Bortezomib Resistance Research Articles

TRNA methyltransferase DNMT2 promotes hepatocellular carcinoma progression and enhances Bortezomib resistance through inhibiting TNFSF10

The tRNA methyltransferase DNMT2 (TRDMT1) plays a crucial role in various biological functions; however, its role in cancer, particularly in liver cancer, remains incompletely understood. In this study, we demonstrate that high DNMT2 expression is negatively correlated with prognosis in clinical liver cancer patients. A series of in vitro and in vivo experiments showed that DNMT2 promotes the proliferation, colony formation, and metastasis of hepatocellular carcinoma cells. We identified the pro-apoptotic gene TNFSF10 (TRAIL) as a downstream target of DNMT2, regulated by the N6-methyladenosine (m6A) demethylase FTO. Epigenetically, DNMT2 deletion increased FTO expression, leading to a reduction in m6A methylation levels. FTO upregulated TNFSF10 expression, significantly reducing the proliferation and metastasis of DNMT2-deficient hepatocellular carcinoma cells. Furthermore, DNMT2 deletion was shown to significantly upregulate chemokine expression in tumors. Finally, we demonstrated that the NF-κB inhibitor Bortezomib further enhances DNMT2 deletion-induced apoptosis in hepatocellular carcinoma cells. This study reveals DNMT2's role in liver cancer and presents a new therapeutic target for future treatments.

Overexpression of Pin1 regulated by TOP2A, which subsequently stabilizes Pyk2 to promote bortezomib resistance in multiple myeloma.

Multiple myeloma (MM), a hematological malignancy of plasma cells, has remained largely incurable owing to drug resistance and disease relapse, which requires novel therapeutic targets and treatment approaches. Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) acts as an oncoprotein linked to the development of various tumors. However, the functional consequence of Pin1 overexpression in modulating MM biology has not been established. In the present study, we show that Pin1 expression is highly variable in myeloma cell lines and primary MMs and that high Pin1 expression is associated with poor survival of MM patients. Next, TOP2A is identified to be a Pin1 promoter-binding protein and CK2 activates TOP2A to promote the expression level of Pin1. Additionally, we demonstrate that Pin1 positively modulates the stability and function of Pyk2 to enhance bortezomib resistance in MM. Pin1 recognizes three phosphorylated Ser/Thr-Pro motifs in Pyk2 via its WW domain and increases the cellular levels of Pyk2 in an isomerase activity-dependent manner by inhibiting the ubiquitination and proteasomal degradation of Pyk2. Moreover, Pin1 inhibition combined with Pyk2 inhibition decreases myeloma burden both in vitro and in vivo. Altogether, our findings reveal the tumor-promoting role of Pin1 in MM and provide evidence that targeting Pin1 could be a therapeutic strategy for MM.

Acquired Bortezomib Resistance in Multiple Myeloma:From Mechanisms to Strategy.

Multiple myeloma (MM) is a heterogeneous plasma cell tumor with a survival period of several months to over ten years. Despite the development of various new drugs, MM is still incurable and recurs repeatedly. Bortezomib, a landmark event in the history of MM treatment, has dramatically improved the prognosis of patients with MM. Although proteasome inhibitors (PIs) represented by bortezomib, have greatly prolonged MM survival, unfortunately, almost all MM will develop bortezomib resistance, leading to relapse with a shorter survival. It has been reported that both the tumor microenvironment and myeloma cells drive bortezomib resistance. Multiple treatment methods have been attempted to overcome bortezomib resistance, but unfortunately, there has been no breakthrough. It is believed that the key resistance mechanism has not yet been discovered. A deeper understanding of the mechanism of bortezomib resistance and strategies to overcome it can help identify key resistance mechanisms and further improve the prognosis of MM.

Identification and Validation of a novel Circulating miRNA Predictive Biomarkers for acquired Bortezomib Resistance in Multiple Myeloma patients at Southeast Medical Center

Abstract Introduction/Objective Bortezomib is used to treat multiple myeloma but some patients become resistant to it. MiRNAs, small non-coding RNAs that regulate gene expression, may serve as biomarkers for drug resistance in cancer. Dysregulation of miRNA expression is common in cancer development. We investigated whether plasma miRNAs can serve as clinical indicators for bortezomib resistance in multiple myeloma. We hypothesize that there is a significant difference in miRNA signature between sensitive and resistant Bortezomib multiple myeloma samples derived from African American male patients. Methods/Case Report Specific Aim 1: Discovery: To identify miRNAs using five BTZ-resistant and five sensitive African American male bone marrow samples using RNA sequencing technology. Using log2fold change ≥ 1.3, p ≤ 0.05, and known function as screening criteria of statistically significant upregulated miRNAs. Specific Aim 2: Analytical validation: Quantitative reverse-transcription PCR (qRT-PCR) technology using bone African American Male Bone Marrow samples to confirm the differential expression of candidate miRNAs identified in Aim 1. Using log2fold change ≥ 1.3, p ≤ 0.05, and Two-tailed student T-Test known function as screening criteria of statistically significant upregulated miRNAs. Specific Aim 3: Clinical Validation: To be performed using patients’ serum and qRT-PCR technology. Using log2fold change ≥ 1.3, p ≤ 0.05, known function, and Two-tailed student T-Test as the screening criteria of statistically significant upregulated miRNAs. Results (if a Case Study enter NA) We identified upregulated and downregulated miRNAs from African American males with MM in 5-BTZ-resistant and 5-BTZ-sensitive bone marrow samples using RNA sequencing. Four statistically significant upregulated miRNAs were identified and validated using RT-qPCR and bioinformatic target prediction (has-miR-18a-3p, has-miR-18a-5p, has-miR-20a-5p, has-mir-664a-5p). Using log2fold change ≥ 1.3, p ≤ 0.05, and Two-tailed student T-Test, and known function as screening criteria, has-miR-18a-5p and has-miR-20a-5p were validated as statistically significant upregulated miRNAs among the BTZ-resistant bone marrow samples. These miRNAs will now undergo clinical validation. Conclusion According to the qRT-PCR analysis, significant differential expression was observed between the Bortezomib-sensitive and Bortezomib-resistant groups was has-miR-18a-5p (p ≤ 0.02) and has-miR-20a-5p (p ≤ 0.04) as statistically significant differential expressed miRNAs and are associated with promoting carcinogenesis.

Correlation between Bone Marrow Microvascular Density, Angiogenesis Factors and Bortezomib Resistance in Multiple Myeloma

To investigate the correlation between bone marrow microvascular density, angiogenesis factors and bortezomib resistance in multiple myeloma (MM). The data of 200 patients with MM treated in our hospital from January 2020 to August 2023 were retrospectively analyzed, and the patients with MM were divided into drug-resistant group(n=68) and non-drug-resistant group(n=132) according to their drug resistance during bortezomib treatment. The univariate and multivariate logistic analysis were used to screen the independent influencing factors of bortezomib resistance in MM patients during treatment. The receiver operating characteristic (ROC) curve and clinical decision curve (DCA) were used to evaluate the predictive performance and clinical application value of the risk prediction model, the consistency between the actual incidence rate and the predicted incidence rate was judged by validating the calibration chart, and the goodness-of-fit of the model was judged by H-L test. 68 of the 200 MM patients developed resistance and poor clinical efficacy during bortezomib treatment, and the clinical resistance rate of bortezomib was 34.0%. The results of multivariate analysis showed that high bone marrow microvessel density (MVD) and high bone marrow supernatant VEGF, HGF, and bFGF expression levels were independent risk factors for bortezomib resistance in MM patients (P < 0.05). The area under the ROC curve (AUC) of the model jointly constructed by bone marrow MVD, serum VEGF, HGF, bFGF and TNF-α levels was 0.924, and its sensitivity and specificity were 92.6% and 78.8%, which were higher than those of the bone marrow MVD model (AUC=0.743) and the vasogenesis factor model (AUC=0.878). The calibration curve of the joint prediction model was close to the standard curve, indicating that the model is more consistent. The results of H-L goodness -of - fit test showed χ2=14.748， P =0.164, the joint prediction model had a good fit. The DCA curve showed that the clinical net benefit of intervention in the range of 0.0~1.0 was greater than that of full intervention and no intervention. The prediction model based on bone marrow MVD and vasogenesis factors (VEGF, HGF, bFGF) in MM patients has higher clinical evaluation performance and predictive value.

