Normal Bone Marrow Stem Cells Research Articles

Protein Structure Inspired Discovery of a Novel Inducer of Anoikis in Human Melanoma.

Drug discovery historically starts with an established function, either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function, we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds, we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures, we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast, prostate, colon, or lung cancer, and for toxicity on normal human bone marrow stem cells, both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound, Dxr2-017, exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays, Dxr2-017 had an IC50 of 12 nM against melanoma cells, while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis, a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable.

Noggin promotes osteogenesis in human adipose-derived mesenchymal stem cells via FGFR2/Src/Akt and ERK signaling pathway

The balance between Noggin and bone morphogenetic proteins (BMPs) is important during early development and skeletal regenerative therapies. Noggin binds BMPs in the extracellular space, thereby preventing BMP signaling. However, Noggin may affect cell response not necessarily through the modulation of BMP signaling, raising the possibility of direct Noggin signaling through yet unspecified receptors. Here we show that in osteogenic cultures of adipose-derived stem cells (ASCs), Noggin activates fibroblast growth factor receptors (FGFRs), Src/Akt and ERK kinases, and it stabilizes TAZ proteins in the presence of dexamethasone. Overall, this leads ASCs to increased expression of osteogenic markers and robust mineral deposition. Our results also indicate that Noggin can induce osteogenic genes expression in normal human bone marrow stem cells and alkaline phosphatase activity in normal human dental pulp stem cells. Besides, Noggin can specifically activate FGFR2 in osteosarcoma cells. We believe our findings open new research avenues to further explore the involvement of Noggin in cell fate modulation by FGFR2/Src/Akt/ERK signaling and potential applications of Noggin in bone regenerative therapies.

Reducing genotoxicity of cyclophosphamide by hydro-coholic leaves extract of allium porrum in normal mouse bone marrow stem cells

This paper evaluates percentage of chromosomal aberration (CA )and mitotic index (MI) in mice bone marrow as a method for assessing the ability of hydro-ethanolic leaves extract of leek ( Allium Porrum) to reduce genotoxicity of Cyclophosphamide (CP) . Mice were allocated in to 4 groups, two negative controls ,the first group receive ( 0.2 ml) D.W. and the second group was treated with (0.1ml) DMSO, the positive control group, (CP) 40mg/kg was given single intraperitoneally dose for 24 hr , while the treatment groups were given different doses of extract (75, 150,200and 250 mg/kg ) for 5 consecutive days and the same dose of CP . Experimental data was analyzed using (one –way ANOVA). The results demonstrated the intermediate dose (150 mg/kg) of extract increased in MI significantly and reduced CA (P<0.01) significantly .This study concluded that Allium porrum leaves were able to reduce genotoxicity of CP and protect normal proliferating cells in bone marrow from the effect of CP.

Delineation of target expression profiles in CD34+/CD38− and CD34+/CD38+ stem and progenitor cells in AML and CML

AbstractIn an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38− and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38− and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38− LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably expressed “aberrant” membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication–mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38− LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.

Catechins-Modified Selenium-Doped Hydroxyapatite Nanomaterials for Improved Osteosarcoma Therapy Through Generation of Reactive Oxygen Species.

Osteosarcoma is the most common bone cancer with limited therapeutic options. It can be treated by selenium-doped hydroxyapatite owing to its known antitumor potential. However, a high concentration of Se is toxic toward normal and stem cells whereas its low concentration cannot effectively remove cancer cells. Therefore, the current study was aimed to improve the anticancer activity of Se-HAp nanoparticles through catechins (CC) modification owing to their high cancer therapeutic value. The sequentially developed catechins modified Se-HAp nanocomposites (CC/Se-HAp) were characterized for various physico-chemical properties and antitumor activity. Structural analysis showed the synthesis of small rod-like single phase HAp nanoparticles (60 ± 15 nm), which effectively interacted with Se and catechins and formed agglomerated structures. TEM analysis showed the internalization and degradation of CC/Se-HAp nanomaterials within MNNG/HOS cells through a non-specific endocytosis process. Cell toxicity analysis showed that catechins modification improved the antitumor activity of Se-HAp nanocomposites by inducing apoptosis of human osteosarcoma MNNG/HOS cell lines, through generation of reactive oxygen species (ROS) which in turn activated the caspase-3 pathway, without significantly affecting the growth of human normal bone marrow stem cells (hBMSCs). qPCR and western blot analyses revealed that casp3, p53, and bax genes were significantly upregulated while cox-2 and PTK-2 were slightly downregulated as compared to control in CC/Se-HAp-treated MNNG/HOS cell lines. The current study of combining natural biomaterial (i.e., catechins) with Se and HAp, can prove to be an effective therapeutic approach for bone cancer therapy.

Potential Involvement of BIRC5 in Maintaining Pluripotency and Cell Differentiation of Human Stem Cells.

