New Research Identifies W491 as a Potential Therapeutic Target in Essential Thrombocythemia

In patients with familial essential thrombocythemia (ET) negative for JAK2, CALR, or MPL W515 mutation (triple-negative), newly identified MPL mutations elucidate a broad mechanism of thrombopoietin receptor (TpoR) activation that is shared by the small molecule eltrombopag. This is according to a study in Blood, which found that the activation of TpoR by the novel L498W-H499C or H499Y-S505N mutations relies on upstream amino acid W491. W491 is also the key residue required for activation by the W515 mutation pathway and the activation by eltrombopag.1 Together, these results suggest that this new target structure within the TpoR could serve as a therapeutic target in ET.

“If W491 would be targeted and prevented to stabilize the dimeric interface of the mutants described in this paper, this would lead to inhibition of oncogenic activity,” said corresponding study author Stefan Constantinescu, MD, of the Université Catholique de Louvain in Belgium. The same would occur for more frequent mutants of MPL, W515K, and S505N, he added. “But the interesting part is that W491 is in the extracellular region above the membrane and is accessible to antibodies and molecules that would not need to enter the cell via the plasma membrane,” he noted.

The study was an analysis of 2 patients with ET who tested negative for JAK2, CALR, and MPL W515 mutations – common driver mutations for myeloproliferative neoplasms. Each patient harbored either L498W-H499C or H499Y- S505N, defined as double cis mutations in the MPL gene, which encodes for TpoR protein. According to the authors of this study, this is the first report of these combination mutations in patients with ET.

The human TpoR is mainly a cell-surface monomer that dimerizes, in the presence of either Tpo or the small-molecule agonist eltrombopag or when mutated (S505N, W515K), via a structural change in the region around H499. To test their hypothesis that L498W and H499C would induce persistent dimerization of TpoR, the investigators generated cell lines to express the TpoR mutations alone or in combination.

In their analysis, the authors found that both single mutations L498W or H499C led to ligand-independent STAT5 transcriptional activity, although L498W had a stronger effect, and the combination was even stronger. Dimerization of TpoR H499C was equivalent to or greater than that observed in L498W and there was an increase in cell-surface localization and stability of H499C. However, H499C alone “dimerizes TpoR in a conformation not optimal for signaling.”

The investigators then truncated TpoR upstream of position 489 and subsequently moved the mutations in the receptor construct. Compared with the WT truncated receptor, single and double mutants demonstrated more signaling, suggesting that the mutations act on transmembrane and cytosolic domains. When the researchers treated the cells with eltrombopag, the TpoR WT was activated, but not the truncated TpoR mutants in agreement with a requirement for extra amino acid residues upstream of H499.

In a second ET patient, the authors identified the double mutation H499Y-S505N (in cis). Although the H499Y mutant was found to be active alone, the double mutant exhibited significantly enhanced activation of STAT5 over S505N. Both H499C and H499Y were specific enhancers of their counterparts L498W and S505N. They were unable to enhance signaling by other TpoR mutants such as W515K.

Furthermore, the authors used a structural approach and identified W491 as a key upstream residue necessary to stabilize mutations in the Tpo receptor as well as allowing a response to eltrombopag.

When W491A mutation was induced this was associated with impairment of eltrombopag-mediated activation as well as the transmembrane or W515 mutations. Thus, W491 is crucial for the changes in tilt and orientation required for activation. As W491 might be accessible in the juxtamembrane region compared with the transmembrane mutations, this residue could be targeted clinically to reduce the activity of TpoR mutations.

In an accompanying editorial, Ian Hitchcock, PhD, of the University of York in the U.K., noted that several key questions about TpoR activity remain.2 First, a comprehensive understanding of how TpoR is activated remains elusive, since a complete structure of the receptor has yet to be identified. He added that, even if a structure is identified, it will not offer answers to all key questions because JAK2 will play a role in stabilization, dimerization, and localization of the receptor and will also be involved in interactions with the plasma membrane.

“Moreover,” Dr. Hitchcock wrote, “receptor density at the plasma membrane will alter the ability of the activating mutations to drive receptor dimerization, highlighting one of the issues faced by all of us using TpoR-overexpressing cell lines to model receptor activity.”

The authors report no relevant conflicts of interest.


  1. Levy G, Carillo S, Papoular B, et al. MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491. Blood. 2020;135:948-953.
  2. Hitchcock IS. Novel ET mutations: stuck in the MPL with you. Blood. 2020;135:889-890.