Carfilzomib Triggers Cell Death in Chronic Lymphocytic Leukemia by Inducing Proapoptotic and Endoplasmic Reticulum Stress Responses

Chronic lymphocytic leukemia (CLL; ref. 1) is an incurable malignancy that is characterized by the progressive accumulation of mature B cells in the peripheral blood, lymph nodes, bone marrow, spleen, and liver. CLL typically affects the elderly population, many of whom have preexisting conditions that restrict the use of the current standard treatment, which involves the combination of chemotherapeutic agents (fludarabine and cyclophosphamide) and an antibody (rituximab; refs. 2, 3). Therefore, combination therapy that targets distinct biologic pathways in CLL pathogenesis represents a significant step outside of the conventional chemotherapy-based approach. Accordingly, we recently identified carfilzomib, an FDA-approved second-generation ubiquitin–proteasome pathway (UPP) inhibitor (4), as a potent pharmacologic agent that causes cytotoxicity in CLL cells before or after treatment with ibrutinib (5–9), an irreversible inhibitor of Bruton's tyrosine kinase (10).

The UPP, which has been validated clinically as a therapeutic target (11), regulates many vital cellular processes including cell-cycle progression, transcriptional regulation, signal transduction, apoptosis, immune response, and elimination of damaged proteins. Formation of a K48-linked polyubiquitin chain is the first step in the UPP that permits recognition of a protein targeted for degradation by the proteasome (12). The 26S proteasome is a proteolytic complex with caspase-like activity (beta 1 subunit), trypsin-like activity (beta 2 subunit), and chymotrypsin-like activity (beta 5 subunit; ref. 13). Carfilzomib (also known as PR-171) is a tetrapeptide epoxyketone-based analogue of the natural compound epoxomicin, which selectively and irreversibly inhibits the chymotrypsin-like activity of the proteasome (14–16).

Structurally, chemically, and mechanistically, carfilzomib differs from bortezomib, the first proteasome inhibitor approved by the FDA, because bortezomib is a synthetic peptide borate that binds reversibly to the proteasome. Carfilzomib targets more selectively the chymotrypsin-like activity of the proteasome than bortezomib (i.e., carfilzomib has less selectivity to the caspase- and trypsin-like activity of the proteasome than bortezomib) in multiple myeloma (17) and earlier phase I clinical trials in multiple myeloma indicated that carfilzomib is a more robust inhibitor of the chymotrypsin-like activity than bortezomib (18, 19). Moreover, in multiple myeloma in vivo and in vitro studies indicated that carfilzomib can circumvent bortezomib resistance because some bortezomib-resistant patients (20) and bortezomib-resistant cell lines (21) remain responsive to carfilzomib therapeutic effects. In CLL, earlier preclinical studies indicated that in vitro treatment of CLL cells with bortezomib resulted in significant cell death by apoptosis (22, 23); however, in a phase II clinical trial conducted with bortezomib in fludarabine-refractory CLL patients, no patients achieved complete or partial responses. Though, some biologic activity was observed (e.g., ≥50% decrease in absolute lymphocyte count, lymph nodes, and spleen size was achieved in some of the patients; ref. 24). Subsequently, it was shown that CLL refractory effect to bortezomib was due to its boronate moiety interaction with plasma components (25–27). Nevertheless, the biologic activities observed with bortezomib in this high-risk factors and severe treatment-resistant group of CLL patients indicated that proteasome inhibitors with better efficacy and safety profiles in combination with agents with different toxicity profiles are warranted.

