PMC:3940921 / 17987-27019 JSONTXT

{"project":"2_test","denotations":[{"id":"24590311-21195056-79065528","span":{"begin":543,"end":545},"obj":"21195056"},{"id":"24590311-20305692-79065529","span":{"begin":547,"end":549},"obj":"20305692"},{"id":"24590311-20068223-79065530","span":{"begin":551,"end":553},"obj":"20068223"},{"id":"24590311-21282974-79065531","span":{"begin":555,"end":557},"obj":"21282974"},{"id":"24590311-21816374-79065532","span":{"begin":865,"end":867},"obj":"21816374"},{"id":"24590311-23138275-79065533","span":{"begin":1243,"end":1245},"obj":"23138275"},{"id":"24590311-12023956-79065534","span":{"begin":2113,"end":2115},"obj":"12023956"},{"id":"24590311-11003655-79065535","span":{"begin":2425,"end":2427},"obj":"11003655"},{"id":"24590311-9585502-79065536","span":{"begin":2684,"end":2686},"obj":"9585502"},{"id":"24590311-9585502-79065537","span":{"begin":2730,"end":2732},"obj":"9585502"},{"id":"24590311-18632609-79065538","span":{"begin":3238,"end":3240},"obj":"18632609"},{"id":"24590311-7590236-79065539","span":{"begin":3242,"end":3244},"obj":"7590236"},{"id":"24590311-15591787-79065540","span":{"begin":3246,"end":3248},"obj":"15591787"},{"id":"24590311-18632609-79065541","span":{"begin":3261,"end":3263},"obj":"18632609"},{"id":"24590311-7590236-79065542","span":{"begin":3467,"end":3469},"obj":"7590236"},{"id":"24590311-20068223-79065543","span":{"begin":4518,"end":4520},"obj":"20068223"},{"id":"24590311-19157685-79065544","span":{"begin":4522,"end":4524},"obj":"19157685"},{"id":"24590311-11003655-79065545","span":{"begin":4635,"end":4637},"obj":"11003655"},{"id":"24590311-20068223-79065546","span":{"begin":4708,"end":4710},"obj":"20068223"},{"id":"24590311-12454739-79065547","span":{"begin":5875,"end":5877},"obj":"12454739"},{"id":"24590311-15902298-79065548","span":{"begin":5896,"end":5898},"obj":"15902298"},{"id":"24590311-15665113-79065549","span":{"begin":5914,"end":5916},"obj":"15665113"},{"id":"24590311-18728023-79065550","span":{"begin":5933,"end":5935},"obj":"18728023"},{"id":"24590311-19157685-79065551","span":{"begin":6454,"end":6456},"obj":"19157685"},{"id":"24590311-21816374-79065552","span":{"begin":6458,"end":6460},"obj":"21816374"},{"id":"24590311-15590391-79065553","span":{"begin":6462,"end":6464},"obj":"15590391"},{"id":"24590311-19671918-79065554","span":{"begin":6756,"end":6758},"obj":"19671918"},{"id":"24590311-19671918-79065555","span":{"begin":6887,"end":6889},"obj":"19671918"},{"id":"24590311-17616698-79065556","span":{"begin":6891,"end":6893},"obj":"17616698"},{"id":"24590311-18181098-79065557","span":{"begin":7333,"end":7335},"obj":"18181098"},{"id":"24590311-16778216-79065558","span":{"begin":7337,"end":7339},"obj":"16778216"},{"id":"24590311-17024115-79065559","span":{"begin":7341,"end":7343},"obj":"17024115"},{"id":"24590311-19671918-79065560","span":{"begin":7372,"end":7374},"obj":"19671918"}],"text":"Discussion\nAlthough the introduction of TKIs into the clinic has provided a major advancement in the treatment of CML, the development of drug resistance and persistence of TKI-resistant stem cells remain obstacles to eradicating the disease. The proteasome has become a validated target for cancer therapy, particularly in hematological malignancies. A number of reports demonstrate that bortezomib induces apoptosis in BCR-ABL expressing cells, including those resistant to imatinib and suggest that PIs may have significant activity in CML.18, 19, 20, 31 To date, only one study has investigated the clinical activity of bortezomib in CML. The pilot study demonstrated minimal efficacy of bortezomib as a single agent in imatinib-refractory CML and suggested that future trials should focus on alternative use of PIs in CML, such as combination-based treatments.32 In support of this, a recent report by Dewer et al.,31 demonstrates that a bortezomib-based regimen resulted in a complete hematological, cytogenetic and molecular remission in BCR-ABL+ acute lymphoblastic leukemia. Until recently, bortezomib was the only PI licensed for use in the clinic and its use can be restricted by limiting side effects such as peripheral neuropathy.17 However, the next generation PI carfilzomib has received FDA approval for the treatment of relapsed/refractory myeloma and appears to exhibit less toxicities than bortezomib. In this study, we have analyzed the effect of carfilzomib, both on its own and in combination with TKIs, in cell lines that reflect a wide spectrum of imatinib resistance and in CD34+38− populations, which represent the TKI-resistant stem cell population.