Multiple myeloma had been successfully treated by combining lenalidomide and bortezomib with reports suggesting benefits of such a combination even in relapsed/refractory cases. Recently, it was demonstrated that Ikaros degradation by lenalidomide happens via proteasome-dependent pathway and this process is critical for the eradication of myeloma cells. On the basis of this, an antagonistic effect should be observed if a combination of both these agents were used, which however is not the observation seen in the clinical setting. Our study demonstrates that when these agents are combined they exhibit a synergistic activity against myeloma cells and degradation of Ikaros happens by a proteasome-independent calcium-induced calpain pathway. Our study identifies the crucial role of calcium-induced calpain pathway in inducing apoptosis of myeloma cells when this combination or lenalidomide and bortezomib is used. We also report that this combination enhanced the expression of CD38 compared with lenalidomide alone. Thus, data from our study would establish the rationale for the addition of daratumumab along with this combination to further enhance therapeutic activity against multiple myeloma.
Lenalidomide and bortezomib combination degrades IKZF1 in multiple myeloma through a calcium-dependent calpain and caspase pathway.
Multiple myeloma represents a spectrum of B-cell–derived neoplasm that accounts for approximately 13% of all hematologic malignancies (1). In the younger patients, over the past 2 decades, high-dose chemotherapy along with autologous stem cell transplantation has been the standard of care (1, 2). In the past decade, introduction of immunomodulatory drugs (IMiD) and proteasome inhibitors along with dexamethasone have been shown to increase the rate of complete response without increased toxicity and were able to increase progression-free survival and overall survival in patients with newly diagnosed multiple myeloma (3), as well as improved partial response in two third of patients with relapsed/refractory multiple myeloma (4).
Recent evidences suggests that the mechanism of action of lenalidomide in multiple myeloma depends upon its ability to interact with cereblon E3 ubiquitin ligase to induce the degradation of IKZF1 and IKZF3 proteins via proteasome machinery (5, 6). It was also noted that this IKZF1 degradation axis remains central to the efficacy of lenalidomide in multiple myeloma. This degradation of IKZF1 and IKZF3 results in the downregulation of c-MYC and IRF4 and as a result the multiple myeloma cells undergo apoptosis (7). Recently, it was also shown that when multiple myeloma cells are treated with lenalidomide, it interfere with CD147 and MCT1 protein interaction by binding to cereblon, which acts as chaperon for CD147 maturation (8). Although inhibition of CD147–MCT1 complex induces cell death in multiple myeloma, the IKZF1 degradation by lenalidomide still remains central for disease clearance (9). Recently, it has been shown that IKZF1 can suppress the expression of CD38 antigen in myeloma cells and degradation of IKZF1 by IMiDs can induce CD38 expression (10) thereby facilitating daratumumab-mediated cytotoxicity. Furthermore, pretreating the cells with bortezomib a proteasome inhibitor, resulted in the accumulation of IKZF1, thereby reducing the efficacy of lenalidomide (9). However, even prior to this understanding of the mechanism of action of lenalidomide in the treatment of multiple myeloma, the combination of lenalidomide and bortezomib was routinely used in combination in the clinic with reported synergy on combining them (3, 4, 11). The mechanism of synergy and more importantly the fate of IKZF1 on combining these drugs are not well understood. In this article, we undertook a study to evaluate the fate of IKZF1 in multiple myeloma when a combination of lenalidomide and bortezomib was administered.
Materials and Methods
The human myeloma cell line MM.1S and U266 were obtained from the ATCC and were used in their early passages. The cell lines were periodically characterized phenotypically by flow cytometry. Mycoplasma detection was done once in every 6 months and the cell lines used were free from Mycoplasma contamination (Universal Mycoplasma detection Kit, ATCC).
Reagents and antibodies
Chemicals such as lenalidomide, bortezomib, MG132, bafilomycin A1, hydroxychloroquine, PD150606, Calpeptin, BAPTA, Ionomycin, E64, pepstatinA, and zVAD.fmk were obtained from Sigma and were used in this study. Antibodies against Actin, p62, IKZF1, BIM, Bcl2, cIAP2, and XIAP1 (Santa Cruz Biotechnology), LC3, Caspase-3, and PARP (Cell Signaling Technology), IRF4 and IKZF3 (Abcam), CD38 FITC conjugate (BD Biosciences), and anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology), and with Alexa Fluor 594 (Invitrogen) were also used for Western blotting, immunofluorescence, and flow cytometry assays.
Assays for apoptosis
Myeloma cell lines were treated with drugs for indicated time. After incubation at 37°C in CO2 incubator, the leukemic cells were carefully pipetted out and their viability was measured using Annexin V/7AAD Apoptosis Assay Kit (BD Pharmingen) as per the manufacturer's protocol. The flow data were analyzed using Kaluza Software (Beckman Coulter) and FlowJo v10 (FlowJo LLC).
