Synuclein is a group of 3 small acidic proteins of 140 amino acids (α-synuclein), 134 amino acids (β-synuclein), and 127 amino acids (γ-synuclein). These proteins contain an N-terminal lysine-rich amphipathic α-helix domain, the central hydrophobic non-amyloid β component (NAC), and a C-terminal structureless acidic region. The N-terminal domain of synucleins is positively charged and capable of lipid binding at the cell membrane. The NAC domain is an antiparallel β-sheet structure and is involved in fibril formation and aggregation. The structureless acidic region interacts with the N-terminal domain to prevent synuclein aggregation. The C-terminal region is also responsible for protein interaction. While α-synuclein and β-synuclein was first purified from the human brain, with both enriched in presynaptic terminals of neurons, the γ-synuclein was first identified as the breast cancer-specific gene 1, due to its overexpression in these tumors.
Synucleins are mostly known for their roles in a group of neurodegenerative disorders termed synucleinopathies. Mutations of SNCA (encoding α-synuclein) or increased copies of the wild-type SNCA lead to early-onset autosomal dominant Parkinson’s disease, characterized by prominent loss of dopaminergic neurons in the substantia nigra. Two mutations of SYNB (encoding β-synuclein), the P123H and the V70M, are associated with dementia with Lewy bodies, a disorder closely related to Parkinson’s disease. Lewy bodies are protein aggregates in neurons that are pathological hallmarks in synucleinopathies, with α-synuclein being a major component.
The role of synucleins in cancer is implicated by their overexpression and their association with disease progression and/or therapeutic response. α-synuclein is overexpressed in meningioma, pancreatic cancer, glioblastoma, ovarian cancer, colorectal cancer, and melanoma [1, 2]. α-synuclein is not expressed in grade 1 meningioma but expressed in grade 2 and 3 meningiomas, consistent with a role of α-synuclein in disease progression [3]. In pancreatic cancer, α-synuclein expression was correlated to neurotropism, a sign of tumor progression [4]. Evidence of the β-synuclein in cancer is scarce, although its expression is seen in brain tumors including ependymoma, astrocytoma, oligodendroglioma, and medulloblastoma [5]. γ-synuclein is overexpressed in many tumors including breast, ovary, colon, liver, and cervical cancer, and the expression level is higher in more advanced cancers [6, 7]. In animal models and in vitro assays, γ-synuclein is essential in promoting tumor invasion and metastasis. Recent studies suggest that tumors with γ-synuclein overexpression are more resistant to chemotherapy and radiotherapy [8, 9].
We describe here a novel β-synuclein rearrangement in a pediatric T-ALL, with the SYNB fused to a well-known leukemic gene ETV6. By searching the TCGA database, an additional case of squamous lung cancer was found to have an SYNB rearrangement. To our knowledge, these are the first described tumors with SYNB rearrangement.
A 5-year-old girl presented with a neck mass for 2 months. Physical examination revealed lymphadenopathy on both sides of her neck. Complete blood counts showed WBC 1.39 x 109/L, Hb 91 g/L, PLT 111 x 109/L, 31.7% neutrophils, 65% lymphocytes, 0% monocytes, 0% eosinophils, and 0% basophils. A neck lymph node biopsy showed an effaced node with diffuse infiltration of blasts positive for CD3 (dim), CD7, TdT, CD99, PAX5, Ki-67 (40%) and negative for MPO, CD20, CD21 (Fig. 1). The bone marrow smear showed 65% of blasts of variable sizes with small amounts of bluish-grey cytoplasm, dispersed nuclear chromatin, and inconspicuous nucleoli. Cytochemistry was positive for periodic acid-Schiff (PAS) and negative for myeloperoxidase. The bone marrow flow cytometry showed 58% of CD45+ cells with cCD3+, CD5+, CD7+, CD33dim, CD34dim, CD99+, CD1a-, CD2-, sCD3-, CD4-, CD8-, CD13-, CD19-, cCD79a-, MPO-, TdT-. A diagnosis of T-ALL was made. Cytogenetic analysis of bone marrow showed a complex karyotype 45,XX,t(5;12)(q35;p13),t(7;9)(q34;q22),add(12)(p13),der(13)del(13)(q14q32)t(12;13) (p13;q34),-21[16]/46,XX[4] (Fig. 1B). Notably, both copies of chromosome 12p13 (ETV6 locus) were rearranged. FISH assays showed that one locus of the ETV6 was rearranged while the other was partially deleted (the 3’end of ETV6). The rearranged 5’ETV6 was observed at the 5q terminal, consistent with the translocation between chromosomes 5 and 12 (Fig. 1C). A targeted RNA-NGS assay was performed to identify the ETV6 fusion partner gene, which showed 2 in-frame fusion transcripts. The first transcript was consisting of the first exon of ETV6 and the last 2 exons (exon 5 and 6) of SNCB. The second fusion transcript contained sequences from 3 genes, including the first exon of ETV6, the exon 5 of SNCB, and the last exon (exon 2) of the GPRIN1. Because the GPRIN1 is located immediately 3’ to the SNCB, the 3 gene fusion transcript is likely a read-through product of the ETV6::SYNC rearrangement. Both fusion transcripts were further confirmed by RT-PCR with gene-specific primers and subsequent Sanger sequencing (Fig. 2A-D). We believe this is the fusion transcript involving 3 genes observed in the tumor.
