ULK/Atg1: phasing in and out of autophagy.

Autophagy - a highly regulated intracellular degradation process - is pivotal in maintaining cellular homeostasis. Liquid-liquid phase separation (LLPS) is a fundamental mechanism regulating the formation and function of membrane-less compartments. Recent research has unveiled connections between LLPS and autophagy, suggesting that phase separation events may orchestrate the spatiotemporal organization of autophagic machinery and cargo sequestration. The Unc-51-like kinase (ULK)/autophagy-related 1 (Atg1) family of proteins is best known for its regulatory role in initiating autophagy, but there is growing evidence that the functional spectrum of ULK/Atg1 extends beyond autophagy regulation. In this review, we explore the spatial and temporal regulation of the ULK/Atg1 family of kinases, focusing on their recruitment to LLPS-driven compartments, and highlighting their multifaceted functions beyond their traditional role.

ULK1 and ULK2 Regulate Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97.

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.

Receptor interacting protein kinase 2-mediated mitophagy regulates inflammasome activation during virus infection.

NOD2 receptor and the cytosolic protein kinase RIPK2 regulate NF-κB and MAP kinase signaling during bacterial infections, but the role of this immune axis during viral infections has not been addressed. We demonstrate that Nod2(-/-) and Ripk2(-/-) mice are hypersusceptible to infection with influenza A virus. Ripk2(-/-) cells exhibited defective autophagy of mitochondria (mitophagy), leading to enhanced mitochondrial production of superoxide and accumulation of damaged mitochondria, which resulted in greater activation of the NLRP3 inflammasome and production of IL-18. RIPK2 regulated mitophagy in a kinase-dependent manner by phosphorylating the mitophagy inducer ULK1. Accordingly, Ulk1(-/-) cells exhibited enhanced mitochondrial production of superoxide and activation of caspase-1. These results demonstrate a role for NOD2-RIPK2 signaling in protection against virally triggered immunopathology by negatively regulating activation of the NLRP3 inflammasome and production of IL-18 via ULK1-dependent mitophagy.

An ULK1/2-PXN mechanotransduction pathway suppresses breast cancer cell migration.

The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well-characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN-dependent mechanotransduction.

The Noncanonical Role of ULK/ATG1 in ER-to-Golgi Trafficking Is Essential for Cellular Homeostasis.

ULK1 and ULK2 are thought to be essential for initiating autophagy, and Ulk1/2-deficient mice die perinatally of autophagy-related defects. Therefore, we used a conditional knockout approach to investigate the roles of ULK1/2 in the brain. Although the mice showed neuronal degeneration, the neurons showed no accumulation of P62(+)/ubiquitin(+) inclusions or abnormal membranous structures, which are observed in mice lacking other autophagy genes. Rather, neuronal death was associated with activation of the unfolded protein response (UPR) pathway. An unbiased proteomics approach identified SEC16A as an ULK1/2 interaction partner. ULK-mediated phosphorylation of SEC16A regulated the assembly of endoplasmic reticulum (ER) exit sites and ER-to-Golgi trafficking of specific cargo, and did not require other autophagy proteins (e.g., ATG13). The defect in ER-to-Golgi trafficking activated the UPR pathway in ULK-deficient cells; both processes were reversed upon expression of SEC16A with a phosphomimetic substitution. Thus, the regulation of ER-to-Golgi trafficking by ULK1/2 is essential for cellular homeostasis.

Too sweet for autophagy: hexokinase inhibition of mTORC1 activates autophagy.

In this issue, Roberts et al. (2014) describe how hexokinase and mTORC1 cooperate to sense disequilibrium between glucose uptake and utilization and direct the balance of anabolism and catabolism to ensure the appropriate use of cellular resources.

Sestrin2 Phosphorylation by ULK1 Induces Autophagic Degradation of Mitochondria Damaged by Copper-Induced Oxidative Stress.

Selective autolysosomal degradation of damaged mitochondria, also called mitophagy, is an indispensable process for maintaining integrity and homeostasis of mitochondria. One well-established mechanism mediating selective removal of mitochondria under relatively mild mitochondria-depolarizing stress is PINK1-Parkin-mediated or ubiquitin-dependent mitophagy. However, additional mechanisms such as LC3-mediated or ubiquitin-independent mitophagy induction by heavy environmental stress exist and remain poorly understood. The present study unravels a novel role of stress-inducible protein Sestrin2 in degradation of mitochondria damaged by transition metal stress. By utilizing proteomic methods and studies in cell culture and rodent models, we identify autophagy kinase ULK1-mediated phosphorylation sites of Sestrin2 and demonstrate Sestrin2 association with mitochondria adaptor proteins in HEK293 cells. We show that Ser-73 and Ser-254 residues of Sestrin2 are phosphorylated by ULK1, and a pool of Sestrin2 is strongly associated with mitochondrial ATP5A in response to Cu-induced oxidative stress. Subsequently, this interaction promotes association with LC3-coated autolysosomes to induce degradation of mitochondria damaged by Cu-induced ROS. Treatment of cells with antioxidants or a Cu chelator significantly reduces Sestrin2 association with mitochondria. These results highlight the ULK1-Sestrin2 pathway as a novel stress-sensing mechanism that can rapidly induce autophagic degradation of mitochondria under severe heavy metal stress.

