Effects of Clinically Relevant MPL Mutations in the Transmembrane Domain Revealed at the Atomic Level through Computational Modeling

BACKGROUND: Mutations in the thrombopoietin receptor (MPL) may activate relevant pathways and lead to chronic myeloproliferative neoplasms (MPNs). The mechanisms of MPL activation remain elusive because of a lack of experimental structures. Modern computational biology techniques were utilized to explore the mechanisms of MPL protein activation due to various mutations. RESULTS: Transmembrane (TM) domain predictions, homology modeling, ab initio protein structure prediction, and molecular dynamics (MD) simulations were used to build structural dynamic models of wild-type and four clinically observed mutants of MPL. The simulation results suggest that S505 and W515 are important in keeping the TM domain in its correct position within the membrane. Mutations at either of these two positions cause movement of the TM domain, altering the conformation of the nearby intracellular domain in unexpected ways, and may cause the unwanted constitutive activation of MPL's kinase partner, JAK2. CONCLUSIONS: Our findings represent the first full-scale molecular dynamics simulations of the wild-type and clinically observed mutants of the MPL protein, a critical element of the MPL-JAK2-STAT signaling pathway. In contrast to usual explanations for the activation mechanism that are based on the relative translational movement between rigid domains of MPL, our results suggest that mutations within the TM region could result in conformational changes including tilt and rotation (azimuthal) angles along the membrane axis. Such changes may significantly alter the conformation of the adjacent and intrinsically flexible intracellular domain. Hence, caution should be exercised when interpreting experimental evidence based on rigid models of cytokine receptors or similar systems

Elucidating the Mechanism of Action of the Attributed Immunomodulatory Role of Eltrombopag in Primary Immune Thrombocytopenia: An In Silico Approach

Intel·ligència artificial; Eltrombopag; Trombocitopènia immune primàriaInteligencia artificial; Eltrombopag; Trombocitopenia inmune primariaArtificial intelligence; Eltrombopag; Primary immune thrombocytopeniaEltrombopag is a thrombopoietin receptor (MPL) agonist approved for the treatment of primary immune thrombocytopenia (ITP). Recent evidence shows that some patients may sustain platelet counts following eltrombopag discontinuation. The systemic immunomodulatory response that resolves ITP in some patients could result from an increase in platelet mass, caused either by the direct action of eltrombopag on megakaryocytes through MPL stimulation, or potential MPL-independent actions on other cell types. To uncover the possible mechanisms of action of eltrombopag, in silico analyses were performed, including a systems biology-based approach, a therapeutic performance mapping system, and structural analyses. Through manual curation of the available bibliography, 56 key proteins were identified and integrated into the ITP interactome analysis. Mathematical models (94.92% mean accuracy) were obtained to elucidate potential MPL-dependent pathways in non-megakaryocytic cell subtypes. In addition to the effects on megakaryocytes and platelet numbers, the results were consistent with MPL-mediated effects on other cells, which could involve interferon-gamma, transforming growth factor-beta, peroxisome proliferator-activated receptor-gamma, and forkhead box protein P3 pathways. Structural analyses indicated that effects on three apoptosis-related proteins (BCL2L1, BCL2, BAX) from the Bcl-2 family may be off-target effects of eltrombopag. In conclusion, this study proposes new hypotheses regarding the immunomodulatory functions of eltrombopag in patients with ITP.This research was funded and promoted by Novartis, which contributed to setting up fundamental questions relevant for the study and reviewing the manuscript. However, the company did not have a role on study design, data collection, analysis, or interpretation

