Proteasome Inhibitors In Multiple Myeloma Research Articles

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.

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.

Disrupting Autophagy Sensitizes Human Acute Myeloid Leukemia Cells to Proteasome Inhibition By Disrupting Protein Homeostasis

Proteasome inhibitors are clinically approved and highly effective for treating multiple myeloma in the first line setting, but exhibit little effectiveness for treatment of other hematological malignancies. In this study, we set out to determine why acute myeloid leukemia (AML) is largely refractory to proteasome inhibition and to determine if we could sensitize human AML to this class of therapeutics. Efficacy of proteasome inhibitors in multiple myeloma partly depends on disruption of protein homeostasis (proteostasis). This is driven by an accumulation of misfolded proteins that overwhelm the endoplasmic reticulum and activate the unfolded protein response (UPR) - part of the integrated stress response (ISR) pathway. Due to their highly secretory nature, myeloma cells exhibit relatively elevated baseline endoplasmic reticulum stress, and are thus preferentially sensitive to proteasome inhibition as they readily activate a terminal ISR. We hypothesized that AML cells are better able to cope with proteasome inhibition by activating alternative stress response pathways that rebalance proteostasis. We determined that proteasome inhibition fails to significantly disrupt proteostasis in human AML cells. Instead, RNA-sequencing studies suggested that AML cells respond to proteasome inhibition by activating at least two distinct stress response pathways, including autophagy - a key pathway utilized by normal hematopoietic stem cells to preserve proteostasis. Using a GFP-LC3-RFP fluorescent reporter system, we found that proteasome inhibition significantly increased autophagic flux in multiple human AML cell lines in a dose-dependent manner. Dual, concurrent pharmacological proteasome and autophagy inhibition was required to disrupt proteostasis across multiple human AML cell lines, as it resulted in a profound accumulation of unfolded protein. Proteostasis disruption caused by concurrent proteasome and autophagy inhibiton resulted in activation of a terminal ISR response in AML cells, marked by activation of eIF2a signaling and its downstream effectors ATF4 and CHOP. Consistent with this activation, dual proteasome and autophagy inhibition dramatically attenuated protein synthesis and synergistically reduced viability in multiple human AML cell lines (Figure 1A, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001). In addition, dual proteasome and autophagy inhibition reduced proliferation and induced massive apoptosis in these human AML cell lines and in primary AML patient samples. Surprisingly, this activity was not driven by the canonical PERK ( EIF2AK3)-mediated endoplasmic reticulum UPR, but was instead driven by an alternative eIF2a kinase PKR ( EIF2AK2). Treatment with a small molecule inhibitor to PERK had no significant effect on apoptosis while an inhibitor to PKR partially rescued the increased apoptosis and decreased protein synthesis induced by dual proteasome and autophagy inhibition. This suggests that the proteostasis network is uniquely remodeled in AML. Furthermore, in contrast to AML cells, dual proteasome and autophagy inhibition was well tolerated by healthy human CD34+ hematopoietic stem and progenitor cells, suggesting a tractable therapeutic window for treating AML. Indeed, dual proteasome and autophagy inhibition was tolerated in vivo, and decreased leukemia burden in mice xenografted with human AML cell lines (Figure 1B). Overall, this study revealed that AML cells rewire the proteostasis network to promote growth, survival, and therapeutic resistance. Targeting the unique configuration of the proteostasis network is uncovering new therapeutic strategies to eliminate AML.

