Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2025 Jan 1.
Published in final edited form as: J Immunol. 2024 Jan 1;212(1):154–164. doi: 10.4049/jimmunol.2300212

The proteasome inhibitor bortezomib induces p53 dependent apoptosis in activated B cells

Trini A Ochoa 1, Amy Rossi 2, E Steve Woodle 3, David Hildeman 2, David Allman 1
PMCID: PMC10872551  NIHMSID: NIHMS1942592  PMID: 37966267

Abstract

The proteasome inhibitor bortezomib (BTZ) is proposed to deplete activated B cells and plasma cells. However, a complete picture of the mechanisms underlying BTZ-induced apoptosis in B-lineage cells remains to be established. Here, using a direct in vitro approach we show that deletion of the tumor suppressor and cell cycle regulator p53 rescues recently activated mouse B cells from BTZ-induced apoptosis. Further, BTZ treatment elevated intracellular p53 levels, and p53 deletion constrained apoptosis as recently stimulated cells first transitioned from the G1 to S phase of the cell cycle. Moreover, combined inhibition of the p53-associated cell cycle regulators and E3-ligases MDM2 and APC/C induced cell death in post-division B cells. Our results reveal that efficient cell cycle progression of activated B cells requires proteasome-driven inhibition of p53. Consequently, BTZ-mediated interference of proteostasis unleashes a p53-dependent cell cycle-associated death mechanism in recently activated B cells.

Introduction

B-lineage cells play pivotal roles in host-protection. B cells and antibody-secreting plasma cells however can also cause a variety of pathologies including in autoimmunity and transplant rejection, and in multiple myeloma and associated plasma cell dyscrasias (1–4). Proteasome inhibitors (PIs) such as bortezomib (BTZ) have emerged as an approach to deplete problematic plasma cells due to their capacity to induce a terminal unfolded protein response (5–7). Consequently, BTZ is commonly used to combat multiple myeloma, but has also been considered for depleting non-malignant plasma cells secreting self-reactive or transplant-specific antibodies (5, 8–10). Other work however suggests that BTZ depletes activated B cells including those within germinal centers (GCs) (5, 11, 12), unique T cell dependent anatomical structures enriched for activated B cells undergoing somatic hypermutation and affinity-based selection (13). Yet, although BTZ causes depletion of GC B cells in vivo and activated B cells in vitro (5, 11, 12), the mechanisms whereby BTZ induces apoptosis in activated B cells are unknown.

The 26S proteasome is a multi-protein enzyme complex that degrades most intracellular proteins (14). Ubiquitinated substrates are recruited by the 19S regulatory cap and degraded within the active sites of the 20S core subunit, the latter of which is targeted by BTZ and the second-generation PI carfilzomib (15, 16). In cancer cell lines, PIs can induce cell death by stabilizing factors with pro-apoptotic potential (Myc, Bax, p53), inhibiting protein synthesis, or inducing mitotic arrest or ER-stress (6, 15, 17–19). Consequently, the pleiotropic effects of PIs make it challenging to predict which pathways are operative in diverse cell types and contexts including for activated and differentiating B-lineage cells.

The p53 pathway is chiefly associated with the induction of cell cycle arrest, apoptosis, and DNA repair pathways in malignant or pre-malignant cells (20, 21). Once activated, p53 promotes cell cycle arrest and the expression of pro-apoptotic proteins from the Bcl-2 family, modulating cell intrinsic apoptotic pathways to promote programmed cell death. p53 is subject to negative regulation by the E3-ligase MDM2, which maintains the p53 protein at low and even undetectable levels by targeting p53 for proteasomal-mediated degradation (22–24). However, certain cellular stressors such as genotoxic drugs, activated oncogenes, and UV irradiation can augment p53 activity (20). Non-genotoxic drugs such as nutlin3a, which binds the MDM2 p53-binding pocket, also promote p53 activity to promote the expression of canonical p53 targets (25, 26). Similarly, PIs disrupt MDM2-mediated degradation of p53, leading to activation of the pathway (22). Multiple studies have identified p53-dependent (27–29), as well as p53-independent (18, 30, 31), roles for PI-induced apoptosis; however, the relevance of these observations to primary B cells remains uncertain.

Here, we show that activated primary B cells and newly formed plasma cells possess largely comparable sensitivity to BTZ-driven apoptosis. Additionally, in B cells BTZ induces apoptosis by interfering with cell cycle initiation, thus unleashing a p53-dependent death pathway that can be further augmented via p53 potentiation with nutlin3a. These results provide new insights into the impact of BTZ on humoral immune cells.

Materials and Methods

Mice

C57BL/6 (7–9 weeks old) and p53−/− adults (Strain #: 002101) were purchased from The Jackson Laboratory and housed in our colony for at least 1–2 weeks before immunizations or treatments. B6.Blimp1+/GFP mice (32) were bred and housed in our colony.

Cell culture and stimulation

Spleens were harvested and processed into a single cell suspension by grating it against the frosted ends of glass slides. The cells were then lysed with ACK lysis buffer and passed through Nitex Nylon (64 μM). Splenocytes were labeled with the CellTrace Violet Proliferation Kit. Splenocytes were stained with CD23 biotin and streptavidin microbeads, and follicular B-cells (CD23+) were then isolated by magnetic assisted cell sorting using an LS column and a QuadroMACS Separator.

Follicular B-cells were plated in complete growth media (RPMI 1640, 10% FBS, 1 mM sodium pyruvate, 1x Pen-Strep, 2 mM L-glutamine, 10 mM HEPES, 1x MEM non-essential amino acids, and 55 μM 2-mercaptoethanol) at a concentration of 1×106 cells/ml in a 96-well flat-bottom microplates (Nunclon Delta-treated) at 200 μl/well. B-cell differentiation into plasma cells was stimulated with 100 nM CpG (s-ODN-1826), 100 ng/ml murine IL-4 (PeproTech), and 100 ng/ml murine IL-5 (PeproTech). Alternatively, cells were stimulated with 5 μg/ml anti-CD40 (clone HM40–3; BD) and 100 ng/ml murine IL-4 (PeproTech). Cells were cultured in an incubator at 37°Celsius, 5% CO2. BTZ, nutlin3a, and proTAME were purchased from Cayman Chemical and dissolved in DMSO. All reagents were further diluted in PBS for a final DMSO concentration of 0.5% or less.

Flow cytometry

The following reagents were used: TO-PRO-3 for nucleic acid stain (Thermo Fisher Molecular Probes), anti-CD138-BV605 (Biolegend). For intracellular staining we used anti-p53-AF647 (1C12, Cell Signaling), anti-CC3-AF488 (Cell Signaling) and we fixed cells with 4% PFA and then utilized the eBioscience FoxP3 Transcription Factor Staining Buffer Set as per the manufacturer’s protocol. Full wells were run for in vitro experiments. Cell cycle staining was performed as previously described (33). Briefly, cells were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and then fixed with 10% ethanol for 1 hour. Cells were then washed twice with PBS and stained with 7-AAD (Invitrogen) for 20 minutes, followed by staining with Pyronin Y for 10 minutes and immediate analysis by flow cyometry. Singlets were identified based on Forward Scatter-Area (FSC-A) by Forward Scatter-Height (FSC-H) signals. For cell cycle experiments, singlets were gated on FSC-A vs FSC-H, followed by gating on live cells, and then single DNA events based on 7-AAD-Area vs 7-AAD-Height. For BrdU pulsing, we utilized the FITC BrdU FlowKit (BD, 559619) as per the manufacturer’s protocol. Cells were analyzed with a Symphony A3 Lite, a BD LSR II, or an LSR Fortessa. Data was analyzed with FlowJo 10.2 software.

Statistics

Statistical analyses were performed with GraphPad Prism 9 software. All data are represented as the mean ± SEM. Statistical differences between two groups were calculated using a two-tailed unpaired Student’s t-test with Welch’s correction. One-way or two-way ANOVA tests were utilized for multigroup or multifactor comparisons, respectively. A p-value of less than .05 or .033 was considered statistically significant, accordingly.

Study Approval

All experiments in this study were performed in accordance with IACUC protocols and were approved by the Office of Regulatory Affairs at the University of Pennsylvania.

Results

BTZ affects activated B cells and newly formed plasma cells

We used a well characterized in vitro system wherein we could titrate BTZ into cultures containing activated B cells at key time points before and after early cell cycle progression or subsequent plasma cell differentiation. With this approach, follicular B cells are stimulated with the TLR9 ligand CpG together with IL-4 and IL-5 which causes the vast majority of cells to experience a coordinated proliferative burst followed by division-dependent plasma cell differentiation (34, 35). We purified CD23+ follicular splenic B cells from either normal B6 adults or B6.Blimp1+/GFP mice harboring a GFP reporter gene in the 3-prime UTR of the Blimp1 locus (32). Thus, we distinguished induced plasma cells versus activated B cells via surface CD138 expression or Blimp1-GFP expression. Importantly, with this system proliferative B cells revealed by dilution of cell proliferation dyes such as cell trace violet (CTV) and plasma cells are first detected at 44- and 66-hours post-stimulation, respectively (Suppl. Fig. 1). These results are consistent with past data showing that CpG-stimulated resting B cells do not initially enter S-phase of the cycle until 32 hours post-stimulation (36).

To compare PI sensitivity of activated B cells versus plasma cells directly, we titrated BTZ into cultures of stimulated CTV-labeled B6.Blimp1+/GFP follicular B cells at 72-hours post-stimulation, then evaluated numbers of viable Blimp1− and Blimp1+ cells 22 hours later. As shown (Fig. 1A), whereas 10 nM BTZ was sufficient to deplete Blimp1+ cells, 20 nM BTZ was required to deplete activated Blimp1− B cells. Nearly identical results were obtained when we evaluated CD138+ cells in cultures initiated with B6 WT B cells (not shown). Furthermore, adding BTZ at 44 hours post-stimulation revealed that lower BTZ concentrations, such as 5 nM, inhibited the emergence of CD138+ cells without impacting the viability of CD138− cells (Fig. 1B).

Figure 1. Differential BTZ sensitivity for B and plasma cells.

Figure 1.

Open in a new tab

Purified CTV-labeled splenic follicular B cells from B6.Blimp1+/GFP or C57BL/6 adults were stimulated with CPG, IL-4, and IL-5. (A) 72 hours post-stimulation cultures were supplemented with vehicle or the indicated final concentration of BTZ and viability analyzed 22 hours later. Plots gated on viable (TOPRO-3−) singlets. (B) Follicular B cells were treated with BTZ at 44 hours and analyzed at 66 hours post-stimulation for numbers of viable (TOPRO-3−) B cells (CD138−) and plasma cells (CD138+). (C) Follicular B cells were treated with PBS or BTZ at 22 hours post-stimulation and analyzed at 45 hours post-stimulation. Graph shows numbers of viable cells within each division peak. Gated on CD138− TOPRO-3− cells. (D) Follicular B cells were treated with PBS or BTZ at 22 hours and analyzed at 45 hours post-stimulation. Gated on CD138− cells. (E) Follicular B cells were treated with BTZ at 22 hours and analyzed at 40 hours post-stimulation. Representative plots show impact of 20 nM BTZ on CC3 levels. Gated on CD138− cells. Bars indicate means with error bars showing SD for technical replicates. p-values were calculated with a one-way ANOVA with Dunnett’s multiple comparisons test or Tukey’s multiple comparisons, a two-way ANOVA with Sidak’s multiple comparisons test, or a two-tailed unpaired T-test with Welch’s correction, *<.033, **<.002, ***<.001. These results are representative of at least 3 separate experiments.

We sought to understand the action of BTZ on very recently stimulated B cells. To this end, we added 20 nM BTZ to stimulated B6 follicular B cells at 22 hours post-stimulation before emergence of proliferative cells, then evaluated CTV dilution and viability at 44 hours. Whereas a fraction of control cells had achieved 1–3 divisions at 44 hours, all viable BTZ treated cells remained undivided (Fig. 1C). Further and as expected, early exposure to BTZ increased numbers of apoptotic cells as revealed by quantification of B cells that stained with the viability dye TOPRO-3 or for cleaved caspase-3 (CC3) (Fig. 1D-E).

p53 drives apoptosis in response to BTZ and nutlin3a in pre-proliferative B cells

We showed previously that newly activated T cells experience a cell cycle-connected p53-regulated genomic stress responses (37). Because many elements of the cell cycle are controlled by the proteasome (38), we hypothesized that BTZ-induced apoptosis of activated B cells is regulated by p53. Consistent with this hypothesis, activated B cells possess elevated levels of p53 protein (39), and p53 function has been connected to PI-induced apoptosis in transformed and non-transformed mammalian cell lines (15, 28, 40). Accordingly, we treated activated B cells derived from wild-type (p53+/+) or p53 knock-out (p53−/−) mice (21) with 20 nM BTZ at 22 hours (“early activated”) post-stimulation and analyzed viability in these cultures 22 hours later (44 hours post-stimulation). Interestingly, we found that p53 deletion increased numbers of viable B cells as found by TOPRO-3 exclusion (Fig 2A). To probe further for p53-driven apoptosis in early activated B cells, we treated stimulated cells with the MDM2 inhibitor nutlin3a, which potentiates p53 activity by preventing its ubiquitination and degradation by inhibiting p53-MDM2 interactions (22, 23, 25). We found that single treatment with BTZ or nutlin3a induced cell death in early activated B cells in a p53-dependent manner. This loss of viability was further increased with a combination of these agents (Fig 2B).

Figure 2. BTZ and nutlin3a induce p53-dependent apoptosis in recently stimulated B cells.

Figure 2.

Open in a new tab

(A) CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. 22 hours later cultures were supplemented with PBS or the indicated final concentration of BTZ and analyzed at 44 hours post-stimulation. Graphs indicate total percent (left) and numbers (right) of recovered viable B cells for each BTZ dose. Gated on CD138− cells. (B) CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. 22 hours later cultures were supplemented with vehicle or the indicated concentration of nutlin3a and/or BTZ and analyzed at 44 hours. Gated on CD138− cells. p-values were calculated using technical replicates and a two-way ANOVA with Sidak’s multiple comparison test, *<.033, **<.002, ***<.001. These results are representative of at least 2 separate experiments.

We also quantified p53 levels (gMFI) in activated WT and p53−/− B cells by flow cytometry as described by Watanabe and colleagues (41). Here, we evaluated cells that were stimulated for 22 hours before adding BTZ, nutlin3a, or both. p53 levels were then evaluated 3-hours (Fig. 3A) or 22-hours (Fig. 3B) later. Notably, at the 3-hour treatment timepoint BTZ and nutlin3a caused discernable increases in the frequencies of p53-positive cells (Fig. 3A), and at 22-hours a large fraction of cells given BTZ or BTZ and nutlin3a were p53-positive (Fig. 3B). We were also able to detect p53 protein in cells treated with nutlin3a alone, though importantly under these conditions p53 levels appeared to increase earlier and were routinely lower compared to BTZ-exposed cells (Fig. 3A, 3B). Further, the combination of BTZ and nutlin3a appeared to stabilize p53 levels at both time points, suggesting a synergistic effect. In addition, as expected p53 deletion led to decreased CC3+ cells (Fig. 3C), further cementing the rescue effect of p53 deletion.

Figure 3. BTZ and nutlin3a stabilize p53 in primary B cells.

Figure 3.

Open in a new tab

(A) CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. 22 hours later cultures were supplemented with PBS or the indicated final concentration of BTZ and/or nutlin3a and analyzed 3 hours later (25 hours post-stimulation). Graphs show the percentages and protein expression levels (gMFI) for p53. Gated on CD138− TOPRO-3− events. (B) Shows percentages and protein expression levels for p53. Cells were treated at 22 hours post-stimulation and analyzed 22 hours later (total 44 hours post-stimulation). Gated on CD138− TOPRO-3− cells. (C) Percentages and protein expression levels (gMFI) for CC3. Cells were treated at 22 hours post-stimulation and analyzed at 44 hours. Gated on CD138− cells. p-values were calculated using a two-way ANOVA with Sidak’s multiple comparison test, *<.033, **<.002, ***<.001.

BTZ promotes cell cycle arrest in early activated B cells via p53

To characterize further the impact of BTZ, we interrogated the cell cycle status of stimulated WT and p53−/− B cells in which BTZ was added prior to cell division. To identify cells at each phase of the cell cycle we examined intracellular RNA and DNA levels using Pyronin Y and 7-AAD: Whereas 7-AAD stains for and saturates DNA, Pyronin Y only stains for RNA (33). In this manner, G0 cells are 7-AAD− Pyronin Y−, G1 cells are 7-AAD− Pyronin Y+, S-phase cells are 7-AADintermediate Pyronin Y+, and G2/M cells are 7-AAD+ Pyronin Y+ (see Fig. 4A).

Figure 4. BTZ causes cell cycle arrest in early activated B cells via p53.

Figure 4.

Open in a new tab

(A) Phases of the cell cycle revealed with 7-AAD and Pyronin Y. (B) Splenic follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. 22 hours later cultures were supplemented with vehicle or 20 nM BTZ and analyzed at 44 hours post-stimulation. Gated on LD-NIR− CD138− cells. Viable cells were identified with LD-NIR. (C) The graphs show the percentages of viable cells and the percentages of cells in the cell cycle. (D) BTZ was added to follicular B cells 22-hours post-stimulation. Cells were then pulsed with 20 μM BrdU at 43-hours post-stimulation, then stained with anti-BrdU antibodies and 7-AAD one hour later. Gated on LD-NIR− CD138− cells. (E) The graphs show the percentages of viable cells and the percentages of cells in the cell cycle. p-values were calculated using a two-way ANOVA with Tukey’s multiple comparison test, *<.033, **<.002, ***<.001. These results in panel B are representative of 3 separate experiments.

