Normal Hemopoietic Stem Cells Research Articles

The N6-methyladenosine RNA modification in acute myeloid leukemia.

In recent years, the N6-methyladenosine (m6A) modification of RNA has been shown to play an important role in the development of acute myeloid leukemia (AML) and the maintenance of leukemic stem cells (LSCs). In this review we summarise the recent findings in the field of epitranscriptomics related to m6A and its relevance in AML. Recent studies have focused on the role of m6A regulators in the development of AML and their potential as translational targets. The writer Methyltransferase Like 3 and its binding partner Methyltransferase Like 14, as well as the reader YTH domain-containing family protein 2, were shown to be vital for LSC survival, and their loss has detrimental effects on AML cells. Similar observations were made with the demethylases fat mass and obesity-associated protein and AlkB homologue 5 RNA demethylase. Of importance, loss of any of these genes has little to no effect on normal hemopoietic stem cells, suggesting therapeutic potential. The field of epitranscriptomics is still in its infancy and the importance of m6A and other RNA-modifications in AML will only come into sharper focus. The development of therapeutics targeting RNA-modifying enzymes may open up new avenues for treatment of such malignancies.

Telomere and a Final Verdict for Cellular Senescence

Telomeres are specialized DNA complexes found at the end of all chromosomes. Human, as a member of eukaryotic cells, requires telomeres to maintain the length and the stability of chromosomes. Telomeres lose their non-coding DNA sequence to protect the genetic information on the chromosomes. Shortening of telomeres occurs in most somatic cells after sufficient cell division in a human lifetime. Normal haemopoietic cells or stem cells possess telomerase enzyme to restore telomeres and allow further replication. Telomere dysfunction is the origin of several degenerative disorders and also predispose to cancer. Roles of telomere in carcinogenesis and ageing related disorders are reviewed.

S124 IDENTIFICATION OF THE DOPAMINE AND SEROTONIN TRANSPORTERS SLC6A3 AND SLC6A4 AS NOVEL SURVIVAL FACTORS IN LEUKEMIC STEM CELLS IN CHRONIC MYELOID LEUKAEMIA

Background: In chronic myeloid leukaemia (CML), BCR-ABL tyrosine kinase inhibitors (TKI) fail to eliminate leukaemia stem cells (LSC) which can serve as a reservoir to drive relapse, TKI resistance and promote disease progression. Therefore, identification of pathways/targets that promote LSC survival is essential for the development of curative therapies. Our previous transcriptomics analysis pointed to neurotransmitter pathways as being aberrantly expressed in LSC compared to normal haemopoietic stem cells. Aims: This led us to hypothesise that LSCs require neurotransmitter pathways to maintain their stem cell potential. The aim of our study was therefore to understand whether modulating these pathways would affect LSC survival. Methods: To identify drug-able targets within these pathways, we performed compound screens in BCR-ABL-/+ cell lines and primary CML CD34+ cells using 658 compounds known to modulate neurotransmitter signalling, many of which are used clinically to treat neuro-psychiatric disorders. Hits selective for BCR-ABL-expressing cells were further evaluated in phenotypic assays using primary CML CD34+ versus non-CML CD34+ cells. Western blotting and FACs analysis was used to determine the effects that these compounds had on cellular pathways known to affect LSC survival. Results: 3-CPMT, a selective dopamine reuptake inhibitor (SDRI), and paroxetine, a clinical grade selective serotonin reuptake inhibitor (SSRI), that target SLC6A3 and SLC6A4 respectively, emerged as compounds that selectively reduced CML CD34+ cell counts (P = 0.0007 and 0.04 respectively) and inhibited CFC outputs (P = 0.001 and 0.04, respectively). FACs analysis has shown that LSC and CML cell lines express 3–4-fold higher levels of SLC6A3 and SLC6A4 than their non-CML counterparts, with expression differences for SLC6A4 reaching significance (CML CD34+CD38- versus non-CML CD34+CD38- cells, P = 0.04; BaF3 BCR-ABL+ versus wild type BaF3, P = 0.04). Targeting of SLC6A3 or SLC6A4 with 3-CPMT or paroxetine +/- nilotinib inhibits a number of known LSC survival factors including PRKCH, FOXO3a, p38 MAPK and c-Myc. Furthermore, we have demonstrated that paroxetine in combination with nilotinib is highly effective at eradicating CD34+CD38-CD90+ LSC compared to nilotinib alone in patient-derived xenograft mouse models (P = 0.01). Summary/Conclusion: In conclusion, our pre-clinical data suggest that repurposing drugs that target neurotransmitter transporters may represent a novel way to target LSC in CML patients.

