YM155 potently kills acute lymphoblastic leukemia cells through activation of the DNA damage pathway

Abstract

Background: Novel-targeted therapies are in rapid development for the treatment of acute lymphoblastic leukemia (ALL) to overcome resistance and decrease toxicity. Survivin, a member of the inhibitor of apoptosis gene family and chromosome passenger complex, is critical in a variety of human cancers, including ALL. A well-established suppressor of survivin has been the small molecule, YM155. Reports are identifying other mechanisms of action for YM155. Therefore, we sought to investigate the mode of action and role of YM155 for therapeutic use in the context of ALL.

Methods: Primary ALL samples and ALL cell lines were interrogated with YM155 to identify drug sensitivity. Ph(+)ALL harboring the BCR-ABL1 oncogene were tested for any interaction with YM155 and the multi-kinase inhibitor dasatinib. Representative ALL cell lines were tested to identify the response to YM155 using standard biochemical assays as well as RNA expression and phosphorylation arrays.

Results: ALL samples exhibited significant sensitivity to YM155, and an additive response was observed with dasatinib in the setting of Ph(+)ALL. ALL cells were more sensitive to YM155 during S phase during DNA replication. YM155 activates the DNA damage pathway leading to phosphorylation of Chk2 and H2AX. Interestingly, screening of primary patient samples identified unique and exquisite YM155 sensitivity in some but not all ALL specimens.

Conclusion: These results are the first to have screened a large number of primary patient leukemic samples to identify individual variations of response to YM155. Our studies further support that YM155 in ALL induces DNA damage leading to S phase arrest. Finally, only subsets of ALL have exquisite sensitivity to YM155 presumably through both suppression of survivin expression and activation of the DNA damage pathway underscoring its potential for therapeutic development.

Figure 1

Response to YM155 of primary ALL and AML patient samples. Primary patient and xenografted samples were collected as previously described [14]. (A) Samples were then incubated with increasing concentrations of YM155 (0 nM to 1 μM) and IC₅₀ were calculated using a second-order polynomial. (Filled triangle) ALL samples without a recurring cytogenetic abnormality; (filled diamond) ALL with t(9;22); (filled circle) ALL with 11q23 rearrangement or MLL rearrangement; (filled square) ALL with <44 chromosomes or hypodiploid; (open triangle) ALL with t(1;19); (open square) ALL with t(12;21); (open circle) ALL with hyperdiploid; (grey square) total ALL; (grey triangle) total AML samples. Statistical significance of p < 0.05 by Student’s t-test between total ALL and AML samples. Samples with IC₅₀s within the shaded area are within levels achievable in phase 1 pharmacokinetics [30]. Horizontal line denotes median IC₅₀ of all tested samples. (B) Dose–response curves of two AML-resistant samples (red filled circle, red filled square), one ALL resistant sample (red filled triangle), one ALL intermediate sample (purple open square), and two sensitive ALL samples (black filled diamond, black filled circle). Numbers in parentheses denote patient number (Additional file 2: Table S1 and S2).

Figure 2

Ph⁺ALL samples are sensitive to YM155. (A) The Ph⁺ALL cell line SUPB15 was tested for sensitivity to dasatinib (top left) and YM155 (Top right) with increasing concentrations of drug (0 nM to 10 μM). The IC₅₀ for each drug was approximately 10 nM. Combination of YM155 and dasatinib on SUPB5 cells showed a decrease in IC₅₀ suggestive of synergy/additive by isobologram analysis [34]. Dose–response of SUPB15 cells with YM155 was carried out with increasing concentrations of dasatinib (bottom left). IC₅₀ of individual drug alone was used as the reference value of 1 and subsequent IC₅₀ of combination of drugs were compared (bottom right). Points on the red dotted line would be additive, while points left of the line would be synergistic and points on the right would be antagonistic. (B) Sensitivity to YM155 and dasatinib was further tested on primary Ph⁺ALL patient samples (10–668) and xenograft samples (10–668 xenograft, ICN1, SFO2, TXL3, LAX2, BLQ5, x10-378). Red samples signify dasatinib-resistant T315I mutants. (C) Knockdown of BCR-ABL1 expression. SUPB15 were treated with either non-specific (NS) or ABL1 siRNA. The cells were then incubated in increasing concentrations of YM155 (0 to 1 μM) for 4 days and then assayed for viability with MTS. Top panel describes the viability of the cells normalized to NS without YM155 exposure. Bottom panel describes the viability normalized to with NS or ABL1 without YM155 exposure. An aliquot of electroporated cells were used for immunoblot analysis of BCR-ABL1 knockdown 3 days after electroporation. (D) P53 Ser-15 phospho-flow after treatment with YM155. Each cell line (REH, RCH, HAL01, and SUPB15) and xenograft samples (SFO2, BLQ5) were treated with 100 nM YM155 for 24 h. (Red peak) Control signal for ser15-phospho-p53. (Blue peak) YM155 treatment. REH and RCH cells showed a distinctive increase in phosphorylation of p53. In contrast, the Ph⁺ALL cells showed minimal increase in p53 phosphorylation.