Tumor Suppressive Mir-7 Targeting PSME3 Improves the Efficacy of Histone Deacetylases Inhibitors in Multiple Myeloma

Background: Histone deacetylases (HDACs) play a tumor-promoting role in multiple myeloma (MM), contributing to disease progression and treatment resistance. Panobinostat, an HDAC inhibitor, is clinicallyrelapsed or refractory MM. However, HDAC inhibitors have several limitations, such as drug resistance, poor tolerance, and limited use of combination drugs. Our previous comprehensive analysis of microRNAs modulated by HDAC inhibitors revealed new molecular pathogenesis and potential therapeutic targets in malignant lymphomas. However, the relationship between HDAC inhibitors and microRNAs in MM remains largely unknown. Aim and methods: We treated the MM cell lines (MM.1S and KMS-11) with HDAC inhibitors (10 nM panobinostat and 1 µM vorinostat), collected RNA 48 h later, and conducted a comprehensive microarray analysisFurthermore, we explored the novel clinical impact of HDAC inhibitors via functional analysis of microRNAs. Results: Microarray analysis showed that no microRNAs were commonly upregulated; however, only hsa-miR-7-5p (miR-7) was commonly downregulated in MM.1S and KMS-11 cells treated with both HDAC inhibitors. After exposing MM cell lines (MM.1S, KMS-11, RPMI-8226, H929, and U266) and patient samples to 10 nM panobinostat and 1 µM vorinostat for 48 h, we performed quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and observed significant downregulation in the expression of miR-7. Next, we examined the function of miR-7 in MM by introducing miR-7 into MM cell lines. Introducing miR-7 significantly suppressed the proliferation of MM.1S and KMS-11 cells. We hypothesized that the introduction of miR-7 enhances the effects of HDAC inhibitors. We introduced miR-7 into MM.1S and cultured them for 48 h with panobinostat, followed by an apoptosis assay. The introduction of miR-7 significantly enhanced the apoptosis-inducing effect of panobinostat, suggesting a tumor-suppressive role of miR-7 in MM. Furthermore, we examined the regulatory mechanisms of miR-7 in MM cells. Because it has been reported that MYC promotes miR-7 transcription, we focused on the role of MYC in miR-7 expression in MM. After exposing MM.1S and KMS-11 cells to HDAC inhibitors for 24 h, western blot analysis revealed that MYC was downregulated. After exposing MM.1S and KMS-11 cells to the BRD4 inhibitor JQ1, which suppresses MYC, at concentrations of 50 and 100 nM for 48 h, qRT-PCR showed that the expression of miR-7 was significantly downregulated in these cell lines. Similarly, introducing siRNA for MYC into MM.1S cells downregulated the expression of miR-7. These results suggest that HDAC inhibitors downregulate miR-7 expression by suppressing MYC expression. In addition, we examined the effect of bortezomib, a proteasome inhibitor, in combination with MM on the expression of miR-7. After exposing MM.1S, KMS-11, and H929 cells to 5 nM and 10 nM bortezomib for 48 h, we performed qRT-PCR and observed a significant upregulation in the expression of miR-7. Therefore, anti-myeloma effects of panobinostat by restoring miR-7 expression. Finally, we investigated miR-7 targets that mediate the anti-myeloma effects. We introduced miR-7 into MM.1S cells via electroporation, collected RNA 48 h later, and performed microarray analysis. As a result, 26 genes were identified, and their expression was downregulated by less than one-third. Among these, four genes ( SLC6A9, LRRC59, EXOSC2, and PSME3) possess conserved binding sites for miR-7. PSME3 (proteasome activator subunit 3：PA28ɤ) has been reported as an oncogene in MM, and we focused on PSME3. Introducing miR-7 into MM.1S cells via electroporation significantly suppressed PSME3 expression. Furthermore, when MM.1S cells were exposed to HDAC inhibitors, PSME3 expression was significantly upregulated. It has been reported that the inhibition of PSME3 increases sensitivity to proteasome inhibitors in MM. The favorable combination effect of bortezomib with HDAC inhibitors could be due to the modulation of the miR-7- PSME3 axis. Conclusion: We revealed that miR-7 exerts anti-myeloma effects and that PSME3 is one of the targets of miR-7. miR-7 is an interesting microRNA because it is positively regulated by MYC, even though it is tumor-suppressive. Elucidating the role of miR-7 may lead to the re-discovery of HDAC inhibitors in MM therapeutic strategies.

