Quantitative Proteomic Analysis Implicates An Altered B-Cell Differentiation State as a Mechanism Underlying GC-Resistance in An Acute Lymphoblastic Leukaemia (ALL) Cell Line Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1736-1736
Author(s):  
Lindsay Nicholson ◽  
Caroline Evans ◽  
Elizabeth Matheson ◽  
Lynne Minto ◽  
Christopher Keilty ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Acute Lymphoblastic Leukaemia ◽  
B Cell ◽  
Cell Line ◽  
Mismatch Repair ◽  
Cell Lines ◽  
Lymphoblastic Leukaemia ◽  
B Cell Differentiation ◽  
Protein Levels ◽  
Resistant Phenotype

Abstract Abstract 1736 Poster Board I-762 Introduction Glucocorticoids (GC) are pivotal agents used in the treatment of childhood acute lymphoblastic leukaemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome in childhood ALL, but the underlying molecular basis remains unclear. Previous studies using cell lines have identified mutation/deletion of the glucocorticoid receptor (GR) as a mechanism of GC-resistance. However, genetic aberration of the GR is rare in clinical samples1. This disparity may be due to the mismatch repair deficient status of many ALL cell lines which consequently have a greater likelihood of acquiring mutations under GC-selection. We have used a discovery proteomics approach for hypothesis generation on potential mechanisms for resistance. To achieve this, we compared a well-characterized mismatch repair proficient GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9) both bearing wildtype GR, in a comparative proteomics experiment using 4-channel isobaric tagging for relative and absolute quantitation (the iTRAQ approach). Methods Cells were treated with either vehicle control or 0.1μM dexamethasone for 24 hours and subjected to subcellular fractionation to prepare a nuclear fraction. Each sample was labelled with a distinct isobaric tag for relative quantification and analysed by 2-dimensional liquid chromatography/ tandem mass spectrometry. The proteins were identified and relatively quantified using Protein Pilot software (Applied Biosystems). Ratios were calculated for dexamethasone-treated ‘versus’ control vehicle for each cell line and an ITRAQ ratio of greater than or equal to ± 1.2 or less than 0.8 fold change were considered to be differentially expressed. Results The comparative dataset highlighted two transcription factors which are involved in B-cell differentiation, PAX5 and IRF4, to be differentially expressed in the PreB 697 compared to the R3F9 cell line. The GC-resistant R3F9 cell line had reduced PAX5 and IRF4 protein expression compared to the parental cell line and this was further validated in other GC-resistant sub-clones derived from the PreB 697 cell line by western blot analysis. The reduced PAX5 level in the GC-resistant cell lines was not due to monoallelic loss, as measured by a QRT-PCR method or mutation as determined by DHPLC analysis of ‘hot-spot’ exons. In addition, PAX5 mRNA levels were not significantly altered, thus suggestive of a post-transcriptional mechanism for PAX5 protein reduction. To test the direct role of PAX5 in GC-resistance, we reduced PAX5 mRNA and protein levels using RNA interference in the parental GC-sensitive, PreB 697 cell line. PAX5 protein levels were reduced by at least 80% and were maintained for 48 hours post-transfection. The PreB 697 cell line was transfected with siRNA directed to PAX5 using electroporation, the cells were allowed to recover for 24 hours and the levels of cell kill were assessed in response to a 48 hour incubation with 1 μM dexamethasone by Annexin V staining and the MTS assay. Paradoxically, PAX5 knockdown increased GC-sensitivity (mean 60.4% apoptosis, S.D. 16.8, N=3) in comparison to a non-specific siRNA (mean 31.0% apoptosis, S.D. 5.2, N=3) but did not influence sensitivity to either vincristine or daunorubicin. Thus, this response was specific to glucocorticoids. Conclusion Using a proteomic approach we have shown alterations in PAX5 protein levels are associated with a GC-resistant phenotype which an mRNA-based technology would fail to detect. Modulation of PAX5 in ALL cells may influence the response to GC-therapy. It is known that GC-sensitivity alters during B-cell development, with early lymphoid precursors being highly sensitive and more mature B cells being highly resistant to GC-induced apoptosis. We propose that reduced PAX5 protein levels may reflect an altered differentiation state of the sub-clones of PreB 697 which are associated with a GC resistant phenotype. 1Irving et al, Cancer Res, 2005 2Schmidt et al, FASEB, 2006 Disclosures No relevant conflicts of interest to declare.

scholarly journals G protein subunit G alpha 16 expression is restricted to progenitor B cells during human B-cell differentiation

Blood ◽  
1995 ◽  
Vol 85 (7) ◽  
pp. 1836-1842 ◽  
Author(s):  
MY Mapara ◽  
K Bommert ◽  
RC Bargou ◽  
C Leng ◽  
C Beck ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Cell Phenotype ◽  
Low Grade ◽  
B Cell Differentiation ◽  
Human B Cell

Recently G alpha 16, a new guanosine triphosphate (GTP) binding protein alpha subunit has been described to be specifically expressed in human hematopoietic cells. Expression of G alpha 16 was observed in human cell lines of myelomonocytic and T-lymphocytic origin, but not in human B-cell lines Raji and IM9. We studied the expression of G alpha 16 in human B cells corresponding to different stages of B-cell differentiation by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The human Burkitt's lymphoma cell lines Raji, Ramos, BJAB, the lymphoblastoid cell line SKW6.4, and the plasmocytoma cell line U266 were devoid of G alpha 16. In contrast, G alpha 16 was detected in the human progenitor B cell lines Reh and Nalm-6. Using the mu+, k-cell line BLIN-1 (pre-B cell phenotype) and its derived subclone 1E8 (surface mu+, k+; B-cell phenotype) G alpha 16 expression was found to disappear on transition from pre-B to B-cell differentiation stage. The analysis of a broad panel of human neoplastic B lymphocytes ranging from progenitor B-acute lymphatic leukemia (pre-pre-B-ALL), common acute leukemias (cALL), pre-B-ALL, mature B-ALL to low grade B-cell lymphoma (chronic lymphocytic leukemia of B-cell type, leukemic centrocytic non-Hodgkins lymphoma [NHL], hairy cell leukemia) showed that G alpha 16 expression is limited to progenitor and pre-B-ALL cells. Therefore, we conclude that within B-cell differentiation, G alpha 16 is expressed solely during early B cell ontogeny and downregulated during differentiation. Thus, G alpha 16 might be an important regulator involved in signaling processes in progenitor B cells.

Lack of PRDM1α Expression in Hodgkin/Reed-Sternberg Cells Is Likely Mediated by a Translational or Post-Translational Mechanism.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4367-4367
Author(s):  
Wayne Tam ◽  
Leonard Tan ◽  
Mario Gomez ◽  
Shaoan Fan ◽  
Yifang Liu ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Line ◽  
Cell Fate ◽  
Cell Lines ◽  
Western Blotting ◽  
Plasma Cell ◽  
Zinc Finger Protein ◽  
Cell Phenotype

Abstract Hodgkin/Reed Sternberg (H/RS) cells are the neoplastic cells in classical Hodgkin lymphoma (HL). They are thought to resemble post-germinal center (GC) B cells with expression of markers associated with late stage of B-cell differentiation, for example, interferon regulatory factor -4/multiple myeloma-1 (IRF4/MUM1) and syndecan 1 (CD138). The PR (PRDI-BF1-RIZ) domain zinc finger protein 1 (PRDM1), a transcription repressor with a master regulatory role in plasma cell differentiation, is normally co-expressed with IRF-4/MUM-1 in plasma cells and in a subset of activated GC cells committed to plasma cell fate. We studied expression of PRDM1α, the functional isoform of PRDM1, in 14 classical HL cases [including 3 positive for Epstein-Barr-virus (EBV)] and 4 HL cell lines by immunohistochemistry and Western blotting, respectively. H/RS cells in primary HL cases are negative for PRDM1α, implying a desynchrony in expression between IRF-4/MUM1 and PRDM1. While the myeloma cell line U266 expresses relatively abundant PRDM1α, it was undetectable by Western Blotting in all HL cell lines tested, except for the EBV-positive HL cell line L591 which, unlike in vivo H/RS cells, has a Type III EBV latency pattern. PRDM1α expression in L591 but not in vivo H/RS cells suggests that PRDM1 expression may be modulated by latency type-specific EBV-encoded gene products or the B-cell phenotype exhibited by the cell line. The lack of PRDM1α protein in H/RS cells is not due to impaired gene transcription, since real-time quantitative PCR revealed similarly abundant PRDM1α transcripts in the HL cell lines as U266. In the absence of mutation in the PRDM1 coding region, these results suggest that failure to accumulate PRDM1α protein in H/RS cells is likely due to abnormal translation repression or protein turnover. Loss of functional PRDM1 as a result of translational or post-translational deregulation may represent a novel molecular lesion in HL.

CtBP1 Is a Novel and Essential Corepressor for BCL6 Autoregulation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3581-3581
Author(s):  
Lourdes M. Mendez ◽  
Jose Polo ◽  
Melissa Krupski ◽  
Jessica Yu ◽  
Ari M. Melnick ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Lines ◽  
Cell Biology ◽  
Target Genes ◽  
Point Mutations ◽  
B Cell Lymphoma ◽  
B Cell Differentiation ◽  
Wild Type ◽  
Negative Autoregulation

Abstract BCL6 is POZ/BTB transcription repressor that is required for the germinal center (GC)- stage of B cell development and its deregulated expression underlies the development of many GC-derived B cell lymphomas such as diffuse large B cell lymphoma (DLBCL). BCL6 carries out its biological function by repressing target genes involved in various aspects of B cell biology such as DNA damage response, cell-cycle regulation and plasma cell differentiation. Recent publications indicate that BCL6 differentially utilizes its corepressor partners to silence target genes involved in different biological processes. Negative autoregulation of BCL6 is likely to play an important role in B-cell differentiation, and is frequently disrupted in DLBCL due to translocation or point mutation of the BCL6 promoter. However, from a mechanistic standpoint, it is not known how BCL6 mediates negative autoregulation. BCL6 is reported to repress its target genes through binding of the SMRT, NCoR and BCoR corepressors to its N-terminal POZ domain and through binding of the MTA3 and HDAC2 corepressors to its second repression domain. However, a BCL6 mutant unable to bind these corepressors retained near wild-type repression activity on the BCL6 promoter. The expression of endogenous BCL6 was unchanged in DLBCL cell lines treated with BCL6 Peptide Inhibitor, which selectively disrupts the association between BCL6 and its POZ domain corepressors, or with MTA3 siRNA. This led us to consider the possibility that BCL6 autoregulation proceeds through a novel corepressor. Several POZ transcription factors can interact with CtBP as their corepressor. We found BCL6 and CtBP can interact in both the ectopically expressed and endogenous settings in DLBCL cells. Moreover, our ChIP experiments demonstrate that CtBP is present in the 5′UTR of BCL6 at sites that were previously shown by us and others to mediate BCL6 negative autoregulation. Nearly half of DLBCL patients are estimated to carry translocations and “activating” point mutations in the 5′UTR of BCL6 which allow negative autoregulation to be bypassed. In DLBCL cell lines carrying BCL6 promoter mutations or translocations, CtBP was preferentially bound to the wild-type BCL6 allele. Moreover, CtBP siRNA specifically derepressed the wild-type allele sparing the translocated BCL6 allele driven by heterologous promoters. This allelic analysis of BCL6 is consistent with a model in which BCL6 recruits CtBP to carry out negative autoregulation. Tiling ChIP-on-chip of BCL6 target genes showed colocalization of CtBP in a BCL6 repression complex at only a subset of target genes, including BCL6. However, the BCL6 locus was the only target dependent exclusively on CtBP for repression. In an effort to address the corepressor requirements of BCL6 autoregulation, we have uncovered a novel BCL6 corepressor, CtBP. Our results substantiate the growing body of evidence that BCL6’s mechanism of repression is dynamic, selectively calling upon corepressors to silence different cohorts of target genes perhaps reflecting segregation of biological functions. Our study provides new insight into normal BCL6-driven biology and also informs BCL6-targeted lymphoma therapies.

Characterization of two novel pre-B-cell lines (LK63 and LiLa-1): Potential models of pre-B-cell differentiation

1992 ◽  
Vol 16 (6-7) ◽  
pp. 655-663 ◽  
Author(s):  
Evelyn Salvaris ◽  
Jürgen R. Novotny ◽  
Karen Welch ◽  
Lynda Campbell ◽  
Andrew W. Boyd
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Differentiation ◽  
Potential Models

A role for calcitonin gene related peptide (CGRP) in the regulation of early B lymphocyte differentiation

1995 ◽  
Vol 73 (7) ◽  
pp. 1057-1064 ◽  
Author(s):  
Joseph P. McGillis ◽  
Vidya Rangnekar ◽  
John R. Ciallella
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Line ◽  
Inhibitory Effect ◽  
Calcitonin Gene Related Peptide ◽  
Mouse Bone Marrow ◽  
B Cell Differentiation ◽  
Mobility Shift ◽  
Related Peptide ◽  
Calcitonin Gene

In previous studies we identified high affinity adenylyl cyclase linked receptors for calcitonin gene related peptide (CGRP) on rat T and B cells, on lymphocyte cell lines including the mouse pre-B cell line 70Z/3, and on cells in mouse bone marrow. The effect of CGRP on early B cell differentiation has been examined using the 70Z/3 cell line. CGRP inhibits the lipopolysaccharide (LPS) induction of surface immunoglobulin (sIg) protein expression in 70Z/3 cells, an effect that is associated with a decrease in the steady-state levels of Ig heavy (μ) and light (κ) chain mRNA. In this report, experiments are described that provide further information on the mechanism by which CGRP inhibits sIg expression. The kinetics of CGRP inhibition of LPS-induced sIg expression was examined in 70Z/3 cells. An optimal window for the inhibitory effect of CGRP on sIg induction occurs at least 24 h after the cells are treated with LPS. To determine whether the inhibitory effects of CGRP on sIg expression are mediated by an inhibition of NFκ-B translocation to the nucleus, electrophoretic mobility shift assays were performed using nuclear proteins from 70Z/3 cells. There was no difference in NFκ-B binding activity in cells that had been treated with LPS or LPS + CGRP, suggesting that the inhibitory effect of CGRP is not mediated by an inhibition of NFκ-B activity. These studies provide further evidence that CGRP plays an inhibitory role in early B cell differentiation. Finally, a model is proposed that describes an integrated role for CGRP in the homeostatic regulation of early B cell differentiation.Key words: neuropeptides, immune, immunoglobulin, neuroimmunology, calcitonin gene related peptides.

scholarly journals Exploring Pre-B Cell Receptor Signalling As a Therapeutic Target in Acute Lymphoblastic Leukaemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4066-4066
Author(s):  
Ali Alhammer ◽  
Marian Case ◽  
Helen J Blair ◽  
Josef Vormoor ◽  
Julie Irving
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Acute Lymphoblastic Leukaemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukaemia ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Great Promise ◽  
Receptor Signalling

Abstract Introduction: Acute lymphoblastic leukaemia (ALL) is a clonal disorder of developing lymphocytes and is the most common malignancy in children and adolescents. While cure rates are high, treatment is associated with significant morbidity and relapsed ALL remains one of the leading causes of cancer-related deaths in children. New, less toxic therapies are clearly needed for refractory ALL. There are a number of lines of evidence to suggest that ALL cells hijack components of precursor-B cell receptor (Pre-BCR) signalling and this dependency may be amenable to therapeutic exploitation. There are a number of tyrosine kinase inhibitors (TKIs) targeting Pre-BCR signalling that are showing great promise in the clinic for other leukemia subtypes which warrant preclinical evaluation in childhood ALL.[1] These include CAL-101 (PI3K-δ inhibitor), Ibrutinib (BTK inhibitor), Fostamatinib R406 (SYK inhibitor) and Dasatinib (BCR-ABL/SRC inhibitor). Methods: TKIs were evaluated in ALL cells, including cell lines (PreB 697 and its glucocorticoid resistant descendant, R3F9; Nalm-6 and Reh) and 25 patient derived xenograft samples (PDX) from 12 predominantly high risk/relapse children ALLs. Resazurin was used to assess cell viability. Flow cytometry was used to detect Pre-BCR expression (µHc, Vpreb and λ5) and functionality using a Calcium flux assay. Phospho-flow cytometry was performed to monitor constitutive phosphorylation and response to Pre-BCR activation and to assess pharmacodynamic drug action (p-AKT, p-BLNK, p-BTK, p-SYK, p-ERK, and p-PLC-ϒ2). GILZ expression was measured by RQ-PCR. Cell cycle and apoptosis were determined by flow cytometry using Propidium Iodide and Annexin V staining. Results: ALL cell lines were resistant to CAL-101 (mean GI50 52.08 µM, range 25 µM-77.83 µM) and Ibrutinib (mean GI50 15.9 µM, range 11.47 µM-18.3 µM). However, modest sensitivity was seen to R406 (mean GI50 4.32 µM, range 2.88 µM-5.83 µM) and Dasatinib (mean GI50 5.33 µM, range 2.45 µM-12.5 µM). CAL-101 and Dasatinib were shown to be cytostatic, causing G1 arrest but no significant apoptosis, while Ibrutinib and R406 were associated with cell cycle arrest and significant apoptosis after 72 hours incubation with GI50 concentrations (16.81±1.71 % and 31.34±5.78 % apoptosis, respectively). Pre-B receptor positive cells were more sensitive to Dasatinib and Fostamatinib. Pharmacodynamic assessment using Phospho-flow cytometry and Western blotting showed inhibition of the relevant targets at the GI50 concentrations. PDX ALL cells were generally more sensitive than the cell lines; CAL-101 (mean GI50 25.56 µM, range 76 nM-100 µM; 2 out of 12 patient samples <2µM); Ibrutinib (mean GI50 14.23 µM, range 490 nM-100 µM; 3 out of 12 patient samples <5µM); R406 (mean GI50 11.52 µM and range 56 nM-25 µM, 4 out of 12 patient samples <4µM); Dasatinib (mean GI50 25.56 µM, range 76 nM-100 µM; 3 out of 12 patient samples <0.5µM). ALLs sensitive to Dasatinib were Ph+ or Pre-BCR positive and the latter were also sensitive to Fostamatinib. Synergism was seen after co-treatment of TKIs with the glucocorticoid (GC), Dexamethasone (CAL-101 CI mean 0.7, range 0.056-1.34; Ibrutinib CI mean 0.71, range 0.41-0.97; R406 CI mean 0.27, range 0.1-0.6 and Dasatinib CI mean 0.63, range 0.14-1.29). No synergism was observed in the glucocorticoid receptor negative, Reh cell line. For R406 and Dasatinib, co- exposure was strongly synergistic and was associated with increased apoptosis in PreB 697 and the GC resistant lines, Nalm-6 and R3F9. Synergism was associated with a significant increase in expression of the GR target, GILZ and an enhanced downregulation of R406 and Dasatinib targets (p-SYK for R406 and p-BTK, p-SYK for Dasatinib). Conclusion: We have identified significant sensitivity of TKIs impacting on Pre-BCR signalling in ALL cells at clinically relevant concentrations; Pre-BCR positive ALLs were associated with Dasatinib and Fostamatinib sensitivity; Pre-BCR negative ALL cells were also sensitive to some TKIs, although predictive biomarkers remain to be established. Marked synergism was observed in combination with dexamethasone, even in GC resistant cells. In vivo preclinical confirmation of these data may offer new therapies for refractory ALL. 1. Young, R.M. and L.M. Staudt, Targeting pathological B cell receptor signalling in lymphoid malignancies. Nature Reviews Drug Discovery, 2013. 12(3): p. 229-243. Disclosures No relevant conflicts of interest to declare.