For B cells stimulated 22-hours before BTZ treatment, 22 hours after adding BTZ (44 hours altogether) p53+/+ B cells exhibited decreased overall viability and loss of cells in the G1-phase as well as all downstream phases of the cell cycle, whereas p53−/− cells exhibited more modest changes in the percentages of cells in G1-, S-, and G2/M phase due to BTZ exposure including a readily discernible decline in G1 phase cells (Fig. 4B-C). At earlier time points an increased fraction of p53+/+ B cells treated with BTZ were found in the Sub-G0 gate, indicative of DNA fragmentation (Suppl. Fig. 2A), although at later time points frequencies of WT and p53−/− cells in the Sub-G0 gate were more variable with comparable percentages (Fig. 4B-C) including in some samples higher frequencies for p53+/+ B cells (Fig. 4D-E, Suppl. Fig. 2B). These data suggested that upon BTZ exposure p53 induces apoptosis at the G1-phase and arrest of cell cycle progression upstream of entry of cells into the S phase of the cell cycle. Moreover, combined BTZ and nutlin3a treatment led to increased frequencies of p53+/+ cells with DNA fragmentation, in comparison to p53−/− cells (Fig. 4B-C). Of note, whereas nutlin3a reduced frequencies of viable activated B cells in a p53-dependent manner (Fig. 4B-C), small numbers of cells survived and were able to progress into the cell cycle with increased accumulation at the G1-phase. This result is consistent with our previous observation that nutlin3a treatment causes lower levels of p53 compared to BTZ treatment (Fig. 3B) and previous work suggesting that relatively low p53 levels may foster cell cycle arrest as opposed to apoptosis (for review see (20)).

Additionally, we examined the impact of BTZ on cell cycle progression by pulsing activated cells with BrdU. Here, cells were treated with BTZ at 22 hours post-stimulation, pulsed with BrdU at 43 hours post-stimulation, and analyzed one hour later. As shown, (Fig. 4D-E), frequencies of cells progressing into S-phase (BrdU+ 7-AAD+) were reduced substantially by BTZ pretreatment in p53+/+ B cells, with comparably significant higher percentages in S-phase cells for treated p53−/− B cells. Altogether we conclude that BTZ prevents the progression of activated B cells from the G0-G1-S phases of the cell cycle via a p53-dependent mechanism.

Stabilization of p53 with nutlin3a induces cell death for B cells post-division

Surprisingly, p53 deletion failed to improve B cell survival when BTZ was added at 44 hours post-stimulation (Fig. 5A), after which many cells have fully entered the cell cycle (see controls in Fig. 4B-C). Based on these data we reasoned that p53 is either irrelevant to BTZ-driven apoptosis in post-division (“late activated”) B cells or that p53 remains operative but in parallel with other pro-apoptotic pathways. Consistent with an ongoing role for p53, titration of nutlin3a alone into cultures of late activated B cells caused a dose-dependent p53-dependent cell death (Fig. 5B, Suppl. Fig. 3). Furthermore, co-delivery of BTZ and nutlin3a augmented p53-dependent death in late activated B cells over each compound alone (Fig. 5B). Of note, BTZ and nutlin3a treatment was also able to increase frequencies of non-viable p53−/− B cells. This result is consistent with the notion that nutlin3a interferes with additional relevant pathways such as those regulated by p73 and/or E2F1 (42). Finally, single treatment with lower concentrations of BTZ or nutlin3a had very little or no effect on late activated B cells but combining these two inhibitors at these concentrations had additive killing effects for CD138− cells (Fig. 5C). The latter results reveal the possibility that combinations of these inhibitors at low concentrations may preferentially inhibit early plasma cell differentiation.

Figure 5. p53 is dispensable for BTZ induced apoptosis in late activated B cells but can be potentiated by nutlin3a.

Figure 5.

Open in a new tab

(A) CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. 44 hours later cultures were supplemented with PBS or the indicated final concentration of BTZ and analyzed at 66 hours post-stimulation. Graphs indicate frequencies (left) and numbers (right) of recovered viable B cells at each BTZ dose. Gated on CD138− cells. (B) CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. 44 hours later cultures were supplemented with vehicle or the indicated concentrations of nutlin3a and/or BTZ and analyzed at 66 hours. Gated on CD138− cells. (C) CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. 44 hours later cultures were supplemented with vehicle or the indicated concentrations of nutlin3a and/or BTZ and analyzed at 66 hours. Gated on TOPRO-3− cells. p-values were calculated using a two-way ANOVA with Sidak’s multiple comparison test, *<.033, **<.002, ***<.001. These results are representative of 2 or more separate experiments.

Combined p53 potentiation and APC/C inhibition

Based on our results we hypothesized that BTZ causes B cell apoptosis by disturbing interactions between p53 and other key elements of the cell cycle. The anaphase promoting complex/cyclosome (APC/C) is one of two main E3 ligases involved in cell cycle regulation (43, 44). APC/C promotes progression from metaphase to anaphase and G1 phase re-entry by ubiquitinating cyclins and other cell cycle-related substrates which targets them for proteasome-mediated degradation. Given the impact of BTZ on cell cycle entry, we hypothesized that APC/C inhibition would recapitulate the effects of BTZ. To test this hypothesis, we first titrated increasing concentrations of the APC/C inhibitor proTAME (45) into cultures of early activated or late activated B cells (not shown). proTAME induced B cell death in a dose-dependent manner, though when added to dividing cells many post-division B cells and newborn plasma cells were readily evident at higher doses (not shown), in contrast to optimal BTZ dosing. However, when we added nutlin3a and proTAME together to late activated B cells at 44 hours post-stimulation, before the emergence of plasma cells, we observed highly reduced numbers of viable B cells and newly formed plasma cells (Fig. 6). Notably, although the effect of nutlin3a and proTAME treatment was less pronounced in p53−/− cells, we observed some killing effects for p53−/− cells, suggesting additional mechanisms independent of p53. We conclude that combined p53 potentiation and APC/C inhibition synergize to deplete activated B cells and thus reduce the generation of newborn plasma cells.

Figure 6. Nutlin3a and proTAME synergize to induce apoptosis in activated B cells.

Figure 6.