KS18, a Mcl-1 inhibitor, improves the effectiveness of bortezomib and overcomes resistance in refractory multiple myeloma by triggering intrinsic apoptosis.

Despite a record number of clinical studies investigating various anti-myeloma treatments, the 5-year survival rate for multiple myeloma (MM) patients in the US is only 55%, and almost all patients relapse. Poor patient outcomes demonstrate that myeloma cells are "born to survive" which means they can adapt and evolve following treatment. Thus, new therapeutic approaches to combat survival mechanisms and target treatment resistance are required. Importantly, Mcl-1, anti-apoptotic protein, is required for the development of MM and treatment resistance. This study looks at the possibility of KS18, a selective Mcl-1 inhibitor, to treat MM and overcome resistance. Our investigation demonstrates that KS18 effectively induces cell death in MM by dual regulatory mechanisms targeting the Mcl-1 protein at both transcriptional and post-translational levels. Specifically, KS18 suppresses Mcl-1 activation via STAT-3 pathway and promotes Mcl-1 phosphorylation/ubiquitination/proteasome-dependent protein degradation (UPS). Significantly, KS18 triggered caspase-dependent apoptosis in MM patient samples and bortezomib-resistant cells, synergizing with venetoclax to boost apoptosis. KS18 promises to overcome bortezomib and venetoclax resistance and re-sensitize myeloma cells to chemotherapy. Furthermore, the study shows the tremendous impact of KS18 in inhibiting colony formation in bortezomib-resistant cells and demonstrates significant tumor shrinkage in KS18-treated NSG mice without notable toxicity signs after 4weeks of therapy with a single acceptable dose each week, indicating its powerful anti-neoplastic and anti-resistance characteristics. This study strongly implies that KS18 may treat MM and provide new hope to patients who are experiencing recurrence or resistance.

Discovery of novel 20S proteasome subunit β5 PROTAC degraders as potential therapeutics for pharyngeal carcinoma and Bortezomib-resistant multiple myeloma

Resistance to proteasome inhibitors like Bortezomib is a major challenge in the treatment of multiple myeloma (MM). Proteolysis targeting chimeras (PROTACs), an emerging therapeutic approach that induces selective degradation of target proteins, offer a promising solution to overcome drug resistance. In this study, we designed and synthesized novel small-molecule PROTACs that induce 20S proteasome subunit β5 degradation as a strategy to overcome Bortezomib resistance. These 20S proteasome subunit β5 PROTACs demonstrated considerable binding affinity to 20S proteasome subunit β5 and cereblon (CRBN), effectively induced 20S proteasome subunit β5 degradation, and exhibited potent antiproliferative activity against a panel of cancer cell lines. Notably, PROTACs 12f and 14 displayed robust antitumor effects against both the pharyngeal carcinoma cell line FaDu and the Bortezomib-resistant MM cell line KM3/BTZ in vitro and in vivo with excellent safety profiles. Taken together, our findings highlight the potential of PROTACs 12f and 14 as novel 20S proteasome subunit β5-degrading agents for the treatment of pharyngeal carcinoma and overcoming Bortezomib resistance in MM.

Multi-omics analysis identifies repurposing bortezomib in the treatment of kidney-, nervous system-, and hematological cancers

Repurposing of FDA-approved drugs is a quick and cost-effective alternative to de novo drug development. Here, we identify genes involved in bortezomib sensitivity, predict cancer types that may benefit from treatment with bortezomib, and evaluate the mechanism-of-action of bortezomib in breast cancer (BT-474 and ZR-75–30), melanoma (A-375), and glioblastoma (A-172) cells in vitro. Cancer cell lines derived from cancers of the blood, kidney, nervous system, and skin were found to be significantly more sensitive to bortezomib than other organ systems. The in vitro studies confirmed that although bortezomib effectively inhibited the β5 catalytic site in all four cell lines, cell cycle arrest was only induced in G2/M phase and apoptosis in A-375 and A-172 after 24h. The genomic and transcriptomic analyses identified 33 genes (e.g. ALDH18A1, ATAD2) associated with bortezomib resistance. Taken together, we identified biomarkers predictive of bortezomib sensitivity and cancer types that might benefit from treatment with bortezomib.

Ginsenoside Rg1 inhibits multiple myeloma and overcomes bortezomib resistance through AMPK-mTOR pathway

BackgroundThe resistance of multiple myeloma (MM) to bortezomib (BTZ) has brought multiple challenges to its clinical use. Numerous ginsenosides have potential anti-tumor effects, however, the research on the role of Rg1 in MM has not been reported. ObjectiveTo examine the inhibitory impact of Rg1 on the growth of MM and reduce the drug resistance of MM to BTZ through in vivo and in vitro experiments, and to explore their potential mechanism. MethodsBTZ drug-resistant cell line RPMI8226R was constructed. Mouse tumor-bearing model was developed by abdominal subcutaneous injection of MM cells. MM cells were treated with AMPK inhibitor Compound C or autophagy inhibitor Chloroquine together with Rg1. RPMI8226R cells were treated with BTZ and Rg1. Cell multiplication was detected using Methylthiazolyldiphenyl-tetrazolium bromide assay. Apoptosis was assessed using flow cytometry. Immunofluorescence assay was employed to assess the autophagy markers LC3. Western blot was utilized to assess the protein expression. Immunohistochemistry was used to detect cell proliferation and apoptosis in tumor tissues. ResultsIn vitro experiments demonstrated that Rg1 could hinder the proliferation of MM cells, promote apoptosis and enhance autophagy. Rg1 could also increase the sensitivity of RPMI8226R to BTZ. In vivo experiments illustrated that Rg1 could hinder the development of MM cells in mice, weaken the proliferation of tumor cells and enhance their apoptosis. Further study found that the anti-MM impact of Rg1 was linked to AMPK-mTOR pathway, the autophagy degree of RPMI8226R was higher than that of RPMI8226, and that Rg1 could inhibit MM and overcome drug resistance through autophagy induced by AMPK-mTOR pathway. ConclusionRg1 has significant anti-MM effect and can overcome BTZ resistance, and its potential mechanism is related to the regulation of autophagy induced by AMPK-mTOR pathway. Rg1 is a promising adjuvant drug for the treatment of MM.

NCX1/Ca2+ promotes autophagy and decreases bortezomib activity in multiple myeloma through non-canonical NFκB signaling pathway

Although bortezomib (BTZ) is the cornerstone of anti-multiple myeloma (MM) therapy, the inevitable primary and secondary drug resistance still seriously affects the prognosis of patients. New treatment strategies are in need. Sodium-calcium exchanger 1 (NCX1) is a calcium-permeable ion transporter on the membrane, and our previous studies showed that low NCX1 confers inferior viability in MM cells and suppressed osteoclast differentiation. However, the effect of NCX1 on BTZ sensitivity of MM and its possible mechanism remain unclear. In this study, we investigated the effect of NCX1 on BTZ sensitivity in MM, focusing on cellular processes of autophagy and cell viability. Our results provide evidence that NCX1 expression correlates with MM disease progression and low NCX1 expression increases BTZ sensitivity. NCX1/Ca2+ triggered autophagic flux through non-canonical NFκB pathway in MM cells, leading to attenuated the sensitivity of BTZ. Knockdown or inhibition of NCX1 could potentiate the anti-MM activity of BTZ in vitro and vivo, and inhibition of autophagy sensitized NCX1-overexpressing MM cells to BTZ. In general, this work implicates NCX1 as a potential therapeutic target in MM with BTZ resistance and provides novel mechanistic insights into its vital role in combating BTZ resistance.

Interleukin-33 increases the sensitivity of multiple myeloma cells to the proteasome inhibitor bortezomib through reactive oxygen species-mediated inhibition of nuclear factor kappa-B signal and stemness properties.

The proteasome inhibitor bortezomib (BTZ) is the first-line therapy for multiple myeloma (MM). BTZ resistance largely limits its clinical application in MM. Interleukin-33 (IL-33) exerts antitumor effects through various mechanisms, including enhancing antitumor immunity and promoting the apoptosis of cancer cells. Here, the synergistic anti-MM effect of IL-33 and BTZ was verified, and the underlying mechanisms were elucidated. Bioinformatic analysis indicated that IL-33 expression levels were downregulated in MM, and that BTZ-treated MM patients with high IL-33 levels had better prognosis than those with low IL-33 levels. Moreover, the patients with high IL-33 levels had a better treatment response to BTZ. Further immune analysis suggested that IL-33 can enhance the anti-MM immunity. IL-33 and BTZ synergistically inhibited proliferation and induced apoptosis of MM cells, which was mediated by the excessive accumulation of cellular reactive oxygen species (ROS). Furthermore, increased ROS hindered the nuclear translocation of NF-κB-p65, thereby decreasing the transcription of target stemness-related genes (SOX2, MYC, and OCT3/4). These effects induced by the combination therapy could be reversed by eliminating ROS by N-acetylcysteine. In conclusion, our results indicated that IL-33 enhanced the sensitivity of MM to BTZ through ROS-mediated inhibition of nuclear factor kappa-B (NF-κB) signal and stemness properties.

Identification of KDM4C as a gene conferring drug resistance in multiple myeloma.

Bortezomib (BTZ), a proteasome inhibitor, is a promising therapeutic option for multiple myeloma (MM) patients. However, drug resistance often occurs, leading to disease relapse and poor prognosis. In this study, we aimed to identify novel genes associated with drug resistance and investigate their roles in BTZ resistance. Through the screening of 26 genes frequently associated with chemosensitivity or drug resistance, we discovered that KDM4C, a histone demethylase, exhibited increased expression in BTZ-resistant MM cells compared to their sensitive counterparts. Overexpression of KDM4C enhanced the tolerance of a MM cell line to the drug, whereas the knockdown of KDM4C, using shRNA, increased the sensitivity of resistant cells to BTZ treatment. This suggests that KDM4C plays a pivotal role in conferring BTZ resistance. Our study offers fresh insights into BTZ resistance in MM and highlights KDM4C as a potential target for overcoming drug resistance.

MiR-34c-5p Inhibition Affects Bax/Bcl2 Expression and Reverses Bortezomib Resistance in Multiple Myeloma Cells.

Developing resistance to anticancer drugs complicates the clinical treatment of multiple myeloma patients. Previous studies revealed a link between the unfolded protein response (UPR) and miRNAs with acquired drug resistance. This study aimed to determine the expression profile of XBP1, hsa-miR-34c-5p, hsa-miR-214, and hsa-miR-30c-2* in resistant and sensitive multiple myeloma cell lines to a proteasome inhibitor, bortezomib. After establishing bortezomib-resistant cells, the expression level of XBP1, hsa-miR-214, hsa-miR-34c-5p, and hsa-miR-30c-2* in both cell lines were assessed by qRT-PCR. Hsa-miR-34c-5p was suppressed to study its effect on the expression profile of Bax/Bcl-2. Statistical analysis was done by t-test in two clinically resistant and sensitive cells to bortezomib. MTT assay confirmed the creation of the resistant cell line. The qRT-PCR screening showed a significant difference between XBP1 and miR-34c-5p levels in resistant and sensitive cells. Following hsa-miR-34c-5p blockage, while Bax was overexpressed, Bcl-2 expression was reduced in the resistant cell line, overcoming cells resistant to bortezomib. Our findings demonstrate miR-34c-5p is differentially expressed between bortezomib-sensitive and -resistant MM cells. Inhibiting miR-34c-5p re-sensitized resistant cells to bortezomib by modulating Bax/Bcl-2 expression, suggesting this miRNA regulates apoptosis and drug resistance and may be a promising therapeutic target for overcoming proteasome inhibitor resistance in MM.

#38. Cholesterol increases bone marrow tumour burden in myeloma in vivo and promotes myeloma cell viability and bortezomib resistance ex vivo

#38. Cholesterol increases bone marrow tumour burden in myeloma in vivo and promotes myeloma cell viability and bortezomib resistance ex vivo

N6-methyladenosine-modified circ_0000337 sustains bortezomib resistance in multiple myeloma by regulating DNA repair.

Studies have shown that bortezomib resistance in multiple myeloma (MM) is mediated by the abnormalities of various molecules and microenvironments. Exploring these resistance mechanisms will improve the therapeutic efficacy of bortezomib. In this study, bone marrow tissues from three patients with MM, both sensitive and resistant to bortezomib, were collected for circRNA high-throughput sequencing analysis. The relationship between circ_0000337, miR-98-5p, and target gene DNA2 was analyzed by luciferase detection and verified by RT-qPCR. We first found that circ_0000337 was significantly upregulated in bortezomib-resistant MM tissues and cells, and overexpression of circ_0000337 could promote bortezomib resistance in MM cells. circ_0000337 may act as a miR-98-5p sponge to upregulate DNA2 expression, regulate DNA damage repair, and induce bortezomib resistance. Furthermore, it was determined that the increased circ_0000337 level in bortezomib-resistant cells was due to an increased N6-methyladenosine (m6A) level, resulting in enhanced RNA stability. In conclusion, the m6A level of circ_0000337 and its regulation may be a new and potential therapeutic target for overcoming bortezomib resistance in MM.

Abstract 4670: KS18, an Mcl-1 inhibitor, triggers cell death and enhances the therapeutic efficacy of venetoclax in bortezomib-resistant multiple myeloma cells

Abstract A critical clinical challenge in the management of multiple myeloma (MM) is the development of both innate and acquired resistance to the treatment with bortezomib. Our prior research showed that the overexpression of the anti-apoptotic Bcl-2 family protein Mcl-1 can result in resistance to the chemotherapy drugs bortezomib and venetoclax. In order to overcome the issues posed by resistance, this study investigates the possibility of a small chemical called KS18, a novel Mcl-1 inhibitor. KS18 outperforms other Mcl-1 inhibitors such as S63845, VU661013, and AZD5991, according to the findings of our research, which shows that it is extraordinarily effective against MM cells that have become resistant to bortezomib. We establish the selectivity of KS18 through tests employing Mcl-1 knock-down (KD), and we find that it is both effective and efficient. In addition, we used immunoprecipitation (IP) and protein-protein interaction analysis (PPIs) to investigate the dynamic connections that exist between Mcl-1 and pro-apoptotic proteins. The KS18 therapy exhibits an outstanding ability to cause complete apoptosis, as seen by the remarkable 88.8% apoptosis rate recorded in MM patient samples. Not only does KS18 restore apoptosis in bortezomib-resistant MM cells, but it also synergizes with venetoclax to enhance the apoptotic response. This is an important finding. It has been demonstrated through both in vitro and in vivo research that KS18 can re-sensitize MM cells to the effects of bortezomib, which represents a potential step forward in the treatment of the disease. In bortezomib-resistant MM cells, treatment with KS18 led to a remarkable 92% reduction in colony formation compared to untreated controls. In vivo experiments that were performed on mice models that were resistant to bortezomib provide additional evidence that KS18 is effective. The extraordinary reduction in tumor weight of 55% that occurred as a direct result of treatment with KS18 at a dose of 1 mg/kg for a period of 15 days exemplifies the powerful anti-resistance characteristics of this compound. These intriguing findings highlight the potential value of KS18 as an adjunct to therapies aimed at overcoming bortezomib resistance in MM and other associated cancers. Bortezomib-resistant malignancies provide a challenge for patients, but additional research into the clinical value of KS18 holds the potential to improve treatment outcomes and broaden the range of therapeutic alternatives available to these patients. Citation Format: Omar S. Al Odat, Osama Aloudat, Emily Nelson, Tulin Budak-Alpogan, Subash Jonnalagadda, Yong Chen, Manoj Pandey. KS18, an Mcl-1 inhibitor, triggers cell death and enhances the therapeutic efficacy of venetoclax in bortezomib-resistant multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4670.

Abstract 1233: Novel inhibitor targeting triple integrated stress response kinase HRI, PERK, and GCN2 provides new insights into overcoming resistance to proteasome inhibitors in multiple myeloma

Abstract The introduction of novel treatments for Multiple myeloma (MM), particularly proteasome inhibitors (PI) such as bortezomib (BTZ), has greatly improved outcomes. However, many patients eventually experience relapse. Therefore, new approaches for managing patients with resistance to PI should be explored. The integrated stress response (ISR) is an evolutionarily conserved intracellular signaling network that is activated in response to both intrinsic and extrinsic stresses. Stresses are sensed by four specialized kinases: HRI, PERK, PKR, and GCN2, which converge on the phosphorylation of eIF2α. This phosphorylation triggers the translation of ATF4, thus promoting cell survival and homeostasis. Recent studies investigating the contribution of the four ISR kinases in overcoming resistance to PI treatment, have revealed the potential of a selective GCN2 inhibitor to overcome this resistance. Here, we observed that PI simultaneously induced mitochondrial stress, ER stress, and amino acid starvation, leading to the activation of HRI, PERK, and GCN2, respectively. However, the contribution of crosstalk among ISR kinases has not been considered thus far. To gain a comprehensive understanding of ISR kinases in BTZ resistance, we generated single or combination patterns of double and triple KO of the four ISR kinases and reevaluated their contribution to BTZ treatment. Sensitivity screening using the HAP1 single, double, and triple KO cell library showed that HRI primarily, and to a lesser extent, PERK and GCN2, contribute to BTZ resistance. Consistent with the HAP1 KO screening, MM cell line-derived KO cells revealed a similar dependency on these three kinases. Building upon our observation of the significance of the HRI/PERK/GCN2 triple kinase, we developed a novel and orally available small molecule, CRD-1367799, which inhibits these triple kinases in cells. CRD-1367799 effectively dampened the activity of these ISR kinases, leading to a complete inhibition of eIF2α phosphorylation and ATF4 activation. Additionally, we observed a distinct anti-growth effect when combined with BTZ in vitro and in vivo, which was not replicated by the GCN2 or PERK-selective inhibitor. Furthermore, we uncovered the mechanism of cell death induction in combination with proteasome inhibitors. Notably, the apoptosis inducers NOXA and CHOP were upregulated, but the mechanism did not seem to rely on previously known processes such as ATF4-mediated transcriptional induction. In summary, our novel findings on the contribution of the triple ISR kinase provides us with new insights for overcoming resistance to proteasome inhibitors with HRI/PERK/GCN2 inhibitor. To the best of our knowledge, the use of a triple kinase inhibitor in this context has not been reported thus far, making CRD-1367799 a new therapeutic strategy for MM with PI. Citation Format: Yasumichi Inoue, Koki Hikami, Shunsuke Ebara, Midori Sugiyama, Robert Oda, Akio Mizutani, Misaki Yoshida, Hiroko Yamakawa, Shigehiro Yagishita, Akinobu Hamada, Hidetoshi Hayashi, Masaki Ri, Shinsuke Iida, Daisuke Morishita. Novel inhibitor targeting triple integrated stress response kinase HRI, PERK, and GCN2 provides new insights into overcoming resistance to proteasome inhibitors in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1233.

Autophagy and oxidative stress modulation mediate Bortezomib resistance in prostate cancer.

Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom's macroglobulinemia, and mantle cell lymphoma, based on the cancer cell's susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders.

Epigenetic Alterations as Vital Aspects of Bortezomib Molecular Action.

Bortezomib (BTZ) is widely implemented in the treatment of multiple myeloma (MM). Its main mechanism of action is very well established. BTZ selectively and reversibly inhibits the 26S proteasome. More precisely, it interacts with the chymotryptic site of the 20S proteasome and therefore inhibits the degradation of proteins. This results in the intracellular accumulation of misfolded or otherwise defective proteins leading to growth inhibition and apoptosis. As well as interfering with the ubiquitin-proteasome complex, BTZ elicits various epigenetic alterations which contribute to its cytotoxic effects as well as to the development of BTZ resistance. In this review, we summarized the epigenetic alterations elicited by BTZ. We focused on modifications contributing to the mechanism of action, those mediating drug-resistance development, and epigenetic changes promoting the occurrence of peripheral neuropathy. In addition, there are therapeutic strategies which are specifically designed to target epigenetic changes. Herein, we also reviewed epigenetic agents which might enhance BTZ-related cytotoxicity or restore the sensitivity to BTZ of resistant clones. Finally, we highlighted putative future perspectives regarding the role of targeting epigenetic changes in patients exposed to BTZ.

Epigenetic dysregulation of eukaryotic initiation factor 3 subunit E (eIF3E) by lysine methyltransferase REIIBP confers a pro-inflammatory phenotype in t(4;14) myeloma.

REIIBP is a lysine methyltransferase aberrantly expressed through alternative promoter usage of NSD2 locus in t(4;14)-translocated multiple myeloma (MM). Clinically, t(4;14) translocation is an adverse prognostic factor found in approximately 15% of MM patients. The contribution of REIIBP relative to other NSD2 isoforms as a dependency gene in t(4;14)-translocated MM remains to be evaluated. Here, we demonstrated that despite homology with NSD2, REIIBP displayed distinct substrate specificity by preferentially catalyzing H3K4me3 and H3K27me3, with little activity on H3K36me2. Furthermore, REIIBP was regulated through microRNA by EZH2 in a Dicer-dependent manner, exemplifying a role of REIIBP in SET-mediated H3K27me3. Chromatin immunoprecipitation sequencing revealed chromatin remodeling characterized by changes in genome-wide and loci-specific occupancy of these opposing histone marks, allowing a bidirectional regulation of its target genes. Transcriptomics indicated that REIIBP induced a pro-inflammatory gene signature through upregulation of TLR7, which in turn led to B-cell receptor-independent activation of BTK and driving NFkB-mediated production of cytokines such as IL-6. Activation of this pathway is targetable using Ibrutinib and partially mitigated bortezomib resistance in a REIIBP xenograft model. Mechanistically, REIIBP upregulated TLR7 through eIF3E, and this relied on eIF3E RNA-binding function instead of its canonical protein synthesis activity, as demonstrated by direct binding to the 3'UTR of TLR7 mRNA. Altogether, we provided a rationale that co-existence of different NSD2 isoforms induced diversified oncogenic programs that should be considered in the strategies for t(4;14)-targeted therapy.