Functional interplay of SP family members and nuclear factor Y is essential for transcriptional activation of the human Calreticulin gene.

Calreticulin (CALR) is a highly conserved, multifunctional protein involved in a variety of cellular processes including the maintenance of intracellular calcium homeostasis, proper protein folding, differentiation and immunogenic cell death. More recently, a crucial role for CALR in the pathogenesis of certain hematologic malignancies was discovered: in clinical subgroups of acute myeloid leukemia, CALR overexpression mediates a block in differentiation, while somatic mutations have been found in the majority of patients with myeloproliferative neoplasms with nonmutated Janus kinase 2 gene (JAK2) or thrombopoietin receptor gene (MPL). However, the mechanisms underlying CALR promoter activation have insufficiently been investigated so far. By dissecting the core promoter region, we could identify a functional TATA-box relevant for transcriptional activation. In addition, we characterized two evolutionary highly conserved cis-regulatory modules (CRMs) within the proximal promoter each composed of one binding site for the transcription factors SP1 and SP3 as well as for the nuclear transcription factor Y (NFY) and we verified binding of these factors to their cognate sites in vitro and in vivo.

Activation of the unfolded protein response in human acute myeloid leukemia.

There is accumulating evidence for the involvement of the unfolded protein response (UPR) in the pathogenesis of many tumor types in humans. This is particularly the case in rapidly growing solid tumors in which the demand for oxygen and nutrients can exceed the supply until new tumor-initiated blood vessels are formed. In contrast, the role of the UPR during leukemogenesis remains largely unknown. Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by the accumulation of somatic mutations in hematopoietic progenitor cells that alter the physiological regulation of self-renewal, survival, proliferation, or differentiation. The CCAAT/enhancer-binding protein alpha (CEBPA) gene is a key myeloid transcription factor and a frequent target for disruption in AML. In particular, translation of CEBPA mRNA can be specifically blocked by binding of the chaperone calreticulin (CALR), a well-established effector of the UPR, to a stem loop structure within the 5' region of the CEBPA mRNA. The relevance of this mechanism was first elucidated in certain AML subtypes carrying the gene rearrangements t(3;21) or inv(16). In our recent work, we could demonstrate the induction of key effectors of the UPR in leukemic cells of AML patients comprising all subtypes (according to the French-American-British (FAB) classification for human AML). The formation of the spliced variant of the X-box binding protein (XBP1s) was detectable in 17.4% (17 of 105) of AML patients. Consistent with an activated UPR, this group had significantly increased expression of the UPR target genes CALR, the 78 kDa glucose-regulated protein (GRP78), and the CCAAT/enhancer-binding protein homologous protein (CHOP). Consistently, in vitro studies confirmed that calreticulin expression was upregulated via activation of the ATF6 pathway in myeloid leukemic cells. As a consequence, CEBPA protein expression was inhibited in vitro as well as in leukemic cells from patients with activated UPR. We therefore propose a model of the UPR being involved in leukemogenesis through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation and cell-cycle deregulation which represent key features of the leukemic phenotype. From a more clinical point of view, the presence of activated UPR in AML patient samples was found to be associated with a favorable disease course.

Inhibition of tissue transglutaminase sensitizes TRAIL-resistant lung cancer cells through upregulation of death receptor 5.

Tissue transglutaminase (TG2) is implicated in cellular processes such as apoptosis and cell migration. Its acyl transferase activity cross-links certain proteins, among them transcription factors were described. We show here that the TG2 inhibitor KCC009 reversed resistance to tumor necrosis factor-related apoptosis-inducing factor (TRAIL) in lung cancer cells. Sensitization required upregulation of death receptor 5 (DR5) but not of death receptor 4. Upregulation of DR5 involved the first intron of the DR5 gene albeit it was independent from p53 and nuclear factor kappa B. In conclusion, inhibition of tissue transglutaminase provides an interesting strategy for sensitization to TRAIL-induced apoptosis in p53-deficient lung cancer cells.

Unfolded protein response suppresses CEBPA by induction of calreticulin in acute myeloid leukaemia.

The unfolded protein response (UPR) is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The role of the UPR during leukemogenesis is unknown so far. Here, we studied the induction of mediators of the UPR in leukaemic cells of AML patients. Increased expression of the spliced variant of the X-box binding protein 1 (XBP1s) was detected in 17.4% (16 of 92) of AML patients. Consistent with activated UPR, this group also had increased expression of ER-resident chaperones such as the 78 kD glucose-regulated protein (GRP78) and of calreticulin. Conditional expression of calreticulin in leukaemic U937 cells was found to increase calreticulin binding to the CEBPA mRNA thereby efficiently blocking translation of the myeloid key transcription factor CEBPA and ultimately affecting myeloid differentiation. Consequently, leukaemic cells from AML patients with activated UPR and thus increased calreticulin levels showed in fact suppressed CEBPA protein expression. We identified two functional ER stress response elements (ERSE) in the calreticulin promoter. The presence of NFY and ATF6, as well as an intact binding site for YY1 within these ERSE motifs were essential for mediating sensitivity to ER stress and activation of calreticulin. Thus, we propose a model of the UPR being activated in a considerable subset of AML patients through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation.

Complexity of miR-223 regulation by CEBPA in human AML.

microRNA-223 (miR-223) can trigger normal granulopoiesis. miR-223 expression is regulated by two distinct CEBPA (CCAAT/enhancer binding protein-alpha) sites. Here, we report that miR-223 is largely suppressed in cells from acute myeloid leukemia (AML) patients. By sequencing, we found that miR-223 suppression in AML is not caused by DNA sequence alterations, nor is it mediated by promoter hypermethylation. The analysis of the individual contribution of both CEBPA sites to miR-223 regulation identified the site upstream of the miR-223 primary transcript as the predominant regulatory element. Our results suggest that miR-223 suppression in AML is caused by impaired miR-223 upstream factors.

Activation of the unfolded protein response is associated with favorable prognosis in acute myeloid leukemia.

PURPOSE: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far. EXPERIMENTAL DESIGN: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box-binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA. RESULTS: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022). CONCLUSIONS: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease.

Genomic analysis of single cytokeratin-positive cells from bone marrow reveals early mutational events in breast cancer.

Chromosomal instability in human breast cancer is known to take place before mammary neoplasias display morphological signs of invasion. We describe here the unexpected finding of a tumor cell population with normal karyotypes isolated from bone marrow of breast cancer patients. By analyzing the same single cells for chromosomal aberrations, subchromosomal allelic losses, and gene amplifications, we confirmed their malignant origin and delineated the sequence of genomic events during breast cancer progression. On this trajectory of genomic progression, we identified a subpopulation of patients with very early HER2 amplification. Because early changes have the highest probability of being shared by genetically unstable tumor cells, the genetic characterization of disseminated tumor cells provides a novel rationale for selecting patients for targeted therapies.

SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples.

Global genome amplification from formalin-fixed tissues is still problematic when performed with low cell numbers. Here, we tested a recently developed method for whole genome amplification termed "SCOMP" (single cell comparative genomic hybridization) on archival tissues of different ages. We show that the method is very well suited for formalin-fixed paraffin-embedded samples obtained by nuclei extraction or laser microdissection. The polymerase chain reaction (PCR) products can be used for subsequent comparative genomic hybridization, loss of heterozygosity studies, and DNA sequencing. To control for PCR-induced artifacts we amplified genomic DNA isolated from 20 nuclei of archival formalin-fixed, paraffin-embedded nonpathological lymph nodes. Subsequent comparative genomic hybridization revealed the expected balanced profiles. For loss of heterozygosity analysis by microsatellite PCR 60 to 160 cells were sufficient. In comparative experiments the approach turned out to be superior to published degenerated oligonucleotide-primed-PCR protocols. The method provides a robust and valuable tool to study very small cell samples, such as the genomes of dysplastic cells or the clonal evolution within heterogeneous tumors.