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Identification of markers that functionally define a quiescent multiple myeloma cell sub-population surviving bortezomib treatment
- Alfred Adomako†1,
- Veronica Calvo†1,
- Noa Biran1, 3,
- Keren Osman1, 3,
- Ajai Chari1, 3,
- James C Paton5,
- Adrienne W Paton5,
- Kateri Moore4,
- Denis M Schewe1 and
- Julio A Aguirre-Ghiso1, 2, 3, 4Email author
© Adomako et al.; licensee BioMed Central. 2015
Received: 1 August 2014
Accepted: 21 May 2015
Published: 30 May 2015
The mechanisms allowing residual multiple myeloma (MM) cells to persist after bortezomib (Bz) treatment remain unclear. We hypothesized that studying the biology of bortezomib-surviving cells may reveal markers to identify these cells and survival signals to target and kill residual MM cells.
We used H2B-GFP label retention, biochemical tools and in vitro and in vivo experiments to characterize growth arrest and the unfolded protein responses in quiescent Bz-surviving cells. We also tested the effect of a demethylating agent, 5-Azacytidine, on Bz-induced quiescence and whether inhibiting the chaperone GRP78/BiP (henceforth GRP78) with a specific toxin induced apoptosis in Bz-surviving cells. Finally, we used MM patient samples to test whether GRP78 levels might associate with disease progression. Statistical analysis employed t-test and Mann-Whitney tests at a 95% confidence.
We report that Bz-surviving MM cells in vitro and in vivo enter quiescence characterized by p21CIP1 upregulation. Bz-surviving MM cells also downregulated CDK6, Ki67 and P-Rb. H2B-GFP label retention showed that Bz-surviving MM cells are either slow-cycling or deeply quiescent. The Bz-induced quiescence was stabilized by low dose (500nM) of 5-azacytidine (Aza) pre-treatment, which also potentiated the initial Bz-induced apoptosis. We also found that expression of GRP78, an unfolded protein response (UPR) survival factor, persisted in MM quiescent cells. Importantly, GRP78 downregulation using a specific SubAB bacterial toxin killed Bz-surviving MM cells. Finally, quantification of Grp78high/CD138+ MM cells from patients suggested that high levels correlated with progressive disease.
We conclude that Bz-surviving MM cells display a GRP78HIGH/p21HIGH/CDK6LOW/P-RbLOW profile, and these markers may identify quiescent MM cells capable of fueling recurrences. We further conclude that Aza + Bz treatment of MM may represent a novel strategy to delay recurrences by enhancing Bz-induced apoptosis and quiescence stability.
The overall survival of patients with multiple myeloma continues to improve, in large part due to proteasome inhibitors (PIs) and immunomodulatory agents [1, 2]. However, the majority of patients treated with these drugs inevitably relapse after variable remission periods . Much effort has been spent in understanding how PIs induce pathways that regulate cell death during the acute treatment of these patients . Similar effort has been placed in finding ways to maximize PI effectiveness and duration of response. However, less is known about the biology of residual MM cells that survive therapy, how to identify them, and how they persist after treatment [5, 6]. Currently, there are no universal criteria for identifying and tracking residual cells in MM patients in remission . Understanding the biology and characteristics of MM residual disease, thus, represents a key avenue to prevent relapses.
PIs induce MM cell death by regulating several tumor cell intrinsic and stromal pathways . Among these pathways, PIs are powerful activators of the unfolded protein response (UPR). This pathway has the ability to induce cell death but it also can induce growth arrest and survival as a first response to endoplasmic reticulum (ER) stress. We previously showed that acute exposure to bortezomib (Bz) treatment activated a canonical PERK-eIF2α-CHOP pathway that resulted in the majority of MM cells entering cell death . However, MM cells surviving Bz treatment downregulated eIF2α phosphorylation, upregulated the survival chaperone BiP/GRP78 and entered a prolonged G0-G1 cell cycle arrest. Dephosphorylation of eIF2α in quiescent surviving MM cells was key for survival because inhibition of GADD34/PP1C, an eIF2α phosphatase, killed almost all surviving MM cells . While these studies identified a survival mechanism for MM cells that persist after Bz treatment, they did not explain what cell cycle machinery components regulated the prolonged growth arrest and survival after Bz treatment. Further, the role of BiP/GRP78, an HSP70 family member for which inhibitors are in development , in Bz-surviving MM cells was also unknown.
Here, we show that MM cells that survive proteasome inhibitors display a GRP78HIGH/p21HIGH/CDK6LOW/P-RbLOW profile. We also provide preliminary evidence that higher levels of GRP78 detected in MM patient bone marrow biopsies may be present in patients with more aggressive disease and that GRP78 downregulation potentiated Bz killing. Thus, these markers may pinpoint quiescent MM cells with the ability to persist after treatment and sensitivity to Grp78 inhibition. We also show that apoptosis can be potentiated and quiescence extended by a sequential 5-azadeoxycitidine and Bz treatment. This drug combination schedule might represent a novel strategy to potentiate Bz efficacy in MM disease treatment.
Reagents, cell lines, tissue culture and quantitative reverse transcription-PCR
Antibodies: Anti-BiP/GRP78 [610979, BD]; Anti-CD138 [sc-5632, Santa Cruz]; Anti-Ki67 [9449, Cell Sig.]; Anti-P-Rb (Ser807/811) [8516, Cell Sig.]; Anti-P-Rb (Ser249/Thr252) [sc-377528, Santa Cruz]; Anti-p21 [2947, Cell Sig]; Alexa Fluor® 488 Goat Anti-Mouse, [A-11001; Invitrogen]; Alexa Fluor® 568 Goat Anti-Rabbit, [A-11008; Invitrogen]). Vectastain ABC kit and DAB peroxidase substrate kit was used for IHC developing [Vector lab]. Bortezomib (S1013, Selleck Chemicals) was used to treat RPMI8226 (CCL-155, ATCC) and U266 (TIB-196, ATCC) cells at 4 nmol/L or 8 nmol/L Bz for 24 h. The drug was removed by washing 3x with PBS and then re-plated in fresh medium (RPMI-1640 with 10% FBS). Cells were cultured according to ATCC recommendations. In 5-azacytidine (Aza) (A2385, Sigma) experiments, the cells were pre-treated for 4 days with 500 nmol/L Aza (and replaced every 48 h) before Bz treatment. Total RNA was extracted using Trizol. Primers used are in [Additional file 1: Table S1].
Mouse xenograft studies
Institutional Animal Care and Use Committees (IACUC) at Mount Sinai School of Medicine (MSSM) approved all animal studies. Protocol ID: 11-0032PRYR1. ATCC-derived RPMI8226 and U266 cells were expanded and pulsed for 24 h with 8 nM Bz or DMSO vehicle control. Cells were then washed and viability was assessed by Trypan blue exclusion assay. Equal number of live (1 × 106) cells was then resuspended in PBS with 50 % Matrigel (356231, BD), and injected s.c. into 4- to 6-week-old male NSG mice (Charles River). Tumor volumes were measured and calculated using the formula (D × d2)/2, where D is the longest and d is the shortest diameter. All points represent independent biological samples with error bars representing standard deviations and statistical significance determined using a Mann–Whitney test.
Nuclear and chromatin extraction and western blots
After drug treatments, cells were washed with PBS and resuspended in 2 mL of Buffer A (10 mM HEPES pH = 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34M sucrose, 10 % glycerol) with 1 mM DTT, protease inhibitors, and 0.1 % Triton X-100 on ice for 7 min. The cells were then spun at 4,000 rpm and 4°C for 4 min. The pellets, containing the nuclear fractions, were resuspended in 300 μL of 2× Laemmli sample buffer per 10 × 106 cells and then heated to 95°C for 10 min for western blotting. For chromatin fractions, the nuclear extracts were treated with “no salt buffer” (3 mM EDTA and 0.2 mM EGTA) before addition of 2× Laemmli sample buffer. For whole-cell lysates, cells were lysed for 30 min with lysis buffer containing 1 % Triton X-100, 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM orthovanadate, 1 mM NaFl, and protease inhibitors. Western blots were performed as previously described  and imaged using Image Quant LAS (GE).