The patient was enrolled in a clinical trial of the Chinese Children’s Cancer Group (CCCG) and was treated with the CCCG-2020 protocol, which contains a 4-day-pretreatment with dexamethasone, induction with a combination of dexamethasone, vincristine, daunorubicin, peg-asparaginase, cyclophosphamide, cytarabine, and 6-mercaptopurine, additional early intensification therapy with cyclophosphamide, cytarabine, 6-mercaptopurine, vincristine, and peg-asparaginase, and consolidation therapy with high-dose methotrexate followed by triple intrathecal therapy every other week and daily 6-mercaptopurine for 4 courses. The patient has been in remission for one year. Both ETV6::SYNC and ETV6::SYNC::GPRIN1 were used to monitor the therapeutic response. On the 19th day of the treatment, the ETV6::SYNC disappeared but the ETV6::SYNC::GPRIN1 stayed positive in bone marrow, consistent with minimal residual disease (MRD). On the 46th day of the treatment, both ETV6::SYNC and ETV6::SYNC::GPRIN1 disappeared (Fig. 2A). These RT-PCR results were consistent with clinical remission, negative histology findings, and the MRD status by flow cytometry.
A search of 10,565 cancer genomes from TCGA database found a lung squamous cell carcinoma with LDLRAD3::SNCB (TCGA-85-A50M-01A). The first 4 exons (exons 1-4) of the LDLRAD3, which encodes low-density lipoprotein receptor class A domain containing 3, were fused with the last 4 exons of SYNB (exons 4-7). The reading frame is intact, which encodes the domains 1-3 (D1-3) of the LDLRAD3 and the partial N-terminal, the NAC domain, and the C-terminal of the β-synuclein.
ETV6 is one of the promiscuity genes involved in many tumors including leukemia, lymphoma, secretory carcinoma of the breast, infantile fibrosarcoma, congenital mesoblastic nephroma, and thyroid cancer. A thorough literature search found 38 fusion partners of ETV6, with the SNCB being the 39th fusion partner (Fig. 2E). Rearranged ETV6 has a variety of mechanisms for oncogenesis [10]. One of the more commonly rearranged ETV6, the ETV6::RUNX1, is found in approximately 25% of pediatric B-ALL and is responsible for the deregulation of genome-wide gene expression. The wildtype ETV6 can dimerize with ETV6::RUNX1 to reduce its transforming activity and, therefore, the wildtype ETV6 is often lost in these tumors to accelerate the malignant process. We observed a similar loss of the ETV6 in our case with the ETV6::SNCB rearrangement.
14-3-3s are a group of small proteins playing an essential role in programmed cell death. α-synuclein binds to 14-3-3s and both α-synuclein and 14-3-3s are found in Lewy bodies in patients with Parkinson’s disease. The interaction between α-synuclein and 14-3-3s may be a double-edged sword on the fate of the neurons.14-3-3s function as a chaperone to reduce α-synuclein aggregation and thus could exert neuroprotective activity. On the other hand, α-synuclein may sequester 14-3-3s and release the apoptotic BAD and BAX, leading to apoptosis. Due to extensive homology between α-synuclein and the β-synuclein, a potential oncogenic mechanism of the ETV6::SNCB or LDLRAD3::SNCB could involve the overexpression of the C-terminal synuclein, driven by their fusions with ETV6 or LDLRAD3, both ubiquitously and highly expressed genes, and bind and sequester 14-3-3s to compromise their anti-apoptotic signaling. Additional mechanisms, including p53-dependent and Akt-related apoptotic pathways, could also be involved [11, 12].
The evidence of the involvement of synucleins in cell proliferation mainly comes from γ-synuclein. Overexpression of γ-synuclein leads to increased cell proliferation in H175 (squamous cell carcinoma) by binding and activating the AKT. γ-synuclein is a nicotine-responsive protein and is essential for nicotine-induced cancer cell proliferation [13]. Diabetes is a risk factor for lung cancer. Glucose-induced lung cancer cell proliferation is mediated by γ-synuclein [14]. In ovarian cancer cell lines A2780 and OVCAR5, forced expression of the γ-synuclein increased cell proliferation by activating mitogen-activated kinase (MAPK) signaling and attenuated the chemotherapeutic drug-induced apoptosis by blocking the c-Jun N-terminal kinase (JNK) signaling [8]. The ETV6::SNCB only retains the C-terminal acidic region of the β-synuclein, while the LDLRAD3::SNCB contains partial N-terminal α-helix domain, the NAC domain, and the C-terminal acidic region of the β-synuclein. It will be interesting to investigate whether these fusion proteins affect cell proliferation and/or apoptosis differently.
In summary, we describe the first cases of synuclein rearrangement in tumors. Further evaluation of these naturally occurring chimeric proteins may shed light on not only tumorigenesis but also the physiologic function of β-synuclein, a critical protein for us to understand synucleinopathies.