CRLF2 and JAK2 Mutations: Occurrence and Function In Pre-B ALL Cell Lines

Abstract Abstract 5109 Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells promotes early B-cell development and activates dendritic cells. It has recently been reported that a subset of B-cell precursor acute lymphoblastic leukemia (pre-B ALL) overexpresses the TSLP receptor CRLF2. CRLF2 overexpression is linked to translocations between sex chromosomes – localizing CRLF2 – and the immunoglobulin heavy chain locus on chromosome 14, or to an interstitial deletion on the gonosomes. Both events, translocation and deletion juxtapose CRLF2 to a different promoter (IgH or P2RY8). Performing quantitative real-time PCR we tested pre-B ALL and acute myeloid leukemia (AML) cell lines for overexpression of CRLF2. AML cell lines were included in the screening because we knew from an earlier TSLP project that the AML cell line MUTZ-3 is TSLP-responsive, and thus positive for the cytokine receptor. Three of 63 (5%) pre-B ALL cell lines tested (INC, MHH-CALL4, MUTZ-5) overexpressed CRLF2 mRNA. CRLF2-high cell lines carry a t(14;Y). With respect to the 58 AML cell lines tested: some expressed CRLF2 mRNA, but none of them rivalled the aforementioned pre-B cell lines. Pre-B ALL cell lines show the association between chromosomal CRLF2 aberrations and JAK2 pseudokinase domain mutations that has been described for primary pre-B ALL cells: cell lines MHH-CALL4 (JAK2I682F) and MUTZ-5 (JAK2R683G) and – newly described - also the CRLF2-high pre-B ALL cell line INC express a mutated version of Janus kinase 2 (JAK2R683G). We established a PCR based assay system that allowed for the rapid detection of the JAK2R683G mutation: none of the CRLF2-low or –negative pre-B ALL cell lines exhibited this mutation. All three CRLF2-high/JAK2mu cell lines showed high phosphorylation levels of the JAK2 downstream target STAT5. Inhibition of the JAK kinase led to dephosphorylation of STAT5. However, repression of 3H-thymidine uptake and induction of apoptosis by inhibition of the JAK2/STAT5 pathway was weaker in the JAK2mu pre-B ALL cell lines than in the JAK2V617F positive essential thrombocythemia-derived cell line SET-2. Provided that these results reflect the situation in primary cells, mutated JAK2 seems to be of lesser importance for growth and survival of pre-B ALL cells than for cells from myeloproliferative neoplasms. The CRLF2-high/JAK2mu cell lines INC, MHH-CALL4 and MUTZ-5 are promising model systems for the study of the roles of high-level CRLF2 expression and of JAK2 mutations in pre-B ALL. Disclosures: No relevant conflicts of interest to declare.

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Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽

10.1182/blood.v114.22.1737.1737 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1737-1737

Author(s):

David M. Lucas ◽

Ellen J. Sass ◽

Ryan B. Edwards ◽

Li Pan ◽

Gerard Lozanski ◽

...

Keyword(s):

Drug Resistance ◽

T Cells ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Multi Drug Resistance ◽

Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

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SALL4 Is a Key Survival Factor in Acute B-Cell Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2428.2428 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2428-2428

Author(s):