The BIRC5 gene encodes a survivin protein belonging to class III of inhibitors of apoptosis, IAP. This protein serves a dual role. First, it regulates cell death, and second, it is an important regulator of mitosis progression, although its physiological regulatory function has not been fully understood. Many studies have shown and confirmed that survivin is practically absent in mature tissues in nature, while its overexpression has been reported in many cancerous tissues. There is little information about the significance of BIRC5 expression in normal adult human stem cells. This paper presents the study and analysis of survivin expression at the transcription level using qPCR method, in hematopoietic stem cells from peripheral blood mobilized with a granulocyte growth factor, adherent cells derived from the umbilical cord, and normal bone marrow stem cells. The expression of this gene was also examined in the blood of normal healthy individuals. The results of the analysis have shown that the more mature the cells are, the lower the expression of the BIRC5 gene is. The lowest expression has been found in peripheral blood cells, while the highest in normal bone marrow cells. The more the CD34+ and CD105 cells in the tested material are, the higher the BIRC5 expression is. Stem cells from cell culture show higher BIRC5 expression. The study confirms the involvement of BIRC5 from the IAP family in many physiological processes apart from apoptosis inhibition. The possible effect of BIRC5 on cell proliferation; involvement in cell cycle, cell differentiation, survival, and maintenance of stem cells; and the possible effect of IAP on the antineoplastic properties of mesenchymal stem cells have been demonstrated. Our research suggests that BIRC5 may be responsible for the condition of stem cell pluripotency and its high expression may also be responsible for the dedifferentiation of tumor cells.

Comparison of the Transcriptomic Signature of Pediatric Vs. Adult CML and Normal Bone Marrow Stem Cells

IntroductionPediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with myeloid leukemia and 2 to 9% of all pediatric leukemias. Prior to the discovery of tyrosine kinase inhibitors (TKI) such as imatinib, stem cell transplantation was the only curative treatment for both adults and children with CML. However, due to the small numbers of patients, standardized treatment approaches for pediatric CML have not been established. There are several unique characteristics of CML diagnosed in children and adolescents, and young adults (AYA; 16-29 years), compared to adults. Children and AYA with CML present with a higher white blood count and have larger spleens, higher peripheral blast counts, and lower hemoglobin levels, suggesting that the biology of pediatric CML is different than adult CML. In addition, potential side effects of TKIs unique to pediatric CML patients include impaired bone growth, fertility and immune function, however none have been extensively studied. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that are absent in adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells.MethodsCD34+ cells were isolated from pediatric CML (n=7), adult CML (n=8), pediatric normal (n=2) and adult normal (n=3) bone marrow samples. Total RNA was isolated from cells, and then cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. We aligned reads using the HISAT2 alignment software, and mapped to genes with HT-Seq. We removed genes that had zero reads across all the samples, resulting in a set of 4,696 genes that were detected in one or more samples. In case of technical replicates, we used mean of replicates. We performed three differential expression comparisons with edgeR: (1) Pediatric CML vs Adult CML, (2) Adult CML vs Adult Normal, and (3) Pediatric CML vs Pediatric Normal. We used a False Discovery Rate (FDR) of £ 20% and absolute log2 fold-change ³ 1 for selecting differentially expressed genes in each comparison. We used Fisher's exact test to identify significant KEGG pathways for the differentially expressed genes in each comparison.ResultsPediatric CML vs Adult CMLWe found 24 differentially expressed genes (15 over- and 9 under-expressed). Though no pathway was found to be significant at the false discovery rate (FDR) £ 20%, we identified a number of sub-pathways that are relevant. For example, the Chemokine Signaling pathway shows at the top of the list (ordered by raw p-value) because of two genes, XCR1 and HCK, associated with VEGF and MAPK pathways involved in cell proliferation, angiogenesis, DNA repair, and cancer pathogenesis.Adult CML vs Adult NormalWe found 60 genes (30 over- and 30 under-expressed) differentially expressed when comparing adult CML patients to normal adults. Ten genes overlapped with 24 genes we identified when comparing pediatric and adult CML patients. We found 11 pathways as significant at FDR £ 10%. Multiple pathways, including Cell adhesion, allograft rejection, Graft versus Host Disease, and Type I diabetes pathways, showed downregulation of MHC, with subsequent downstream reduction in expression of apoptosis-related genes. The IL-17 pathway makes sense, as MAPK, well-known to be associated with various cancers, is down-regulated. Lastly, in the NK pathway the gene DAP12 is up-regulated. This gene is known as a tyrosine kinase binding protein, and although tyrosine kinase inhibitors are the standard treatment for CML, the role of DAP12 in relation to leukemia has not yet been described.Pediatric CML vs Pediatric NormalWe found 509 genes (350 over- and 159 under-expressed) differentially expressed in pediatric CML patients compared to normal. Interestingly, transcriptional regulators are differentially enriched in the hematopoietic stem cell differentiation function group including GATA1, GATA2, KLF1 and KLF2. RFC is down-regulated. RFC is a mismatch repair gene known to be involved in colorectal cancer. Many of the significant pathways are involved in glucose and fatty acid metabolism.Our pilot study identified novel molecular features of pediatric CML bone marrow stem cells, providing new insights into the novel biomarkers and pathogenesis of pediatric CML. DisclosuresGotlib:Blueprint Medicines: Consultancy, Honoraria, Research Funding; Promedior: Research Funding; Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Study of TIM-3 in Pathogenesis of Myelodysplastic Syndrome

