Chromothripsis orchestrates leukemic transformation in blast phase MPN through targetable amplification of DYRK1A.

Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a common event in cancer. Whether chromothripsis might constitute an actionable molecular event amenable to therapeutic targeting remains an open question. We describe recurrent chromothripsis of chromosome 21 in a subset of patients in blast phase of a myeloproliferative neoplasm (BP-MPN), which alongside other structural variants leads to amplification of a region of chromosome 21 in ∼25% of patients ('chr21amp'). We report that chr21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. The chr21amp event is highly clonal and present throughout the hematopoietic hierarchy. DYRK1A , a serine threonine kinase and transcription factor, is the only gene in the 2.7Mb minimally amplified region which showed both increased expression and chromatin accessibility compared to non-chr21amp BP-MPN controls. We demonstrate that DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development, including DNA repair, STAT signalling and BCL2 overexpression. DYRK1A is essential for BP-MPN cell proliferation in vitro and in vivo , and DYRK1A inhibition synergises with BCL2 targeting to induce BP-MPN cell apoptosis. Collectively, these findings define the chr21amp event as a prognostic biomarker in BP-MPN and link chromothripsis to a druggable target.

Ectopic sphenoidal ACTH-secreting adenoma revealed by 11C Methionine PET scan: case report.

BACKGROUND: Ectopic ACTH pituitary adenomas (EAPA), located outside the sella turcica and deriving from cellular remnants of Rathke's pouch are a very rare cause of Cushing's syndrome (CS). The diagnosis is often difficult and delayed, even after comprehensive work-up. To our knowledge, we report for the first time an ectopic corticotroph tumor of the posterior wall of the sphenoid sinus, leading to false positive results of bilateral inferior petrosal sinus sampling (BIPPS) and which was finally localized by a co-registered11 C Methionine PET/MR imaging. CASE PRESENTATION: A 48-year-old woman was referred for a high clinical suspicion of ACTH-dependent CS. Biological testing comprising low dose dexamethasone suppression and CRH stimulation tests were indicative of pituitary Cushing's disease, but comprehensive pituitary MRI did not reveal any pituitary adenoma. BIPSS confirmed however a central origin of ACTH secretion (central-to-peripheral ACTH ratio > 100) and revealed a significant right-to-left gradient (6.2), leading to a first right-sided exploratory hypophysectomy, that did not cure the patient. BIPSS images were reviewed and revealed preferential drainage of the left pituitary to the right petrosal sinus, leading us to a left sided exploratory hypophysectomy, which was again unsuccessful. A11 C Methionine PET/MRI was performed and revealed a hypermetabolic lesion adjacent to the posterior wall of the sphenoidal sinus. After surgical resection, this polypoid mass was identified as an ectopic ATCH-secreting pituitary adenoma expressing ACTH and T-Pit and complete remission of hypercortisolism was observed. CONCLUSIONS: In conclusion, we report a case of ACTH-dependent Cushing's syndrome, caused by an ectopic corticotroph adenoma located in the sphenoidal sinus, which perfectly mimicked the biological features of a classical pituitary ACTH adenoma on a comprehensive hormonal evaluation including BIPPS, and the features of a benign naso-sinusal polyp at MRI. We report for the first time a key role of11 C Methionine PET co-registered to high resolution MRI for localizing ectopic adenomas, efficiently guiding surgical removal and leading to complete remission of hypercortisolism.

Conservative management of complicated Rathke's cleft cyst mimicking pituitary apoplexy.

