Gossypol acetic acid regulates leukemia stem cells by degrading LRPPRC via inhibiting IL-6/JAK1/STAT3 signaling or resulting mitochondrial dysfunction

BACKGROUND Leukemia stem cells(LSCs)are found to be one of the main factors contributing to poor therapeutic effects in acute myeloid leukemia(AML),as they are protected by the bone marrow microenvironment(BMM)against conventional therapies.Gossypol acetic acid(GAA),which is extracted from the seeds of cotton plants,exerts anti-tumor roles in several types of cancer and has been reported to induce apoptosis of LSCs by inhibiting Bcl2.AIM To investigate the exact roles of GAA in regulating LSCs under different microenvironments and the exact mechanism.METHODS In this study,LSCs were magnetically sorted from AML cell lines and the CD34+CD38-population was obtained.The expression of leucine-rich pentatricopeptide repeat-containing protein(LRPPRC)and forkhead box M1(FOXM1)was evaluated in LSCs,and the effects of GAA on malignancies and mitochondrial RESULTS LRPPRC was found to be upregulated,and GAA inhibited cell proliferation by degrading LRPPRC.GAA induced LRPPRC degradation and inhibited the activation of interleukin 6(IL-6)/janus kinase(JAK)1/signal transducer and activator of transcription(STAT)3 signaling,enhancing chemosensitivity in LSCs against conventional chemotherapies,including L-Asparaginase,Dexamethasone,and cytarabine.GAA was also found to downregulate FOXM1 indirectly by regulating LRPPRC.Furthermore,GAA induced reactive oxygen species accumulation,disturbed mitochondrial homeostasis,and caused mitochondrial dysfunction.By inhibiting IL-6/JAK1/STAT3 signaling via degrading LRPPRC,GAA resulted in the elimination of LSCs.Meanwhile,GAA induced oxidative stress and subsequent cell damage by causing mitochondrial damage.CONCLUSION Taken together,the results indicate that GAA might overcome the BMM protective effect and be considered as a novel and effective combination therapy for AML.

Resveratrol-downregulated Phosphorylated Liver Kinase B1 Is Involved in Senescence of Acute Myeloid Leukemia Stem Cells

Summary： Senescence is an important obstacle to cancer development. Engaging a senescent response may be an effective way to cure acute myeloid leukemia （AML）. The aim of this study was to examine the effect of resveratrol-downregulated phosphorylated liver kinase B1 （pLKB1） on the senescence of acute myeloid leukemia （AML） stem cells. The protein expressions of pLKB 1 and Sirtuin 1 （SIRT1）, a regulator ofpLKB1, were measured in CD34＋CD38-KGla cells treated with resveratrol （40 μmol/L） or not by Western blotting. Senescence-related factors were examined, including p21 mRNA tested by real-time PCR, cell morphology by senescence-associated β-galactosidase （SA-β-gal） staining, cell pro- liferation by MTT assay and cell cycle by flow cytometry. Besides, apoptosis was flow cytometrically determined. The results showed that pLKB1 was highly expressed in CD34＋CD38- KGla cells, and resveratrol, which could downregulate pLKB1 through activation of SIRT1, induced senescence and apoptosis of CD34＋CD38- KGla cells. It was concluded that resveratrol-downregulated pLKB1 is in- volved in the senescence of AML stem cells.

Update on acute myeloid leukemia stem cells:New discoveries and therapeutic opportunities

The existence of cancer stem cells has been wellestablished in acute myeloid leukemia. Initial proof of the existence of leukemia stem cells(LSCs) was accomplished by functional studies in xenograft models making use of the key features shared with normal hematopoietic stem cells(HSCs) such as the capacity of self-renewal and the ability to initiate and sustain growth of progenitors in vivo. Significant progress has also been made in identifying the phenotype and signaling pathways specific for LSCs. Therapeutically, a multitude of drugs targeting LSCs are in different phases of preclinical and clinical development. This review focuses on recent discoveries which have advanced our understanding of LSC biology and provided rational targets for development of novel therapeutic agents. One of the major challenges is how to target the selfrenewal pathways of LSCs without affecting normal HSCs significantly therefore providing an acceptable therapeutic window. Important issues pertinent to the successful design and conduct of clinical trials evaluating drugs targeting LSCs will be discussed as well.

Bone marrow niche-mediated survival of leukemia stem cells in acute myeloid leukemia: Yin and Yang

Acute myeloid leukemia （AML） is characterized by the accumulation of circulating immature blasts that exhibit uncontrolled growth, lack the ability to undergo normal differentiation, and have decreased sensitivity to apoptosis. Accumulating evidence shows the bone marrow （BM） niche is critical to the maintenance and retention of hematopoietic stem cells （HSC）, including leukemia stem cells （LSC）, and an increasing number of studies have demonstrated that crosstalk between LSC and the stromal cells associated with this niche greatly influences leukemia initiation, progression, and response to therapy. Undeniably, stromal cells in the BM niche provide a sanctuary in which LSC can acquire a drug-resistant phenotype and thereby evade chemotherapy- induced death. Yin and Yang, the ancient Chinese philosophical concept, vividly portrays the intricate and dynamic interactions between LSC and the BM niche. In fact, LSC-induced microenvironmental reprogramming contributes significantly to leukemogenesis. Thus, identifying the critical signaling pathways involved in these interactions will contribute to target optimization and combinatorial drug treatment strategies to overcome acquired drug resistance and prevent relapse following therapy. In this review, we describe some of the critical signaling pathways mediating BM niche-LSC interaction, including SDFI/CXCL12, Wnt/β-catenin, VCAM/VLA-4/NF-κB, CD44, and hypoxia as a newly-recognized physical determinant of resistance, and outline therapeutic strategies for overcoming these resistance factors.

Signaling pathways governing the behaviors of leukemia stem cells

Leukemia is a malignancy in the blood that develops from the lymphatic system and bone marrow.Although various treatment options have been used for different types of leukemia,understanding the molecular pathways involved in the development and progression of leukemia is necessary.Recent studies showed that leukemia stem cells(LSCs)play essential roles in the pathogenesis of leukemia by targeting several signaling pathways,including Notch,Wnt,Hedgehog,and STAT3.LSCs are highly proliferative cells that stimulate tumor initiation,migration,EMT,and drug resistance.This review summarizes cellular pathways that stimulate and prevent LSCs'self-renewal,metastasis,and tumorigenesis.

Molecular mechanisms for survival regulation of chronic myeloid leukemia stem cells

Studies on chronic myeloid leukemia(CML)have served as a paradigm for cancer research and therapy.These studies involve the identifi cation of the fi rst cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors(TKIs)that inhibit BCR-ABL kinase activity in CML.It becomes clear that leukemia stem cells(LSCs)in CML which are resistant to TKIs,and eradication of LSCs appears to be extremely difficult.Therefore,one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies.Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis,in which a rare stem cell population with limitless self-renewal potential gives rise to progenies that lack such potential.LSCs also possess biological features that are different from those of normal hematopoietic stem cells(HSCs)and are critical for their malignant characteristics.In this review,we summarize the latest progress in CML field,and attempt to understand the molecular mechanisms of survival regulation of LSCs.

Molecular mechanisms for stemness maintenance of acute myeloid leukemia stem cells

Human acute myeloid leukemia(AML)is a fatal hematologic malignancy characterized with accumulation of myeloid blasts and differentiation arrest.The development of AML is associated with a serial of genetic and epigenetic alterations mainly occurred in hematopoietic stem and progenitor cells(HSPCs),which change HSPC state at the molecular and cellular levels and transform them into leukemia stem cells(LSCs).LSCs play critical roles in leukemia initiation,progression,and relapse,and need to be eradicated to achieve a cure in clinic.Key to successfully targeting LSCs is to fully understand the unique cellular and molecular mechanisms for maintaining their stemness.Here,we discuss LSCs in AML with a focus on identification of unique biological features of these stem cells to decipher the molecular mechanisms of LSC maintenance.

Lower Phosphorylation of p38 MAPK Blocks the Oxidative Stress-induced Senescence in Myeloid Leukemic CD34^+CD38^- Cells

Leukemia seems to depend on a small population of ＂leukemia stem cells （LSCs）＂ for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species （ROS） induces cellular senescence through DNA damage and phophorylation of p38 MAPK （p38）, in myeloid leukemic CD34＋CD38- cells. Bone marrow samples were obtained from patients with acute myeloid leukemia （AML, n=11） and chronic myeloid leukemia （CML, n=9）. CD34＋CD38- cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells （two kinds of myeloid leukemia cell lines） by mini-magnetic activated cell sorting. Hematopoietic stem cells （HSCs） from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluo-rescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34＋CD38- cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34＋CD38- cells as compared with HSCs and H2O2-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34＋CD38- cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.

HDAC I/IIb selective inhibitor Purinostat Mesylate combined with GLS1 inhibition effectively eliminates CML stem cells

Purinostat Mesylate(PM)is a novel highly selective and active HDAC I/IIb inhibitor,and the injectable formulation of PM(PMF)based on the compound prescription containing cyclodextrin completely can overcome PM’s poor solubility and improves its stability and pharmacokinetic properties.Here,we showed that PM effectively repressed the survival of Ph+leukemia cells and CD34+leukemia cells from CML patients in vitro.In vivo studies demonstrated that PMF significantly prevented BCR-ABL(T315I)induced CML progression by restraining leukemia stem cells(LSCs),which are insensitive to chemotherapy and responsible for CML relapse.Mechanism studies revealed that targeting HDAC I/IIb repressed several important factors for LSCs survival including c-Myc,β-Catenin,E2f,Ezh2,Alox5,and mTOR,as well as interrupted some critical biologic processes.Additionally,PMF increased glutamate metabolism in LSCs by increasing GLS1.The combination of PMF and glutaminase inhibitor BPTES synergistically eradicated LSCs by altering multiple key proteins and signaling pathways which are critical for LSC survival and self-renewal.Overall,our findings represent a new therapeutic strategy for eliminating LSCs by targeting HDAC I/IIb and glutaminolysis,which potentially provides a guidance for PMF clinical trials in the future for TKI resistance CML patients.

Allogeneic peripheral blood stem cell transplantation for leukemia

Objective To observe engraftment kinetics, the incidence and severity of graft-versus-host disease (GVHD), and clinical outcome on 40 recipients undergoing allogeneic peripheral blood stem cell transplantation (allo-PBSCT).Methods From June 1997 to May 1999, forty leukemia patients with a median age of 35 years underwent allo-PBSCT. PBSC were mobilized with G-CSF at a dose of 5 μg/kg s.c. every 12 hours for 5 days. A median of 7.7 (2.0 - 16.8) × 106 CD34+ cells/kg was infused into the recipients. Busulfancyclophosphamide (BU-CY) was used as the conditioning regimen. All patients received cyclosporine A and either methotrexate ( n = 34) or methylprednisolone ( n = 6) for GVHD prophylaxis.Results Engraftment of neutrophils and platelets was achieved at a median of 13 days (9- 28 days) and 12 days (7- 60 days) respectively. Patients receiving ≥4×106 CD34+ cells/kg or given G-CSF post transplant had significantly accelerated neutrophil and platelet engraftment. Acute GVHD occurred in 17 of 40 patients (42.5％), with grade Ⅱ-Ⅳ acute GVHD in 10 patients (25％). Chronic GVHD developed in 21 (9 extensive, 12 limited) out of 30 evaluable patients (21/30, 70％) with a median follow up of 380 days (180-900 days). Transplant related mortality was 17.5％ end the relapse rate was 10％. The probability of leukemia free survival at 3 years was 72.5％.Conclusion Allo-PBSCT can provide rapid hematopoietic reconstitution without an increased incidence of acute GVHD, but may be associated with a high risk of chronic GVHD.

CD34^(+)CD38^(-)subpopulation without CD123 and CD44 is responsible for LSC and correlated with imbalance of immune cell subsets in AML

Acute myeloid leukemia(AML)is regarded as a stem cell disease.However,no one unique marker is expressed on leukemia stem cells(LSC)but not on leukemic blasts nor normal hematopoietic stem cells(HSC).CD34^(+)CD38^(-)with or without CD123 or CD44 subpopulations are immunophenotypically defined as putative LSC fractions in AML.Nevertheless,markers that can be effectively and simply held responsible for the intrinsical heterogeneity of LSC is still unclear.In the present study,we examined the frequency of three different LSC subtypes(CD34^(+)CD38^(-),CD34^(+)CD38^(-)CD123^(+),CD34^(+)CD38^(-)CD44^(+))in AML at diagnosis.We then validated their prognostic significance on the relevance of spectral features for diagnostic stratification,immune status,induction therapy response,treatment effect maintenance,and long^(-)term survival.In our findings,high proportions of the above three different LSC subtypes were all significantly characterized with low complete remission(CR)rate,high relapse/refractory rate,poor overall survival(OS),frequent FLT3^(-)ITD mutation,the high level of regulatory T cells(Treg)and monocytic myeloid^(-)derived suppressor cells(M^(-)MDSC).However,there was no significant statistical difference in all kinds of other clinical performance among the three different LSC groups.It was demonstrated that CD34^(+)CD38^(-)subpopulation without CD123 and CD44 might be held responsible for LSC and correlated with an imbalance of immune cell subsets in AML.

Targeting metabolic vulnerabilities to overcome resistance to therapy in acute myeloid leukemia

Malignant hematopoietic cells gain metabolic plasticity, reorganize anabolic mechanisms to improve anabolic output and prevent oxidative damage, and bypass cell cycle checkpoints, eventually outcompeting normal hematopoietic cells. Current therapeutic strategies of acute myeloid leukemia (AML) are based on prognostic stratification that includes mutation profile as the closest surrogate to disease biology. Clinical efficacy of targeted therapies, e.g., agents targeting mutant FMS-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1 or 2, are mostly limited to the presence of relevant mutations. Recent studies have not only demonstrated that specific mutations in AML create metabolic vulnerabilities but also highlighted the efficacy of targeting metabolic vulnerabilities in combination with inhibitors of these mutations. Therefore, delineating the functional relationships between genetic stratification, metabolic dependencies, and response to specific inhibitors of these vulnerabilities is crucial for identifying more effective therapeutic regimens, understanding resistance mechanisms, and identifying early response markers, ultimately improving the likelihood of cure. In addition, metabolic changes occurring in the tumor microenvironment have also been reported as therapeutic targets. The metabolic profiles of leukemia stem cells (LSCs) differ, and relapsed/refractory LSCs switch to alternative metabolic pathways, fueling oxidative phosphorylation (OXPHOS), rendering them therapeutically resistant. In this review, we discuss the role of cancer metabolic pathways that contribute to the metabolic plasticity of AML and confer resistance to standard therapy;we also highlight the latest promising developments in the field in translating these important findings to the clinic and discuss the tumor microenvironment that supports metabolic plasticity and interplay with AML cells.

Effect of Compound Zhebei Granule(复方浙贝颗粒)Combined with Chemotherapy on Surface Markers of Leukemia Stem Cell in Patients with Acute Myeloid Leukemia

Objective： To observe the effects of Compound Zhebei Granule （复方浙贝颗粒, CZBG） combined with chemotherapy on surface markers of leukemia stem cell （LSC） in the bone marrow of patients with acute myeloid leukemia （AML）. Methods： Seventy-eight patients with AML received bone marrow aspiration and the percentages of CD34＋CD123＋ and CD33＋CD123＋ cells were tested using flow cytometry method. A total of 24 refractory or relapsed AML patients were enrolled and treated with one cycle of standard chemotherapy combined with CZBG. Bone marrow samples were obtained before and after treatment, and the percentages of CD34＋CD123~ and CD33＋CD123＋ cells were examined by flow cytometry. Results： Compared with refractory or relapsed AML patients, patients achieved remission had a significant lower percentage of CD34＋CD123＋ cells （P〈0.01） and CD33＋CD123＋ cells （P〈0.01）, indicating that controlling the LSC percentage may be important for patients with AML to achieve sustainable remission. Compared with those before treatment, the expression levels of CD34＋CD123＋ were significantly decreased after CZBG combined with chemotherapy treatment （P〈0.01）. The percentages of CD34＋CD123＋ cells and CD33＋CD123＋ in patients achieving complete remission after CZBG combined with chemotherapy treatment were both significantly lower than those in patients with non- remission （P〈0.01）. Conclusion： CZBG combining chemotherapy could reduce the percentages of CD34＋CD123＋ and CD33＋CD123＋ LSC, which might improve the clinical efficacy of refractory or relapsed AML.

Allo-hematopoietic stem cell transplantation is a potential treatment for a patient with a combined disorder of hereditary spherocytosis

Both human hereditary spherocytosis （HS） and chronic myelogenous leukemia （CML） are life threatening. Herein we have reported the case of a woman with a combined disorder of HS and CML who underwent the matched sibling allogeneic stem cell transplantation. The complete donor erythroid cells were obtained. The red blood cell counts significantly improved throughout life comparing with pre-hematopoietic stem cell transplantation （HSCT）. Reticulocyte counts normalized, and BCR-ABL was cleared away. The total bilirubin level was also corrected in this recipient. Our case is a rare example with a combined disorder of HS and CML following allogeneic stem cell transplantation. HS was not a contraindication for patient in the matched sibling transplant setting.