Differential mRNA Expression Levels of Human  Histone-Modifying Enzymes in Normal Karyotype B Cell Pediatric Acute Lymphoblastic Leukemia

Abstract Purpose: Study was by intention to screen serum autoantibodies that may contribute to the early detection of B-cell acute lymphoblastic leukemia (B-ALL) in children.Patients and methods: The total protein from three pooled B-ALL cell lines(NALM-6, REH and BALL-1 cells) was separated using two-dimensional gel electrophoresis(2-DE), which was followed by Western blot by mixed serum from B-ALL patients (n=20) or healthy children(n=20). We obtained and analyzed the images of 2-D gel and Western blot by PDQuest software,and then identify the spots of immune responses in B-ALL samples compared with those in control samples.The proteins from spots were identified using mass spectrometry (MS). The autoantibodies against α-enolase and voltage-dependent anion-selective channel protein 1(VDAC1) were further validated on the use of enzyme-linked immunosorbent assay(ELISA). The protein expression levels of the candidate antigens α-enolase and VDAC1 in B-ALL were thoroughly studied by immunohistochemical analysis.Results: Six protein dots were identified with MS as Aconitase,apoptosis-inducing factor(AIF),dihydrolipoamide dehydrogenase(DLD), α-enolase,medium-chain acyl-CoA dehydrogenase(MCAD) and VDAC 1.The frequencies of autoantibodies against α-enolase and VDAC1 in children with B-ALL were 27% and 23%, respectively, which were significantly higher than those in normal controls(4% and 0). Immunohistochemical analysis showed the expression of α-enolase and VDAC1 was positive in 95% and 85% of B-ALL patients, respectively, but negative expression levels were showed in the control group. Conclusion: This study incidates that α-enolase and VDAC1 may be the antigen associated with B-ALL .α-enolase and VDAC1 autoantibodies may develop into potential serological markers of B-ALL in children.Other proteins also need to be confirmed in a large number of serum samples.

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Abstract 1576: Identification of six novel candidate miRNAs associated with early relapse in B-cell pediatric acute lymphoblastic leukemia

10.1158/1538-7445.sabcs18-1576 ◽

2019 ◽

Author(s):

Ernest K. Amankwah ◽

Kelly W. Maloney ◽

Eric C. Larsen ◽

Meenakshi Devidas ◽

David T. Teachey ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Early Relapse ◽

Candidate Mirnas

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Different Activation of WNT Signaling Pathway in B-Cell and T-Cell Acute Leukemias.

Blood ◽

10.1182/blood.v108.11.4317.4317 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4317-4317

Author(s):

Muge Sayitoglu ◽

Ozden Hatirnaz ◽

Yucel Erbilgin ◽

Fatmahan Atalar ◽

Ugur Ozbek

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Wnt Signaling ◽

B Cell ◽

Signaling Pathway ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Wnt Signaling Pathway ◽

Hematopoietic Cells ◽

Expression Levels

Abstract WNT signaling pathway proteins function as hematopoietic growth factors and regulate proliferation in normal T-cell and B-cell development. Recent experimental evidence demonstrated that oncogenic transformation in leukemias of both lymphoid and myeloid lineages is dependent on WNT signaling. Not much is known about activation of WNT signaling pathway, its ligands and receptors in hematopoiesis and leukemia pathogenesis. To define its role in leukemia, we aimed to determine mRNA levels of the critical members of WNT pathway (WNT5A, WNT10B, FZ5, β catenin, APC, TCF-1 and LEF-1) by using quantitative real time PCR in acute lymphoblastic leukemia (ALL) patients (T-cell n=42, B-cell n=46 and pre B-cell n=30) and normal hematopoietic cells (bone marrow n=6, peripheral blood n=10, and CD19+ cells from peripheral blood). These genes expressed varying levels in B-cells, preB-cells and T-cells. In the B-cell leukemia patients, WNT5A was expressed notably (OR=58.05 CI 95% 1.63–1219.55, p>0,001). WNT5A directs Ca++ dependent signaling by PKC and a G protein dependent manner which is an alternative pathway for beta-catenin mediated signaling. Also LEF-1 levels were higher in B-ALL patients and APC expression was down regulated when compared to normal tissue (OR=18.81 CI 95% 0.34–5703, p>0.001 and OR=0.212 CI 95% 0.006–8.816, p=0.001, respectively). It is known that LEF-1 blocks APC mediated β catenin nuclear export and activates transcription of various transforming genes, including cyclin, D1, c-myc, MMP7, and LEF-1 itself. WNT5A or WNT10B proteins were not found to be up regulated in preB-ALL whereas APC and LEF-1 gene expressions were increased compared to normal hematopoietic cells (OR=32.97 CI 95% 0.27–1281, 38 p>0.001 and OR=5.57 CI 95% 0.28–89.51, p=0.01, respectively). We found increased TCF-1 expression (7.4 fold) without any β catenin accumulation in T-ALL patients. It is known that TCF-1 in absence of β catenin functions as a tumor suppressor gene. WNT5A, APC and LEF-1 gene expression levels were also different between T-cell, B-cell and preB cell ALL cases. WNT5A expression had the highest levels in B-ALL compared to T-ALL cases, whereas the highest APC expression levels were observed in preB and T-ALL patients. Also LEF-1 expression levels were significantly different between preB and T-cell ALL patients. Taken together these results indicate that WNT signaling genes have abnormal expression and are active in acute lymphoblastic leukemia. This data suggests different WNT activation mechanisms exist in the leukemic transformation in different hematopoietic cells.

