Paraskevi Diamanti

Expression of CD133 on leukemia-initiating cells in childhood ALL

Optimization of therapy for childhood acute lymphoblastic leukemia (ALL) requires a greater understanding of the cells that proliferate to maintain this malignancy because a significant number of cases relapse, resulting from failure to eradicate the disease. Putative ALL stem cells may be resistant to therapy and subsequent relapses may arise from these cells. We investigated expression of CD133, CD19, and CD38 in pediatric B-ALL. Cytogenetic and molecular analyses demonstrated that karyotypically aberrant cells were present in both CD133(+)/CD19(+) and CD133(+)/CD19(-) subfractions, as were most of the antigen receptor gene rearrangements. However, ALL cells capable of long-term proliferation in vitro and in vivo were derived from the CD133(+)/CD19(-) subfraction. Moreover, these CD133(+)/CD19(-) cells could self-renew to engraft serial nonobese diabetic-severe combined immunodeficient recipients and differentiate in vivo to produce leukemias with similar immunophenotypes and karyotypes to the diagnostic samples. Furthermore, these CD133(+)/CD19(-) ALL cells were more resistant to treatment with dexamethasone and vincristine, key components in childhood ALL therapy, than the bulk leukemia population. Similar results were obtained using cells sorted for CD133 and CD38, with only the CD133(+)/CD38(-) subfraction demonstrating xenograft repopulating capacity. These data suggest that leukemia-initiating cells in childhood B-ALL have a primitive CD133(+)/CD19(-) and CD38(-) phenotype.

Comparison of childhood leukemia initiating cell populations in NOD/SCID and NSG mice

Leukemia initiating cells (LICs) are defined by their ability to generate leukemias in animal models and to self-renew by repopulating serial recipients. Recent findings suggest that multiple sub-populations of acute leukemia cells are capable of engrafting immune deficient mice raise important questions about the biology of these diseases.1, 2, 3 If several cell populations have LIC properties, what are the relationships of these stem cell populations to each other and which populations are most important to target with therapy? To address these questions, it is crucial to utilize assays that are capable of identifying any leukemia cells that possess stem cell properties. The recently developed NOD/LtSz-scid IL-2Rγc null (NSG) mouse is now considered a more permissive host for the growth and development of normal and acute myeloid leukemia cells than the nonobese diabetic severe combined immune deficient (NOD/SCID) strain.4, 5 In acute lymphoblastic leukemia (ALL) engraftment of high-risk cases has been reported to be slower in NOD/SCID than NSG but otherwise leukemias developed in both strains.6 However, another study reported that engraftment of T-ALL cells was improved using NSG mice.7 Observations that different populations of cells can engraft different mouse strains raise questions as to how the reports that used only NOD/SCID mice should be interpreted. NSG and NOD/SCID strains have not previously been directly and quantitatively compared for engraftment potential of pediatric LIC sub-populations using the same source of cells. Such studies should provide clear information as to the permissiveness of each strain and which sub-populations have LIC potential.

Parthenolide eliminates leukemia initiating cell populations and improves survival in xenografts of childhood acute lymphoblastic leukemia

Approximately 20% of children with acute lymphoblastic leukemia (ALL) relapse because of failure to eradicate the disease. Current drug efficacy studies focus on reducing leukemia cell burden. However, if drugs have limited effects on leukemia-initiating cells (LICs), then these cells may expand and eventually cause relapse. Parthenolide (PTL) has been shown to cause apoptosis of LIC in acute myeloid leukemia. In the present study, we assessed the effects of PTL on LIC populations in childhood ALL. Apoptosis assays demonstrated that PTL was effective against bulk B- and T-ALL cells, whereas the CD34(+)/CD19(-), CD34(+)/CD7(-), and CD34(-) subpopulations were more resistant. However, functional analyses revealed that PTL treatment prevented engraftment of multiple LIC populations in NOD/LtSz-scid IL-2Rγ(c)-null mice. PTL treatment of mice with established leukemias from low- and high-risk patients resulted in survival and restoration of normal murine hemopoiesis. In only 3 cases, disease progression was significantly slowed in mice engrafted with CD34(+)/CD19(-) or CD34(+)/CD7(-) and CD34(-) cells, but was not prevented, demonstrating that individual LIC populations within patients have different responses to therapy. These observations indicate that PTL may have therapeutic potential in childhood ALL and provide a basis for developing effective therapies that eradicate all LIC populations to prevent disease progression and reduce relapse.

