Resistance of t(17;19)-acute lymphoblastic leukemia cell lines to multiagents in induction therapy

Abstract

t(17;19)(q21-q22;p13), responsible for TCF3-HLF fusion, is a rare translocation in childhood B-cell precursor acute lymphoblastic leukemia(BCP-ALL). t(1;19)(q23;p13), producing TCF3-PBX1 fusion, is a common translocation in childhood BCP-ALL. Prognosis of t(17;19)-ALL is extremely poor, while that of t(1;19)-ALL has recently improved dramatically in intensified chemotherapy. In this study, TCF3-HLF mRNA was detectable at a high level during induction therapy in a newly diagnosed t(17;19)-ALL case, while TCF3-PBX1 mRNA was undetectable at the end of induction therapy in most newly diagnosed t(1;19)-ALL cases. Using 4 t(17;19)-ALL and 16 t(1;19)-ALL cell lines, drug response profiling was analyzed. t(17;19)-ALL cell lines were found to be significantly more resistant to vincristine (VCR), daunorubicin (DNR), and prednisolone (Pred) than t(1;19)-ALL cell lines. Sensitivities to three (Pred, VCR, and l-asparaginase [l-Asp]), four (Pred, VCR, l-Asp, and DNR) and five (Pred, VCR, l-Asp, DNR, and cyclophosphamide) agents, widely used in induction therapy, were significantly poorer for t(17;19)-ALL cell lines than for t(1;19)-ALL cell lines. Consistent with poor responses to VCR and DNR, gene and protein expression levels of P-glycoprotein (P-gp) were higher in t(17;19)-ALL cell lines than in t(1;19)-ALL cell lines. Inhibitors for P-gp sensitized P-gp-positive t(17;19)-ALL cell lines to VCR and DNR. Knockout of P-gp by CRISPRCas9 overcame resistance to VCR and DNR in the P-gp-positive t(17;19)-ALL cell line. A combination of cyclosporine A with DNR prolonged survival of NSG mice inoculated with P-gp-positive t(17;19)-ALL cell line. These findings indicate involvement of P-gp in resistance to VCR and DNR in Pgp positive t(17;19)-ALL cell lines. In all four t(17;19)-ALL cell lines, RAS pathway mutation was detected. Furthermore, among 16 t(1;19)-ALL cell lines, multiagent resistance was usually observed in the cell lines with RAS pathway mutation in comparison to those without it, suggesting at least a partial involvement of RAS pathway mutation in multiagent resistance of t(17;19)-ALL.

Keywords: chemotherapy; hematalogical cancer; leukemia; pediatric cancer.

Figure 1

Prospective minimal residual disease (MRD) analysis in a newly diagnosed t(17;19)‐ALL case and t(1;19)‐ALL cases by real‐time RT‐PCR targeting of TCF3‐HLF and TCF3‐PBX1 chimeric mRNA, respectively. Levels of TCF3‐HLF and TCF3‐PBX1 chimeric mRNA in bone marrow aspirates were monitored at diagnosis and on days 15, 29, and 43 in induction therapy in a t(17;19)‐ALL case (red bold line) and 16 t(1;19)‐ALL cases, respectively, treated with the TCCSG L0416 high risk protocol

Figure 2

Sensitivities to agents in induction therapy in the t(17;19)‐ALL and t(1;19)‐ALL cell lines. (A) Dose response curves to daunorubicin (DNR) in t(17;19)‐ALL (top panel) and t(1;19)‐ALL (bottom panel) cell lines. Horizontal and vertical axis respectively indicates log concentration of DNR and cell viability determined by alamarBlue cell viability assay. (B, C, and D) Comparison of IC50s for DNR (B), VCR (B), Pred (C), Dex (C), L‐Asp (D), and Maf (D) between four t(17;19)‐ALL cell lines and 16 t(1;19)‐ALL cell lines. Pvalues in Mann‐Whitney test are shown

Figure 3

Multi‐agent resistance in t(17;19)‐ALL cell lines. Total score of sensitivities to three (Pred, VCR, and LAsp), four (Pred, VCR, L‐Asp, and DNR), and five (Pred, VCR, L‐Asp, DNR, and MAF) agents were compared between t(17;19)‐ALL and t(1;19)‐ALL cell lines. P‐values in Mann‐Whitney test are indicated

