PMC:3940921 / 5693-17986 JSONTXT

{"project":"2_test","denotations":[{"id":"24590311-11003655-79065513","span":{"begin":617,"end":619},"obj":"11003655"},{"id":"24590311-9585502-79065514","span":{"begin":768,"end":770},"obj":"9585502"},{"id":"24590311-18632609-79065515","span":{"begin":1315,"end":1317},"obj":"18632609"},{"id":"24590311-17591945-79065516","span":{"begin":3695,"end":3697},"obj":"17591945"},{"id":"24590311-19671918-79065517","span":{"begin":7281,"end":7283},"obj":"19671918"},{"id":"24590311-17616698-79065518","span":{"begin":7285,"end":7287},"obj":"17616698"},{"id":"24590311-19157685-79065519","span":{"begin":7877,"end":7879},"obj":"19157685"},{"id":"24590311-17616698-79065520","span":{"begin":8216,"end":8218},"obj":"17616698"},{"id":"24590311-18181098-79065521","span":{"begin":8220,"end":8222},"obj":"18181098"},{"id":"24590311-19671918-79065522","span":{"begin":8981,"end":8983},"obj":"19671918"},{"id":"24590311-16778216-79065523","span":{"begin":9220,"end":9222},"obj":"16778216"},{"id":"24590311-18181098-79065524","span":{"begin":10080,"end":10082},"obj":"18181098"},{"id":"24590311-16778216-79065525","span":{"begin":10084,"end":10086},"obj":"16778216"},{"id":"24590311-17024115-79065526","span":{"begin":10088,"end":10090},"obj":"17024115"},{"id":"24590311-19671918-79065527","span":{"begin":10953,"end":10955},"obj":"19671918"}],"text":"Results\n\nEffect of carfilzomib on key signaling pathways in CML\nCell lines and primary cells were pulsed with carfilzomib at IC50 doses for 1 h and returned to fresh medium for 24 h before protein lysates were prepared and immunoblot analysis was performed to determine the effect of carfilzomib on Bcr-Abl signaling pathways. Carfilzomib treatment resulted in a decrease of p-ERK by 52±11% (P\u003c0.01), with no effect on STAT5 or PI3K signaling pathways (Figure 1a). We next looked at the effect of carfilzomib treatment on ABI 1/2 proteins, which have been reported to inhibit ERK activation that was induced by v-Abl.22 ABI 1/2 proteins have been shown to be stable in normal cells but rapidly degraded via the ubiquitin–proteasome pathway in Bcr-Abl expressing cells.23 We observed increased expression of ABI 1/2 in cells in which BCR-ABL was knocked down using siRNA and also in NBM CD34+38−-enriched cells compared with CML counterparts (Supplementary Figure 1). Treatment with carfilzomib at IC50 levels increased the expression of ABI 1/2 in cell lines and primary CD34+38−-enriched cells (range 2.2–6.8 fold, Figure 1b) and this was associated with an accumulation of polyubiquitinated ABI 1/2 (Figure 1c). ABI 2 has been reported to be tagged for degradation by the E3 ligase TRIM32 in head and neck cancer.24 Figure 1d demonstrates an association of ABI 2 with TRIM32 in the CML cell lines, which appears to be enhanced following treatment with carfilzomib, suggesting that TRIM32 also acts as an E3 ligase to target ABI proteins for degradation in Bcr-Abl expressing cells.\n\nAntiproliferative and apoptotic effects of carfilzomib in models of imatinib-sensitive and -resistant CML\nCML cell lines were treated either continuously or pulsed for 1 h with increasing concentrations of carfilzomib (1–1000 nM) for up to 72 h. A time- and dose-dependent decrease in viability was observed in all cell lines with an IC50 of \u003c15 nM at 24 h cultured continuously in the presence of carfilzomib (Figure 2a). To mimic the in vivo pharmacokinetics of carfilzomib, cell lines were pulsed for 1 h with the same concentrations of carfilzomib, followed by growth in drug-free medium for up to 72 h. This treatment also induced a time- and dose-dependent decrease in viability, although higher concentrations were required to achieve IC50 (20–79 nM, 24 h) (Figure 2b). Under both conditions, imatinib-resistant cell lines displayed equal or greater sensitivity to carfilzomib as their imatinib-sensitive counterparts.\nCD34+38− cells were enriched from three CML patient samples and normal bone marrow (NBM). As numbers of this enriched cell population are small, cells were exposed to a median dose of 50 nM carfilzomib for 1 h before being transferred to drug-free medium. After 24 h, the viability of CML CD34+38− cells was reduced by 39±1 % compared with 19±2 % in NBM (P=0.02; Figure 2c). To assess the effect of carfilzomib on longer-term stem cell proliferation, CD34+ cells were enriched from CML (n=3) and NBM samples (n=2), pulsed for 1 h with 50 nM carfilzomib and transferred to LTC-IC assays. Colony formation was reduced by 58±11% in CML stem cells compared with 44±7% in normal stem cells (P=0.04; Figure 2d).