The mitochondrial pyruvate carrier complex potentiates the efficacy of proteasome inhibitors in multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy that emerges from antibody-producing plasma B cells. Proteasome inhibitors, including the US Food and Drug Administration-approved bortezomib (BTZ) and carfilzomib (CFZ), are frequently used for the treatment of patients with MM. Nevertheless, a significant proportion of patients with MM are refractory or develop resistance to this class of inhibitors, which represents a significant challenge in the clinic. Thus, identifying factors that determine the potency of proteasome inhibitors in MM is of paramount importance to bolster their efficacy in the clinic. Using genome-wide CRISPR-based screening, we identified a subunit of the mitochondrial pyruvate carrier (MPC) complex, MPC1, as a common modulator of BTZ response in 2 distinct human MM cell lines invitro. We noticed that CRISPR-mediated deletion or pharmacological inhibition of the MPC complex enhanced BTZ/CFZ-induced MM cell death with minimal impact on cell cycle progression. In fact, targeting the MPC complex compromised the bioenergetic capacity of MM cells, which is accompanied by reduced proteasomal activity, thereby exacerbating BTZ-induced cytotoxicity invitro. Importantly, we observed that the RNA expression levels of several regulators of pyruvate metabolism were altered in advanced stages of MM for which they correlated with poor patient prognosis. Collectively, this study highlights the importance of the MPC complex for the survival of MM cells and their responses to proteasome inhibitors. These findings establish mitochondrial pyruvate metabolism as a potential target for the treatment of MM and an unappreciated strategy to increase the efficacy of proteasome inhibitors in the clinic.

Proteasome Inhibitors Silence Oncogenes in Multiple Myeloma through Localized Histone Deacetylase 3 (HDAC3) Stabilization and Chromatin Condensation.

Proteasome inhibitors have become the standard of care for multiple myeloma (MM). Blocking protein degradation particularly perturbs the homeostasis of short-lived polypeptides such as transcription factors and epigenetic regulators. To determine how proteasome inhibitors directly impact gene regulation, we performed an integrative genomics study in MM cells. We discovered that proteasome inhibitors reduce the turnover of DNA-associated proteins and repress genes necessary for proliferation through epigenetic silencing. Specifically, proteasome inhibition results in the localized accumulation of histone deacetylase 3 (HDAC3) at defined genomic sites, which reduces H3K27 acetylation and increases chromatin condensation. The loss of active chromatin at super-enhancers critical for MM, including the super-enhancer controlling the proto-oncogene c-MYC, reduces metabolic activity and cancer cell growth. Epigenetic silencing is attenuated by HDAC3 depletion, suggesting a tumor-suppressive element of this deacetylase in the context of proteasome inhibition. In the absence of treatment, HDAC3 is continuously removed from DNA by the ubiquitin ligase SIAH2. Overexpression of SIAH2 increases H3K27 acetylation at c-MYC-controlled genes, increases metabolic output, and accelerates cancer cell proliferation. Our studies indicate a novel therapeutic function of proteasome inhibitors in MM by reshaping the epigenetic landscape in an HDAC3-dependent manner. As a result, blocking the proteasome effectively antagonizes c-MYC and the genes controlled by this proto-oncogene.

An mTORC1 to HRI signaling axis promotes cytotoxicity of proteasome inhibitors in multiple myeloma

Multiple myeloma (MM) causes approximately 20% of deaths from blood cancers. Notwithstanding significant therapeutic progress, such as with proteasome inhibitors (PIs), MM remains incurable due to the development of resistance. mTORC1 is a key metabolic regulator, which frequently becomes dysregulated in cancer. While mTORC1 inhibitors reduce MM viability and synergize with other therapies in vitro, clinically, mTORC1 inhibitors are not effective for MM. Here we show that the inactivation of mTORC1 is an intrinsic response of MM to PI treatment. Genetically enforced hyperactivation of mTORC1 in MM was sufficient to compromise tumorigenicity in mice. In vitro, mTORC1-hyperactivated MM cells gained sensitivity to PIs and hypoxia. This was accompanied by increased mitochondrial stress and activation of the eIF2α kinase HRI, which initiates the integrated stress response. Deletion of HRI elevated the toxicity of PIs in wt and mTORC1-activated MM. Finally, we identified the drug PMA as a robust inducer of mTORC1 activity, which synergized with PIs in inducing MM cell death. These results help explain the clinical inefficacy of mTORC1 inhibitors in MM. Our data implicate mTORC1 induction and/or HRI inhibition as pharmacological strategies to enhance MM therapy by PIs.

Overexpression of NIMA related kinase 2 in multiple myeloma and its relevance with disease features and prognosis to bortezomib treatment.

NIMA related kinase 2 (NEK2) promotes the malignant transformation and enhances the chemoresistance to proteasome inhibitor in multiple myeloma (MM) cell lines. The current study aimed to further investigate its correlation with clinical features and responsiveness to bortezomib treatment in MM patients. Totally, 76 MM patients and 30 health donors (HDs) were enrolled to collect bone marrow plasma cells for NEK2 detection using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Meanwhile, NEK2 siRNA was transfected into the RPMI-8226 and KMS-11 cells, subsequently their cell viability was evaluated using Cell Counting Kit-8 reagent after treatment with different doses of bortezomib. NEK2 expression was higher in MM patients compared with HDs (Z=-5.123, p < 0.001). Besides, elevated NEK2 expression was associated with the occurrence of the bone lesion (χ2 =4.610, p=0.032) and t (4; 14) (χ2 =3.971, p=0.046). Additionally, elevated NEK2 expression was correlated with declined objective response rate (ORR) (χ2 =4.808, p=0.028), but not with complete response (CR) (χ2 =2.341, p=0.126). More importantly, elevated NEK2 expression was correlated with shorter progression-free survival (PFS) (χ2 =8.352, p=0.039), but not with overall survival (OS) (χ2 =5.624, p=0.131), What is more, NEK2 silence decreased the cell viability under bortezomib treatment and the inhibitory concentration (IC50 ) value of bortezomib in RPMI-8226 and KMS-11 cell lines (all p < 0.05). NEK2 overexpression links with occurrence of bone lesion, t (4; 14), and poor prognosis to bortezomib treatment in MM patients.

Abstract 5439: MDM2 inhibitor alrizomadlin (APG-115) stabilizes p53 and synergizes with proteasome inhibitors in multiple myeloma