Bone marrow aspirates (BMA) from multiple myeloma patients were collected in heparinized tubes following an Icahn School of Medicine Institutional Review Board approved protocol (Number: MSSM HS 10-00105). The BMAs were then subjected to density gradient centrifugation using Ficoll-Paque Plus (17-1440-02, GE). The isolated bone marrow mononuclear cells were then incubated with CD138 MicroBeads (130-090-503, Miltenyi) and separated using autoMACS separator (Miltenyi). CD138-positive cells were fixed and spun onto slides. To test for enrichment, mononuclear cells before and after separation were stained for CD138. RPMI8226 cells were stained as a positive control. The percentage of CD138-positive cells increased from about 8 % pre-separation to 97 % post-separation in all patients. The patient slides were stained with BiP/GRP78 primary antibody overnight and Alexa Fluor® 488 goat anti-mouse secondary antibody the next day. For controls, slides were stained with the secondary antibody alone. The slides were imaged using Leica DM6000 and quantified with ImageJ (NIH).
Immunofluorescence (IF) and Immunohistochemistry (IHC)
For IF analysis of cytospins, cells were separated by density gradient centrifugation using Ficoll-Paque Plus after treatment to remove dead cells. The live cells were then fixed in 4 % paraformaldehyde in PBS (15 mins) and cyto-spun onto slides. Slides were then washed, permeablized using 0.5 % Triton X-100 and blocked for an hour with 3 % normal goat serum and 3 % BSA in PBS. The slides were then incubated with primary antibodies or diluent (1 % BSA in PBS) overnight at 4°C. After washing, the slides were incubated with secondary antibodies. The slides were then washed and mounted with Prolong gold anti-fade reagent with DAPI [P36931, Invitrogen]. Slides were images using a Leica DM5500B microscope and analyzed using MetaMorph®. For IHC analysis of tumors, tissues were fixed in 4 % PFA for 24 h, and then transferred to 70 % ethanol until processing for paraffin embedding and sectioning into 4-um-thick slices. Slides were deparaffinized and rehydrated through xylene and ethanol washes, and antigen unmasking was performed by heat-induced retrieval in citrate buffer. Quenching of endogenous peroxidase activity was done with 3 % H2O2. After blocking with 3 % normal goat serum in 3 % BSA/PBS for 1h, slides were incubated with primary antibodies overnight at 4°C. After washing, either fluorophor-conjugated secondary antibody was used and then mounted or an avidin/biotin peroxidase system was used and developed with peroxidase substrate kit [Vector lab]. In the latter case, VectaMount mounting media was used [Vector lab]. For quantification purposes, at least 10 randomly selected 20x fields were counted.
Generation of the H2B-GFP tagged line and label retention assay
The Tet-inducible H2B-GFP construct was a kind gift from Dr. Kateri Moore . The plasmid was transfected in 293T cells. Lentiviral particles were harvested from 293T cells and used to infect RPMI8226 cells. The infected cells were selected for stable expression using puromycin (1 ug/mL). Upon induction of H2B-GFP with doxycycline (1 ug/ml), high expressers were sorted using FACSAriaII (BD). For label retention experiments, the cells were induced with doxycycline for 6 days and released at the time of Bz treatment. Label retention was analyzed using FACS LSR Fortessa (BD). For viability assessment, Trypan blue exclusion assay was performed.
Bortezomib-surviving MM cells display a CDK6LOW/p21HIGH quiescent profile
Analysis of the viable quiescent Bz-surviving MM cells using immunofluorescence and western blot (nuclear fraction) showed that these cells were enriched for the cyclin-dependent kinase inhibitor p21CIP1 mRNA and nuclear protein [Fig. 1d-f]. The CDK inhibitors p15 and p16 mRNAs were also induced but p21CIP1 mRNA showed the strongest induction [Additional file 3: Figure S2E], which could be followed by Western blot and IF. This was observed at the end of the acute phase (Day 0) and at 3, 6 and 7 days after washout of the 24 h pulse of Bz [Fig. 1d-f]. Furthermore, H2B-GFPHIGH cells expressed significantly more p21CIP1 in Bz-treated cells compared to DMSO controls [Fig. 1e-f]. Arguing for a G0-G1 arrest, p21CIP1 induction correlated with the downregulation of CDK6 protein as measured by Western blot [Fig. 1g] and with decreased levels of cyclin-D3 and CDK4 protein levels in proteasome inhibitor-pulsed cells [Additional file 2: Figure S1B]. This is in agreement with our data from [Fig. 1a-c] showing the existence of a deeply quiescent population in vitro.
A Low-dose 5-aza-cytidine treatment potentiates Bz-induced cell death and deep quiescence
Upregulation of GRP78 in Bz-surviving MM cells is associated with disease progression in patients and therapy-mediated cell death
Characteristics for the patients whose BM samples were tested for BiP levels in CD138+ cells
Age at diagnosis
Ouant Immunoglobulins and Serum immunofixation at collection
Myeloma status at time of Collection
lgG 2371, monoclonal protein in gamma region
IgG 282, IgA 5083, IgM 16, Two IgA lambda monoclonal band seen, representing 85 %
IgG 2333, IgA 12, 1gM 20, monoclonal protein seen in gamma region
Progression of Disease (increase in M-spike)
IgG 4656, IgA 27, 1 gM 53, monoclonal protein in gamma region
Minimal response/stable disease
IgG 4099, monoclonal band in gamma region
IgG 314, 1 gM <5, IgA <5, faint free kappa band
Progression of Disease
IgG 453, 1 gM 7, IgA 132, faint IgG lambda band
partial response/stable disease
lgG 840, IgA 9, 1 gM 9, lgG kappa monoclonal spike seen, representing all of monoclonal protein
Remission (very good partial response)
lgG 255, 1 gM 12, IgA 19, faint free Lambda band seen
Progression of Disease
lgG 188, IgA 10, 1gM 12, Free monoclonal lambda light chain, normal Igs greatly diminished
Progression of Disease
lgG 6589, Iga 8, 1 gM <5, lgG kappa monoclonal representing all of total
Progression of Disease
lgG 5543, IgA 11, 1 gM 19, lgG kappa monoclonal protein
Progression of Disease
We next depleted the GRP78 protein to assess whether cells hypomorphic for this chaperone were unable to survive Bz-induced cell death. To this end, we used subtilase cytotoxin (SubAB), a bacterial AB5 toxin that has been shown to specifically cleave GRP78 (18, 19) [Fig. 4g]. As a control, we used a non-functional mutant toxin termed SubAA272B. Inhibition of GRP78 using the IC50 for SubAB significantly decreased the viability of the MM cells after Bz treatment compared to the non-functional mutant SubA272B [Fig. 4h]. This suggests that GRP78 is a major survival factor in residual Bz-surviving cells and a potential target to eradicate these residual cells.
Multiple myeloma cells synthesize and secrete large amounts of immunoglobulins  and thus possess a very tightly regulated ER quality control system. The proteasome inhibitor bortezomib was the first in its class to be FDA-approved for treatment of MM patients [20, 21] and second generation agents are now also available . While proteasome inhibition is a standard of care for MM, patients invariably relapse. This suggests that a small fraction of neoplastic cells can escape this treatment through poorly understood mechanisms. We hypothesized that by exploring the biology of the residual surviving MM cells we may identify markers for residual cells and survival mechanisms to target and prevent MM relapse.