In vitro cytotoxicity assay
In vitro cytotoxicity of drugs was determined using the MTT assay as described previously (12). Combination index between drugs was calculated using CalcuSyn Software (Biosoft).
Total RNA was extracted using TRizol Reagent (Invitrogen Carlsbad). Five-hundred nanogram of the extracted RNA was converted into cDNA using superscript II cDNA Kit (Invitrogen Carlsbad). The expression of genes was studied using SYBR green method (Finnzymes F410L, Thermo Fisher Scientific). The Ct values were normalized with ACTB and the fold differences were calculated using 2−ΔΔCt method. Primer details are provided in Table 1.
|S. No. .||Primer .||Sequence .|
|S. No. .||Primer .||Sequence .|
Coimmunoprecipitation and immunoblots
Myeloma cells lines (MM.1S and U266) were treated with drugs for indicated time and the homogenates were obtained by cell lysis in RIPA Buffer (Sigma), with complete protease inhibitors (Roche). Coimmunoprecipitation was performed using Co-IP Kit (Thermo Pierce) according to the manufacturer's protocol. The lysates and elutes were analyzed in SDS-PAGE. After protein transfer to nitrocellulose membrane, membranes were blocked with BSA (5%, 2 hours) followed by incubation with primary antibodies overnight. The protein bands were detected by standard chemiluminescence method (Thermo Pierce Femto).
This was done as previously reported by us (13). In summary, the myeloma cells were treated with drugs for 24 hours and cytospun slides were made. The cells were fixed in 4% paraformaldehyde followed by blocking using 5% goat serum. It was further incubated with primary antibodies such as IKZF1 (Santa Cruz Biotechnology) overnight at 4°C. The slides were rinsed with PBS thrice and incubated with secondary antibodies (anti-mouse) conjugated with Alexa Fluor 594, (Invitrogen) for 1 hour. The slides were again washed, air dried, and counterstained with DAPI containing mountant (Vectashield). The images were acquired in fluorescence microscope (Axioimager M1, Carl Zeiss) at 100 × with oil immersion and images were analyzed using ISIS Metasystem (Metasystems GmbH).
Induction of autophagy was assessed using CYTO-ID Autophagy Detection Kit (Enzo Life Sciences). The assays were carried out as per the manufacturer's instructions post 24 hours of drug treatments.
Calcium flux assay
Relative estimate of intracellular calcium was analyzed using Fura2-Am Reagent (Molecular Probes). Briefly, the cells were treated with drugs for 6 hours followed by incubation with Fura2-Am reagent (1 μmol/L) for 1 hour. The cells were washed and incubated further for 30 minutes. The fluorescence intensity was measured as ratio of values at an excitation at 340 nm and 380 nm with an emission at 510 nm, using Spectramax M4 (Molecular Devices).
Calpain activity assay
The cells were treated with different drugs for 24 hours and were resuspended in 100 μL extraction buffer and homogenized by pipetting. Protein concentration was determined by the Bradford assay. Calpain activity was measured using a kit from Abcam according to the manufacturer's instructions. The fluorescence intensity (calpain activity) was measured at an excitation at 400 nm and with an emission at 505 nm, using Spectramax M4 (Molecular Devices).
Statistical significance was calculated using Student t test (two tailed t test) or one sample t test. The values are denoted as mean ± SD. The P values are denoted as *, P < 0.02; **, P = 0.001; ***, P = 0.0001; NS, not significant.
Lenalidomide and bortezomib combination induces apoptosis synergistically in multiple myeloma cell lines
We initially tested the efficacy of lenalidomide on two multiple myeloma cell lines (MM.1S and U266). We evaluated the viability on day 5 post-treatment of lenalidomide and found that MM.1S cell line is comparatively more sensitive to lenalidomide compared with U266 (Fig. 1A). We also checked for the effect of bortezomib on these cell lines. Again we found that MM.1S cell lines were comparatively more sensitive to bortezomib compared with U266 (Fig. 1B). These results were consistent with previous reports (14). Next, we showed a significantly increased kill, when bortezomib was combined with increasing concentration of lenalidomide and vice versa (Fig. 1C and D; Supplementary Fig. S1). The synergism was well documented with a combination index of 0.5 for U266 and 0.7 for MM.1S. The induction of apoptosis was also confirmed by a Western blot where a decrease in antiapoptotic proteins and increase in apoptotic proteins were observed in MM.1S cell line (Supplementary Fig. S2). Next, we assessed the function of proteasome when lenalidomide was combined with bortezomib. We observed that combining these two agents does not interfere with bortezomib action in inhibiting proteasome complex (Fig. 2A). As a result of this proteasome inhibition, we also observed an accumulation of ubiquitinated proteins in the bortezomib- and lenalidomide + bortezomib–treated cells when compared with lenalidomide alone treated and control cells (Fig. 2B).