Abstract Multiple myeloma (MM) accounts for about 2% of all cancers and 18% of all hematologic malignancies in the US. Newly developed treatments (e.g., immunomodulators, proteasome inhibitors, monoclonal antibodies) have significantly improved outcomes, but MM inevitably relapses and is considered incurable. Genomic analysis shows that the TP53 gene encoding tumor suppressor protein p53 is infrequently mutated in patients with MM, of whom about 82% retain wild-type (WT) TP53. Mouse double minute 2 (MDM2) is an E3 ubiquitin ligase that inhibits p53 via proteasome degradation. Proteasome inhibitors might help to stabilize p53 and synergize with MDM2 inhibitors. Therefore, MDM2 inhibitors that activate p53 might constitute an attractive pharmacologic approach to MM. Alrizomadlin (APG-115) is an investigational, new small molecule targeting the p53/MDM2 interaction and is in clinical development for solid and hematologic cancers. This study aimed to evaluate whether alrizomadlin can potentiate the antitumor effects of proteasome inhibitors in MM. In cell-based antiproliferation studies, alrizomadlin demonstrated selective activity against WT TP53 MM cell lines, including MOLP-8, H929, and MM1S, with IC50 values of 0.495 ± 0.132 µM, 0.259 ± 0.251 µM, and 0.325 ± 0.105 µM, respectively. In contrast, alrizomadlin was inactive against TP53-mutant MM cell lines (IC50 &amp;gt;10 µM). In MM cell lines, APG-115 exhibited synergistic activity with anti-MM agents (e.g., carfilzomib, lenalidomide, melphalan, selinexor) and other targeted agents (e.g., panobinostat, bromodomain inhibitor OTX-015, tumor necrosis factor-related apoptotic-inducing ligand). In vivo studies showed that coadministration of alrizomadlin with proteasome inhibitor bortezomib or carfilzomib enhanced tumor regression (vs. single agents) in H929 xenograft models. As to mechanism, alrizomadlin likely disrupts MDM2/p53 interactions and induces accumulation of p53, MDM2, and downstream proteins 4 hours after treatment. The proteasome inhibitors induced dose-related increases in p53 protein expression 24 hours after treatment. The combination of carfilzomib with alrizomadlin further promoted accumulation of p53 and MDM2 and increased phosphorylation of p53. As a transcription factor, p53: (1) activated expression of p21 to cause cell cycle arrest (2) augmented downstream BCL-2-associated X protein (BAX) and p53-upregulated modulator of apoptosis (PUMA), (3) increased cleavage of caspase-3 and poly [ADP-ribose] polymerase 1 (PARP-1; hallmarks of apoptosis), and (4) increased apoptosis and associated antitumor effects. In conclusion, our results demonstrate that the combination of MDM2 inhibitor APG-115 and proteasome inhibitors have synergistic antitumor effects on MM tumors harboring WT TP53. These data lay a foundation for clinical trials of a new and novel therapeutic option for patients with refractory MM. Citation Format: Guangfeng Wang, Eric Liang, Chunyang Tang, Li Rui, Ping Min, Jing Lv, Yangfeng Ge, Fei Zhang, Lvcheng Wang, Jingjin Shang, Dajun Yang, Yifan Zhai. MDM2 inhibitor alrizomadlin (APG-115) stabilizes p53 and synergizes with proteasome inhibitors in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5439.

The aspartyl protease DDI2 drives adaptation to proteasome inhibition in multiple myeloma

Proteasome inhibitors, such as bortezomib, are first-line therapy against multiple myeloma (MM). Unfortunately, patients frequently become refractory to this treatment. The transcription factor NRF1 has been proposed to initiate an adaptation program that regulates proteasome levels. In the context of proteasome inhibition, the cytosolic protease DDI2 cleaves NRF1 to release an active fragment that translocates to the nucleus to promote the transcription of new proteasome subunits. However, the contribution of the DDI2-NRF1 pathway to bortezomib resistance is poorly understood. Here we show that upon prolonged bortezomib treatment, MM cells become resistant to proteasome inhibition by increasing the expression of DDI2 and consequently activation of NRF1. Furthermore, we found that many MM cells became more sensitive to proteasome impairment in the context of DDI2 deficiency. Mechanistically, we demonstrate that both the protease and the HDD domains of DDI2 are required to activate NRF1. Finally, we show that partial inhibition of the DDI2-protease domain with the antiviral drug nelfinavir increased bortezomib susceptibility in treated MM cells. Altogether, these findings define the DDI2-NRF1 pathway as an essential program contributing to proteasome inhibition responses and identifying DDI2 domains that could be targets of interest in bortezomib-treated MM patients.

Epigenomic reprogramming via HRP2-MINA dictates response to proteasome inhibitors in multiple myeloma with t(4;14) translocation.

The chromosomal t(4;14) (p16;q32) translocation drives high expression of histone methyltransferase nuclear SET domain–containing 2 (NSD2) and plays vital roles in multiple myeloma (MM) evolution and progression. However, the mechanisms of NSD2-driven epigenomic alterations in chemoresistance to proteasome inhibitors (PIs) are not fully understood. Using a CRISPR/Cas9 sgRNA library in a bone marrow–bearing MM model, we found that hepatoma-derived growth factor 2 (HRP2) was a suppressor of chemoresistance to PIs and that its downregulation correlated with a poor response and worse outcomes in the clinic. We observed suppression of HRP2 in bortezomib-resistant MM cells, and knockdown of HRP2 induced a marked tolerance to PIs. Moreover, knockdown of HRP2 augmented H3K27me3 levels, consequentially intensifying transcriptome alterations promoting cell survival and restriction of ER stress. Mechanistically, HRP2 recognized H3K36me2 and recruited the histone demethylase MYC-induced nuclear antigen (MINA) to remove H3K27me3. Tazemetostat, a highly selective epigenetic inhibitor that reduces H3K27me3 levels, synergistically sensitized the anti-MM effects of bortezomib both in vitro and in vivo. Collectively, these results provide a better understanding of the origin of chemoresistance in patients with MM with the t(4;14) translocation and a rationale for managing patients with MM who have different genomic backgrounds.

