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Background T cell acute lymphoblastic leukemia is a fatal hematological malignancy. Despite the treatment progress, no targeted therapy is available currently, which urges to deepen the understanding of the underlying mechanism of T-ALL cell growth/survival. Autophagy is a conserved cellular process, which plays a dual role in human cancers. Nevertheless, many aspects of the involvement of autophagy in T-ALL are not fully understood. Methods T-ALL patient cells and normal control cells were subjected to RT‒qPCR analysis. Gene silence and overexpression was used to study the function of ATG4B and sestrin 3 (SESN3) in T-ALL cells. Atg4b deficient mice were used to study the role of Atg4b in normal hematopoietic cells and T cell development. The efficacy of S130, an ATG4B inhibitor to suppress T-ALL cell growth was evaluated in xenograft models. Results The results showed that the expression of several autophagy-related genes (especially ATG4B ) was significantly higher in T-ALL patient cells than control cells. ATG4B ablation decreased autophagic flux and inhibited T-ALL cell growth. In contrast, Atg4b depletion had mild effects on normal hematopoiesis and T cell development. RNA-seq data and subsequent studies revealed a novel regulatory circuit of ATG4B and SESN3, and the results indicated that SESN3 hampered T-ALL cell growth via the inhibition of both mTOR/S6K/protein synthesis pathway and autophagy. Importantly, S130 exhibited anti-leukemia activity in xenograft models. Conclusions The present study demonstrates that a novel ATG4B-SESN3 regulatory circuit plays a crucial role in T cell leukemogenesis, which suggests that targeting ATG4B is a promising strategy for T-ALL treatment.

Autophagy is closely related to the growth and drug resistance of cancer cells, and autophagy related 4B (ATG4B) performs a crucial role in the process of autophagy. The long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) promotes the progression of hepatocellular carcinoma (HCC), but it is unclear whether the tumor-promoting effect of CRNDE is associated with the regulation of ATG4B and autophagy. Herein, we for the first time demonstrated that CRNDE triggered autophagy via upregulating ATG4B in HCC cells. Mechanistically, CRNDE enhanced the stability of ATG4B mRNA by sequestrating miR-543, leading to the elevation of ATG4B and autophagy in HCC cells. Moreover, sorafenib induced CRNDE and ATG4B as well as autophagy in HCC cells. Knockdown of CRNDE sensitized HCC cells to sorafenib in vitro and in vivo . Collectively, these results reveal that CRNDE drives ATG4B-mediated autophagy, which attenuates the sensitivity of sorafenib in HCC cells, suggesting that the pathway CRNDE/ATG4B/autophagy may be a novel target to develop sensitizing measures of sorafenib in HCC treatment.

Purpose Previous studies indicate that breast cancer molecular subtypes differ with respect to their dependency on autophagy, but our knowledge of the differential expression and prognostic significance of autophagy-related biomarkers in breast cancer is limited. Methods Immunohistochemistry (IHC) was performed on tissue microarrays from a large population of 3992 breast cancer patients divided into training and validation cohorts. Consensus staining scores were used to evaluate the expression levels of autophagy proteins LC3B, ATG4B, and GABARAP and determine the associations with clinicopathological variables and molecular biomarkers. Survival analyses were performed using the Kaplan–Meier function and Cox proportional hazards regression models. Results We found subtype-specific expression differences for ATG4B, with its expression lowest in basal-like breast cancer and highest in Luminal A, but there were no significant associations with patient prognosis. LC3B and GABARAP levels were highest in basal-like breast cancers, and high levels were associated with worse outcomes across all subtypes (DSS; GABARAP: HR 1.43, LC3B puncta: HR 1.43). High ATG4B levels were associated with ER, PR, and BCL2 positivity, while high LC3B and GABARAP levels were associated with ER, PR, and BCL2 negativity, as well as EGFR, HER2, HER3, CA-IX, PD-L1 positivity, and high Ki67 index ( p  < 0.05 for all associations). Exploratory multi-marker analysis indicated that the combination of ATG4B and GABARAP with LC3B could be useful for further stratifying patient outcomes. Conclusions ATG4B levels varied across breast cancer subtypes but did not show prognostic significance. High LC3B expression and high GABARAP expression were both associated with poor prognosis and with clinicopathological characteristics of aggressive disease phenotypes in all breast cancer subtypes.

