Pivotal role of PDK1 in megakaryocyte cytoskeletal dynamics and polarization during platelet biogenesis

Geue, Sascha; Aurbach, Katja; Manke, Mailin-Christin; Manukjan, Georgi; Münzer, Patrick; Stegner, David; Brähler, Caroline; Walker-Allgaier, Britta; Märklin, Melanie; Borst, Carla E.; Quintanilla-Fend, Leticia; Rath, Dominik; Geisler, Tobias; Salih, Helmut R.; Seizer, Peter; Läng, Florian; Nieswandt, Bernhard; Gawaz, Meinrad; Schulze, Harald; Pleines, Irina

doi:10.1182/blood.2019000185

Cited by 26 publications

(27 citation statements)

References 53 publications

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“…On the other hand, altered Cof1 phosphorylation has been observed in several thrombocytopenic mouse lines with deficiencies in cytoskeletal-regulatory genes, including Cdc42 and Pak2, classic upstream regulators of Cof1 activity, 49,50 as well as Twf2a and Pdk1. 27,51 Furthermore, aberrant regulation of Cof1 activity was functionally associated with thrombocytopenia in a mouse model for von Willebrand disease type 2B, 52 as well as with defective PPF in MKs from a patient with platelet-type von Willebrand disease. 53 Together, these findings emphasize the importance of Cof1 as a master regulator of cytoskeletal dynamics in MKs.…”

Section: Discussionmentioning

confidence: 95%

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

et al. 2020

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Rearrangements of the microtubule (MT) and actin cytoskeleton are pivotal for platelet biogenesis. Hence, defects in actin- or MT-regulatory proteins are associated with platelet disorders in humans and mice. Previous studies in mice revealed that loss of the actin-depolymerizing factor homology (ADF-H) protein Cofilin1 (Cof1) in megakaryocytes (MKs) results in a moderate macrothrombocytopenia but normal MK numbers, whereas deficiency in another ADF-H protein, Twinfilin1 (Twf1), does not affect platelet production or function. However, recent studies in yeast have indicated a critical synergism between Twf1 and Cof1 in the regulation of actin dynamics. We therefore investigated platelet biogenesis and function in mice lacking both Twf1 and Cof1 in the MK lineage. In contrast to single deficiency in either protein, Twf1/Cof1 double deficiency (DKO) resulted in a severe macrothrombocytopenia and dramatically increased MK numbers in bone marrow and spleen. DKO MKs exhibited defective proplatelet formation in vitro and in vivo as well as impaired spreading and altered assembly of podosome-like structures on collagen and fibrinogen in vitro. These defects were associated with aberrant F-actin accumulation and, remarkably, the formation of hyperstable MT, which appears to be caused by dysregulation of the actin- and MT-binding proteins mDia1 and adenomatous polyposis coli. Surprisingly, the mild functional defects described for Cof1-deficient platelets were only slightly aggravated in DKO platelets suggesting that both proteins are largely dispensable for platelet function in the peripheral blood. In summary, these findings reveal critical redundant functions of Cof1 and Twf1 in ensuring balanced actin/microtubule crosstalk during thrombopoiesis in mice and possibly humans.

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Section: Discussionmentioning

confidence: 95%

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

et al. 2020

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“…Circulating platelet counts in PDK1 null mice are about 25% reduced . A recent study has shown that PDK1 plays a critical role in platelet production by regulating cytoskeletal dynamics . Our lab and others have shown that regulated protein synthesis is critical for proplatelet formation by mature megakaryocytes .…”

Section: Resultsmentioning

confidence: 87%

“…A recent study published while our manuscript was under review demonstrated that PDK1 mediates platelet production by regulating cytoskeletal dynamics. In mice, PDK1 deletion caused defects in actin polymerization with disrupted F‐actin assembly, reduced podosome numbers, and impaired spreading . Previous studies in other cells have shown that MARCKs plays an important role in cytoskeletal development and regeneration as well as actin crosslinking …”

Section: Discussionmentioning

confidence: 99%

Phospho‐inositide‐dependent kinase 1 regulates signal dependent translation in megakaryocytes and platelets

