all have highly conserved DBD and LBD. 8 Fusions and aberrations of RARs contributed to hematopoietic differentiation arrests at promyelocytes stage and constitute the basis for therapeutic response of ATRA-induced differentiation therapy. Although very rare, translocations involving RARB (TBL1XR1-RARB) 9 and RARG (NUP98-RARG, PML-RARG, and CPSF6-RARG) [1][2][3][4] have been reported in APL. As in PML-RARA and other RARA fusions, RARB and RARGrearrangements in reported cases preserve both DBD and LBD 1-4,9 (Fig. S2b, e). In the present case, deletion of RARG exon 10 caused 25 amino acids loss of LBD thus may result in impaired ATRA binding affinity (Fig. S2f). On the other hand, the fusion partner of RARG is NPM1 rather than PML may make the patient resistant to As 2 O 3 due to lack of As 2 O 3 binding site. 10 These are in line with the clinical resistance of ATRA and As 2 O 3 of the patient.

CONCLUSION
We report NPM1 as a partner gene of RARG in a patient morphologically resembling APL but lacking response to ATRA and As 2 O 3 therapy for the first time and identify a unique NPM1-RARG-NPM1 chimeric fusion. RARG fusion with different partners is rare but recurrent in APL. Further investigation in larger cohorts is expected to assess frequency, clinical characteristics and outcomes of RARG-translocation in APL.