Carfilzomib cytotoxic activity in CLL has been described previously by us (10) and Gupta and colleagues (25); however, the molecular mechanism by which carfilzomib triggers cell death in CLL has not been elucidated. Furthermore, in our investigation, a bimodal distribution of cytotoxicity was observed in response to carfilzomib treatment: limited or substantial cell death (10). Therefore, in the current report, we first evaluated the cytotoxic effect of carfilzomib in 30 CLL patient samples at five different concentrations. We then used these CLL patient samples and additional B-cell lines to further examine the intracellular pathways implicated in carfilzomib-induced cell death. Finally, we investigated the molecular differences that could potentially be responsible for the heterogenic cytotoxic response to carfilzomib between patients. Thus, the aim of this study was to gain a better understanding of the molecular networks affected by carfilzomib, which could help identify CLL patients with a higher probability of responding to carfilzomib and carfilzomib-based combination therapies who could participate in further clinical studies. Our investigation identified the proapoptotic transcription factor CCAAT/enhancer-binding protein (C/EBP) homology protein (CHOP) as a biomarker that could predict sensitivity to carfilzomib in CLL.

The source and location of all reagents, cell lines, lentiviral vectors, and antibodies that were used in this study are described in Supplementary Table S1.

Peripheral blood samples were collected from CLL patients after written informed consent was obtained in accordance with the Declaration of Helsinki and under a protocol approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center (Houston, TX). All patients and clinical characteristics are summarized in Table 1. Only 5 of 30 patients received prior therapies (patient 085: (1) chlorambucil; (2) fludarabine + rituximab; (3) rituximab; (4) fludarabine + rituximab; (5) bendamustine + rituximab; patient 514: ibrutinib; patient 195: fludarabine + cyclophosphamide + rituximab; patient 575: (1) fludarabine + cyclophosphamide + rituximab; (2) ofatumumab + revlimid; patient 622: ibrutinib).

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll–Hypaque density centrifugation (Atlanta Biologicals). PBMCs were cultured at 1 × 10⁷/mL in RPMI supplemented with 10% human serum. The CLL cell lines (MEC1 and MEC2) and the mouse embryonic fibroblasts (MEF) were cultured in RPMI1640 supplemented with 20% FBS and DMEM supplemented with 10% FBS, respectively. Cells were left untreated or were treated with either the vehicle (DMSO) or the indicated concentrations of carfilzomib for the indicated times. HEK293 supernatants containing lentiviral particles were obtained and used to infect the MEC1 cell line as described previously (28). Carfilzomib-resistant MEC1 was generated by exposing the cells to gradually increasing nontoxic concentrations of carfilzomib (0.5–13 nmol/L) for a period of 4 months. Selection was stopped for 10 days before experimental treatment.

Cell lysates preparation, immunoprecipitation assays, pull-down assays, and immunoblot analyses were performed as described previously (28).

Cells double positive for annexin V/propidium iodide (PI) were identified as described previously (29). Cell viability was also evaluated with use of the CellTiter-Glo Luminescent Cell Viability Assay (Promega Corporation) according to the manufacturer's procedure.

Where shown, values are expressed as mean ± SDs. To evaluate changes between experimental groups, an unpaired two-tailed Student t test was used. P values <0.05 were considered statistically significant.

The cytotoxic effect of carfilzomib was evaluated in PBMCs isolated from 30 CLL patients (Table 1). Apoptosis assessed by Annexin V/PI double positivity showed a concentration-dependent response effect after carfilzomib treatment for 16 hours, with cytotoxic response variability between patient samples (Fig. 1A). For instance, the cytotoxic median response was 11.6% (range: 0.3%–60%) and 27.5% (range: 5.1%–68.4%) with 50 nmol/L and 100 nmol/L carfilzomib (Fig. 1B), respectively. Next, we investigated correlations between clinical and prognostic markers and the cytotoxic effect of carfilzomib. A strong association between IgVH unmutated status (unfavorable biologic marker) and lower cytotoxic effect of carfilzomib was observed at both 50 nmol/L (P = 0.0157) and 100 nmol/L (P = 0.0070; Fig. 1C). Similar associations could not be made with other prognostic factors (data not shown).