\nBcr-Abl activates multiple downstream signaling pathways, including ERK, PI3K and STAT5 pathways, which contribute to leukemic cell proliferation and survival. We analyzed the effect of carfilzomib on these signaling pathways and found that exposure to carfilzomib resulted in a downregulation of phosphorylated ERK; this effect was replicated across all cell line models and primary CD34+38− cells. A previous study by Orlowski et al.,33 has also suggested inhibition of ERK signaling as a key factor in proteasome inhibitor-mediated apoptosis. Following on from this, we investigated the effect of carfilzomib on ABI proteins that have been reported to negatively regulate cell growth and transformation by specifically targeting the ERK pathway.22 ABI proteins were originally identified as binding partners of the Abl tyrosine kinase and two highly homologous members of this family, ABI-1 and ABI-2, were shown to be rapidly degraded through the ubiquitin–proteasome system in Bcr-Abl-expressing cells.23 In keeping with the findings by Dai et al.,23 we found that ABI 1/2 levels were higher in normal compared with CML CD34+38− cells and also that ABI 1/2 expression increased in CML cell lines in which BCR-ABL was knocked down. Treatment with carfilzomib resulted in a similar increase in ABI 1/2 protein expression by preventing the degradation of these proteins through the proteasome, as proved by the accumulation of polyubiquitinated ABI 1/2. ABI-2 in particular, has been documented to act as a tumor suppressor and an inhibitor of cell migration.24, 34, 35 Kano et al.,24 demonstrated that the E3 ligase TRIM32 is responsible for mediating the degradation of ABI-2 in head and neck squamous cell carcinoma, thereby promoting tumor cell growth, metastasis and drug resistance.34 In this study, we have demonstrated a protein–protein interaction between ABI-2 and TRIM32 and observed an accumulation of this protein interaction following treatment with carfilzomib, suggesting that TRIM32 may also act as an E3 ligase for ABI-2 in Bcr-Abl expressing cells. The role of Bcr-Abl in promoting the degradation of ABI-2 through TRIM32 is yet to be fully delineated. Collectively, these results demonstrate a novel mechanism of action for PIs in CML.\nThe effect of carfilzomib on the proliferation of imatinib-sensitive and -resistant cell populations was evaluated using either continuous treatment, or 1-h pulse treatment, which is more reflective of the in vivo pharmacokinetics of carfilzomib. Both methods induced a time- and dose-dependent decrease in proliferation, with equal or greater sensitivity to carfilzomib exhibited by imatinib-resistant cell lines compared with their imatinib-sensitive counterparts. This is in agreement with earlier studies demonstrating that imatinib resistance does not affect sensitivity to PIs.20, 21 The IC50 values obtained for carfilzomib in CML cell lines are comparable to those reported for MM cell lines,22 suggesting that these doses may be clinically relevant. Heaney et al.,20 previously demonstrated that primitive CML stem cells are sensitive to the apoptotic effects of bortezomib. Here we also demonstrate an antiproliferative and apoptotic effect of carfilzomib on the imatinib-resistant stem cell population. Furthermore, we found that the stem cells enriched from CML samples displayed greater sensitivity to carfilzomib than those enriched from nonmalignant samples.