Induction of autophagy and autophagy-dependent apoptosis in diffuse large B-cell lymphoma by a new antimalarial artemisinin derivative, SM1044.
ABSTRACT: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. R-CHOP is currently the standard therapy for DLBCL, but the prognosis of refractory or recurrent patients remains poor. In this study, we synthesized a new water-soluble antimalarial drug artemisinin derivative, SM1044. The treatment of DLBCL cell lines with SM1044 induces autophagy-dependent apoptosis, which is directed by an accelerated degradation of the antiapoptosis protein Survivin, via its acetylation-dependent interaction with the autophagy-related protein LC3-II. Additionally, SM1044 also stimulates the de novo synthesis of ceramide, which in turn activates the CaMKK2-AMPK-ULK1 axis, leading to the initiation of autophagy. Our findings not only elucidate the mechanism of autophagy-dependent apoptosis in DLBCL cells, but also suggest that SM1044 is a promising therapeutic molecule for the treatment of DLBCL, along with R-CHOP regimen.
Project description:Here we show that bortezomib induces effective proteasome inhibition and accumulation of poly-ubiquitinated proteins in diffuse large B-cell lymphoma (DLBCL) cells. This leads to induction of endoplasmic reticulum (ER) stress as demonstrated by accumulation of the protein CHOP, as well as autophagy, as demonstrated by accumulation of LC3-II proteins. Our data suggest that recruitment of both ubiquitinated proteins and LC3-II by p62 directs ubiquitinated proteins, including I-?B?, to the autophagosome. Degradation of I-?B? results in increased NF-?B nuclear translocation and transcription activity. Since bortezomib treatment promoted I-?B? phosphorylation, ubiquitination and degradation, this suggests that the route of I-?B? degradation was not via the ubiquitin-proteasome degradation system. The autophagy inhibitor chloroquine (CQ) significantly inhibited bortezomib-induced I-?B? degradation, increased complex formation with NF-?B and reduced NF-?B nuclear translocation and DNA binding activity. Importantly, the combination of proteasome and autophagy inhibitors showed synergy in killing DLBCL cells. In summary, bortezomib-induced autophagy confers relative DLBCL cell drug resistance by eliminating I-?B?. Inhibition of both autophagy and the proteasome has great potential to kill apoptosis-resistant lymphoma cells.
Project description:Overexpression of the anti-apoptotic protein BCL-2 is characteristic of human follicular lymphoma (FL) and some cases of diffuse large B cell lymphoma (DLBCL). We aimed to determine autophagy status in primary FL and DLBCL samples and the BCL-2+/BCL-2- lymphoma cell lines using both autophagy PCR array and tissue microarray (TMA). A greater number of autophagy machinery genes were up-regulated in the BCL-2+ Su-DHL4 cell line compared with BCL-2- Su-DHL8 cells, at both the basal level and in response to autophagic stress. The autophagy-related gene expression profiles were determined in purified and unpurified malignant human lymph node biopsies. Seven autophagy machinery genes were up-regulated in purified FL B-cells compared with reactive B-cells. Only 2 autophagy machinery genes were up-regulated in DLBCL B-cells. In unpurified tissue biopsies, 20 of 46 genes in FL and 2 of 5 genes in DLBCL with increased expression were autophagy machinery genes. Expression of autophagy substrates p62 and LC3 were determined by TMAs. FL samples showed significantly decreased levels of both p62 and LC3 compared with reactive and DLBCL, indicative of an increased autophagy activity in FL. In summary, these results demonstrate that FL showed increased basal autophagy activity, regardless of overexpression of BCL-2 in this disease.
Project description:Autophagy promotes tumor growth by generating nutrients from the degradation of intracellular structures. Here we establish, using shRNAs, a dominant-negative mutant, and a pharmacologic inhibitor, mefenamic acid (MFA), that the Transient Receptor Potential Melastatin 3 (TRPM3) channel promotes the growth of clear cell renal cell carcinoma (ccRCC) and stimulates MAP1LC3A (LC3A) and MAP1LC3B (LC3B) autophagy. Increased expression of TRPM3 in RCC leads to Ca(2+) influx, activation of CAMKK2, AMPK, and ULK1, and phagophore formation. In addition, TRPM3 Ca(2+) and Zn(2+) fluxes inhibit miR-214, which directly targets LC3A and LC3B. The von Hippel-Lindau tumor suppressor (VHL) represses TRPM3 directly through miR-204 and indirectly through another miR-204 target, Caveolin 1 (CAV1).
Project description:Autophagy is induced during differentiation of human monocytes into macrophages that is mediated by CSF1/CSF-1/M-CSF (colony stimulating factor 1 [macrophage]). However, little is known about the molecular mechanisms that link CSF1 receptor engagement to the induction of autophagy. Here we show that the CAMKK2-PRKAA1-ULK1 pathway is required for CSF1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2RY6 to the induction of autophagy, and we decipher the signaling network that links the CSF1 receptor to P2RY6-mediated autophagy and monocyte differentiation. In addition, we show that the physiological P2RY6 ligand UDP and the specific P2RY6 agonist MRS2693 can restore normal monocyte differentiation through reinduction of autophagy in primary myeloid cells from some but not all chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for PRKAA1-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML.
