Proteomic Analysis Reveals Autophagy as Pro-Survival Pathway Elicited by Long-Term Exposure with 5-Azacitidine in High-Risk Myelodysplasia

Abstract

Azacytidine (5-AZA) is the standard first-choice treatment for high-risk myelodysplasia (MDS) patients. However, the clinical outcome for those patients who interrupt treatment or whose disease failed to respond is very poor. In order to identify the cellular pathways that are modified by long-term exposure to 5-AZA, we evaluated key proteins associated with the autophagy pathway by reverse-phase microarray (RPPA). Comparing bone marrow mononucleated cells (BMMCs) obtained from 20 newly-diagnosed patients and after four 5-AZA cycles we found an increased autophagy signaling. We then evaluated ex-vivo the effect of the combination of 5-AZA with autophagy inhibitors chloroquine (CQ) and leupeptin. Since 5-AZA and CQ showed synergism due to an increase of basal autophagy after 5-AZA exposure, we adopted a sequential treatment treating BMMCs with 5 μM 5-AZA for 72 h followed by 10 μM CQ for 24 h and found increased apoptosis, associated to a reduction of G2M phase and increase in G0-G1 phase. Long-term exposure to 5-AZA induced the reduction of the autophagic marker SQSTM1/p62, reversible by CQ or leupeptin exposure. In conclusion, we identified autophagy as a compensatory pathway occurring in MDS-BM after long-term exposure to 5-AZA and we provided evidences that a sequential treatment of 5-AZA followed by CQ could improve 5-AZA efficacy, providing novel insight for tailored therapy in MDS patients progressing after 5-AZA therapy.

Autophagy Promoted Neural Differentiation of Human Placenta-derived Mesenchymal Stem Cells

“…In addition to the inhibition of Notch signaling, expression of autophagy genes (LC3I/II, BECLIN) was enhanced upon DAPT treatment, in a dose-dependent manner. Most recent data show that bone marrow mononucleated cells exposed to 5-Azacytidine express increased proteins associated with autophagy (46). Hence, it could be hypothesized that autophagy plays a role in neuronal differentiation, once Notch singling is inhibited.…”

Anti-apoptotic proteins in the autophagic world: an update on functions of XIAP, Survivin, and BRUCE

“…Upregulation of autophagy has been shown to promote the survival of cancer and cancer-related cells treated with a variety of therapeutics including tamoxifen, paclitaxel, epirubicin, and azacytidine [14][15][16][17][18]. Autophagy is also known to assist homologous recombination, which is a type of DNA repair mechanisms, in cells treated with DNA damaging agents and UV radiation [91][92][93].…”

“…Autophagy is currently one of the hottest topics in cancer research. Despite intensive research has been conducted in the past decade to better understand the process of autophagy [13][14][15][16][17][18][19][20][21][22][23], the detailed regulatory mechanism and cellular effects are still not yet fully understood. Generally, autophagy is a dynamic catabolic process used for removing unnecessary or dysfunctional proteins and organelles in cells.…”

“…Generally, autophagy is a dynamic catabolic process used for removing unnecessary or dysfunctional proteins and organelles in cells. Pathologically, dysregulation of autophagy promotes tumorigenesis and upregulation of autophagy has widely been shown to provide extra survival signals in both normal and cancer cells exposed to various internal and external stresses [14][15][16][17][18][19][20][21][22]. For example, hypoxia-induced autophagy process might contribute to the resistance to chemotherapeutic agent, cisplatin, in non-small cell lung cancer [19].…”

Moving beyond hydroxychloroquine: the novel lysosomal autophagy inhibitor ROC-325 shows significant potential in preclinical studies

“…In a subsequent study, we investigated the potential benefit of autophagy inhibition as a new approach to increase the anti-leukemic activity of the hypomethylating agent azacitidine (AZA) [8]. Recent studies have shown that autophagy plays a role in resistance to AZA, which is frequently used in the management of patients with myelodysplastic syndromes (MDS) and AML [9, 10]. We showed that the combination of ROC-325 and AZA blocked AZA-induced autophagy and yielded significantly improved activity against AML cell lines and primary AML blasts.…”