Because of its inhibition of the proteasome, carfilzomib treatment resulted in a concentration-dependent accumulation of polyubiquitinated proteins and the stabilization of short-lived proteins (e.g., β-catenin and p-IκBα; refs. 30, 31; Fig. 1D). We next evaluated carfilzomib treatment effect on the levels of pro- and antiapoptotic proteins in CLL samples (n = 7) representing a wide range of cytotoxic profile. Bcl-xL, Bcl-2, cIAP1, cIAP2, p65, and Puma protein expression levels were not affected by carfilzomib treatment (Fig. 1E). At higher concentrations of carfilzomib, however, some of these proteins showed decreased expression, likely due to their cleavage by caspases; for example, the appearance of a 23-kDa cleaved form of Bcl-2, previously shown to be generated by caspase activation and blocked by a pan caspase inhibitor (32), was observed (Fig. 1E). In response to carfilzomib treatment, MCL-1 (Fig. 1D) and Noxa (Fig. 1D and E) protein levels increased in a concentration-dependent manner, with the exception of that in the CLL-871 sample that was highly sensitive to carfilzomib at higher concentrations, in which decrease of MCL-1 protein was likely due to its cleavage by caspases (33).

Carfilzomib treatment activated the endoplasmic reticulum (ER) stress pathway, as indicated by increased protein expression of two transcription factors, activator of transcription 4 (ATF4), and CHOP (34) (Fig. 1D). Furthermore, both the intrinsic and extrinsic apoptosis pathways were activated by carfilzomib in a concentration-dependent manner as determined by the accumulation of the active forms of caspase-9 and caspase-8, respectively (Fig. 1E). Activation of the initiator caspases resulted in activation of the downstream executioner caspase-3 and accumulation of cleaved PARP and cleaved Bcl-2 (Fig. 1E–D).

To dissect the molecular mechanism that triggers the cytotoxic response of carfilzomib, we chose to use the MEC1 and MEC2 CLL cell lines (35). Carfilzomib induced a similar cytotoxic and molecular response in these cell lines. Carfilzomib treatment for 24 hours was cytotoxic to MEC cell lines in a concentration-dependent manner (Fig. 2A). Molecular responses such as accumulation of polyubiquitinated proteins (Fig. 2B) and stabilization of the short-lived protein β-catenin (Fig. 2C) were comparable with those observed in primary CLL lymphocytes. Changes in pro- and antiapoptotic protein expression indicated a similar response to those in the CLL patient samples (Fig. 2B and C). The appearances of a 23-KDa, 55-KDa, and 24-KDa cleaved form of Bcl-2, Hsp90, and MCL-1, respectively, previously shown to be generated by caspase activation and blocked by a pan caspase inhibitor (32, 33, 36), were observed (Fig. 2C and D). Moreover, carfilzomib treatment did not affect the expression of the proapoptotic proteins Bax and Bak (Fig. 2C).

The unfolded protein response (UPR) is triggered when the ER function is disturbed (34) and consists of three signaling pathways controlled by transcription factors ATF4, ATF6, and X-box binding protein 1 (XBP1s; ref. 34). ATF6 protein expression level did not change after carfilzomib treatment (Fig. 2B), and XBP1s protein was not affected in MEC1, but showed a slight increase in MEC2 (Fig. 2B). Similar to what we observed in CLL patient samples, carfilzomib treatment triggered a comparable ER stress response, as noted by the increased protein level of ATF4 and CHOP (Fig. 2B). ATF3 protein, which is also regulated by the ATF4 UPR arm (37), was also increased by carfilzomib treatment (Fig. 2B). These results suggest that ATF4 and CHOP are the prevalent UPR transcription factors induced after carfilzomib treatment in CLL cells. Finally, similar to findings in primary CLL lymphocytes, carfilzomib treatment resulted in activation of the intrinsic and extrinsic apoptotic pathways in both cell lines (Fig. 2B and C).