Similarity of regulatory network between leukemia stem cells and normal hemopoietic stem cells

AbstractTo reveal the biology of AML, we compared gene-expression profiles between normal hematopoietic cells from 38 healthy donors and leukemic blasts (LBs) from 26 AML patients. We defined the comparison of LB and unselected BM as experiment 1, LB and CD34+ isolated from BM as experiment 2, LB and unselected PB as experiment 3, and LB and CD34+ isolated from PB as experiment 4. Then, protein–protein interaction network of DEGs was constructed to identify critical genes. Regulatory impact factors were used to identify critical transcription factors from the differential co-expression network constructed via reanalyzing the microarray profile from the perspective of differential co-expression. Gene ontology enrichment was performed to extract biological meaning. The comparison among the number of DEGs obtained in four experiments showed that cells did not tend to differentiation and CD34+ was more similar to cancer stem cells. Based on the results of protein–protein interaction network,CREBBP,F2RL1,MCM2, andTP53were respectively the key genes in experiments 1, 2, 3, and 4. From gene ontology analysis, we found that immune response was the most common one in four stages. Our results might provide a platform for determining the pathology and therapy of AML.

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.

Deregulated hedgehog pathway signaling is inhibited by the smoothened antagonist LDE225 (Sonidegib) in chronic phase chronic myeloid leukaemia.

Targeting the Hedgehog (Hh) pathway represents a potential leukaemia stem cell (LSC)-directed therapy which may compliment tyrosine kinase inhibitors (TKIs) to eradicate LSC in chronic phase (CP) chronic myeloid leukaemia (CML). We set out to elucidate the role of Hh signaling in CP-CML and determine if inhibition of Hh signaling, through inhibition of smoothened (SMO), was an effective strategy to target CP-CML LSC. Assessment of Hh pathway gene and protein expression demonstrated that the Hh pathway is activated in CD34+ CP-CML stem/progenitor cells. LDE225 (Sonidegib), a small molecule, clinically investigated SMO inhibitor, used alone and in combination with nilotinib, inhibited the Hh pathway in CD34+ CP-CML cells, reducing the number and self-renewal capacity of CML LSC in vitro. The combination had no effect on normal haemopoietic stem cells. When combined, LDE225 + nilotinib reduced CD34+ CP-CML cell engraftment in NSG mice and, upon administration to EGFP+ /SCLtTA/TRE-BCR-ABL mice, the combination enhanced survival with reduced leukaemia development in secondary transplant recipients. In conclusion, the Hh pathway is deregulated in CML stem and progenitor cells. We identify Hh pathway inhibition, in combination with nilotinib, as a potentially effective therapeutic strategy to improve responses in CP-CML by targeting both stem and progenitor cells.

Clinical significance of acquired somatic mutations in aplastic anaemia.

Aplastic anaemia (AA) is frequently associated with other disorders of clonal haemopoiesis such as paroxysmal nocturnal haemoglobinuria (PNH), myelodysplastic syndrome (MDS) and T-large granular lymphocytosis. Certain clones may escape the immune attack within the bone marrow environment and proliferate and attain a survival advantage over normal haemopoietic stem cells, such as trisomy 8, loss of heterozygosity of short arm of chromosome 6 and del13q clones. Recently acquired somatic mutations (SM), excluding PNH clones, have been reported in around 20-25% of patients with AA, which predispose to a higher risk of later malignant transformation to MDS/acute myeloid leukaemia. Furthermore, certain SM, such as ASXL1 and DNMT3A are associated with poor survival following immunosuppressive therapy, whereas PIGA, BCOR/BCORL1 predict for good response and survival. Further detailed and serial analysis of the immune signature in AA is needed to understand the pathogenetic basis for the presence of clones with SM in a significant proportion of patients.

The role of the bone morphogenetic proteins in leukaemic stem cell persistence.

CML (chronic myeloid leukaemia) is characterized by the presence of the oncogenic tyrosine kinase fusion protein BCR (breakpoint cluster region)-Abl, responsible for driving the disease. Current TKI (tyrosine kinase inhibitor) therapies effectively inhibit BCR-Abl to control CML in the majority of patients, but do not eliminate the LSC (leukaemic stem cell) population, which becomes quiescent following treatment. Patients require long-term treatment to sustain remission; alternative strategies are therefore required, either alone or in combination with TKIs to eliminate the LSCs and provide a cure. The embryonic morphogenetic pathways play a key role in haemopoiesis with recent evidence suggesting LSCs are more dependent on these signals following chemotherapy than normal HSCs (haemopoietic stem cells). Recent evidence in the literature and from our group has revealed that the BMP (bone morphogenetic protein) pathway is differentially expressed in CML patients compared with normal donors. In the present review, we explore the role that BMP signalling plays in oesteoblast differentiation, HSC maintenance and the implication of altered BMP signalling on LSC persistence in the BM (bone marrow) niche. Overall, we highlight the BMP pathway as a potential target for developing LSC-directed therapies in CML in the future.

Does Expression Of CD200 Provide a Target For LIC In Childhood ALL?

