Liangchun Yang

PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis

Increasing evidence suggests the important role of metabolic reprogramming in the regulation of the innate inflammatory response, but the underlying mechanism remains unclear. Here, we provide evidence to support a novel role for the pyruvate kinase M2 (PKM2)-mediated Warburg effect, namely aerobic glycolysis, in the regulation of high mobility group box 1 (HMGB1) release. PKM2 interacts with hypoxia-inducible factor 1α (HIF1α) and activates the HIF-1α-dependent transcription of enzymes necessary for aerobic glycolysis in macrophages. Knockdown of PKM2, HIF1α, and glycolysis-related genes uniformly decreases lactate production and HMGB1 release. Similarly, a potential PKM2 inhibitor, shikonin, reduces serum lactate and HMGB1 levels and protects mice from lethal endotoxemia and sepsis. Collectively, these findings shed light on a novel mechanism for metabolic control of inflammation by regulating HMGB1 release and highlight the importance of targeting aerobic glycolysis in the treatment of sepsis and other inflammatory diseases.

microRNA 30A promotes autophagy in response to cancer therapy

microRNAs (miRNAs) are a class of small regulatory RNAs that regulate gene expression at the post-transcriptional level. miRNAs play important roles in the regulation of development, growth, and metastasis of cancer, and in determining the response of tumor cells to anticancer therapy. In recent years, they have also emerged as important regulators of autophagy, a lysosomal-mediated pathway that contributes to degradation of a cells own components. Imatinib, a targeted competitive inhibitor of the BCR-ABL1 tyrosine kinase, has revolutionized the clinical treatment of chronic myelogenous leukemia (CML). We demonstrate that MIR30A-mediated autophagy enhances imatinib resistance against CML including primary stem and progenitor cells. MIR30A, but not MIR101, is a potent inhibitor of autophagy by selectively downregulating BECN1 and ATG5 expression in CML cells. MIR30A mimics, as well as knockdown of BECN1 and ATG5, increases intrinsic apoptotic pathways. In contrast, the antagomir-30A increases autophagy and inhibits intrinsic apoptotic pathways, confirming that autophagy serves to protect against apoptosis. Taken together, these data clarify some of the underlying molecular mechanisms of tyrosine kinase inhibitor-induced autophagy.

PKM2-dependent glycolysis promotes NLRP3 and AIM2 inflammasome activation

Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2–EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis.

Chloroquine Inhibits HMGB1 Inflammatory Signaling and Protects Mice from Lethal Sepsis

Sepsis is caused by an overwhelming immune response to bacterial infection. The discovery of high mobility group box 1 (HMGB1) as a late mediator of lethal sepsis has prompted investigation into the development of new therapeutics which specifically target this protein. Here, we show that chloroquine, an anti-malarial drug, prevents lethality in mice with established endotoxemia or sepsis. This effect is still observed even if administration of chloroquine is delayed. The protective effects of chloroquine were mediated through inhibition of HMGB1 release in macrophages, monocytes, and endothelial cells, thereby preventing its cytokine-like activities. As an inhibitor of autophagy, chloroquine specifically inhibited HMGB1-induced Iκ-B degradation and NF-κB activation. These findings define a novel mechanism for the anti-inflammatory effects of chloroquine and also suggest a new potential clinical use for this drug in the setting of sepsis.

Up-regulated autophagy by endogenous high mobility group box-1 promotes chemoresistance in leukemia cells