\nHaving established an effect for carfilzomib as a single agent in cell line models of imatinib-sensitive and -resistant CML, we also looked at the effect of carfilzomib in combination with the TKIs imatinib and nilotinib. In imatinib-sensitive cell lines, all combinations of carfilzomib with either TKI resulted in synergistic effects. Notably, we also observed that pretreatment with carfilzomib sensitizes imatinib-resistant cells to TKIs. The mechanism behind this synergistic effect is unclear; however, we demonstrate that it is independent of Bcr-Abl activity. Similar effects have been documented in a number of other studies using inhibitors of signaling pathways downstream of Bcr-Abl. For example, the combination of imatinib with chemotherapeutic agents,36 a Hsp90 inhibitor,37 PDK-1 inhibitor38 and leptomycin B39 have all been shown to produce synergistic activity in imatinib-resistant cell lines. The synergism between carfilzomib and TKIs was not replicated in the CML-quiescent stem cell population, nonetheless carfilzomib has a significant effect as a single agent in these cells.\nTotal levels of proteasome activity were found to be significantly higher in primary CML cells compared with normal, supporting previous observations by our group and others that demonstrate an increase in proteasome activity in BCR-ABL+ cells.21, 32, 40 Primary cells and cell lines exhibited both constitutive proteasome and immunoproteasome subunits. Primary cells were found to express a higher percentage of the LMP7 subunit than CML cell lines. This pattern of higher immunoproteasome activity in primary cells has also been observed in MM.26 Carfilzomib has been reported to selectively inhibit the CT-L subunits (β5 and LMP7) of the proteasome in MM and lymphoma cells.26, 27 In keeping with this, we show that carfilzomib inhibits CT-L subunits with at least threefold selectivity compared with T-L and C-L subunits.\nFinally, we investigated whether the presence of immunoproteasome subunits in CML cells influences the sensitivity of the cells to carfilzomib. Our group, along with others, have reported that sensitivity of tumor cells to PIs is associated with proteasome activity levels and subunit composition.28, 29, 30 In addition, Parlati et al.,26 demonstrated that inhibition of either the β5 or LMP7 subunit alone was insufficient to produce an antitumor response in hematopoietic cells. In this study, we found that the CML cell line K562 exhibited little to no immunoproteasome activity and, although carfilzomib was a potent inhibitor of the β5 subunit in these cells, the IC50 for carfilzomib in K562 cells was \u003efivefold higher than that for the immunoproteasome-expressing cell lines. Induction of immunoproteasome expression in K562 cells by IFN-γ resulted in an increase in sensitivity to carfilzomib, and this effect was also replicated in the lung carcinoma cell line A549. Conversely, knockdown of LMP7 expression reduced sensitivity to carfilzomib in KCL22S and LAMA84S cells. These results suggest that both CT-L subunits are required for optimal effect and that proteasome composition may predict the response to carfilzomib.\nIn summary, this work shows that carfilzomib effectively reduces proliferation and induces apoptosis in CML cell lines and primary cells, regardless of their sensitivity to imatinib. We have shown for the first time that proteasome inhibition in CML cells is associated with a decrease in ERK signaling and increased expression of the tumor suppressor ABI-2. Combination of carfilzomib with TKIs has been demonstrated to generate synergistic effects, even in imatinib-resistant cells, and the presence of immunoproteasome subunits influences sensitivity to carfilzomib. Taken together, these findings suggest that carfilzomib could be a useful therapeutic agent in CML, and its role in resensitizing imatinib-resistant cells to TKIs warrants further investigation."}