Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia

Cui, L.; Moraga, I.; Lerbs, T.; Neste, C. V.; Wilmes, S.; Tsutsumi, N.; Trotman-Grant, A. C.; Gakovic, M.; Andrews, S.; Gotlib, J.; Darmanis, S.; Enge, M.; Quake, S.; Hitchcock, I. S.; Piehler, J.; Garcia, C.; Wernig, G.

doi:10.1101/2020.09.23.290593

bioRxiv · 10.1101/2020.09.23.290593

Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia

Cui, L.·Moraga, I.·Lerbs, T.·Neste, C. V.·Wilmes, S.·Tsutsumi, N.·Trotman-Grant, A. C.·Gakovic, M.·Andrews, S.·Gotlib, J.·Darmanis, S.·Enge, M.·Quake, S.·Hitchcock, I. S.·Piehler, J.·Garcia, C.·Wernig, G.

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Abstract

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL)) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable, both experimentally and clinically. We have developed a series of surrogate protein-ligands for TPO-R, in the form of diabodies, that homodimerize the TPO-R on the cell surface in different geometries, in effect tuning downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism and antagonism, thus decoupling the dual functions of TPO/TPO-R. Using scRNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in Essential Thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R. Significance StatementThe TPO cytokine, which signals through its receptor c-MPL (or TPO-R), is essential for megakaryocyte differentiation and maintenance of hematopoietic stem cells (HSCs). Its signaling is deregulated in Essential Thrombocythemia (ET). Here, we engineered diabodies (DBs) against the TPO-R as surrogate TPO ligands to manipulate TPO-R signaling, from full to partial to antagonism, thus decoupling the dual functions of TPO/TPO-R (i.e, HSC maintenance versus megakaryopoiesis). We subsequently discovered that partial agonistic DBs, by reducing the strength of the TPO-R signal, not only preserved HSCs in culture, but also blocked oncogenic signaling in ET. This finding has the potential to improve HSC cultures for transplants, as well as serve as a unique therapeutic approach for ET.

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Cui, L., Moraga, I., Lerbs, T., Neste, C. V., Wilmes, S., Tsutsumi, N., Trotman-Grant, A. C., Gakovic, M., Andrews, S., Gotlib, J., Darmanis, S., Enge, M., Quake, S., Hitchcock, I. S., Piehler, J., Garcia, C., Wernig, G.. 2020-09-23. Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia. https://doi.org/10.1101/2020.09.23.290593

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