No abstract

• Atg7 expression is associated with shorter remission duration in AML.• Atg7 inhibition is a proapoptotic phenotype and enhances sensitivity to chemotherapy.Autophagy is a cellular adaptive mechanism to stress, including that induced by chemotherapeutic agents. Reverse phase protein array suggested that high expression of the essential autophagy-related protein, Atg7, was associated with shorter remission in newly diagnosed acute myeloid leukemia (AML) patient samples, indicating a role in chemoresistance. Knockdown of Atg7 in AML cells using short hairpin RNA markedly increased apoptosis and DNA damage following treatment with cytarabine and idarubicin. Interestingly, coculture of AML cells with stromal cells increased autophagy and chemoresistance in the AML cells exposed to chemotherapeutic agents, and this was reversed following Atg7 knockdown. This effect was further enhanced by concomitant knockdown of Atg7 in both AML and stromal cells. These findings strongly suggest that Atg7, and likely microenvironment autophagy in general, plays an important role in AML chemoresistance. Mechanistic studies revealed that Atg7 knockdown induced a proapoptotic phenotype in AML cells, which was manifested by an increased NOXA expression at the transcriptional level. Finally, in a mouse model of human leukemia, Atg7 knockdown extended overall survival after chemotherapy. Thus, the inhibition of Atg7 appears to be a valid strategy to enhance chemosensitivity, and it could indeed improve outcomes in AML therapy. (Blood. 2016;128(9):1260-1269

Therapeutic inhibition of macroautophagy/autophagy is expected to increase chemosensitivity of cancers and alter tumor-stroma interdependence. The hypoxic, metabolically challenged bone marrow microenvironment confers chemoresistance to leukemia cells. The impact of autophagy inhibition in the context of microenvironment-mediated resistance in leukemia is less explored. Our recent studies demonstrated that co-culture of acute myelogenous leukemia (AML) cells with marrow-derived mesenchymal stromal cells (MSC) induces autophagy in AML cells and increases resistance to genotoxic agents (cytarabine and idarubicin). Genetic silencing of ATG7 in AML enhances the sensitivity to these genotoxic agents, an effect that was more pronounced with concomitant silencing of ATG7 in AML and MSCs. Mechanistically, the increased sensitivity of AML cells to genotoxic agents is associated with alteration of BCL2 family proteins, particularly transcriptional upregulation of PMAIP1/NOXA. In a disseminated AML model in immunocompromised mice, ATG7 knockdown in AML cells results in better survival compared to control mice when treated with chemotherapy. Our studies support the therapeutic role of autophagy inhibition, specifically ATG7 inhibition, in AML.

Oncolytic adenoviruses, such as Delta-24-RGD, are replication-competent viruses that are genetically engineered to induce selective cancer cell lysis. In cancer cells, Delta-24-RGD induces massive autophagy, which is required for efficient cell lysis and adenoviral spread. Understanding the cellular mechanisms underlying the regulation of autophagy in cells treated with oncolytic adenoviruses may provide new avenues to improve the therapeutic effect. In this work, we showed that cancer cells infected with Delta-24-RGD undergo autophagy despite the concurrent activation of the AKT/mTOR pathway. Moreover, adenovirus replication induced sustained activation of JNK proteins in vitro. ERK1/1 phosphorylation remained unchanged during adenoviral infection, suggesting specificity of JNK activation. Using genetic ablation and pharmacological inactivation of JNK, we unequivocally demonstrated that cells infected with Delta-24-RGD required JNK activation. Thus, genetic co-ablation of JNK1 and JNK2 genes or inhibition of JNK kinase function rendered Delta-24-RGD–treated cells resistant to autophagy. Accordingly, JNK activation induced phosphorylation of Bcl-2 and prevented the formation of Bcl-2/Beclin 1 autophagy suppressor complexes. Using an orthotopic model of human glioma xenograft, we showed that treatment with Delta-24-RGD induced phosphorylation and nuclear translocation of JNK, as well as phosphorylation of Bcl-2. Collectively, our data identified JNK proteins as an essential mechanistic link between Delta-24-RGD infection and autophagy in cancer cells. Activation of JNK without inactivation of the AKT/mTOR pathway constitutes a distinct molecular signature of autophagy regulation that differentiates Delta-24-RGD adenovirus from the mechanism used by other oncolytic viruses to induce autophagy and provides a new rationale for the combination of oncolytic viruses and chemotherapy.

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide-induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs. [Cancer Res 2009;69(21):8356-65]