Combination of lenalidomide and bortezomib degrades IKZF1 through a proteasomal-independent pathway
Next, we looked for the fate of IKZF1 in myeloma cells treated with lenalidomide and bortezomib combination. We observed a degradation of IKZF1 in both MM.1S (Fig. 2B and C) and U266 cells (Supplementary Fig. S3A and S3B) in lenalidomide alone and lenalidomide + bortezomib–treated cells. This was further validated by downregulation of IRF4 (transcriptionally regulated by IKZF1) by Western blot analysis and qPCR in MM.1S cell line (Fig. 2D). We also noted that along with IKZF1, IKZF3 was also degraded in the combination-treated cells (Fig. 2B). We were able to demonstrate that before degradation, IKZF1 was ubiquitinated by an immunoprecipitation assay when a combination of lenalidomide + bortezomib was used (Supplementary Fig. S4). A similar effect of degrading IKZF1 and IKZF3 was observed when another irreversible proteasome inhibitor (MG132; Fig. 2B) was used along with lenalidomide. This suggested that the observed phenomenon is an after effect of proteasome inhibition. Taken together, we were able to show that in spite of significant proteasome complex inhibition IKZF1 was degraded, which suggested involvement of alternative pathway for its degradation.
IKZF1 is not degraded by autophagy pathway which is upregulated because of proteasome inhibition
From our previous article (15), it was evident that autophagy can act as an alternative pathway to degrade an onco-protein meant to be degraded via proteasome complex. We hypothesized, that autophagy pathway induced because of proteasome complex inhibition could degrade IKZF1 (ubiquitinated by lenalidomide). Hence, we looked for the expression of autophagy-associated proteins. As expected and as previously reported by us (15), we observed an induction of autophagy pathway, evidenced through increased generation of LC3II bands along with p62 and poly-ubiquitinated protein accumulation and degradation at time points (Fig. 3A), which correlated with time points at which there was an increased intensity of autophagosome staining measured by Cyto-ID (Fig. 3B). To support our hypothesis that IKZF1 is degraded by autophagy in the absence of proteasome complex, we pretreated myeloma cells with autophagy inhibitors [hydroxychloroquine and bafilomycin (BAFA1), inhibition of autophagy was confirmed by cyto-ID and other autophagy markers; Supplementary Fig. S5] followed by treatment with lenalidomide and bortezomib. However, we did not observe an accumulation of IKZF1 proteins when autophagy was inhibited (Fig. 3C; Supplementary Fig. S6). Taken together, these data indicate that IKZF1 is degraded by an alternative pathway independent of the proteasome and autophagy pathways when lenalidomide and bortezomib are combined.
IKZF1 is degraded by calcium-dependent calpain and caspase pathway by the combination in multiple myeloma cells
Previous reports had demonstrated that inducing apoptosis by kinase inhibitors in myeloma cells can degrade IKZF1 (16). It was also well recognized that lenalidomide degrades IKZF1 in MDS cells through activation of calcium-activated calpain pathway (17) and bortezomib can also induce a transient calcium flux in cells (18, 19). We hypothesized, that when lenalidomide and bortezomib were combined, it induces an increased calcium flux followed by apoptosis where IKZF1 can be degraded via calpain (activated by calcium flux) and caspase-dependent pathways. Toward this, we measured the intracellular calcium levels of myeloma cell line using Fura2-Am post 6 hours of drug treatment. We noted that there was an increased calcium level in the myeloma cells when a combination of lenalidomide and bortezomib was used (Fig. 4A). We also noted an increased activity of calpain in cells treated with lenalidomide + bortezomib (Fig. 4B). Furthermore, chelating calcium by BAPTA or inhibiting calpain (calpastatin or PD150606) or caspase (zVAD.fmk) resulted in the accumulation of IKZF1 (Fig. 4C and D; Supplementary Fig. S7) in the presence of lenalidomide and bortezomib and no accumulation of IKZF1 was noted when other protease inhibitors (E64 and Pepstatin A) were used (data not shown). This suggested that specific protease system activated because of calcium flux in myeloma cells degrades IKZF1 protein in the absence of proteasome machinery. This inhibition of calpain or chelation of calcium also resulted in the rescue of myeloma cells from apoptosis induced by the combination drugs (Fig. 4C–E; Supplementary Fig. S8). Furthermore, modulation of calcium flux with ionomycin induced IKZF1 degradation in ionomycin alone or in combination with lenalidomide treatment (Supplementary Fig. S9). This suggests a crucial role of calcium flux which can activate calpain and caspase to degrade IKZF1 and induce apoptosis in myeloma cells by bortezomib and lenalidomide.
