Identification of prognostic protein biomarkers in childhood acute lymphoblastic leukemia (ALL)
Graphical abstract
Research highlights
► We performed proteomic analysis on PRED-sensitive and resistant ALL cell lines. ► Several differentially expressed proteins were identified in these cells. ► PCNA was validated in paired patient bone marrows (at diagnosis and after PRED). ► PCNA was identified to have prognostic value.
Introduction
Childhood acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer throughout the world; it alone accounts for more than 30% of childhood cancers diagnosed every year. Currently, using risk stratified, multi-agent chemotherapy, more than 80% of children with ALL in developed countries are cured [1].
Many of the ALL treatment protocols including ours Malaysia-Singapore (Ma-Spore) and the widely adopted German Berlin-Frankfurt-Munster (BFM) clinical trial utilize Day 8 prednisolone (PRED) response for risk stratification [2], [3], [4]. This comprises of 7 days of PRED and one intrathecal dose of methotrexate. Patients with PRED poor response (PPR) with Day 8 blast count more than 1000/μl, have poor 38–50% event-free survival (EFS) compared to patients with PRED good response (PGR) who achieve 80% EFS. Similarly, in our Ma-Spore ALL 2003 study, PPR patients (n = 50) have 70.6% EFS compared to PGR patients (n = 422) who have 83.7% EFS (p = 0.001). Poor PRED responders now receive intensified therapy in order to improve their treatment outcome. Using in vitro 4-day drug cytotoxicity assay, cellular resistance to PRED is also associated with unfavorable treatment outcome in pediatric ALL [5]. The reason behind why the D8 in vivo and D4 in vitro PRED responses are prognostic of eventual outcome is a mystery.
Little is known about the mechanisms of PRED resistance in leukemic blasts. It may be due to the misregulation of glucocorticoid receptors (GR) [6] or defects in the glucocorticoid response genes and their cross talks. Some studies showed that multidrug-resistant gene (MDR1) and genes involved in cell cycle, drug metabolism, DNA repair, and intrinsic apoptosis pathway are involved in glucocorticoid resistance in ALL [7], [8], [9].
In this study, we aimed to identify prognostic biomarkers and potential protein regulators that underlie the prognostic significance of Day 8 PRED response by comparing the changes in the proteome of four clinically important cells lines (pre- and post-PRED treatment) representing the 4 most common subtypes of ALL: REH [ETV6-RUNX1], 697 [TCF3-PBX1], Sup-B15 [BCR-ABL1] and RS4;11[MLL-AF4]. We then validated our findings in 43 paired bone marrow samples of ALL patients at diagnosis (Day 0) and after 7 days of PRED treatment (Day 8). We surmised that the prognostic biomarkers identified will provide insights in developing novel drug target to reverse glucocorticoid resistance or used as prognostic markers to risk-stratify patients in the future.
Section snippets
Cell lines
Four clinically important cells lines REH, 697, Sup-B15 and RS4;11 were used in this study. The 697 cell line was purchased from German Collection of Microorganisms and Cell Cultures (DSMZ). The other three cell lines were purchased from the American Type Culture Collection (ATCC). They were cultured and maintained according to the manufacturer's protocol.
Patients samples
We randomly selected paired bone marrow samples at diagnosis (Day 0) and after 7 days of PRED (Day 8) from 43 children with newly diagnosed
Precursor-B ALL cell lines showed differential sensitivity to PRED treatment
Four clinically important leukemia cell lines (REH, 697, Sup-B15 and RS4;11) were treated with 1.0 μg/ml PRED for 24 h and 48 h. REH was resistant to PRED treatment (100% cell viability). The other three cell lines (697, Sup-B15 and RS4;11) were sensitive with approximately 80% of cells survived at 24 h time point, and 20% survived at 48 h time point, Fig. 1A. Co-treatment of Ru486, a GR antagonist, significantly inhibited the cell death induced by 10.0 μg/ml PRED at 24 h time point in all three
Discussion
PRED is a glucocorticoid which is widely used in the treatment of childhood ALL. Interestingly, despite its widespread usage in clinics and its important role in treatment of childhood ALL, the mechanism of PRED-induced apoptosis is still unclear. In this study, we used a systematic proteomic-based approach to define the differences in the changes in proteome of PRED-sensitive and PRED-resistant cell lines after PRED. We wanted a biomarker that reflects the irreversible commitment of the
Conclusion
Using proteomics approach, proteins from four clinically important ALL cell lines before and after PRED treatment were successfully separated by 2-DE and 94 differentially expressed proteins of interests have been identified. Proteins that involved in cell cycle regulation may play critical roles in regulating PRED-induced cell death, and may act as a distinct signature for PRED response clinically. We have further identified and validated a highly promising universal marker of PRED response,
Acknowledgements
We would like to thank Singapore Cancer Syndicate/A*STAR (SCS-EN35) and National Medical Research Council (NMRC/CSA/003/2008) for funding this project work.
