Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines

Abstract

Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.

Fig 1. Anti-leukemic activity of bortezomib.

(A) Dose-response curve of bortezomib sensitivity in representative cell lines. The vertical axis indicates % viability in alamarBlue cell viability assay and the horizontal axis indicates log concentration of bortezomib (nM). Phenotype, deletion of IKZF1 and CDKN2A, and IC50 of each cell is indicated. Abbreviations: del, deletion; -, no deletion; hm, homozygous deletion; ht, heterozygous deletion; nt, not tested. (B) Induction of apoptotic cell death by bortezomib. Three cell lines (KOPN34, YCUB2 and Reh) were cultured in the presence or absence of 20 nM of bortezomib for 17 hours, and analyzed with Annexin V-binding (horizontal axis) and 7AAD-staining (vertical axis) using flow cytometry. The percentages of living cells (Annexin V-negative/7AAD-negative) and early (Annexin V-positive/ 7AAD-negative) and late (Annexin V-positive/ 7AAD-positive) apoptotic cells are indicated. (C) Induction of CHOP and NOXA expression. Highly sensitive cell lines (RS4;11 and Reh) and moderately sensitive cell lines (KOPN1 and YCUB2) were cultured in the presence or absence of bortezomib at 20 nM for eight hours, and immunoblotting was performed using Tubulin as an internal control. (D) Bortezomib sensitivity in 79 BCP-ALL, 9 T-ALL, and two MM cell lines. The vertical axis indicates the IC50 value for bortezomib. The p values determined by a Mann-Whitney test and boxplots are indicated.

Fig 2. Anti-leukemic activity of bortezomib in BCP-ALL cell lines.

(A) Association of bortezomib sensitivity with different types of translocation in BCP-ALL cell lines. The vertical axis indicates the IC50 value for bortezomib. Boxplot of each type is indicated. The p value determined by a Mann-Whitney test is indicated when < 0.1. (B-E) Associations of IKZF1 deletion (B and D) and homozygous CDKN2A deletion (C and E) with bortezomib sensitivity in BCP-ALL cell liens (B and C) or in Ph-negative BCP-ALL cell lines (D and E). The vertical axis indicates the IC50 value for bortezomib. The p value determined by a Mann-Whitney test and boxplots are indicated.

Fig 3. Cross-resistance of bortezomib and representative chemotherapeutic agents in 79 BCP-ALL cell lines.

(A and B) Cross-resistance between two of four chemotherapeutic agents. Each vertical or horizontal axis indicates the log IC50 value for either of daunorubicin, vincristine, L- asparaginase, or dexamethasone. Correlation coefficients and p values are shown at the top of each panel. (C and D) Cross-resistance between bortezomib and the four chemotherapeutic agents. The vertical axes indicate the log IC50 values for daunorubicin (C), vincristine (C), L-asparaginase (D), and dexamethasone (D), whereas the horizontal axis indicates those for bortezomib. Correlation coefficients and p values are shown at the top of each panel.

Fig 4. Anti-leukemic activity of carfilzomib.

(A) Induction of apoptotic cell death by carfilzomib. Three cell lines (KOPN34, YCUB2 and Reh) were cultured in the presence or absence of 20 nM of carfilzomib for 17 hours, and analyzed with Annexin V-binding (horizontal axis) and 7AAD-staining (vertical axis) using flow cytometry. The percentages of living cells (Annexin V-negative/7AAD-negative) and early (Annexin V-positive/ 7AAD-negative) and late (Annexin V-positive/ 7AAD-positive) apoptotic cells are indicated. (B) Dose-response curve of carfilzomib sensitivity in representative cell lines. The vertical axis indicates % viability and the horizontal axis indicates the log concentration of carfilzomib (nM). Phenotype, deletion of IKZF1 and CDKN2A, and IC50 of each cell is indicated. Abbreviations: del, deletion; -, no deletion; hm, homozygous deletion; ht, heterozygous deletion; nt, not tested. (C) Correlation of anti-leukemic activity of carfilzomib with that of bortezomib in 79 BCP-ALL cell lines and 9 T-ALL cell lines. The vertical axes indicate the log IC50 values for carfilzomib, whereas the horizontal axis indicates that for bortezomib. Correlation coefficient and p value are shown at the top of panel. (D) Carfilzomib sensitivity in 79 BCP-ALL cell lines and 9 T-ALL cell lines. The vertical axis indicates the IC50 value for carfilzomib. The p value determined by a Mann-Whitney test and boxplots are indicated.

