BackgroundSomatic calreticulin (CALR), Janus kinase 2 (JAK2), and thrombopoietin receptor (MPL) mutations essentially show mutual exclusion in myeloproliferative neoplasms (MPN), suggesting that they activate common oncogenic pathways. Recent data have shown that MPL function is essential for CALR mutant-driven MPN. However, the exact role and the mechanisms of action of CALR mutants have not been fully elucidated.MethodsThe murine myeloid cell line 32D and human HL60 cells overexpressing the most frequent CALR type 1 and type 2 frameshift mutants were generated to analyze the first steps of cellular transformation, in the presence and absence of MPL expression. Furthermore, mutant CALR protein stability and secretion were examined using brefeldin A, MG132, spautin-1, and tunicamycin treatment.ResultsThe present study demonstrates that the expression of endogenous Mpl, CD41, and the key megakaryocytic transcription factor NF-E2 is stimulated by type 1 and type 2 CALR mutants, even in the absence of exogenous MPL. Mutant CALR expressing 32D cells spontaneously acquired cytokine independence, and this was associated with increased Mpl mRNA expression, CD41, and NF-E2 protein as well as constitutive activation of downstream signaling and response to JAK inhibitor treatment. Exogenous expression of MPL led to constitutive activation of STAT3 and 5, ERK1/2, and AKT, cytokine-independent growth, and reduction of apoptosis similar to the effects seen in the spontaneously outgrown cells. We observed low CALR-mutant protein amounts in cellular lysates of stably transduced cells, and this was due to accelerated protein degradation that occurred independently from the ubiquitin-proteasome system as well as autophagy. CALR-mutant degradation was attenuated by MPL expression. Interestingly, we found high levels of mutated CALR and loss of downstream signaling after blockage of the secretory pathway and protein glycosylation.ConclusionsThese findings demonstrate the potency of CALR mutants to drive expression of megakaryocytic differentiation markers such as NF-E2 and CD41 as well as Mpl. Furthermore, CALR mutants undergo accelerated protein degradation that involves the secretory pathway and/or protein glycosylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0275-0) contains supplementary material, which is available to authorized users.

Mutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.

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DEK is a biochemically distinct, conserved nonhistone protein that is vital to global heterochromatin integrity. In addition, DEK can be secreted and function as a chemotactic, proinflammatory factor. Here we show that exogenous DEK can penetrate cells, translocate to the nucleus, and there carry out its endogenous nuclear functions. Strikingly, adjacent cells can take up DEK secreted from synovial macrophages. DEK internalization is a heparan sulfate-dependent process, and cellular uptake of DEK into DEK knockdown cells corrects global heterochromatin depletion and DNA repair deficits, the phenotypic aberrations characteristic of these cells. These findings thus unify the extracellular and intracellular activities of DEK, and suggest that this paracrine loop involving DEK plays a role in chromatin biology. cellular biology | cancer | autoimmunity | juvenile arthritis S ince its initial cloning as part of the t(6;9) translocation in a subset of patients with acute myelogenous leukemia (1, 2), DEK has been shown to affect global heterochromatin integrity (3), mRNA splicing (4, 5), transcriptional control (both negative and positive) (6-8), DNA damage repair and susceptibility (9, 10), DNA replication (11), cellular differentiation (12, 13), cell viability (8), apoptosis (14), and senescence (15). DEK also plays a key role in the biology of hematopoietic and muscle stem cells (12,16).DEK overexpression occurs in various prevalent and difficult-totreat neoplasms, including hepatocellular carcinoma (17), glioblastoma (18), melanoma (8,19), bladder cancer (20), retinoblastoma (21, 22), breast cancer (23), and T-cell large granular lymphocytic leukemia (24). DEK is degraded by the F-box/tryptophan-aspartic acid (WD) repeat-containing protein 7 (Fbxw7) tumor suppressor, which affects cell division and splicing of messenger RNA (25). Elevated levels of DEK can interfere with cellular differentiation, apoptosis, senescence, and the response to chemotherapy, justifying the classification of DEK as a bona fide oncogene that plays a role in central pathways promoting tumor growth and survival (8,19,23).In addition to its roles in tumor biology, which appear to be related mainly to its intracellular functions, DEK also has been implicated in the pathogenesis of autoimmune disorders, functions more attributable to its extracellular activities (discussed below). In fact, circulating autoantibodies to DEK have been identified in the serum of patients with various autoimmune diseases, including juvenile idiopathic arthritis (JIA), sarcoidosis, and systemic lupus erythematosus (SLE) (26). Furthermore, autoantibodies to DEK, as well as DEK protein itself, have been detected in synovial fluids of children with JIA (27). The presence of DEK protein and DEK autoantibodies in the extracellular space suggests a proinflammatory role for DEK. In fact, on activation, macrophages secrete DEK, which in turn can act as a chemotactic factor for neutrophils, natural killer cells, and cytotoxic T lymphocytes in the extracellular milieu (28). Th...

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