A Senescence-Like Cell-Cycle Arrest Occurs During Megakaryocytic Maturation: Implications for Physiological and Pathological Megakaryocytic Proliferation

During normal megakaryocyte development, in response to thrombopoetin, mature cells enter a senescence-like state in which they shed platelets; this state, characterized by cell cycle arrest, is defective in malignant megakaryocytes

Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its natural ER/Golgi maturation pathway. In contrast to cells expressing the parental MPLW515L, MPLW515L-KDEL-expressing FDC-P1 cells were unable to grow autonomously and to produce tumors in nude mice. When observed, tumor nodules resulted from in vivo selection of cells leaking the receptor at their surface. JAK2 co-immunoprecipitated with MPLW515L-KDEL but was not phosphorylated. We generated disulfide-bonded MPLW515L homodimers by the S402C substitution, both in the normal and KDEL context. Unlike MPLW515L-KDEL, MPLW515L-S402C-KDEL signaled constitutively and exhibited cell surface localization. These data establish that MPLW515L with appended JAK2 matures through the ER/Golgi system in an inactive conformation and suggest that the MPLW515L/JAK2 complex requires membrane localization for JAK2 phosphorylation, resulting in autonomous receptor signaling

New mutations of MPL in primitive myelofibrosis: only the MPL W515 mutations promote a G1/S-phase transition.

International audienceMPL (or thrombopoietin receptor, TPO-R) 515 mutations have recently been described in 5-10% of primitive myelofibrosis (PMF) cases as decisive oncogenic events capable of triggering the disease. Here we report additional mutations located in exon 10 of MPL in PMF patients. We investigated whether these new mutations also lead to cell transformation. MPL exon 10 was systematically sequenced in 100 PMF patients. Seven different mutations were found in eight patients. We introduced each MPL mutant in Ba/F3 cells to determine whether they correspond to gain-of-function mutations. Only MPL W515 mutations induced (1) Ba/F3 proliferation independently of growth factors, (2) tumorigenesis in nude mice, (3) spontaneous activation of JAK/STAT, RAS/MAPK and PI3K transduction pathways and (4) increased S phase of cell cycle. Similar to all other myeloproliferative disorder oncogenic events identified to date, these results demonstrate that only the detected MPL W515 mutations trigger spontaneous MPL activation leading to a G(1)/S transition activation. The other mutations are devoid of significant transforming activity but may synergize with JAK2 V617F or other not yet characterized molecular events

Hypermethylator Phenotype and Ectopic GIP Receptor in GNAS Mutation-Negative Somatotropinomas

Abstract Context Besides GNAS gene mutations, the molecular pathogenesis of somatotroph adenomas responsible for gigantism and acromegaly remains elusive. Objective To investigate alternative driver events in somatotroph tumorigenesis, focusing on a subgroup of acromegalic patients with a paradoxical increase in growth hormone (GH) secretion after oral glucose, resulting from ectopic glucose-dependent insulinotropic polypeptide receptor (GIPR) expression in their somatotropinomas. Design, Setting, and Patients We performed combined molecular analyses, including array-comparative genomic hybridization, RNA/DNA fluorescence in situ hybridization, and RRBS DNA methylation analysis on 41 somatotropinoma samples from 38 patients with acromegaly and three sporadic giants. Ten patients displayed paradoxical GH responses to oral glucose. Results GIPR expression was detected in 13 samples (32%), including all 10 samples from patients with paradoxical GH responses. All GIPR-expressing somatotropinomas were negative for GNAS mutations. GIPR expression occurred through transcriptional activation of a single allele of the GIPR gene in all GIPR-expressing samples, except in two tetraploid samples, where expression occurred from two alleles per nucleus. In addition to extensive 19q duplications, we detected in four samples GIPR locus microamplifications in a certain proportion of nuclei. We identified an overall hypermethylator phenotype in GIPR-expressing samples compared with GNAS-mutated adenomas. In particular, we observed hypermethylation in the GIPR gene body, likely driving its ectopic expression. Conclusions We describe a distinct molecular subclass of somatotropinomas, clinically revealed by a paradoxical increase of GH to oral glucose related to pituitary GIPR expression. This ectopic GIPR expression occurred through hypomorphic transcriptional activation and is likely driven by GIPR gene microamplifications and DNA methylation abnormalities

Adrenal GIPR expression and chromosome 19q13 microduplications in GIP-dependent Cushing’s syndrome

International audienceGIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing's syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations

Single-cell multi-omics defines the cell-type-specific impact of splicing aberrations in human hematopoietic clonal outgrowths

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3' splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3' splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.</p

Anatomic position determines oncogenic specificity in melanoma

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults