A novel <i>HNRNPC‐RARA</i> fusion in acute promyelocytic leukaemia lacking <i>PML‐RARA</i> rearrangement, sensitive to venetoclax‐based therapy

Abstract

The vast majority of acute promyelocytic leukaemia (APL) is characterised by the balanced translocation t (15;17) (q22;q12) resulting in the fusion transcript promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA). Rare cases with APL are found to bear translocations with RARA fused with other partner genes including promyelocytic leukaemia zinc finger protein (PLZF), nucleophosmin 1 (NPM1), nuclear mitotic apparatus protein (NUMA), signal transducer and activator of transcription 5b (STAT5b), STAT3, protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A), factor interacting with PAPOLA and CPSF1 (FIP1L1), B-cell lymphoma 6 (BCL-6) co-repressor (BCOR), oligonucleotide/oligosaccharide-binding fold-containing protein 2A (OBFC2A), transducin beta-like 1 X-linked receptor 1 (TBLR1), general transcription factor IIi (GTF2I), interferon regulatory factor 2 binding protein 2 (IRF2BP2) and fibronectin type III domain containing 3B (FNDC3B).1-3 In the present study, we report a novel fusion gene involving the heterogeneous nuclear ribonucleoprotein C (HNRNPC) gene and the RARA gene identified in an APL patient lacking t(15;17) (q22;q12)/PML-RARA fusion. The study was approved by the Ethics Committee of Sir Run Run Shaw Hospital at Zhejiang University School of Medicine. The patient gave her written informed consent. In December 2019, a 29-year-old woman was admitted to our hospital due to fever and lower back pain. Her blood analysis showed a white blood cell count of 10·5 × 109/l, a haemoglobin level of 57 g/l, a platelet count of 65 × 109/l and 5% blasts. Prothrombin time and activated partial thromboplastin time were within the normal range. Fibrinogen and D-dimer levels were 0·7 g/l (reference range 2–4 g/l) and 6·9 μg/ml (reference <0·5 µg/ml) respectively. A bone marrow (BM) smear showed an infiltration by 91% of atypical hypergranular promyelocytes, and no Auer rod was found in the patient’s promyelocytes (Fig 1A). Further flow cytometry revealed that these cells were positive for cluster of differentiation (CD)13, CD33, CD117 and cytoplasmic myeloperoxidase (cMPO), partially expressed CD64, human leukocyte antigen-DR isotype (HLA-DR), CD9 and CD123 and were negative for CD16, CD11b, CD34 and other T- or B-lymphoid-related markers, which is consistent with the phenotype of abnormal promyelocytes. The presumptive diagnosis of this patient was APL. However, interphase fluorescence in situ hybridisation (FISH) using the PML-RARA dual-colour dual-fusion translocation probes, nuc ish (PML, RARA)×2(PML con RARA×1), failed to detect the PML-RARA rearrangement in 400 nuclei of the patient’s BM cells (Fig 1B). The karyotype analysis revealed 46, XX [20] (Fig 1C). Moreover, detection of PML-RARA or PLZF-RARA fusion by fluorescent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was also negative. The multiplex fluorescent qualitative RT-PCR, targeting 51 leukaemia-related translocations/chromosomal abnormalities (Table SI) including the PML-RARA (bcr1, bcr2, bcr3), BCOR-RARA, FIP1L1-RARA, PLZF-RARA, NUMA1-RARA, nucleoporin 98 and 96 precursor (NUP98)-RARA and STAT5B-RARA fusion transcripts, were all negative. To search for potential rearrangements involving the RARA or retinoic acid receptor-β (RARB) or retinoic acid receptor-γ (RARG) genes, we used the patient’s BM sample collected at diagnosis to perform whole transcriptome RNA sequencing (RNA-Seq) analysis using Illumina HiSeq X (llumina, San Diego, CA, USA). Two types of novel RARA fusion transcripts were detected. The major fusion transcript involved the HNRNPC gene and the RARA gene. RNA-Seq results revealed one breakpoint in intron 3 of the HNRNPC gene and one breakpoint in intron 2 of the RARA gene (Fig 1D). The 5′ region of the HNRNPC gene (from exon 1 to exon 3) (NM_031314) was fused in frame with the 5′ region of the RARA gene (from exon 3 to exon 9) (NM_000964.4) (Fig 1D). Another minor fusion transcript involving the RARA gene and the HNRNPC pseudogene 2 (HNRNPCP2). RNA-Seq results revealed one breakpoint in intron 8 of the RARA gene and the 5′ region of the RARA gene (from exon 1 to exon 8) (NM_000964.4) was fused in frame with the 5′ region of the HNRNPCP2 gene [from nucleotide sequences 409–1722 base pairs (bp)] (NG_006653) (Fig 1E). For validation of two novel fusions, RT-PCR using complementary DNA was performed, and the following primers were designed to amplify HNRNPC-RARA mRNA: forward (at HNRNPC exon 3), 5′-ATTGGGAATCTCA A-3′, and reverse (at RARA exon 3), 5′-TCAGGCTACCACTATGGG-3′. The following primers were used to amplify RARA-HNRNPCP2 mRNA: forward (at RARA exon 8), 5′-GGTCTACGTGCGGAAGC-3′, and reverse (at HNRNPCP2: c.568–585), 5′-CAGCCCGAGCAATAGGAG-3′. The expected bands of approximately 300 bp (HNRNPC-RARA) and 270 bp (RARA- HNRNPCP2) were visualised by electrophoresis (Fig 1F), and the PCR product was analysed by Sanger sequencing and confirmed HNRNPC-RARA and RARA-HNRNPCP2 fusion transcripts (Fig 1G) in the patient’s cells. HNRNPC 5′-region encodes an RNA-recognition motif (RRM), and the main domains of the RARA gene encoding a DNA-binding domain (DBD) and a ligand-binding domain (LBD) were preserved on the HNRNPC-RARA fusion protein (Fig 1H). The DBD and LBD of RARA were also preserved on the RARA-HNRNPCP2 fusion protein. The function of the pseudogene HNRNPCP2 is unknown, the following functional analysis of the fusion protein focus on HNRNPC-RARA. A customised next-generation sequencing (NGS) panel using Illumina Next500 (Illumina) to target single nucleotide variations (SNVs) and insertiona and deletions (indels) in 66 haematological malignancy related genes (Table SII) was also performed, and the X-linked plant homeodomain finger protein 6 (PHF6) gene mutation (NM_001015877:exon2: c.96dupA: p.L32fs) with a variant frequency of 27·59% was detected. Other common gene mutations in haematological malignancies, including ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1), additional sex combs like-1 (ASXL1), ataxia-telangiectasia mutated (ATM), FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), tumour protein p53 (TP53), NPM1, NRAS proto-oncogene, GTPase (NRAS) and so on, were undetected. The sequencing depth of the targeted NGS panel was ×1912. The diagnostic BM mononuclear lysates from the patient were analysed by Western blot with a rabbit monoclonal (EPR 23871-271) to RARA antibody (from Abcam, Cambridge, UK). As shown in Fig 2A, in addition to the wild-type RARA band (50 kDa), a higher molecular weight band (54 kDa) corresponding to the HNRNPC-fused RARA was seen, which had 21 more amino acids than the wild type. We then detected the subcellular distribution feature of the novel RARA fusion protein. In the present study, we demonstrated that the HNRNPC protein was found in nucleus and the RARA in nucleus, so HNRNPC-RARA was predominantly found in nucleus by immunofluorescence analysis (Fig 2B and Data S1). We further performed RT-qPCR to detect the expression levels of RARA fusion targeted-genes, which have been reported to be involved in the pathophysiology of APL including RARB, RARG, E74-like ETS transcription factor 4 (ELF4), PML-RARA-regulated adaptor molecule 1 (PRAM1), tumour necrosis factor alpha-induced protein 2 (TNFAIP2) and TNF receptor 2 (TNFR2),4 in the patient’s BM sample obtained at diagnosis compared with those in BM mononuclear cells obtained from healthy volunteers, blasts obtained from patients with APL with PML/RARA fusion, blasts obtained from patients with non-APL acute myeloid leukaemia (AML) and APL cell line NB4 cells. The detailed information of the primers of these respective genes are described in Table SIII. The RT-qPCR results validated that compared with cells from healthy volunteers or patients with non-APL AML, there were significant down-expressions of targeted-genes of RARA fusion protein in the patient’s cells, which was comparable to those in APL cells carrying