Shikiko Ueno ◽

Jiayun Lu ◽

He Jie ◽

Ailing Li ◽

XiaoXian Zhang ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mrna Level ◽

Down Regulation ◽

Immunodeficient Mice ◽

Donor Cells ◽

Reh Cells ◽

And Control

Abstract Abstract 2428 SALL4 is a zinc-finger transcriptional factor and a member of the SALL gene family. It plays an essential role in the maintenance of ESC pluripotent and self-renewal properties by interacting with other two key regulators in ESCs, Nanog and Oct4. We previously have shown that stem cell factor SALL4 is aberrantly expressed in 75% of acute B-cell lymphoblastic leukemia (B-ALL). We have also shown that SALL4 is aberrantly expressed in AML, and down-regulation of SALL4 in AML leads to significant cell death. In this study, we focused on investigating the functional role of SALL4 in human B-ALL leukemogenesis. We first assessed the SALL4 mRNA level in four B-ALL cell lines (REH, Nalm6, 697, Blin-1) and five primary patient samples by qRT-PCR. We observed SALL4 mRNA in these four cell lines increased when compared to normal human CD34 negative BM cells. Moreover 4 of 5 primary samples showed high level expression of SALL4, suggesting that SALL4 might play a role in B-ALL pathogenesis. Then, we selected a SALL4 expressing B-ALL cell line (REH and Nalm6) and attenuated SALL4 expression through GFP-labeled shRNA approach in these cell lines. We monitored the growth of SALL4 knockdown and control REH and Nalm6 cells through MTS assay. SALL4 knockdown cells had a decreased growth rate compared to that of the control cells. We also stained SALL4 knockdown and control cells with Annexin V and 7-AAD by flow cytometric quantitation of apoptotic cells. The percentages of apopotic cells in SALL4 knockdown cells were much higher than these in controls. These data demonstrated that inhibition of SALL4 in REH cells and Nalm6 cells led to reduced proliferation and increased apoptosis. We then examined the oncogenesis ability of SALL4 knockdown REH cells in a mouse xenotransplantation model. SALL4 knockdown or control REH cells were injected intravenously into immunodeficient mice. All the recipients succumbed to fatal leukemia within 4 to 6 weeks post transplantation. In both BM and spleen of SALL4 knockdown recipients the engrafted proportion of GFP+ cells was significantly decreased compared to the initial donor cells. Whereas, in both BM and spleen control recipients the percentage of GFP+ REH cells engrafted was similar to that of initial donor cells. This suggests that down-regulation of SALL4 is essential for B-ALL engraftment. To rule out the observed engraftment defect was due to homing, we next performed homing assay. SALL4 knockdown or control cells were injected intravenously into immunodeficient mice as well. Three hours of the injection, mice were sacrificed and analyzed the percentage of GFP+ cells in BM and spleen by flow cytometry. There was no difference among SALL4 knockdown and the control. Furthermore, we performed gene expression profiling on apoptosis-related genes in SALL4 knockdown and control REH cells. The result showed that in SALL4-knockdown REH, TNF mediated cell apoptosis pathways was up-regulated as well as multiple caspase members. The expression of Caspase 3, Caspase 8, FADD was up-regulated in both SALL4 knockdown REH and Nalm6 when compared to these controls, and was verified by real time RT-PCR. This suggests that SALL4 could repress apoptosis through the TNF signal pathway. In summary, we report a novel SALL4/TNF pathway in maintaining cell survival in B-ALL. Disclosures: No relevant conflicts of interest to declare.

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Enzastaurin Induces Apoptosis and Cell Cycle Arrest in B-Cell Acute Lymphoblastic Leukemia Cell Lines Through AKT Pathway Inhibition and ß-Catenin Accumulation

Blood ◽

10.1182/blood.v120.21.1350.1350 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1350-1350

Author(s):

Nakhle Saba ◽

Magdalena Angelova ◽

Patricia Lobelle-Rich ◽

Laura S Levy

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Growth Inhibition ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Time Dependent ◽