Combination of lenalidomide and bortezomib also induces CD38 expression in multiple myeloma cells
Finally, we also looked for the expression of CD38 in myeloma cells posttreatment with lenalidomide and bortezomib. Here, we used a lower concentration of bortezomib (1 nmol/L) because the other concentration used was shown to kill almost 90% of the myeloma cells by itself (Supplementary Fig. S10) and we had also demonstrated that at these lower concentrations, IKZF1 was still degraded (Fig. 2B). We noted that upon combination of these two agents, there was an increased expression of CD38 in MM.1S cells compared with either of the drug treated MM.1S cells (Fig. 5). This further validates our finding that IKZF1 is degraded by the combination and also suggests a rationale for using daratumumab along with lenalidomide and bortezomib to further enhance efficacy in multiple myeloma.
Multiple myeloma had been treated successfully with lenalidomide and bortezomib along with dexamethasone, which had significantly improved the clinical outcomes even in older patients, not eligible for high-dose chemotherapy and autologous stem cell transplantation. Recently, it has been demonstrated that the mechanism of action of lenalidomide includes degradation of IKZF1 and IKZF3, which act as a central transcription factor regulating various genes involved in the survival of myeloma cells. The degradation was brought by the activation of cereblon protein which can ubiquitinate the IKZF1 and IKZF3 proteins and makes them a substrate for proteasome complex. Hence, this proteasome complex–mediated degradation of IKZF1 and IKZF3 would appear to be central for the clearance of this disease. Previously it was also demonstrated that combining lenalidomide and proteasome inhibitor inhibited the degradation of IKZF1 (9). However, these experiments were done at short time points (3 hours and 5 hours posttreatment), which may not be adequate for demonstration of biological activity. As seen in the clinic, we were able to demonstrate a synergistic activity of these two drugs on multiple myeloma cell lines. We were able to demonstrate that both IKZF1 and IKZF3 were degraded at 12 and 24 hours when these drugs were combined. We also checked for the induction of apoptosis and found there was an increase in the induction of apoptosis; however, we could not find a significant difference between Caspase-3 and BIM expression between bortezomib alone and in combination. Furthermore, we were able to demonstrate that this combination does not interfere with the degradation of IKZF1, in spite of significant proteasome inhibition. We also report that calcium-dependent calpain, as well as caspases activated during apoptosis of myeloma cells can act as an alternative pathway to proteasome machinery by which IKZF1 is degraded with this combination. We believe that the calpain activated upon the drug treatment can induce apoptosis by altering mitochondrial membrane potential of myeloma cells as demonstrated by decrease in BCL2 proteins and cleavage of autophagic proteins (20) induced during drug treatment. This in turn can induce apoptosis. To validate this, we had further showed that inhibiting calpain restored the viability (as shown by pro-caspase 3 levels and viability assay), which suggests that activation of calcium-induced calpain determines the efficacy of this combination in inducing apoptosis in multiple myeloma cells. These observations validate the existing report where it has been shown that the sensitivity of myeloma cells to lenalidomide relies on its ability to decompose H2O2 (21), which can generate reactive oxygen species which can also be the effect of increased calcium flux (22).
Thus, our results demonstrate a significant in vitro synergism between lenalidomide and bortezomib as seen in the clinic and mechanistically explain how IKZF1 degradation happens even in the presence of proteasome inhibitor. Furthermore, we also demonstrated that this combination enhanced the expression of CD38 in myeloma cells suggesting that a triple therapy combining daratumumab with lenalidomide and bortezomib would be rationale way to further enhance efficacy in multiple myeloma.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: S. Ganesan, H.K. Palani, V. Mathews
Development of methodology: S. Ganesan, H.K. Palani, N. Balasundaram, B. George, V. Mathews
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Ganesan, H.K. Palani, N. Balasundaram, A.J. Devasia, B. George, V. Mathews
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Ganesan, H.K. Palani, N. Balasundaram, S. David, A.J. Devasia, V. Mathews
Writing, review, and/or revision of the manuscript: S. Ganesan, H.K. Palani, B. George, V. Mathews
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Ganesan, H.K. Palani, N. Balasundaram, S. David
Study supervision: V. Mathews
This study was supported by a Wellcome-DBT India Alliance research grant (IA/S/11/2500267) and DBT-COE grant (BT/COE/34/SP13432/2015). V. Mathews was supported by senior fellowship program of Wellcome-DBT India Alliance IA/S/11/2500267 and IA/CPHS/18/1/503930. S. Ganesan, H.K. Palani, and S. David were supported by senior research fellowship from Council for Scientific and Industrial Research. We acknowledge Intas Pharmaceutical Ltd, and NATCO Pharmaceutical Ltd, for kindly providing us API of pharmaceutic drugs for this study.
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.