Although the survival of children with acute lymphoblastic leukaemia (ALL) has improved considerably, there are still around 20% of cases that go on to relapse. These patients do not respond well to current therapies and there is a need to develop treatment strategies to directly target the cells that initiate the leukaemia to allow eradication of the disease. Leukaemia initiating cells (LIC) have been shown to be present in several immunophenotypic subpopulations evaluated in NSG assays. Identifying a marker that is expressed by all LIC populations may allow their discrimination from normal haemopoietic stem cells and more specific targeting of these cells. Whole genome expression studies have recently highlighted genes, including CD58, CD97 and CD99 that were over expressed in B-ALL cases when compared to normal CD19+/CD10+ cells. The addition of these markers to the minimal residual disease (MRD) flow cytometric panel increased the sensitivity of detection by 10-fold to one leukaemia cell in 105 bone marrow (BM) cells. We have previously investigated the functional capacity of ALL cells sorted for CD58, CD97 and CD99 in combination with CD34 in vivo. However, none of these antigens were specific for LIC. CD200 has been described as another marker that improved MRD sensitivity by flow cytometry and consequently we investigated its expression in LIC populations and the functional ability of these cells in vivo. Expression of CD34, CD19 and CD200 was assessed in 12 B-ALL cases (6 pre B-ALL, 6 c-ALL) and 6 cord blood samples (CB) by flow cytometry. Whole genome microarray analysis was also performed on unsorted cells and sorted CD34+/CD19+, CD34+/CD19-, CD34-/CD19+ and CD34-/CD19- subpopulations to compare expression of genes between these LIC and the bulk leukaemia. Cells from 5 of these patients were also sorted on the basis of expression of CD34 and CD200 and the functional ability of the sorted subpopulations was assessed in NSG mice. The microarray analyses demonstrated that CD200 was over expressed in all the subpopulations and was especially high in the CD34+/CD19+ and the CD34-/CD19+ subpopulations. CD200 was significantly over expressed in the CD34+/CD19+ subpopulation when compared to its expression in the CD34+/CD19- and CD34-/CD19- subpopulations (p=0.009, F=6.3, Fcritical=4.5). Flow cytometric analyses confirmed that CD200 was over expressed in unsorted B-ALL cells compared to unsorted CB (54.6%±8.1% vs 0.07±0.09% respectively, p=0.0002) and expression was higher in CD34+/CD19+ (68.4%±10.8% p=0.0006), CD34+/CD19- (39%±9.2% p=0.01), CD34-/CD19+ (48.4%±8.6% p=0.002) and CD34-/CD19- (5%±1.8% p=0.3) subpopulations compared to CD34+/CD38- CB cells (2%±1.1%). Over expression of CD200 on these ALL subpopulations indicates that it may be useful in characterisation of LIC. When cells from 5 of these cases were sorted, the CD34+/CD200+ subpopulation accounted for the greatest proportion of cells (60.5%±16.1%). Expression of CD19 was also high in this subpopulation (65±11%). CD34+/CD200- cells represented the smallest subpopulation (0.5%±0.1%) and these cells had low expression of CD19 (4±7%). The CD34-/CD200+ and CD34-/CD200- subpopulations represented 18%±11.4% and 21%±120.6% of the bulk cells, respectively. CD19 expression was higher in the CD34-/CD200+ subpopulation (30±14%) than in CD34-/CD200- cells (7±5%). Results from 4 cases to date indicate that engraftment was achieved with the CD34+/CD200+ subpopulation in every case (4-92% human leukaemia). In 3 cases, comparable engraftment levels were also achieved using CD34-/CD200+ cells (10.4-86.1%). No engraftment was detected using the CD34+/CD200- or CD34-/CD200- subpopulations, with the exception of 1 case where engraftment levels of 18-48% were achieved. These levels were lower than those achieved with CD200+ cells from the same patient. Interestingly when BM from mice engrafted with CD200- cells from this patient were analysed they now expressed CD200 (12-35%), indicating cell differentiation had occurred in vivo. Assessment of self-renewal capability of these LIC populations is ongoing. These data suggest that CD200 may be a useful marker for discriminating LIC cells from normal haemopoietic cells, irrespective of expression of CD34 and CD19 in these cases. Disclosures:No relevant conflicts of interest to declare.

Do Proteasome and Heat Shock Protein Inhibitors Eliminate Leukaemia Initiating Cell Populations In Childhood ALL?