Objective The biological characteristics of TIM3+ stem cells were further explored from the aspects of cell morphology, cytogenetics, and animal models, providing a theoretical basis for the early identification of malignant MDS clones.Methods The subjects of the study were 44 newly diagnosed MDS patients who were admitted to the General Hospital of Tianjin Medical University from July 2016 to February 2018 and 17 normal controls. TIM3+ and TIM3-stem cells were sorted from the bone marrow of 10 unteated patients with MDS, CD34+ stem cells were sorted out from the bone marrow of four normal controls. We subjected them to Wright's staining, colony-forming unit culture, and fluorescence in situ hybridization (FISH) respectively. The TIM3+ , TIM3- stem cells, and MDSC in the bone marrow of 8 unteated patients with MDS were sorted. TIM-3 inhibitors, Gal-9 inhibitors, and both of the two inhibitors were added into the three kinds of cells separately and they were cultured in groups. We examined the apoptosis of TIM3+ stem cells and TIM3- stem cells, and then analyzed the role of TIM-3/Gal-9 pathway in TIM3+ stem cells and MDSC in patients with MDS. We sorted TIM3+ , TIM3-stem cells, and MDSC from the bone marrow of 11 untreated patients with MDS and sorted CD34+ stem cells from the bone marrow of 5 normal controls. The above cells were transplanted into 32 NCG mice. 10-12 weeks later, the differentiation of human-derived cells was detected by flow cytometry.Results The TIM3+ and TIM3- stem cells were stained by Wright's stain respectively and observed under high magnification are same. The CFU assay showed that the average number of BFU-E formed by TIM3+ stem cells, TIM3- stem cells and normal bone marrow stem cells was (6 vs 11 vs 12), and the CFU-E was (3 vs 6 vs 9), CFU-GM was (5 vs 17 vs 23). The expression of Gal-9 on MDSC was significantly higher than that of normal controls (0.62±0.64% vs 0.22±0.18%, P<0.05). The proportion of apoptotic TIM3+ stem cells in the TIM3+ stem cells mixed MDSC group was significantly lower than that in the two inhibitor groups and that of apoptotic TIM3-stem cells in TIM3- mixed MDSC group [12.59(5.12, 31.24) vs 42.54(29.41, 58.39), 65.86(24.44, 99.43), P<0.05]. In the animal transplant model of our study, the expression rate of hCD45 in TIM3+ stem cells mixed MDSC group was significantly higher than that in TIM3+ , TIM3- stem cells group and normal control CD34+ stem cells group (4.99±5.47% vs 0.65±0.22%, 1.68±2.38%, 0.55±0.40%, P<0.05). The expression rate of hCD19 in TIM3+ stem cells group was significantly lower than that in TIM3-stem cells group and normal control CD34+ stem cells group (8.68±9.34% vs 27.46±18.06%, 37.38±16.14%, P<0.01); The expression rate of hCD19 in TIM3+ stem cells mixed MDSC group was significantly lower than that in TIM3-stem cells group and normal control CD34+ stem cells group (4.76±4.78% vs 27.46±18.06%, 37.38±16.14%, P<0.05). The expression rate of hCD33 in TIM3+ stem cells group was significantly lower than that in TIM3- stem cells group (11.34±14.70 % vs 22.45±8.73%, P<0.05). The expression of hCD33 in TIM3+ stem cells mixed MDSC group was significantly lower than that in TIM3-stem cells group (5.07±5.29% vs 22.45±8.73%, P<0.05).Conclusions(1) The TIM3+ and TIM3- stem cells of MDS patients are difficult to distinguish morphologically, but the colony forming ability of the two are obviously different, and the karyotype abnormalities are also different. Therefore the TIM3+ stem cells may be the earlier karyotype anomaly “malignant” stem cells.(2) The expression rate of Gal-9 on MDSC cells in bone marrow of MDS patients was significantly increased. The apoptotic rate of TIM3+ stem cells in TIM3+ stem cells and MDSC mixed culture group was significantly lower than that of TIM3- stem cells in TIM3-stem cells and MDSC mixed culture group. Blocking the TIM-3/Gal-9 pathway can inhibit the anti-apoptotic effect of MDSC on TIM3+ stem cells, providing a new target for the clearance of malignant clones in patients with MDS.(3) The number of human-derived cells detected was the largest. And MDSC can promote the expansion of TIM3+ stem cells. Human-derived cells in the TIM3+ stem cell group and the TIM3+ stem cell and MDSC mixed group cannot undergo normal myeloid and lymphoid differentiation as well as the TIM3- stem cell group or the normal control CD34+ stem cell group. It is speculated that TIM3+ stem cells may have clonal growth, while MDSC has a promoting effect. DisclosuresNo relevant conflicts of interest to declare.

Efficiency of Cell Therapy to GC-Induced ONFH: BMSCs with Dkk-1 Interference Is Not Superior to Unmodified BMSCs.

Glucocorticoid-induced osteonecrosis of the femoral head (ONFH) is a hip disorder, and it threatens patients who require megadose of steroid therapies. Nowadays, no valid therapies can reverse the development of GC-induced ONFH once it occurs. Stem cell therapy to GC-induced ONFH would be a promising choice. Although the pathogenesis of GC-induced ONFH is not yet fully clear, Dickkopf-1 (Dkk-1) upregulated by excessive GC use, which hinders the canonical Wnt pathway, could be an explanation. Thus, the aim of the present work lies in investigating the efficiency of the allograft bone marrow stem cells (BMSCs) with Dkk-1 interference in preventing the progression of the GC-induced ONFH. Lentivirus-meditated Dkk-1 RNAi was introduced into BMSCs which was exposed to dexamethasone (10−6 mol/L) in vitro. This interference blocked Dkk-1 overexpression by GC and afterwards prompted the transduction of Wnt/β-catenin in which the Runx2 and PPARγ were upregulated and downregulated, respectively. Thus, the osteogenesis was promoted while adipogenesis was inhibited. In vivo, GC-induced ONFH rats were treated by allotransplantation of BMSCs with Dkk-1 interference, and the progression of the disease was prevented. However, the effects were not significantly superior to treatment with nongenetically modified or normal BMSCs.

