Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion.

BACKGROUND: Somatic 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: These 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.

[Differential diagnosis of myeloproliferative neoplasms. Quantitative NF-E2 immunohistochemistry for differentiating between essential thrombocythemia and primary myelofibrosis]. / Differenzialdiagnose myelproliferativer Neoplasien. Quantitative NF-E2-Immunhistochemie zur Unterscheidung zwischen essenzieller Thrombozythämie und primärer Myelofibrose.

BACKGROUND: Besides essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) the myeloproliferative neoplasms (MPN) defined by the World Health Organization (WHO) comprise the entity of unclassifiable MPNs (MPN, U). The exact differential diagnosis of the specific MPN entities can be challenging particularly at early stages of the diseases. So far, pathologists have had to rely only on histomorphological evaluation of bone marrow biopsies in combination with laboratory data because helpful ancillary tests are not yet available. Even molecular tests, such as JAK2 mutation analysis are not helpful particularly in the differential diagnosis of ET and PMF because both entities are associated with the V617F mutation in 50 % of the cases. Recently overexpression of the transcription factor NF-E2 in MPN was described. MATERIALS AND METHODS: A collective of samples consisting of 163 bone marrow biopsies including 139 MPN cases was stained immunohistochemically for NF-E2 and analyzed regarding the subcellular localization of NF-E2 in erythroid progenitor cells. The results were compared between the MPN entities as well as the controls and statistical analyses were conducted. RESULTS AND DISCUSSION: This study showed that NF-E2 immunohistochemistry and analysis of the proportion of nuclear positive erythroblasts of all erythroid precursor cells can help to distinguish between ET and PMF even in early stages of the diseases. An MPN, U case showing a proportion of more than 20 % nuclear positive erythroblasts can be classified as a PMF with 92 % accuracy.

Increased BTB-Kelch type substrate adaptor protein immunoreactivity associates with advanced stage and poor differentiation in renal cell carcinoma.

Renal cell carcinoma (RCC) is a malignancy with increasing incidence. Despite the well-known prognostic factors - the stage, grade and histological subtype - the clinical course of RCC can seem quite random. The aim of this study was to evaluate markers of the oxidative system as candidate prognostic factors for RCC. Our study population consisted of 152 patients who underwent operation for RCC between 1990 and 1999. The tumours were examined with three immunohistochemical markers of the oxidative system, thioredoxin (Trx), NF-E2-related factor (Nfr2) and BTB-Kelch type substrate adaptor protein (Keap1). Cytoplasmic Keap1 staining was related to poorer prognosis in renal cancer-specific survival. The difference was statistically significant (P=0.02). Keap1 staining was associated with a more advanced stage and a higher nuclear grade. Cytoplasmic Trx staining was associated with a trend of better prognosis in renal cancer-specific survival. Nfr2 staining was not a prognostic factor in renal cancer-specific survival. In RCC, Keap1 is associated with a more advanced disease, a higher grade and a poorer prognosis.