Outcomes for patients with childhood acute lymphoblastic leukaemia (ALL) are increasingly successful. Nevertheless, 20% of cases relapse due to failure to eradicate the disease, emphasising the need for more effective treatment. We and others have shown that multiple ALL subpopulations contain leukaemia initiating cells (LIC) that have the capacity to engraft and generate leukaemias in immune deficient mice. In addition, we have shown that some of these populations are resistant to current therapies and these cells may be responsible for disease relapse. Therefore, future therapies should be designed to target and eradicate all LIC populations. Preliminary results from our whole genome microarray analyses on bulk B-ALL cells and LIC subpopulations have indicated that certain key proteasome pathway and Hsp90 genes are similarly expressed across the LIC subpopulations. Therefore, inhibitors of these genes may be effective on all LIC subpopulations. Proteasome and heat shock protein (Hsp) 90 inhibitors are promising agents in cancer therapy. Bortezomib, a specific proteasome inhibitor, currently used in multiple myeloma, disrupts various signalling pathways leading to cell cycle arrest and apoptosis. Hsp90 is involved in the maturation and stabilisation of a wide range of oncogenic client proteins involved in survival and tumour progression. Hsp90 inhibitors, such as 17-DMAG and Celastrol have been shown to suppress B- and T-ALL cells, while sparing normal haemopoietic stem cells (HSC). 17-DMAG targets the ATP-binding domain of Hsp90 and Celastrol disrupts the Cdc37-Hsp90 complex, so when used in combination their potential to target leukaemia cells might be improved. The aim of this study was to investigate the effects of Bortezomib and the Hsp90 inhibitor combination 17-DMAG and Celastrol (Hsp90i) in childhood ALL. The viability and functional capacity of specific LIC subpopulations from 4 B-ALL cases and 5 CD34+/CD38- HSC samples following drug treatment were assessed by flow cytometry and functional assays. Cells were stained with Annexin V, 7AAD and antibodies against CD34 and CD19 (B-ALL) or CD34 and CD38 (normal cells). Following treatment with 1nM Bortezomib, the proportions of surviving bulk ALL cells, CD34+/CD19+, CD34+/CD19- and CD34-/CD19+ LIC subpopulations were significantly reduced to ≤23.6±21% compared to untreated cells (P≤0.025). The CD34-/CD19- subpopulation demonstrated resistance to Bortezomib with 61.6±28% remaining viable (P=0.144). Encouragingly, all LIC subpopulations responded to Hsp90i treatment (0.1nM Celastrol + 10nM 17-DMAG) with the proportions surviving ranging from 3.0±4% to 10.5±5% compared to untreated cells (P≤0.002). Normal cells were largely unaffected by Bortezomib and Hsp90i (75.9±15% and 90.8±29% surviving, respectively P=0.22). When ALL subpopulations were sorted, treated with Bortezomib then set up in long-term culture, viable cell numbers decreased significantly compared to untreated samples, even in the CD34-/CD19- subpopulation (up to 725-fold from 11.6±16x105 cells to 2.4±1x103, P=0.013). Treatment with Hsp90i also resulted in a significant reduction in viable cells after 4 weeks in culture (up to 161-fold to 8.0±9x103, P=0.015). The ex vivo effects of these drugs on LIC proliferation have also been assessed using NOD/LtSz-scid IL-2Rγc null (NSG) mice. High levels of leukaemia engraftment were observed in NSG with untreated bulk T-ALL cells and LIC subpopulations (60.3%-86.8% leukaemia). Results to date indicate that treatment with Bortezomib, prior to inoculation, prevented engraftment of bulk and all LIC in most cases or suppressed it to only 1.3±2.3% leukaemia cells in 1 NSG inoculated with bulk ALL (P=0.02). Similarly, treatment with Hsp90i prevented engraftment in bulk and LIC subpopulations in 2 of 3 cases. In the third case, a relapse sample, engraftment of some LIC was prevented and significantly reduced in NSG injected with bulk (68.73%), CD34+/CD7+ (24.06%) and CD34-/CD7+ (48.36%) cells (P=0.02). None of the treatment conditions have significantly affected the engrafting capacity of normal HSC (16.21±13.7% with untreated cells, 5.3±9% with Bortezomib P=0.11 and 5.8±7% with Hsp90i P=0.12). In conclusion, proteasome and Hsp90i show promising results in the killing of most LIC in B-ALL and T-ALL and further in vivo studies are warranted to explore their full potential. Disclosures:No relevant conflicts of interest to declare.

Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL

Abstract 1882Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures:No relevant conflicts of interest to declare.

An In Vitro model of the Bone Marrow in Paroxysmal Nocturnal Hemoglobinuria Showing a Direct Effect of T-Cells within the Bone Marrow Allowing Clonal Expansion

