Proteomic tools and new insights for the study of B-cell precursor acute lymphoblastic leukemia

Figures & data

Figure 1. Proteomic tools as powerful approach in the investigation of BCP-ALL and the discovery of new molecular biomarkers. Proteomics involves a series of tools to study the proteome from organisms, tissues, body fluids or cells (left panel). The most common proteomics tools include mass spectrometry (MS), mass cytometry (CyTOF), two-dimensional polyacrylamide electrophoresis (2D-PAGE), flow cytometry, and protein arrays (middle panel). Because of the extensive data produced by proteomics, the use of bioinformatic tools is necessary for the analysis of hundreds of molecules. Next, analytical and clinical validations are the last steps in testing the utility of these molecules in the clinical context (right panel).

Table 1. Current markers with the clinical application for the classification of BCP-ALL and monitoring of BCP-ALL MRD.

Biomarkers	Functions and/or cell type	Clinical application in B-ALL	References
CD10	c-ALL, lymphatic precursor cells, neutrophils subset of mature B cells	It is the hallmark of childhood most common ALL and of BCP-ALL MRD	[21,90]
CD19	B-cell signaling / Precursor B-cells, B-cells	Diagnosis of BCP-ALL and subtype classification also it is a marker for BCP-ALL MRD	[21,91] [20,92–95]
CD20	Subpopulation of precursor B-cells, B-cells	Marker for mature B-cell differentiation and for BCP-ALL MRD	[21,28]
CD22	Regulates B-cell function and migration / Surface expression on B-cells, cytoplasmic expression in precursor B-cells	It is used for diagnosis of B-ALL and subtype classification. It is strongly associated with the B-lineage, but is not B-cell specific	[21,96] [29,35–38]
CD34	Attachment of SC to extracellular matrix in bone marrow / Myeloid and lymphoid precursor cells	It is used for B-ALL diagnosis and it is a marker of BCP-ALL MRD	[21,97] [29,98]
CD45	All leukocytes	Marker for BCP-ALL MRD	[21]
CD123	It is expressed on a subset of B-ALL	Marker for BCP-ALL MRD	[99]
CyIgμ	PreB and B-lymphocytes	Marker for pre-B stage of B-III ALL	[100]
SmIgκ	Surface expression on B-lymphocytes	Marker of BCP-ALL	[21]
CyISmIgλ	Surface expression on B-lymphocytes	Marker of BCP-ALL	[26]
NuTdT	Nuclear expression in lymphoid precursor cells	Marker for distinction from normal B-cell development patterns	[21]

Table 2. Examples of potential molecular biomarkers for diagnosis, prognosis, and targeted therapy of BCP-ALL.

Biomarkers	Functions	Applications in B-ALL	References
CD79a	B-cell activation.	Diagnosis of B-ALL and subtype classification.	[29,35–38]
CD38	Catalyzes the synthesis from cADP ribose to ADP ribose.	It is used for B-ALL diagnosis.	[30–32]
TP53, JAK2	Tumor suppressor-transcription factor, Participates in intracellular signaling upon activation of cytokine receptors.	Both molecules have been tested as potential biomarker of poor outcome in B-ALL.	[101–103] [39–41]
RAS	GTPase involved in protein cell signaling.	Unfavorable prognosis and predictive biomarker for treatment.	[104,105] [42,43]
LRG1, CLU, F2, SERPIND1	Participates in vascularization by modulating TGF-β signaling, Promotes or inhibits tumorigenesis based in the cellular context, Anticoagulant and proinflammatory, Coagulating process.	This cluster of proteins have the potential to be used for B-ALL diagnosis and treatment evaluation.	[52,106,107] [44–46]
CSRP2	Participates in breast cancer cell invasiveness.	Diagnosis/treatment monitoring for B-ALL.	[47–108]
ADA1	Implicated in purine salvage pathway.	Diagnosis/treatment monitoring for B-ALL.	[48–109]
IKZF1, CRLF2	Participates in the regulation of hematopoiesis, Proto-oncogene in B-ALL.	Both molecules can be used as markers for poor prognosis in high-risk ALL.	[49,50,110,111]
Hsp90	Participates for folding and regulation of cellular proteins.	Diagnosis/treatment monitoring for B-ALL.	[51,52,112,113]
A20	Inhibits activation of NF-κβ and apoptosis.	Target for B-ALL treatment.	[53,114]

Table 3. Proteomic tools in studies of BCP-ALL.

Tool	Type of study	Objective	Findings	References
LC-ESI-MS	In vitro	To analyze the differential protein expression in the pediatric B-cell line CCRF-SB during vincristine treatment.	Vincristine treatment led to the expression of 135 proteins participating in Toll receptor signaling pathway, Ras Pathway, B and T cell activation, CCKR signaling map, cytokine-mediated signaling pathway, and oxidative phosphorylation.	[83]
MFC	Case-control	To analyze the expression of B-, myeloid, T-lymphoid and NK-cell antigens in 200 blood or bone marrow samples of pretreated children and adult patients with B-ALL.	Aberrant expression of myeloid-associated antigens was observed in 86.5% of cases and differential expression between children and adult patients with B-ALL.	[33]
MFC	Case-control	To analyze the differential expression of TdT, CD34, CD45, CD10, CD38, CD20, and CD22 in CD10^+ and Cd10^- subpopulations of B-ALL cells and hematogones.	Differential expression was observed between CD10^+ in CD10^- subpopulations. CD10+ blasts exhibited higher levels of TdT, CD22, CD34, and CD20. Conversely, CD10- blasts showed higher expression of CD45 than CD10+ blasts.	[32]
MFC	Case-control	To identify the malignant clone harboring clone-specific genomic markers in 53 bone marrow samples derived from 28 children diagnosed with B-ALL for B-ALL MRD monitoring.	FC could identify 93% true-positive (leukemic) and 93% true-negative (normal) cell populations and four discrepant cases for MRD monitoring in B-ALL cases.	[115]
MFC	Case-control	To analyze the immunomodulation of cell surface-antigen in MRD-positive samples of children with B-ALL for MRD detection.	A downregulation of CD10, CD19, and CD34 and upregulation of CD20 were observed after 15 and 33 days of treatment, but no changes in the expression of CD38, CD58 and CD45 were noticed.	[38]
MFC	Case-control	To standardize an immunophenotyping protocol for MRD monitoring and for sensitivity improvement in 319 bone marrow samples of children with B-ALL after induction therapy.	A standardized FC protocol showed a high percentage of concordance (93%) in comparison with RQ-PCR-based MRD detection.	[39]
MFC	Case-control	To standardize a multicolor FC protocol to evaluate the MRD in 263 bone marrow samples derived from childhood relapsed B-ALL.	FC analysis by using a set of B-cell markers showed a consistently high overall concordance (P < .001) and, under optimal conditions, sensitivity comparable to the PCR method for MRD detection.	[40]
MFC	Case-control	To analyze the expression of CD73 in normal young hematogones cells and B-ALL cells with or without MRD^+ of bone marrow for MRD monitoring.	CD73 expression was 6-fold greater in MRD-positive B cells in comparison with MRD negative ones. In all, 41.82% MRD-positive B-ALL cases expressed high CD73 and sensitivity MRD detection reached 10^−4.	[116]
MFC	Case-control	To analyze the expression of Neurophilin-1 (NRP-1)/CD304 as an MRD and prognosis marker in bone marrow samples derived from 70 children with B-ALL and 40 controls.	CD304 expression was higher in 40% of B-ALL (CD304+) cells at diagnosis compared with controls. However, at day 28 of induction therapy, 57.1% of CD304+ patients transitioned to a CD304- phenotype.	[41]
LC-MS/MS	Case-control	To analyze protein expression for risk stratification in BM samples derived from B-ALL patients. The patients were classified into low /medium and high-risk groups.	86 differently expressed proteins were identified in childhood high-risk ALL patients. They found the aberrant evens happened in pre-mRNA splicing, DNA damage response, and stress response.	[54]
AC-MS	Case-control	To analyze the protein expression profile and biomarker discovery in 10 samples derived from B-ALL patients.	A panel of molecules (i.e. LRG1, CLU, F2, SERPIND1, A2M, SERPINF2, SERPINA1, CFB, and C3) were identified as potential biomarkers for early diagnosis of B-ALL.	[52]
LC-MS/FC	In situ/in vivo	To analyze the protein expression profile in immunodeficient mice xenotransplanted with B-ALL human cells.	A total of 713 proteins were detected in B-ALL cases. From all these proteins, CD97, CD157, CD63, and CD305 were identified as the most promising makers to distinguish between normal and malignant cells.	[53]
SELDI-ToF-MS	Case-control	To analyze the protein expression profile and biomarker discovery in samples derived from ALL pediatric patients.	PF4, CTAP-III, and two fragments of C3a were differentially expressed and used to distinguish pediatric BCP-ALL patients from healthy controls, and pediatric AML patients.	[117]
LC-MS/MS	Case-control	To analyze the role of IKZF1 in B-ALL leukemogenesis.	Ikzf1 and Arf alterations contribute to leukemogenesis by promoting the development of an aggressive lymphoid leukemia.	[50]
2D-PAGE/ MALDI-ToF-MS/MS	Case-control	To analyze protein expression profiles and protein functions in B-ALL patient samples.	A total of 79 differentially regulated proteins were identified in B-ALL cells, participating in proteostasis, cytoskeletal organization, redox homeostasis, and signal transduction pathways relevant to leukemogenesis.	[51]
SDS-PAGE/WB/RPPA	Case-control	To analyze the expression and function of Aurora B kinases in 172 B-ALL pediatric patients.	Expression of AURKB and AURKA was mostly observed in E2A-PBX1-translocated B-ALL cases. AURKB inhibition resulted in proliferation arrest and apoptosis.	[69]
RPPA	Case-control/in vitro	To analyze the protein expression profiles for identification of new therapeutic targets in 129 samples derived from Ph^+ B-ALL patients and by using Z-181 and Z-119 cell lines.	Increased phosphorylation of ErbB2 was observed in 56% of Ph^+ ALL. ErbB2 kinase activity inhibition by canertinib resulted in increased expression of the proapoptotic protein Bim, caspase-3 activation, and cell death.	[70]
RPPA	Case-control	To analyze the expression of cathepsin G (CG) in 130 samples derived from B-ALL patients.	CG was overexpressed in B-ALL samples and was proposed as a poor prognostic marker. Endogenous expression analysis demonstrated that CG can also be taken up by different B-ALL cell lines (SB, RS4, Nalm6, and HMY).	[72]
RPPA	Case-control/in vivo	To analyze the role of the STAT5 signaling pathway in leukemogenesis by using samples derived from B-ALL patients and the Stat-CA mouse model.	Increased phosphorylation of STAT5, which was correlated with poor prognosis in BCR-ABL^+ ALL patients.	[73]
RPPA	Case-control	To analyze the role of PAX5-JAK2 fusion protein in B-ALL	It was observed that the PAX5-JAK2 fusion protein led to phosphorylation of STAT1, 3, and 5. Additionally JACK2 inhibition led to the death of transformed cells.	[74]
RPPA	In vitro/in vivo	To analyze the role of the Wnt signaling pathway in leukemogenesis by using the Reh cell line and a mouse leukemia model.	Marrow stromal cells (MSC) induced activation of the Wnt signaling pathway of leukemic cells to induce drug resistance and cell survival.	[81]
RPPA	Case control/in vitro	To analyze the role of the PI3K-Akt-mTOR signaling pathway in leukemogenesis by using 72 samples derived from B-ALL patients and the Reh cell line.	It was demonstrated that reduced expression of the CD200 protein was associated with increased activation of the PI3K-Akt-mTOR signaling pathway in B-ALL patients, characterized by increased phosphorylation of Akt.	[82]
RPPA	Case-control/in vitro	To analyze the role of TCF3-PBX1 fusion in cell proliferation and differentiation in samples derived from 19 TCF3-rearranged and 113 non-TCF3 B-ALL pediatric patients and by using the MHH-CALL3 cell line.	TCF3 + B-ALL samples showed significant expression of elements of the pre-BCR signaling complex (ZAP70, SLP65, BLNK, BTK, PI3K-p110δ, and IRF4). BTK inhibition led to decreased phosphorylation of Erk1/2 and reduction of growth and cell viability.	[75]
RPPA	In vitro	To analyze the role of hypoxia in leukemic cell survival chemoresistance to methotrexate (MTX) and prednisolone (PRD) in the Nalm-6 and Reh B-ALL cell lines.	Hypoxia induced chemoresistance by increasing the expression of anti-apoptotic proteins (XIAP, Mcl-1, and Bcl-2) and decreasing the expression of pro-apoptotic proteins (cleaved caspase 3, Bax, and Bim).	[80]
RPPA	Case-control/in vitro	To analyze the role of the Erk1/2 signaling pathway in leukemogenesis by using 192 samples derived from B-ALL patients and leukemic pre-B cells.	Increased expression of phospho-ERK-T^202/Y^204, DUSP6, SPRY2, and ETV5 was observed in CD19^+ bone marrow pre-B cells and patient-derived pre-B ALL. DUSP6 inhibition produced hyperactivation of Erk but increased ROS levels and cell death.	[76]
CyTOF	Cases	To identify characteristics of expanded leukemic populations sufficient to predict relapse at diagnosis of BCP-ALL.	Were identified 6 features of expanded leukemic populations sufficient to predict patient relapse at diagnosis. These features implicated pro-BII cells with activated mTOR signaling, and pre-BI cells with activated and unresponsive pre-B-cell receptor signaling.	[118]
CyTOF	Cases/in vitro	To identify additional activated networks in BCP-ALL primary patient samples by single-cell mass cytometry.	SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in hiCRLF2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.	[119]
CyTOF	Case-control	To analyze the depth of single-cell mass cytometry and an algorithm developed to predict the developmental path de novo of human B cell lymphopoiesis.	Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development. This trajectory revealed nascent fractions of B cell progenitors and aligned them with developmentally-cued, regulatory signaling including IL-7/STAT5.	[120]

Note: Proteomics tools such as FC, 2D-PAGE, MS, and RPPA have aroused great interest in research and the discovery of new biomarkers for the diagnosis, prognosis, and monitoring of MRD and for the identification of new therapeutic targets for BCP-ALL.

Figure 2. A protein microarray (protein chip) is a high throughput method used to track the interactions and activities of proteins. Protein microarrays are typically prepared by immobilizing proteins onto a microscope slide using a standard contact spotter or non-contact microarray. There are three types of protein microarrays that are currently used to study the biochemical activities of proteins: Analytical protein microarrays are mostly represented by antibody arrays and focus on protein detection. In this class of microarrays, targeted proteins can be detected either by direct labeling or using a reporter antibody in sandwich assay format. Functional protein microarrays have broad applications in studying protein interactions, including protein binding and enzyme-substrate reactions. Reverse-phase protein microarrays provide a different array format by immobilizing many different lysate samples on the same chip. There are five major areas where protein arrays are being applied: diagnostics, proteomics, protein functional analysis, antibody characterization and treatment.

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