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Wilms Tumor 1 Gene Mutations in Adult Acute T-Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v112.11.2516.2516 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2516-2516

Author(s):

Sandra Heesch ◽

Nicola Goekbuget ◽

Jutta Ortiz Tanchez ◽

Cornelia Schlee ◽

Stefan Schwartz ◽

...

Keyword(s):

Mrna Expression ◽

Wilms Tumor ◽

Lymphoblastic Leukemia ◽

Single Amino Acid ◽

Newly Diagnosed ◽

Expression Levels ◽

Wilms Tumor 1 ◽

Standard Risk ◽

Wt1 Protein ◽

Mrna Expression Levels

Abstract The wilms tumor 1 gene (WT1) encodes a transcriptional regulator involved in normal hematopoietic development. The role of WT1 in acute leukemia has been underscored by the finding of WT1 overexpression in subsets of patients (pts) associated with an increased relapse risk. In addition mutations of WT1 have been found in about 10–15% of acute myeloid leukemia (AML) pts and have recently shown to predict inferior survival. Thus far, larger studies have not yet determined the frequency and impact of WT1 mutations in acute T-lymphoblastic leukemia (T-ALL). Herein, we have analyzed WT1 mutations and WT1 mRNA expression levels in a large cohort of T-ALL including 239 newly diagnosed adult pts treated on the GMALL protocols 0699 and 0703. Diagnostic bone marrow specimens were studied for WT1 mutations by DNA sequencing. In addition, samples were immunophenotyped, and mRNA expression of the molecular markers HOX11, HOX11L2, ERG, BAALC, as well as WT1 were determined by real-time RT-PCR. Twenty (8%) of the 239 analyzed T-ALL pts had WT1 mutations (WT1mut) [20 pts had mutations in exon 7 (WT1mut7), with 2 pts having coexisting mutations in exon 9 (WT1mut9)]. WT1mut7 were frameshift or nonsense mutations predicted to result in a truncated WT1 protein, whereas WT1mut9 were missense mutations leading to single amino-acid substitutions. WT1mut and WT1 wildtype (WTwt) pts did not significantly differ with respect to clinical parameters at diagnosis (e. g. age, leukocyte count, and sex). WT1mut cases were characterized by immature features such as an early immunophenotype (45% of WT1mut showed an early T-ALL immunophenotype as compared to only 25% of WT1wt), and WT1mut also showed higher levels of CD34 expression as determined by flow cytometry (WT1mut median: 46% vs. WT1wt median: 2 %; P=0.03). Moreover, WT1mut had significantly higher WT1 mRNA expression levels [WT1mut median: 0.05 (range: 0–0.395) vs. WT1wt median: 0 (range: 0–0.15); P<0.001]. Significant differences were not observed in the complete remission rate nor overall survival or relapse free-survival (RFS) between WT1mut and WT1wt pts. However, in the standard risk group of thymic T-ALL 80% (4/5) of WT1mut relapsed as compared to 28% (25/89) of WT1wt thymic pts [P=0.01; RFS at 18 months: 20% (SE: ±18) for thymic WT1mut vs. 82% (SE: ±4) for thymic WT1wt pts; P=0.008]. In conclusion, in adult T-ALL WT1 mutations are present in 8% of newly diagnosed pts and are located in the same region as reported in AML expected to impair the DNA binding ability of the WT1 protein. Similar to findings in AML, WT1mut cases are characterized by immature features pinpointing to a genetic hit in hematopoietic progenitors likely harboring bilineage potential. The prognostic implications of WT1 mutations in standard risk thymic T-ALL will have to be further validated in independent studies and may in future direct molecularly-based treatment stratification.