DPPIV (CD26) as a novel stem cell marker in Ph+ chronic myeloid leukaemia.

The concept of leukaemic stem cells (LSCs) has been developed to explain the complex cellular hierarchy and biology of leukaemias and to screen for pivotal targets that can be employed to improve drug therapies through LSC eradication in these patients. Some of the newly discovered LSC markers seem to be expressed in a disease-specific manner and may thus serve as major research tools and diagnostic parameters. A useful LSC marker in chronic myeloid leukaemia (CML) appears to be CD26, also known as dipeptidylpeptidase IV. Expression of CD26 is largely restricted to CD34(+) /CD38(-) LSCs in BCR/ABL1(+) CML, but is not found on LSCs in other myeloid or lymphoid neoplasms, with the exception of lymphoid blast crisis of CML, BCR/ABL1p210 + acute lymphoblastic leukaemia, and a very few cases of acute myeloid leukaemia. Moreover, CD26 usually is not expressed on normal bone marrow (BM) stem cells. Functionally, CD26 is a cytokine-targeting surface enzyme that may facilitate the mobilization of LSCs from the BM niche. In this article, we review our current knowledge about the biology and function of CD26 on CML LSCs and discuss the diagnostic potential of this new LSC marker in clinical haematology.

Difference in glycerol levels between leukemia and normal bone marrow stem cells.

Aquaglyceroporin 9 (AQP9) is considered to be involved in numerous types of carcinogenic processes, particularly in liver carcinoma. AQP9 expression is significantly decreased in the human hepatocellular carcinoma when compared with the non-tumourigenic liver, which leads to increased resistance to apoptosis. In addition, AQP9 is permeable to glycerol and urea. The involvement of AQP9 in leukemia has not been fully delineated. It is proposed that abnormal proliferation of hematopoietic stem cells (HSCs) contributes to leukemia carcinogenesis. Therefore, the present study aimed to investigate the possible roles of AQP9 in HSCs and its effect on the intracellular glycerol content. HSCs and non-HSCs (nHSCs) were isolated via magnetic-activated cell sorting and then subjected to flow cytometry for evaluation of purity. White blood cells (WBCs) were isolated from peripheral blood from healthy volunteers. Furthermore, AQP9 expression was examined at the mRNA and protein levels using western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The glycerol content of HSCs, nHSCs and WBCs was evaluated by ELISA. Finally, in order to observe the morphology of HSCs and nHSCs, a blood smear was conducted and the cells were observed with Wright-Giemsa staining. The results indicated that the glycerol content in the HSCs was markedly greater than that in the nHSCs. AQP9 mRNA and protein expression was not detected in the HSCs and nHSCs, but was identified in the WBCs. Moreover, the HSC morphological characteristics included round or oval cells with round, slightly oval or irregularly shaped nuclei. Additionally, the nuclei occupied almost the entire cell, were located in the middle or were biased toward one side, and were stained light purple or red. Overall, our results indicated that intracellular glycerol is involved in HSC proliferation, despite the fact that glycerol is not mediated by AQP9. Hence, our findings may be useful in further understanding the mechanism of leukemia carcinogenesis, and these data may be valuable in developing future therapeutic strategies.

Phenotyping Of Leukemic Stem Cells In Ph+ ALL and Ph- ALL Reveals Unique Profiles Of Markers and Targets In Distinct Disease Variants

