Page 242 - 2021_04-Haematologica-web

LETTERS TO THE EDITOR
Concomitant constitutive LNK and NFE2 mutation with loss of sumoylation in a case of hereditary thrombocythemia
The vast majority of patients with myeloproliferative neoplasms (MPN), polycythemia vera, essential throm- bocythemia (ET), and primary myelofibrosis acquire driv- er mutations in the JAK2, MPL or CALR gene. Clustering of MPN is seen in select families, but in most pedigrees the MPN-predisposing change has not been determined and affected individuals somatically acquire one of the three above-mentioned driver mutations. In contrast, a small number of individuals with hereditary thrombo- cythemia (HT) carry constitutive alterations, e.g., in the TPO or the LNK (SH2B3) gene.1-4 Acquired mutations in LNK, a negative regulator of JAK2 signaling, rarely occur in both sporadic and familial MPN cases.1,2 In the latter, they do not segregate with disease phenotype and dis- eased individuals acquire a concomitant MPN driver mutation.2 It, therefore, appears unlikely that mutant LNK acts as a driver in MPN.
We have shown that the transcription factor Nuclear Factor Erythroid-derived-2 (NFE2) is overexpressed in the vast majority of MPN patients, independent of other molecular aberrations.5 In addition, we have identified NFE2 mutations in MPN and acute myeloid leukemia (AML) patients, that enhance the activity of wild-type (WT) NFE2.6,7 In several murine models, elevated NFE2 activity causes pathognomonic features of MPN.6-8 The molecular mechanisms by which NFE2 mutants exert their effect remain unclear since the regulation of NFE2 transcriptional activity is poorly understood. Various post-translational modifications have been described, including ubiquitination, phosphorylation, and sumoyla- tion but their functional role remains elusive.9
Here, we identified a 74-year-old female patient given the diagnosis of ET by World Health Organization crite- ria (Online Supplementary Table S1), who tested negative for the three MPN driver mutations, JAK2, CALR, and MPL. Sequencing 36 myeloid neoplasms associated genes (Online Supplementary Tables S2 and S3) revealed both a previously described p.E208Q point-mutation in LNK as well as a novel mutation in NFE2 (c.1102A>T) that pre- maturely truncates the protein at lysine 368 (p.K368X, Figure 1A). Buccal swab DNA analysis determined that both mutations were heterozygously present in the germline. Because of the constitutive nature of both mutations, this patient should be designated as having hereditary thrombocythemia.
The p.K368X mutation leaves almost the entire NFE2 protein, including the bZIP domain and the N-terminal activation domain, intact. Only the terminal 4 amino acids are lost. Notably, this mutation deletes the ψKXE sumoylation consensus motif identified at lysine 368 and shown to be sumoylated by SUMO1 in vitro and in vivo.10 The LNK p.E208Q mutation retains near-complete inhibitory capacity and did not confer significantly higher TPO-hypersensitivity in cell proliferation assays than WT-LNK, suggesting only a subtle loss of function.3 Therefore, we hypothesized that the loss of sumoylation increases NFE2 activity, which, in co-operation with mutant LNK, drives thrombocytosis in this patient.
To test whether NFE2-K368X retains binding to its cognate DNA motif, we performed an electromobility shift assay (EMSA) using a consensus NFE2 binding site from the human PBGD promoter. DNA binding is pre-
served in the NFE2-K368X mutant (Figure 1B), consistent with retention of the complete DNA binding and het- erodimerization domains.
We subsequently examined the ability of NFE2-K368X to transactivate transcription using a luciferase reporter assay. Heterodimerization with MafG is required for opti- mal NFE2 activity (Online Supplementary Figure S1). The NFE2-K368X mutant was more than twice as active at promoting reporter gene expression than WT-NFE2 (Figure 1C). Because transcription off a plasmid DNA template does not model intact chromatin, we used endogenous gene activation in a cell line as a second read-out. CB3 cells are devoid of NFE2 expression due to viral integration but express β-globin upon re-introduc- tion of NFE2. We therefore lentivirally transduced CB3 cells with either WT-NFE2 or NFE2-K368X and deter- mined β-globin expression by quantitative reverse tran- scriptase-polymerase chain reaction (qRT-PCR). Again, NFE2-K368X, present in the same amount, was two times more active than its WT counterpart in directing transcription, demonstrating that the mutation results in a protein with supraphysiological activity on intact chro- matin (Figure 1D). NFE2-K368X thus constitutes a novel Type Ia mutation, DNA–binding and activating, accord- ing to the classification of NFE2 mutations we proposed.7
To investigate sumoylation of the NFE2-K368X mutant, we conducted in vitro sumoylation assays using recombinantly expressed proteins. SUMO is attached to proteins by hierarchical action of the E1-activating enzyme Aos1/Uba2, the E2-conjugating enzyme UBC9, and a substrate-specific E3-ligase. Sumoylation of WT- NFE2 with SUMO1 has been shown in assays that con- tained Aos1/Uba2 and UBC9 but lacked an E3-ligase.10 Under these conditions, we could not detect SUMO1 modification of NFE2 (Online Supplementary Figure S2). Substrate recognition is accomplished by UBC9, but E1 and E2 enzymes have poor transfer efficiency, which is stimulated by E3-ligases. Addition of the E3-ligases IR1+M,11 PIAS1,12 or ZNF451-N,13 led to sumoylation of GST-NFE2-WT but not the NFE2-K368X mutant (Figure 2, top).
IR1+M, the catalytic core domain of the E3-enzyme RanBP2, has high SUMO ligase activity but low substrate specificity.11 Unphysiological in vitro conditions can facili- tate sumoylation of non-canonical lysine residues, which may facilitate both the observed IR1+M mediated sumoylation of WT-NFE2 (Figure 2, lane 1, marked*) as well as unspecific modification of the NFE2-K368X mutant (Figure 2, lane 6, marked**). The RanBP2 frag- ment RanBP2DFG has a higher substrate specificity,11 and did not sumoylate either GST-NFE2-WT or NFE2-K368X (Figure 2, lanes 2 and 7). These data suggest that while NFE2 sumoylation is facilitated by the minimal catalytic activity of the IR1+M fragment,11 NFE2 is not a substrate of the RanBP2 E3-ligase itself. Presence of the small sub- unit MafG does not influence the sumoylation efficacy of NFE2 by IR1+M with SUMO1 (Online Supplementary Figure S3).
Two additional E3-ligases modified NFE2: PIAS1 and ZNF451-N, both with either SUMO1 or SUMO2/3 (Figure 2, top and bottom, lanes 3 and 4). The ZNF451-N ligase has been described as specific for SUMO2/3,13 but modified GST-NFE2-WT with SUMO1 in our study. This activity may result from the high concentration of the components and the unphysiological conditions in vitro. PIAS1 is a member of the PIAS family class of E3-ligases and was described to facilitate MafG sumoylation by
1158
haematologica | 2021; 106(4)