Early emergence of PNH-like T cells after allogeneic stem cell transplants utilising CAMPATH-1H for T cell depletion

Summary:CAMPATH-1H (C-1H) is widely used in vivo and / or in vitro for T cell depletion in hematopoietic SCT. This humanised monoclonal antibody is specific for CD52, a marker coexpressed on the majority of human lymphocytes with CD48 and other glycosylphosphatidyl-inositol (GPI) anchored proteins. We detected CD52 / CD48 dual expression on >99% of CD3+ lymphocytes from normal individuals and all 15 post-SCT patients whose transplants did not utilise C-1H. By contrast, 23 / 26 patients with transplants involving C-1H (in vivo, in vitro or both) exhibited populations lacking CD52 expression that accounted for 49.7% (4.2–86.2%) of the CD3+ lymphocytes (median and range) in samples evaluated at a median of 2 months post-SCT. Most CD52− cells also lacked CD48 expression. These GPI− T cells were of either donor or mixed donor / recipient origin. They were predominant in the early months after SCT at times of profound lymphopenia and inversely correlated with the recovery of the absolute lymphocyte count (r= − 0.663, P<0.0001). The presence of CD52− cells has been correlated previously with clinical outcome after CAMPATH therapy for both malignant and nonmalignant diseases.

Functionalized Triblock Copolymer Vectors for the Treatment of Acute Lymphoblastic Leukemia

The chemotherapeutic Parthenolide is an exciting new candidate for the treatment of acute lymphoblastic leukemia, but like many other small-molecule drugs, it has low aqueous solubility. As a consequence, Parthenolide can only be administered clinically in the presence of harmful cosolvents. Accordingly, we describe the synthesis, characterization, and testing of a range of biocompatible triblock copolymer micelles as particle-based delivery vectors for the hydrophobic drug Parthenolide. The drug-loaded particles are produced via an emulsion-to-micelle transition method, and the effects of introducing anionic and cationic surface charges on stability, drug sequestration, biocompatibility, and efficacy are investigated. Significantly, we demonstrate high levels of efficacy in the organic solvent-free systems against human mesenchymal stem cells and primary T-acute lymphoblastic leukemia patient cells, highlighting the effectiveness of the delivery vectors for the treatment of acute lymphoblastic leukemia.

Biodegradable, Drug-Loaded Nanovectors via Direct Hydration as a New Platform for Cancer Therapeutics

The stabilization and transport of low-solubility drugs, by encapsulation in nanoscopic delivery vectors (nanovectors), is a key paradigm in nanomedicine. However, the problems of carrier toxicity, specificity, and producibility create a bottleneck in the development of new nanomedical technologies. Copolymeric nanoparticles are an excellent platform for nanovector engineering due to their structural versatility; however, conventional fabrication processes rely upon harmful chemicals that necessitate purification. In engineering a more robust (copolymeric) nanovector platform, it is necessary to reconsider the entire process from copolymer synthesis through self-assembly and functionalization. To this end, a process is developed whereby biodegradable copolymers of poly(ethylene glycol)-block-poly(trimethylene carbonate), synthesized via organocatalyzed ring-opening polymerization, undergo assembly into highly uniform, drug-loaded micelles without the use of harmful solvents or the need for purification. The direct hydration methodology, employing oligo(ethylene glycol) as a nontoxic dispersant, facilitates rapid preparation of pristine, drug-loaded nanovectors that require no further processing. This method is robust, fast, and scalable. Utilizing parthenolide, an exciting candidate for treatment of acute lymphoblastic leukemia (ALL), discrete nanovectors are generated that show strikingly low carrier toxicity and high levels of specific therapeutic efficacy against primary ALL cells (as compared to normal hematopoietic cells).

Dual targeting of Hsp90 in childhood acute lymphoblastic leukaemia

Survival rates for children with acute lymphoblastic leukaemia (ALL) have improved considerably to over 90% in recent years but despite these advances, 15–20% of patients relapse. Current chemotherapeutic regimens are designed around the properties of bulk leukaemia cells, which differ from those of the leukaemia-initiating cell (LIC) populations (Cox et al, 2009). If drugs have no effect on LIC, these cells may proliferate and cause relapse. Given that several populations in childhood ALL have been shown to have LIC properties (Cox et al, 2009; Diamanti et al, 2013) the development of therapies that are effective against all leukaemia cells, with minimal toxicity to normal cells, is of utmost importance. Efforts to uncover the biological pathways that mediate drug resistance and promote cell survival have lead to the targeting of heat shock protein 90 (Hsp90). Hsp90 is a molecular chaperone protein involved in the maturation and stabilisation of a range of oncogenic client proteins, such as Bcr-Abl, Akt and IKK, that are known to be mutated and/or overexpressed in leukaemias (Mjahed et al, 2012). Targeting Hsp90 could have an impact on several oncogenic pathways and the use of Hsp90 inhibitors is a promising approach for cancer therapy (Hassane et al, 2008; Hertlein et al, 2010; Lancet et al, 2010; Hong et al, 2013). Alvespimycin (17-DMAG) targets the binding site of ATP in Hsp90 and has shown clinical activity in acute myeloid leukaemia (AML)(Lancet et al, 2010; Mjahed et al, 2012). Celastrol has been shown to increase tumour necrosis factorinduced apoptosis (Sethi et al, 2007) and disrupt the Hsp90/ Cdc37 complex (Zhang et al, 2008). Celastrol significantly impairs viability and engraftment of AML LIC by inhibiting nuclear factor-jB survival signals and inducing oxidative stress (Hassane et al, 2008). However, there are no reports on the efficacy of alvespimycin or celastrol in childhood ALL. The aim of this study was to examine the effects of these structurally and functionally distinct Hsp90 inhibitors (Hsp90i) on primary ALL cells and evaluate their potential when used in combination. Cells from 3 B-cell precursor (BCP)-ALL, 3 T-cell ALL (TALL) cases and 3 cord blood (CB) samples were incubated with alvespimycin for 24 h and celastrol for 48 h then compared for survival (Fig 1A). Clinical characteristics of ALL samples are shown in Table SI. The 50% inhibitory concentration (IC50) for alvespimycin was 10 2 nmol/l in

Investigating CD99 Expression in Leukemia Propagating Cells in Childhood T Cell Acute Lymphoblastic Leukemia

A significant number of children with T-lineage acute lymphoblastic leukemia (T-ALL) fail to respond to therapy and experience early relapse. CD99 has been shown to be overexpressed on T-ALL cells and is considered to be a reliable detector of the disease. However, the relevance of CD99 overexpression in ALL has not been investigated in a functional context. The aim of this study was to investigate the functional capacity of CD99+ cells in childhood ALL and determine the suitability of CD99 as a therapeutic target. Flow cytometric analyses confirmed higher expression of CD99 in ALL blasts (81.5±22.7%) compared to normal hemopoietic stem cells (27.5±21.9%) and T cells (3.1±5.2%, P≤0.004). When ALL cells were sorted and assessed in functional assays, all 4 subpopulations (CD34+/CD99+, CD34+/CD99-, CD34-/CD99+ and CD34-/CD99-) could proliferate in vitro and establish leukemia in NSG mice. Leukemia propagating cell frequencies ranged from 1 in 300 to 1 in 7.4x104 but were highest in the CD34+/CD99- subpopulation. In addition, all four subpopulations had self-renewal ability in secondary NSG mice. Cells in each subpopulation contained patient specific TCR rearrangements and karyotypic changes that were preserved with passage through serial NSG transplants. Despite high levels of CD99 antigen on the majority of blast cells, leukemia initiating capacity in vivo was not restricted to cells that express this protein. Consequently, targeting CD99 alone would not eliminate all T-ALL cells with the ability to maintain the disease. The challenge remains to develop therapeutic strategies that can eliminate all leukemia cells with self-renewal capacity in vivo.

Investigating chemoresistance to improve sensitivity of childhood T-cell acute lymphoblastic leukemia to parthenolide

Current therapies for childhood T-cell acute lymphoblastic leukemia have increased survival rates to above 85% in developed countries. Unfortunately, some patients fail to respond to therapy and many suffer from serious side effects, highlighting the need to investigate other agents to treat this disease. Parthenolide, a nuclear factor kappa (κ)B inhibitor and reactive oxygen species inducer, has been shown to have excellent anti-cancer activity in pediatric leukemia xenografts, with minimal effects on normal hemopoietic cells. However, some leukemia initiating cell populations remain resistant to parthenolide. This study examined mechanisms for this resistance, including protective effects conferred by bone marrow stromal components. T-cell acute leukemia cells co-cultured with mesenchymal stem cells demonstrated significantly enhanced survival against parthenolide (73±11%) compared to cells treated without mesenchymal stem cell support (11±9%). Direct cell contact between mesenchymal cells and leukemia cells was not required to afford protection from parthenolide. Mesenchymal stem cells released thiols and protected leukemia cells from reactive oxygen species stress, which is associated with parthenolide cytotoxicity. Blocking cystine uptake by mesenchymal stem cells, using a small molecule inhibitor, prevented thiol release and significantly reduced leukemia cell resistance to parthenolide. These data indicate it may be possible to achieve greater toxicity to childhood T-cell acute lymphoblastic leukemia by combining parthenolide with inhibitors of cystine uptake.