Acute lymphoblastic leukemia (ALL) is a life-threatening hematopoietic neoplasm characterized by abnormal growth and accumulation of lymphatic blast cells in various hematopoietic tissues. In a substantial number of patients, the Philadelphia (Ph) chromosome and the related oncoprotein BCR/ABL, are detectable. Despite recent advances in the management and therapy of patients with ALL, including the use of BCR/ABL1 tyrosine kinase inhibitors (TKI), the prognosis is still poor. Therefore, several attempts have been made to improve targeted treatment approaches in ALL. One strategy is to identify markers and targets expressed on leukemic stem cells (LSC) in these patients and to apply targeted drugs in order to eliminate LSC. In patients with Ph+ ALL, the leukemia-initiating cell-population is considered to reside within a CD34+/CD38- fraction of the clone. In the present study, we examined the expression of various stem cell markers and target antigens in CD34+/CD38- stem cells and in more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL (n=12), Ph- ALL (n=13), Ph+ CML (n=20), and in control bone marrow (BM) samples (unexplained cytopenia, n=10). Surface expression of target antigens was analyzed by multicolor flow cytometry, and mRNA expression levels by qPCR. As assessed by flow cytometry, CD34+/CD38- cells were found to co-express CD19, the stem cell-homing receptor CD44, the Campath-1 antigen (CD52), AC133 (CD133), FLT3 (CD135), and CXCR4 (CD184) in all ALL patients examined. In a majority of the ALL patients tested (14/25), LSC also expressed Siglec-3 (CD33). In CML, LSC were found to express a similar profile of antigens, including CD33, CD44, CD52, CD133, CD135, and CXCR4, but these cells did not express CD19. In control BM samples, CD34+/CD38- cells expressed a similar phenotype, but the levels of CD33 and CD52 were lower compared to LSC in ALL and CML. The IL-1RAP was found to be expressed on LSC in patients with Ph+ CML and Ph+ ALL, but not on LSC in Ph- ALL or in normal BM stem cells. By contrast, the SCF receptor KIT (CD117) was found to be expressed on LSC in Ph+ CML but was hardly detectable on LSC in patients with Ph+ ALL or Ph- ALL. The IL-2RA (CD25) and the SDF-1-degrading surface enzyme dipeptidyl-peptidase IV (DPPIV=CD26) were expressed on LSC in patients with CML and in all patients with Ph+ ALL exhibiting BCR/ABL-p210, whereas in Ph+ ALL with BCR/ABL-p190, LSC variably expressed CD25, and did not express CD26. In patients with Ph- ALL and in the normal BM, CD34+/CD38- cells did not express CD25 or CD26. The target receptor CD20 was detectable on ALL LSC in 7/18 patients examined. All target receptors tested were also detectable on more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL and Ph- ALL. In consecutive studies, expression of target antigens was confirmed at the mRNA level by qPCR analyses of highly enriched ALL LSC. Finally, we were able to show that the CD52-targeting drug alemtuzumab induces rapid lysis of CD34+/CD38- ALL LSC in all patients examined (Figure). In summary, our data show that LSC in Ph+ ALL and Ph- ALL express a unique phenotype, including clinically relevant cytokine receptors and cell surface target antigens, including the Campath-1 antigen, CD52. In Ph+ ALL with BCR/ABL-p210, the phenotype of ALL LSC largely resembles the phenotype of LSC in Ph+ CML, confirming the close relationship and similar pathogenesis of these two types of leukemias. [Display omitted] Ficoll-isolated MNC of 4 patients with Ph+ ALL were incubated in control medium (Co) or in various concentrations of alemtuzumab (10-300 µg/ml) in RPMI 1640 medium in the presence of 30% human serum at 37°C for 1 hour. After washing, cells were stained with fluorochrome-conjugated mAb against CD34, CD38 and CD45 for 15 minutes. DAPI-staining was used to evaluate the percentage of viable cells. Cells were analysed using a FACSCanto II and FlowJo software. Results show the numbers of viable CD34+/CD38- cells and are expressed as percent of control (Co). Values represent the mean±S.D. of four independent experiments. Asterisk (*): p<0.05 compared to control. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

TIM-3, a Leukemia Stem Cell Marker, Plays a Role In Leukemic Transformation Through Autocrine Stimulatory Signaling By Its Ligand, Galectin-9

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs) (Kikushige et al, Cell Stem Cell, 2010). We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38- LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although CD34+CD38- normal bone marrow stem cells do not express TIM-3, TIM-3 is expressed in the CD34+CD38- population in MDS, and is further up-regulated with progression into leukemia. The average percentages of TIM-3+ cells in the CD34+CD38- population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is a type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. To understand the function of TIM-3, we investigated the interaction between TIM-3 and its ligand galectin-9 in AML LSCs. We found that AML patients showed significantly higher serum galectin-9 concentration than healthy individuals (healthy controls: 18.3+4.3 pg/ml, AML patients: 139.1+33.4 pg/ml, P<0.05). Unexpectedly, we found that leukemic cells expressed a high level of galectin-9 protein, as compared to other hematopoietic cells including T cells, B cells and monocytes. Using KASUMI-3 (TIM-3+ AML cell line) and primary AML samples, we confirmed that AML cells could secrete galectin-9 after TLR stimulation in vitro. Furthermore, microarray analysis demonstrated that TIM-3 stimulation by the physiological concentration of galectin-9 induced significant gene expression changes toward pro-survival axis including up-regulation of MCL-1, the important survival factor for HSCs and LSCs. These results collectively suggest that AML cells can produce and secrete galectin-9, and galectin-9 can bind and stimulate TIM-3-expressing AML cells including LSCs in an autocrine manner to support their survival or leukemia progression. Disclosures:Miyamoto:Kyushu University Hospital: Employment.

The Oncogenic Transcription Factor STAT5 Triggers Aberrant Expression Of CD25 (IL-2RA) In Neoplastic Stem Cells In Ph+ CML