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In-Vitro Efficacy of the Deoxyguanoside Analogs Forodesine (BCX-1777) and ARA-G in Pediatric Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.2038.2038 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2038-2038

Author(s):

Irene Homminga ◽

Michel C. Zwaan ◽

Amel Seghouani ◽

Chantal Y. Manz ◽

Shanta Bantia ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Nucleoside Transporter ◽

Nucleotide Synthesis ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Expression Levels ◽

Lc50 Value ◽

Pediatric All

Abstract Abstract 2038 Poster Board II-15 Purine nucleoside phosphorylase (PNP) deficiency in humans is associated with elevated deoxyguanosine (dGuo) plasma levels. DGuo is converted into dGTP inducing apoptosis in T-cells and this provides the rationale for the development of deoxyguanosine analogues as a potential treatment option for T-cell malignancies. Forodesine (BCX-1777; BioCryst-Mundipharma) is an efficient blocker of PNP activity, thereby boosting the conversion of dGuo into dGTP and raising intracellular dGTP levels. AraG (9-b-D-arabinofuranosyl-guanine) is a compound that is resistant to PNP-mediated degradation that is efficiently converted into AraGTP. AraGTP becomes incorporated in the DNA, blocking DNA synthesis and promoting apoptosis. In a phase II clinical trial, the AraG prodrug Nelarabine enforced a complete remission rate of 55% for pediatric T-ALL patients at 1st relapse. (Berg, JCO 2005). Clinical data of Forodesine treatment in pediatric ALL patients are not yet available. As tested on primary pediatric acute lymphoblastic leukemia (ALL) patient samples (4 T-ALL, 2 BCP-ALL), 1μM of Forodesine is sufficient to completely block PNP and abolish rapid dGuo degradation resulting in a median 7.9 (range 0.5-378) fold raise of intracellular dGTP levels. Accumulation of dGTP is comparable for T-ALL (n=31) and BCP-ALL (n=11) patient samples. This reflects equal intrinsic ability of salvage nucleotide synthesis for both T-ALL and BCP-ALL cells. Cytotoxic effect of Forodesine was tested on primary leukemia cells from newly diagnosed pediatric ALL patients in-vitro by incubating cells with Forodesine (1μM) in the presence of increasing concentrations of dGuo (0.001-50μM). In accordance with selective T-cell toxicity, T-ALL cells were more sensitive to Forodesine/dGuo treatment (median T-ALL LC50 value: 1.1μM dGuo/1μM Forodesine, n=27, p=0.001) compared to BCP-ALL cells, which had a median LC50 value of 8.8μM dGuo/1μM Forodesine (n=30). All patients that responded demonstrated dGTP accumulation (1.5-222.1 fold), although the raise of dGTP levels did not correlate with Forodesine/dGuo toxicity (r2= 0.10, p=0.22). Studying in-vitro responsiveness to AraG, T-ALL cells were more sensitive compared to BCP-ALL cells (p=0.0002) with a median AraG LC50 value of 20.5μM for T-ALL samples (n=24) versus 48.3μM for BCP-ALL samples (n=20). Remarkably, TELAML1 positive BCP-ALL cases were insensitive to AraG treatment (median LC50 value >50μM, n=9). No correlation was identified between in-vitro Forodesine/dGuo and AraG cytotoxicities (r2=0.05, p=0.29). Most patient samples that displayed AraG resistance still responded to Forodesine/dGuo treatment. This may be explained by the fact that the uptake of both drugs may be facilitated by different transporters. Using RQ-PCR we could demonstrate that AraG toxicity, in contrast to Forodesine, was significantly associated with ENT1 (equilibrative nucleoside transporter 1) expression levels (p=0.008), which was previously identified as strong predictor for AraC cytotoxicity in pediatric ALL (Stam RW. et al., Blood 2003). AraG cytotoxicity strongly correlated with AraC cytotoxicity (r2=0.71, p<0.0001). We found no significant correlation between Forodesine sensitivity and the expression levels of other nucleoside transporters (CNT1, CNT2, CNT3, ENT2), kinases (dCK, dGK), nucleotidases (NT5C1A, NT5C2, PNI) or other enzymes that are involved in dGuo metabolism (PNP, RRM1, RRM2). In conclusion, T-ALL cells are more sensitive to Forodesine/dGuo treatment in-vitro than BCP-ALL cells that have nearly 8 fold higher dGuo LC50 values. Resistance to AraG treatment does not preclude responsiveness to Forodesine treatment and vice versa, indicating that Forodesine and AraG rely on different cellular mechanisms for cytotoxicity, possibly involving differences in dependence on the nucleoside transporter ENT1. Disclosures: No relevant conflicts of interest to declare.