PML-RARA fusion and those in NB4 cells (Fig 2C). Western blot analysis also confirmed decreased levels of ELF4, PRAM1 and TNFR2 proteins in BM cells of our patient with HNRNPC-RARA fusion and that of the patient with classical APL with PML/RARA fusion (Fig 2D, and the densitometry data in Figure S1). A more important question is whether the HNRNPC-RARA fusion protein was sensitive to all-trans retinoic acid (ATRA) or not. Then, we expressed haemagglutinin (HA)-tagged HNRNPC-RARA vector in 293T cells. Whereas unlike other RARA fusion proteins, we found that HNRNPC-RARA expression was not responsive to ATRA treatment, but down-regulated by RARα agonist (tamibarotene) treatment or BCL-2 inhibitor (ABT-199) treatment by immunofluorescence analysis (Fig 2E) and Western blot analysis (Fig 2F, and the densitometry data in Figure S2). Strikingly, the patient in our present study showed no blast differentiation after induction therapy with ATRA for 2 weeks. Then, she received induction chemotherapy composed of idarubicin and cytarabine (IA). After the first course of induction, BM aspiration revealed 3% myeloblasts and promyelocytes, and flow cytometry suggested the BM minimal residual disease (MRD) was 2·3%. The patient received a second course of IA chemotherapy. After that, it remained at 2% myeloblasts and promyelocytes in the BM, with a MRD of 0·9% detected by flow cytometry. Considering that the patient did not achieve complete remission (CR) after two courses of IA induction chemotherapy, the patient underwent a new chemotherapy induction following a scheme of azacitidine (75 mg/m2 for 7 days) combined with a BCL-2 inhibitor (venetoclax, 100 mg daily with posaconazole). The patient achieved MRD-negative CR detected by flow cytometry. Then, she underwent HLA-matched unrelated donor haematopoietic stem cell transplantation (URD-HSCT) with a myeloablative conditioning regimen. The follow-up was regular for ~12 months, and haematological CR persisted with complete donor chimerism. The patient also achieved molecular remission by detection of HNRNPC-RARA and RARA-HNRNPCP2 fusion transcripts by RT-qPCR. The HNRNPC gene is located at 14q11.2 and encodes hnRNP C protein, an abundant and ubiquitously expressed nuclear RNA-binding protein responsible for pre-mRNA processing including RNA splicing, polyadenylation, stability, RNA expression, transport and translation mediated by internal ribosome entry site.5-7 Although the roles of elevated expression of HNRNPC in promoting the oncogenesis of solid tumours are well-studied,8, 9 the potential contribution of deregulated HNRNPC expression to leukaemia is largely unknown. Recently, Su et al.10 reported a novel HNRNPC-RARG fusion gene and its reciprocal in a patient with APL-like leukaemia. We identified a HNRNPC-RARA fusion in APL for the first time, which suggests that HNRNPC may be as a new partner gene other than PML in APL and the detailed biological function of HNRNPC-RARA/RARG needs to be investigated in the future. More importantly, compared with traditional APL, most patients with PLZF-RARA, STAT3-RARA, STAT5B-RARA or cleavage and polyadenylation specific factor 6 (CPSF6)-RARG fusion transcripts have a poor response to ATRA and significantly worse overall survival (OS) (3-year OS: 26·7% vs. 92·1%) and leukaemia-free survival (LFS) (3-year LFS: 20·0% vs. 86·5%).11 Our present patient and the above mentioned patient with HNRNPC-RARG fusion gene both showed no response or a resistance to ATRA treatment. Although combined chemotherapy was administrated during induction therapy for patients with APL carrying RARA rearranged with partner genes other than PML or APL-like patients with translocations involving RARB or RARG genes. Rare cases benefited from conventional combined chemotherapy. This highlights the appeal of incorporating newer agents in the treatment of such rare cases. Disruption of the RARα-related signalling pathway may represent a potential therapeutic target for AML with RARA dysregulation. McKeown et al.12 recently