Proteasome Inhibitor Research Articles

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3

Decoy oligodeoxynucleotides (ODNs) allow targeting undruggable transcription factors, such as STAT3, but their limited potency and lack of delivery methods hampered translation. To overcome these challenges, we conjugated a STAT3-specific decoy to thalidomide, a ligand to cereblon in E3 ubiquitin ligase complex, to generate a proteolysis-targeting chimera (STAT3DPROTAC). STAT3DPROTAC downregulated STAT3 in target cells, but not STAT1 or STAT5. Computational modeling of the STAT3DPROTAC ternary complex predicted two surface lysines, K601 and K626, in STAT3 as potential ubiquitination sites. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibition or cereblon deletion, alleviated STAT3DPROTAC effect. Next, we conjugated STAT3DPROTAC to a CpG oligonucleotide targeting Toll-like receptor 9 (TLR9) to generate myeloid/B cell-selective C-STAT3DPROTAC. Naked C-STAT3DPROTAC was spontaneously internalized by TLR9+ myeloid cells, B cells, and human and mouse lymphoma cells but not by Tcells. C-STAT3DPROTAC effectively decreased STAT3 protein levels and also STAT3-regulated target genes critical for lymphoma cell proliferation and/or survival (BCL2L1, CCND2, and MYC). Finally, local C-STAT3DPROTAC administration to human Ly3 lymphoma-bearing mice triggered tumor regression, while control C-STAT3D and C-SCR treatments had limited effects. Our results underscore the feasibility of using a PROTAC strategy for cell-selective, decoy oligonucleotide-based STAT3 targeting of and potentially other tumorigenic transcription factors for cancer therapy.

Abstract B019: The bone microenvironment's impact on the heterogeneity of resistance to proteasome inhibitors in multiple myeloma

Abstract Multiple myeloma (MM) is an osteolytic plasma cell malignancy that, despite being responsive to therapies such as proteasome inhibitors, frequently relapses. Understanding the mechanism and the niches where resistant disease evolves remains of major clinical importance. Cancer cell intrinsic mechanisms and bone ecosystem factors are known contributors to the evolution of resistant MM but the exact contribution of each is difficult to define with current in vitro and in vivo models. Using a novel bmathematical model that incorporates key cellular species of the bone ecosystem that control normal bone remodeling and, in MM, yields a protective environment we studied how, under therapy, the bone ecosystem contributes to the evolutionary dynamics of resistant MM under control and proteasome inhibitor treatment. Our results show that Environmentally-Mediated Drug Resistance (EMDR) might not be sufficient to explain resistant disease. But our results highlight the importance of EMDR in facilitating the emergence of resistance, and more importantly facilitating the survival of tolerant cells, allowing the tumor multiple routes for resistant disease. Our results also provide evidence that intervention with therapies targetting the bone ecosystem would significantly improve the ability of treatment to increase the time to progression for patients with multiple myeloma and, potentially, other diseases where EMDR plays a role. Citation Format: David Basanta, Ryan Bishop, Anna Miller, Conor C. Lynch, Matthew Froid, Ariosto S. Silva, Kenneth H. Shain. The bone microenvironment's impact on the heterogeneity of resistance to proteasome inhibitors in multiple myeloma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B019.

Presentation and Outcome of Patients with Multiple Myeloma (MM), Single Centre Experience from Windsor Essex Regional Cancer Centre.

Outcomes for patients with multiple myeloma has significantly improved through the years. This is mainly related to the use of novel agents. This is a retrospective study that reviewed presentation and outcome of 139 patients with multiple myeloma at the Windsor Essex Regional Cancer Centre from Jan. 1, 2015 to Dec. 31, 2019. Median age was 71 years and most patients had higher risk disease (65.5% either R ISS stage II or III). 30% had high risk FISH for myeloma including del.17P, t (4:14), t (14:16) and Gain (1q21). In terms of presentation, 38.8% had anemia (hemoglobin <100g/L), 18.7% had hypercalcemia, 74.1% had skeletal lytic lesions, 38.8% had pathologic fracture and 17.3% had plasmacytoma. Almost all (92%) of the patients were treated using at least one novel agent (proteasome inhibitor or immunomodulators [ImiDs]). Cyclophosphamide, bortezomib, and dexamethasone (CyBorD) was the most used treatment regimen (48.9%) followed by bortezomib, melphalan and prednisone (BMP) at 28.8% and lenalidomide, dexamethasone (LenDex) at 14.4%. With respect to response to therapy, 51.8% had at least Very good partial response (VGPR), while 9.4% had progressive disease. 33% had autologous stem cell transplant. After a median follow up of 2.4 years, median overall survival was 3.7 years. 2 years overall survival and relapse-free survival were 70% and 83%, respectively. Our study showed comparable outcome for patients with multiple myeloma despite older age and higher risk disease. Outcome is expected to improve with the introduction of more novel agents.

Effectiveness of combined plasma cell therapy and costimulation blockade based desensitization regimen in heart transplant candidates.

Desensitization is one of the strategies to reduce antibodies and facilitate heart transplantation in highly sensitized patients. We describe our center's desensitization experience with combination of plasma cell (PC) depletion therapy (with proteasome inhibitor or daratumumab) and costimulation blockade (with belatacept). We reviewed five highly sensitized patients who underwent desensitization therapy with plasma cell depletion and costimulation blockade. We evaluated the response to therapy by measuring the changes in cPRA, average MFI, and number of positive beads>5000MFI. Five patients, mean age of 56 (37-66) years with average cPRA of 98% at 5000 MFI underwent desensitization therapy. After desensitization, mean cPRA decreased from 98% to 70% (p=.09), average number of beads>5000 MFI decreased from 59 to 37 (p=.15), and average MFI of beads>5000 MFI decreased from 16713 to 13074 (p=.26). Combined PC depletion and CoB could be a reasonable strategy for sustained reduction in antibodies in highly sensitized patients being listed for heart transplantation.

Isatuximab-pomalidomide-dexamethasone versus pomalidomide-dexamethasone in patients with relapsed and refractory multiple myeloma: final overall survival analysis.

The primary and pre-specified updated analyses of ICARIA-MM (NCT02990338) demonstrated improved progression-free survival and a benefit in overall survival (OS) was reported with the addition of isatuximab, an anti-CD38 monoclonal antibody, to pomalidomide-dexamethasone (Pd) in patients with relapsed/refractory multiple myeloma. Here, we report the final OS analysis. This multicenter, randomized, open-label, phase 3 study included patients who had received and failed ≥2 previous therapies, including lenalidomide and a proteasome inhibitor. Between January 10, 2017, and February 2, 2018, 307 patients were randomized (1:1) to isatuximab-pomalidomide- dexamethasone (Isa-Pd; n = 154) or Pd (n = 153), stratified based on age (3). At data cutoff for the final OS analysis after 220 OS events (January 27, 2022), median follow-up duration was 52.4 months. Median OS (95% confidence interval) was 24.6 months (20.3-31.3 months) with Isa-Pd and 17.7 months (14.4-26.2 months) with Pd (hazard ratio = 0.78; 95% CI, 0.59-1.02; 1-sided P = 0.0319). Despite subsequent daratumumab use in the Pd group and its potential benefit on PFS in the first subsequent therapy line, median PFS2 was significantly longer with Isa-Pd vs. Pd (17.5 vs. 12.9 months; log-rank 1-sided P = 0.0091). In this analysis, Isa-Pd continued to be efficacious and well tolerated after follow-up of approximately 52 months, contributing to a clinically meaningful, 6.9-month improvement in median overall survival in patients with relapsed/refractory multiple myeloma.

Proteasome maturation and inhibitor design

Proteasome maturation and inhibitor design

Complex Topology of Ubiquitin Chains Mediates Lysosomal Degradation of MrgC Proteins.

Activation of Mas-related G protein-coupled receptor C (MrgC) receptors relieves pain, but also leads to ubiquitination of MrgC receptors. Ubiquitination mediates MrgC receptor endocytosis and degradation. However, MrgC degradation pathways and ubiquitin-linked chain types are not known. N2a cells were treated with cycloheximide (CHX, protein synthesis inhibitor), Mg132 (proteasome inhibitor), 3-Methyladenine (3MA, autophagy lysosome inhibitor) and Chloroquine (CQ, autophagy lysosome inhibitor) to observe the half-life and degradation pathway of MrgC. The location of internalized MrgC receptors and lysosomes (Lyso-Tracker) was observed by immunofluorescence staining. N2a cells were transfected with Myc-MrgC and a series of HA-tagged ubiquitin mutants to study the ubiquitin-linked chain type of MrgC. The amount of MrgC protein decreased with time after CHX treatment of N2a cells. Autophagy lysosome inhibitors can inhibit the degradation of MrgC. The amount of MrgC protein decreased with time after CHX treatment of N2a cells. 3-MA and CQ inhibited the degradation of MrgC protein, whereas Mg-132 did not inhibit it. Partially internalized MrgC receptors were co-labeled with lysosomes. MrgC proteins have multiple topologies of ubiquitin-modified chains. As a member of the G protein-coupled receptor family, MrgC receptors can be degraded over time. The complex topology of the ubiquitin-linked chain mediates the lysosomal degradation of MrgC proteins.

Subsequent therapy and outcomes in patients with newly diagnosed multiple myeloma experiencing disease progression after quadruplet combinations.

The combination of anti-CD38 monoclonal antibodies to a proteasome inhibitor, an immunomodulatory agent and dexamethasone (quadruplet-QUAD) in sequence with autologous stem cell transplantation (ASCT) leads to deep and durable responses in newly diagnosed multiple myeloma (NDMM). Disease progression in the first year post-QUADs is uncommon. We analysed 274 consecutive NDMM patients treated with QUADs + ASCT. After a median follow-up of 21.3 months, 20 patients had disease progression <18 months and 21 had progression ≥18 months after the onset of a QUAD regimen. All patients received subsequent anti-MM therapy, and 38 were evaluated for response. Nine (22.0%) received T-cell redirecting therapy as the next treatment, and 21 (51.2%) at some point in the treatment course. Response to next therapy was 26.3% for patients with progression <18 months and 52.6% for those with progression ≥18 months after the onset of a QUAD regimen. Median PFS on the next therapy was 2.5 months (95% CI 1.5-3.4) for those with progression <18 months and 7.0 months (95% CI 3.6-10.5) for those with progression ≥18 months. Efforts should focus on the early deployment of therapies with new mechanism of action for patients experiencing treatment failure after QUADs.

Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is associated with poor prognosis and limited treatment options due to the lack of important receptors (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2 [HER2]) used for targeted therapy. However, high-throughput in vitro drug screening of cell lines is a powerful tool for identifying effective drugs for a disease. Here, we determine the intrinsic chemosensitivity of TNBC cell lines to proteasome inhibitors (PIs), thereby identifying potentially potent 2-drug combinations for TNBC. Eight TNBC cell lines (BT-549, CAL-148, HCC1806, HCC38, HCC70, MDA-MB-436, MDA-MB-453, and MDA-MB-468) and two controls (MCF-10A and MCF-7) were first exposed to 18 drugs (11 PIs and 7 clinically relevant chemotherapeutic agents) as monotherapy, followed by prediction of potent 2-drug combinations using the IDACombo pipeline. The synergistic effects of the 2-drug combinations were evaluated with SynergyFinder in four TNBC cell lines (CAL-148, HCC1806, HCC38, and MDA-MB-468) and three controls (BT-474, MCF-7, and T47D) in vitro, followed by further evaluation of tumor regression in zebrafish tumor models established using HCC1806 and MCF-7 cells. Monotherapy identified nine effective drugs (bortezomib, carfilzomib, cisplatin, delanzomib, docetaxel, epoxomicin, MLN-2238, MLN-9708, and nedaplatin) across all cell lines. PIs (e.g., bortezomib, delanzomib, and epoxomicin) were highly potent drugs in TNBC cells, of which bortezomib and delanzomib inhibited the chymotrypsin-like activity of the 20 S proteasome by 100% at 10 µM. Moreover, several potent 2-drug combinations (e.g., bortezomib+nedaplatin and epoxomicin+epirubicin) that killed virtually 100% of cells were also identified. Although HCC1806- and MCF-7-derived xenografts treated with bortezomib+nedaplatin and carboplatin+paclitaxel were smaller, HCC1806 cells frequently metastasized to the trunk region. Taken together, we show that PIs used in combination with platinum agents or topoisomerase inhibitors exhibit increased efficiency with almost 100% inhibition in TNBC cell lines, indicating that PIs are therefore promising compounds to use as combination therapy for TNBC.

Activity-Guided Proteomic Profiling of Proteasomes Uncovers a Variety of Active (and Inactive) Proteasome Species

Proteasomes are multisubunit, multicatalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided profiling approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20SBlm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of nonmature and therefore inactive proteasomal protease subunits β2 and β5 in the fully assembled proteasomes.

Imaging Very Late Antigen-4 on MOLT4 Leukemia Tumors with Cysteine Site-Specific 89Zr-Labeled Natalizumab Immuno-Positron Emission Tomography.

Very late antigen-4 (VLA4; CD49d) is a promising immune therapy target in treatment-resistant leukemia and multiple myeloma, and there is growing interest in repurposing the humanized monoclonal antibody (Ab), natalizumab, for this purpose. Positron emission tomography with radiolabeled Abs (immuno-PET) could facilitate this effort by providing information on natalizumab's in vivo pharmacokinetic and target delivery properties. In this study, we labeled natalizumab with 89Zr specifically on sulfhydryl moieties via maleimide-deferoxamine conjugation. High VLA4-expressing MOLT4 human T cell acute lymphoblastic leukemia cells showed specific 89Zr-natalizumab binding that was markedly blocked by excess Ab. In nude mice bearing MOLT4 tumors, 89Zr-natalizumab PET showed high-contrast tumor uptake at 7 days postinjection. Biodistribution studies confirmed that uptake was the highest in MOLT4 tumors (2.22 ± 0.41%ID/g) and the liver (2.33 ± 0.76%ID/g), followed by the spleen (1.51 ± 0.42%ID/g), while blood activity was lower at 1.12 ± 0.21%ID/g. VLA4-specific targeting in vivo was confirmed by a 58.1% suppression of tumor uptake (0.93 ± 0.15%ID/g) when excess Ab was injected 1 h earlier. In cultured MOLT4 cells, short-term 3 day exposure to the proteasome inhibitor bortezomib (BTZ) did not affect the α4 integrin level, but BTZ-resistant cells that survived the treatment showed increased α4 integrin expression. When the effects of BTZ treatment were tested in mice, there was no change of the α4 integrin level or 89Zr-natalizumab uptake in MOLT4 leukemia tumors, which underscores the complexity of tumor VLA4 regulation in vivo. In conclusion, 89Zr-natalizumab PET may be useful for noninvasive monitoring of tumor VLA4 and may assist in a more rational application of Ab-based therapies for hematologic malignancies.

Systematic identification of 20S proteasome substrates

For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology.

ERCC1 abundance is an indicator of DNA repair-apoptosis decision upon DNA damage

DNA repair is essential for successful propagation of genetic material and fidelity of transcription. Nucleotide excision repair (NER) is one of the earliest DNA repair mechanisms, functionally conserved from bacteria to human. The fact that number of NER genes vary significantly between prokaryotes and metazoans gives the insight that NER proteins have evolved to acquire additional functions to combat challenges associated with a diploid genome, including being involved in the decision between DNA repair and apoptosis. However, no direct association between apoptosis and NER proteins has been shown to date. In this study, we induced apoptosis with a variety of agents, including oxaliplatin, doxorubicin and TRAIL, and observed changes in the abundance and molecular weight of NER complex proteins. Our results showed that XPA, XPC and ERCC1 protein levels change during DNA damage-induced apoptosis. Among these, ERCC1 decrease was observed as a pre-mitochondria depolarisation event which marks the “point of no return” in apoptosis signalling. ERCC1 decrease was due to proteasomal degradation upon lethal doses of oxaliplatin exposure. When ERCC1 protein was stabilised using proteasome inhibitors, the pro-apoptotic activity of oxaliplatin was attenuated. These results explain why clinical trials using proteasome inhibitors and platinum derivatives showed limited efficacy in carcinoma treatment and also the importance of how deep understanding of DNA repair mechanisms can improve cancer therapy.

A phase 1-2 trial of DA-EPOCH-R plus ixazomib for MYC-aberrant lymphoid malignancies: the DACIPHOR regimen

AbstractMYC-aberrant non-Hodgkin lymphoma (NHL) is associated with poor outcomes with conventional chemotherapy. Ixazomib is an orally bioavailable proteasome inhibitor that targets drivers of MYC expression and has demonstrated preclinical activity in aggressive MYC-aberrant NHL. We conducted a phase 1/2 study evaluating the safety and efficacy of DA-EPOCH-R with adjunctive ixazomib in aggressive MYC-aberrant NHL. For induction, patients received 6 cycles of DA-EPOCH-R with ixazomib administered twice per 21-day cycle; responders continued weekly ixazomib maintenance for up to 1 year. Primary objectives were to determine the maximum tolerated dose in phase 1 and efficacy of DA-EPOCH-R with ixazomib as measured by 12-month progression-free survival (PFS) rate in phase 2. Thirty-six patients were evaluable for response. Median age was 63 years (range, 31-77) and 44% had double-hit lymphoma (DHL)/triple-hit lymphoma (THL). In phase 1, 3 mg of ixazomib was established as recommended phase 2 dose. Twenty-nine (76.3%) patients completed 6 cycles of DA-EPOCH-R and 25 (65.8%) underwent dose escalations. The ORR after induction was 97% (95% confidence interval, 81-100) with a CR rate of 69%. At median follow-up of 18.8 months, the 12-month PFS and overall survival (OS) rates were 78% and 86%, respectively. For DHL/THL vs dual expressor lymphomas (DEL), 12-month PFS rates were 53% vs 95% and 12-month OS rates were 65% vs 100%, respectively. Grade ≥3 toxicities were predominantly hematologic. Twenty-seven (75%) of patients experienced neuropathy, nearly all low-grade. DA-EPOCH-R induction with adjunctive ixazomib is feasible and appears effective in patients with DEL. This trial was registered at www.clinicaltrials.gov as #NCT02481310.

Metformin and chidamide synergistically suppress multiple myeloma progression and enhance lenalidomide/bortezomib sensitivity.

Multiple myeloma (MM) is a common hematological malignancy, and patients with MM are recommended to take immunomodulatory drugs such as lenalidomide along with proteasome inhibitors such as bortezomib to extend survival. However, drug resistance influences the efficacy of treatment for MM. In our study, we found that metformin and chidamide both suppressed MM cell growth in a concentration- and time-dependent way (p < .001). Moreover, combined therapy with metformin and chidamide exhibited enhanced inhibition of the growth of MM cells compared with monotherapy (p < .05). Additionally, the triple-drug combination of metformin and chidamide with lenalidomide or bortezomib was used to stimulate the MM cells, and the results revealed that metformin and chidamide treatment sensitized MM cells to lenalidomide and bortezomib. As a result, the apoptosis (p < .001) together with cell cycle arrest at G0/G1 phase (p < .05) was stimulated by lenalidomide and bortezomib, and showed significant elevation in the triple-drug combination group compared with the lenalidomide or bortezomib treatment alone group (p < .05). Furthermore, the impacts of different drugs on glycolysis in MM cells were examined. We found that metformin and chidamide combined treatment significantly promoted glucose uptake and reduced energy production in MM cells treated with lenalidomide and bortezomib (p < .001), suggesting that metformin and chidamide affected glycolysis in MM cells and enhanced the sensitivity of lenalidomide and bortezomib in MM by regulating glucose metabolism. In conclusion, metformin and chidamide synergistically hindered MM cell growth and sensitized cells to lenalidomide/bortezomib. The findings of this study might provide novel clues to improve MM therapy.

MiR-383 sensitizes osteosarcoma cells to bortezomib treatment via down-regulating PSMB5.

Proteasome inhibition is a promising strategy for cancer therapy. Bortezomib, which primarily targets the chymotrypsin-like activity of PSMB5, has demonstrated efficacy in various tumors. However, there is variable sensitivity to bortezomib, which could be attributed, in part, to variations in the expression of proteasome subunits. In this study, we investigated whether miR-383 affects the expression of proteasome subunits in osteosarcoma (OS) cells, and if so, whether OS cells display differential sensitivity to bortezomib concerning miR-383 expression. We detected a decreased miR-383 expression in OS cells and tissues. Then we found a negative correlation between the cytotoxicity of bortezomib and the expression level of the proteasome 20S core particle subunit β5 (PSMB5). Intriguingly, we identified PSMB5 as a direct target of miR-383. Increased expression of miR-383 resulted in decreased PSMB5 expression and increased sensitivity to bortezomib in OS cells. In summary, our findings present the initial comprehensive analysis of the function of miR-383 in OS. The outcomes indicate that miR-383 may augment the anticancer effect of bortezomib through PSMB5 repression, offering a novel therapeutic approach in OS and a fresh pathway for proteasome regulation.

Inhibition of mitochondrial cyclophilin D, a downstream target of glycogen synthase kinase 3α, improves sperm motility.

Cyclophilin D (CypD) negatively regulates ATP production by opening of the mitochondrial permeability transition pore. This study aimed to understand the role of CypD in sperm motility regulation. Changes in CypD during sperm capacitation and its interaction with glycogen synthase kinase 3α (GSK3α), a key kinase regulating sperm motility, were examined in mouse spermatozoa. The effects of CypD inhibitor cyclosporin A (CsA) and GSK3 inhibitor 6-bromo-indirubin-3'-oxime (BIO) on sperm motility, p-GSK3α(Ser21), mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), and ATP production were examined. The effect of proteasome inhibitor MG115 on the cellular levels of CypD was examined. In cauda epididymal spermatozoa, GSK3α was found in both cytosolic and mitochondrial fractions whereas CypD was primarily found in the mitochondrial fraction together with ATP synthase F1 subunit alpha (ATP5A), a mitochondrial marker. GSK3α and CypD were co-localized in the sperm midpiece. Interaction between GSK3α and CypD was identified in co-immunoprecipitation. CsA, a CypD inhibitor, significantly increased sperm motility, tyrosine phosphorylation, mPTP closing, MMP, and ATP levels in spermatozoa, suggesting that CypD acts as a negative regulator of sperm function. Under capacitation condition, both GSK3α and CypD were decreased in spermatozoa but ATP5A was not. The GSK3 inhibitor BIO markedly increased p-GSK3α(Ser21) and decreased CypD but significantly increased mPTP closing, MMP, ATP production, and motility of spermatozoa. This suggests that inhibitory phosphorylation of GSK3α is coupled with degradation of CypD, potentiating the mitochondrial function. Degradation of CypD was attenuated by MG115, indicative of involvement of the ubiquitin proteasome system. During sperm capacitation, CypD act as a downstream target of GSK3α can be degraded via the ubiquitin proteasome system, stimulating mitochondrial function and sperm motility.

Acute accumulation of PIM2 and NRF2 and recovery of β5 subunit activity mitigate multiple myeloma cell susceptibility to proteasome inhibitors.

Resistance to proteasome inhibitors (PIs) has emerged as an important clinical issue. We investigated the mechanisms underlying multiple myeloma (MM) cell resistance to PIs. To mimic their pharmacokinetic/pharmacodynamic (PK/PD) profiles, MM cells were treated with bortezomib and carfilzomib for 1h at concentrations up to 400 and 1,000nM, respectively. Susceptibility to these PIs markedly varied among MM cell lines. Pulsatile treatments with PIs suppressed translation, as demonstrated by incorporation of puromycin at 24h in PI-susceptible MM.1S cells, but not PI-resistant KMS-11 cells. Inhibition of β5 subunit activity decreased at 24h in KMS-11 cells, even with the irreversible PI carfilzomib, but not under suppression of protein synthesis with cycloheximide. Furthermore, the proteasome-degradable pro-survival factors PIM2 and NRF2 acutely accumulated in MM cells subjected to pulsatile PI treatments. Accumulated NRF2 was trans-localized into the nucleus to induce the expression of its target gene, HMOX1, in MM cells. PIM and Akt inhibition restored the anti-MM effects of PIs, even against PI-resistant KMS-11 cells. Collectively, these results suggest that increased synthesis of β5 proteasome subunit and acute accumulation of PIM2 and NRF2 reduce the anti-MM effects of PIs.

Mouse transient receptor potential melastatin 2 (TRPM2) isoform 7 attenuates full-length mouse TRPM2 activity through reductions in its expression by targeting it to ER-associated degradation.

Transient receptor potential melastatin 2 (TRPM2) assembles into tetramers to function as an oxidative stress-sensitive Ca2+ channel at the surface membrane. Limited information is currently available on the 10 protein isoforms of mouse TRPM2 (mTRPM2) identified. This study investigated whether these isoforms function as Ca2+ channels and examined their effects on full-length mTRPM2 activity using the HEK 293 cell exogenous expression system. Only full-length mTRPM2, isoform 1 localized to the surface membrane and was activated by oxidative stress. Isoform 7 was clearly recognized by protein quality control systems and degraded by endoplasmic reticulum-associated degradation after transmembrane proteolysis. In the co-expression system, the activation and expression of full-length mTRPM2 were attenuated by its co-expression with isoform 7, but not with the other isoforms. This decrease in the expression of full-length mTRPM2 was recovered by the proteasomal inhibitor. The present results suggest that isoforms other than isoform 1 did not function as oxidative stress-sensitive channels and also that only isoform 7 attenuated the activation of full-length mTRPM2 by targeting it to endoplasmic reticulum-associated degradation. The present study will provide important information on the functional nature of mTRPM2 isoforms for the elucidation of their roles in physiological and patho-physiological responses in vivo using mouse models.

Characterizing differential efficacy and phenotypic response to proteasome and survivin inhibitors in colorectal cancers using a high throughput organoid assay.

153 Background: Conventional monolayer cell cultures and xenograft models, while useful and economical in early drug discovery, cannot predict clinical efficacy. Further, preclinical screening assays that rely on differential metabolic activity between separate control and treated wells are incapable of capturing phenotypic response and could overstate efficacy for cells with high rates of proliferation. Consequently, over 95% of anticancer agents that show efficacy in preclinical studies, fail in clinical trials. Recently, patient-derived organoid (PDO) models have been utilized in developing platforms to predict clinical efficacy of preclinical formulations. If successful, such predictive ex vivo technologies could revolutionize cancer treatment by reducing cost and time-to-market for new, more effective therapeutics. Objective: Characterize a novel bioprinted organoid tumor (BOT) high-throughput screening ex vivo platform for drug response prediction (DRP) with known proteosome and survivn inhibitors in colorectal cancer. Methods: Bioink for 3D printing BOTs was prepared with HT-29 cells, an established NCI-60 human colorectal adenocarcinoma cell line with known sensitivity to proteosome and survivin inhibitors. Bioink was deposited layer-by-layer on multiple substrates, in various geometrical configurations, and cured in stages to allow cells and matrix to self-assemble with limited degrees of freedom. BOTs were screened 24h and 48h after printing with proteosome inhibitor Bortezomib and survivin inhibitor YM-155. BOTs were evaluated 48h and 72h after treatment using immunofluorescence live/dead assay. Morphological phenotypic changes resulting from treatment were also recorded. Results: Proteasome and survivin inhibitors have been reported to inhibit proliferation and induce cell death in colorectal cancer cells. A dose dependent response was observed for both agents in our novel BOT HTS thereby validating the platform. In addition, characteristic self-assembly of HT-29 cells was observed to be disrupted at effective doses and at certain concentrations below the effective dose. Traditional ATP assays are incapable of recording such phenotypic modulation. Further, a higher proliferation profile was observed in untreated BOTs suggesting that use of independent control wells in traditional assays could overstate efficacy of treatment. Conclusions: Functional high-throughput ex vivo DRP technologies have the potential to transform cancer treatment – from bench to bedside – along the drug discovery to market roadmap for much needed novel anticancer agents.