- The 32Dp210-T315I cells were resistant to imatinib and dasatinib but sensitive to ponatinib and axitinib.
The 32Dp210 and 32Dp210-T315I cells stably express BCR-ABL and BCR-ABL T315I proteins, respectively. To test the sensitivity of these cells to different TKIs, we used the CCK-8 cell proliferation/toxicity detection kit to analyze cell viability after 48-h treatments with different TKIs at different concentrations, and calculated the IC50 values. Consistent with previous studies, 32Dp210-T315I cells were resistant to imatinib and dasatinib, with IC50 values of 11046nM and 4876 nM at 48 h, respectively, which are hundreds- and thousands-fold higher than 32Dp210 cells (IC50 values of 58.7 nM and 1 nM for imatinib and dasatinib, respectively) (Fig. 1a). Both 32Dp210-T315I and 32Dp210 cells were sensitive to ponatinib with similar IC50 values (4.8 nm and 4.3 nm, respectively), indicating the extensive potency of ponatinib in CML treatment (Fig. 1a, b). The IC50 values of axitinib in 32Dp210-T315I and 32Dp210 cells were 43.1 nm and 124.5 nm, respectively, indicating that 32Dp210 cells were insensitive to axitinib. These results confirmed that 32Dp210-T315I cells were resistant to imatinib and dasatinib, but sensitive to ponatinib and axitinib.
- HCQ potentiated the effects of ponatinib and axitinib on 32Dp210-T315I cells.
- HCQ enhanced ponatinib- and axitinib-induced apoptosis in 32Dp210-T315I cells
To test whether HCQ enhanced ponatinib- and axitinib-induced apoptosis in 32Dp210-T315I cells, flow cytometry and an annexin V-7AAD apoptosis kit were used to detect apoptosis. Annexin V-positive cells were defined as apoptotic cells. The rates of apoptosis in 32Dp210-T315I and 32Dp210 cells treated with ponatinib and axitinib with or without HCQ for 24 h and 48 h are shown in Figure 2. Compared with the control group, HCQ did not increase apoptosis levels in 32Dp210-T315I cells(fig.2a,b). Ponatinib and axitinib induced apoptosis in 32Dp210-T315I cells in a time dependent manner; increasing the ponatinib treatment time from 24 h to 48 h was associated with an increased apoptosis rate (24 h: 15.4%±1.4%; 48 h: 39.7%±6.3%); similar results were observed in the axitinib group (24 h: 10.35%±1.6%; 48 h: 42.7%±6.9%)(fig2b). Combinatorial treatment with HCQ significantly increased ponatinib- and axitinib-induced apoptosis in 32Dp210-T315I cells (fig2a, b). After 24-h treatment, the apoptosis rate in the ponatinib group was 15.4%±1.4%, which increased to 55.7%±4.5% in the HCQ + ponatinib group. After 48-h treatment, HCQ increased the ponatinib-induced apoptosis rate from 39.7%±6.3% to 80.6%±4.96% (fig2b). Similar results were found in the axitinib and axitinib+HCQ groups. After 24-h and 48-h treatments, HCQ increased the rate of axitinib-induced apoptosis in 32Dp210-T315I cells from 10.35%±1.6% to 34.2%±2.2% and from 42.7%±6.9% to 67.8%±7.7%, respectively (Fig. 2b).
Finally, we detected the rate of apoptosis in 32Dp210 cells. Neither HCQ nor axitinib induced apoptosis in 32Dp210 cells, while both ponatinib and dasatinib could induce apoptosis. HCQ significantly enhanced the rate of apoptosis in 32Dp210 cells treated with ponatinib and dasatinib, but not for those treated with axitinib (Fig. 2c,d).
- HCQ enhanced the inhibitory effect of ponatinib and axitinib on the clonality of 32Dp210-T315I cells.
To test the effect of HCQ combined with either ponatinib or axitinib on cell proliferation, we inoculated the same number of 32Dp210-T315I cells (500/well) onto a semi-solid medium and observed the number of cell colonies after 7 d. As shown in Figure 3, both ponatinib and axitinib inhibited the clonality of 32Dp210-T315I cells, and the number of colonies was significantly lower than that of the control group (fig3a, b). HCQ alone had little effect on the clonality of 32Dp210-T315I cells, but HCQ significantly enhanced the effects of ponatinib and axitinib. After 7 d, almost no colonies were observed in the HCQ+ponatinib and HCQ+axitinib groups (fig3a, b).
We also performed colony formation assays for 32Dp210 cells. The results showed that HCQ alone had no effect on colony formation in wild-type BCR-ABL expressing cells. Ponatinib inhibited the clonality of 32Dp210 cells, and HCQ further enhanced this effect of ponatinib. Axitinib could not inhibit colony formation in 32Dp210 cells, and HCQ did not modulate axitinib efficacy (fig3b).
- HCQ did not promote ponatinib- or axitinib-induced cell cycle arrest in 32Dp210-T315I cells.
The induction of cell cycle arrest in CML cells is an important aspect of TKI treatment. We next investigated whether HCQ enhanced the cell cycle arrest induced in 32Dp210-T315I cells by ponatinib and axitinib. To this end, cellular DNA was stained with propidium iodide, and stages of the cell cycle were detected by flow cytometry. Consistent with the active proliferation of tumor cells, most of the 32Dp210-T315I cells in the control group were in S phase (60.25%±5.2%), some were in G0/G1 (32.86%±4.24%), and a few were in G2/M phase (6.89±2.1%) (Fig. 4a, b). After 48-h treatment with ponatinib or axitinib, a large number of 32dp210-T315I cells were blocked in G0/G1 phase (60.42%±3.9% and 57.45%±1.64%, respectively); the number of cells in S phase was also significantly reduced (36.15%±2.9% and 38.2%±4.28%, respectively). Compared with the control group, HCQ alone had no effect on cell cycle progression. Additionally, HCQ did not enhance the cell cycle inhibitory effects of ponatinib or axitinib on 32Dp210-T315I cells; there were no significant differences in the cells treated with or without HCQ (Fig. 4a, b).
When studying 32Dp210 cells, we found that ponatinib significantly blocked cell cycle progression. The proportion of cells in G0/G1 increased from 31.8%±8.86% to 60.68%±5%. Additionally, the ratio of S phase cells decreased to 17.09% from 56.03%±7.7%. Axitinib had no effect on the cell cycle progression of 32Dp210 cells. HCQ did not enhance the efficacy of either ponatinib or axitinib on 32Dp210 cells (Fig. 4b).
- HCQ did not increase ponatinib- and axitinib-induced senescence in 32Dp210-T315I cells.
Inducing senescence is the primary mechanism of many anti-tumor drugs. Whether ponatinib and axitinib can induce senescence in 32Dp210-T315I cells, and whether HCQ can further enhance the cell senescence induced by these TKIs have not been evaluated. We assayed for cell senescence by staining with β-galactosidase. Similar to many other tumor cells, senescent cells account for a low proportion of all 32Dp210-T315I and 32Dp210 cells; we found that the rates of senescence in control cells were 1.2%±0.9% and 1.775%±0.56%, respectively(Fig5). While ponatinib and axitinib significantly increased the levels of senescent cells, HCQ did not influence their effects. After treatment with ponatinib and axitinib for 48 h, the proportions of senescent 32Dp210-T315I cells increased from 1.2%±0.9% to 4.97%±1.07% and 5.6%±1.2%, respectively. The proportions of senescent 32Dp210-T315I cells treated with ponatinib and axitinib combined with HCQ were 5.9%±1.5% and 5.7%±1.03%, respectively. These results confirmed HCQ did not increase ponatinib- and axitinib-induced the senescence in 32Dp210-T315I cells (Fig5) .
Axitinib did not induce senescence in 32Dp210 cells, but ponatinib increased the rate of senescent 32Dp210 cells from 1.78%±0.56% to 12.17%±4.56%. Adding HCQ did not significantly alter the proportion of senescent 32Dp210 cells treated with ponatinib alone (10.77±2.21% and 12.17%±4.56%, respectively) (Fig5).
- HCQ inhibited ponatinib- and axitinib-induced autophagy in 32Dp210-T315I cells.
Many studies have revealed that HCQ potentiates the anti-tumor effects of other drugs by inhibiting autophagy. To further understand the mechanism through which HCQ achieves the previously defined effects in 32Dp210-T315I cells, we must answer two questions: (1) do ponatinib and axitinib induce autophagy in 32Dp210-T315I cells; and (2) does HCQ inhibit ponatinib- and axitinib-induced autophagy in 32dp210-T315I cells?
- Ponatinib and axitinib induced autophagy in 32Dp210-T315I cells
LC3II is specifically expressed on autophagic vacuole membranes, and the quantity of LC3II protein reflects the number of autophagosomes. We tested the expression of LC3II protein in 32Dp210-T315I cells by western blot after 12-h treatment with ponatinib and axitinib. The results showed that compared with the control group, LC3II levels were significantly increased in the ponatinib and axitinib groups (Fig6a). We also labeled LC3 protein with a fluorescent antibody and observed the number of autophagosomes under confocal fluorescence microscopy. This showed that while autophagosomes were rarely seen in 32Dp210-T315I cells in the control group, there were a significantly increased number of autophagosomes in the ponatinib group and axitinib groups (Fig. 6b).
The increased levels of LC3II protein expression and number of autophagosomes do not demonstrate induction of autophagy, because autophagy is a dynamic process. Conditions of either autophagy induction or blocking autophagosome degradation will cause LC3II levels to increase. To further identify whether ponatinib and axitinib induced autophagy or blocked autophagic flux, we assayed for LC3II expression in the control group after blocking autophagic flux with HCQ as well as in the ponatinib and axitinib groups. LC3II levels should be unchanged between the three groups if ponatinib and axitinib block autophagosome degradation, whereas if ponatinib and axitinib induce autophagy, LC3II expression should be significantly higher in the treated cells compared with the control group. The results shown in Figure 6c demonstrated that LC3II expression was higher in the ponatinib and axitinib groups than in the control group. These results confirmed that both ponatinib and axitinib induced autophagy in 32Dp210-T315I cells.
We also found that LC3II expression was increased in 32Dp210 cells in the ponatinib group and dasatinib group compared with the control group, but this was not true for the axitinib group (Fig. 6a). After autophagosome degradation was blocked with HCQ, we found that ponatinib and dasatinib could still induce autophagy in 32Dp210 cells, while axitinib could not (Fig. 6c).
- HCQ inhibited ponatinib- and axitinib-induced autophagy in 32Dp210-T315I cells.
We next addressed whether HCQ could inhibit ponatinib- and axitinib-induced autophagy in 32Dp210-T315I cells. ponatinib or axitinib was added with or without HCQ, and then LC3II expression were tested after 6 h. Compared with the ponatinib group, LC3II expression was significantly increased in the ponatinib+HCQ 32Dp210-T315I cells. Similarly, LC3II expression was significantly increased in the axitinib+HCQ group compared with the axitinib group (Fig.7). These results suggested that HCQ inhibited ponatinib- and axitinib-induced autophagy in 32Dp210-T315I cells. We also confirmed HCQ inhibited ponatinib- and dasatinib-induced autophagy in 32Dp210 cells.(Fig7).
- Inhibiting autophagy enhances the killing effect of ponatinib and axitinib on 32Dp210-T315I cells.
- Knocking-down ATG7 inhibited ponatinib- and axitinib- induced autophagy in 32Dp210-T315I cells.
Thus far, we have confirmed that HCQ inhibits ponatinib- and axitinib-induced autophagy, but it remained unclear whether inhibiting autophagy was the mechanism by which HCQ enhanced the killing effect ponatinib and axitinib on 32Dp210-T315I cells. ATG7 is an important autophagy regulator, and it has been reported that knocking down ATG7 can specifically block autophagy. We knocked down ATG7 using a shRNA to inhibit autophagy, and then analyzed whether inhibiting autophagy enhanced the killing effect of ponatinib and axitinib on 32Dp210-T315I cells.
We designed and synthesized a lentiviral plasmid vector with shRNA-ATG7 and GFP to knockdown ATG7 protein expression in 32Dp210-T315I and 32Dp210 cells. Five days after lentiviral infection, the proportion of GFP-positive cells was detected by flow cytometry, and the results showed that the proportion of GFP positive cells was >90% (Fig. 8a). Cells were also collected to detect ATG7 protein expression by western blot. This revealed that shRNA-ATG7-1 and shRNA-ATG7-2 significantly inhibited ATG7 protein expression compared with the control shRNA (Fig. 8b ). After knocking-down the expression of ATG7 in 32Dp210-T315I cells, we tested whether ponatinib and axitinib could still induce autophagy. As seen in Figure8b, after ponatinib and axitinib treatment, LC3II expression was much higher in the control group than in the ATG7-knockdown groups, with almost undetectable LC3II levels in ATG7-knockdown cells. This result showed that knocking down ATG7 blocked ponatinib- and axitinib-induced autophagy in 32Dp210-T315I cells. Similar results were seen in 32Dp210 cells (Fig.8b). After treatment with ponatinib, LC3II expression was lower in ATG7-knockdown 32Dp210 cells compared with control cells (Fig.8b). Thus, knocking down ATG7 blocked ponatinib-induced autophagy in 32Dp210 cells.