POST: A framework for set-based association analysis in high-dimensional data.

Evaluating the differential expression of a set of genes belonging to a common biological process or ontology has proven to be a very useful tool for biological discovery. However, existing gene-set association methods are limited to applications that evaluate differential expression across k⩾2 treatment groups or biological categories. This limitation precludes researchers from most effectively evaluating the association with other phenotypes that may be more clinically meaningful, such as quantitative variables or censored survival time variables. Projection onto the Orthogonal Space Testing (POST) is proposed as a general procedure that can robustly evaluate the association of a gene-set with several different types of phenotypic data (categorical, ordinal, continuous, or censored). For each gene-set, POST transforms the gene profiles into a set of eigenvectors and then uses statistical modeling to compute a set of z-statistics that measure the association of each eigenvector with the phenotype. The overall gene-set statistic is the sum of squared z-statistics weighted by the corresponding eigenvalues. Finally, bootstrapping is used to compute a p-value. POST may evaluate associations with or without adjustment for covariates. In simulation studies, it is shown that the performance of POST in evaluating the association with a categorical phenotype is similar to or exceeds that of existing methods. In evaluating the association of 875 biological processes with the time to relapse of pediatric acute myeloid leukemia, POST identified the well-known oncogenic WNT signaling pathway as its top hit. These results indicate that POST can be a very useful tool for evaluating the association of a gene-set with a variety of different phenotypes. We have developed an R package named POST which is freely available in Bioconductor.

Hsp90-Cdc37 chaperone complex regulates Ulk1- and Atg13-mediated mitophagy.

Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.

Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates.

Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington's disease.

Ulk1-mediated autophagy plays an essential role in mitochondrial remodeling and functional regeneration of skeletal muscle.

Autophagy is a conserved cellular process for degrading aggregate proteins and dysfunctional organelle. It is still debatable if autophagy and mitophagy (a specific process of autophagy of mitochondria) play important roles in myogenic differentiation and functional regeneration of skeletal muscle. We tested the hypothesis that autophagy is critical for functional regeneration of skeletal muscle. We first observed time-dependent increases (3- to 6-fold) of autophagy-related proteins (Atgs), including Ulk1, Beclin1, and LC3, along with reduced p62 expression during C2C12 differentiation, suggesting increased autophagy capacity and flux during myogenic differentiation. We then used cardiotoxin (Ctx) or ischemia-reperfusion (I/R) to induce muscle injury and regeneration and observed increases in Atgs between days 2 and 7 in adult skeletal muscle followed by increased autophagy flux after day 7 Since Ulk1 has been shown to be essential for mitophagy, we asked if Ulk1 is critical for functional regeneration in skeletal muscle. We subjected skeletal muscle-specific Ulk1 knockout mice (MKO) to Ctx or I/R. MKO mice had significantly impaired recovery of muscle strength and mitochondrial protein content post-Ctx or I/R. Imaging analysis showed that MKO mice have significantly attenuated recovery of mitochondrial network at 7 and 14 days post-Ctx. These findings suggest that increased autophagy protein and flux occur during muscle regeneration and Ulk1-mediated mitophagy is critical for recovery for the mitochondrial network and hence functional regeneration.

Mito-protective autophagy is impaired in erythroid cells of aged mtDNA-mutator mice.

Somatic mitochondrial DNA (mtDNA) mutations contribute to the pathogenesis of age-related disorders, including myelodysplastic syndromes (MDS). The accumulation of mitochondria harboring mtDNA mutations in patients with these disorders suggests a failure of normal mitochondrial quality-control systems. The mtDNA-mutator mice acquire somatic mtDNA mutations via a targeted defect in the proofreading function of the mtDNA polymerase, PolgA, and develop macrocytic anemia similar to that of patients with MDS. We observed an unexpected defect in clearance of dysfunctional mitochondria at specific stages during erythroid maturation in hematopoietic cells from aged mtDNA-mutator mice. Mechanistically, aberrant activation of mechanistic target of rapamycin signaling and phosphorylation of uncoordinated 51-like kinase (ULK) 1 in mtDNA-mutator mice resulted in proteasome-mediated degradation of ULK1 and inhibition of autophagy in erythroid cells. To directly evaluate the consequence of inhibiting autophagy on mitochondrial function in erythroid cells harboring mtDNA mutations in vivo, we deleted Atg7 from erythroid progenitors of wild-type and mtDNA-mutator mice. Genetic disruption of autophagy did not cause anemia in wild-type mice but accelerated the decline in mitochondrial respiration and development of macrocytic anemia in mtDNA-mutator mice. These findings highlight a pathological feedback loop that explains how dysfunctional mitochondria can escape autophagy-mediated degradation and propagate in cells predisposed to somatic mtDNA mutations, leading to disease.