Project description:Autophagy is an evolutionary conserved process that degrades subcellular constituents. Unlike starvation-induced autophagy, the molecular mechanism of genotoxic stress-induced autophagy has not yet been fully elucidated. In this study, we analyze the molecular mechanism of genotoxic stress-induced autophagy and identify an essential role of dephosphorylation of the Unc51-like kinase 1 (Ulk1) at Ser637, which is catalyzed by the protein phosphatase 1D magnesium-dependent delta isoform (PPM1D). We show that after exposure to genotoxic stress, PPM1D interacts with and dephosphorylates Ulk1 at Ser637 in a p53-dependent manner. The PPM1D-dependent Ulk1 dephosphorylation triggers Ulk1 puncta formation and induces autophagy. This happens not only in mouse embryonic fibroblasts but also in primary thymocytes, where the genetic ablation of PPM1D reduces the dephosphorylation of Ulk1 at Ser637, inhibits autophagy, and accelerates apoptosis induced by X-ray irradiation. This acceleration of apoptosis is caused mainly by the inability of the autophagic machinery to degrade the proapoptotic molecule Noxa. These findings indicate that the PPM1D-Ulk1 axis plays a pivotal role in genotoxic stress-induced autophagy.
Project description:Currently, there is limited understanding about hormonal regulation of mitochondrial turnover. Thyroid hormone (T3) increases oxidative phosphorylation (OXPHOS), which generates reactive oxygen species (ROS) that damage mitochondria. However, the mechanism for maintenance of mitochondrial activity and quality control by this hormone is not known. Here, we used both in vitro and in vivo hepatic cell models to demonstrate that induction of mitophagy by T3 is coupled to oxidative phosphorylation and ROS production. We show that T3 induction of ROS activates CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, ?) mediated phosphorylation of PRKAA1/AMPK (5' AMP-activated protein kinase), which in turn phosphorylates ULK1 (unc-51 like autophagy activating kinase 1) leading to its mitochondrial recruitment and initiation of mitophagy. Furthermore, loss of ULK1 in T3-treated cells impairs both mitophagy as well as OXPHOS without affecting T3 induced general autophagy/lipophagy. These findings demonstrate a novel ROS-AMPK-ULK1 mechanism that couples T3-induced mitochondrial turnover with activity, wherein mitophagy is necessary not only for removing damaged mitochondria but also for sustaining efficient OXPHOS.
Project description:Expression of the anti-apoptotic B-cell lymphoma 2 (BCL-2) protein in patients with diffuse large B-cell lymphoma (DLBCL) strongly correlates with resistance to standard therapy with cyclophosphamide, vincristine, doxorubicin, prednisolone, and rituximab (R-CHOP). Although studies focus mainly on the contribution of BCL-2, here we also investigate the contribution of other anti-apoptotic proteins to CHOP-therapy resistance in DLBCL. Functional dynamic BCL-2 homology (BH)3 profiling was applied to DLBCL cell lines upon CHOP treatment or single CHOP compounds. Cell-specific anti-apoptotic dependencies were validated with corresponding BH3-mimetics. We found high expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL in DLBCL cell lines and patients. CHOP treatment resulted in both enhanced and altered anti-apoptotic dependency. Enhanced sensitivity to different BH3-mimetics after CHOP treatment was confirmed in specific cell lines, indicating heterogeneity of CHOP-induced resistance in DLBCL. Analysis of single CHOP compounds demonstrated that similar changes could also be induced by doxorubicin or vincristine, providing evidence for clinical combination therapies of doxorubicin or vincristine with BH3-mimetics in DLBCL. In conclusion, we show for the first time that CHOP treatment induces increased anti-apoptotic dependency on MCL-1 and BCL-XL, and not just BCL-2. These results provide new perspectives for the treatment of CHOP-resistant DLBCL and underline the potential of BH3 profiling in predicting therapy outcomes.
Project description:Autophagy is a multistage process. Progress within the field has led to the development of agents targeting both early (initiation) and late (fusion) stages of this process. The specific stage of autophagy targeted may influence cancer treatment outcomes. We have previously shown that central nervous system (CNS) tumors with the BRAFV600E mutation are autophagy dependent, and late-stage autophagy inhibition improves the response to targeted BRAF inhibitors (BRAFi) in sensitive and resistant cells. Drugs directed toward initiation of autophagy have been shown to reduce tumor cell death in some cancers, but have not been assessed in CNS tumors. We investigated early-stage inhibition for autophagy-dependent CNS tumors. BRAFi-sensitive and resistant AM38 and MAF794 cell lines were evaluated for the response to pharmacologic and genetic inhibition of ULK1 and VPS34, two crucial subunits of the autophagy initiation complexes. Changes in autophagy were monitored by western blot and flow cytometry. Survival was evaluated in short- and long-term growth assays. Tumor cells exhibited a reduced autophagic flux with pharmacologic and genetic inhibition of ULK1 or VPS34. Pharmacologic inhibition reduced cell survival in a dose-dependent manner for both targets. Genetic inhibition reduced cell survival and confirmed that it was an autophagy-specific effect. Pharmacologic and genetic inhibition were also synergistic with BRAFi, irrespective of RAFi sensitivity. Inhibition of ULK1 and VPS34 are potentially viable clinical targets in autophagy-dependent CNS tumors. Further evaluation is needed to determine if early-stage autophagy inhibition is equal to late-stage inhibition to determine the optimal clinical target for patients.
Project description:Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51-like kinase 1 (ULK1) is a conserved serine-threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold.
Project description:Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.