- Knocking down ATG7 was nontoxic to 32Dp210-T315I and 32Dp210 cells
We next examined the effects of knocking down ATG7 on apoptosis, cell cycle progression, and colony formation. Annexin V and 7AAD were used to detect apoptosis 15d after lentiviral infection. Annexin V positive celles were thought apoptosis. As shown in Figur9a, the apoptosis rate in ATG7-knockdown cells did not increase compared with controls. The cell cycle analysis showed that there was no significant difference in cell cycle distribution between the ATG7-knockdown group and the control group, with most cells in S and G1 phase, and few in G2/M phase (Fig. 9b). We also found that ATG7 knockdown had no effect on colony formation in 32Dp210-T315I and 32Dp210 cells. After in vitro culture for 7 d, the colony formation rates in the control group were 89.8±14.2%, and 86.9%±17.4% and 90.2%±18% in ATG7-knockdown 32Dp210-T315I cells (Fig.9c). In 32Dp210 cells, the colony formation rates of control and ATG7 knockout groups were 91.8±10.4%, 88.8±13.1% and 87.4±9.6%, respectively (Fig9c). Together, these results suggested that ATG7 knockdown was nontoxic to 32Dp210-T315I and 32Dp210 cells.
- ATG7 knockdown enhanced ponatinib- and axitinib-induced apoptosis in 32Dp210-T315I cells.
We next addressed whether inhibiting autophagy by knocking-down ATG7 enhanced ponatinib- and axitinib-induced apoptosis in 32Dp210-T315I cells. After treatment with ponatinib and axitinib for 48 h, we assayed for apoptosis in ATG7-knockdown 32Dp210-T315I cells. Compared with the control group, ATG7-knockdown 32Dp210-T315I cells were more sensitive to ponatinib and axitinib (Fig10a, b). The apoptosis rates in the control and ATG7-knockdown groups were 32.9%±7.4%, 68.2%±8.2%, and 59.4%±7.3%, respectively, after ponatinib treatment; the apoptosis rates were 45.8%±7.96%, 77.0%±6.3%, and 71.9%±9.1%, respectively, after axitinib treatment (Fig10a,b). Our results also showed that ATG7 knockdown increased the sensitivity of 32Dp210 cells to ponatinib and dasatinib, but did not increase the sensitivity of 32Dp210 cells to axitinib (Fig. 10b).
- ATG7 knockdown did not influence the ponatinib- and axitinib-induced cell cycle arrest of 32Dp210-T315I cells.
To test whether inhibiting autophagy promoted ponatinib- and axitinib-induced cell cycle arrest in 32Dp210-T315I cells, cell cycle distributions were evaluated in ATG7-knockdown and control 32Dp210-T315I cells after treatment with these drugs for 48 h. Both ponatinib and axitinib had similar effects on the ATG7-knockdown and control groups; most cells were blocked in G0/G1 phase, and there were few cells in S phase and in G2/M phase (Fig. 11a, b). There were no statistical difference between ATG7-knockdown group and control groups. After ponatinib, dasatinib and axitinib treatment, the cell cycle distributions of ATG7-knockdown and control 32dp210 cells were similar (Fig. 11 b).
- HCQ enhanced the killing effect of axitinib on 32Dp210-T315I cells in vivo.
Currently, studys on the toxicity of axitinib to BCR-ABL expressing cells with the T315I mutation were limited to only in vitro experiments. Therefore, we next tested whether axitinib could kill cells with the T315I mutation and whether HCQ could enhance this activity using an in vivo model. To answer these questions we established a model of subcutaneous neoplasia with 32Dp210-T315I cells in nude mice. Twenty-four female nude mice aged 5-weeks-old were subcutaneously injected with 4×106 32Dp210-T315I cells .After 2 weeks, tumors had formed under the skin, and the volumes were 0.49–0.52 cm3 (Fig. 12a, b). Nude mice were randomly divided into four groups with six mice in each group: the control group; HCQ group; axitinib group; and HCQ+axitinib group. There was no statistical difference in tumor volume among groups before treatment (Fig. 12b). Ten days after treatment, the mice were sacrificed, and tumors were removed to compare weights. The mean tumor weights were similar in the HCQ and control groups, and were the highest among the four groups. The mean tumor weight in the HCQ+axitinib group was the lowest, while that in the group treated with axitinib alone was the second lowest (Fig 12c). These results confirmed that axitinib could kill 32Dp210-T315I cells in vivo and were consistent with the in vitro results that showed HCQ could further enhance the anti-tumor effects of axitinib.