B prolymphocytlc leukemia cell lines: phenotype and response to B cell differentiation factor

Pathology ◽  
1988 ◽  
Vol 20 (1) ◽  
pp. 10-14 ◽  
Author(s):  
Heddy Zola ◽  
Virginia Furness ◽  
Anthony Nikoloutsopoulos ◽  
John Bradley ◽  
Junia V. Melo ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cell ◽  
Cell Lines ◽  
Leukemia Cell ◽  
B Cell Differentiation ◽  
Differentiation Factor ◽  
Leukemia Cell Lines ◽  
Cell Differentiation Factor

Frequent Loss of the SLP-65 Adapter Protein in Childhood Acute B-Precursor Lymphoblastic Leukemia (ALL) with TEL/AML1 Rearrangement.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 741-741 ◽  
Author(s):  
Arndt Borkhardt ◽  
Christine Damm-Welk ◽  
Thomas Wossning ◽  
Bettina Storch ◽  
Uta Fuchs ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
B Cells ◽  
Tumor Suppressor ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Sh2 Domain ◽  
B Cell Differentiation ◽  
Cell Leukemia ◽  
B Cell Leukemia

Abstract The adaptor protein SLP-65 plays an essential role during B cell differentiation. A crucial consequence of SLP-65 deficiency in mice is a high incidence of pre-B-cell leukemia, suggesting a tumor suppressor role for SLP-65 in pre-B-cells. While the link between SLP-65 deficiency and leukemia development is established in mice, experiments mainly using microarrays for gene expression profiling suggested normal expression of SLP-65 in human precursor B-cell ALL. This analysis however does not discriminate between normal and aberrant SLP-65 transcripts with the latter being unable to generate functional protein. To examine the correlation between SLP-65 deficiency and childhood precursor B-cell ALL, we determined SLP-65 expression in 119 precursor B-cell ALL samples by both RNA and protein methods. The expression of SLP-65 was compared to clinical and laboratory findings, cytogenetics as well as to the outcome data within this uniformly treated cohort of patients. Loss of slp-65 protein was significantly associated with the occurrence of the TEL/AML1 rearrangement (p=0.026) but not with any other clinical or cytogenetic feature. We found a profound disconnection between slp-65 mRNA and protein expression in 38 out of the 119 leukemic samples pointing to a posttranscriptional regulation of slp-65 (Table). To confirm that SLP-65 transcript expression does not automatically correlate with its protein expression, we analyzed a panel of human cell lines derived from precursor B-cell ALL patients. The cell lines HPB-NULL and BV-173 showed a deficiency in SLP-65 protein expression, although SLP-65 transcripts can easily be detected in both lines. Together, the data suggest that SLP-65 expression might be regulated at the posttranscriptional level and that the presence of SLP-65 transcripts does not necessarily lead to SLP-65 protein and function. In one particular patient, we found a truncated slp-65 transcript and the predicted slp-65 protein lacks its SH2 domain. We tested whether this SLP-65 protein lacking the SH2 domain is functional in pre-B cells. To this end, we transfected murine SLP-65 −/− pre-B cells with retroviral constructs for either wild-type (wt SLP-65) or truncated SLP-65 (SLP-65delSH2) and analysed pre-BCR downregulation, Ca2+ release and pre-B cell differentiation. The results showed that, in contrast to wt SLP-65, SLP-65delSH2 failed to induce any effects in the performed experiments. Together with previous findings showing that SLP-65-deficient mice develop pre-B cell leukemia, the data suggest that SLP-65 acts as a tumor suppressor that limits pre-B cell proliferation by inducing differentiation. Disconnection between slp-65 transcripts and protein expression total slp-65 protein+ (51 patients) slp-65 protein weak (19 patients) slp-65 protein- (49 patients) PCR+ 108 51(9 TEL/AML+, 42 TEL/AML-) 19 (9 TEL/AML+, 10 TEL/AML-) 38 (15 TEL/AML+, 23 TEL/AML-) PCR- 11 0 0 11 (T-ALL)

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 ◽  
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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