After 24 hours of incubation, carfilzomib induced the accumulation of MCL-1 and Noxa protein in a concentration-dependent manner in both CLL patient samples and cell lines (Figs. 1D and 2B and C). Treatment with carfilzomib (50 and 100 nmol/L) showed that in the MEC1 line, MCL-1 and Noxa proteins visibly accumulated within 6 hours and 8 hours, respectively (Fig. 3A), whereas the Bak protein level was not affected (Fig. 3A). We next correlated accumulation of MCL-1 and Noxa proteins with their posttranslational modification by the UPP. Carfilzomib treatment of MEC1 and MEC2 resulted in the accumulation of high molecular weight ubiquitinated forms of MCL-1 (Fig. 3B and Supplementary Fig. S1A); however, in a similar assay, accumulation of ubiquitinated forms of Noxa was less obvious (Supplementary Fig. S1B). However, a slower migrating band was present in the Noxa immunoblot, which likely represented a ubiquitinated form of Noxa (Supplementary Fig. S1B, asterisk). To confirm that carfilzomib induces the accumulation of polyubiquitinated Noxa, we performed a pull-down assay with GST-TUBE 2 that specifically binds to polyubiquitinated chains (Fig. 3C and Supplementary Fig. S1C, top); this assay revealed the presence of higher molecular weight forms recognized by anti-Noxa antibody, specifically in carfilzomib-treated cells (Fig. 3C and Supplementary Fig. S1C, bottom). Together, these results show that carfilzomib treatment leads to the stabilization and polyubiquitination of both MCL-1 and Noxa.

MCL-1 exercises its antiapoptotic role by sequestering the proapoptotic Bak and does so by blocking its ability to oligomerize and form pores in the mitochondrial outer membrane (38). The BH3-only protein Noxa competes for MCL-1 binding, which results in the dissociation of the MCL-1/Bak complex, releasing the apoptotic protein Bak (39). Immunoprecipitation assays indicated that MCL-1 formed complexes with both Noxa and Bak in untreated or vehicle-treated cells (Fig. 3D, U and D lanes). After carfilzomib treatment, the major anti-MCL-1 immunoprecipitation complex was between accumulated MCL-1 and Noxa proteins (Fig. 3D anti-Noxa panel and 3E), and less Bak was found in the anti-MCL-1 immunoprecipitation complex (Fig. 3D anti-Bak panel and 3E). These results indicated that carfilzomib favored the formation of a proapoptotic complex formed by MCL-1 and Noxa versus an antiapoptotic complex formed by MCL-1 and Bak.

Our results indicated that carfilzomib treatment affects the dynamic between the prosurvival protein MCL-1 with the BH3-only protein Noxa and the proapoptotic executioner protein Bak. Therefore, we evaluated the role of Noxa and Bak in carfilzomib-induced cell death. We silenced Noxa protein by using a lentivirus expressing a shRNA-targeting mRNA of Noxa (sh-Noxa) in MEC1 cells. Among the five shRNAs tested, only sh-Noxa(63) and sh-Noxa(64) resulted in decreased Noxa protein at baseline (Supplementary Fig. S2A), which correlated with a protective effect against the cytotoxicity induced by carfilzomib (Fig. 4A and Supplementary Fig. S2B). Furthermore, after a concentration-dependent response of carfilzomib, accumulation of Noxa protein was diminished in MEC1 expressing sh-Noxa(64) compared with MEC1 expressing sh-control (Fig. 4B), which correlated with a delay in accumulation of the cleaved form of caspase-3 (Fig. 4B) and protection against cell death (Fig. 4A). Because of the fact that shRNA approaches do not lead to complete protein knockdown, we limited our incubation time to 24 hours with 25 nmol/L carfilzomib.

Bak and Bax are crucial executioners of the intrinsic apoptotic pathway because their activation and oligomerization result in mitochondrial outer membrane permeabilization. As a corollary, Bax and Bak double–deficient cells are resistant to apoptotic stimuli that disrupt mitochondria integrity (40). To evaluate the importance of the carfilzomib-induced cytotoxic effect through the intrinsic apoptotic pathways, we used MEFs deficient in both Bax and Bak (DKO-Bax/Bak; Fig. 4D). Compared with wild-type (WT) MEFs, the inhibitory effect of carfilzomib on the proteasome or ER stress response were not affected in DKO-Bax/Bak MEFs, as shown by accumulation of polyubiquitinated proteins and an increase in ATF4 and CHOP proteins (Fig. 4E). Notably, DKO-Bax/Bak MEFs, compared with WT MEFs, were less sensitive to carfilzomib-induced cell death in a concentration-dependent manner (Fig. 4C), and consequently, activation of caspase-3 was significantly attenuated (Fig. 4E). Taken together, these results indicated that carfilzomib induced an intrinsic apoptotic response that is dependent on the BH3-only protein Noxa and on the proapoptotic proteins Bak and Bax.

Disruption of ER function triggers a stress signaling network through the UPR in an attempt to restore protein homeostasis. However, an unresolved ER stress condition results in UPR-induced apoptosis. Besides activation of both extrinsic and intrinsic apoptotic pathways by carfilzomib treatment, we also observed increased expression of the transcription factor CHOP, a key mediator in ER stress–induced apoptosis (41, 42). To determine the role of CHOP in carfilzomib cytotoxic response, we silenced CHOP protein by using a lentivirus expressing a shRNA-targeting mRNA of CHOP (sh-CHOP) in MEC1 cells. Because CHOP protein is not present at baseline in MEC1 cells (Fig. 2B), but its protein level increases after carfilzomib treatment (Fig. 2B), we treated MEC1 cells that stably expressed different sh-CHOPs with varying concentrations of carfilzomib. Because of the fact that shRNA approaches do not lead to complete protein knockdown, we limited our incubation time to 24 hours with 25 nmol/L carfilzomib. Compared with MEC1 sh-control cells, a decrease of CHOP protein was clearly noted in cells from the four MEC1 cell lines expressing respective sh-CHOP; sh-CHOP(93) was the most effective (Fig. 5B). Notably, downregulation of CHOP protein correlated with protection against the cytotoxic effect of carfilzomib (Fig. 5A) and consequently, attenuation of activated caspase-3 was observed (Fig. 5B). Knockdown of CHOP protein did not affect accumulation of polyubiquitinated proteins, ATF4 and MCL-1, after carfilzomib treatment (Fig. 5C). Comparison of MEC1 sh-control and MEC1 sh-CHOP(93) cells treated with carfilzomib revealed a delay in protein accumulation of Noxa, cleaved form of caspase-3 and PARP, and cleavage of pro-caspase-8 in MEC1 sh-CHOP(93) cells (Fig. 5C). In addition, the cytotoxic effect of carfilzomib was also attenuated in MEC1 sh-CHOP(93) (Fig. 5C, bottom numbers). These results indicate that CHOP, a critical transcription factor in the unresolved ER stress response, plays a significant role in carfilzomib-induced cell death.

Cytotoxic profiling of carfilzomib in 30 CLL samples indicated high heterogeneity responses between patient samples (Fig. 1A and B). Our preclinical investigations using shRNA knockdown approaches and genetically deficient cells indicated that the cytotoxic effect of carfilzomib is dependent on MCL-1 through the proapoptotic proteins Noxa and Bak and also involves a proapoptotic ER stress response through the transcription factor CHOP. Therefore, we decided to compare the expression level of the above proteins in five CLL patient samples with a low cytotoxicity profile (CLL-504, -315, -820, -002, -425) versus five CLL patient samples with a high cytotoxic profile (CLL-575, -835, -622, -176, -417) in response to 50 and 100 nmol/L carfilzomib, respectively (Fig. 6B). All patient samples responded to carfilzomib, as indicated by accumulation of polyubiquitinated proteins (Fig. 6A). In all patient samples, carfilzomib treatment resulted in an apoptotic response, as indicated by accumulation of the cleaved form of caspase-3 and PARP (Fig. 6A). After carfilzomib treatment, Noxa protein accumulation was noticeable with no clear differences between both sets of patient samples (Fig. 6A), whereas the Bak protein level was not affected (Fig. 6A). Noticeably, after carfilzomib treatment, the CHOP protein level was significantly higher in patient samples with high cytotoxic profiles than in those with low cytotoxic profiles (Fig. 6A). After carfilzomib treatment, MCL-1 protein accumulation was noticeable in patients with low cytotoxic profiles but not in patients with high cytotoxic profiles, probably due to excessive cleavage of MCL-1 by caspases (Fig. 6A). Furthermore, to determine whether there were differences in MCL-1, Bak, Noxa, and CHOP protein levels at baseline, and in MCL-1/Noxa and MCL-1/Bak ratios, we quantified the immunoblot band corresponding to these proteins in untreated samples for each patient (Fig. 6C). Differences in MCL-1/Noxa (P = 1.000) and MCL-1/Bak (P = 0.9696) ratios were not significant between both sets of patients. Differences in protein expression at baseline between both sets of patients were significant only for CHOP (P = 0.0488), with the set of patients with high cytotoxicity showing the highest level of CHOP protein (Fig. 6D). These results identified the CHOP protein level as a biomarker to predict sensitivity to the cytotoxic effect of carfilzomib in CLL.

We next generated carfilzomib-resistant MEC1 to investigate the outcome on Noxa and CHOP protein expression after carfilzomib treatment. Carfilzomib-resistant MEC1 (MEC1^CR) was less sensitive to carfilzomib cytotoxic effect than MEC1 subjected to no treatment (MEC1) or DMSO (MEC1^Veh; Fig. 6E). As compared with MEC1^Veh, higher concentrations of carfilzomib were required in MEC1^CR to induce accumulation of polyubiquitinated protein, β-catenin, MCL-1, Noxa, ATF4, CHOP, and activation of the intrinsic and extrinsic apoptotic pathways (Fig. 6F). These results indicated that carfilzomib-induced accumulation of Noxa and CHOP proteins was altered in carfilzomib-resistant cells. Therefore, this model strongly indicates that less sensitivity to carfilzomib cytotoxicity is correlated with less Noxa protein accumulation and weaker ER stress responses.

Taken together, our results indicate that the extent of Noxa protein buildup and the strength of the ER stress response seem to be determinants for carfilzomib-mediated cytotoxicity response. On the basis of the results presented in this report, we propose a signaling model to summarize the different pathways involved in carfilzomib-induced cell death in CLL (Fig. 6G).

CLL is the most common leukemia of the Western world and primarily affects the elderly population, who are often not healthy enough to undergo chemotherapy. Recently, we showed that carfilzomib, a selective and irreversible proteasome inhibitor, is a potent cytotoxic agent in CLL samples isolated from patients treated with ibrutinib, a well-tolerated, orally bioavailable Bruton's tyrosine kinase inhibitor (10). While our current article was under preparation, a phase I trial conducted in a group of 19 patients with relapsed/refractory CLL showed modest activity of carfilzomib (43). In this study, the patient population was high risk with very unfavorable clinical and prognostic factors (i.e., 84% had a Rai stage between 3 and 4, 85% were IgVH unmutated, 47% had del(17p), 21% had del(11q), 68% were refractory to fludarabine, and 70% had complex karyotype), which probably contributed to the poor response observed with carfilzomib. For instance, a recent report indicated that complex karyotype and fludarabine-refractory disease showed poor outcome for CLL patients treated with ibrutinib-based regimens (i.e., shorter event-free survival and overall survival) (44). Nonetheless, this clinical trial opens up a question of what are the determinants of cytotoxicity for carfilzomib in CLL.

From the established prognostic markers in CLL (45–47), we report for the first time a correlation between IgVH unmutated status (unfavorable biologic marker) and lower sensitivity to carfilzomib's cytotoxic effect in CLL patient samples. Correlation between ZAP-70 positivity (unfavorable biologic marker) and the carfilzomib cytotoxic effect was not significant at 50 nmol/L (P = 0.2028) or 100 nmol/L (P = 0.1417). A recent report showed that the cytotoxic effect of carfilzomib was irrespective of del(17p) (25), which is associated with loss of p53 and poor outcome with chemotherapy. However, our report could not illustrate a correlation between the cytotoxic effect of carfilzomib and del(11q), which is associated with loss of ataxia telangiectasia mutated and progressive disease, or del (17p) because only 13% and 10% of the patients, respectively, presented these unfavorable genetic abnormalities. Furthermore, the authors of the phase I trial with carfilzomib did not investigate the cytotoxic effect of carfilzomib ex vivo at baseline to assess any correlation between the carfilzomib-induced cytotoxic effects ex vivo versus in vivo (43). This is particularly relevant because here we used 30 CLL patient samples and showed high interpatient variability in response to the cytotoxic effect of carfilzomib. Notably, using carfilzomib-sensitive and carfilzomib-resistant CLL samples, we identified the protein level of CHOP as a biomarker that could predict sensitivity to carfilzomib in CLL. Nevertheless, this phase I trial showed that carfilzomib was well tolerated, with no concentration-limiting toxicity reported up to an amount of 56 mg/m² and indicated that carfilzomib could be used in combination therapy with other agents. For instance, we previously demonstrated that carfilzomib caused potent cytotoxicity in CLL cells isolated from peripheral blood of patients undergoing ibrutinib therapy (10).

In 2012, carfilzomib was approved by the FDA for treatment of multiple myeloma (4, 48), and several preclinical studies have focused on its mode of action in this B-cell neoplasm. At the apex of carfilzomib-induced apoptotic sequelae is inhibition of chymotrypsin-like activity (49). However, investigation of downstream events of this action has suggested changes in several pathways leading to cell demise. For instance, activation of JNK (50), ER stress response (51), and oxidative stress (52) have been shown to be induced by carfilzomib preceding cell death in multiple myeloma. Carfilzomib also improves multiple myeloma–induced tumor burden on bone homeostasis by targeting osteoblasts and osteoclasts, possibly through β-catenin and IκBα protein stabilization, respectively (53–55). While our article was under review, Narita and colleagues showed a correlation between shorter progression-free survival and lower mRNA levels of ATF3 and ATF4, key players involved in ER stress response, at baseline and during therapy with bortezomib and dexamethasone in patients with refractory/relapsed multiple myeloma (56). However, similar studies need to be conducted to establish any correlation between ER stress response and clinical outcome during carfilzomib therapy.

Noxa protein accumulation (but not Noxa mRNA) after carfilzomib treatment in CLL patient samples has been reported previously (10, 25); however, the biologic significance of this observation has not been investigated. In our study, carfilzomib treatment resulted in accumulation of both the proapoptotic BH3-only protein Noxa and the antiapoptotic protein MCL-1. Accumulated Noxa and MCL-1 were polyubiquitinated and preferentially formed a complex, causing a shift in the balance between the proapoptotic executioner Bak and MCL-1. Depletion of Noxa, Bak, and Bax proteins provided protection against carfilzomib's cytotoxic effect. Notably, in contrast to this study and to our previous report (10), Gupta and colleagues (25) observed no changes in markers associated with activation of the ER stress response. Nonetheless, we showed that carfilzomib preferentially engaged the ATF4 branch of the UPR. The unresolved ER stress response resulted in the accumulation of the transcription factor CHOP, and its depletion had a protective effect against carfilzomib-induced cell death. Taken together, our results confirm the importance of the ER stress pathway and specifically the induction of proapoptotic proteins CHOP and Noxa in the cytotoxic activity of carfilzomib.