Abstract 731Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder of hemopoietic stem cells (HSCs). Affected individuals experience intravascular hemolysis and have a predisposition to thromboembolism, renal impairment and pulmonary hypertension. These symptoms vary in severity, but in general, the higher the proportion of PNH blood cells produced, the more severe the symptoms. The molecular pathogenesis in PNH is known to relate to a single gene mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins leading to the symptoms of the disease. The recent development of eculizumab therapy in PNH has had a dramatic impact in reducing both morbidity and mortality in the disease but PNH remains incurable. A sub-optimal response to eculizumab, certainly when assessed by transfusion requirements, is often due to the underlying bone marrow failure that is considered to be universally present in PNH. The factors that lead from the development of a mutant clone to clonal expansion and symptomatic disease are poorly understood but are the key to improving responses and potentially to move towards a cure.Bone marrow failure appears to provide the environment necessary for expansion of PNH clones and small PNH clones are often detected in aplastic anemia and less commonly in myelodysplasia. There is no evidence that PNH HSCs have an intrinsic proliferative advantage compared to normal HSCs and an immune-mediated extrinsic suppression of normal hematopoiesis with a selective advantage for the PNH cells over residual normal stem cells is likely to explain the preferential development of PNH clones concurrent with bone marrow failure.To gain a better understanding of clonal expansion in PNH we have developed an in vitro PNH bone marrow culture model using a stromal cell line which allows PNH stem cells to be maintained in long term cultures and their capacity to form progenitor cells in myeloid colony forming assays to be assessed. We have evaluated bone marrow from 11 patients with PNH (median age 47 years, median granulocyte clone size 95.3%) and 10 normal controls (median age 42 years) within these long term bone marrow culture experiments. Unmanipulated bone marrow mononuclear cells (MNCs), CD34 selected cells and MNCs with their T-cell component depleted were used in this model. This in vitro model provides the environment for PNH stem cells to be maintained for up to eight weeks and, unlike previous studies, produce progenitor cells. When the patient's MNC's are used to seed the culture system there is poor growth of the culture which is only maintained for a median of 2 weeks. However if CD34 selected cells are used then the cultures are maintained for up to 8 weeks (similar to the normal controls) suggesting that there is a component within the MNC's that is responsible for suppressing the marrow culture which is removed by CD34 select. We next selectively removed the T-cells from the PNH MNC's and demonstrated that the marrow cultures now survived to the extent of the controls (see Figure). This demonstrates that the immune suppression in PNH resides in the T-cells. The progenitor cells produced in both the CD34 selected or the T-cell depleted MNCs over the course of the long term culture experiments show an increase in the proportion of normal progenitors the longer the cultures are maintained. This supports the hypothesis that PNH stem cells have no intrinsic proliferative advantage over normal HSCs and that T-cells are the critical cell suppressing the normal hematopoiesis in PNH. We are now examining the specific cell type that causes the myelosuppression in PNH and which will facilitate a targeted approach to the treatment of bone marrow failure in PNH and related disorders.In conclusion we have developed an in vitro PNH bone marrow culture model that allows the immune insult in PNH to be evaluated. Furthermore, this work confirms the important role that T-cells play in the etiology of PNH and provides a model with which to define the exact mechanism of suppression of hematopoiesis in PNH (and aplastic anemia). This information is essential to develop targeted therapies for the marrow failure seen in PNH and aplastic anemia. [Display omitted] Disclosures:Kelly:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Arnold:Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

A Spontaneous Reduction of Clone Size in Paroxysmal Nocturnal Hemoglobinuria Patients Treated with Eculizumab for Greater Than 12 Months.

Abstract 1992Poster Board I-1014Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells that is characterized by bone marrow failure, intravascular hemolysis and venous thrombosis. Eculizumab is a humanized monoclonal antibody that specifically binds to the complement protein C5 preventing its cleavage thereby inhibiting the formation of the terminal components of the complement cascade. Eculizumab was approved by the FDA in 2007 after clinical trials showed it was efficacious in treating patients with hemolytic PNH. Prior to eculizumab therapy treatment options were mainly supportive in nature. Historical data shows that a third of patients who survive greater than 10 years undergo spontaneous recovery.We present data on 38 patients with hemolytic PNH treated at a single centre with eculizumab for 12 months or longer. Thirty six of these patients were treated with a loading dose of 600mg every week for 4 doses followed by 900mg the following week and then a maintenance dose of 900mg dose every 14 day. The other 2 patients required a higher maintenance dose of eculizumab, 1200mg every 14 days, due to symptomatic intravascular hemolysis on the standard regime. All our patients had a high PNH granulocyte clone size at the initiation of eculizumab treatment from 52.90% to 99.95% with a median of 96.38%. The duration of eculizumab therapy varied from 12 to 84 months with a median treatment duration of 50 months.Granulocyte clone size was used as it is not subject to as much variation as the erythrocyte clone size which changes both due to blood transfusions and to the extent of intravascular hemolysis present. The proportion of PNH granulocytes probably most accurately reflects the true size of the PNH clone. Seven out of these 38 patients (18.4%) have had a 10% or greater reduction in their granulocyte clone size during the course of their eculizumab treatment. These patients have had a steady and continued decline in their granulocyte clone size throughout their treatment with eculizumab. This may actually be due to an increase in the residual normal cells in some patients (see Table). Two of these patients (U.P.N. 5 and 7) have had such a dramatic reduction in their clone size that they have been able to stop their eculizumab treatment without any observed detriment to their health.TableChange in PNH clones in patients on eculizumabU.P.N.Months on eculizumabNeutrophils PNH clone size (%)Normal neutrophils (%)Pre-treatmentMost recent on treatmentPre-treatmentMost recent on treatment15097.242.82.85724878.063.222.036.335596.484.13.615.941592.577.07.523.051261.732.438.367.664788.362.511.737.578552.98.547.191.5Two of these 7 patients were treated with ciclosporin for underlying aplasia as compared to 3 of the 31 of those who haven't had a decrease in their clone size. There was no difference in the white cell or platelet count in these 7 patients from when they started eculizumab treatment to the present day indicating that the degree of bone marrow failure present has not changed dramatically during this time course. 5 of the 7 patients had neutrophil clone sizes of less than the median perhaps indicating that recovery requires a certain number of residual normal stem cells to be present. There were no other observed differences to distinguish between patients whose clone size fell and those that did not.It is unlikely that eculizumab has a direct effect on clone size in hemolytic PNH. A more probable hypothesis is that the immune selection in favour of the PNH clone expires over time allowing normal hemopoietic stem cells to repopulate the bone marrow. Whether eculizumab has any influence on this rather than just allowing patients to survive and remain well until recovery occurs is not clear. Our data suggests that there needs to be some normal hematopoietic activity in order for the normal marrow cells to expand and clone size under 95% predicts for recovery.In conclusion, a significant minority of patients with PNH on eculizumab have a progressive reduction in the size of their PNH clone during therapy and in some of these patients the clone falls to a level at which eculizumab can safely be stopped. Disclosures:Kelly:Alexion Pharmaceuticals: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria. Hill:Alexion: Honoraria. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Effect of Parthenolide on Stem Cells in Childhood Acute Lymphoblastic Leukaemia.

