The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway.

Accumulation of unfolded and potentially toxic proteins in the endoplasmic reticulum (ER) activates a cell stress adaptive response, which involves a reprogramming of general gene expression. ATF4 is a master stress-induced transcription factor that orchestrates gene expression in cells treated with various ER stress inducers including those used to treat cancers. ER stress-induced ATF4 expression occurs mainly at the translational level involving the activity of the phosphorylated (P) translation initiation factor (eIF) eIF2α. While it is well established that under ER stress PeIF2α drives ATF4 expression through a specialised mode of translation re-initiation, factors (e.g. RNA-binding proteins and specific eIFs) involved in PeIF2α-mediated ATF4 translation remain unknown. Here we identified the RNA-binding protein named DDX3 as a promotor of ATF4 expression in cancer cells treated with sorafenib, an ER stress inducer used as a chemotherapeutic. Depletion experiments showed that DDX3 is required for PeIF2α-mediated ATF4 expression. Luciferase and polyribosomes assays showed that DDX3 drives ER stress-induced ATF4 mRNA expression at the translational level. Protein-interaction assays showed that DDX3 binds the eIF4F complex, which we found to be required for ER stress-induced ATF4 expression. This study thus showed that PeIF2α-mediated ATF4 mRNA translation requires DDX3 as a part of the eIF4F complex.

Epithelial-to-mesenchymal transition (EMT) is a process by which cancer cells gain the ability to leave the primary tumor site and invade surrounding tissues. These metastatic cancer cells can further increase their plasticity by adopting an amoeboid-like morphology, by undergoing mesenchymal-to-amoeboid transition (MAT). We found that adhering cells produce spreading initiation centers (SICs), transient structures that are localized above nascent adhesion complexes, and share common biological and morphological characteristics associated with amoeboid cells. Meanwhile, spreading cells seem to return to a mesenchymal-like morphology. Thus, our results indicate that SIC-induced adhesion recapitulates events that are associated with amoeboid-to-mesenchymal transition (AMT). We found that polyadenylated RNAs are enriched within SICs, blocking their translation decreased adhesion potential of metastatic cells that progressed through EMT. These results point to a so-farunknown checkpoint that regulates cell adhesion and allows metastatic cells to alter adhesion strength to modulate their dissemination.

Epithelial-to-mesenchymal transition (EMT) is a process by which cancer cells gain the ability to leave the primary tumor site and invade surrounding tissues. These metastatic cancer cells can further increase their plasticity by adopting an amoeboid-like morphology, by undergoing mesenchymal-to-amoeboid transition (MAT). We found that adhering cells produce spreading initiation centers (SICs), transient structures that are localized above nascent adhesion complexes, and share common biological and morphological characteristics associated with amoeboid cells. Meanwhile, spreading cells seem to return to a mesenchymal-like morphology. Thus, our results indicate that SIC-induced adhesion recapitulates events that are associated with amoeboid-to-mesenchymal transition (AMT). We found that polyadenylated RNAs are enriched within SICs, blocking their translation decreased adhesion potential of metastatic cells that progressed through EMT. These results point to a so-farunknown checkpoint that regulates cell adhesion and allows metastatic cells to alter adhesion strength to modulate their dissemination.

To the editor:Homologous recombination of wild-type JAK2, a novel early step in the development of myeloproliferative neoplasmTransformation of hematopoietic cells depends on the acquisition of genetic events leading to cytokine independence, typically associated with acquisition of an autocrine cytokine loop or/and increased expression or/and mutation of JAK genes. 1 Rearrangement of the JAK2 gene, which presumably alters JAK2 transcription, is reported in hematopoietic cells. 2 Murine models of myeloproliferative neoplasms (MPN) demonstrated that the polycythemia vera (PV) phenotype requires the combination of high expression and activation of Jak2. 3 Indeed, expression of both wild-type (WT) and mutant JAK2 transcripts can be high in PV. 4 PV is characterized by a high frequency of the JAK2 46/1 (GGCC) haplotype (represented in Figure 1A) predisposing to the JAK2V617F mutation. 5,6 The JAK2V617F mutation facilitates the acquisition of homozygous status for the JAK2V617F by mitotic homologous recombination (HR) occurring between the JAK2WT and JAK2V617F alleles, resulting in chromosome 9p uniparental disomy (9pUPD). 7,8 Here we report 2 cases where high JAK2 mRNA expression was associated with a novel early step in MPN development, HR preceding JAK2 mutation. Patients Na1061 and Na1253 presented with a high hematocrit, slightly elevated leukocyte counts, normal (Na1061) or elevated (Na1253) platelet counts, aquagenic pruritus, absence of splenomegaly, and presence of JAK2V617F (20.7% for Na1061, 30.0% for Na1253), and were diagnosed with PV (see supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Sequencing and allele-specific qPCR analysis in granulocyte DNA of marker rs12343867 (C/T) in intron 14 of JAK2, characteristic of the 46/1 haplotype, revealed rs12343867 ratios sharply different from JAK2V617F ratios: 80% C-alleles for Na1061 and 100% T-alleles for Na1253 (Figure 1B-C). For both patients, CD3 ϩ lymphocytes were unambiguously heterozygous for rs12343867 ( Figure 1C). This indicated granulocyte acquisition of homozygosity for rs12343867 but not for the V617F mutation. In other words, the acquisition of homozygosity for rs12343867 must have preceded JAK2 mutation in these patients. This was confirmed by further analysis of JAK2 in granulocytes and CD3 ϩ lymphocytes ( Figure 1C), and of chromosome 9p using SNP arrays ( Figure 1D). These studies showed that the DNA regions recombined involved JAK2 exons 6-25 for Na1061, and the complete 46/1 haplotype for Na1253. Moreover, SNP array studies revealed the presence of 1 subclone for Na1253 (28.24 Mb) or 2 subclones for Na1061 (5.7 and 24.54 Mb) with partial 9pUPD (supplemental Figures 2-3 and Figure 1E). Sequencing of the complete JAK2 cDNA excluded any mutation other than V617F.These first cases of HR of JAK2WT led us to propose a new model for MPN: the 46/1 haplotype may predispose carriers to diverse alteration of JAK2 including early HR of wild-type JAK2, associated or not with mut...