We were the first to report that carfilzomib treatment in CLL patient samples resulted in the activation of both the intrinsic and extrinsic apoptotic pathways (10), which we confirmed in the current study. Bcl-2 family members play an important role in the engagement of the intrinsic apoptotic pathways (57). Particularly, equilibrium between proapoptotic and antiapoptotic members is crucial to maintain the integrity of the outer membrane of the mitochondria and prevent the release of cytochrome c, a key component of the apoptosome complex responsible for the activation of the initiator caspase-9 (58). Our study indicated that carfilzomib treatment resulted in upregulation of a protein complex between the proapoptotic BH3-only protein Noxa and the antiapoptotic protein MCL-1, which probably contributed to the unleashing of Bak. Consequently, depletion of Noxa, or depletion of Bax and Bak, the two executioners of mitochondrial outer membrane permeabilization, resulted in a protective effect against the carfilzomib cytotoxic effect. In this study, we showed interplay between Noxa, MCL-1, and Bak; however, because we used cells deficient in both Bak and Bax, we could not determine whether Bak is the preferred apoptotic executioner. Furthermore, higher cytotoxic concentrations of carfilzomib were correlated with a decreased MCL-1 protein level, probably due to its cleavage by caspases that could also contribute to the release of Bak. Furthermore, while our article was under submission, Cheng and colleagues used biochemical assays as well as quadruple knockout cells deficient in Puma/Bim/Bid/Noxa to show that Noxa, like Puma, Bim, and tBid can directly activate Bak and Bax (59).

Carfilzomib treatment also resulted in activation of the extrinsic pathway that is activated by engagement of cell-surface death receptors by their respective ligands (60). Death receptor activation results in the formation of the death-inducing signaling complex, where the apical initiator pro-caspase-8 is recruited and activated and triggers the apoptotic signal. Consequently, caspase-8 activation by carfilzomib indicates activation of death receptor signaling pathways. Because Death Receptor 5 (DR5) transcription has been shown to be regulated by CHOP (61, 62), we hypothesized that DR5 may be responsible for carfilzomib-induced caspase-8 activation. In addition, carfilzomib-induced cell surface expression of DR5 was recently reported in solid tumors (63). Moreover, implication of the extrinsic pathway is also suggested by the fact that deficiency in Bax and Bak proteins did not confer complete protection against carfilzomib-induced cell death.

In conclusion, by using primary CLL lymphocytes as well as cell lines, we showed that the cytotoxic effect of carfilzomib induced a proapoptotic response that involved the Bcl-2 family members Noxa, MCL-1, Bax, and Bak as well as an unresolved ER stress response that resulted in upregulation of the proapoptotic transcription factor CHOP. Accordingly, carfilzomib-induced cytotoxic, apoptotic, and ER stress responses were significantly affected in carfilzomib-resistant cells. Importantly, we identified the CHOP protein level as an important determinant in the cytotoxic efficacy of carfilzomib in CLL.

No potential conflicts of interest were disclosed.

Conception and design: B. Lamothe, V. Gandhi

Development of methodology: B. Lamothe, V. Gandhi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): B. Lamothe, W.G. Wierda, V. Gandhi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B. Lamothe, M.J. Keating, V. Gandhi

Writing, review, and/or revision of the manuscript: B. Lamothe, W.G. Wierda, M.J. Keating, V. Gandhi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): B. Lamothe, V. Gandhi

Study supervision: B. Lamothe, V. Gandhi

The authors thank Dr. Bryant G. Darnay and Tamara K. Locke for critical comments and reading of the manuscript, Ben Hayes and Mark Nelson for coordinating CLL sample distribution, and Xioyan Shao for maintenance of the CFZ resistant MEC1 lines.

This work was supported in part by grant CLL P01 CA81534 from the National Cancer Institute, Department of Health and Human Services, and The University of Texas MD Anderson Moon Shot Program.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.