Current therapies for the treatment of childhood acute lymphoblastic leukaemia (ALL) have resulted in vastly improved survival rates of around 80% in recent years. Despite these successes, around 15% of patients die of the disease and relapse is the most common cause of treatment failure. Intensification of treatment to prevent or treat relapse may not be a feasible approach due to an increased risk of significant adverse effects. It is possible that ALL may be maintained by a subpopulation of stem cells that are resistant to regimens designed to kill the bulk population and subsequent relapses may arise from these stem cells. Consequently, there is a need to assess the efficacy of therapeutic agents on ALL stem cells. We have previously shown that ALL cells that are capable of initiating and sustaining the disease in serial xenografts have a CD34+/CD19− phenotype. Furthermore, these putative ALL stem cells were resistant to treatment in vitro with dexamethasone and vincristine, two agents routinely used in the treatment of paediatric leukaemia. In this investigation we have examined the effects of the sesquiterpene lactone parthenolide (PTL), a natural compound which induces oxidative stress and inhibits NF-kB. PTL effectively eradicates stem cells in AML and B-CLL in vitro while sparing normal heamopoietic cells. Unsorted leukaemia cells from 11 cases, with mixed prognostic subgroups, were co-cultured with and without PTL at a range of 0–10μM for 18–24 hours. Cell viability and apoptosis were evaluated by flow cytometry using annexin V and propidium iodide staining. Four out of the 11 cases were relatively resistant to treatment with PTL with only small reductions in viability (<5%) and no significant effect on apoptosis, even at the highest dose evaluated. There was no correlation between the prognostic risk group and the response to PTL. Primary cells from the 4 resistant cases were sorted into CD34+/CD19+ and CD34+/CD19− subfractions to assess the effect of PTL on these cells. The effects of PTL on the CD34+/CD19+ population were similar to that observed with the unsorted leukaemia cells. The CD34+/CD19− population was completely resistant to treatment with PTL, with more cells surviving treatment than the unsorted cells (P=0.03). In the 7 responding cases, the viability of the unsorted cells decreased to 28.4±7.1% and 38±12% were apoptotic following treatment. Very similar effects were observed with the CD34+/CD19+ subfraction in these responding cases with viability reduced to 33.4±6% and 35.9±14% were apoptotic. In contrast, the CD34+/CD19− cells from these 7 cases were significantly resistant to PTL with viabilities >75% at all concentrations evaluated (P<0.003). Apoptosis was 2.6-fold lower at 10μM PTL in the CD34+/CD19− subfraction compared to the unsorted cells (P=0.05). FISH analyses were performed on the viable cells at the end of the time-course and confirmed that leukaemia cells were surviving treatment with PTL. CD34+/CD38− cells from normal peripheral blood samples were also found to be resistant to treatment with PTL and survival of these cells at 10μM was not significantly different to that observed in the CD34+/CD19− ALL population in all 11 cases (P=0.38). Studies to assess the functional capacity of PTL-treated ALL cells are ongoing. These data demonstrate that while PTL shows promising effects on the bulk leukaemia population in some ALL cases, it had no significant effect on the putative ALL stem cell population. In each case examined, the CD34+/CD19− cells were resistant to short term exposure to PTL and responded in a similar manner to normal haemopoietic stem cells. These findings highlight the importance of evaluating therapeutic agents in the context of leukaemia stem cell populations and not just on the bulk leukaemia. Future studies are warranted to gain insight into how the drug sensitivity of ALL stem cells may be mediated.