All authors declare that there are no financial/commercial conflicts of interest.
References (34)
- et al.
Acute lymphoblastic leukaemia
Lancet
(2008) - et al.
Prednisone response is the strongest predictor of treatment outcome in infant acute lymphoblastic leukemia
Blood
(1999) - et al.
Philadelphia chromosome-positive (Ph+) childhood acute lymphoblastic leukemia: good initial steroid response allows early prediction of a favorable treatment outcome
Blood
(1998) - et al.
The relationship of blast cell glucocorticoid receptor levels to response to single-agent steroid trial and remission response in children with acute lymphoblastic leukemia
Leuk Res
(1984) - et al.
Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466
Mol Cell Proteomics
(2002) - et al.
PCNA, the maestro of the replication fork
Cell
(2007) - et al.
Emerging pathways of non-genomic glucocorticoid (GC) signalling in T cells
Immunobiology
(2010) Therapeutic implications of non-genomic glucocorticoid activity
Lancet
(2000)- et al.
Non-genomic glucocorticoid effects to provide the basis for new drug developments
Mol Cell Endocrinol
(2006) - et al.
Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling
Cancer Cell
(2002)
VDAC is a conserved element of death pathways in plant and animal systems
Biochim Biophys Acta
Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors
Trends Cell Biol
In vivo characterization of the proteasome regulator PA28
J Biol Chem
Childhood acute lymphoblastic leukemia: prognostic value of initial peripheral blast count in good responders to prednisone
J Pediatr Hematol Oncol
Patient stratification based on prednisolone–vincristine–asparaginase resistance profiles in children with acute lymphoblastic leukemia
J Clin Oncol
Relapse in childhood acute lymphoblastic leukemia is associated with a decrease of the Bax/Bcl-2 ratio and loss of spontaneous caspase-3 processing in vivo
Leukemia
Polymorphisms in genes encoding drugs and xenobiotic metabolizing enzymes, DNA repair enzymes, and response to treatment of childhood acute lymphoblastic leukemia
Clin Cancer Res
Cited by (63)
Insights into glucocorticoid responses derived from omics studies
2021, Pharmacology and TherapeuticsCitation Excerpt :Among those proteins, proliferating cell nuclear antigen (PCNA) showed decreased expression in the glucocorticoid-sensitive cell lines and was validated in 43 paired bone marrow samples from children with newly diagnosed ALL versus 7 days after prednisolone treatment. The difference in PCNA expression was highly predictive of prednisolone response in patients independent of their molecular subtypes, suggesting that it could be a universal prognostic marker for treatment outcome (Jiang et al., 2011). Another study that applied an MS-based approach to compare proteomes of the glucocorticoid-sensitive cell line PreB 697 versus its glucocorticoid-resistant sub-clone R3F9 when exposed to dexamethasone versus vehicle control found that paired box 5 (PAX5), a transcription factor critical to B-cell development, had significantly decreased expression in the glucocorticoid-resistant cell line, as did its transcription target CD19 (Nicholson et al., 2015).
REDD1 (regulated in development and DNA damage 1) modulates the glucocorticoid receptor function in keratinocytes
2023, Experimental DermatologyA new insight updates in diagnosis and management of acute lymphoblastic leukemia, cytogenetics, immunophenotyping, and proteomic profile
2023, Electronic Journal of General Medicine