Sending the Right Message: Optimizing Cytokine-receptor Signaling for Therapeutic Impact

Cytokines govern the fate and function of all hematopoietic cells and are the key mediators of cell signaling. All cytokines, to varying degrees, are “pleiotropic,” meaning they can have different effects depending on the cellular and molecular context.¹  Thrombopoietin (TPO) — acting via its receptor myeloproliferative leukemia protein (MPL) — is an exemplar pleiotropic cytokine, acting as a master regulator of hematopoietic stem cell (HSC) maintenance²  while also directing megakaryocyte differentiation and platelet production.³ 

The central importance of TPO/MPL signaling in hematopoiesis is apparent from the consequences of genetic perturbations in the receptor and its downstream signaling pathways, and from the significant impact of agonist therapies. TPO binds to the MPL receptor expressed on HSCs and megakaryocyte lineage cells, activating JAK-STAT signaling. Somatic mutations affecting MPL/JAK2/STAT signaling cause persistent, cytokine-independent MPL activation. This results in clonal expansion and an overproduction of megakaryocytes, causing a myeloproliferative neoplasm (MPN). Some patients develop a severe phenotype called myelofibrosis, in which a pronounced inflammatory and fibrotic reaction in the bone marrow leads to bone marrow failure.⁴ –⁷  Conversely, loss-of-function TPO and MPL mutations can cause multi-lineage aplasia and bone marrow failure as well as thrombocytopenia due to the loss of TPO-driven HSC maintenance. Pharmacological TPO receptor agonists are now widely used clinically, not only to restore platelet counts in patients with immune thrombocytopenia but also to expand the HSC pool and reconstitute trilineage hematopoiesis in aplastic anemia.

Although TPO receptor agonists and inhibitors of JAK2 are now widely deployed in the clinic, the precise mechanics of TPO/MPL signaling have been elusive. Several questions have remained: Is it possible to more precisely control MPL signaling by, for example, boosting stem cell function without causing excess thrombopoiesis and potentially myelofibrosis,^{8, 9}  or to increase platelet counts without causing expansion of HSCs? Further, is it possible to selectively inhibit oncogenic MPL signaling in cells bearing MPN driver mutations while preserving TPO/MPL signaling in healthy cells?

This year, two seminal papers have moved this field forward — with one team targeting the structure of TPO and the other focusing on the structure of MPL — exploring the different dimeric conformations that result from activation by TPO or in the context of a JAK2^V617F mutation.

Naotaka Tsutsumi, PhD, and colleagues published an elegant study in Cell that revealed the structure of the TPO/MPL receptor complex, showcasing how modifications to the TPO molecule can alter its impact on hematopoiesis.¹⁰  The team used cryo-electron microscopy to uncover the structure of the extracellular TPO-MPL complex, visualizing how TPO bridges two MPL chains through high- and low-affinity interfaces. They found that certain loss-of-function mutations in MPL that cause thrombocytopenia impair TPO binding, while others alter receptor trafficking and reduce membrane expression, shedding light on the mechanism of disease and the differential impact of specific disease-associated mutations.

Armed with this information, they then designed modified TPO molecules (TPO^mods) with different charges and receptor affinities. As demonstrated in their study, these TPO^mods could differentially activate MPL and alter downstream signaling by reducing phosphorylation of JAK2 and STAT5 without altering CREB and AKT (or vice versa). Using a live-cell imaging technique (Förster resonance energy transfer [FRET]) to examine the effects of neutral antagonists, partial agonists, and super-agonists they had engineered, Dr. Tsutsumi’s team showed that partial agonist TPO^mods led to reduced MPL dimerization when compared with that observed for native TPO.

They next examined how modified TPOs affected hematopoiesis in vivo, treating mice with either native TPO or murine equivalents of the partial agonist TPO^mod. While a murine TPO^mod did expand the HSC pool — albeit to a lesser extent than “normal” TPO — and increase platelet counts, it had no impact on the number of megakaryocytes or megakaryocyte progenitors. Finally, experiments using human HSCs revealed that TPO^mods could promote HSC maintenance without inducing HSC proliferation and differentiation. Taken together, their findings beautifully highlight how it is possible to functionally uncouple the impact of cytokines by slightly adjusting their structure in a manner that alters receptor dynamics. This sets the stage for increasing the range of TPO receptor agonists deployed in the clinic, enabling a “fine-tuning” of hematopoiesis for patients with disorders affecting stem cell and platelet production.

Also published this year, a detailed study by Nicolas Papadopoulos, PhD, and colleagues, focused not on TPO but on the MPL receptor, exploring how human MPL is differentially activated by wild-type JAK2 (JAK2^WT) and JAK2^V617F, the most common mutation occurring in MPNs.¹¹  Their strategy involved generating multiple MPL fusion proteins to study different possible dimeric orientations of the receptor. Comparisons between JAK2^WT and JAK2^V617F cells showed that the two cell types require different dimeric conformations of MPL for their activity. Most notably, discovering the specific dimeric confirmation responsible for activation of MPL in complex with JAK2^V617F led them to design modifications to the receptor that selectively disrupted mutant but not wild-type signaling. Verification experiments performed in a mouse model were successful: In the absence of TPO and against the background of MPL knockout and JAK2^V617F, colony formation was reduced for all inhibitory mutants when compared with that observed for wild-type MPL.

Although further work is required to confirm that structural modifications of MPL can be translated to the development of mutant-specific pharmacological inhibitors, these studies exemplify how structural insights into cytokine-receptor biology can lead directly to the design of novel therapies that manipulate blood cell production. These insights bring us one step closer to the selective manipulation of pleiotropic cytokine signaling in the clinic, with broad implications for strategic manipulation of communication networks across hematology, immunology, and other physiological settings.