Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning

Abstract

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), 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− essential thrombocythemia and primary myelofibrosis 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 “noncanonical” 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 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 7 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 We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

Structural and functional characterisation of the thrombopoietin receptor and its negative regulator, LNK in myeloid malignancies

“…L498W, V501A, V501S, and L502S [394]. These oncogenic substitutions are dependent on the position and amino acid residues that are mutated [394]. Consistent with these results, the V501A mutation was found in MPN patients and recently reported to confer constitutive receptor activation [403].…”

“…and S505N have shown that the latter conferred stronger or similar cytokine-independent proliferative activity as the W515K mutant [227,394]. In the current study, the addition of puromycin did not continue after selection.…”

“…The varying clinical phenotypes caused by the different mutations may be explained by the ability of TPOR TMD to adopt several orientations that confer its pleiotropic role in haematopoiesis [61], which when dysregulated due to mutations, can result in the different clinical phenotypes observed in patients. Furthermore, in a collaborative study (with the groups headed by Dr Melissa Call and Associate Professor Matthew Call, The Walter and Eliza Hall Institute), several novel mutations (L498W, V501A, V501S, and L502S) in the TMD that result in constitutive signalling of the JAK-STAT and MAPK pathway were identified by utilising the deep mutational scanning approach [394]. This collaborative work also showed that oncogenic TPOR mutations are dependent on the position and amino acid residues that are mutated [394].…”

“…L498W, V501A, V501S, and L502S [394]. These oncogenic substitutions are dependent on the position and amino acid residues that are mutated [394].…”

Next Generation Sequencing in MPNs. Lessons from the Past and Prospects for Use as Predictors of Prognosis and Treatment Responses

“…The remaining 2% of PV patients carry somatic driver mutations in JAK2 exon 12 [ 43 , 44 ]. Another driver mutation, the thrombopoietin receptor gene MPL , is found in up to 5 or 10% of ET or PMF patients, respectively, with MPLW515L/K being the most common mutation [ 14 , 15 , 18 , 45 , 46 , 47 ]. As the JAK2V617F mutation, the MPL mutation confers constitutive, cytokine-independent activation of the JAK-STAT pathway [ 14 ].…”