Cell Growth Inhibition ◽

Downstream Target ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 1350 Precursor B-Cell acute lymphoblastic leukemia (B-ALL) is the most common leukemia in children and accounts for 20% of acute leukemia in adults. The intensive induction–consolidation–maintenance therapeutic regimens used currently have improved the 5-year disease free survival to around 80% in children and to 25%-40% in adults. The poorer response in adults is due to the inability to tolerate the intensive chemotherapy, and to the biology of adult disease which is associated with poor-risk prognostic factors. In the present era of target-specific therapy, protein kinase C beta (PKCß) targeting arose as a new, promising, and well-tolerated treatment strategy for a variety of neoplasms, especially in B-cell malignancies. The most frequently examined drug candidate to date is enzastaurin (LY317615.HCl) (ENZ), an acyclic bisindolylmaleimide that is orally administered and selectively inhibits PKCß. PKCß plays a major role in B-cell receptor signaling, but studies describing the role of PKCß in B-ALL are primitive. In the present study, we investigate the effect of ENZ on a variety of B-ALL cells representing the wide spectrum of the disease. Seven B-ALL cell lines were studied: RS4;11 and SEM-K2 [both Pro-B ALL with t(4;11)(q21;q23)], TOM-1 and SUP-B15 [both Ph-positive Pro-B ALL with t(9;22)(q34;q11)], HB-1119 [Pre-B ALL with t(11;19)(q23;p13)], NALM-6 [Pre-B ALL with t(5;12)(q33;p13)], and Reh [Pre-B ALL with t(12;21)(p13;q22)]. Cells were tested against serial dilutions of ENZ (final concentrations: 0.5–20μM) for 24, 48, and 72 hours in flat bottom 96-well plates. MTS assay was performed to quantify cell viability. ENZ induced a dose and time-dependent cell growth inhibition in B-ALL cell lines. RS4;11, SEM-K2, and HB-1119 (all with translocations involving the MLL gene) showed the greatest sensitivity to ENZ, with statistically significant cell growth inhibition starting at 1 μM, a concentration easily achieved in-vivo. TOM-1 and SUP-B15, both Ph-positive ALL, showed the lowest sensitivity to ENZ. The mechanism of ENZ cell growth inhibition was shown by flow cytometric TUNEL assay to involve apoptotic induction and cell cycle inhibition. Because of its relatively high sensitivity to inhibition among B-ALL cells, RS4;11 was selected for further analysis of the effect of ENZ on phosphorylation of AKT and its downstream target GSK3ß. RS4;11 cells were treated with the corresponding IC50 of ENZ for 0.5, 1, 2, 4, 24, and 48 hours. Treatment resulted in a time-dependent loss of AKT phosphorylation, at both ser473 and thr308, and a decrease in GSK3ß phosphorylation starting after 30 minutes and continuing to 48 hours. No effect on total AKT and GSK3ß was observed. By activating GSK3ß, its downstream target ß-catenin was expected to be diminished secondary to phosphorylation and proteasomal degradation. Surprisingly, ENZ induced a rapid and sustained ß-catenin accumulation, in both its nuclear and cytoplasmic forms. This was explained by a transient loss of ß-catenin phosphorylation at ser33-37; no effect on the proteasome activity was observed. Similar effect on total and phosphorylated ß-catenin was observed in all other cell lines. ß-catenin represents a central component of Wnt/ß-catenin canonical pathway which is found to be implicated in ALL pathogenesis. To investigate the effect of ENZ on Wnt/ß-catenin pathway, total RNA (1 μg) from RS4;11 treated for 24 hours with ENZ was profiled on RT2 Profiler™ PCR Array Human WNT Signaling Pathway (SABiosciences) and compared to untreated control. There were 8 genes whose expression changed >3-fold, most prominently c-Myc, c-Jun, and several genes encoding Wnt proteins. This was confirmed by western blot analysis showing that treatment with ENZ resulted in decreased c-Myc and increased c-Jun proteins expression. The latter showed a preliminary effect on p73, a p53 homologue, and is a subject for further investigation. These results indicate that PKCß plays an important role in the malignant process in B-cell ALL, and suggest that ENZ should be considered as a potential treatment, whether in combination or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCß inhibition, explain the contribution of ß-catenin accumulation to the cytotoxic effect of ENZ, and possible relationships between PKCß signaling and 11q23 translocation. Disclosures: No relevant conflicts of interest to declare.

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Characterization of lincRNA BALIR-6 in MLL rearranged B-lymphoblastic leukemia

Blood ◽

10.1182/blood.v122.21.3730.3730 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3730-3730

Author(s):

Norma Iris Rodriguez-Malave ◽

Weihong Yan ◽

Giuseppe Basso ◽

Martina Pigazzi ◽

Dinesh S. Rao

Keyword(s):

Gene Expression ◽

B Cells ◽

B Cell ◽

Cell Lines ◽

Microarray Data ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Regulate Gene Expression ◽

In Cis ◽

Regulate Gene

Abstract A new class of non-coding RNA, known as long intergenic non-coding RNAs (lincRNAs), has only recently been described. These lincRNAs have been found to play a role in various molecular processes within the cell including gene regulation, acting as sinks for microRNAs, and regulating splicing, implicating them in development and oncogenic processes. B lymphoblastic leukemia (B acute lymphoblastic leukemia; B-ALL), a malignancy of precursor B-cells, harbors mutations and translocations that result in a dysregulated gene expression. Interestingly, dysregulated expression of lincRNAs has been found in various cancers, but has not yet been described in B-ALL. Recently, we completed a gene expression profiling study in human B-ALL samples, which showed differential lincRNA expression in samples with particular cytogenetic abnormalities. This led us to hypothesize that lincRNAs may be related to disease pathogenesis. Here, we describe a promising lincRNA from our microarray data designated B-ALL associated long intergenic RNA 6 (BALIR-6). Expression of BALIR-6 is highest in patient samples carrying the MLL rearrangement (n=16; when compared to patients with TEL-AML1-translocated, n=39; E2A-PBX1-translocated, n=8; BCR-ABL-translocated, n=3; and cytogenetically normal cases, n=56; 1-way ANOVA p<0.0001) and showed significant variance in the expression level based on the immunophenotype (1-way ANOVA p=0.0004). BALIR-6 is located on chromosome 3p24.3 in humans, and exists in a syntenic gene block in with neighboring genes SATB1 and TBC1D5, and is conserved in mammals. Rapid Amplification of cDNA Ends (RACE) uncovered multiple transcript isoforms; from these, three were cloned out and sequenced, corresponding to the genomic locus as predicted. In B-ALL cell lines, BALIR-6 expression was highest in RS411 cells, which carry the MLL rearrangement, when compared to other B-ALL cell lines. This suggests that the cell lines may show a similar expression pattern to human B-ALL samples. To study the functional role of BALIR-6 we utilized siRNA in a mmu-miR-155 expression cassette to knockdown the transcript. In RS411 cells we observed a reduction in proliferation by MTS assay. Additionally, we observed an increase Sub-G0 cells and a decrease in G2-M phase cells by propidium iodide staining, suggesting an increase in apoptosis. Conversely, overexpression of BALIR-6 in a mouse pre-B cell line (70Z/3), leads to an increase in proliferation. Interestingly, during normal B cell development, BALIR-6 is dynamically expressed, with high expression in pre-B cells and subsequent downregulation, suggesting that a normal role during development is being hijacked in patients with B-ALL. Mechanistically, a few recent studies have described that lincRNAs can regulate gene expression in cis. To explore whether BALIR 6 regulates surrounding genes in cis, we analyzed microarray data of MLL rearranged B-ALL samples, finding that expression of BALIR-6 correlates with expression of surrounding genes SATB1 and TBC1D5. Interestingly for SATB1, this correlation is also seen in human B cell developmental stages. Altering BALIR-6 expression by siRNA mediated knockdown or overexpression causes an effect on the expression of surrounding genes SATB1 and TBC1D5. Previous findings have shown that dysregulated SATB1 has been seen in a variety of malignancies, suggesting a mechanism for how BALIR-6 may produce the changes in cell growth and apoptosis described above. Altogether, these results identify a novel and interesting RNA transcript with the potential to regulate gene expression and pathogenesis in B-ALL with MLL rearrangement, suggesting novel diagnostic, prognostic, and therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

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Spontaneous Mutations of Bcor and Jak1/2 genes Lead to an Aggressive Leukemia of B-1 Progenitor B Cells

Blood ◽

10.1182/blood.v124.21.3573.3573 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3573-3573

Author(s):

Sheryl M Gough ◽

Liat Goldberg ◽

Marbin Pineda ◽

Robert L Walker ◽

Yuelin J Zhu ◽

...

Keyword(s):

High Risk ◽

B Cell ◽

Progenitor Cell ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Hot Spot ◽

Lymphoblastic Leukemia ◽

Hematologic Malignancies ◽

Janus Kinase ◽

Hematopoietic Stem

Abstract NUP98 gene fusions, generated by non-random chromosomal translocations, are associated with a wide spectrum of high risk hematologic malignancies and have been shown to alter normal hematopoietic stem and progenitor cell (HSPC) gene expression programs. A recurrent t(11;17)(p15;p13) translocation in patients with AML leads to the production of a NUP98–PHF23 (NP23) fusion gene. The consequent NP23 fusion protein retains the PHD domain, known to bind H3K4me3, and is thought to have aberrant chromatin regulation properties. We have generated a transgenic mouse model of the NUP98-PHF23 gene fusion which develops a range of hematologic malignancies, most commonly pre-T LBL and AML. However, approximately 10% of NP23 mice develop an aggressive B-1 progenitor acute lymphoblastic leukemia (pro B-1 ALL). B-1 and B-2 lymphocytes have distinct developmental pathways and are thought to represent arms of the innate and adaptive immune systems, respectively. Mature B-2 lymphocytes predominate in the peripheral circulation, and are characterized by expression of B220; whereas B-1 lymphocytes are more prevalent in the pleural and peritoneal cavities, and do not express B220. Murine B cell malignancies typically stain positive for B220, and represent transformed B-2 cells. In the present study, NP23 progenitor ALLs displayed an immunophenotype (Lin-B220- CD19+ AA4.1+) that was identical to that of the recently described B-1 progenitor cell. All B-1 progenitor ALLs exhibited clonal rearrangements of the IgH gene locus. Specifically, these rearrangements involve favored usage of 3’ VH regions, similar to observations with fetal B-1 progenitor cells, further supporting the notion that these are leukemias of B-1 progenitors. Using whole exome sequencing, we found acquired mutations in the BCL6 interacting corepressor (Bcor) gene in 5 out of 7 B-1 progenitor leukemias. The mutations were all frame shift or nonsense mutations, and were located within a 9 bp “hot spot” in Bcor exon 8. In addition, 4 of 7 cases had somatic mutations of Janus kinase 1 (Jak1) or 2 (Jak2), and 7/7 cases showed hyperphosphorylation of Stat3 or Stat5, consistent with the contention that the Jak1/2 mutations are activating mutations, and leading to a hypothesis that the NP23 pro B-1 ALLs which do not harbor Jak1/2 mutations may have acquired an unidentified mutation in the Jak-Stat pathway. Of note, Jak1/2 mutations have previously been identified in a subset of high-risk pediatric B-cell precursor ALL patients. The striking correlation between Bcor and Jak1/2 mutations, occurring specifically in a subset of NP23 leukemias, implies that these three mutations (NP23, Bcor, and Jak1/2) collaborate and provide the oncogenic setting for B-1 progenitor transformation. Disclosures No relevant conflicts of interest to declare.

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New LMO2 Translocations in T-Cell Acute Lymphoblastic Leukemia, Involving STAG2 at Xq25 and MBNL1 at 3q25: A Positive Change?.

Blood ◽

10.1182/blood.v114.22.2641.2641 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2641-2641

Author(s):

Suning Chen ◽

Stefan Nagel ◽

Bjoern Schneider ◽

Maren Kaufmann ◽

Ursula R. Kees ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

B Cell Lymphoma ◽

Small Sample ◽

Upstream Region ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2641 Poster Board II-617 Background: In T-cell acute lymphoblastic leukemia (T-ALL) the LMO2 transcription factor locus is juxtaposed with T-cell receptor (TCR) genes by a recurrent chromosome translocation, t(11;14)(p13;q11). Recent molecular cytogenetic data indicate that unlike classical TCR rearrangements, t(11;14) operates synonymously with submicroscopic del(11)(p13p13) by removing a negative upstream LMO2 regulator (Dik et al., Blood 2007;110:388). The combined incidence of both LMO2 rearrangements is ∼10-15% (Van Vlierberghe and Huret, Atlas Genet Cytogenet Oncol Haematol, November 2007). However, aberrant LMO2 expression occurs in nearly half of all T-ALL cases, a discrepancy which may indicate a significant contribution by cryptic chromosome alterations. We attempted the extended characterization of the LMO2 genomic region in T-ALL cell lines to look for such rearrangements. Cells and Methods: We investigated a panel of 26 well characterized and authenticated T-ALL cell lines using parallel fluorescence in situ hybridization (FISH) with a tilepath BAC/fosmid contig and both conventional and quantitative reverse transcriptase (Rq)-PCR. Global gene expression was additionally measured in some cell lines by Affymetrix array profiling. Results: LMO2 rearrangements were detected in 5/26 (19.2%) cell lines including both established rearrangements, t(11;14) and del(11)(p13p13) in one cell line apiece (3.8%). Interestingly, we found two novel LMO2 translocations: t(X;11)(q25;p13) in 2/26 (7.7%), and t(3;11)(q25;p13) in 1/26 (3.8%) cell lines, respectively. Comparing transcription levels in cell lines with and without genomic rearrangements showed that LMO2 expression was significantly higher in T-ALL cell lines carrying LMO2 rearrangements (P<0.001). Rq-PCR revealed that 5 of the top 10 (50%) LMO2 expressing cell lines carry cytogenetic rearrangements at this locus, compared to 0/16 remaining examples. Loss of a recently defined LMO2 negative regulatory element was identified in the del(11)(p13p13) cell line but no other deletions were detected. Two genes STAG2 at Xq25 and MBNL1 at 3q25 were identified as novice LMO2 partners in t(X;11) and t(3;11), respectively. In both genes breakpoints lay at intron 1 close to deeply conserved noncoding regulatory regions. Both t(X;11) cell lines displayed conspicuous silencing of the ubiquitously expressed STAG2 gene highlighting the transcriptional significance of the region displaced. Unlike t(11;14)/del(11)(p13p13) both new rearrangements carry LMO2 breakpoints in the far upstream region (at minus 80–150 Kbp), and appear to result in upregulation of LMO2 by juxtaposition rather than via covert deletion. STAG2 is a component of the chromosomal cohesin complex which acts as a transcriptional coactivator, and which has been recently identified as a potential driver of oncogene transcription in acute myeloid leukemia (Walter et al., Proc Natl Acad Sci U S A. 2009;106:1295). MBNL1 controls RNA splicing and is a rare BCL6 partner gene in B-cell lymphoma, but this is the first report of its involvement in T-ALL. Conclusion: Given their frequency and variety in a small sample, we propose that cryptic chromosome rearrangements targeting LMO2 upregulation may be significantly more frequent than hitherto appreciated in T-ALL. Unlike canonical LMO2 rearrangements, both t(X;11) and t(3;11) would appear to function positively by upregulation of LMO2 via juxtaposition with noncoding driver elements within these novel partner genes. Perspectives: Future work will address the regulatory potential of candidate enhancer sequences embedded within conserved noncoding intronic sequences of MBNL1 and STAG2. Cytogenetically inconspicuous cell lines displaying LMO2 upregulation will be subjected to more detailed scrutiny using high density genomic SNP arrays. Disclosures: No relevant conflicts of interest to declare.

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Anti-Leukemic Property of Sulphoraphane In Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.3974.3974 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3974-3974

Author(s):

Koramit Suppipat ◽

Xiao Zhu ◽

Chun Shik Park ◽

H. Daniel Lacorazza

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Dependent Manner ◽

Carcinogenic Activity ◽

Childhood All

Abstract Abstract 3974 Acute lymphoblastic leukemia (ALL) is the most common form of hematologic malignancy in children. In spite of significant advances achieved in the treatment of childhood ALL, one fifth of these patients still relapse after the standard treatment. Moreover, relapse ALL is the second most common cause of cancer-related deaths in children. The development of novel therapies is prevented by a limited understanding of the exact pathobiology. There are emerging evidences that the transcription factor KLF4 has a tumor suppressor property in ALL. Recently, a gene expression classifier study in pediatric precursor B-cell ALL (pre-B ALL) showed that KLF4 expression was significantly reduced in high risk ALL patients with positive MRD after induction. This finding suggests a possible role of this cell cycle inhibitor on the development, expansion and drug-resistant of leukemic cells. Several studies demonstrate that overexpression of KLF4 in normal B cells and BCR transformed B cells show increased apoptosis and reduced proliferation. Furthermore, we recently described that KLF4 inhibits proliferation of naïve lymphocytes by activating p21 (Yamada, et al, 2009). Sulphoraphane (SF; 4-methylsulfonylbutyl isothiocyanate) is a dietary compound derived from Cruciferae vegetables with anti-carcinogenic activity in colon cancer by upregulating KLF4 and p21 among other genes. Thus, we hypothesized that SF could also exhibit anti-leukemic activity in human-derived acute lymphoblastic leukemia cells via the activation of KLF4. The pre-B ALL cell lines (Nalm6, REH, RS-4, SUP-B15) and an EBV transformed B cell line were treated with different concentrations of SF (0-40 μM) for 24–48 hours. Then, cell number was estimated using an ATP-based viability method. Flow cytometric analysis of ANNEXIN-V/7-AAD binding as well as CFSE dilution was used to measure apoptosis and proliferation respectively. We found that SF induced cytotoxicity in Nalm-6, REH and RS-4 cell lines in a dose and time dependent manner. This cytotoxic effect was less pronounced in EBV-transformed B cell line. SF treatment led to increased ANNEXIN-V and 7-AAD positive cells (82% apoptotic cells in SF-treated versus 9% in DMSO control). Further, SF-treated cells displayed significantly less proliferation in comparison to DMSO controls thus suggesting that SF inhibits cellular proliferation. Preliminary data also suggest that SF-mediated apoptosis is caused by upregulation of KLF4. In conclusion, Sulphoraphane exhibits an anti-leukemic property by inducing apoptosis and abrogating proliferation in pre-B ALL cell lines. Thus, sulphoraphane could potentially be used as an adjunctive therapy in a subgroup of pre-B ALL patients who have decreased expression of KLF4. Disclosures: No relevant conflicts of interest to declare.

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Quantitative Proteomic Analysis Reveals Maturation As a Mechanism Underlying Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia and PAX5 As a Re-Sensitising Therapeutic Target

Blood ◽

10.1182/blood.v118.21.1411.1411 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1411-1411

Author(s):

Lindsay Nicholson ◽

Caroline Evans ◽

Elizabeth C Matheson ◽

Lynne Minto ◽

Christopher Keilty ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Viability ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Therapeutic Target ◽

Proteomic Analysis ◽

Lymphoblastic Leukemia ◽

Childhood All ◽

Resistant Phenotype

Abstract Abstract 1411 Glucocorticoids (GC), such as prednisolone and dexamethasone, are an integral component of the multi-agent treatment of childhood acute lymphoblastic leukemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome and remains a clinical problem, with the underlying mechanisms still unclear. Mutation or loss of the primary mediator of GC-action, the glucocorticoid receptor (GR), underlies the GC-resistant phenotype in several commonly used leukemic cell lines. However, these events are rare in primary leukemic cells, with relatively few examples in vivo. This suggests that it may be possible to reverse the GC-resistant phenotype pharmacologically. We have used an iTRAQ proteomics approach for hypothesis generation of potential mechanisms for GC-resistance in childhood ALL. To achieve this, we compared a well-characterized GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9), both bearing wildtype GR, in a comparative proteomic experiment using 4-channel isobaric tagging for relative and absolute quantification (iTRAQ). A comparison of protein profiles before and after dexamethasone exposure of the two cell lines identified two transcription factors involved in B-cell differentiation, PAX5 and IRF4, to be differentially upregulated in the PreB 697 compared to the R3F9 cell line in response to GC. Experimentally, there was approximately 50% reduction in PAX5 basal protein expression in R3F9 compared to its GC-sensitive parent, a finding which was also evident in four other resistant sub-lines. This was accompanied by a decreased expression of CD19 and CD10, indicative of an increased B-cell maturation state. The reduced PAX5 level in the GC-resistant cell lines was not due to mono-allelic loss or mutation and mRNA levels were not significantly altered, suggestive of a post-transcriptional mechanism for PAX5 protein reduction. Paradoxically, knockdown of PAX5 reversed the GC-resistant phenotype of the R3F9 cell line such that the apoptotic response to dexamethasone was similar to that of the GC-sensitive parent line as measured by Annexin V staining (R3F9: mean 52.22%, SD 12.54%, n=3; PreB 697: mean 67.23%, SD 9.96%, n=3) and cell viability assays. This chemosensitization after PAX5 knockdown was specific to GC, with no difference in cell viability observed in either cell line after exposure to daunorubicin, vincristine or L-asparaginase when compared to negative siRNA or mock controls. This increase in GC-sensitivity was coupled with a significant upregulation of GR and its transcriptional target, GILZ. We also showed an enhanced GC response after PAX5 knockdown in two out of eight primary, diagnostic pre-B lineage ALL patient samples. Thus, in this ALL cell line model, quantitative proteomic analysis revealed increased maturation as a recurrent mechanism underlying GC-resistance and identifies PAX5 as a possible therapeutic target to fully re-sensitise GC-response in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

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Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

Blood ◽

10.1182/blood.v120.21.4865.4865 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4865-4865 ◽

Cited By ~ 1

Author(s):

Aradhana Awasthi Tiwari ◽

Janet Ayello ◽

Carmella van de Ven ◽

Danielle Glassman ◽

Anthony Sabulski ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Dismal Prognosis

Abstract Abstract 4865 Background: Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective: To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods: Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results: GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1) GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion: Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures: No relevant conflicts of interest to declare.

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Drug-Induced, NKG2D-Dependent Sensitization of ALL Cell Lines to NK Recognition.

Blood ◽

10.1182/blood.v114.22.2024.2024 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2024-2024

Author(s):

Laura Spence ◽

Sophie Hambleton ◽

Venetia Bigley ◽

Sarah Pagan ◽

Matthew Collin

Keyword(s):

Drug Treatment ◽

Cell Line ◽

Cell Lines ◽

Sodium Valproate ◽

Histone Deacetylase Inhibitors ◽

Nk Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Published Data ◽

Drug Induced

Abstract Abstract 2024 Poster Board II-1 The combination of tumor-sensitizing drugs with NK cell infusion is beginning to emerge as a novel anti-tumor therapy. A growing body of in vitro studies show that drugs such as proteosome inhibitors, histone deacetylase inhibitors and thiazolidinediones are able to sensitize tumor cells but not their healthy counterparts to NK-mediated lysis. Drug induced NK-sensitization has shown promise in acute myeloid leukemias but no studies have yet proven this principle in acute lymphoblastic leukemia (ALL); a tumor phenotype reported to be relatively NK-resistant. The mechanisms underlying sensitization have not been fully identified but up regulation of ligands for TRAIL and the NK activating receptor NKG2D: MICA MICB and the UL16-binding proteins, may have a role. We set out to explore ALL susceptibility to NK cytotoxicity and whether this could be modulated by drug treatment. In contrast to published data, untreated ALL cell lines were positive for surface expression of MICB and ULBP2. Median fluorescence intensity ratios (mean ± SD; n = 6) for MICB detection on the cell lines 697, NALM-6, BV173 and SEM were: 3.2 ± 0.9; 3.8 ± 1.3; 4.0 ± 0.5; 2.5 ± 0.9, respectively and for ULBP-2: 2.3 ± 0.4; 55 ± 4.9; 2.9 ± 0.2; 1.8 ± 0.4, respectively. NALM-6 was also positive for ULBP1 (3.3 ± 0.6) while all were negative for MICA and ULBP3. Susceptibility of untreated ALL lines to NK mediated killing was assessed by chromium release assay using an IL-2 stimulated primary NK cell line. At effector to target ratio 40:1, specific release was 2.3% with cell line 697, 12% with NALM-6, 36% with BV173 and 63% with SEM. These results correlated with CD107a exposure in a degranulation assay using IL-2 stimulated peripheral blood lymphocytes: specific degranulation (% CD107a+ target with effector minus %CD107a+ effector alone) was 0.68% (697), 7.1% (NALM-6), 10% (BV173) and 17% (SEM). There was no correlation between baseline expression of NKG2DL and susceptibility to NK killing. Bortezomib, sodium valproate and troglitazone were added to cell cultures at sub-IC50 doses for 48 hours and compared with equimolar vehicle controls. Surface NKG2DL expression was measured by flow cytometry. On NALM-6 troglitazone treatment increased ULBP1 MFI by 2.0 ± 0.33 fold compared with vehicle control and increased percentage of ULBP1 positive cells by 39.6% (paired t-test: p=0.063). Sodium valproate increased MICA expression by 2.91 ± 1.18 fold and percentage of MICA positive cells by 12.3% (p=0.0382). On BV173, sodium valproate treatment increased ULBP2 MFI by 1.55 ± 0.07 fold and percentage of ULBP2 positive cells by 8.6% (p=0.04). There were no significant ligand changes after drug treatment on cell line 697. No NKG2DL changes were seen after Bortezomib treatment on any cell line. The functional significance of NKG2DL changes was assessed by CD107a degranulation assay. NALM-6 treated for 48 hours with drugs yielded the following fold increases in specific degranulation of NK cells compared to NALM-6 vehicle controls: 5.02 ± 5.98 for Bortezomib (mean ± SD), 2.4 ± 0.67 for Troglitazone and 1.44 ± 0.13 for Valproate. Levels of NK degranulation with 697 were very low (<5%) and drug treatment had no effect. Finally, we demonstrated that sensitization of NALM-6 was at least partly dependent on NKG2DL recognition, since blocking antibody to NKG2D reduced CD107a exposure by all three drugs. Compared with controls, blocking reduced CD107a expression by 59 ± 12% for Bortezomib, 47 ± 1.1% for Troglitazone and 48 ±11% for valproate-treated cells. This result was unexpected for Bortezomib as no changes in surface NKG2DL expression were detected after drug treatment. However, we are investigating the possibility that Bortezomib may down-regulate HLA class I expression, thus reducing inhibitory signaling upon NALM-6/NK interaction and unmasking an activation pathway that signals through NKG2D. In conclusion, we found basal levels of expression of NKG2DL on ALL cell lines. There was no correlation between NKG2DL expression and susceptibility to NK lysis, although this was to be expected given that a wealth of other activating and inhibitory receptors that contribute to NK activation. Bortezimib, valproate and troglitazone induced NKG2DL expression and sensitization to NK recognition in a cell line-specific manner. These drugs may therefore be useful to augment conventional chemotherapy or immunotherapeutic approaches to ALL. Disclosures: No relevant conflicts of interest to declare.

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