Autophagy is a widespread physiological process in the body, which also protects the host by degrading invading pathogens and harmful substances during pathological conditions. Nevertheless, Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, has evolved strategies to subvert autophagy by modulating microRNA (miRNA) expression, enabling its escape from host defenses. In this study, we established an in vitro model using the human macrophage cell line infected with the highly virulent MTB strain H37Rv. Through RNA sequencing and bioinformatic analysis post H37Rv infection, we screened 14 differentially expressed miRNAs. We predicted and demonstrated that miR-30c-1-3p inhibits autophagy and promotes MTB survival by targeting ATG4B and ATG9B during the infection process. The results showed that miR-30c-1-3p expression was gradually increased before 12 h of H37Rv infection, followed by a decrease. Overexpression of miR-30c-1-3p suppressed autophagic activity. We also identified the targeting of miR-30c-1-3p to ATG4B and ATG9B for the first time, and overexpression of both ATG4B and ATG9B, alone or together, on the basis with upregulation of miR-30c-1-3p reversed the inhibition of autophagy. Autophagy levels were analyzed at different levels by western blot, immunofluorescence, and transmission electron microscopy, all of which showed that upregulation of miR-30c-1-3p inhibited autophagy during H37Rv infection. Additionally, the intervention of miR-30c-1-3p mimics resulted in an increased bacterial load in macrophages, suggesting that MTB achieves immune evasion by upregulating miR-30c-1-3p during infection. In conclusion, our study provides a valuable target for the development of host-directed anti-tuberculosis therapy as well as a new diagnostic marker.

Autophagy is a lysosome-mediated degradation pathway that enables the degradation and recycling of cytoplasmic components to sustain metabolic homoeostasis. Recently, autophagy has been reported to have an astonishing number of connections to cancer, as tumor cells require proficient autophagy in response to metabolic and therapeutic stresses to sustain cell proliferation. Autophagy-related gene 4 (ATG4) is essential for autophagy by affecting autophagosome formation through processing full-length microtubule-associated protein 1A/1B-light chain 3 (pro-LC3) and lipidated LC3. An increasing amount of evidence suggests that ATG4B expression is elevated in certain types of cancer, implying that ATG4B is a potential anticancer target. In this review, we address the central roles of ATG4B in the autophagy machinery and in targeted cancer therapy. Specifically, we discuss how pharmacologically inhibiting ATG4B can benefit cancer therapies.

Autophagy plays important but complex roles in aging, affecting health and longevity. We found that, in the general population, the levels of ATG4B and ATG4D decreased during aging, yet they are upregulated in centenarians, suggesting that overexpression of ATG4 members could be positive for healthspan and lifespan. We therefore analyzed the effect of overexpressing Atg4b (a homolog of human ATG4D) in Drosophila, and found that, indeed, Atg4b overexpression increased resistance to oxidative stress, desiccation stress and fitness as measured by climbing ability. The overexpression induced since mid-life increased lifespan. Transcriptome analysis of Drosophila subjected to desiccation stress revealed that Atg4b overexpression increased stress response pathways. In addition, overexpression of ATG4B delayed cellular senescence, and improved cell proliferation. These results suggest that ATG4B have contributed to a slowdown in cellular senescence, and in Drosophila, Atg4b overexpression may have led to improved healthspan and lifespan by promoting a stronger stress response. Overall, our study suggests that ATG4D and ATG4B have the potential to become targets for health and lifespan interventions.

Autophagy related gene 4B (ATG4B) plays a central role in autophagy machinery, but its clinical relevance to AAA remains unknown. In this study, 205 AAA patients and 205 age- and sex-matched controls were included to detect the serum ATG4B levels. Meanwhile, abdominal aortic specimens from 24 AAA patients and 6 human organ donors were collected to evaluate the mRNA and in situ protein expression of ATG4B. We observed significantly higher ATG4B mRNA and protein expression levels in AAA group compared to those in control group, with a positive correlation between mRNA levels and serum/in situ protein levels (serum, r  = 0.518, P =  0.010; in situ, r  = 0.453, P =  0.026). Serum ATG4B exhibited the diagnostic potential for AAA (AUC = 0.702, sensitivity = 75.6%) and intraluminal thrombus recognition (AUC = 0.602, sensitivity = 67.9%). Logistic regression revealed a significant association between elevated serum ATG4B and an increased risk of AAA and intraluminal thrombus formation. Deceased patients displayed higher baseline serum ATG4B levels, which could predict postoperative mortality (HR = 1.028, 95%CI = 1.007–1.049, P  = 0.009, AUC = 0.612, sensitivity = 84.6%). The bioinformatics analysis suggested that ATG4B may modulate cellular autophagy and influence pathways associated with inflammation, lipid metabolism, or apoptosis, thereby contributing to the occurrence and development of AAA. The drug-gene interaction network identified 13 potential therapeutic drugs targeting ATG4B. In conclusion, ATG4B may serve as a promising biomarker for the diagnosis and prognostic assessment of AAA patients and play a key role in the pathogenesis of AAA.