Summary: Complicated Rathke's cleft cyst (RCC) is a rare occurrence of symptomatic bleeding or growth of a previously asymptomatic (and often undiagnosed) intrasellar cyst derived from remnants of Rathke's pouch, situated on the midline between the adeno- and neurohypophysis. Symptoms may be identical to those of pituitary apoplexy: acute onset of headache, hypopituitarism, and neurological disturbances. Both syndromes may also exhibit a similar appearance of a large haemorrhagic sellar mass at initial radiological evaluation. We report on two patients who presented with headache and complete hypopituitarism. Based on the initial MRI, they were first diagnosed with pituitary apoplexy but managed conservatively with hormone therapy alone because of the absence of severe visual or neurological threat. Upon follow-up at 4 months, clinical evolution was good in both patients but their pituitary mass had not reduced in size and, after careful radiologic reviewing, was more indicative of a large midline complicated RCC. In conclusion, the diagnosis of complicated RCC is challenging because it can mimic pituitary apoplexy clinically, biologically, and radiologically. Clinicians should distinguish between the two entities using specific radiological signs or evolution of the mass at MRI if the patient does not undergo surgery. To our knowledge, we report conservative management of this rare condition for the first time, though it seems appropriate in the absence of neurological compromise or visual compression. Long-term follow-up is however mandatory. Learning points: Complicated Rathke's cleft cyst can mimic pituitary apoplexy, presenting with sudden onset of headache, hypopituitarism, and visual and neurological compromise in the most severe cases. At diagnosis, pituitary MRI may not be able to differentiate between the two entities, showing a large haemorrhagic mass inside the sella, with little or no normal pituitary tissue visible. Patients are often diagnosed with apoplexy at this stage and may undergo pituitary surgery. When surgery has not been performed initially in these patients, repeat imaging at 3-6 months is unchanged and does not show the expected involution usually seen after adenoma apoplexy. Conservative management with hormonal replacement seems a valid option in the absence of visual or neurological deficits that would require trans-sphenoidal surgery.

Rethinking JAK2 inhibition: towards novel strategies of more specific and versatile Janus kinase inhibition.

This corrects the article DOI: 10.1038/leu.2017.43.

Rethinking JAK2 inhibition: towards novel strategies of more specific and versatile Janus kinase inhibition.

Janus kinases (JAKs) are required for cytokine receptor signaling. Since the discovery of the highly prevalent JAK2 V617F mutation in myeloproliferative neoplasms (MPNs), JAK2 became a prime target for inhibition. Only one approved JAK2 inhibitor exists, with positive, but not curative effects in MPNs, and promising effects in autoimmune diseases and cancer. On the basis of recent advances in the structural features regulating both normal and mutant JAKs, as well as in small-molecule targeting, we review the current state of JAK2 inhibitor development and present novel avenues of selecting JAK2 inhibitors, with broad and narrow specificities and extend these approaches to other JAKs.

PDGFRB mutants found in patients with familial infantile myofibromatosis or overgrowth syndrome are oncogenic and sensitive to imatinib.

Recently, germline and somatic heterozygous mutations in the platelet-derived growth factor receptor ß (PDGFRB) have been associated with familial infantile myofibromatosis (IM), which is characterized by soft tissue tumors, and overgrowth syndrome, a disease that predisposes to cancer. These mutations have not been functionally characterized. In the present study, the activity of three PDGFRB mutants associated with familial IM (R561C, P660T and N666K) and one PDGFRB mutant found in patients with overgrowth syndrome (P584R) was tested in various models. The P660T mutant showed no difference with the wild-type receptor, suggesting that it might represent a polymorphic variant unrelated to the disease. By contrast, the three other mutants were constitutively active and able to transform NIH3T3 and Ba/F3 cells to different extents. In particular, the germline mutant identified in overgrowth syndrome, P584R, was a stronger oncogene than the germline R561C mutant associated with myofibromatosis. The distinct phenotypes associated with these two mutations could be related to this difference of potency. Importantly, all activated mutants were sensitive to tyrosine kinase inhibitors such as imatinib, nilotinib and ponatinib. In conclusion, the PDGFRB mutations previously identified in familial IM and overgrowth syndrome activate the receptor in the absence of ligand, supporting the hypothesis that these mutations cause the diseases. Moreover, imatinib seems to be a promising treatment for patients carrying these mutations. To our knowledge, these are the first confirmed gain-of-function point mutations of PDGFRB in human cancer.

Persistent STAT5 activation in myeloid neoplasms recruits p53 into gene regulation.

