High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation.

The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains.

New pathogenic mechanisms induced by germline erythropoietin receptor mutations in primary erythrocytosis.

Primary familial and congenital polycythemia is characterized by erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the erythropoietin receptor gene. All mutations so far described lead to the truncation of the C-terminal receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the erythropoietin hypersensitivity induced by a new erythropoietin receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing erythropoietin receptor dimerization, stability and cell-surface localization, causes pre-activation of erythropoietin receptor and JAK2, constitutive signaling and hypersensitivity to erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with erythropoietin receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to erythropoietin receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of erythropoietin receptor mutation.

Germline duplication of ATG2B and GSKIP predisposes to familial myeloid malignancies.

No major predisposition gene for familial myeloproliferative neoplasms (MPN) has been identified. Here we demonstrate that the autosomal dominant transmission of a 700-kb duplication in four genetically related families predisposes to myeloid malignancies, including MPN, frequently progressing to leukemia. Using induced pluripotent stem cells and primary cells, we demonstrate that overexpression of ATG2B and GSKIP enhances hematopoietic progenitor differentiation, including of megakaryocytes, by increasing progenitor sensitivity to thrombopoietin (TPO). ATG2B and GSKIP cooperate with acquired JAK2, MPL and CALR mutations during MPN development. Thus, the germline duplication may change the fitness of cells harboring signaling pathway mutations and increases the probability of disease development.

Germ-line JAK2 mutations in the kinase domain are responsible for hereditary thrombocytosis and are resistant to JAK2 and HSP90 inhibitors.

The main molecular basis of essential thrombocythemia and hereditary thrombocytosis is acquired, and germ-line-activating mutations affect the thrombopoietin signaling axis. We have identified 2 families with hereditary thrombocytosis presenting novel heterozygous germ-line mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, whereas the other presents 2 JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of Janus kinase 2 (JAK2) R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-thrombopoietin receptor (MPL) but not of Ba/F3-human receptor of erythropoietin cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed thrombopoietin-independent phosphorylation of signal transducer and activator of transcription 1. The JAK2 R867Q and S755R/R938Q proteins had significantly longer half-lives compared with JAK2 V617F. The longer half-lives correlated with increased binding to the heat shock protein 90 (HSP90) chaperone and with higher MPL cell-surface expression. Moreover, these mutants were less sensitive to JAK2 and HSP90 inhibitors than JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL while inducing a decreased sensitivity to clinically available JAK2 inhibitors.