The mutation landscape of 20 MDS patients
As EMS is often initially diagnosed as MDS, we performed DNA and RNA sequencing for a cohort of 20 MDS patients. The median age of these patients is 59 years (range, 13-78 years), and the male:female ratio is 1:1. Clinical data for all patients are available in Additional files 1: Table S1. For Case 1 who was diagnosed as MDS with abnormal monoclonal B-cell lymphocytosis, RNA-seq detected a novel in-frame HOOK3-FGFR1 fusion gene (Additional files 1: Table S4). Thus, we also performed WES to detect additional mutations in Case 1, upon the target DNA panel including 38 commonly mutated genes in myeloid hematologic malignancies (Additional files 1: Table S2). The mutations identified in all patients are presented in Fig 1A with further details provided in Additional files 1: Table S3. In our MDS cohort, 19 different genes were mutated: ASXL1 (9/20, 45%); U2AF1 (6/20, 30%); TET2 (4/20, 20%); RUNX1 (3/20, 15%); PHF6 (3/20, 15%); SF3B1 (2/20, 10%), CBL (2/20, 10%), TP53 (2/20, 10%), EZH2 (2/20, 10%), PPM1D (2/20, 10%) and CCND1, DNMT3A, IDH1, KMT2A, SETBP1, STAG2, BCOR, ZRSR2 and SRSF2 (1/20, 5% each) (Fig. 1B). In our cohort, we identified nine mutations in the ASXL1 gene (six frameshift, one missense, and two stopgain mutations), although no mutations were located in the functional domain of ASXL1 (Fig. 1C). In contrast, all six variants of U2AF1 were located in the key zinc finger domain (Fig. 1C). The recurrently mutated genes involve in ASXL1 and U2AF1 in our MDS cohort which is consistent with previous study .
Clinical presentation of one EMS patient with ring chromosome 8
The clinical course of Case 1 was shown in Fig. 2A. This patient is a 58-year-old woman who suddenly presented with unconsciousness after initial complaints of fatigue and chest tightness dyspepsia, abdominal distention, and early satiety lasting for 1 month. Blood tests revealed a leukocytosis (white blood cell: 4×109/L), anemia (hemoglobin: 29 g/L), and thrombocytopenia (platelet: 4×109/L). The BM wright-stained smear showed decreased myelodysplasia, with 3.5% myeloblasts (Fig. 2B). Cytogenetic analysis revealed the following complex karyotype: 46,xx,add(1)(p36),-5,-8,add(9)(p13),del(10)(q24),add(11)(q23),-15,+22,+marl,+mar2/47,idem,+X/46,idem,-marl,+mar3/46,idem,+r,-marl (Fig. 2C). We also observed an abnormal ring chromosome (RC), which indicates poor prognosis (Fig. 2C). Immunophenotyping by flow cytometry (FCM) identified 2.43% myeloblasts (positive for CD34 and CD117) and 11% monoclonal B-lymphoid cells (positive for CD19 and cLambda) (Fig. 2D). Multiplex RT-PCR analysis for 43 leukemia-related fusion genes showed a negative result (data not shown). Target DNA sequencing of 38 genes (Additional files 1: Table S2) and WES revealed mutations in the TET2, ASXL1, KMT2A, RUNX1, TP53, and CCND1 genes (Fig. 1A, Additional files 1: Table S4). RNA-seq detected a novel HOOK3-FGFR1 fusion gene involved in 8p11 locus, thus this patient was diagnosed as EMS based on the WHO 2016 criteria.
Initially, the patient received standard induction chemotherapy of the BHA regimen (bortezomib, homobarringtonie, and cytarabine), but showed no remission (NR) with 2.6% myeloid blasts and 7.5% monoclonal B-lymphoid blasts (Fig. 2A). The patient was sequentially treated with rituximab 200 mg and rituximab 600 mg within one week. The FCM results indicated the disappearance of monoclonal B-lymphoid blasts, but 6.17% myeloid blasts remained (Fig. 2A). As further treatment, one course of B+5-AZA+AAG regimen (bortezomib, 5-azacytidine, pirarubicin, cytarabine, and granulocyte colony stimulating factor (G-CSF)) was given, and 3.9% myeloid blasts indicated NR (Fig. 2A). As ponatinib has been proven to be effective in the treatment of FGFR1 fusion positive patients [36-38], ponatinib treatment was started. However, this patient failed to respond to 1 week of ponatinib treatment (Fig. 2A). The patient died of pulmonary infection on October 2, 2020.