\nThe effect of 1-h exposure to carfilzomib on the induction of apoptosis in CML cell lines and primary samples was investigated. Pulse treatment with carfilzomib at IC50 doses induced an increase in caspase-3 activity along with an associated increase in the number of sub G0/G1 events, indicative of apoptosis (Supplementary Figure 2). Further analysis demonstrated induction of apoptosis through both the caspase-8 and -9 pathways, consistent with previous reports (Figure 2e).25\n\nSynergistic activity of carfilzomib with TKIs imatinib and nilotinib\nThe combination of carfilzomib and imatinib or nilotinib was assessed in imatinib-sensitive and -resistant cell lines. Combinations were set up with all drugs using IC12.5, IC25, IC50 and IC75 values for imatinib-sensitive cell lines and drugs were combined in three ways: (i) carfilzomib and imatinib/nilotinib were added to cells simultaneously; (ii) cells were treated with carfilzomib at t=0 h, followed by imatinib/nilotinib at t=24 h; (iii) cells were treated with imatinib/nilotinib at t=0 h, followed by carfilzomib at t=24 h. Proliferation was assessed 48 h after first drug treatment. Calcusyn software was used to calculate a combination index (CI), whereby a value less than, equal to or greater than 1 indicates synergy, additivity or antagonism, respectively.\nIn imatinib-sensitive cell lines, all combinations of carfilzomib with the TKIs significantly reduced cell viability and proliferation compared with either drug alone (P\u003c0.05) and were found to be synergistic (CI 0.241–0.941; IC50 values). There was no significant difference dependent on the order in which the drugs were added or the TKI used. To assess synergy in imatinib-resistant cell lines, IC50 values derived for the TKIs in imatinib-sensitive cell lines were used. Both, the simultaneous combination of carfilzomib and a TKI and the addition of either TKI before carfilzomib resulted in antagonistic effects (CI 1.053–4.553; IC50). Pretreatment with carfilzomib for 24 h followed by addition of a TKI resulted in a significant reduction in viability and proliferation compared with carfilzomib alone in 4 out of 5 imatinib-resistant cell lines (P⩽0.04; P=0.06 for Ba/F3 H396P cells) and synergy in all imatinib-resistant cell lines (CI 0.609–0.895; IC50). Using p-CRKL as a marker of Bcr-Abl kinase activity, we show that the sequential combination of carfilzomib and a TKI had no effect on p-CRKL, therefore demonstrating that the synergy observed is independent of Bcr-Abl inhibition (Figure 3c). Cell viability following combinations of carfilzomib and imatinib is represented graphically in Figures 3a and b, and CI values and cell proliferation for all combinations using IC50 values are given in Supplementary Table 1 and Supplementary Figure 3.\nWe next investigated if the combination of carfilzomib and a TKI demonstrated a beneficial effect in CML cells. CD34+ cells were enriched from primary CML samples (n=3) and treated as follows: 50 nM carfilzomib, 1 μM imatinib or 30 nM nilotinib used as single agents, or carfilzomib combined simultaneously and sequentially at the prior indicated concentrations. Cells treated with single agent or combined simultaneously were exposed to inhibitors for 24 h; for sequential treatment the cells were exposed to carfilzomib for 24 h followed by either TKI for 24 h. LTC-IC assays were then performed on all cell treatments. No further decrease in colony formation was observed when carfilzomib was combined simultaneously with either TKI, compared with carfilzomib as a single agent. A reduction in colony formation from 42% with carfilzomib treatment alone, to 35%, and 40% of control following sequential treatment with imatinib and nilotinib was observed (Figure 3d); however, the difference was not significant (P=0.06 and P=0.19, respectively).\n\nProteasome profiles and subunit selectivity of carfilzomib in CML\nCarfilzomib has been demonstrated to primarily target CT-L subunits of both the constitutive proteasome (β5) and immunoproteasome (LMP7) in MM and lymphoma cells.26, 27 To evaluate the inhibitory effect of carfilzomib in CML cells, we quantified the levels of constitutive proteasome and immunoproteasome subunits in primary CML cells, normal mononuclear cells and imatinib-sensitive and -resistant CML cell lines (Supplementary Table 2). In primary cells, the amount of active proteasome subunits per total protein concentration was significantly higher in CML cells compared with normal donors (24.05±1.38 ng/μg vs 