Abstract Autophagy has recently attracted increasing attention for its role in conferring resistance to various commonly used anticancer therapies. Whereas its activities are known primarily to be under regulation of the high mobility group box-1 (HMGB1) gene, the expression of HMGB1 and its function in leukemia cells still remain unclear. In this study, we found that HMGB1 was expressed abundantly in various kinds of both leukemia and non-blood cancer cell-lines, and its expression was positively correlated with clinical status in childhood leukemia. In leukemia cells, when endogenous HMGB1 increased starvation-induced autophagy, this reaction was inhibited by the suppression of HMGB1. While the use of autophagy inhibitor, 3-methyladenine (3-MA), blocked the autophagic reaction and increased leukemia cell sensitivity to chemotherapy, enhancing HMGB1 expression decreased this sensitivity. Notably, suppressing HMGB1 expression also increased leukemia cell chemosensitivity. Furthermore, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway was found to be functionally connected with HMGB1. HMGB1 gene transfection increased the LC3-II level and inhibited phosphorylation of Akt and p70S6K levels. Knockdown of HMGB1 expression blocked the association between mTOR and raptor in the setting of enhanced autophagy. When class I PI3K was inhibited by PI3K-I shRNA, it decreased the PI3K-I expression level. Knockdown of HMGB1 expression had no further effects on LC3-II. These results suggest that endogenous HMGB1 is an intrinsic regulator of autophagy in leukemia cells and it enhances leukemia cell chemoresistance likely through the PI3K/Akt/mTORC1 pathway.

The ferroptosis inducer erastin enhances sensitivity of acute myeloid leukemia cells to chemotherapeutic agents

Acute myeloid leukemia (AML) is the most common type of leukemia in adults. Development of resistance to chemotherapeutic agents is a major hurdle in the effective treatment of patients with AML. The quinazolinone derivative erastin was originally identified in a screen for small molecules that exhibit synthetic lethality with expression of the RAS oncogene. This lethality was subsequently shown to occur by induction of a novel form of cell death termed ferroptosis. In this study we demonstrate that erastin enhances the sensitivity of AML cells to chemotherapeutic agents in an RAS-independent manner. Erastin dose-dependently induced mixed types of cell death associated with ferroptosis, apoptosis, necroptosis, and autophagy in HL-60 cells (AML, NRAS_Q61L), but not Jurkat (acute T-cell leukemia, RAS wild type), THP-1 (AML, NRAS_G12D), K562 (chronic myelogenous leukemia, RAS wild type), or NB-4 (acute promyelocytic leukemia M3, KRAS_A18D) cells. Treatment with ferrostatin-1 (a potent ferroptosis inhibitor) or necrostatin-1 (a potent necroptosis inhibitor), but not with Z-VAD-FMK (a general caspase inhibitor) or chloroquine (a potent autophagy inhibitor), prevented erastin-induced growth inhibition in HL-60 cells. Moreover, inhibition of c-JUN N-terminal kinase and p38, but not of extracellular signal-regulated kinase activation, induced resistance to erastin in HL-60 cells. Importantly, low-dose erastin significantly enhanced the anticancer activity of 2 first-line chemotherapeutic drugs (cytarabine/ara-C and doxorubicin/adriamycin) in HL-60 cells. Collectively, the induction of ferroptosis and necroptosis contributed to erastin-induced growth inhibition and overcame drug resistance in AML cells.

S100A8 Contributes to Drug Resistance by Promoting Autophagy in Leukemia Cells

Autophagy is a double-edged sword in tumorigenesis and plays an important role in the resistance of cancer cells to chemotherapy. S100A8 is a member of the S100 calcium-binding protein family and plays an important role in the drug resistance of leukemia cells, with the mechanisms largely unknown. Here we report that S100A8 contributes to drug resistance in leukemia by promoting autophagy. S100A8 level was elevated in drug resistance leukemia cell lines relative to the nondrug resistant cell lines. Adriamycin and vincristine increased S100A8 in human leukemia cells, accompanied with upregulation of autophagy. RNA interference-mediated knockdown of S100A8 restored the chemosensitivity of leukemia cells, while overexpression of S100A8 enhanced drug resistance and increased autophagy. S100A8 physically interacted with the autophagy regulator BECN1 and was required for the formation of the BECN1-PI3KC3 complex. In addition, interaction between S100A8 and BECN1 relied upon the autophagic complex ULK1-mAtg13. Furthermore, we discovered that exogenous S100A8 induced autophagy, and RAGE was involved in exogenous S100A8-regulated autophagy. Our data demonstrated that S100A8 is involved in the development of chemoresistance in leukemia cells by regulating autophagy, and suggest that S100A8 may be a novel target for improving leukemia therapy.