Figure 3

YM155 causes an S phase arrest. (A) Cell cycle arrest with YM155 treatment. An asynchronous population of cells (REH, RCH, and SUPB15) were treated with 100 nM YM155 for 24 h and assayed for DNA content by propidium iodide. (Grey line filled) Control asynchronous population. (Blue line) YM155 treatment. Bars indicate the cell cycle (subG1, G1, S, G2/M). All three cell lines show an increase in S phase and a slight increase in the sub G1 phase within 24 h of treatment. (B) YM155 treatment has a greater effect when exposed to cells that are in S phase of the cell cycle. An asynchronous REH population was sorted by DNA content for G1, S, and G2/M and treated with increasing concentration of YM155 (0 to 100 nM) for 24 h, then assessed for viability using an MTS colorimetric assay (middle panel) or for Annexin V staining (right panel).

Figure 4

YM155 activates DNA damage response. (A) YM155 has multiple effects on RNA expression. REH (wild-type p53), SUPB15 (wild-type p53), and K562 (mutant p53) cells were treated with 100 nM YM155 or vehicle for 24 h and mRNA expression levels of 84 genes were evaluated using the P53 RT² Array. Treatment with YM155 caused about a twofold decrease in survivin mRNA (BIRC5) in the p53 intact cells, but had minimal impact on survivin expression in K562 cells despite potent effects of YM155 on K562 cell growth/viability (IC₅₀ of <10nM [13]). Meanwhile, other genes such as JUN and GADD45A involved in DNA damage response exhibit increased expression after YM155 treatment in all three cell lines. (B) YM155 treatment greatly enhances phosphorylation of Chk2. REH, SUPB15, and HAL01 cells were treated with either vehicle or 100 nM YM155 for 24 h, and protein phosphorylation patterns were assessed using Proteome Profiler Arrays. Values were quantified and normalized to untreated control for each site. REH cells show p53 and Chk2 with the largest change in phosphorylation. SUPB15 shows only Chk2 with the largest change in phosphorylation. HAL01 cells, known to be resistant to YM155 showed minimal change in phosphorylation.

Figure 5

YM155 activates DNA damage response with S phase arrest. (A) Immunoblot verifies the increase in threonine 68 phosphorylation of Chk2. REH, RCH, and SUPB15 cells were treated with increasing concentrations of YM155 (0, 10, 100 nM), doxorubicin (0.1 μg/ml) or dasatinib (100 nM) for 24 h. Whole cell lysates were subjected to immunoblot using antibodies specific for phospho-T68 Chk2, total Chk2, survivin, and Aurora B kinase. (B) Treatment with YM155 causes an increase in γH2AX. REH, RCH, and SUPB15 cells were treated with vehicle or YM155 (10 and 100nM) for 24 h and then stained with γH2AX-FITC and Vibrant DyeCycle Violet Stain. The cells were quantified using FACS/AriaIII for DNA content and FITC. Treatment with YM155 in each cell line showed a dose-dependent increase in γH2AX-FITC, mostly in G1 and S. (C) Comet assays of leukemia cell lines identify DNA damage. REH, RCH, SUPB15, and K562 cells were treated with 100 nM YM155 and 100 nM Dasatinib (excluding REH) for 24 h. Top panel is the box-whisker plot of the percent of DNA in the comet tail after treatment. Bottom panels are photographic representations of the comet assays for each cell line.