demonstrated that in non-APL AML, the presence of super-enhancers (SEs) at the RARA gene locus and concomitant high levels of RARA mRNA were found to be sensitive to SY-1425 (tamibarotene), a potent and selective RARα agonist, in preclinical models; however, ATRA failed to show efficacy in RARA-high models. Further study suggested that hypomethylating agents (HMAs) and SY-1425 exerted synergistic anti-proliferative effects in AML models with RARA pathway activation in vitro and in vivo, which supports clinical trials to explore the safety and efficacy of SY-1425 in combination with HMAs in patients with AML.13 Tamibarone is currently approved in Japan for the treatment of patients with relapsed/refractory APL. On the other hand, defects in the cell growth and apoptotic pathways are responsible for both disease pathogenesis and treatment resistance. Therefore, pro-apoptotic agents are potential candidates for APL treatment. Dos Santos et al.14 reported the vitamin E derivative (+)alpha-tocopheryl succinate is as effective as arsenic trioxide or ATRA in classic PML/RARA murine APLs, which is a pro-apoptotic agent and induces an early dissipation of the mitochondrial membrane potential and the inhibition of mitochondrial respiratory chain complex I. Niu et al.15 found that APL cell lines and primary AML samples with the APL phenotype are especially sensitive to single-agent ABT-199, which suggested a possible role for BCL-2-selective inhibitor in patients with APL who do not respond to ATRA and/or arsenic trioxide-based therapies. Consistent with this, our present results suggested that HNRNPC-RARA expression was refractory to ATRA treatment, but down-regulated by RARα agonist or ABT-199 in vitro, and our patient showed no response to ATRA treatment and achieved CR with venetoclax combined with azacitidine treatment. In summary, we identified HNRNPC-RARA as another novel RARA fusion in APL. However, it is also worth noting that pseudogene transcripts may often be involved in regulating translation and transcript availability. The function of pseudogene HNRNPCP2 and RARA-HNRNPCP2 fusion transcript in APL may deserve further study. Our present study highlighted the importance of comprehensive molecular analysis to characterise AML with APL-like morphology. A better knowledge of RA signalling in different leukaemia contexts represents a novel target for rational drug combinations with ATRA or analogues. This work was supported by the National Natural Science Foundation of China (No. 81870136). Haowen Xiao and He Huang designed the research study; Meng Liu, Zijun Qian, Mengbao Du and Li-Mengmeng Wang performed the experiments; Meng Liu, Xiujie Zhao, Wenjue Pan and Haowen Xiao analysed the data; Haowen Xiao wrote the paper and He Huang revised the paper. The authors declare no conflict of interest. Data S1. Supplemental methods. Table SI. The multiplex fluorescent qualitative RT-PCR targeting 51 leukaemia-related translocations/chromosomal abnormalities. Table SII. A customised next-generation sequencing (NGS) panel using targeting single nucleotide variations (SNVs) and insertion and deletions (indels) in 66 haematological malignancy related genes. Table SIII. The detailed information of the primers to detect the expression levels of the RARB and RARG genes and RARA targeted-genes in RT-PCR. Fig S1. Relative optical density of ELF4, PRAM1 and TNFR2 to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in BM cells from the patient with HNRNPC-RARA fusion, blasts from a patient with classical APL with PML/RARA fusion and BM mononuclear cells obtained from healthy volunteer. Fig S2. Relative optical density of HNRNPC-RARA to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in transfected 293T cells after treatment with 1 μmol/l of ATRA, or 1 μmol/l of RARα agonist (tamibarotene), or 5 μmol/l of BCL-2 inhibitor (ABT-199), or dimethyl sulphoxide (DMSO; vehicle control) for 48 h. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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