We had previously found that Bz-surviving MM cells entered quiescence and silenced specific components of the UPR signaling that commonly induce cell death [6, 17, 23, 24]. However, what genes may mark quiescent cells with enhanced survival properties and what components of the UPR might promote survival was unknown. Here we show that after a Bz pulse, the residual cells are for the most part slow-cycling as expected by the growth arrest propelled by high eIF2α phosphorylation . However, we also found a deeply quiescent and viable fraction of cells that were marked by p21HIGH levels and prolonged H2B-GFP label retention. In addition to p21 upregulation, which appeared to be transcriptional, Bz-surviving MM cells showed loss of CDK6 and consequently loss of P-Rb protein , which could explain the G0-G1 cell cycle arrest in MM cells. These data argue that while slow-cycling is a main response to Bz, a small fraction is capable of entering a deeper quiescence. Importantly, these cells were preferentially enriched for GRP78 arguing they may be prone to enhanced survival. It is possible that with repeated cycles of PI treatments used in the clinic more of the deep quiescent MM cells that survive the treatment accumulate creating a population that escapes Bz treatment and anti-proliferative drugs, eventually fueling relapses. That these cells may become “professional” ER stress tolerant is suggested by the upregulation of GRP78 that was also found in MM cells from patients with progressive disease. Our in vivo data using U266 MM cells argues that p21HIGH MM cells can be found and may persist without expanding for ~90 days (~1 year in humans) after a 24 h pulse with Bz. The lack of apoptosis in these dormant lesions and the upregulation of p21 coupled to no net increase in tumor mass argues against continuous apoptosis and in favor of long-term quiescence as a mechanism to explain de prolonged time to take of these MM cells in vivo. We propose that in the bone marrow of patients a specific MM cell subpopulation (CDK6LOW/P-RbLOW/p21HIGH) may be found dormant after Bz treatment.
Many common quiescence regulators such as the tumor suppressors p15INK4B  and p16INK4A are epigenetically silenced in cancer . Our data shows that mRNA induction of p15INK4B, p16INK4A and p21CIP1 (and protein) in surviving MM cells is not greatly increased by an Aza pre-treatment and Bz pulse. However, the initial apoptosis and later prolonged growth arrest phase in vitro is more than doubled in cells treated with Aza and Bz and this correlated with p21CIP1, Ki67 and P-Rb levels in viable growth-arrested cells. While we have not performed detailed gene promoter methylation analysis to determine the targets influenced by the Aza treatment, our data suggested that “reprogramming” with Aza might be amenable to be used as a way to maximizing the apoptosis but also quiescence induction effects of Bz.
Our work also tested the role of GRP78, a well-characterized survival component of the UPR  that is upregulated and promotes drug resistance of quiescent squamous cell carcinoma (HNSCC) cells . Here we found that Bz-surviving and quiescent (viable H2B-GFP label- retaining) MM cells maintained high levels of GRP78 for many days after drug washout, arguing these quiescent cells may selectively upregulate this ER chaperone. This suggests that GRP78 is important for cell survival during PI-mediated UPR activation in the quiescent MM cell population. Importantly, targeted depletion of GRP78 enhanced Bz-mediated cell death, justifying further studies to test if this chaperone might be an amenable therapeutic target in the resistant residual disease. Overexpression of GRP78 was correlated with clinical progression in other cancer models [29–31]. We found GRP78 upregulation might be associated to disease progression in MM patient samples. Because our patient sample size is small, we cautiously propose that either in residual MM or recurrent MM cells, GRP78 is likely to mark a subpopulation with enhanced survival. Our analysis of patient samples was a pilot study and larger cohorts of patients tested for GRP78 expression in their MM samples may prove useful to determine whether this chaperone of the ER is indeed a marker to distinguish persistent Bz-refractory and/or recurrent disease.
We conclude that Bz-surviving MM cells display a GRP78HIGH/p21HIGH/CDK6LOW/P-RbLOW profile. These markers may pinpoint quiescent MM cells capable of fueling recurrences. We further conclude that upregulation of GRP78 allows specifically quiescent tumor cells to survive for prolonged periods and this may be an amenable target to kill residual MM cells. Although the mechanisms are incompletely understood, we also conclude that the combination of Aza and Bz treatments may represent a novel strategy to delay MM recurrences by enhancing Bz-induced apoptosis and the stability of the quiescence program.
We acknowledge Y. Estrada for his contribution to generating the H2B-GFP cell line and especially the members of the Aguirre-Ghiso lab for useful discussions and comments. Grant Support: Samuel Waxman Cancer Research Foundation Tumor Dormancy Program and NIH/National Cancer Institute (CA109182) to J.A.A-G. A.A was supported by NIH T32 HL094283 training grant.
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