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 chemotherapyinduced 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 SDF1/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.

Identification and targeting leukemia stem cells: The path to the cure for acute myeloid leukemia

Accumulating evidence support the notion that acute myeloid leukemia(AML) is organized in a hierarchical system, originating from a special proportion of leukemia stem cells(LSC). Similar to their normal counterpart, hematopoietic stem cells(HSC), LSC possess selfrenewal capacity and are responsible for the continued growth and proliferation of the bulk of leukemia cells in the blood and bone marrow. It is believed that LSC are also the root cause for the treatment failure and relapse of AML because LSC are often resistant to chemotherapy. In the past decade, we have made significant advancement in identification and understanding the molecular biology of LSC, but it remains a daunting task to specifically targeting LSC, while sparing normalHSC. In this review, we will first provide a historical overview of the discovery of LSC, followed by a summary of identification and separation of LSC by either cell surface markers or functional assays. Next, the review will focus on the current, various strategies for eradicating LSC. Finally, we will highlight future directions and challenges ahead of our ultimate goal for the cure of AML by targeting LSC.

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

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 p LKB1 and Sirtuin 1(SIRT1), a regulator of p LKB1, were measured in CD34+CD38– KG1 a cells treated with resveratrol(40 μmol/L) or not by Western blotting. Senescence-related factors were examined, including p21 m RNA tested by real-time PCR, cell morphology by senescence-associated β-galactosidase(SA-β-gal) staining, cell proliferation by MTT assay and cell cycle by flow cytometry. Besides, apoptosis was flow cytometrically determined. The results showed that p LKB1 was highly expressed in CD34+CD38– KG1 a cells, and resveratrol, which could downregulate p LKB1 through activation of SIRT1, induced senescence and apoptosis of CD34+CD38– KG1 a cells. It was concluded that resveratrol-downregulated p LKB1 is involved 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.

Targeting leukemia stem cells:The new goal of therapy in adult acute myeloid leukemia

The most popular view of hematopoietic cell lineage organization is that of complex reactive or adaptative systems.Leukemia contains a subpopulation of cells that display characteristics of stem cells.These cells maintain tumor growth.The properties of leukemia stem cells indicate that current conventional chemotherapy, directed against the bulk of the tumor,will not be effective.Leukemia stem cells are quiescent and do not respond to cell cycle-specific cytotoxic agents used to treat leukemia and thus contribute to treatment failure. New strategies are required that specifically target this malignant stem cell population.

A novel genetic screen for leukemia stem cell immortalization genes

Leukemia,like many other cancers,is thought to arise from a small population of stem cells that have the capacity to self-renewal extensively and to initiate,sustain or regenerate the disease.Elimination of the leukemia stem cells(LSCs) will likely be essential,and probably sufficient,for curing this disease.Recent studies have shown that LSCs can be derived from early hematopoietic progenitors as well as more differentiated derivatives;the key feature being these cells have acquired an increased proliferative capacity and the ability to self-renew extensively.Genes that make this possible are attractive drug targets for treating leukemia.In our laboratory we have developed a novel in vitro genetic screen that uses retroviral insertional mutagenesis as a tool for identifying genes that are able to convert both normal hematopoietic progenitors and committed myeloid progenitor cells into cells that resemble LSCs.Interestingly,the genes identified in this screen are either known human leukemia genes or genes thought to regulate them?Recently,insertional mutations in these same genes were identified in the blood of X-linked granulomatous disease patients following retroviral gene therapy and likely reinforced the therapeutic efficacy in this trial.The insertional mutations were associated with a 3-4 expansion in the myeloid compartment of gene-corrected patients,indicating that mutations in these genes also have an effect on the self-renewal of human hematopoietic cells.Currently,we are determining whether this screen can be extended to other cells types(i.e.,epithelial cells) and whether we can use this information to develop better methods for killing LSCs.

