Lack of Protein Kinase C Alpha Is Associated with Poor Prognosis in Pediatric T-Lineage Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 744-744
Author(s):  
Gloria Milani ◽  
Benedetta Accordi ◽  
Marco Giordan ◽  
Silvia Bresolin ◽  
Luisa Galla ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Lines ◽  
Gene Expression Analysis ◽  
Pediatric Patients ◽  
Lymphoblastic Leukemia ◽  
Kinase C

Abstract Abstract 744 Background: T-lineage Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of pediatric ALL. Although the outcome of T-ALL has improved with current therapies, T-ALL pediatric patients remain at high risk for early relapse. It is therefore very important to identify new prognostic biomarkers and to develop new therapeutic approaches for these patients. More specific and less toxic therapies might be developed through the identification of molecular targets, such as aberrantly activated phosphoproteins, that offer the possibility to use specific kinase inhibitors. In this research we first explored the phosphoproteomic profile of pediatric T-ALL patients at diagnosis, through Reverse Phase Protein Arrays (RPPA), and we further investigated the origin and the functional significance of Protein Kinase C alpha (PKCα) downregulation in patients that are liable to relapse. Methods: We analyzed 98 pediatric T-ALL patients at diagnosis using RPPA. This innovative approach allows to profile phosphorylation modifications and to characterize the activation state of cellular protein networks. The whole proteome of each patient was immobilized on nitrocellulose coated glass slides and each slide was stained with one of 53 different antibodies included in the study. Protein expression/activation was compared between patients subgroups defined by clinical/molecular features through statistical analyses. Gene expression analysis was performed by means of HG-U133 Plus 2.0 array. Permission for this study was obtained following the tenets of the Declaration of Helsinki. A set of commercially available human T-ALL cell lines was studied by Western Blot and cells were treated with Ro-32-0432, a commercial PKC inhibitor. Results: The phosphoproteomic profile of T-ALL patients was compared between patients subgroups at diagnosis. Comparison between relapsed vs non relapsed patients samples revealed the downregulation of PKCα(S657) in relapsed patients (t test with BH multiplicity corrections p=0.02) and Relapse Free Survival analysis through Kaplan-Meier estimates showed that patients with low PKCα(S657) have a high probability to relapse. Notably, 54.5% of patients with low PKCα(S657) relapsed whereas no relapses were observed among patients with high PKCα activity. Multivariate analysis showed no correlation between PKCα(S657) levels and other parameters currently used for clinical characterization of T-ALL patients. Differences in PKCα(S657) between relapsed and non relapsed patients were confirmed by Western Blot in two independent sets of T-ALL patients. Analysis of PKCα total protein form revealed concordance between total protein expression and PKCα(S657) levels. Furthermore, gene expression analysis revealed a significant difference in PKCα gene expression levels, comparable with levels of protein expression among T-ALL patients studied by RPPA. Gene expression data were validated by Sybr Green Real-Time Quantitative PCR. Moreover, gene pathway analysis in a larger cohort of T-ALL pediatric patients, divided by high and low PKCα mRNA levels, revealed the phosphatidylinositol signalling pathway as the most important deregulated pathway. Of note, this pathway is involved in the production of DAG and Ca2+, the PKCα cofactors that play an essential role in mediating its translocation from the cytosol to the plasmatic membrane and subsequent activation. PKCα and PKCα(S657) were then analysed by Western Blot in five T leukemic cell lines (DND41, P12 ICHIKAWA, MOLT3, TALL1 and CEM). These cell lines were treated with a PKCα inhibitor and PKCα inhibition effects were studied by the means of MTT assay and Trypan Blue cell count. An increase in cell proliferation was observed in each treated cell line 9 hours after PKCα inhibition. Conclusions: PKCα is differentially expressed in T-ALL pediatric patients. Low levels of PKCα(S657) are associated with a higher probability to relapse and downregulation or absence of PCKα seems to be related to a more aggressive T-ALL phenotype. Ongoing studies are needed to better define its role as a new prognostic marker. Disclosures: No relevant conflicts of interest to declare.