Frailty in childhood cancer survivors.

Young adult childhood cancer survivors are at an increased risk of frailty, a physiologic phenotype typically found among older adults. This phenotype is associated with new-onset chronic health conditions and mortality among both older adults and childhood cancer survivors. Mounting evidence suggests that poor fitness, muscular weakness, and cognitive decline are common among adults treated for childhood malignancies, and that risk factors for these outcomes are not limited to those treated with cranial radiation. Although the pathobiology of this phenotype is not known, early cellular senescence, sterile inflammation, and mitochondrial dysfunction in response to initial cancer or treatment-related insults are hypothesized to play a role. To the authors' knowledge, interventions to prevent or remediate frailty among childhood cancer survivors have not been tested to date. Pharmaceutical, nutraceutical, and lifestyle interventions have demonstrated some promise.

Potent obatoclax cytotoxicity and activation of triple death mode killing across infant acute lymphoblastic leukemia.

Survival in infants younger than 1 year who have acute lymphoblastic leukemia (ALL) is inferior whether MLL is rearranged (R) or germline (G). MLL translocations confer chemotherapy resistance, and infants experience excess complications. We characterized in vitro sensitivity to the pan-antiapoptotic BCL-2 family inhibitor obatoclax mesylate in diagnostic leukemia cells from 54 infants with ALL/bilineal acute leukemia because of the role of prosurvival BCL-2 proteins in resistance, their imbalanced expression in infant ALL, and evidence of obatoclax activity with a favorable toxicity profile in early adult leukemia trials. Overall, half maximal effective concentrations (EC50s) were lower than 176 nM (the maximal plasma concentration [Cmax] with recommended adult dose) in 76% of samples, whether in MLL-AF4, MLL-ENL, or other MLL-R or MLL-G subsets, and regardless of patients' poor prognostic features. However, MLL status and partner genes correlated with EC50. Combined approaches including flow cytometry, Western blot, obatoclax treatment with death pathway inhibition, microarray analyses, and/or electron microscopy indicated a unique killing mechanism involving apoptosis, necroptosis, and autophagy in MLL-AF4 ALL cell lines and primary MLL-R and MLL-G infant ALL cells. This in vitro obatoclax activity and its multiple killing mechanisms across molecular cytogenetic subsets provide a rationale to incorporate a similarly acting compound into combination strategies to combat infant ALL.

Physiological functions of ULK1/2.

UNC-51-like kinases 1 and 2 (ULK1/2) are serine/threonine kinases that are best known for their evolutionarily conserved role in the autophagy pathway. Upon sensing the nutrient status of a cell, ULK1/2 integrate signals from upstream cellular energy sensors such as mTOR and AMPK and relay them to the downstream components of the autophagy machinery. ULK1/2 also play indispensable roles in the selective autophagy pathway, removing damaged mitochondria, invading pathogens, and toxic protein aggregates. Additional functions of ULK1/2 have emerged beyond autophagy, including roles in protein trafficking, RNP granule dynamics, and signaling events impacting innate immunity, axon guidance, cellular homeostasis, and cell fate. Therefore, it is no surprise that alterations in ULK1/2 expression and activity have been linked with pathophysiological processes, including cancer, neurological disorders, and cardiovascular diseases. Growing evidence suggests that ULK1/2 function as biological rheostats, tuning cellular functions to intra and extra-cellular cues. Given their broad physiological relevance, ULK1/2 are candidate targets for small molecule activators or inhibitors that may pave the way for the development of therapeutics for the treatment of diseases in humans.

Associations between mitochondrial copy number, exercise capacity, physiologic cost of walking, and cardiac strain in young adult survivors of childhood cancer.