Chronic myeloid leukemia (CML) is a stem cell neoplasm characterized by the Philadelphia (Ph) chromosome and the related oncoprotein, BCR/ABL. Despite the availability of novel BCR/ABL-targeting tyrosine kinase inhibitors (TKI), many patients relapse which may be due to resistance of neoplastic stem cells (NSC) against TKI. So far, little is known about specific markers and targets expressed on CML NSC. We here report that CML NSC express the interleukin-2R alpha-chain CD25 in a specific (aberrant) manner. Whereas normal bone marrow (BM) stem cells were found to express only low amounts or did not express CD25, CD34+/CD38− NSC in CML were found to express CD25 in almost all patients examined (31/33=95%), independent of the phase of disease. CML NSC were also found to express IL-1RAP, DPPIV (CD26), Siglec-3 (CD33), Campath-1 (CD52), KIT (CD117) and IL-3RA (CD123). We were also able to show that highly purified CD25+ NSC express BCR/ABL and engraft irradiated NOD-SCID-IL-2Rg-/- (NSG) mice with BCR/ABL+ cells, whereas CD34+/CD38-/CD25-/CD26- cells from the same patients failed to express BCR/ABL, and engrafted NSG mice with BCR/ABL-negative cells. To define signalling-molecules contributing to the expression of CD25 in CML NSC, we employed primary murine hematopoietic cells infected with bcr/abl-p210 in combination with a retrovirus encoding for STAT5A or STAT5B. Enforced expression of either STAT5A or STAT5B resulted in enhanced expression of CD25 in Lin-/Sca-1+/Kit+ mouse BM stem cells (LSK cells), regardless of the presence or absence of bcr/abl. In fact, the CD25-promoting effect of STAT5 was seen in normal stem cells as well as in bcr/abl-transformed LSK cells. Correspondingly, shRNA against STAT5 was found to downregulate expression of CD25 in the human CML cell line KU812. The BCR/ABL-targeting TKI imatinib, nilotinib and ponatinib were also found to inhibit expression of CD25 on KU812 cells. In primary CML NSC, ponatinib was found to induce marked suppression of CD25, whereas imatinib and nilotinib showed only weak effects. In consecutive experiments, we found that the MEK inhibitors RDEA119 and PD032509, the STAT5-targeting drug pimozide and the dual PI3 kinase/mTOR blocker BEZ235 all inhibit 3H-thymidine uptake and thus proliferation in KU812 cells. However, whereas RDEA119 and PD032509 as well as pimozide were found to downregulate expression of CD25 in KU812 cells, BEZ235 was found to even upregulate expression of CD25 on KU812 cells. We next applied an shRNA against CD25. The shRNA-induced knock-down of CD25 in KU812 resulted in an increased proliferative capacity, suggesting that CD25 may act as a potential tumor suppressor in CML stem- and progenitor cells. Together, our data show that CD25 is a novel functionally relevant marker-antigen and potential tumor suppressor of CML NSC. Expression of CD25 in CML cells appears to be triggered by MEK and STAT5 activity, and may be negatively regulated by the PI3 kinase/mTOR pathway. Whether modulation of CD25 can be exploited clinically and used as basis for the development of novel NSC-targeting treatment concepts in CML remains to be determined in future studies. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

In This Issue

HomeCirculation ResearchVol. 111, No. 1In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published22 Jun 2012https://doi.org/10.1161/RES.0b013e3182628bafCirculation Research. 2012;111:2Hyperhomocysteinemia and Vascular Inflammation (p 37)Genetically engineered mice created by Zhang et al reveal how hyperhomocysteinemia contributes to atherosclerosis.Download figureDownload PowerPointIt has been known for some time that hyperhomocysteinemia—a condition in which blood plasma levels of the amino acid homocysteine are significantly raised—is a risk factor for cardiovascular disease and stroke. How the condition contributes to cardiovascular problems has not been clear, partly because treatments aimed at lowering blood homocysteine in humans have given mixed results. To resolve the confusion, Zhang et al created genetically engineered, atherosclerosis-prone mice in which raising and lowering homocysteine levels could be tightly controlled by diet. They showed that hyperhomocysteinemia was not just a biomarker of cardiovascular disease but could indeed promote atherosclerosis. High homocysteine levels were associated with an increase in proinflammatory monocyte numbers in the blood, which in turn resulted in the accumulation of these cells in vessel walls and systemic vascular inflammation. The team also showed that lowering homocysteine levels, by increasing folate in the diet, could prevent both hyperhomocysteinemia-induced monocyte differentiation and atherosclerosis. The results suggest that a similar vitamin fix might be a preventative measure against cardiovascular disease in humans with hyperhomocysteinemia.Fibroblast Reprogramming by GMT Is Inefficient (p 50)Contrary to previous findings, Chen et al find it difficult to reprogram fibroblasts into cardiomyocytes.Download figureDownload PowerPointIn 2010, an article by Ieda et al appeared in Cell that claimed transfecting mouse fibroblasts with 3 transcription factors—Gata4, Mef2c, and Tbx5—could reprogram the cells to become spontaneously beating cardiomyocytes. Not only that, but the reprogrammed cells could survive when transplanted into mouse hearts in vivo. Given the immense potential of such a technique for future heart reparative therapies, Chen et al hoped to establish the methods in their own lab. The team successfully transfected mouse fibroblasts with the 3 factors, which were expressed robustly. However, signs that the cells were converting to cardiomyocytes were distinctly lacking. Some cardiac genes were induced in some cells, whereas many other important genes were not induced. Furthermore, even cells that did switch on certain cardiac genes remained morphologically indistinguishable from fibroblasts and exhibited no spontaneous beating activity. Transplanting the transfected cells into mouse hearts, in the hope that the in vivo environment would support transformation, was also unsuccessful. The authors say that much more work must be done to improve transformation efficiency by this strategy.Pim-1 Bone Marrow Cells Improve Cardiac Structure (p 77)Pim-1 gives bone marrow cells a boost to help fix injured hearts, reports Quijada et al.Download figureDownload PowerPointResearchers have analyzed a number of different stem and progenitor cell types for their abilities to mend injured heart tissues after myocardial infarction. The hope is that either the cells themselves will give rise to new myocardial tissue in the injured heart or that they will secrete factors that promote endogenous cells to do so. Bone marrow stem cells (BMCs) are believed to do the latter and are a popular choice for use in therapies because of the relative ease with which they are acquired. They have also been shown to be safe and relatively efficacious in promoting myocardial repair. However, the majority of BMCs transferred to injured hearts are rapidly lost. Therefore, researchers must find a way to make the cells stick around and prolong their beneficial effects. Quijada et al now suggest that BMCs genetically engineered to express Pim-1 might be just the solution. Pim-1 is a kinase that promotes cell proliferation and survival, and, when introduced into BMCs, it improved not only their survival in culture but also their persistence in injured mouse hearts. This persistence translated to better hemodynamic performance and increased anterior wall thickness in the injured hearts compared with hearts that received normal BMCs. Thus, boosting Pim-1 levels in BMCs might be a beneficial approach for enhancing the clinical efficacy of these cells. Previous Back to top Next FiguresReferencesRelatedDetails June 22, 2012Vol 111, Issue 1 Advertisement Article InformationMetrics © 2012 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e3182628baf Originally publishedJune 22, 2012 PDF download Advertisement

Abstract 844: Tumor cell detection in autologous stem cell harvests in patients with high risk neuroblastoma

Abstract Introduction The presence of minimal residual disease (MRD) detected by real-time quantitative (RQ)-PCR in autologous stem cell harvests (ASCH) in children with high risk neuroblastoma (NBL) seems to be associated with an unfavourable outcome. However, to date only small studies have been performed, which suffered from lack of specificity of the assay due to background amplification in normal bone marrow (BM) and peripheral blood stem cells (PBSC). We previously developed a new panel of specific neuroblastoma RQ-PCR markers for MRD testing. The goal of this study was to determine the incidence of harvest contamination using this panel of markers and to investigate whether reinfusion of contaminated harvests was associated with poor outcome. Methods In this single center study, RQ-PCR was performed with six NBL-specific markers: homeobox2b (PHOX2B), tyrosine hydroxylase (TH), dopamine decarbohydroxylase (DDC), growth associated protein 43 (GAP43), cholinergic receptor3 (CHRNA3), and dopamine beta-hydroxylase (DBH) on 40 BM and 38 PBSC harvests of 70 high risk patients. The prognostic impact of MRD in reinfused autologous harvests (n=50) was assessed using Kaplan-Meier plots and log-rank tests. Results NBL mRNA was detected in 42% (18/40) of BM harvests and in 13% (5/38) of PBSC harvests of which in total 50 harvests were reinfused. In reinfused patients, 30% (15/50) received a MRD positive harvest, which was associated with poor outcome (5-y-OS 20±11.8 versus 62.3%±8.8;p=0.02). This association was more pronounced in patients reinfused with BM harvests than in patients reinfused with PBSC harvests, since more BM harvests were MRD positive. Remarkably, overall there was no difference in survival between patients reinfused with PBSC or BM harvest (p=0.7). Conclusion The presence of NBL mRNA in stem cell harvests, as reliably measured by a panel of NBL specific-PCR targets, is significantly associated with an unfavourable outcome. This could either mean that the amount of tumor cells reinfused in the graft influences relapse rate or that the stem cell graft reflects remission status. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 844.

PHOX2B Is a Novel and Specific Marker for Minimal Residual Disease Testing in Neuroblastoma

Polymerase chain reaction (PCR)-based detection of minimal residual disease (MRD) in neuroblastoma can be used to monitor therapy response and to evaluate stem cell harvests. Commonly used PCR markers, tyrosine hydroxylase (TH) and GD2 synthase, have expression in normal tissues, thus limiting MRD detection. To identify a more specific MRD marker, we tested PHOX2B. To determine PHOX2B, TH, and GD2 synthase expression in normal tissues, it was measured by real-time quantitative PCR in samples of normal bone marrow (BM; n = 51), peripheral blood (PB; n = 37), and peripheral-blood stem cells (PBSCs; n = 24). Then, 289 samples of 101 Dutch patients and 47 samples of 43 German patients were tested for PHOX2B and TH; these samples included 52 tumor, 214 BM, 32 BM, and 38 PBSC harvests. Of the 214 BM samples, 167 were compared with cytology, and 47 BM samples were compared with immunocytology (IC). In contrast to TH and GD2 synthase, PHOX2B was not expressed in any of the normal samples. In patient samples, PHOX2B was detected in 32% cytology-negative and in 14% IC-negative samples and in 94% of cytology-positive and in 90% of IC-positive BM samples. Overall, PHOX2B was positive in 43% compared with 31% for TH. In 24% of all samples, TH expression was inconclusive, which is similar to expression found in normal tissues. In 42% of these samples, PHOX2B expression was positive. PHOX2B is superior to TH and GD2 synthase in specificity and sensitivity for MRD detection of neuroblastoma by using real-time quantitative PCR. We propose to include PHOX2B in additional prospective MRD studies in neuroblastoma alongside TH and other MRD markers.