TYK2-STAT1 Pathway Positively Regulates BCL2 Gene Expression in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1470-1470
Author(s):  
Takaomi Sanda ◽  
Jeffrey W Tyner ◽  
Alejandro Gutierrez ◽  
Vu N Ngo ◽  
Jason M Glover ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Protein Expression ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bcl2 Protein ◽  
Bcl2 Expression

Abstract Abstract 1470 To discover oncogenic pathways that are characteristically deregulated in T-cell acute lymphoblastic leukemia (T-ALL), we performed RNA interference screens both in T-ALL cell lines and primary specimens. We found that the JAK tyrosine kinase family member, TYK2, and its downstream effector, STAT1, are each required for the survival of T-ALL cells. To identify the effector molecules downstream of the TYK2-STAT1 pathway in T-ALL, we analyzed global gene expression profiles in TYK2-dependent T-ALL cell lines after silencing of TYK2 or STAT1. As expected, gene set enrichment analysis revealed that genes downregulated by TYK2 knockdown were generally also downregulated by knockdown of STAT1. Importantly, we found that expression of the anti-apoptotic gene BCL2 was significantly downregulated after silencing of both TYK2 and STAT1. Analysis by quantitative PCR of additional T-ALL cell lines revealed that silencing of TYK2 resulted in significant reductions of BCL2 mRNA expression in multiple TYK2-dependent cell lines. Expression of the wild-type but not the kinase-dead TYK2 protein was sufficient to rescue BCL2 protein expression and to prevent apoptosis after knockdown of endogenous TYK2, indicating that the tyrosine kinase activity of TYK2 is required for BCL2 upregulation. Similarly, expression of the shRNA-resistant wild-type STAT1A protein partially rescued BCL2 protein expression and prevented apoptosis, while a variant of STAT1A (Y701F) that is incapable of becoming phosphorylated on a requisite tyrosine residue did not rescue BCL2 levels. Taken together, our findings indicate that aberrant activation of a TYK2-STAT1 pathway upregulates BCL2 expression in T-ALL cells, and that the T-ALL cells develop pathway dependence, in that they require these sustained high levels BCL2 expression for survival. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HMGB1 Interacts with the MLL-AF4 Fusion Complex to Regulate Pro-Leukemic Gene Transcription in Infant Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2602-2602
Author(s):  
Liana M Toia ◽  
Erica Lynne Braverman ◽  
Jinno Antonio Magno ◽  
Jessica C. Shand
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Western Blot ◽  
Mrna Expression ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Mll Gene ◽  
Gel Shift

Abstract Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target. CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens. Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level. Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot. Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. Disclosures No relevant conflicts of interest to declare.

High CD45 (PTPRC) Expression Is Associated with An Overall Poor Outcome in Childhood Acute Lymphoblastic Leukemia Treated on the ALL-BFM 2000 Protocol and Exerts An Especially Pronounced Effect in Intermediate Risk Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 742-742
Author(s):  
Gunnar Cario ◽  
Rita Mitlohner ◽  
Martin Zimmermann ◽  
Renja Romey ◽  
Peter Rhein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Protein Expression ◽  
Lymphoblastic Leukemia ◽  
Treatment Resistance ◽  
Intermediate Risk ◽  
Prognostic Relevance ◽  
Risk Patients ◽  
Low Expression

Abstract Abstract 742 Further improvement of outcome in childhood acute lymphoblastic leukemia (ALL) could be achieved by identifying additional high-risk (HR) patients who then may benefit from an intensified treatment. In trial ALL-BFM 2000, the HR group was defined by inadequate initial response to induction treatment [poor prednisone response on treatment day eight (PPR), non remission on treatment day 33, and/or a high load of minimal residual disease (MRD, ≥10E-3) after 12 weeks of treatment (TP2)] and/or by positive cytogenetics for a t(4;11) or t(9;22). No MRD already on treatment day 33 defined standard risk (SR) patients, a measurable MRD at a low level characterized the intermediate risk (IR) group. Of importance, the majority of relapses occurred within this heterogeneous group of patients. In order to identify potential new stratification markers we earlier compared gene expression profiles of MRD resistance (HR) and sensitive (SR) ALL in a case-control setting (Cario et al, Blood 2005). Subsequently, we aimed at confirming the potential prognostic relevance of genes identified and their respective proteins in representative study populations. CD45 (also PTPRC, protein-tyrosine phosphatase, receptor-type, C) was one of these candidate genes. In order to assess its prognostic relevance, CD45 gene expression was first analyzed by quantifiable RT-PCR in a set of 555 precursor B-ALL (pB-ALL); its protein expression subsequently in 422 pB-ALL patients by flow cytometry. About one third of patients were included in both study sets. Normalization of protein expression was done by assessing the density of surface expression relative to its density on normal lymphocytes. The 90th percentile was used as a cut-off to distinguish a CD45-high from a CD45-low expression group in both analyses. In gene expression analysis we observed a significant association of a high CD45 expression with a high white blood cell count at diagnosis (WBC) (P = 0.0004), NCI-HR (P = 0.03) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, a high CD45 expression was associated with in-vivo treatment resistance as defined by MRD (P = 0.0025). Analyzing CD45 protein expression confirmed the association of a high expression with a high WBC (P < 0.0001), NCI-HR (P = 0.0002) as well as presence of the MLL-AF4 rearrangement (P < 0.0001). Moreover, although the association to treatment resistance was lower (P = 0.055), patients with a high CD45 expression had a significantly worse 5-years EFS probability of 62±8% compared to 82±2% for those in the low-expression group (P=0.002). Focussing on the IR group, patients with a high CD45 expression had a very poor outcome (EFS 45±15%) as compared to those with a low expression (EFS 86±3%, P < 0.0001). This effect was mainly related to a higher cumulative relapse incidence (55±16% vs. 13±3%, P < 0.0001). Of interest, no significant differences in EFS were seen in HR patients. Based on our results, consideration of CD45 protein expression may serve as additional stratification tool in BFM-based protocols to further refine true non-high-risk patients with a low risk of relapse by identifying additional patients at high relapse risk. Of importance, in view of the fact that CD45 expression was not prognostic in the high-risk group, patients with a high CD45 expression currently treated on non high risk arms, may potentially benefit from an intensified treatment in the HR arm. Disclosures: No relevant conflicts of interest to declare.

Down-Regulation of Mir-26b and Its Inhibition on the Growth of T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2440-2440
Author(s):  
Tian Yuan ◽  
Yaling Yang ◽  
Jeffrey You ◽  
Daniel Lin ◽  
Kefeng Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Expression Level ◽  
Rt Pcr ◽  
Altered Expression ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. With current chemotherapies and transplantation therapy, there are still 25-50% T-ALL patients that suffer from relapse and have a poor outcome. MicroRNAs (miRNAs or miRs) are endogenous small non-coding RNAs (containing about 22 nucleotides in length). miRs function at posttranscriptional level as negative regulators of gene expression and exert their regulatory function through binding to target mRNAs and silencing gene expression. To better understand the pathogenesis and develop the new therapeutic targets of T-ALL, we have developed a Pten tumor suppressor knockout T-ALL mouse model and profiled miRs from the mouse Pten deficient T-ALL. miR-26b was one of the miRs that were found down-regulated in the mouse Pten deficient T-ALL. Recent studies showed that the aberrant expression of miR-26b is implicated in several types of cancer. The expression level of miR-26b and its role of in T-ALL, however, are unknown. We investigated if the expression level of miR-26b is aberrant in T-ALL and the effect of potentially altered expression on the growth of human T-ALL cells. Methods: We conducted miR array profiling to identify differentially expressed miRs in the mouse Pten deficient T-ALLs compared with preneoplastic thymocyte controls. We validated expression levels of several miRs, including miR-26b, that are differentially expressed in mouse and human T-ALL cells using quantitative RT-PCR. We also overexpressed miR-26b using a lentivirus based vector in human T-ALL cell lines to assess its effect on cell growth and apoptosis. Results: Employing miR array profiling, we identified a subset of miRs that exhibited marked altered expression in the mouse Pten deficient T-ALL cells. Quantitative RT-PCR validated that the expression level of miR-26b in the mouse Pten deficient T-ALL cells was markedly lower in comparison to that of preneoplastic thymocytes. To determine if miR-26b expression level is also altered in human T-ALL, we performed quantitative RT-PCR on a panel of human T-ALL cell lines. Indeed, the expression level of miR-26b is significantly lower in the human T-ALL cell lines when compared with that of normal thymocytes. To functionally assess if miR-26b plays a role in the cell growth of human T-ALL cells, we expressed exogenous miR-26b in a panel of human T-ALL cell lines. We demonstrated that the expression of exogenous miR-26b significantly reduced the proliferation and promoted apoptosis of several human T-ALL cell lines. Conclusions: Our results demonstrated that miR-26b is down-regulated in T-ALL and the expression of exogenous miR-26b elicits deceased cell proliferation and increased apoptosis of human T-ALL. These results suggest that miR-26b may function as a tumor suppressor in the development of T-ALL and further characterization of the target and regulation of miR-26b may have therapeutic implications. Disclosures No relevant conflicts of interest to declare.

scholarly journals Precise Modeling of IKZF1 Alterations in Human B-Cell Acute Lymphoblastic Leukemia Cell Lines Reveals Distinct Chemosensitivity, Homing, and Engraftment Properties

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 549-549
Author(s):  
Jason H. Rogers ◽  
Rohit Gupta ◽  
Jaime M. Reyes ◽  
Lorenzo Brunetti ◽  
Michael C. Gundry ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Sanger Sequencing ◽  
Lymphoblastic Leukemia ◽  
Clonal Cell ◽  
Ribonucleoprotein Complexes ◽  
Clonal Cell Lines ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Around 20% of pediatric and the majority of adults with B-cell acute lymphoblastic leukemia (B-ALL) suffer relapse, and prognosis after relapse is very poor. Therefore, identifying those at risk for treatment failure and improving their outcome is imperative. In B-ALL, deletions and mutations of the gene IKAROS family zinc finger 1 (IKZF1) are associated with an increased risk of relapse. IKZF1 encodes the IKAROS protein, which is a master lymphoid regulatory transcription factor and chromatin remodeler. Somatic IKZF1 lesions are thought to be secondarily acquired, arising in lymphoblasts with existing driver genetic lesions, most commonly co-occurring with BCR-ABL1 fusion, activating kinase fusions of Ph-like disease and deregulated DUX4 and ERG. In B-ALL, mono- or bi-allelic deletions of the entire gene, as well as intragenic deletions occur. One of the most common perturbations of IKZF1 in B-ALL is an intragenic deletion of a 50-kilobase (kb) region containing exons 4-7, resulting in the expression of a dominant-negative isoform, IK6. Recently published clinical data show potentially conflicting results over the benefits of therapy intensification in IKZF1-mutant cases (Clappier et al., 2015; Hinze et al., 2017; & Yeoh, et al., 2018). Human cell models of these deletions are needed, as there may be unknown functional differences among mutation types, and the available body of data relies on clinical statistical associations, in vitro RNA interference, viral overexpression of IK6, and mouse models. We used the CRISPR/Cas9 system in the human B-ALL cell lines Nalm-6 and REH by electroporation with sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to generate IKZF1-mutant clones. We identified single cell-derived clonal lines with IKZF1 frameshift mutations in one or both alleles by Sanger sequencing and TIDE decomposition. We confirmed ablation of protein expression by immunoblotting. We treated the IKZF1-mutant clonal cell lines with chemotherapeutic agents commonly used to treat B-ALL and calculated the IC50 by Annexin V/7-AAD double-negative population after 48-72 hour treatment. Compared to IKZF1-wild type Nalm-6 cells, Nalm-6 IKZF1-/- clones exhibited profound resistance to dexamethasone and modest but significant resistance to most other chemotherapeutics tested including vincristine, asparaginase, and daunorubicin. In contrast, these cell lines were more sensitive to the nucleoside analog, cytarabine (Panel A). We next analyzed gene expression profiles by RNA-seq and observed that IKZF1-/- clones are characterized by a stem cell-like gene expression signature and activation of the JAK/STAT pathway (Panel B). Transplantation into immunodeficient NOD scid gamma (NSG) mice demonstrated that IKZF1 deletion leads to enhanced engraftment, significantly increased bone marrow homing, and reduced survival time (Panel D). We also employed a novel CRISPR/Cas9 homology-directed repair (HDR) strategy to generate clonal cell lines expressing IK6 under control of the endogenous promoter, which represents a significant advantage to many previous studies utilizing viral overexpression. We electroporated the cells with sgRNA-Cas9 RNPs along with a 3kb commercially synthesized double-stranded DNA HDR template that knocks-in exon 8 with a GFP tag directly following exon 3. Using this strategy, we were able to isolate heterozygous clones (IKZF1IK6/+) from both Nalm-6 (Panel C) and REH cell lines using flow cytometry sorting for GFP-positive cells. We confirmed precise HDR by Sanger sequencing and immunoblotting. When transplanted into immunodeficient mice, IKZF1IK6/+cells showed delayed engraftment and disease onset, but profound splenic infiltration, consistent with a more indolent, infiltrative disease phenotype (Panels D & E). Ongoing drug treatment assays suggest the chemosensitivity profiles of IKZF1IK6/+ and IKZF1IK6/-clonal cell lines are distinct from their isogenic IKZF1-/-counterparts. Our data support clinical studies reporting that IKZF1-mutated B-ALL is an aggressive, infiltrative, and treatment-resistant disease. Notable differences in drug response and in vivo dynamics in xenografts exist between IKZF1-/-cells and IKZF1IK6/+cells. Detailed delineation of the exact IKZF1 status in ALL patients at diagnosis may be informative in more accurately determining risk stratification and the most effective therapeutic regimen. Disclosures No relevant conflicts of interest to declare.

scholarly journals Engineered Bcor Mutations Lead to Acute Lymphoblastic Leukemia of Progenitor B-1 Lymphocyte Origin in a Sensitized Background

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1331-1331
Author(s):  
Mianmian Yin ◽  
Yang Jo Chung ◽  
R. Coleman Lindsley ◽  
Yeulin Zhu ◽  
Robert L. Walker ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Regulatory Elements ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Indel Mutation

Abstract Chromosomal translocations resulting in NUP98 fusion genes have been associated with a wide spectrum of hematologic malignancies, including MDS, AML, T-ALL, and B cell precursor (BCP) ALL. Based on gene expression profiles and murine transplantation experiments, it is thought that NUP98 fusions can confer aberrant self-renewal potential to hematopoietic cells. Approximately 90% of mice that express a NUP98-PHF23 (NP23) fusion in the hematopoietic compartment, under the control of Vav1 regulatory elements develop AML and/or T-ALL. However, approximately 10% of NP23 mice develop an aggressive acute lymphoblastic leukemia of B1-lymphocyte progenitor origin (pro B-1 ALL). Whole exome sequencing demonstrated that all NP23 pro-B1-ALL had acquired somatic frameshift mutations of the BCL6 co-repressor (Bcor) gene, and most had acquired mutations in the Jak/Stat pathway. To determine whether experimentally engineered Bcor mutations would lead to pro B-1 ALL, we used CRISPR-Cas9 to introduce Bcor indel mutations into NP23 hematopoietic stem and progenitor cells through the use of Bcor single guide RNAs (Bcor sgRNA). Recipient mice transplanted with NP23 bone marrow (BM) or fetal liver (FL) cells that had been transduced with a Bcor sgRNA developed pro B-1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. In addition, similar to some human BCP ALL, the Bcor sgRNA/NP23 murine pro B-1 ALL had acquired somatic mutations in Jak kinase genes. A distinct subset of pediatric BCP ALL are characterized by rearrangement and overexpression of the CRLF2 gene (designated CRLF2r); the CRLF2 gene is the receptor for thymic stromal lymphopoietin (TSLP), a cytokine that plays a role in normal progenitor B1 cell development. The NP23 pro-B1 ALL are similar to CRLF2r BCP ALL in terms of a preferential V heavy chain (VH) usage, gene expression profile, and propensity for acquired JAK/STAT pathways mutations. JAK inhibitors (ruxolitinib and tofacitinib) induced apoptosis and inhibited the growth of pro B-1 ALL cell lines established from Bcor sgRNA/NP23 recipients, at clinically achievable concentrations (10-100 nM). Taken together, these findings demonstrate that a CRISPR-induced Bcor frameshift collaborates with an NP23 transgene to predispose B-1 progenitors to leukemic transformation. These two events are unlikely to be sufficient for leukemic transformation, as we detected spontaneous Jak pathway mutations that were required for continued growth of the leukemic cells. This constellation of mutations (NP23 expression leading to increased stem cell self-renewal, Bcor frameshift leading to impaired B cell differentiation, and Jak pathway mutations leading to dysregulated proliferation) is similar to that seen in human BCP ALL patients, and suggests that the NP23/Bcor mutant mice and cell lines will be a useful model for human pro-B1 ALL. Disclosures Aplan: NIH Office of Technolgy Transfer: Employment, Patents & Royalties: NUP98-HOXD13 mice.