Open in a new tab

CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with CPG, IL-4, and IL-5. Cells were treated with vehicle or the indicated concentrations of proTAME and/or nutlin3a at 44 hours and analyzed at 66 hours post-stimulation. Gated on TOPRO-3− cells. p-values were calculated using a two-way ANOVA with Sidak’s multiple comparison test, *<.033, **<.002, ***<.001.

Anti-CD40 and IL-4 stimulation yields comparative results as T-independent type stimulation

To establish whether BTZ induces a p53-regulated death pathway for B cells experiencing T-cell associated activation cues, we examined the combined impact of p53 deletion and BTZ-addition in B cells stimulated with IL-4 and anti-CD40 antibody. Here we stimulated cells for 22 hours, then added BTZ and/or nutlin3A and evaluated cell division and viability, p53 protein levels, and CC3 levels 22 hours later (44 hours post-stimulation). p53 deletion rescued early activated B cells from single treatment with BTZ and nutlin3a, as well as from the combined exposure to both inhibitors (Fig. 7A). Exposure to BTZ and nutlin3a or BTZ alone led to increased percentages of cells with readily detectable levels of p53 protein (Fig. 7B), whereas p53 levels were much lower in cells exposed to nutlin3a alone, likely owing to the relatively late time point examined here (compared to the earlier timepoint evaluated in Fig. 3A). p53 deletion led to decreased frequencies of CC3+ cells after treatment with BTZ, nutlin3a, or both (Fig. 7C), and BrdU pulsing experiments confirmed that p53 deletion rescued anti-CD40 + IL-4 stimulated B cells from BTZ-induced apoptosis and cell cycle arrest (Fig. 7D). Finally, deletion of p53 had a major rescue effect in late activated B cells that were treated with proTAME and nutlin3a (Fig. 7E). In conclusion, B cells that were stimulated with anti-CD40 and IL-4 respond to BTZ, nutlin3a, and proTAME in a similar p53-dependent fashion as to B cells that were stimulated with CPG and IL-4.

Figure 7. Anti-CD40 and IL-4 stimulation yields comparative results as T-independent type stimulation.

Figure 7.

Open in a new tab

(A) CTV-labeled follicular B cells from p53+/+ or p53−/− mice were stimulated with IL-4 and anti-CD40 antibodies. 22 hours later cultures were supplemented with vehicle or the indicated concentrations of nutlin3a and/or BTZ and analyzed at 44 hours. Gated on CD138− cells. (B) Shows percentages and protein expression levels for p53. Cells were treated at 22 hours post-stimulation and analyzed at 44 hours post-stimulation. Gated on CD138−, TOPRO-3−. (C) Shows percentages and protein expression levels (gMFI) for CC3. Cells were treated at 22 hours post-stimulation and analyzed at 44 hours. Gated on CD138− cells. (D) BTZ was added to follicular B cells 22-hours post-stimulation. Cells were then pulsed with 20 μM BrdU at 43-hours post-stimulation, then stained with anti-BrdU antibodies and 7-AAD one hour later. Gated on LD-NIR− CD138− cells. (E) Cells were treated with vehicle or the indicated concentrations of proTAME and/or nutlin3a. Gated on TOPRO-3− cells. p-values were calculated using a two-way ANOVA with Sidak’s multiple comparison test, *<.033, **<.002, ***<.001.

Discussion

Reducing pools of pathogenic B-lineage cells and associated antibodies remains an ongoing challenge. Our results show that activated B cells are nearly as sensitive to BTZ as newly formed plasma cells. Furthermore, in contrast to plasma cells where BTZ is thought to cause ER-stress-associated apoptosis (5, 6), we found that activated B cells succumb to BTZ via p53-driven apoptosis. Deletion of p53 in early activated pre-division B cells promoted survival in response to BTZ. This rescue effect due to p53 deletion was associated with increased percentages of B cells in the G1, S- and G2/M phases of the cell cycle, indicating a p53 dependent block within the G1-phase similar to the effects of proteasome inhibition observed in human fibroblast cells and colon cancer cells (46). In addition, p53 deletion reduced frequencies of early activated B cells with DNA fragmentation and reduced numbers of B cells possessing cleaved caspase-3. Overall, our data suggests that BTZ blocks proliferation and differentiation in recently activated B cells, at least in part by inducing p53-regulated apoptosis.

Ag-induced B cell differentiation requires the temporal coordination of several distinct processes. Within the first several days of initial antigenic exposure responding B cells undergo a massive clonal burst. Typically, within five days cohorts of proliferative B cells yield early plasma cells (47), while other cells seed GCs where they undergo additional rounds of division accompanied by somatic hypermutation and selection for subclones bearing high-affinity BCRs (48). Our results suggest that the mechanisms whereby proliferative B cells are depleted by BTZ contrast substantially with BTZ-mediated death mechanisms in mature non-proliferative plasma cells. Thus, while BTZ can be used to deplete mature bone marrow plasma cells secreting self-reactive antibodies due do its impact on ER homeostasis (5), our results reveal the possibility that compounds that inhibit proteasome-regulated p53 degradation may also prove useful for depleting proliferative B cells upstream of plasma cell differentiation. Such approaches may warrant consideration in scenarios characterized by chronic B cell activation, especially in autoimmune diseases where activated B cells and early plasma cells play a dominant role in disease.

Our results are consistent with past studies suggesting that activated B and T cells experience increased levels of DNA damage and p53 function (37, 39). Importantly however, other work has shown that in activated T cells p53 protein levels decrease beginning at 48 hours post-activation as a strategy to optimize clonal burst size (41). This result is consistent with our data showing a lack of impact of p53 deletion on BTZ-driven apoptosis in late activated post-division B cells. We note however that p53 potentiation with an MDM2 inhibitor induced apoptosis including for late activated B cells, suggesting that sufficient quantities of p53 are available in post-division cells to override alternative pro-survival pathways and surpass the apoptotic threshold (49). In line with its known transcriptional targets, we speculate that in BTZ-treated B cells p53 promotes increased availability of pro-apoptotic targets of the BCL2 family (26, 50–53).

Disrupting the degradation of p53 and cyclin related proteins with nutlin3a and proTAME reduced numbers of activated B cells and newly formed plasma cells. These results together with our in vitro BTZ data emphasize the need to consider the impact of biochemical processes operative at key phases of the cell cycle on the efficacy of these and related drugs. However, other very recent work has shown that innate-like MZ B cells can yield functional plasma cells despite full cell cycle blockade, in contrast to the bulk of peripheral B cells (35). These results raise the question of whether nutlin3a can also synergize with APC/C inhibitors to prevent division-independent plasma cell differentiation. Similarly, it should be emphasized that intracellular levels of p53 were substantially different for cells experiencing BTZ versus nutlin3a, with BTZ causing substantially higher p53 levels (see Figure 3).

The different outcomes of p53 activation, such as cell cycle arrest or apoptosis, can be influenced by a wide variety of factors including differential availability of particular cell extrinsic factors and cell intrinsic interplay between p53 and cell-type restricted signaling pathways. Indeed, given that p53 is proposed to target the critical plasma cell regulator Blimp1 (PRDM1) in colorectal cancer cells (54), p53-deletion may regulate a variety of processes that are relatively unique to activated B cells. Additionally, the levels of p53 may influence whether cells experience proliferative arrest versus cell death, with higher levels more likely to drive apoptosis (20). Whereas both BTZ and nutlin3a caused dose-dependent losses in primary B cell viability, when compared with BTZ, nutlin3a caused only modest increases in p53 levels and it was easier to visualize progression and arrest of cells at later stages of the cell cycle in these cells (Fig. 3, Fig. 4). We speculate that BTZ is especially suited for promoting apoptosis in primary B cells because of the exceptionally high levels of p53 that occur due to BTZ-mediated proteasome inhibition in these cells.

BTZ use has been limited clinically due to adverse reactions such as peripheral neuropathy (55). Likewise, second generation inhibitors like carfilzomib are associated with increased risk for cardiovascular adverse events (56). Thus, the mechanistic insights in this study may provide avenues to explore more specific therapeutic interventions for inducting apoptosis for activated B cells while avoiding the limitations of PIs. As such, combinatorial approaches (e.g. nutlin3a and BTZ) may be advantageous when used at doses of BTZ that minimize neuropathy yet deplete proliferative B cell pools and consequently arrest plasma cell differentiation. This idea is consistent with our results showing that suboptimal doses of BTZ and nutlin3a worked together to reduce B cell viability and inhibit plasma cell differentiation. However, it should also be emphasized that nutlin3a may target several additional and potentially relevant regulatory pathways such as those controlled by HIF-1α and p73 (42). Future studies focused on PI-resistance mechanisms in B cells and plasma cells may provide additional therapeutic targets that maximally exploit the vulnerabilities of B-lineage cells while avoiding drug toxicity.

Supplementary Material

1

Key points:

  1. BTZ-induced apoptosis in activated B cells is mediated by p53.

  2. p53 regulation of BTZ-induced apoptosis occurs early in the cell cycle.

  3. The combination of BTZ and nutlin3a optimize p53 stabilization.

Acknowledgements

We gratefully thank Drs. Paula Oliver, Michael Cancro, and Avinash Bhandoola for helpful discussions. We also thank the UPenn Flow Cytometry and Cell Sorting facility.

*This work was supported by National Institutes of Health (NIH) grants AI139123 (DA) and AI154932 (DA, DH, SW).

Footnotes

Declaration of Interest

The authors have declared that no conflict of interest exists.

References

  • 1.Akkaya M, Kwak K, and Pierce SK 2020. B cell memory: building two walls of protection against pathogens. Nat Rev Immunol 20: 229–238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Kubiczkova L, Pour L, Sedlarikova L, Hajek R, and Sevcikova S. 2014. Proteasome inhibitors - molecular basis and current perspectives in multiple myeloma. Journal of Cellular and Molecular Medicine 18: 947–961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ludwig RJ, Vanhoorelbeke K, Leypoldt F, Kaya Z, Bieber K, McLachlan SM, Komorowski L, Luo J, Cabral-Marques O, Hammers CM, Lindstrom JM, Lamprecht P, Fischer A, Riemekasten G, Tersteeg C, Sondermann P, Rapoport B, Wandinger KP, Probst C, El Beidaq A, Schmidt E, Verkman A, Manz RA, and Nimmerjahn F. 2017. Mechanisms of Autoantibody-Induced Pathology. Frontiers in immunology 8: 603–645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Woodle ES, Tremblay S, Rossi A, Rojas CC, Alloway R, Roskin K, Allman D, and Hildeman D. 2020. Plasma cell targeting to prevent antibody-mediated rejection. Am J Transplant 20: 33–41. [DOI] [PubMed] [Google Scholar]
  • 5.Neubert K, Meister S, Moser K, Weisel F, Maseda D, Amann K, Wiethe C, Winkler TH, Kalden JR, Manz RA, and Voll RE 2008. The proteasome inhibitor bortezomib depletes plasma cells and protects mice with lupus-like disease from nephritis. Nat Med 14: 748–755. [DOI] [PubMed] [Google Scholar]
  • 6.Obeng EA, Carlson LM, Gutman DM, Harrington WJ Jr., Lee KP, and Boise LH 2006. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood 107: 4907–4916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Woodle ES, Tremblay S, Brailey P, Girnita A, Alloway RR, Aronow B, Dasgupta N, Ebstein F, Kloetzel PM, Lee MJ, Kim KB, Singh H, and Driscoll JJ 2020. Proteasomal adaptations underlying carfilzomib-resistance in human bone marrow plasma cells. Am J Transplant 20: 399–410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Woodle ES, Tremblay S, and Driscoll J. 2017. Targeting Plasma Cells with Proteasome Inhibitors: Principles from Primates. Journal of the American Society of Nephrology : JASN 28: 1951–1953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Rossi AP, Tremblay S, Castro-Rojas CM, Burg AA, Roskin KM, Gehman JM, Rike-Shields A, Alloway RR, Brailey P, Allman D, Hildeman DA, and Woodle ES 2023. Effects of invivo CXCR4 blockade and proteasome inhibition on bone marrow plasma cells in HLA-sensitized kidney transplant candidates. Am J Transplant 23: 759–775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ishii T, Tanaka Y, Kawakami A, Saito K, Ichinose K, Fujii H, Shirota Y, Shirai T, Fujita Y, Watanabe R, Chiu SW, Yamaguchi T, and Harigae H. 2018. Multicenter double-blind randomized controlled trial to evaluate the effectiveness and safety of bortezomib as a treatment for refractory systemic lupus erythematosus. Mod Rheumatol 28: 986–992. [DOI] [PubMed] [Google Scholar]
  • 11.Mulder A, Heidt S, Vergunst M, Roelen DL, and Claas FH 2013. Proteasome inhibition profoundly affects activated human B cells. Transplantation 95: 1331–1337. [DOI] [PubMed] [Google Scholar]
  • 12.Khodadadi L, Cheng Q, Alexander T, Sercan-Alp O, Klotsche J, Radbruch A, Hiepe F, Hoyer BF, and Taddeo A. 2015. Bortezomib Plus Continuous B Cell Depletion Results in Sustained Plasma Cell Depletion and Amelioration of Lupus Nephritis in NZB/W F1 Mice. PLoS One 10: e0135081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Victora GD, and Nussenzweig MC 2022. Germinal Centers. Annu Rev Immunol 40: 413–442. [DOI] [PubMed] [Google Scholar]
  • 14.Rock KL, Colette G, Lisa R, Karen C, Ross S, Lawrence D, Daniel H, and Alfred LG 1994. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on mhc class i molecules. Cell 78: 761–771. [DOI] [PubMed] [Google Scholar]
  • 15.Kisselev AF, and Goldberg AL 2001. Proteasome inhibitors: from research tools to drug candidates. Chem Biol 8: 739–758. [DOI] [PubMed] [Google Scholar]
  • 16.Teicher BA, and Tomaszewski JE 2015. Proteasome inhibitors. Biochem Pharmacol 96: 1–9. [DOI] [PubMed] [Google Scholar]
  • 17.Chen S, Blank JL, Peters T, Liu XJ, Rappoli DM, Pickard MD, Menon S, Yu J, Driscoll DL, Lingaraj T, Burkhardt AL, Chen W, Garcia K, Sappal DS, Gray J, Hales P, Leroy PJ, Ringeling J, Rabino C, Spelman JJ, Morgenstern JP, and Lightcap ES 2010. Genome-wide siRNA screen for modulators of cell death induced by proteasome inhibitor bortezomib. Cancer Res 70: 4318–4326. [DOI] [PubMed] [Google Scholar]
  • 18.Strauss SJ, Higginbottom K, Juliger S, Maharaj L, Allen P, Schenkein D, Lister TA, and Joel SP 2007. The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines. Cancer Res 67: 2783–2790. [DOI] [PubMed] [Google Scholar]
  • 19.Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, and Elliot PJ 1999. Proteasome inhibitors a novel class of potent and effective antitumor agents. Cancer Research 59: 2615–2622. [PubMed] [Google Scholar]
  • 20.Lieschke E, Wang Z, Kelly GL, and Strasser A. 2019. Discussion of some ‘knowns’ and some ‘unknowns’ about the tumour suppressor p53. J Mol Cell Biol 11: 212–223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT, and Weinberg RA 1994. Tumor spectrum analysis in p53-mutant mice. Curr Biol 4: 1–7. [DOI] [PubMed] [Google Scholar]
  • 22.Haupt Y, Maya R, Kazaz A, and Oren M. 1997. Mdm2 promotes the rapid degradation of p53. Nature 387: 296–299. [DOI] [PubMed] [Google Scholar]
  • 23.Kubbutat MHG, Jones SN, and Vousden KH 1997. Regulation of p53 stability by mdm2. Nature 387: 299–303. [DOI] [PubMed] [Google Scholar]
  • 24.Wu X, Bayle H, Olson D, and Levine AJ 1993. The p53 mdm2 autoregulatory feedback loop. Genes and Development 7: 1126–1132. [DOI] [PubMed] [Google Scholar]
  • 25.Vassilev LT, Vu VT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, and Liu EA 2004. In vivo activation of the p53 pathway by small molecule antagonists of mdm2. Science 303: 844–848. [DOI] [PubMed] [Google Scholar]
  • 26.Valente LJ, Aubrey BJ, Herold MJ, Kelly GL, Happo L, Scott CL, Newbold A, Johnstone RW, Huang DC, Vassilev LT, and Strasser A. 2016. Therapeutic Response to Non-genotoxic Activation of p53 by Nutlin3a Is Driven by PUMA-Mediated Apoptosis in Lymphoma Cells. Cell Rep 14: 1858–1866. [DOI] [PubMed] [Google Scholar]
  • 27.Vaziri SA, Grabowski DR, Hill J, Rybicki LR, Burk R, Bukowski RM, Ganapathi MK, and Ganapathi R. 2009. Inhibition of proteasome activity by bortezomib in renal cancer cells is p53 dependent and VHL independent. Anticancer research 29: 2961–2969. [PMC free article] [PubMed] [Google Scholar]
  • 28.Lopes UG, Erhardt P, Yao R, and Cooper GM 1997. p53-dependent induction of apoptosis by proteasome inhibitors. J Biol Chem 272: 12893–12896. [DOI] [PubMed] [Google Scholar]
  • 29.Xue Y, Barker N, Hoon S, He P, Thakur T, Abdeen SR, Maruthappan P, Ghadessy FJ, and Lane DP 2019. Bortezomib Stabilizes and Activates p53 in Proliferative Compartments of Both Normal and Tumor Tissues In Vivo. Cancer Res 79: 3595–3607. [DOI] [PubMed] [Google Scholar]
  • 30.Pandit B, and Gartel AL 2011. Proteasome inhibitors induce p53-independent apoptosis in human cancer cells. Am J Pathol 178: 355–360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Perez-Galan P, Roue G, Villamor N, Montserrat E, Campo E, and Colomer D. 2006. The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status. Blood 107: 257–264. [DOI] [PubMed] [Google Scholar]
  • 32.Kallies A, Hasbold J, Tarlinton DM, Dietrich W, Corcoran LM, Hodgkin PD, and Nutt SL 2004. Plasma cell ontogeny defined by quantitative changes in blimp-1 expression. J Exp Med 200: 967–977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Eddaoudi A, Canning SL, and Kato I. 2018. Flow Cytometric Detection of G0 in Live Cells by Hoechst 33342 and Pyronin Y Staining. In Cellular Quiescence: Methods and Protocols. Lacorazza HD, ed. Humana Press. 49–57. [DOI] [PubMed] [Google Scholar]
  • 34.Gaudette BT, Jones DD, Bortnick A, Argon Y, and Allman D. 2020. mTORC1 coordinates an immediate unfolded protein response-related transcriptome in activated B cells preceding antibody secretion. Nat Commun 11: 723–739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Gaudette BT, Roman CJ, Ochoa TA, Gomez Atria D, Jones DD, Siebel CW, Maillard I, and Allman D. 2021. Resting innate-like B cells leverage sustained Notch2/mTORC1 signaling to achieve rapid and mitosis-independent plasma cell differentiation. J Clin Invest 131: 151975–151992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Dowling MR, Kan A, Heinzel S, Zhou JH, Marchingo JM, Wellard CJ, Markham JF, and Hodgkin PD 2014. Stretched cell cycle model for proliferating lymphocytes. Proc Natl Acad Sci U S A 111: 6377–6382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.McNally JP, Millen SH, Chaturvedi V, Lakes N, Terrell CE, Elfers EE, Carroll KR, Hogan SP, Andreassen PR, Kanter J, Allen CE, Henry MM, Greenberg JN, Ladisch S, Hermiston ML, Joyce M, Hildeman DA, Katz JD, and Jordan MB 2017. Manipulating DNA damage-response signaling for the treatment of immune-mediated diseases. Proc Natl Acad Sci U S A 114: E4782–E4791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Mayor T, Sharon M, and Glickman MH 2016. Tuning the proteasome to brighten the end of the journey. Am J Physiol Cell Physiol 311: C793–C804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Lee H, Haque S, Nieto J, Trott J, Inman JK, McCormick S, Chiorazzi N, and Mongini PK 2012. A p53 axis regulates B cell receptor-triggered, innate immune system-driven B cell clonal expansion. J Immunol 188: 6093–6108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Vaziri SAJ, GRABOWSKI DR, HILL J, RYBICKI LR, BURK R, BUKOWSKI RM, GANAPATHI MK, and GANAPATHI R. 2009. Inhibition of Proteasome Activity by Bortezomib in Renal Cancer Cells Is p53 Dependent and VHL Independent. Anticancer Research 29: 2961–2970. [PMC free article] [PubMed] [Google Scholar]
  • 41.Watanabe M, Moon KD, Vacchio MS, Hathcock KS, and Hodes RJ 2014. Downmodulation of tumor suppressor p53 by T cell receptor signaling is critical for antigen-specific CD4(+) T cell responses. Immunity 40: 681–691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Shen H, and Maki CG 2011. Pharmacologic activation of p53 by small-molecule MDM2 antagonists. Current pharmaceutical design 17: 560–568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Bassermann F, Eichner R, and Pagano M. 2014. The ubiquitin proteasome system - implications for cell cycle control and the targeted treatment of cancer. Biochim Biophys Acta 1843: 150–162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Dang F, Nie L, and Wei W. 2021. Ubiquitin signaling in cell cycle control and tumorigenesis. Cell Death Differ 28: 427–438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Zeng X, Sigoillot F, Gaur S, Choi S, Pfaff KL, Oh DC, Hathaway N, Dimova N, Cuny GD, and King RW 2010. Pharmacologic inhibition of the anaphase-promoting complex induces a spindle checkpoint-dependent mitotic arrest in the absence of spindle damage. Cancer Cell 18: 382–395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Chen F, Chang D, Goh M, Klibanov SA, and Ljungman M. 2000. Role of p53 in cell cycle regulation and apoptosis following exposure to proteasome inhibitors. Cell Growth Differ 11: 239–246. [PubMed] [Google Scholar]
  • 47.Coffey F, Alabyev B, and Manser T. 2009. Initial clonal expansion of germinal center B cells takes place at the perimeter of follicles. Immunity 30: 599–609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Victora GD, and Nussenzweig MC 2012. Germinal centers. Annual review of immunology 30: 429–457. [DOI] [PubMed] [Google Scholar]
  • 49.Kracikova M, Akiri G, George A, Sachidanandam R, and Aaronson SA 2013. A threshold mechanism mediates p53 cell fate decision between growth arrest and apoptosis. Cell Death Differ 20: 576–588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Nakano K, and Vousden KH 2001. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 7: 683–694. [DOI] [PubMed] [Google Scholar]
  • 51.Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer DD, and Green DR 2005. PUMA couples the nuclear and cytoplasmic proapoptotic function of p53. Science 309: 1732–1735. [DOI] [PubMed] [Google Scholar]
  • 52.Green DR, and Kroemer G. 2009. Cytoplasmic functions of the tumour suppressor p53. Nature 458: 1127–1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Aubrey BJ, Kelly GL, Janic A, Herold MJ, and Strasser A. 2018. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell Death Differ 25: 104–113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Liu C, Banister CE, Weige CC, Altomare D, Richardson JH, Contreras CM, and Buckhaults PJ 2018. PRDM1 silences stem cell-related genes and inhibits proliferation of human colon tumor organoids. Proc Natl Acad Sci U S A 115: E5066–E5075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Yamamoto S, and Egashira N. 2021. Pathological Mechanisms of Bortezomib-Induced Peripheral Neuropathy. International journal of molecular sciences 22: 888–902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Waxman AJ, Clasen S, Hwang WT, Garfall A, Vogl DT, Carver J, O’Quinn R, Cohen AD, Stadtmauer EA, Ky B, and Weiss BM 2018. Carfilzomib-Associated Cardiovascular Adverse Events: A Systematic Review and Meta-analysis. JAMA Oncol 4: e174519. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1942592-supplement-1.pdf (1.9MB, pdf)

RESOURCES