PURPOSE: Childhood cancer survivors are at risk for cardiac dysfunction and impaired physical performance, though underlying cellular mechanisms are not well studied. In this cross-sectional study, we examined the association between peripheral blood mitochondrial DNA copy number (mtDNA-CN, a proxy for mitochondrial function) and markers of performance impairment and cardiac dysfunction. METHODS: Whole-genome sequencing, validated by quantitative polymerase chain reaction, was used to estimate mtDNA-CN in 1720 adult survivors of childhood cancer (48.5% female; mean age = 30.7 years, standard deviation (SD) = 9.0). Multivariable logistic regression was performed to evaluate the associations between mtDNA-CN and exercise intolerance, walking inefficiency, and abnormal global longitudinal strain (GLS), adjusting for treatment exposures, age, sex, and race and ethnicity. RESULTS: The prevalence of exercise intolerance, walking inefficiency, and abnormal GLS among survivors was 25.7%, 10.7%, and 31.7%, respectively. Each SD increase of mtDNA-CN was associated with decreased odds of abnormal GLS (adjusted odds ratio (OR) = 0.88, p = 0.04) but was not associated with exercise intolerance (OR = 1.02, p = 0.76) or walking inefficiency (OR = 1.06, p = 0.46). Alkylating agent exposure was associated with increased odds of exercise intolerance (OR = 2.25, p < 0.0001), walking inefficiency (OR = 2.37, p < 0.0001), and abnormal GLS (OR = 1.78, p = 0.0002). CONCLUSIONS: Increased mtDNA-CN is associated with decreased odds of abnormal cardiac function in childhood cancer survivors. IMPLICATIONS FOR CANCER SURVIVORS: These findings demonstrate a potential role for mtDNA-CN as a biomarker of early cardiac dysfunction in this population.

Somatic mitochondrial DNA mutations are a source of heterogeneity among primary leukemic cells.

Somatic mitochondrial DNA (mtDNA) mutations are prevalent in tumors, yet defining their biological significance remains challenging due to the intricate interplay between selective pressure, heteroplasmy, and cell state. Utilizing bulk whole-genome sequencing data from matched tumor and normal samples from two cohorts of pediatric cancer patients, we uncover differences in the accumulation of synonymous and nonsynonymous mtDNA mutations in pediatric leukemias, indicating distinct selective pressures. By integrating single-cell sequencing (SCS) with mathematical modeling and network-based systems biology approaches, we identify a correlation between the extent of cell-state changes associated with tumor-enriched mtDNA mutations and the selective pressures shaping their distribution among individual leukemic cells. Our findings also reveal an association between specific heteroplasmic mtDNA mutations and cellular responses that may contribute to functional heterogeneity among leukemic cells and influence their fitness. This study highlights the potential of SCS strategies for distinguishing between pathogenic and passenger somatic mtDNA mutations in cancer.

Cbfbeta interacts with Runx2 and has a critical role in bone development.

Runx2 (runt-related transcription factor 2, also known as Cbfa1, Osf2 and AML3) is essential for bone development in mice, and mutations in RUNX2 are found in 65-80% of individuals with cleidocranial dysplasia. Although all Runx family members can interact with Cbfbeta (core-binding factor b, encoded by Cbfb), a role for Cbfbeta in bone development has not been demonstrated owing to lethality in Cbfb(-/-) mouse embryos at 12.5 days post coitum (d.p.c.) from hemorrhages and lack of definitive hematopoiesis. Using a 'knock-in' strategy, we generated mouse embryonic stem (ES) cells that express Cbfb fused in-frame to a cDNA encoding green fluorescent protein (GFP). Cbfb(+/GFP) mice had normal life spans and appeared normal, but Cbfb(GFP/GFP) pups died within the first day after birth. The Cbfb(GFP/GFP) mice exhibited a delay in endochondral and intramembranous ossification as well as in chondrocyte differentiation, similar to but less severe than delays observed in Runx2(-/-) mice. We demonstrate that Cbfbeta is expressed in developing bone and forms a functional interaction with Runx2, and that Cbfb(GFP) is a hypomorphic allele. The fusion allele maintains sufficient function in hematopoietic cells to bypass the early embryonic lethality, and identifies a new role for Cbfb in bone development. Our findings raise the possibility that mutations in CBFB may be responsible for some cases of cleidocranial dysplasia that are not linked to mutations in RUNX2.

Hematopoietic stem cell function requires 12/15-lipoxygenase-dependent fatty acid metabolism.

Fatty acid metabolism governs multiple intracellular signaling pathways in many cell types, but its role in hematopoietic stem cells (HSCs) is largely unknown. Herein, we establish a critical role for 12/15-lipoxygenase (12/15-LOX)-mediated unsaturated fatty acid metabolism in HSC function. HSCs from 12/15-LOX-deficient mice are severely compromised in their capacity to reconstitute the hematopoietic compartment in competitive and serial reconstitution assays. Furthermore, we demonstrate that 12/15-LOX is required for the maintenance of long-term HSC quiescence and number. The defect in HSCs is cell-autonomous and associated with a selective reduction in 12/15-LOX-mediated generation of bioactive lipid mediators and reactive oxygen species and with a decrease in canonical Wnt signaling as measured by nuclear beta-catenin staining. These results have implications for development, aging, and transformation of the hematopoietic compartment.