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Targeting Survivin with YM155 As a Potential Therapy in Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2490.2490 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2490-2490

Author(s):

Abdusebur Jemal ◽

Jeffrey W Tyner ◽

Mathew Thayer ◽

Markus Muschen ◽

Brian J. Druker ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

Survivin Expression ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Pediatric Acute Lymphoblastic Leukemia ◽

Pediatric All

Abstract Abstract 2490 Background: Pediatric Acute Lymphoblastic Leukemia (ALL) remains the most common pediatric malignancy. Despite advances in treatment and outcomes, there continue to be subsets of patients that are refractory to standard intensive chemotherapy and hematopoietic stem cell transplant. Therefore, novel gene targets for therapy are needed to further advance treatment for this disease. Survivin, a member of the chromosome passenger complex and inhibitor of apoptosis has been shown to be over-expressed in malignant cells and in relapsed ALL. Therefore, survivin may be a potential target for therapy in pediatric ALL. The selective survivin suppressant, YM155 (Astellas) has been shown to inhibit survivin expression and activate cell death in multiple cell lines. Early phase I studies show promise in both tolerability and possible efficacy in B-cell malignancies. Therefore, this drug may have the potential of improving treatment for pediatric B-cell precursor ALL. Design/methods: Pediatric lymphoblastic cell lines, fresh primary lymphoblast cells from newly diagnosed patients with ALL and xenografted patient samples were used in this study. Cells were incubated in the presence of YM155 at doses ranging from 1nM to 10μM. Viability was measured using a standard methane-thiosulfonate viability assay. Activation of apoptosis was identified using the Guava nexin Annexin V binding assay for cell lines. Results: Treatment of ALL cell lines, primary patient samples and xenograft samples show a dose-dependent sensitivity to YM155 by both activation of apoptosis and by cell viability. IC50 doses for the majority of the samples are in the low nanomolar range (Table). Interestingly, there is some variability amongst patient samples suggesting possible variable responses in vivo. Ectopic expression of survivin in cell lines treated with YM155 rescues the effect of the drug. Further, t(9;22) positive ALL samples, including primary patient, xenograft, and dasatinib resistant samples remain significantly sensitive to YM155. For dasatinib sensitive Ph+ALL samples, combination therapy suggest an additive effect by isobologram analysis. Conclusion: Pediatric ALL samples remain sensitive to treatment with YM155 in cell lines, primary patient and xenografted samples. The results of these experiments will be used as a foundation to develop a comprehensive understanding of the mechanisms of survivin dependence in pediatric ALL. Future studies will also be designed to develop YM155 as an additional therapy for pediatric acute lymphoblastic leukemia. Disclosures: Druker: Cylene:; MolecularMD:; Novartis:; Bristol-Myers-Squibb:.