Fig 5. Anti-leukemic activity and cross-resistance between carfilzomib and four representative chemotherapeutic agents in BCP-ALL cell lines.

(A) Association of carfilzomib sensitivity with different types of translocation in BCP-ALL cell lines. The vertical axis indicates the log IC50 value for carfilzomib. Boxplot of each type is indicated. The p value determined by a Mann-Whitney test is indicated when < 0.1. (B) Association of IKZF1 deletion with carfilzomib sensitivity in Ph+ALL and Ph-negative BCP-ALL cell lines. The vertical axis indicates the log IC50 value for carfilzomib. The p value determined by a Mann-Whitney test is indicated. (C and D) Cross-resistance between carfilzomib and representative chemotherapeutic agents in 79 BCP-ALL cell lines. The vertical axes indicate the log IC50 values for daunorubicin (C), vincristine (C), L-asparaginase (D), and dexamethasone (D), whereas the horizontal axis indicates that for carfilzomib. Correlation coefficients and p values are shown at the top of each panel.

Fig 6. Involvement of P-glycoprotein in specific resistance to carfilzomib.

(A) Comparison of sensitivities to carfilzomib and bortezomib in 79 BCP-ALL cell lines and 9 T-ALL cell lines. IC50 of bortezomib (left) and that of carfilzomib (right) in each cell line is connected by line. The vertical axis indicates concentrations of bortezomib and carfilzomib. Boxplots of 88 samples and the p-value in a paired t-test are indicated. (B) Distribution of ratio of bortezomib IC50 value to carfilzomib IC50 value in 79 BCP-ALL cell lines and 9 T-ALL cell lines. Arrowhead indicates median and asterisks indicate t(17;19)-ALL cell lines. (C) Correlation between cell surface expression level of P-glycoprotein and sensitivities to carfilzomib and bortezomib in 79 BCP-ALL cell lines and 9 T-ALL cell lines. The vertical axis indicates log value of relative P-glycoprotein expression, and the horizontal axes indicated log value of carfilzomib IC50 (left panel) or bortezomib IC50 (right panel). Correlation coefficients and p values are shown at the top of each panel. (D) Correlation between cell surface expression level of P-glycoprotein and sensitivities to carfilzomib and bortezomib in 79 BCP-ALL cell lines and 9 T-ALL cell lines. The vertical axis indicates the log value of relative P-glycoprotein expression, and the horizontal axis indicates the log ratio value of bortezomib IC50 to carfilzomib IC50. Correlation coefficient and p value are shown at the top of each panel.

Fig 7. Effect of P-glycoprotein inhibitors on resistance to carfilzomib.

(A and B) Effects of P-glycoprotein inhibitors on carfilzomib and bortezomib sensitivities in P-glycoprotein-positive (A) and P-glycoprotein-negative ALL cell lines (B). The vertical axes indicate % viability determined by alamarBlue cell viability assay and the horizontal axes indicate the concentration of carfilzomib (upper panel) or bortezomib (lower panel). Solid lines, dotted lines, and gray lines indicate dose response curves of carfilzomib or bortezomib alone, carfilzomib or bortezomib in combination with 0.8 μM of nilotinib, and carfilzomib or bortezomib in combination with 5 μM of verapamil, respectively. Mean values in triplicated analyses are indicated, and asterisk is indicated when p value in t-test is <0.05. Relative expression level of P-glycoprotein (P-gp) in each cell line is indicated at the top of panel. (C) Induction of apoptotic cell death by carfilzomib in combination with verapamil or nilotinib. HALO1 cells (left panels) and RS4;11 cells (right panels) were cultured with 20 nM (HALO1) or 10 nM (RS4;11) of carfilzomib (middle panels) or 10 nM of bortezomib (bottom panels) in the presence or absence of 0.8 μM of nilotinib or 5 μM of verapamil for 18 hours, and analyzed with Annexin V-binding (horizontal axis) and 7AAD-staining (vertical axis) using flow cytometry. The percentages of living cells (Annexin V-negative/7AAD-negative) and early (Annexin V-positive/ 7AAD-negative) and late (Annexin V-positive/ 7AAD-positive) apoptotic cells are indicated. (D) Effect of P-glycoprotein inhibitors on carfilzomib (middle panels) and bortezomib (right panels) sensitivities in P-glycoprotein-positive HALO1 (upper panel) and P-glycoprotein-negative RS4;11 (lower panel) cells. The vertical axis indicates cell viability determined by Annexin V-binding and 7AAD-staining in flow cytometric analyses. Mean ± SD of three independent experiments are indicated. Asterisks indicate significance (*p<0.05) in a paired t-test.

Fig 8. Effect of overexpression and knockout of P-glycoprotein on carfilzomib sensitivity.

(A) Cell surface expression of P-glycoprotein in parental 697 cells and 697R cells. Dotted and solid lines indicate fluorescence intensities of isotype control and anti-P-glycoprotein (P-gp) antibodies in parental cells, respectively. Shade indicates fluorescence intensity of anti-P-glycoprotein antibody in 697R cells. (B) Effects of P-glycoprotein (P-gp) inhibitors on carfilzomib (upper panels) and bortezomib (lower panels) sensitivities in parental 697 cells (left panels) and 697R cells (right panels). The vertical axes indicate % viability determined by alamarBlue cell viability assay and the horizontal axes indicate the concentration of carfilzomib or bortezomib. Blue, red, and green lines indicate dose response curves of carfilzomib or bortezomib alone, carfilzomib or bortezomib in combination with 0.8 μM of nilotinib, and carfilzomib or bortezomib in combination with 5 μM of verapamil, respectively. Mean values in triplicated analyses are indicated, and asterisks are indicated when p value in t-test is <0.01. (C) Induction of apoptotic cell death by carfilzomib in combination with P-glycoprotein (P-gp) inhibitors. Parental 697 cells (left panels) and 697R cells (right panels) were cultured with 20 nM of carfilzomib in the presence or absence of 0.8 μM of nilotinib or 5 μM of verapamil for 18 hours, and analyzed with Annexin V-binding (horizontal axis) and 7AAD-staining (vertical axis) using flow cytometry. The percentages of living cells (Annexin V-negative/7AAD-negative) and early (Annexin V-positive/ 7AAD-negative) and late (Annexin V-positive/ 7AAD-positive) apoptotic cells are indicated. (D) Effect of P-glycoprotein knockout on bortezomib and carfilzomib sensitivities in HALO1 cells. P-glycoprotein-positive HALO1 cells were electroporated with an ABCB1-specific CRISPR/Cas 9 vector that contains cDNA of Cas9 fused to human CD4 cDNA via a 2A peptide sequence. 48 hours after electroporation, CD4-positive cells were harvested using anti-CD4 antibody. Upper middle panel indicates flow cytometric analysis of CD4 expression 3 days after electroporation. Upper left and upper right panels indicate flow cytometric analyses of P-glycoprotein (P-gp) expression in parental and CD4-positive population of HALO1 cells, respectively. Bottom panels indicate two color analysis of P-glycoprotein (P-gp) expression (vertical axis) and Annexin V-binding (horizontal axis) by flow cytometry in the CD4-positive populations of HALO1 cells treated with 20nM of bortezomib and 20nM of carfilzomib for 12 hours. Cell viabilities in P-glycoprotein (P-gp)-positive and negative populations are indicated at the left side of each panel.