Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Novel Drivers and Modifiers of MPL-dependent Oncogenic Transformation Identified by Deep Mutational Scanning (#226)

Jessica L Bridgford 1 2 , Su Min Lee 1 2 , Christine M M Lee 3 , Paola Guglielmelli 4 , Elisa Rumi 5 6 , Daniela Pietra 5 6 , Stephen Wilcox 1 2 , Yash Chhabra 3 , Alan F Rubin 1 2 7 , Mario Cazzola 5 6 , Alessandro M Vannucchi 4 , Andrew J Brooks 3 , Matthew E Call 1 2 , Melissa J Call 1 2
  1. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia
  3. The University of Queensland Diamantina Institute, The University of Queensland, Woolloongabba, QLD, Australia
  4. Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi, Firenza, Italy
  5. Department of Hematology Oncology, UOC Ematologia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
  6. Department of Molecular Medicine, University of Pavia, Pavia, Italy
  7. Bioinformatics and Cancer Genomics Laboratory, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/MPL), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF) patients, respectively, have ‘canonical’ MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other ‘non-canonical’ MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but in almost all cases their functional consequences and relevance to disease are unknown.

Here we used a deep mutational scanning (DMS) approach to evaluate all possible single-amino-acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and seven novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that new mutations reported here have the potential to drive or modify myeloproliferative disease.

With no high-resolution structures of any domain of the TpoR currently available, the DMS approach employed here revealed key structural insights. All constitutively-activating substitutions identified in the transmembrane region were localised to the same face of an alpha-helix, suggesting that they may support formation of structurally similar TpoR TMD interactions. Interestingly, constitutively-activating mutations identified in the juxtamembrane regions, S493L/C in the extracellular juxtamembrane and multiple W515 variants in the intracellular juxtamembrane, were located on a distinct face of an alpha-helix, suggesting the existence of another active interface that may be functionally distinct. Similarly to S505N, all constitutively-activating mutations were highly restricted by position and amino acid type and most substitutions increased the intermolecular hydrogen bonding potential, demonstrating the importance of these interactions for ligand-independent receptor activation. Altogether, this study demonstrates the power of the DMS approach for investigating protein structure and function and additionally provides a valuable diagnostic reference for MPN.

  1. Bridgford JL, Lee SM, Lee CMM, Guglielmelli P, Rumi E, Pietra D, Wilcox S, Chhabra Y, Rubin AF, Cazzola M, Vannucchi AM, Brooks AJ, Call ME and Call MJ. 2019. Novel Drivers and Modifiers of MPL-dependent Oncogenic Transformation Identified by Deep Mutational Scanning. Blood, (online 11/10/2019, official publication date yet to be released).