Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.Irina Pleines ... Benjamin T. Kil

Therapeutic targeting of CK2 in acute and chronic leukemias

Phosphorylation can regulate almost every property of a protein and is involved in all fundamental cellular processes. Thus, proper regulation of phosphorylation events is critical to the homeostatic functions of cell signaling. Indeed, deregulation of signaling pathways underlies many human diseases, including cancer.[1] The importance of phosphorylation makes protein kinases and phosphatases promising therapeutic targets for a wide variety of disorders.[2] CK2, formerly known as casein kinase II, was discovered in 1954, [3] although only recently, and especially over the last two decades, it has become one of the most studied protein kinases, due to its ubiquity, pleiotropy and constitutive activity. In particular, appreciation of its pleiotropy has completely changed our vision of CK2 biology, from an ordinary cell homeostasis-maintaining enzyme to a master kinase potentially implicated in many human physiological and pathological events. CK2 is responsible for about 25% of the phosphoproteome,[4] as it catalyzes the phosphorylation of >300 substrates.[5] This partly explains the CK2 interconnected roles that underlie its involvement in many signaling pathways. However, CK2 prevalent roles are promotion of cell growth and suppression of apoptosis. Accordingly, several lines of evidence support the notion that CK2 is a key player in the pathogenesis of cancer. High levels of CK2 transcript and protein expression, as well as increased kinase activity are associated with the pathological functions of CK2 in a number of neoplasias.[6] It was only over the last decade, after extensive analyses in solid tumors, that basic and translational studies have provided evidence for a pivotal role of CK2 in driving the growth of different blood cancers as well, although the first report demonstrating increased CK2 expression in acute myelogenous leukemia (AML) dates back to 1985.[7] Since then, CK2 overexpression/activity has been demonstrated in other hematological malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML). [8] With the notable exceptions of CML and pediatric ALL, many patients with leukemias still have a poor outcome, despite the development of protocols with optimized chemotherapy combinations. Insufficient response to first-line therapy and unsalvageable relapses present major therapeutic challenges. Moreover, chemotherapy, even if successful, could have deleterious long-term biological and psychological effects, especially in children.[9] Furthermore, CML patients can develop resistance to tyrosine kinase inhibitors (TKIs), while both primary chemoresistant and relapsed pediatric ALL cases still remain an unresolved issue.[9

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.The research participants were enrolled in the Biomedical Research Centres/Units Inherited Diseases Genetic Evaluation (BRIDGE) Bleeding and Platelet Disorders (BPD) study (UK REC10/H0304/66). We are grateful to all the donors who allowed us to use their samples for this study. We thank Sofia Papadia from the NIHR BioResource for organizing the recalls of BRIDGE-BPD participants. The genome sequencing of the BRIDGE-BPD participants was supported by the NIHR BioResource–Rare Diseases (to ET, KD, and WHO). The NIHR BioResource–Rare Diseases is responsible for the delivery of the rare diseases pilot phase of the 100,000 Genomes Project and is funded by the National Institute for Health Research (NIHR; http://www.nihr.ac.uk). Research in the Ouwehand laboratory also receives funding support from the European Commission, NIHR, Wellcome Trust, Medical Research Council (MRC), and British Heart Foundation under numbers RP-PG-0310-1002 and RG/09/12/28096. SKW is supported by an MRC Clinical Training Fellowship (MR/K023489/1). ADM receives support from the Bristol NIHR Biomedical Research Unit for Cardiovascular Disease. This work was supported by a Project Grant (no. 575535), a Program Grant (no. 1016647), a Fellowship (1063008 to BTK and 1058344 to WSA), Project Grants (to PWG and ECH), and an Independent Research Institutes Infrastructure Support Scheme Grant (no. 361646) from the Australian National Health and Medical Research Council; a fellowship from the Sylvia and Charles Viertel Foundation (to BTK); a start-up grant, a fellowship, and a grant from the German Research Foundation (SFB 688, PL707/1-1 and PL707/2-1 to IP); the Kids’ Cancer Project (to PWG); a Fellowship from the European Hematology Association (to MRT) and the British Heart Foundation (PG/13/77/30375 to MRT); NHS Blood and Transplant (to WHO and MRT); the Australian Cancer Research Fund; and a Victorian State Government Operational Infrastructure Support Grant

Tropomyosin controls sarcomere-like contractions for rigidity sensing and suppressing growth on soft matrices

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied via local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micron elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ~2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing