Amos Toren

CD133‐Positive Hematopoietic Stem Cell “Stemness” Genes Contain Many Genes Mutated or Abnormally Expressed in Leukemia

Affymetrix human Hu133A oligonucleotide arrays were used to study the expression profile of CD133+ cord blood (CB) and peripheral blood (PB) using CD133 cell‐surface marker. An unsupervised hierarchical clustering of 14,025 valid probe sets showed a clear distinction between the CD133+ cells representing the hematopoietic stem cell (HSC) population and CD133‐differentiated cells. Two hundred forty‐four genes were found to be upregulated by at least twofold in the CD133‐positive cells of both CB and PB compared with the CD133‐negative cells. These genes represent the hematopoietic “stemness,” whereas the 218 and 304 upregulated genes exclusively in PB and CB, respectively, represent tissue specificity. Some of the stemness genes were also common to HSC genes found to be upregulated in several recently published studies. Among these common stemness genes, we identified several groups of genes that have an important role in hematopoiesis: growth factor receptors, transcription factors, genes that have an important role in development, and genes involved in cell growth. Sixteen selected stemness genes are known to be mutated or abnormally regulated in acute leukemias. It can be suggested that key hematopoietic stemness machinery genes may lead to abnormal proliferation and leukemia upon mutation or change of their expression.

Downregulation of Mir-31, Mir-155, and Mir-564 in Chronic Myeloid Leukemia Cells

Background/Aims MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. However, little is known about the role of miRNAs in chronic myeloid leukemia (CML). Our objective is to decipher a miRNA expression signature associated with CML and to determine potential target genes and signaling pathways affected by these signature miRNAs. Results Using miRNA microarrays and miRNA real-time PCR we characterized the miRNAs expression profile of CML cell lines and patients in reference to non-CML cell lines and healthy blood. Of all miRNAs tested, miR-31, miR-155, and miR-564 were down-regulated in CML cells. Down-regulation of these miRNAs was dependent on BCR-ABL activity. We next analyzed predicted targets and affected pathways of the deregulated miRNAs. As expected, in K562 cells, the expression of several of these targets was inverted to that of the miRNA putatively regulating them. Reassuringly, the analysis identified CML as the main disease associated with these miRNAs. MAPK, ErbB, mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF) were the main molecular pathways related with these expression patterns. Utilizing Venn diagrams we found appreciable overlap between the CML-related miRNAs and the signaling pathways-related miRNAs. Conclusions The miRNAs identified in this study might offer a pivotal role in CML. Nevertheless, while these data point to a central disease, the precise molecular pathway/s targeted by these miRNAs is variable implying a high level of complexity of miRNA target selection and regulation. These deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment.

MiR-30e induces apoptosis and sensitizes K562 cells to imatinib treatment via regulation of the BCR-ABL protein

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cell carrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL fusion gene. Tyrosine kinase inhibitors (TKIs) of the BCR-ABL kinase are the treatment of choice for CML patients. Imatinib was the first TKI used in clinical practice with excellent results. MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. Aberrant miRNA expression profiles have been shown to be characteristic of many cancers. Here, we demonstrate that miR-30e is expressed at low levels in CML cell lines and patient samples. Bioinformatics analysis reveals a putative target site for miR-30e in the 3-untranslated region (UTR) of the ABL gene. In agreement, luciferase assay verified that miR-30e directly targets ABL. Enforced expression of miR-30e in K562 cells suppressed proliferation and induced apoptosis of these cells and sensitized them to imatinib treatment. These findings strongly suggest that miR-30e acts as a tumor suppressor by downregulating BCR-ABL expression. Up-regulation of miR-30e in CML cells may therefore have a therapeutic efficacy against this disease.

Conservative treatment of L-asparaginase-associated lipid abnormalities in children with acute lymphoblastic leukemia.

To determine the incidence and clinical consequences of asparaginase‐associated lipid abnormalities in children with acute lymphoblastic leukemia (ALL).

Transplantation and other uses of human umbilical cord blood and stem cells.

Human umbilical cord blood (CB) has established itself as a legitimate source for hematopoeitic stem cell transplantations. Since the first transplantation was performed in 1988, it is estimated that approximately 4,000 patients, with malignant and non-malignant diseases, were transplanted with CB. Comparing to bone marrow transplants, cord bloods collection is easier and safer. It is also quicker to perform CB transplantation from the time of beginning of donor search. One of the major advantages of it is the naïve nature of newborns immune system. This allows transplantations with less restriction of the HLA system, and with fewer graft versus host disease (GVHD) cases. A true setback of CB transplantations is the slow pace of engraftment. This fact has negative impact on treatment related mortality and is related to the amount of stem cell infused. Since CB has limited nucleated cell dose, transplanting it to heavier patients, namely adults, poses many difficulties. But the skepticism about the possibility that CB might be used in adult hematopoetic stem cell transplantations, can decline after few large scale trials have shown that it is definitely a feasible procedure. Few fields of research might help to improve the outcome of CB transplantations. While some strategies are at different investigational stages, others are at advanced phases of clinical studies. Main strategies are based on expansion of the number of the stem cells in CB grafts, induction of a temporary engraftment with other stem cell sources, or reduction of the toxicity of the conditioning regimens. It is encouraging to witness that the outcome of CB transplantations is improving constantly. Other potential uses of CB are also discussed. It was used for gene transfer for primary immune deficiency, and it was also demonstrated in animal models that its stem cell could serve as regenerative cells in non-hematopoeitic injured tissues. CB has broad spectrum of possible uses, but hematopoeitic stem cell transplantation is still the major indication. In an era where 30-40% of patients will not have a matched related or unrelated donor, CB is a major alternative, which provide a true chance for cure for a wide variety of diseases.

Restoration of miR-424 suppresses BCR-ABL activity and sensitizes CML cells to imatinib treatment

MicroRNAs (miRNAs) are small noncoding RNAs that participate in many biological processes by posttranscriptionally regulating gene expression. Dysregulation of miRNA expression has been shown to be typical of many neoplasms. Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells carrying the Philadelphia (Ph) chromosome and an oncogenic BCR-ABL tyrosine kinase fusion gene. While the development of tyrosine kinase inhibitors (TKIs) like imatinib has revolutionized treatment of CML, it has become increasingly clear in recent years that TKI treatment alone will not be curative in many cases. Thus, further dissection of the regulatory networks that drive BCR-ABL-induced malignant transformation may help to identify other novel therapeutic approaches that complement TKI treatment. In this study we demonstrate that the expression of miR-424 is markedly low in CML cell lines and patient samples at time of diagnosis. With the aid of bioinformatics analysis we revealed a conserved target site for miR-424 in the 3-untranslated region (UTR) of the ABL gene. Via luciferase assays, we showed that miR-424 directly targets BCR-ABL. Overexpression of miR-424 was shown to suppress proliferation and induce apoptosis of K562 cells as well as sensitize these cells to imatinib treatment. These findings strongly suggest that miR-424 acts as a tumor suppressor by downregulating BCR-ABL expression. Up-regulation of miR-424 in CML cells may therefore have a therapeutic effect against this disease.

Human umbilical cord blood biology, transplantation and plasticity

As the significance of hematopoietic stem cell transplantation (HSCT) is constantly rising, the scarcity of matched donors is proving to be a troubling issue. Cord blood (CB) is an important source of stem cells (SC) for transplantation. It has been used in the last two decades for approximately 4500 transplantations. Its collection, cryopreservation, banking and thawing techniques pose unique challenges to clinicians and researchers CB has abundant stem cell with impressive proliferative capacity. On the other hand, CBs immunological system has a naïve and more tolerant nature. Except for the biological aspects, few ethical issues have become a concern for transplantation teams who use CB. There are few advantages of CB over bone marrow, especially the lower rates of acute and chronic graft-versus-host disease (GVHD) after transplantation. On the other hand, there are relatively high rates of early treatment related mortality in cord blood transplantation (CBT). This is related to the small nucleated cell (NC) dose infused from each CB unit. The clinical experience in CBT, especially in children, is encouraging. When using adequate number of NC/kg, results in CBT for malignant and non-malignant diseases are comparable to bone marrow transplantation (BMT). In this article, a comprehensive review of the largest scale studies is presented. There is a continuous search for an optimal way to deal with delayed engraftment of CB and its implication. The current investigational, and also first clinical trials using diverse methods to overcome high rates of TRM are reviewed. Almost twenty years after the first CBT was preformed, many advocate for a routine parallel search, BM and CB, for unrelated donor. Future uses of CB might also be in the field of gene transfer and non hematopoietic injured tissues repair.

Acute Lymphoblastic Leukemia in Early Childhood as the Presenting Sign of Ataxia-Telangiectasia Variant

Ataxia-telangiectasia (A-T), an autosomal recessive disorder is characterized by progressive neurodegeneration, immunodeficiency, sensitivity to ionizing radiation, and predisposition to cancer, especially to lymphoid malignancies. A-T variant is characterized by a milder clinical phenotype and is caused by missense or leaky splice site mutations that produce residual ataxia telangiectasia mutated (ATM) kinase activity. Lymphoid malignancy can precede the diagnosis of A-T, particularly in young children with mild neurological symptoms. We studied a consanguineous family with four A-T variant patients, three of them developed T-ALL at a young age before the diagnosis of A-T was established. ATM mutation analysis detected two new missense mutations both within exon 12: c.1514T>C and c.1547T>C. All four patients are homozygous for the two mutations, while their parents are heterozygous for the mutations. ATM protein level was low in all patients and the response to the radiomimetic agent, neocarzinostatin, was reduced. Leukemic presentation in a young age in three members of consanguineous family led to the identification of a new missense mutation in the ATM gene. The diagnosis of A-T or A-T variant should be considered in children with neurological abnormalities who develop T-ALL at a young age.

Therapy-related acute myeloid leukemia with t(2;11)(q37;q23) after treatment for osteosarcoma.

The survival rate for children with osteosarcoma (OS) has improved dramatically with the introduction of multiagent chemotherapy. As the number of pediatric cancer survivors increases, there is a concern about the development of secondary malignant neoplasms. Secondary acute myeloid leukemia (AML) has been rarely reported after treatment for OS. We describe a 14-year-old boy with OS of the left ileum who developed secondary AML 15 months after completion of treatment. Cytogenetic analysis of the leukemic cells demonstrated deletion 11q23, whereas fluorescence in situ hybridization revealed rearrangement of the MLL gene. Only the addition of the long-distance inverse polymerase chain reaction technique identified the SEPT2 as the MLL fusion partner resulting in t(2;11)(q37;q23) that was reported in a very few secondary AML cases. Because of the cryptic nature of MLL translocations that cannot be detected by conventional cytogenetics or may misinterpreted as deletion, additional molecular techniques are required to identify the precise translocation partner. Because long-distance inverse polymerase chain reaction is not available in most molecular laboratories, the true incidence of t(2;11)(q37;q23) and the involvement of SEPT2 as the MLL translocation partner could be more prevalent in secondary AML.

Analysis of Risk Factors of Cord Blood Transplantation for Children

As cord blood (CB) is being used frequently as a source for heamtopoetic stem cell transplantation defining risk factors for transplantation outcome is an important issue.