The Investigation of HMGA2 Dysregulation and Promoter Mutations in PIG-M in the Molecular Pathogenesis of Paroxysmal Nocturnal Haemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells (HSC) that is characterized by intravascular hemolysis and venous thrombosis. The PNH HSC's (and their progeny) have a somatic mutation of the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins, which in turn produces the symptoms of the disease. PIG-A mutations account for all of classical acquired PNH cases thus far reported as a single acquired PIG-A mutation on the only active X-chromosome results in GPI-deficiency. A novel mutation in the promoter of the phosphatidylinositol glycan class M gene (PIG-M) has recently been reported in two unrelated families exhibiting autosomal recessive inheritance of congenital GPI-deficiency. This point mutation reduces PIG-M transcription and causes partial deficiency of GPI-anchored proteins. It is possible that this point mutation in PIG-M occurs at low frequency in the population but predisposes to mutation of the other PIG-M allele and could result in acquired PNH. To determine the prevalence of the PIG-M in classical acquired PNH the PIG-M promoter region spanning the proposed mutation was sequenced to look for heterozygotes with the C → G substitution at position −270. This mutation was not identified in 36 patients with PNH. There is convincing evidence that GPI-deficient HSC's have no intrinsic proliferative advantage over normal HSC's suggesting that other factors underlie the clonal expansion of PNH cells. The most likely explanation appears to be due to factors which are extrinsic to the PNH clone such as immune attack on normal HSC. Recently secondary molecular events within the PNH clone have been suggested to provide the growth advantage allowing expansion of the PNH clone. One such proposed event is a translocation affecting the HMGA2 gene, which has been described in 2 cases of PNH. HMGA2 is a member of the high motility group of proteins and acts as an architectural transcription factor. HMGA2 proteins are associated with gene activation and are mainly expressed during embryonic development. Rearrangements of HMGA2 commonly occur in benign mesenchymal tumours, but have also been identified as infrequent events in myeloid malignancies. To gain insight into the potential role of HMGA2 deregulation in the pathogenesis of PNH we evaluated the expression of HMGA2 mRNA by means of quantitative RT-PCR in peripheral blood samples of 42 PNH patients (median age: 42, range: 18–81) and ten normal controls. All PNH samples had large granulocyte clones (median size 97.81%) but showed no increase in HMGA2 mRNA. In nine of the 42 PNH patients blood samples underwent additional enrichment for CD15 positive cells to maximize the proportion of PNH granulocytes present. This again showed no aberrant expression of HMGA2 mRNA. This is the largest reported group with PNH evaluated for HMGA2 expression. Despite two case reports suggesting that the deregulation of HMGA2 may be a pathophysiological factor in occasional cases of PNH our data indicates that this mechanism accounts for the growth advantage of PNH cells in, at most, only a small minority of patients. Similarly, the PIG-M promoter mutation observed in congenital GPI-deficiency was not found in any of our patients with classical acquired PNH indicating that it has no or little role in the pathophysiology of PNH.

Effects of structural and “mixed” isomers of Glu-Trp dipeptide on normal hemopoietic stem cells

We studied the effects of optical (dd-, ll-, dl-, and ld-dipeptides with alpha-bond, EW) structural isomers and cyclic (dd-, ll-, dl-, and ld-dipeptides with gamma-bond, iEW) analogs of Glu-Trp synthetic dipeptide on the population of normal hemopoietic stem cells. Dipeptides containing lGlu (lGlu-lTrp, lGlu-dTrp) injected to mice were inert towards committed bone marrow CFU-S; dGlu-containing dipeptides (dGlu-dTrp, dGlu-lTrp) inhibited the growth of CFU-S-8; and LiGlu-dTrp stimulated these cells. Inhibitory or stimulatory effects of optical and chemical isomers of Glu-Trp dipeptide are determined by optical orientation and nature of peptide bond of Glu residue. The effects of cyclic and mixed peptides towards colony formation are similar to those of the corresponding linear dipeptides.

Detection of a CD133+/CD38− SL-IC Population in Childhood B Cell Precursor ALL.

Clonality studies of immunoglobulin rearrangements in B cell precursor acute lymphoblastic leukaemia (BCP ALL) has suggested that the disease may arise in cells already committed to the B cell lineage. In contrast, Ph+ ALL, which has a less favourable prognosis, is thought to arise in a more primitive haemopoietic cell. This was confirmed recently by studies that demonstrated only the CD34++/CD38− subfraction from Ph+ cases could engraft NOD/SCID mice. However, more recently there has been an increasing body of evidence to suggest that pre B and common ALL may also arise in a cell with a primitive phenotype. We have previously demonstrated that in childhood BCP ALL, the cells capable of long term proliferation in vitro in suspension culture and in vivo to engraft NOD/SCID mice are CD34+/CD10−, CD19−. We then attempted to further define these ALL progenitor cells by investigating the expression of CD133, the primitive stem cell marker. ALL cells capable of long term proliferation in vitro and NOD/SCID engrafting capacity were derived from the CD133+/CD19− subfraction only. These cells were capable of secondary NOD/SCID repopulation, demonstrating they had self-renewal ability. Here, we have attempted to further characterise these ALL progenitor cells to address the question as to whether BCP ALL arises in a common lymphoid progenitor cell or in a more primitive haemopoietic cell. ALL cells from five patients were sorted for CD133+/CD38+ and CD133+/CD38− populations, the sorted subfractions were analysed by cytogenetics and their functional ability was assessed in the NOD/SCID mouse model. Cytogenetic analyses by FISH revealed that both CD133+/CD38+ and CD133+/CD38− subfractions contained the BCR/ABL and ETV6/RUNX1 gene fusions, which had been detected in the patients at diagnosis, and in 1 case with del 17p, this deletion was also noted in the sorted subfractions. These sorted ALL subfractions and unsorted cells were injected intravenously into sublethally irradiated NOD/SCID mice. Bone marrow was harvested at 8–10 weeks post inoculation and analysed for the presence of human cells by flow cytometry. Engraftment was achieved in every case using 2.5x106–107 unsorted cells (0.1–4.5% CD45+). There was no evidence of human cell engraftment in recipients of the CD133+/CD38+ subfraction. However, in each case, engraftment was observed with the CD133+/CD38− subfraction, 0.6–3.2% CD45+ using as few as 6x102–4x104 cells. Using this sorting strategy, we were able to enrich NOD/SCID leukaemia engrafting cells by at least 4 logs compared to the bulk ALL population. Cytogenetic analyses demonstrated that the engrafted cells had the same karyotype as the patients at diagnosis, confirming engraftment of leukaemic cells. These findings suggest that the leukaemia has arisen in a cell with a primitive phenotype, similar to that described for normal haemopoietic stem cells and adds further support to the evidence for a primitive cell origin for B cell precursor ALL. Studies are ongoing to determine whether these primitive ALL cells have the same IgH rearrangements that are detected in the bulk ALL population at diagnosis. This primitive ALL population may be resistant to current chemotherapeutic strategies that are targeted against generic properties of the malignant blasts and subsequent relapses may arise from these cells. Hence, identification and characterisation of these putative ALL stem cells is essential for the development of more effective therapeutic strategies.