Manne

Bhatlekar

Middleton

et al. 2020

Journal of Thrombosis and Haemostasis

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Background: Regulated protein synthesis is essential for megakaryocyte (MK) and platelet functions, including platelet production and activation. PDK1 (phosphoinositide-dependent kinase 1) regulates platelet functional responses and has been associated with circulating platelet counts. Whether PDK1 also directly regulates protein synthetic responses in MKs and platelets, and platelet production by MKs, remains unknown. Objective: To determine if PDK1 regulates protein synthesis in MKs and platelets. Methods: Pharmacologic PDK1 inhibitors (BX-795) and mice where PDK1 was selectively ablated in MKs and platelets (PDK1 −/− ) were used. PDK1 signaling in MKs and platelets (human and murine) were assessed by immunoblots. Activation-dependent translation initiation and protein synthesis in MKs and platelets was assessed by probing for dissociation of eIF4E from 4EBP1, and using m7-GTP pulldowns and S 35 methionine incorporation assays. Proplatelet formation by MKs, synthesis of Bcl-3 and MARCKs protein, and clot retraction were employed for functional assays. Results: Inhibiting or ablating PDK1 in MKs and platelets abolished the phosphorylation of 4EBP1 and eIF4E by preventing activation of the PI3K and MAPK pathways. Inhibiting PDK1 also prevented dissociation of eIF4E from 4EBP1, decreased binding of eIF4E to m7GTP (required for translation initiation), and significantly reduced de novo protein synthesis. Inhibiting PDK1 reduced proplatelet formation by human MKs and blocked MARCKs protein synthesis. In both human and murine platelets, PDK1 controlled Bcl-3 synthesis. Inhibition of PDK1 led to complete failure of clot retraction in vitro. Conclusions: PDK1 is a previously unidentified translational regulator in MKs and platelets, controlling protein synthetic responses, proplatelet formation, and clot retraction. K E Y W O R D S platelet activation, platelets, protein translation, signal transduction, thrombosis S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section. How to cite this article: Manne BK, Bhatlekar S, Middleton EA, Weyrich AS, Borst O, Rondina MT. Phospho-inositidedependent kinase 1 regulates signal dependent translation in megakaryocytes and platelets. J Thromb Haemost.

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“…The alteminostat trial (NCT03150316, Phase 1, South Korea) has enrolled 10 RRMM patients treated with median of 2.5 prior lines of therapy in a trial designed to assess alteminostat in combination with lenalidomide and dexamethasone [ 28 ]. Alteminostat is a novel pan-HDAC inhibitor with significant in vitro growth inhibition of multiple myeloma cell lines, especially in reducing the secretion of interleukin 6, which is associated with the proliferation of myeloma cells [ 88 ].…”

Section: Small Molecule Inhibitors and Modulatorsmentioning

confidence: 99%

Emerging therapies for relapsed/refractory multiple myeloma: CAR-T and beyond

2021

J Hematol Oncol

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The pace of innovation of multiple myeloma therapy in recent years is remarkable with the advent of monoclonal antibodies and the approval of novel agents with new mechanisms of action. Emerging therapies are on the horizon for clinical approval with significant implications in extending patient survival and advancing closer to the goal of a cure, especially in areas of immunotherapy such as chimeric antigen receptor T cells, bispecific T cell engager antibodies, antibody drug conjugates, newer generations of monoclonal antibodies, and small molecule inhibitor and modulators. This review provides an update of current myeloma therapeutics in active preclinical and early clinical development and discusses the mechanism of action of several classes of novel therapeutics.

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Pivotal role of PDK1 in megakaryocyte cytoskeletal dynamics and polarization during platelet biogenesis

Abstract: The investigators explore the role of PDK1 (phosphoinositide-dependent protein kinase 1) in the cytoskeletal regulation of platelet production and furnish new insights into megakaryocyte maturation and proplatelet formation.

Cited by 26 publications

References 53 publications

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

Actin/microtubule crosstalk during platelet biogenesis in mice is critically regulated by Twinfilin1 and Cofilin1

Phospho‐inositide‐dependent kinase 1 regulates signal dependent translation in megakaryocytes and platelets

Emerging therapies for relapsed/refractory multiple myeloma: CAR-T and beyond

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