16.69±2.86 ng/μg, P=0.001), consistent with our previous observations that Bcr-Abl activity is associated with increased proteasome activity.21 We found that immunoproteasome subunits were the major constituent of total proteasome in primary cells (59.1–65.4%) and Ba/F3 cell lines (61.1–70.2%), and comprised 16.3–30.1% of total proteasome in KCL22-S and -R and LAMA84-S and -R CML cell lines (Table 1). This is in agreement with previous reports on cells of hematopoietic origin27, 28 and confirms that the immunoproteasome is a substantive component of total proteasome in myeloid as well as lymphoid cells. The amount of CT-L subunits relative to total cellular protein was similar across all cell lines and primary CML cells (6.29±1.04 ng/μg). LMP7 was the predominant form of CT-L subunit expressed in normal mononuclear cells (83.8%), CML mononuclear cells (76.6%) and Ba/F3 cell lines (52.6–65.8%), and accounted for a significant proportion of CT-L activity in human CML cell lines (36.6–51.2%) (Figure 4a). In keeping with previous observations, we found that primary CML cells and KCL22-S and -R and LAMA84-S and -R predominantly express the immunoproteasome subunit LMP7 (12–25.3%) relative to MECL1 (2.2–15.2%) and LMP2 (1.2–18.6%).26 The contribution of individual proteasome subunits in Ba/F3 cell lines differs quite markedly from both primary cells and human CML cell lines. We have previously demonstrated that the proteasome subunit profiles are cell type dependent29 and this difference may reflect the different lineage origin and species of the cell lines.\nTo examine the effect of carfilzomib on individual proteasome activities, cell lines were pulsed with increasing concentrations of carfilzomib (1–1000 nM) for 1 h and analyzed using the ProCISE assay. Whereas there was a dose-dependent inhibition of all proteasome activities (Figures 4b–d), carfilzomib displayed preferential inhibitory activity against CT-L subunits, with IC50 values of ⩽8.32 nM for the β5 subunit and ⩽16.55 nM for LMP7. IC50 values for T-L and C-L subunits were at least threefold higher (Supplementary Table 3).\n\nThe presence of immunoproteasome subunits increases sensitivity to carfilzomib\nWe, along with others, have reported that the sensitivity of tumor cells to PIs may be associated with differential expression of proteasome subunits.28, 29, 30 In this study, we found that in contrast to the proteasome profiles observed for CML cell lines and primary samples above, immunoproteasome subunits made little or no contribution to the overall proteasome activity in K562 cells (Supplementary Table 2). In this regard, the K562 cell line is more similar to a nonhematopoietic cell line. We demonstrate that although carfilzomib is a potent inhibitor of the β5 CT-L subunit in K562 cells (IC50=6.68 nM; Supplementary Table 2), the IC50 value for carfilzomib in reducing the proliferation of this cell line was \u003efivefold higher than the corresponding values for the other CML cell lines. We therefore sought to investigate whether the difference in sensitivity we observed with K562 cells was related to immunoproteasome expression. A549 lung carcinoma cell line, known to express low amounts of immunoproteasome,26 was included as a comparison. K562 and A549 cells were cultured in the presence of 100 U/ml IFN-γ for 72 h to induce immunoproteasome expression, before treatment with carfilzomib (1–1000 nM continuous exposure for 24 h). IFN-γ treatment resulted in an increase in the expression of LMP7 by fourfold in K562 cells and 10-fold in A549 cells (Figures 5a and b); IFN-α treatment produced similar results (Supplementary Figure 4C). The increase in LMP7 expression corresponded with a significantly increased sensitivity to carfilzomib (IC50 value reduced from 608 nM to 179 nM in K562 cells and 92 nM to 10 nM in A549 cells, P⩽0.01; Figures 5c and d). Conversely, we looked at the effect of LMP7 knockdown on the sensitivity of cells to carfilzomib. KCL22S and LAMA84S cells were treated with siRNA targeted against LMP7 before treatment with carfilzomib (1–1000 nM continuous exposure for 24 h). Downregulation of LMP7 (Supplementary Figure 4) was associated with a significant decrease in sensitivity to carfilzomib; the IC50 value for KCL22S cells increased from 5 nM to 16 nM (P=0.01) and LAMA84S cells increased from 7 nM to 20 nM (P=0.04). These results demonstrate that the presence of both the β5 and LMP7 CT-L subunits are required for optimal effect with carfilzomib and pretreatment with IFN-γ can sensitize cells to carfilzomib.\n"}