Bone marrow microenvironment: The guardian of leukemia stem cells

Bone marrow microenvironment (BMM) is the main sanctuary of leukemic stem cells (LSCs) and protects these cells against conventional therapies. However, it may open up an opportunity to target LSCs by breaking the close connection between LSCs and the BMM. The elimination of LSCs is of high importance, since they follow cancer stem cell theory as a part of this population. Based on cancer stem cell theory, a cell with stem cell-like features stands at the apex of the hierarchy and produces a heterogeneous population and governs the disease. Secretion of cytokines, chemokines, and extracellular vesicles, whether through autocrine or paracrine mechanisms by activation of downstream signaling pathways in LSCs, favors their persistence and makes the BMM less hospitable for normal stem cells. While all details about the interactions of the BMM and LSCs remain to be elucidated, some clinical trials have been designed to limit these reciprocal interactions to cure leukemia more effectively. In this review, we focus on chronic myeloid leukemia and acute myeloid leukemia LSCs and their milieu in the bone marrow, how to segregate them from the normal compartment, and finally the possible ways to eliminate these cells.

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.

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.

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 nonremission(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.

Allogeneic hematopoietic stem cell transplantation for patients with acute leukemia

Objective: The purposes of this study were to assess the efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) for acute leukemia (AL) and analyze the factors affecting the prognosis of these patients. Methods: The clinical and follow-up data of 93 AL patients (median age, 30 years) undergoing allogeneic HSCT in Xiangya Hospital over the past 12 years were collected, and the potential factors affecting the efficacy and prognosis of allogeneic HSCT patients were determined. Results: Hematopoietic reconstitution was achieved in 90 patients. At the last follow-up, the incidences of severe acute graft versus host disease (aGvHD) and extensive chronic GvHD (cGvHD) were 14.0% and 20.0%, the 3-year cumulative incidence of transplantation related mortality (TRM) and relapse rate were 16.8%±6.1% and 21.3%±6.7%, and the estimated 3-year overall survival (OS) and disease-free survival (DFS) of the patients were 64.6%±5.4% and 56.5%±5.5%, respectively. Univariate analysis indicated that age older than 40 years, HLA mismatch, and severe lung infection within the first 100 days after transplantation were risk factors for severe aGvHD, age older than 40 years, HLA mismatch, severe lung infection within the first 100 days after transplantation, and severe aGvHD were risk factors for TRM, high-risk AL and lack of cGvHD were risk factors for relapse (all P<0.05). Survival estimation showed that HLA mismatch, severe lung infection occurring within the first 100 days post-transplantation, high-risk AL severe aGvHD and lack of cGvHD were risk factors associated with poor prognosis (all P<0.05). Further multivariate analyses revealed that severe lung infection within the first 100 days post-transplantation, severe aGvHD and lack of cGvHD were independent risk factors for unfavorable outcomes (all P<0.05). Conclusions: Allogeneic HSCT can improve the DFS of AL patients, and severe lung infection within the first 100 days post-transplantation, severe aGvHD and lack of cGvHD are independent risk factors affecting the prognosis.

Leukemia stem cells in drug resistance and metastasis

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NRS2002 assesses nutritional status of leukemia patients undergoing hematopoietic stem cell transplantation

Objective:To discuss whether nutritional risk screening 2002(NRS2002) is appropriate for nutritional risk screening for leukemia patients before and after hematopoietic stem cell transplantation(HSCT),and whether there are risk differences in other conditions,such as age,gender and matching degree;to find the methods and indicators of nutritional risk screening for these patients before and after HSCT,in order to give timely intervention to guarantee the successful completion of the entire transplantation process.Methods:Nutritional risk of 99 leukemia patients was screened with NRS2002 before and after HSCT.The χ2 test was applied to compare the risk differences between groups such as age,gender and matching degree,while the differences of other enumeration data,such as recent(1-3 months) weight loss,reduced food intake within one week and BMI,were compared by continuity correction.Results:Of the 99 leukemia patients,22 cases(22.2%) had nutritional risk before HSCT,while all patients had nutritional risk after HSCT;there is no significant difference in nutritional risk between male and female,and patients of less than 30 years old,not-full matched,recent(1-3 months) weight loss,reduced food intake within a week or BMI <18.5 were more likely to have nutritional risk;and 77 cases(77.8%) had weight loss,among which 49 patients(63.6%) had more than 5% weight loss within one month.Conclusions:This study showed that leukemia patients should receive the nutritional risk screening conventionally before and after HSCT,and NRS2002 was only appropriate for nutritional risk screening before HSCT.More attention should be paid to the patients less than 30 years old or not-full matched.Weight change was one of the important nutritional indicators for patients after HSCT.

Mobilization of CD34^+CD38^- hematopoietic stem cells after priming in acute myeloid leukemia

AIM: To evaluate quantitatively and qualitatively the different CD34+cell subsets after priming by chemotherapy granulocyte colony-stimulating factor(± G-CSF)in patients with acute myeloid leukemia.METHODS: Peripheral blood and bone marrow sampleswere harvested in 8 acute myeloid leukemia patients during and after induction chemotherapy. The CD34/CD38 cell profile was analyzed by multi-parameter flow cytometry. Adhesion profile was made using CXC chemokine receptor 4(CXCR4)(CD184), VLA-4(CD49d/CD29) and CD47.RESULTS: Chemotherapy ± G-CSF mobilized immature cells(CD34+CD38 population), while the more mature cells(CD34+CD38lowand CD34+CD38+populations) decreased progressively after treatment. Circulating CD34+cells tended to be more sensitive to chemotherapy after priming with G-CSF. CD34+cell mobilization was correlated with a gradual increase in CXCR4 and CD47expression, suggesting a role in cell protection and the capacity of homing back to the marrow.CONCLUSION: Chemotherapy ± G-CSF mobilizes into the circulation CD34+bone marrow cells, of which, the immature CD34+CD38-cell population. Further manipulations of these interactions may be a means with which to control the trafficking of leukemia stem cells to improve patients' outcomes.

Murine models based on acute myeloid leukemia-initiating stem cells xenografting

Acute myeloid leukemia(AML) is an aggressive malignant disease defined by abnormal expansion of myeloid blasts. Despite recent advances in understanding AML pathogenesis and identifying their molecular subtypes based on somatic mutations, AML is still characterized by poor outcomes, with a 5-year survival rate of only 30%-40%, the majority of the patients dying due to AML relapse. Leukemia stem cells(LSC) are considered to be at the root of chemotherapeutic resistance and AML relapse. Although numerous studies have tried to better characterize LSCs in terms of surface and molecular markers, a specific marker of LSC has not been found, and still the most universally accepted phenotypic signature remains the surface antigens CD34+CD38- that is shared with normal hematopoietic stem cells. Animal models provides the means to investigate the factors responsible for leukemic transformation, the intrinsic differences between secondary post-myeloproliferative neoplasm AML and de novo AML, especially the signaling pathways involved in inflammation and hematopoiesis. However, AML proved to be one of the hematological malignancies that is difficult to engraft even in the most immunodeficient mice strains, and numerous ongoing attempts are focused to develop "humanized mice" that can support the engraftment of LSC. This present review is aiming to in-troduce the field of AML pathogenesis and the concept of LSC, to present the current knowledge on leukemic blasts surface markers and recent attempts to develop best AML animal models.

NF-κB promotes the stem-like properties of leukemia cells by activation of LIN28B

AIM To examine whether nuclear factor kappa B(NF-κB) activity regulates LIN28 B expression and their roles in leukemia stem cell(LSC)-like properties. METHODS We used pharmacological inhibitor and cell viability assays to examine the relation between NF-κB and LIN28 B. Western blot and q RT-PCR was employed to determine their protein and m RNA levels. Luciferase reporter was constructed and applied to explore the transcriptional regulation of LIN28 B. We manipulated LIN28 B level in acute myeloid leukemia(AML) cells and investigated LSC-like properties with colony forming and serial replating assays. RESULTS This study revealed the relationship between NF-κB and LIN28 B in AML cells through drug inhibition and overexpression experiments. Notably,inhibition of NF-κB by pharmacological inhibitors reduced LIN28 B expression and decreased cell proliferation. We demonstrated that NF-κB binds to the-819 to-811 region of LIN28 B promoter,and transcriptionally regulates LIN28 B expression. LIN28 B protein was significantly elevated in NFκB1 transfected cells compared to vector control. Importantly,ectopic expression of LIN28 B partially rescued the self-renewal capacity impaired by pharmacological inhibition of NF-κB activity. CONCLUSION These results uncover a regulatory signaling,NF-κB/LIN28 B,which plays a pivotal role in leukemia stem cell-like properties and it could serve as a promising intervening target for effective treatment of AML disease.

Decitabine for Relapsed Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation

Relapse after allogeneic hematopoietic stem cell transplantation(allo-HSCT) remains a main question on treatment failure. Current strategies for management that usually include salvage chemotherapy, donor lymphocytic infusion and second transplantation. Our study assessed the efficacy of decitabine(DAC) for treating patients with acute lymphoblastic leukemia(ALL) who relapsed after allogeneic hematopoietic stem cell transplantation(allo-HSCT). We retrospectively analyzed the outcomes of 12 patients with relapsed ALL after allo-HSCT who received DAC therapy. Nine patients received DAC combined with chemotherapy and donor stem cell infusion, and 3 patients received single-agent DAC. Ten of the 12 patients achieved complete remission(CR), 1 achieved a partial remission(PR), and 1 had no response(NR) after treatment at the latest follow-up(LFU), the median survival was 11.2 months(range, 3.8–34, 7 months). The 1-and 2-year overall survival(OS) rates were 50%(6/12) and 25%(3/12), respectively. Five patients were still alive; 4 had maintained CR and 1 was alive with disease. Patients with Philadelphia chromosome-positive ALL had higher survival rate than patients with Philadelphia chromosome-negative ALL(57.1% vs. 20%). No aggravated flares of graft-versus-host disease(GVHD) were observed during DAC treatment. Therefore, DAC may be a promising therapeutic agent for ALL recurrence after allo-HSCT.

A comparison of flow cytometry detection of minimal residual disease and chimerism kinetics in chronic lymphocytic leukemia patients after allogeneic hematopoietic stem cell transplantation

Determination of minimal residual disease (MRD) remains crucial for the follow-up after therapy in chronic lymphocytic leukemia (CLL) patients. Chimerism was assessed by short tandem repeat (STR)-PCR and single nucleotide polymorphisms (SNP)-PCR, and MRD by a multicolor flow cytometric approach in 12 consecutive patients with CLL after they received allogeneic stem cell transplantation (SCT). Overall, 11 patients achieved MRD flow negativity [10 had full donor chimerism (FDC) and one had mixed chimerism (MC)]. Only one patient remained with MRD flow positivity and displayed MC. Fifty-six samples were concomitantly studied by both chimerism and MRD flow. A significant correlation was observed between MRD flow data and chimerism in both PB and BM by using a mixed effect linear regression (p < 0.001). Flow cytometry approach of MRD can be easily combined with chimerism during the follow-up post-allogeneic SCT. Both techniques appeared complementary for guiding post-transplant immunomodulation.

Donor-Derived CD19-Targeted T Cell Infusion Eliminates B Cell Acute Lymphoblastic Leukemia Minimal Residual Disease with No Response to Donor Lymphocytes after Allogeneic Hematopoietic Stem Cell Transplantation

Leukemia relapse is still the leading cause of treatment failure after allogeneic hematopoietic stem cell transplantation (allo-HSCT) for B cell acute lymphoblastic leukemia (B-ALL). Relapsed patients with BALL after allo-HSCT have a very short median survival. Minimal residual disease (MRD) is predictive of forthcoming hematological relapse after hematopoietic stem cell transplantation (HSCT);furthermore, eliminating MRD effectively prevents relapse. Donor lymphoblastic infusion (DLI) is the main established approach to treat B-ALL with MRD after allo-HSCT. However, about one-third of patients with MRD are non-responsive to DLI and their prognosis worsens. Although donor-derived cluster of differentiation (CD)19-directed chimeric antigen receptor-modified (CAR) T cells (CART19s) can potentially cure leukemia, the efficiency and safety of infusions with these cells have not yet been investigated in patients with MRD after HSCT. Between September 2014 and February 2018, six patients each received one or more infusions of CART19s from HSCT donors. Five (83.33%) achieved MRD-negative remission, and one case was not responsive to the administration of CAR T cells. Three of the six patients are currently alive without leukemia. No patient developed acute graft-versus-host disease (aGVHD), and no patient died of cytokine release syndrome. Donor-derived CAR T cell infusions seem to be an effective and safe intervention for patients with MRD in B-ALL after allo-HSCT and for those who were not responsive to DLI.