Long-term Correction of β-Thalassemia With Minimal Cellular Requirement and Transplantation Modalities

Determination of minimal criteria, pre-transplantation regimens, and infusion modalities for effective and reproducible bone marrow (BM) therapy in beta-thalassemia is of fundamental importance for clinical application. In this study, using repopulation assays, we first established the minimal proportion of normal BM stem cells that would result in therapeutic benefit in this red blood cell (RBC) disorder. Eight groups of stable chimeric hemizygous beta-thalassemic (hemi-betathal) mice (10-89%) were systematically subjected to cellular, molecular, and patho-physiologic analyses for approximately 2 years. In the chimeric hemi-betathal groups containing 19-24% normal donor cells, all RBC parameters and consequent erythropoiesis were significantly improved. Mice in the 24% chimeric group and above had marked reduction in organ pathology including iron deposits, and survived to a normal lifespan. Altogether, these results established that a range of 19-24% normal BM cells is sufficient for long-term significant correction of the hemi-betathal phenotype. We also determined concomitantly the minimal myelosuppression radiation doses, the number of cells to be infused, and the number of infusions required in order to attain this therapeutic range in hemi-betathal mice. Importantly, with prior minimal myelosuppression with 1 or 2 Gy, and using cell doses of 40 or 60 millions, 100% of the recipients were successfully engrafted at therapeutic levels, provided the cells were administered in two doses. This study has therefore determined the therapeutic chimeric level as 19-24% of normal cells, and has also defined the minimal transplantation modalities necessary for the stable and successful correction of the hemi-betathal phenotype.

AMD3100 Mobilizes Acute Promyelocytic Leukemia Cells from the Bone Marrow into the Peripheral Blood and Sensitizes Leukemia Cells to Chemotherapy.

CXCR4/SDF-1 axis regulates the trafficking of normal stem cells to and from the bone marrow (BM) microenvironment. SDF-1 is a chemokine widely expressed by many tissues especially BM stromal cells and osteoblasts. AMD3100 (AMD) is a novel bicyclam molecule that is a competitive inhibitor of SDF-1/CXCR4 binding and has been used to enhance stem cell mobilization when combined with G-CSF in mouse, dog and man. We are interested in evaluating whether leukemic cells “mobilize” similar to normal stem cells after treatment with AMD, and if so, whether this mobilization increases the efficacy of chemotherapy. Therefore, we utilized a mouse model of human acute promyelocytic leukemia (APL) in which the PML-RARα transgene was knocked into a single allele of the murine cathepsin G locus. To more efficiently track the leukemic cells, we transduced banked APL tumors with a dual function reporter gene that encodes a fusion protein comprised of click beetle red (CBR) luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (CBR/EGFP). We generated large numbers of CBR/EGFP+ APL cells by isolating EGFP+ cells using a MoFlo cell sorter, and passaging them in secondary syngeneic recipients. Importantly, the secondary recipients developed a rapidly fatal acute leukemia after intravenously (iv) or intraperitoneal injection, which displayed an APL phenotype (CD34/GR1 co-expression) and exhibited luciferase activity. Upon iv injection into syngeneic recipients, the CBR/EGFP+ APL cells rapidly migrated to the BM microenvironment, as evidenced by the significantly increased BLI signal in the femurs, spine, ribs, and skull of recipients at 4 days after injection. Over the next 2–3 days the CBR/EGFP+ cells migrated to the spleen followed rapidly by widespread dissemination and death due to leukostasis by 14–16 days. To our knowledge, this represents the only mouse leukemia model in which leukemia cells home preferentially to the BM microenvironment in a manner that is similar to what is seen in human AML. Therefore, we used this model to study the effect of AMD on the “mobilization” of APL cells into the peripheral blood (PB) and on their sensitivity to chemotherapeutic agents that are known to affect the proliferation of these cells. Surprisingly, injection of AMD (5 mg/kg) immediately at the time of APL infusion had no impact on the engraftment (short term or long term) of either normal BM stem cells or the leukemic cells. However, we observed rapid mobilization of the leukemic cells when AMD was administered 11 days after APL injection. In fact, 40% of mice that received a single dose of AMD on day +11 after APL injection died 2 to 4 hours after AMD injection as a result of the rapid and massive mobilization of blasts. Overall, we found that AMD treatment on day +11 induced a 3-fold increase in total WBC counts with a 10-fold increase in the leukemic blasts into PB. Interestingly, the administration of AMD concomitant with cytarabine (AraC) (200 mg/kg) on day +11 significantly prolonged the overall survival of mice, compared with mice treated only with AraC. In summary, we developed a mouse model to study the APL cell trafficking, and we have shown leukemia cell mobilize from the BM into PB after AMD administration. We propose that CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. In these preliminary results, we observed that AMD sensitizes APL cells to AraC.

Overview of the clinical efficacy of investigational anticancer drugs.

The purpose of this overview was to make a broad inventory of investigational drugs for medicinal cancer treatment and, specifically, to indicate the evidence of clinical efficacy. Information was retrieved from electronic database searches in Medline and CANCERLIT and relevant published reviews. As the most recent findings are first reported as conference abstracts, an important basis for identification of new drugs and clinical results was a hand search of 13,392 abstracts from five major recent cancer conferences. A total of 209 investigational approaches or drugs were identified and classified into one of eight groups according to proposed mechanism of action. For 28 drugs/approaches survival data were available from randomized controlled trials. Statistically significant benefit was observed for only 12. In earlier phases no or modest anticancer activity was reported. It is speculated that the expanding knowledge in tumour biology might not easily translate into new substantially better anticancer drugs.