The confirmation and feature of a novel HOOK3-FGFR1 fusion gene
The STAR-Fusion result for HOOK3-FGFR1 fusion in Case 1 was shown in Fig. 3A (Additional files 1: Table S4). This patient showed the bilineage blasts (2.13% myeloblasts: positive for CD34; 11% monoclonal B-cell blasts: positive for CD19) at diagnosis (Fig. 2A, 2D). Previous studies have reported that FGFR1 fusion was concurrently observed in multiple lineages [39, 40]. We separated the BM sample into two populations (CD19+ and CD19–) by FCM on the basis of CD19 expression, and then used FISH analysis to detect the FGFR1 break-apart signal. In the CD19 negative population, we observed a split green signal consistent with a breakpoint of the FGFR1 gene; this was not observed in the CD19 positive population (Fig. 3B). Further, we identified the positive FGFR1 rearrangement signal using FISH analysis on the specimens (20.08.25) which only including myeloid blasts (Fig. 3B). These results demonstrated that FGFR1 rearrangement of Case 1 only appeared in the myeloid lineage blasts. We also used FISH analysis to confirm that the ring chromosome was chromosome 8 (Fig. 3B). Using qRT-PCR and Sanger sequencing, we further validated the HOOK3-FGFR1 fusion transcript with the breakpoints located at the 11th exon of HOOK3 and the 10th exon of FGFR1 (Fig. 3C). According to the chromosomal position, we inferred that the formation of HOOK3-FGFR1 fusion may be the result of inversion (Fig. 3D). The in-frame HOOK3-FGFR1 fusion transcript is presumed to encode a new kinase protein with 768 amino acids (Fig. 3E). The N-terminal component of the HOOK3-FGFR1 fusion protein consists of HOOK3 exons 1-11 encoding 374 amino acid residues, including a partial coiled-coil dimerization domain (Fig. 3E). The C-terminal component consists of FGFR1 exons 10-18 encoding 394 amino acid residues with only the entire tyrosine kinase domain retained, not the transmembrane (TM) domain (Fig. 3E). Based on these overall findings, we infer that the HOOK3-FGFR1 fusion protein presents constitutive activation of FGFR1 tyrosine kinase and may contribute to the pathogenesis of the Case 1.
Activation of the NF-kappaB pathway induced by the HOOK3-FGFR1 fusion gene
At present, there are no data or studies describing the transcriptomic signature of FGFR1 fusion. First, we compared gene expression profiling between the HOOK3-FGFR1 fusion positive patient and 8 healthy donors. The scatterplot showed the top 10 up-regulated genes including TNF, CCL4 and CXCL3, the top10 down-regulated genes such as MMP9, ANXA3, and LTF (Fig. 4A). The functional annotation found the enrichment of HALLMARK_TNFA_SIGNALING_VIA_NFKB, and KEGG_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION pathways (Fig. 4B). Furthermore, we employed GSEA to compare the expression of the patient with HOOK3-FGFR1 fusion to the other MDS patients. We observed significantly up-regulated enrichment of HALLMARK_TNFA_SIGNALING_VIA_NFKB, PHONG_TNF_TARGETS_UP, and SANA_TNF_SIGNALING_UP (Fig. 4C and Additional files 2: Fig. S1A). These results suggest that HOOK3-FGFR1 fusion may activate the NF-kappaB signaling pathway as an unreported transcriptional feature for HOOK3-FGFR1 fusion.
Previous studies have demonstrated that the FGFR1 fusion protein plays a role in signal activation of FLT3, MYC and STAT5 [28, 41]. However, there is no study reporting activation of NF-kappaB signaling. To further validate whether HOOK3-FGFR1 fusion can trigger NF-kappaB pathway, we firstly constructed the HOOK3-FGFR1 expression vector plasmid and confirmed the presence of chimeric HOOK3-FGFR1 protein, as detected by Western blot analysis with an anti-FLAG antibody (Additional files 2: Fig. S1B). Furthermore, RayBiotech NF-kappaB pathway phosphorylation array was used to measure the phosphorylation level of NF-kappaB signaling proteins. When compared to vecter-control cells, we found that phosphorylated IKB-alpha (Ser32), TAK1 (Ser412), and NF-kappB (p65: Ser536) were increased by 1.62-fold, 1.37-fold and 1.21-fold in HOOK3-FGFR1 cells derived from 293T cells, respectively (Fig. 4D). Taken together, these findings indicate that the HOOK3-FGFR1 fusion protein has potential to trigger NF-kappaB signaling, which may play an important role in the function of the HOOK3-FGFR1 fusion gene.