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Loss of Pigh Expression Frequently Results in a GPI-Negative Subclone Lacking CD52 Membrane Expression, Conferring Alemtuzumab Resistance to B Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.1435.1435 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1435-1435

Author(s):

Floris C. Loeff ◽

Kevin Rijs ◽

Willem H. Zoutman ◽

Esther H.M. Egmond ◽

Maarten H. Vermeer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Mrna Expression ◽

Lymphoblastic Leukemia ◽

Effective Control ◽

Biosynthesis Pathway ◽

Membrane Expression ◽

Gpi Anchor ◽

Empty Vector ◽

Cell Acute Lymphoblastic Leukemia

Abstract To improve treatment outcome of patients with B cell acute lymphoblastic leukemia (B-ALL), several immunotherapeutic approaches have been developed in recent years. E.g., direct targeting of CD19 or CD20 by (bispecific) antibodies or chimeric antigen receptors result in effective control of the disease. In contrast, introduction of alemtuzumab which targets the glycophosphatidylinositol (GPI) anchored CD52 protein has been less successful. Despite its profound lymphodepleting potential, the clinical efficacy was unsatisfactory. Similar to development of escape variants following anti-CD19 and anti-CD20 treatment, this might have been due to the outgrowth of CD52 negative B-ALL escape variants. Indeed, in previous studies (Nijmeijer et al, 2010) we have found outgrowth of CD52 negative escape variants after alemtuzumab treatment in a mouse model engrafted with human B-ALL. Further analysis showed that these variants expressed normal CD52 mRNA levels, but lacked CD52 membrane expression which was found to be due to the loss of GPI anchor expression, as confirmed by loss of staining with the GPI-specific aearolysin FLAER. The aim of the current study was to further unravel the mechanism underlying loss of CD52 membrane expression in adult B-ALL. To study whether the relatively frequent development of these CD52 negative/GPI anchor deficient escape variants during alemtuzumab treatment was the result of outgrowth of pre-existing GPI negative cells, we analyzed 10 Peripheral Blood (PB) and 8 Bone Marrow (BM) samples from B-ALL patients at the moment of diagnosis. GPI negative cells were present in 7 out of 10 (70%) PB and 5 out of 8 (63%) BM samples, and comprised between 0.01% and 4.98% of the B-ALL population as analyzed by flow cytometry using FLAER and CD52 counterstaining (detection limit 100 cells per million). Interestingly, these obvious GPI negative populations were not found in other B cell malignancies such as chronic lymphocytic leukemia (n=5), mantle cell lymphoma (n=5), hairy cell leukemia (n=6), or in healthy donors (n=5). To investigate the mechanism of GPI loss, gene expression analysis was performed for the 26 genes that comprise the GPI anchor biosynthesis pathway. GPI positive and GPI negative B-ALL populations (n=7) were purified by fluorescent activated cell sorting (FACS). Recurrent loss of PIGH mRNA expression, but of none of the other genes involved in GPI anchor biosynthesis pathway, was found in GPI negative cells but not in GPI+ cells in all cases. To validate the relevance of this finding, GPI negative and GPI+ B-ALL cell cultures were generated from diagnosis material (n=2) and subsequently transduced with a retroviral construct encoding PIGH, PIGA (another member of the GPI-N-acetylglucosaminyl-transferase complex, which catalyzes the first step in the GPI synthesis) or an empty vector. Restored GPI anchor expression and coinciding CD52 membrane expression was observed in the GPI negative B-ALL cultures upon transduction with the PIGH, but not PIGA or empty vector. To explore the mechanism underlying the recurrent loss of PIGH mRNA expression, we performed extensive DNA screening of the GPI negative B-ALL cultures to discover possible mutations or indels in the promoter region, gene body, or at splice sites and compared this to GPI+ B-ALL cultures from the same individual. These analyses revealed that both alleles of the PIGH gene were present, unmutated and intact. To investigate whether epigenetic regulation could be the cause of PIGH deficiency, we measured promoter CpG methylation by bisulfite sequencing, comparing GPI negative and GPI+ B-ALL cultures (n=2). This analysis revealed that the region surrounding the transcription start site (-200bp up to +100bp) was heavier methylated in the GPI negative B-ALL cultures compared to the GPI+ counterparts. Also, a 14 day treatment of GPI negative B-ALL cultures with the demethylating agent 5-Azacitidine resulted in re-expression of the GPI anchor. In conclusion, the majority of B-ALL patients presented a CD52/GPI negative, alemtuzumab resistant, B-ALL population in samples taken at diagnosis. These cells lost PIGH expression, a key component in GPI anchor synthesis. This is not due to genomic instability, but rather to epigenetic down regulation. Combining epigenetic modulatory drugs with alemtuzumab might be a promising therapeutic strategy to prevent outgrowth of CD52/GPI negative escape variants in B-ALL. Disclosures No relevant conflicts of interest to declare.

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