STAT (Signal Transducer and Activator of Transcription) transcription factors are constitutively activated in most hematopoietic cancers. We previously identified a target gene, LPP/miR-28 (LIM domain containing preferred translocation partner in lipoma), induced by constitutive activation of STAT5, but not by transient cytokine-activated STAT5. miR-28 exerts negative effects on thrombopoietin receptor signaling and platelet formation. Here, we demonstrate that, in transformed hematopoietic cells, STAT5 and p53 must be synergistically bound to chromatin for induction of LPP/miR-28 transcription. Genome-wide association studies show that both STAT5 and p53 are co-localized on the chromatin at 463 genomic positions in proximal promoters. Chromatin binding of p53 is dependent on persistent STAT5 activation at these proximal promoters. The transcriptional activity of selected promoters bound by STAT5 and p53 was significantly changed upon STAT5 or p53 inhibition. Abnormal expression of several STAT5-p53 target genes (LEP, ATP5J, GTF2A2, VEGFC, NPY1R and NPY5R) is frequently detected in platelets of myeloproliferative neoplasm (MPN) patients, but not in platelets from healthy controls. In conclusion, persistently active STAT5 can recruit normal p53, like in the case of MPN cells, but also p53 mutants, such as p53 M133K in human erythroleukemia cells, leading to pathologic gene expression that differs from canonical STAT5 or p53 transcriptional programs.

JAK/STAT signaling in hematological malignancies.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is central to signaling by cytokine receptors, a superfamily of more than 30 transmembrane proteins that recognize specific cytokines, and is critical in blood formation and immune response. Many of those receptors transmit anti-apoptotic, proliferative and differentiation signals, and their expression and functions are critical for the formation of blood lineages. Several cancers, including blood malignancies, have been associated with constitutive activation of members of the STAT family, which normally require JAK-mediated tyrosine phosphorylation for transcriptional activation. More recently, human myeloproliferative neoplasms were discovered to be associated with a unique acquired somatic mutation in JAK2 (JAK2 V617F), rare exon 12 JAK2 mutations, or thrombopoietin receptor mutations that constitutively activate wild-type JAK2. Prompted by these observations, many studies have explored the possibility that JAKs, cytokine receptors, or other components of the JAK/STAT pathway are mutated or upregulated in several hematological malignancies. This has been observed in certain pediatric acute lymphoblastic leukemias and adult T-cell lymphoblastic leukemias, and overexpression of JAK2 seems to be important in Hodgkin lymphoma. Here we discuss the nature and respective contribution of mutations dysregulating the JAK/STAT pathway in hematological malignancies and present examples in which such mutations drive the disease, contribute to the phenotype, or provide a survival and proliferative advantage. JAK inhibitors are making their way into the therapeutic arsenal (for example, in myelofibrosis), and we discuss the possibility that other hematological diseases might benefit from treatment with these inhibitors in combination with other agents.

Effects of erythropoietin receptors and erythropoiesis-stimulating agents on disease progression in cancer.

Erythropoiesis-stimulating agents (ESAs) increase red blood cell (RBC) production in bone marrow by activating the erythropoietin receptor (EpoR) on erythrocytic-progenitor cells. Erythropoiesis-stimulating agents are approved in the United States and Europe for treating anaemia in cancer patients receiving chemotherapy based on randomised, placebo-controlled trials showing that ESAs reduce RBC transfusions. Erythropoiesis-stimulating agent-safety issues include thromboembolic events and concerns regarding whether ESAs increase disease progression and/or mortality in cancer patients. Several trials have reported an association between ESA use and increased disease progression and/or mortality, whereas other trials in the same tumour types have not provided similar findings. This review thoroughly examines available evidence regarding whether ESAs affect disease progression. Both clinical-trial data on ESAs and disease progression, and preclinical data on how ESAs could affect tumour growth are summarised. Preclinical topics include (i) whether tumour cells express EpoR and could be directly stimulated to grow by ESA exposure and (ii) whether endothelial cells express EpoR and could be stimulated by ESA exposure to undergo angiogenesis and indirectly promote tumour growth. Although assessment and definition of disease progression vary across studies, the current clinical data suggest that ESAs may have little effect on disease progression in chemotherapy patients, and preclinical data indicate a direct or indirect effect of ESAs on tumour growth is not strongly supported.

Contribution of Kunitz protease inhibitor and transmembrane domains to amyloid precursor protein homodimerization.

