ABSTRACT
RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50-70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27-4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.
Subject(s)
B-Lymphocytes/metabolism , Biomarkers, Tumor/genetics , Mutation/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , ras Proteins/genetics , Adolescent , Animals , Cell Line, Tumor , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Mice , Mice, Inbred NOD , Mutation Rate , Oncogene Proteins, Fusion/genetics , Prognosis , Signal Transduction/geneticsABSTRACT
Still 20% of pediatric acute lymphoblastic leukemia (ALL) patients relapse on or after current treatment strategies. Treatment failure is associated with resistance to prednisolone. We aimed to find new druggable targets that modulate prednisolone resistance. We generated microarray gene expression profiles of 256 pediatric ALL patient samples and identified a 3.4-fold increase in epithelial membrane protein 1 (EMP1) expression in in vitro prednisolone-resistant compared with -sensitive patients (P=0.003). EMP1 silencing in six precursor-B ALL (BCP-ALL) and T-ALL cell lines induced apoptosis and cell-cycle arrest leading to 84.1±4.5% reduction in survival compared with non-silencing control transduced cells (non-silencing control short hairpin, shNSC) (P=0.014). Moreover, EMP1 silencing sensitized to prednisolone up to 18.8-fold (P<0.001). EMP1 silencing also abrogated migration and adhesion to mesenchymal stromal cells (MSCs) by 78.3±9.0 and 29.3±4.1% compared with shNSC (P<0.05). We discovered that EMP1 contributes to MSC-mediated prednisolone resistance. Pathway analysis indicated that EMP1 signals through the Src kinase family. EMP1-high BCP-ALL patients showed a poorer 5-year event-free survival compared with EMP1-low patients (77±2 vs. 89±2%, P=0.003). Multivariate analysis taking along white blood cell count, age, prednisolone resistance and subtype identified EMP1 as an independent predictor for poor outcome in BCP-ALL (P=0.004, hazard ratio: 2.36 (1.31-4.25). This study provides preclinical evidence that EMP1 is an interesting candidate for drug development to optimize treatment of BCP-ALL.
Subject(s)
Drug Resistance, Neoplasm , Leukemia/drug therapy , Neoplasm Proteins/physiology , Prednisolone/pharmacology , Receptors, Cell Surface/physiology , Apoptosis , Cell Adhesion , Cell Movement , Cell Proliferation , HEK293 Cells , Humans , Leukemia/mortality , Leukemia/pathology , NF-kappa B/physiology , Neoplasm Proteins/analysis , Prognosis , Receptors, Cell Surface/analysis , src-Family Kinases/physiologySubject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Drug Resistance, Neoplasm/genetics , Orphan Nuclear Receptors/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Animals , Child , Gene Knockdown Techniques , Humans , Mice , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Nuclear Receptor Subfamily 4, Group A, Member 3/genetics , TranscriptomeABSTRACT
MicroRNA-125b (miR-125b), miR-99a and miR-100 are overexpressed in vincristine-resistant acute lymphoblastic leukemia (ALL). Cellular viability of ETV6-RUNX1-positive Reh cells significantly increased in presence of 9 ng/mL vincristine upon co-expression of miR-125b/miR-99a (91 ± 4%), miR-125b/miR-100 (93 ± 5%) or miR-125b/miR-99a/miR-100 (82 ± 17%) compared with miR-125b-transduced cells (38 ± 13%, P<0.05). Co-expression of these miRNAs resulted in downregulation of DNTT, NUCKS1, MALAT1, SNRPE, PNO1, SET, KIF5B, PRPS2, RPS11, RPL38 and RPL23A (fold-change 1.3-1.9, p<0.05). Similarly, 7 out of these genes are lower expressed in vincristine-resistant ALL cells of children (p<0.05). The concerted function of miR-125b in combination with miR-99a and/or miR-100 illustrates the complexity of vincristine-resistant pediatric ALL.