Inhibiting autophagy potentiates the anticancer activity of IFN1@/IFNα in chronic myeloid leukemia cells.

IFN1@ (interferon, type 1, cluster, also called IFNα) has been extensively studied as a treatment for patients with chronic myeloid leukemia (CML). The mechanism of anticancer activity of IFN1@ is complex and not well understood. Here, we demonstrate that autophagy, a mechanism of cellular homeostasis for the removal of dysfunctional organelles and proteins, regulates IFN1@-mediated cell death. IFN1@ activated the cellular autophagic machinery in immortalized or primary CML cells. Activation of JAK1-STAT1 and RELA signaling were required for IFN1@-induced expression of BECN1, a key regulator of autophagy. Moreover, pharmacological and genetic inhibition of autophagy enhanced IFN1@-induced apoptosis by activation of the CASP8-BID pathway. Taken together, these findings provide evidence for an important mechanism that links autophagy to immunotherapy in leukemia.

S100A8-targeting siRNA enhances arsenic trioxide-induced myeloid leukemia cell death by down-regulating autophagy.

Chemoresistance has become a major obstacle to the successful treatment of leukemia. Autophagy, a regulated process of degradation and recycling of cellular constituents, has recently caught increasing attention for its roles in conferring resistance to various commonly used anticancer therapies. Here we showed that the member of the S100 calcium-binding protein family, S100A8, is a critical regulator of chemoresistance in the autophagy process. It positively correlated with the clinical status in childhood acute myeloblastic leukemia (AML) and it was released from leukemia cells after chemotherapy-induced cytotoxicity. Knockdown of S100A8 expression increased the sensitivity of leukemia cells to chemotherapy and apoptosis. Moreover, suppressing S100A8 expression decreased autophagy as evaluated by the increased expression of the autophagic marker microtubule-associated protein light chain 3 (LC3)-II, degradation of SQSTM1/Sequestosome 1 (p62) and formation of autophagosomes. Furthermore, stimuli that enhanced reactive oxygen species (ROS) promoted cytosolic translocation of S100A8 and thereby enhanced autophagy. S100A8 directly interacted with the autophagy protein Beclin1 displacing Bcl-2. These results suggest that S100A8 is a critical pro-autophagic protein that enhances cell survival and regulates chemoresistance in leukemia cells likely through disassociating the Beclin1-Bcl-2 complex.

Hsp27: A novel therapeutic target for pediatric M4/M5 acute myeloid leukemia

Heat shock protein 27 (Hsp27), a member of the heat shock protein (Hsp) family, is critical in the regulation of cancer development, progression and chemotherapy resistance. However, the role of Hsp27 in the pathogenesis of pediatric acute leukemia (AL) remains unknown. In this study, we evaluated the expression levels of Hsp27 in bone marrow samples from 94 children with newly diagnosed acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and 5 leukemia cell lines. Additionally, we transfected a target-specific siRNA duplex against Hsp27 into leukemia cells, and examined the chemosensitivity and cell apoptosis in the response to antitumor drugs. Hsp27 was abundantly expressed in newly diagnosed AML-M4/M5 bone marrow mononuclear cells (BMMCs) and THP-1, OCI/AML-3 leukemia cell lines. Furthermore, its expression was positively correlated with the clinical status in pediatric M4/M5 subtypes. Knockdown of Hsp27 expression increased the chemosensitivity of leukemia cells and the anticancer drug-induced apoptosis. These results support the theory that Hsp27 plays a contributory role in the pathogenesis of pediatric AML-M4/M5. Therefore, Hsp27 may be exploited as a new target for enhancing the efficacy of chemotherapeutic drugs against leukemia.