Figure 4

P‐glycoprotein expression in t(17;19)‐ALL cell lines. (A) Correlation between IC50 of VCR and that of DNR in t(17;19)‐ALL and t(1;19)‐ALL cell lines. (B) Gene expression level of ABCB1 in BCP‐ALL cell lines. ABCB1 gene expression was quantified by real time RT‐PCR using beta‐actin expression as an internal control. P‐values in Mann Whitney‐test are indicated on the top. (C) The cell surface expression of Pglycoprotein (P‐gp) on t(17;19)‐ALL cell lines. In the left pane, representative histograms of P‐gp expression are shown with relative fluorescence index (RFI). Black‐filled histograms represent anti‐P‐gp antibody staining and black‐line histograms represent isotype controls for staining. In the right panel, correlation between RFI of P‐gp expression (vertical axis) and relative expression level of ABCB1 (horizontal axis) in BCP‐ALL cell lines is presented. Red and green circles represent t(17;19)‐ALL cell lines and t(1;19)‐ALL cell lines, respectively, and squares represent other BCP‐ALL cell lines. (D) The cell surface expression of P‐gp in BCP‐ALL cell lines. P‐values in Mann Whitney‐test are indicated on the top

Figure 5

Involvement of P‐gp in DNR and VCR‐resistance in P‐gp‐positive t(17;19)‐ALL cell lines. (A) Increased efflux activity in HALO1, a P‐gp‐positive t(17;19)‐ALL cell line. HALO1 cells stained with calcein AM (CAM) were incubated in the absence or presence of verapamil, cyclosporine A, or nilotinib for 30 min at 37°C and, then, analyzed by flow cytometry. In the top panel, red‐line histograms and blue‐filled histograms represent CAM staining in the absence and presence of an inhibitor, respectively. Relative staining level is shown in each panel. In the bottom panel, the vertical axis indicates relative CAM staining level and the horizontal axis indicates log concentration of inhibitors. (B and C) Induction of apoptotic cell death by DNR (B) or VCR (C) in a combination with nilotinib. HALO1 (left panel) and 697 (right panel), a P‐gp‐negative t(1;19)‐ALL cell line, were cultured in the absence or presence of DNR (20 and 200 ng/mL) or VCR (10 and 100 ng/mL) overnight and then analyzed for Annexin V‐binding (horizontal axis) and actinomycin‐D (ACMD)‐ staining (vertical axis) by flow cytometry. Percentages of living cells (lower left) are pointed out in each panel. (D) Sensitivities to DNR (left panel) and VCR (right panel) in combination with nilotinib in HALO1 and UOCB1, P‐gp‐positive t(17;19)‐ALL cell lines. The vertical axis indicates median cell viability in triplicated alamarBlue cell viability assay. Error bar indicates standard deviation. P‐values in student T‐test between viability of cells treated with DNR or VCR alone and that of cells treated with DNR or VCR in combination with nilotinib. (E) Effect of P‐gp knockout on VCR, DNR, or Dex sensitivities in HALO1 cells. Each panel indicates two‐color analysis of P‐gp expression (vertical axis) and Annexin V‐binding (horizontal axis) by flow cytometry in parental and CD4‐positive populations of HALO1 cells treated with DNR (12.5 ng/mL), VCR (25 ng/mL), or Dex (250 nmol/L) for 72 hours. Cell viabilities in P‐gp‐positive and negative populations are shown at the left side of each panel. (F) Effect of P‐gp inhibitor on DNR sensitivity of t(17;19)‐ALL in vivo. NSG mice inoculated with HAOL1 cells were treated with vehicle, DNR alone, or DNR in combination with CyA for 5 days (n = 5). Vertical axis indicates survival of mice. P value between survival of mice treated with DNR alone and those treated with DNR and CyA in Kaplan‐Meier analysis is shown

Figure 6

Significance of RAS pathway mutation. (A) Genetic landscape of RAS pathway in four t(17;19)‐ALL cell lines and 16 t(1;19)‐ALL cell lines. P value between incidence of mutation in t(17;19)‐ALL cell lines and that in t(1;19)‐ALL cell lines in chi‐square test is shown. (B) Sensitivity to Selumetinib. In left panel, IC50 of Selumetinib was compared between t(17;19)‐ALL cell lines and t(1;19)‐ALL cell lines. In right panel, IC50 of Selumetinib was compared between cell lines with KRAS mutation and those without it. Heptagrams and circles indicate cell lines with KRAS mutation and those without it, respectively. Red and light green symbols indicate t(17;19)‐ALL and t(1;19)‐ALL cell lines, respectively. (C) Multi‐agent resistance of t(1;19)‐ALL cell lines with RAS pathway mutation. Total score of sensitivities to three (Pred, VCR, and L‐Asp), four (Pred, VCR, L‐Asp, and DNR), and five (Pred, VCR, L‐Asp, DNR, and MAF) agents were compared among t(17;19)‐ ALL cell lines, t(1;19)‐ALL cell lines with RAS pathway mutation, and t(1;19)‐ALL cell lines without RAS pathway mutation. P‐values in Mann‐Whitney test are shown.