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Letters to the Editor
Figure 2. In vitro sumoylation assay of NFE2-WT and the NFE2-K368X mutant. Recombinantly expressed GST-NFE2-WT (WT, wild-type) (72 kDa) and NFE2- K368X (45 kDa) were tested for modification with the SUMO1 (top) or the SUMO2/3 (bottom) peptide by different E3 ligases, as indicated. The GST tag was proteolytically cleaved from NFE2-K368X to exclude any potential influence on protein conformation and overall charge. The reaction mixture was subjected to SDS-PAGE and immunoblotting using an anti-NFE2 antibody. Following modification with SUMO2/3, two bands appear as SUMO2/3 is capable of forming chains by self-sumoylation. *The IR1+M mediated modification of GST-NFE2-WT with SUMO1. **The IR1+M mediated modification of NFE2-K368X with SUMO1.
sequestered in PML nuclear bodies, leaving non-sumoy- latable NFE2-K368X available to exert the observed, unphysiologically high transcriptional activity.
To test the physiological effect of the K368X mutation, we employed a murine bone marrow (BM) transplanta- tion model. FVB/N-45.2 donor BM was lentivirally infect- ed to express either NFE2-K368X or NFE2-WT and trans- planted into FVB/N-45.1 recipient mice. No significant difference in CBC between mice expressing NFE2- K368X, NFE2-WT, or an empty control construct was observed, although a trend towards an elevated throm- bocyte count at older age was noted in mice expressing NFE2-K368X (Figure 3A). This observation is consistent with the very late disease onset in our patient, who only manifested clinical signs of thrombocytosis at the age of 74, despite having carried the LNK and NFE2 mutations since birth.
Because of the mild platelet phenotype observed, we morphologically evaluated megakaryopoiesis in histologi- cal BM sections of the transplanted mice. We defined three size categories for megakaryocytes (large, middle and small) and enumerated them in five high power fields
each of 5 mice of each genotype. The total number of megakaryocytes was increased 50% in NFE2-K368X transplanted mice compared to WT controls (Figure 3B). Moreover, the fraction of small and large megakaryocytes was significantly increased compared to WT, while the fraction of middle-sized megakaryocytes decreased, sug- gesting alterations in megakaryocyte maturation (Figure 3B). Polymorphic and pleomorphic megakaryocytes, visi- ble in our murine BM histologies (Figure 3C and D), are also typically observed in BM sections of MPN patients.
In conclusion, this report constitutes the first descrip- tion of a constitutional NFE2 mutation. Since in a murine model, the NFE2-K368X mutant is sufficient to cause thrombocytosis with a long latency similar to that observed in our patient, and since the LNK-E208Q-muta- tion was hypothesized to require co-operation with an MPN-driver mutation to produce an MPN phenotype, we now propose that the NFE2-K368X mutation constitutes such an MPN-driver. Our findings provide a rationale for investigating possible NFE2 mutations in triple-negative MPN patients. The observation that the NFE2-K368X mutation abrogates NFE2 sumoylation by either SUMO1
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haematologica | 2021; 106(4)