The Methylation Status of p21 Is Not Relevant in Adult Acute Lymphoblastic Leukemia Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4552-4552
Author(s):  
Chiara Gregorj ◽  
Fabiana De Cave ◽  
Maria R. Ricciardi ◽  
Maria C. Scerpa ◽  
Cristina M. Precupanu ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Induction Therapy ◽  
Lymphoblastic Leukemia ◽  
Raji Cell ◽  
Methylation Status ◽  
Philadelphia Chromosome ◽  
Normal Peripheral Blood

Abstract Methylation of CpG islands in the 5′ gene region is associated with transcriptional silencing of gene expression. The hypermethylation of tumor suppressor genes has been described in various tumor tissues, as in gastric and pancreatic cancer, as well as in acute myeloid leukemia, suggesting its potential role in tumorigenesis. Among the three members of the Kip/Cip family of cyclin dependent kinase inhibitors (CKI) p21, p27 and p57, little is known of their methylation status in hematological malignancies and contrasting studies have been reported on the role of p21 hypermethylation in the pathogenesis of acute lymphoblastic leukemia (ALL). The aim of our study was to analyze in primary blasts from adult ALL enrolled in the GIMEMA protocols 0496 and LAL2000 the methylation status of p21, defining in addition its protein expression by Western blot using the monoclonal antibody p21-WAF1 (Santa Cruz, CA). Primary samples from 81 untreated ALL patients were processed using a widely accepted method based on bisulfite modification of DNA, followed by the use of methylation-specific PCR assay (MSP). The human lymphoblastic cell lines (Jurkat, RPMI8866 and CEM), the myeloid cell line OCI-AML3 and normal peripheral blood lymphocytes (PBL) from 10 healthy donors were characterized by a consistent p21 promoter unmethylation (negative controls). In contrast, it was weakly methylated in the Raji cell line and strongly methylated in the Rael (Burkitt’s lymphoma) cell line (positive controls). This assay was further validate in vitro by SsI methylase. In the present study we analyzed 54 B-lineage ALLs, 25 T-ALLs and 2 biphenothypic leukemias; the mean WBC value at diagnosis was 125.6x109/L and 20 samples were Philadelphia chromosome positive. 71/81 of patients studied for p21 methylation were evaluated for response: 53 (74.6%) achieved complete remission (CR) after induction therapy, 8 (11.3%) patients were resistance and 10 (14.1%) died during induction therapy. DNA methylation was not observed in any of the adult ALL patients. p21 protein expression was found in OCI-AML3, Raji and RPMI8866 cell lines, while resulted negative in the Jurkat cell line and in normal PBL. Preliminary results obtained in the ALL samples showed that this protein was expressed in 8/29 (27.6%) cases. In summary, we demonstrated in a large number of primary ALL cases studied at presentation that the p21 gene is not methylated in this population and therefore that the status of p21 methylation does not play a role in the pathogenesis of adult ALL.

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