BACKGROUND: The two major isoforms of the human amyloid precursor protein (APP) are APP695 and APP751. They differ by the insertion of a Kunitz-type protease inhibitor (KPI) sequence in the extracellular domain of APP751. APP-KPI isoforms are increased in Alzheimer's disease brains, and they could be associated with disease progression. Recent studies have shown that APP processing to Aß is regulated by homodimerization, which involves both extracellular and juxtamembrane/transmembrane (JM/TM) regions. OBJECTIVE: Our aim is to understand the mechanisms controlling APP dimerization and the contribution of the ectodomain and JM/TM regions to this process. METHODS: We used bimolecular fluorescence complementation approaches coupled to fluorescence-activated cell sorting analysis to measure the dimerization level of different APP isoforms and APP C-terminal fragments (C99) mutated in their JM/TM region. RESULTS: APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain of APP or C99 did not significantly affect fluorescence complementation. CONCLUSION: These findings indicate that the KPI domain plays a major role in APP dimerization. They set the basis for further investigation of the relation between dimerization, metabolism and function of APP.

Aberrant signal transduction pathways in myeloproliferative neoplasms.

The BCR-ABL-negative myeloproliferative neoplasms (MPNs), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), entered the spotlight in 2005 when the unique somatic acquired JAK2 V617F mutation was described in >95% of PV and in 50% of ET and PMF patients. For the very rare PV patients who do not harbor the JAK2 V617F mutation, exon 12 JAK2 mutants were discovered also to result in activated forms of JAK2. A minority of ET and PMF patients harbor mutations that constitutively activate the thrombopoietin receptor (TpoR). In bone marrow reconstitution models based on retroviral transduction, the phenotype induced by JAK2 V617F is less severe and different from the rapid fatal myelofibrosis induced by TpoR W515L. The reasons for these differences are unknown. Exactly by which mechanism(s) one acquired somatic mutation, JAK2 V617F, can promote three different diseases remains a mystery, although gene dosage and host genetic variation might have important functions. We review the recent progress made in deciphering signaling anomalies in PV, ET and PMF, with an emphasis on the relationship between JAK2 V617F and cytokine receptor signaling and on cross-talk with several other signaling pathways.

Clinical implications of JAK2 mutations in myeloproliferative disorders.

The V617F mutation of JAK2 is the key molecular event in 90% of polycythaemia vera (PV), 50% of essential thrombocythaemia (ET) and 50% of primary myelofibrosis (PMF). JAK2 exon 12 and MPLW515 mutations are less frequent. Because JAK2 V617F is specific for myeloid neoplasms, and because it can be detected in peripheral blood granulocytes, it offers a powerful tool that facilitates the diagnosis of these BCR-ABL negative myeloproLiferative disorders. These discoveries provide the rationale for a revision of the current WHO diagnostic criteria for PV, ET and PMF and could ultimately lead to the development of a specific targeted therapy.

JAK kinases overexpression promotes in vitro cell transformation.

Constitutive activation of the JAK-STAT pathway is frequent in cancer and contributes to oncogenesis. Here, we took advantage of the Ba/F3 cell line, a murine proB cell line dependent on IL-3 for growth, to analyse mechanisms of constitutive STAT activation in vitro. Cytokine-independent and tumorigenic Ba/F3 cell lines were derived from a two-step selection process. Cells transfected with a defective IL-9 receptor acquire IL-9 responsiveness during a first step of selection, and progress after a second selection step to autonomously growing tumorigenic cells. Microarray analysis pointed to JAK1 overexpression as a key genetic event in this transformation. Overexpression of JAK1 not only increased the sensitivity to IL-9 but also allowed a second selection step toward cytokine-independent growth with constitutive STAT activation. This progression was dependent on a functional FERM and kinase JAK1 domain. Similar results were observed after JAK2, JAK3 and TYK2 overexpression. All autonomous cell lines showed an activation of STAT5, ERK1-2 and AKT but only TYK2-overexpressing cell lines showed a constitutive activation of STAT3. Thus, JAK overexpression can be considered as one of the oncogenic events leading to the constitutive activation of the JAK-STAT pathway.