Regulation of thrombopoietin receptor expression and function

Of the many cells in the body, the hematopoietic cells are among those with the highest rate of self-renewal and turnover. The production and destruction of these cells are tightly controlled by a number of hematopoietic growth factors, in particular by members of the family of helical cytokines. Studying the thrombopoietin receptor, I focused on two aspects of cytokine receptor signaling: attenuation of signaling by receptor isoforms and the biological function of cytokine receptor target genes. Cytokine receptor signaling has profound effects on cell survival, proliferation and differentiation. It is therefore not surprising that components of the signaling cascade are tightly regulated at the level of expression. An important mechanism for controlling gene expression is alternative splicing. Alternate isoforms have been identified for many cytokine receptors and a regulatory function and/or altered expression in disease have been described for some of these isoforms The cytokine thrombopoietin (TPO) and its cognate receptor c-mpl are the primary regulators of platelet production and also play an important role in hematopoietic stem cell biology. Several isoforms of unknown function exist for both mouse and human mpl and it is possible that they play an important role in modulating mpl signaling. In my thesis work, I have analyzed the function of a truncated receptor isoform (mpl-tr) which is the only alternate mpl isoform conserved between mouse and humans. Although mpl-tr lacks a transmembrane domain, classifying it as a ‘secreted’ or ‘soluble receptor’, it is retained intracellularly. My results provide evidence that mpl-tr acts as a dominant-negative variant of mpl for both proliferation and survival. I also demonstrate that mpl-tr mediates protein degradation of the full-length receptor by a cathepsin-like cysteine protease activity. Due to a shift of the reading frame at a splice acceptor site, the C-terminus of mpl-tr consists of a peptide of unique sequence, 30 amino acids in length. I show that this peptide sequence is essential for the inhibition of TPO-dependent proliferation and for mpl protein degradation mediated by mpl-tr. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function through a proteolytic process directed by a truncated isoform of the same receptor. To test for the in vivo function of alternative mpl isoforms, a c-mpl cDNA was expressed as a transgene in mpl knockout mice. These mice express mpl fulllength as the only mpl isoform and develop severe thrombocytosis with platelet numbers, elevated about five times higher than normal. The reintroduction of the endogenous mpl allele restores normal platelet counts and I attribute this to the in vivo effect of dominant-negative mpl isoforms. A mpl knock-in allele, which does not express mpl-tr but still expresses the second known alternate variant of murine mpl, mpl-II, normalizes platelet numbers, similar to the endogenous mpl allele. This result demonstrates that the absence of mpl-tr is not sufficient to cause thrombocytosis. I propose that mpl-II is an additional dominant-negative mpl isoform and attenuates the expansion of the megakarocytic lineage in vivo. In summary, these results impressively demonstrate the importance of alternate cytokine receptor isoforms in vivo and emphasize the need to study the function of the many uncharacterized cytokine receptor isoforms. In a second project, I studied the role of mpl signaling in regulating the expression of a gene with a potential role in cell differentiation and proliferation. The diversification of cell types is controlled through the use of both lineage-restricted and more widely expressed transcriptional regulators and the combinatorial actions of these regulators specify gene expression. The differentiation of megakaryocyte precursors is dependent on the proper function of the GATA-1 transcription factor. Mice lacking GATA-1 selectively in megakaryocytes have dramatically fewer platelets but more megakaryocytes, altered platelet size and shape and prolonged bleeding times. Further, GATA- 1-null megakaryocytes hyperproliferate in vitro, suggesting that GATA-1 is both a differentiation factor and negative regulator of megakaryocyte cell proliferation. However, GATA-1 regulated genes which are responsible for this growth inhibition are presently unknown. In this thesis work, I describe a novel gene, GASIP (GATA-1 regulated SIAH Interacting Protein), which is dramatically downregulated in mpl-transfected hematopoietic cell lines, identifying mpl as a negative regulator of GASIP expression. The presence of juxtaposed GATA and Ets-binding cis-elements in the GASIP promoter are typical for a megakaryocytic gene. I found that GASIP expression in platelets is indeed robust and correlates with mRNA levels of GATA-1, but not GATA-2 or –3, identifying GATA-1 as a positive regulator of GASIP expression. The finding that mpl and GATA have opposite effects on both proliferation and on GASIP expression, make GASIP a candidate GATA-1 target gene involved in growth inhibition. To investigate the potential role of GASIP in growth regulation, I screened for potential protein binding partners. Interestingly, I identified the p53-inducible tumor suppressor seven in absentia homologe (SIAH) as a GASIP interacting protein. I speculate that GASIP may contribute to the anti-proliferative effect mediated by SIAH

Transmembrane Domain Structure and Function in the Erythropoietin Receptor

a dissertatio

Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.</p

The thrombopoietin receptor : revisiting the master regulator of platelet production

Thrombopoietin (TPO) and its receptor, MPL, are the primary regulators of platelet production and critical for hematopoietic stem cell (HSC) maintenance. Since TPO was first cloned in 1994, the physiological and pathological roles of TPO and MPL have been well characterized, culminating in the first MPL agonists being approved for the treatment of chronic immune thrombocytopenia in 2008. Dysregulation of the TPO-MPL signaling axis contributes to the pathogenesis of hematological disorders: decreased expression or function results in severe thrombocytopenia progressing to bone marrow failure, while hyperactivation of MPL signaling, either by mutations in the receptor or associated Janus kinase 2 (JAK2), results in pathological myeloproliferation. Despite its importance, it was only recently that the long-running debate over the mechanism by which TPO binding activates MPL has been resolved. This review will cover key aspects of TPO and MPL structure and function and their importance in receptor activation, discuss how these are altered in hematological disorders and consider how a greater understanding could lead to the development of better-targeted and more efficacious therapies

Reciprocal t(9;22) ABL/BCR fusion proteins: leukemogenic potential and effects on B cell commitment

Background: t(9;22) is a balanced translocation, and the chromosome 22 breakpoints (Philadelphia chromosome – Ph+) determine formation of different fusion genes that are associated with either Ph+ acute lymphatic leukemia (Ph+ ALL) or chronic myeloid leukemia (CML). The "minor" breakpoint in Ph+ ALL encodes p185BCR/ABL from der22 and p96ABL/BCR from der9. The "major" breakpoint in CML encodes p210BCR/ABL and p40ABL/BCR. Herein, we investigated the leukemogenic potential of the der9-associated p96ABL/BCR and p40ABL/BCR fusion proteins and their roles in the lineage commitment of hematopoietic stem cells in comparison to BCR/ABL. Methodology: All t(9;22) derived proteins were retrovirally expressed in murine hematopoietic stem cells (SL cells) and human umbilical cord blood cells (UCBC). Stem cell potential was determined by replating efficiency, colony forming - spleen and competitive repopulating assays. The leukemic potential of the ABL/BCR fusion proteins was assessed by in a transduction/transplantation model. Effects on the lineage commitment and differentiation were investigated by culturing the cells under conditions driving either myeloid or lymphoid commitment. Expression of key factors of the B-cell differentiation and components of the preB-cell receptor were determined by qRT-PCR. Principal Findings: Both p96ABL/BCR and p40ABL/BCR increased proliferation of early progenitors and the short term stem cell capacity of SL-cells and exhibited own leukemogenic potential. Interestingly, BCR/ABL gave origin exclusively to a myeloid phenotype independently from the culture conditions whereas p96ABL/BCR and to a minor extent p40ABL/BCR forced the B-cell commitment of SL-cells and UCBC. Conclusions/Significance: Our here presented data establish the reciprocal ABL/BCR fusion proteins as second oncogenes encoded by the t(9;22) in addition to BCR/ABL and suggest that ABL/BCR contribute to the determination of the leukemic phenotype through their influence on the lineage commitment

Cooperating Events in Core Binding Factor Leukemia Development: A Dissertation

Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease. In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development. We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b. Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways. Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis