Low Autophagy (ATG) Gene Expression Is Associated with an Immature AML Blast Cell Phenotype and Can Be Restored during AML Differentiation Therapy.

Autophagy is an intracellular degradation system that ensures a dynamic recycling of a variety of building blocks required for self-renewal, homeostasis, and cell survival under stress. We used primary acute myeloid leukemia (AML) samples and human AML cell lines to investigate the regulatory mechanisms of autophagy and its role in AML differentiation. We found a significantly lower expression of key autophagy- (ATG-) related genes in primary AML as compared to healthy granulocytes, an increased autophagic activity during all-trans retinoic acid- (ATRA-) induced neutrophil differentiation, and an impaired AML differentiation upon inhibition of ATG3, ATG4D, and ATG5. Supporting the notion of noncanonical autophagy, we found that ATRA-induced autophagy was Beclin1-independent compared to starvation- or arsenic trioxide- (ATO-) induced autophagy. Furthermore, we identified PU.1 as positive transcriptional regulator of ATG3, ATG4D, and ATG5. Low PU.1 expression in AML may account for low ATG gene expression in this disease. Low expression of the autophagy initiator ULK1 in AML can partially be attributed to high expression of the ULK1-targeting microRNA-106a. Our data clearly suggest that granulocytic AML differentiation relies on noncanonical autophagy pathways and that restoring autophagic activity might be beneficial in differentiation therapies.

MicroRNA-106a targets autophagy and enhances sensitivity of lung cancer cells to Src inhibitors.

OBJECTIVES: Src tyrosine kinase inhibitors (TKIs) significantly inhibit cell migration and invasion in lung cancer cell lines with minor cytotoxic effects. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. Possible resistance mechanisms include the induction of cytoprotective autophagy upon Src inhibition. Autophagy is a cellular recycling process that allows cell survival in response to a variety of stress stimuli including responses to various treatments. MATERIAL AND METHODS: We screened autophagic activity in A549, H460, and H1299 NSCLC cell lines treated with two different Src-TKIs (saracatinib, dasatinib) or shRNA targeting SRC. The autophagy response was determined by LC3B-I to -II conversion, increased ULK1 epxression and increased GFP-LC3B dot formation. Autophagy was inhibited by pharmacological (bafilomycin A, chloroquine) or genetic (ULK1 shRNA) means. Expression of miR-106a and miR-20b was analyzed by qPCR, and we used different lentivral vectors for ectopic expression of either miR-106a mimetics, anti-sense miR-106a or different miR-106a-363 cluster constructs. RESULTS: In the current study we found that Src-TKIs induce autophagy in lung adenocarcinoma cell lines and that a combination of autophagy and Src tyrosine kinase inhibition results in cell death. Moreover, Src-TKI induced autophagy depends on the induction of the key autophagy kinase ULK1. This ULK1 upregulation is caused by downregulation of the ULK1-targeting microRNA-106a. An inverse correlation of miR-106a and ULK1 expression was seen in lung adenocarcinoma. Accordingly, ectopic expression of miR-106a in combination with Src-TKI treatment resulted in significant cell death as compared to control transduced cells. CONCLUSIONS: Autophagy protects lung adenocarcinoma cells from Src-TKIs via a newly identified miR-106a-ULK1 signaling pathway. The combined inhibition of Src and ULK1/autophagy might represent a promising treatment option for future clinical trials. Lastly, our data might challenge the term "oncogenic" miR-106a as it can promote sensitivity to Src-TKIs thereby underlining the context-dependent function of miRNAs.

A Systematic Approach to Defining the microRNA Landscape in Metastasis.

The microRNA (miRNA) landscape changes during the progression of cancer. We defined a metastasis-associated miRNA landscape using a systematic approach. We profiled and validated miRNA and mRNA expression in a unique series of human colorectal metastasis tissues together with their matched primary tumors and corresponding normal tissues. We identified an exclusive miRNA signature that is differentially expressed in metastases. Three of these miRNAs were identified as key drivers of an EMT-regulating network acting though a number of novel targets. These targets include SIAH1, SETD2, ZEB2, and especially FOXN3, which we demonstrated for the first time as a direct transcriptional suppressor of N-cadherin. The modulation of N-cadherin expression had significant impact on migration, invasion, and metastasis in two different in vivo models. The significant deregulation of the miRNAs defining the network was confirmed in an independent patient set as well as in a database of diverse malignancies derived from more than 6,000 patients. Our data define a novel metastasis-orchestrating network based on systematic hypothesis generation from metastasis tissues.

Inhibition of the miR-143/145 cluster attenuated neutrophil differentiation of APL cells.

MicroRNAs can influence hematopoietic cell lineage commitment and aberrant expression of hematopoietic miRNAs contributes to AML pathology. We found that miR-143 and miR-145 expression is significantly repressed in primary AML patient samples as compared to neutrophils of healthy donors. Further analysis revealed impaired neutrophil differentiation of APL cells upon inhibition of miR-145 expression. Lastly, we identified p73 as transcriptional regulator of miR-143/145 during neutrophil differentiation of APL cells. Our data suggest that low miR-145 levels in APL, possibly due to aberrant expression of p73 transcription factors, contribute to the differentiation block seen in this disease.

CLEC5A (MDL-1) is a novel PU.1 transcriptional target during myeloid differentiation.

C-type lectin domain family 5, member A (CLEC5A), also known as myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), is a cell surface receptor strongly associated with the activation and differentiation of myeloid cells. CLEC5A associates with its adaptor protein DAP12 to activate a signaling cascade resulting in activation of downstream kinases in inflammatory responses. Currently, little is known about the transcriptional regulation of CLEC5A. We identified CLEC5A as one of the most highly induced genes in a microarray gene profiling experiment of PU.1 restored myeloid PU.1-null cells. We further report that CLEC5A expression is significantly reduced in several myeloid differentiation models upon PU.1 inhibition during monocyte/macrophage or granulocyte differentiation. In addition, CLEC5A mRNA expression was significantly lower in primary acute myeloid leukemia (AML) patient samples than in macrophages and granulocytes from healthy donors. Moreover, we found activation of a CLEC5A promoter reporter by PU.1 as well as in vivo binding of PU.1 to the CLEC5A promoter. Our findings indicate that CLEC5A expression in monocyte/macrophage and granulocytes is regulated by PU.1.

Dichotomous but stringent substrate selection by the dual-function Cdk7 complex revealed by chemical genetics.

Cdk7 performs two essential but distinct functions as a CDK-activating kinase (CAK) required for cell-cycle progression and as the RNA polymerase II (Pol II) CTD kinase of general transcription factor IIH. To investigate the substrate specificity underlying this dual function, we created an analog-sensitive (AS) Cdk7 able to use bulky ATP derivatives. Cdk7-AS-cyclin H-Mat1 phosphorylates approximately 10-15 endogenous polypeptides in nuclear extracts. We identify seven of these as known and previously unknown Cdk7 substrates that define two classes: proteins such as Pol II and transcription elongation factor Spt5, recognized efficiently only by the fully activated Cdk7 complex, through sequences surrounding the site of phosphorylation; and CDKs, targeted equivalently by all active forms of Cdk7, dependent on substrate motifs remote from the phosphoacceptor residue. Thus, Cdk7 accomplishes dual functions in cell-cycle control and transcription not through promiscuity but through distinct, stringent modes of substrate recognition.