Figure 2.
Figure 2. Relative expression and functional analysis of the KMT2E-ASNS chimera. (A) RT-qPCR analysis of relative expression levels for endogenous KMT2E and ASNS as well as KMT2E-ASNS fusion transcripts in diagnosis and relapse samples of childhood ETP-ALL patients TC0002 and TC0022. Expression levels of KMT2E-ASNS at relapse are 4.6-fold and 9.7-fold higher than endogenous ASNS levels at diagnosis for TC0002 and TC0022, respectively. Expression levels of KMT2E are 6.7-fold and 1.8-fold lower in the relapse samples compared with diagnosis for TC0002 and TC0022, respectively. The vertical bars show 95% confidence intervals according to Student t distribution. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level was used for reference. (B) Schematic representation of the novel KMT2E-ASNS fusion transcript amplified from TC0002s patient cDNA, subcloned into a pLenti vector and transduced in the MOLT-4 cell line. Exon 1 of KMT2E is shown in blue, and ASNS exons 3 to 13 are shown in red. UTRs are drawn thinner than coding regions. The primers used for amplification are depicted as arrow with the genomic position (hg19) of their first (5′) aligned base. (C) RT-qPCR analysis of relative expression levels for KMT2E-ASNS fusion transcripts in cell transduced MOLT4 cell line. Expression levels of ASNS and KMT2E-ASNS in transduced MOLT4 cell line show a fivefold higher KMT2E-ASNS level compared with wild-type and MOLT4 transduced cell line with vector only. The vertical bars show 95% confidence intervals according to Student t distribution. GAPDH expression level was used for reference. ASNS protein level was shown higher in KMT2E-ASNS transduced cell line compared with wild-type and empty vector’s cell lines (D) Apoptosis assay showing close to 30% reduction in apoptosis in KMT2E-ASNS–positive MOLT-4 cells compared with vector only (pLenti) transduced cells. Apoptosis profile of parental (data not shown) and transduced MOLT-4 cells was determined by flow cytometry using annexin V–Allexa488/ propidium iodide (PI). The numbers in each quadrant indicate the percentage of cells from a total of 10 000 cells. Viable cells appear in the lower left-hand quadrant (PI and annexin V negative), whereas cells in the upper left-hand quadrant and in the upper right-hand quadrant denote early and late apoptotic cells, respectively. The lower right-hand quadrant represents dead cells. Camptothecin and doxorubicin were used as negative control to assess the specificity of l-asparaginase. The apoptosis data presented are representative of 3 independent experiments for each of the 2 biological replicates. *P ≤ .05 according to Mann-Whitney U test for the early and late apoptosis of KMT2E-ASNS cell line compared with empty vector cell line.

Relative expression and functional analysis of the KMT2E-ASNS chimera. (A) RT-qPCR analysis of relative expression levels for endogenous KMT2E and ASNS as well as KMT2E-ASNS fusion transcripts in diagnosis and relapse samples of childhood ETP-ALL patients TC0002 and TC0022. Expression levels of KMT2E-ASNS at relapse are 4.6-fold and 9.7-fold higher than endogenous ASNS levels at diagnosis for TC0002 and TC0022, respectively. Expression levels of KMT2E are 6.7-fold and 1.8-fold lower in the relapse samples compared with diagnosis for TC0002 and TC0022, respectively. The vertical bars show 95% confidence intervals according to Student t distribution. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level was used for reference. (B) Schematic representation of the novel KMT2E-ASNS fusion transcript amplified from TC0002s patient cDNA, subcloned into a pLenti vector and transduced in the MOLT-4 cell line. Exon 1 of KMT2E is shown in blue, and ASNS exons 3 to 13 are shown in red. UTRs are drawn thinner than coding regions. The primers used for amplification are depicted as arrow with the genomic position (hg19) of their first (5′) aligned base. (C) RT-qPCR analysis of relative expression levels for KMT2E-ASNS fusion transcripts in cell transduced MOLT4 cell line. Expression levels of ASNS and KMT2E-ASNS in transduced MOLT4 cell line show a fivefold higher KMT2E-ASNS level compared with wild-type and MOLT4 transduced cell line with vector only. The vertical bars show 95% confidence intervals according to Student t distribution. GAPDH expression level was used for reference. ASNS protein level was shown higher in KMT2E-ASNS transduced cell line compared with wild-type and empty vector’s cell lines (D) Apoptosis assay showing close to 30% reduction in apoptosis in KMT2E-ASNS–positive MOLT-4 cells compared with vector only (pLenti) transduced cells. Apoptosis profile of parental (data not shown) and transduced MOLT-4 cells was determined by flow cytometry using annexin V–Allexa488/ propidium iodide (PI). The numbers in each quadrant indicate the percentage of cells from a total of 10 000 cells. Viable cells appear in the lower left-hand quadrant (PI and annexin V negative), whereas cells in the upper left-hand quadrant and in the upper right-hand quadrant denote early and late apoptotic cells, respectively. The lower right-hand quadrant represents dead cells. Camptothecin and doxorubicin were used as negative control to assess the specificity of l-asparaginase. The apoptosis data presented are representative of 3 independent experiments for each of the 2 biological replicates. *P ≤ .05 according to Mann-Whitney U test for the early and late apoptosis of KMT2E-ASNS cell line compared with empty vector cell line.

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