Contactin-associated protein1 (Caspr1) plays an important role in the formation and stability of myelinated axons. In Caspr1 mutant mice, autophagy-related structures accumulate in neurons, causing axonal degeneration; however, the mechanism by which Caspr1 regulates autophagy remains unknown. To illustrate the mechanism of Caspr1 in autophagy process, we demonstrated that Caspr1 knockout in primary neurons from mice along with human cell lines, HEK-293 and HeLa, induced autophagy by downregulating the PI3K/AKT/mTOR signaling pathway to promote the conversion of microtubule-associated protein light chain 3 I (LC3-I) to LC3-II. In contrast, Caspr1 overexpression in cells contributed to the upregulation of this signaling pathway. We also demonstrated that Caspr1 knockout led to increased LC3-I protein expression in mice. In addition, Caspr1 could inhibit the expression of autophagy-related 4B cysteine peptidase (ATG4B) protein by directly binding to ATG4B in overexpressed Caspr1 cells. Intriguingly, we found an accumulation of ATG4B in the Golgi apparatuses of cells overexpressing Caspr1 ; therefore, we speculate that Caspr1 may restrict ATG4 secretion from the Golgi apparatus to the cytoplasm. Collectively, our results indicate that Caspr1 may regulate autophagy by modulating the PI3K/AKT/mTOR signaling pathway and the levels of ATG4 protein, both in vitro and in vivo. Thus, Caspr1 can be a potential therapeutic target in axonal damage and demyelinating diseases. Graphical Abstract

Cysteine protease ATG4B, a key autophagy protein, is an attractive target for colorectal cancer therapy. However, ATG4B inhibitors with higher efficiency, safety, and clear mechanism are still limited. In this study, we discovered ATG4B inhibitors based on the FDA-approved drug library through FRET-based high-throughput screening and gel-based analysis. Among the nine hits, compound Ebselen showed the most potent ATG4B inhibitory activity (IC 50  = 189 nM) and exhibited controllable selectivity and structural optimizable possibility against ATG4A and caspases. We then performed mass spectrometry assay and cysteine mutations to confirm that Ebselen could covalently bind to ATG4B at Cys74. Moreover, Cys292 and Cys361 instead of Cys74 are responsible for the redox-oligomerization and efficient activity inhibition of ATG4B. Ultimately through cell culture and mouse xenograft tumor models, we established the impact of Ebselen on autophagy and tumor suppression via ATG4B inhibition other than apoptosis. These results suggest that old drug Ebselen as an ATG4B inhibitor through oxidative modification may be repurposed as a promising anti-colorectal cancer drug. Highlights High-throughput screening of ATG4B inhibitors based on FDA-approved drug library Ebselen can covalently bind to ATG4B at Cys74 Ebselen can promote ATG4B oligomerization at Cys292 and Cys361 Ebselen suppresses the growth of colorectal cancer cells and xenograft tumors via ATG4B inhibition

Background Oxidative stress in hepatocytes was important pathogenesis of nonalcoholic steatohepatitis (NASH). Autophagy was a cellular process that can remove damaged organelles under oxidative stress, and thus presented a potential therapeutic target against NASH. This work aimed to investigate whether autophagy was participated in the protective effects of dihydromyricetin (DHM) on palmitic acid (PA)-induced oxidative stress in hepatocytes and the underlying mechanism. Methods HepG2 and HHL-5 cell lines were pretreated with DHM (20 μM) for 2 h, followed by PA (0.2 mM) treatment for 16 h. The oxidative stress was assessed by the quantification of intracellular reactive oxygen species (ROS), mitochondrial ROS (mtROS), mitochondrial membrane potential (MMP) and mitochondrial ultrastructural analyses. The protein expressions of SIRT3, LC3I/II, P62 and ATG4B, as well as the acetylation of AGT4B were determined by western blotting using HepG2 and HepG2/ATG4B ± cells with heterozygous knockout of ATG4B . Results Exposure to PA resulted in increased intracellular ROS and mtROS, decreased MMP and aggravated mitochondrial injury in HepG2 cells, which were notably attenuated by DHM treatment. DHM-induced inhibition of oxidative stress was associated with the induction of autophagy, characterized by upregulated ATG4B and LC3 II as well as downregulated P62 levels. Furthermore, the inhibitory effects of DHM on PA-induced autophagy arrest and oxidative stress were eliminated when pretreated with a SIRT3 inhibitor 3-TYP or conducted in HepG2/ATG4B ± cells, suggesting that SIRT3 and ATG4B were involved in DHM-induced benefits. Moreover, DHM treatment increased the protein expression of SIRT3 and SIRT3-dependent deacetylation of ATG4B in HepG2 cells. Conclusion Our results demonstrated that DHM attenuated PA-induced oxidative stress in hepatocytes through induction of autophagy, which was mediated through the increased expression of SIRT3 and SIRT3-mediated ATG4B deacetylation following DHM treatment.