Pterostilbene and 3′-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells

Pterostilbene and 3,5-hydroxypterostilbene are the natural 3,5-dimethoxy analogs of trans-resveratrol and piceatannol, two compounds which can induce apoptosis in tumor cells. In previous studies we demonstrated the importance of a 3,5-dimethoxy motif in conferring pro-apoptotic activity to stilbene based compounds so we now wanted to evaluate the ability of pterostilbene and 3,5-hydroxypterostilbene in inducing apoptosis in sensitive and resistant leukemia cells. When tested in sensitive cell lines, HL60 and HUT78, 3′-hydroxypterostilbene was 50–97 times more potent than trans-resveratrol in inducing apoptosis, while pterostilbene appeared barely active. However, both compounds, but not trans-resveratrol and piceatannol, were able to induce apoptosis in the two Fas-ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate). Of note, pterostilbene-induced apoptosis was not inhibited by the pancaspase-inhibitor Z-VAD-fmk, suggesting that this compound acts through a caspase-independent pathway. On the contrary, 3′-hydroxypterostilbene seemed to trigger apoptosis through the intrinsic apoptotic pathway: indeed, it caused a marked disruption of the mitochondrial membrane potential Δ Ψ and its apoptotic effects were inhibited by Z-VAD-fmk and the caspase-9-inhibitor Z-LEHD-fmk. Moreover, pterostilbene and 3′-hydroxypterostilbene, when used at concentrations that elicit significant apoptotic effects in tumor cell lines, did not show any cytotoxicity in normal hemopoietic stem cells. In conclusion, our data show that pterostilbene and particularly 3′-hydroxypterostilbene are interesting antitumor natural compounds that may be useful in the treatment of resistant hematological malignancies, including imatinib, non-responsive neoplasms.

Optimization of Transplant Regimens for Patients with Myelodysplastic Syndrome (MDS)

Myelodysplastic syndrome (MDS) is a hemopoietic stem cell disorder that is potentially curable by transplantation of normal hemopoietic stem cells. The optimum timing, however, and the best conditioning strategy have remained controversial. Both conventional and reduced-intensity/nonmyeloablative regimens have been used successfully. Among selected patients with less advanced/low-risk MDS (< 5% marrow myeloblasts), 3-year survivals of 65% to 75% are achievable with HLA-matched related and unrelated donors. Among patients with more advanced/ high-risk disease (> or = 5% marrow blasts; high International Prognostic Scoring System [IPSS] scores), the probability of post-transplant relapse ranges from 10% to 40%, and, as a result, relapse-free survival is inferior. In addition to disease stage, co-morbid conditions, pre-transplant chemotherapy, conditioning regimen, source of stem cells, and post-transplant immunosuppression affect transplant outcome. Reduced-intensity conditioning regimens are associated with a decrease in non-relapse mortality and allow for successful hemopoietic stem cell transplants even in patients 60-70 years of age. Graft-versus-host disease, both acute and chronic, and post-transplant relapse remain challenging problems.

Monte Carlo modeling of the effects of injected short-, medium- and longer-range alpha-particle emitters on human marrow at various ages.

The effects of injected short-, medium- and longer-range alpha-particle emitters ((149)Tb, (211)At/(211)Po and (213)Bi/(213)Po, respectively) on the total hemopoietic stem cell population of active normal bone marrow in humans of various ages has been estimated using Monte Carlo modeling. The fraction of the normal hemopoietic stem cells that are hit and survive has been calculated as a first step toward estimating the risk of development of therapy-induced leukemia. The fraction was lowest for the shorter-range alpha-particle emitter ((149)Tb) and highest for the longer-range alpha-particle emitter ((213)Bi/(213)Po), with the value for the medium-range alpha-particle emitter (211)At/(211)Po being intermediate between these. There was little variation in the data with the age of the subject within each alpha-particle emitter. This lack of age dependence provides reassurance that the fraction of cells hit in any subject of any age with normal marrow can be estimated by modeling newborn marrow (which requires little computing time) despite age-related differences in microarchitecture.