Cardiotoxicity as an adverse effect of immunomodulatory drugs and proteasome inhibitors in multiple myeloma: A network meta-analysis of randomized clinical trials.

We aim to determine the cumulative and comparative risk of cardiovascular events associated with different Immunomodulatory Drugs (iMiDs) and Proteasome Inhibitor (PIs) in Multiple Myeloma (MM) patients through pairwise and network meta-analysis. Electronic searches were conducted using Ovid MEDLINE, EMBASE, CINAHL, Web of Science, and Clinical Trial Registry (Clinical Trials.gov) up to May 2021. Phase 3 randomized clinical trials (RCTs) reporting cardiotoxicity in MM patients (newly diagnoses and/or relapsed) treated with iMiD and/or PI. Studies, where iMiD or PI was used alongside the chemotherapy versus placebo or no additional drugs (control) in the other arm were included. The primary outcome was the presence of cardiotoxicity after follow-up. Pairwise meta-analysis and network meta-analysis were performed using the frequentist's approach to estimate the odds ratio (OR). Twenty RCTs with 10,373MM patients were included in this analysis. Eleven studies compared iMiDs with control, seven studies compared PIs with control, and two studies compared bortezomib against carfilzomib. CTACE high-grade (≥grade 3) cardiotoxic events were increased with iMiDs compared to their control counterpart (OR 2.05; 95% CI 1.30-3.26). Similar high-grade cardiotoxicity was also noted more frequently with PI use when compared to the control group (OR 1.67; 95% CI 1.17-2.40). Among the PIs, carfilzomib was associated with a maximum risk of cardiotoxicity (OR 2.68; 95% CI 1.63-4.40). There was no evidence of publication bias among studies. iMiDs and PIs, particularly carfilzomib, appear to be associated with increased risk of high-grade cardiovascular events in MM patients.

Abstract 10860: Cardiotoxicity as an Adverse Effect of Immunomodulatory Drugs and Proteasome Inhibitors in Multiple Myeloma: A Meta-Analysis of Randomized Clinical Trials

Introduction: Combination therapy of Immunomodulatory drugs (iMiDs) (Thalidomide, Lenalidomide, Pomalidomide) and Proteasome Inhibitors (PIs) (Bortezomib, Carfilzomib, Ixazomib) constitutes the backbone of treatment in MM patients. Cardiotoxic side-effect profile of iMiDs is not well-documented. Although Carfilzomib has been associated with cardiotoxicity, whether its adverse effects also mirror the class-specific cardiotoxic profile of PI remain obscure. Proper assessment of cardiotoxicity due to treatment reagents is necessary for optimal patient management. In this pairwise meta-analysis, we explored direct cardiotoxicity with iMiDs and PIs use in patients with multiple myeloma (MM). Hypothesis: Treatment with iMiDs and PIs in MM patients leads to independent cardiotoxicity that is associated with individual class of drugs. Methods: Published randomized control trials (RCTs) were searched up to May 2021 reporting cardiotoxicity (cumulative reported events of ischemia, arrhythmia, failure, stenosis) in MM patients (newly diagnoses and/or relapsed) treated with iMiD and/or PI. Studies, where iMiD or PI were used atop the chemotherapy vs placebo or no additional drugs (control) in the other arm, were included. The primary outcome was presence of cardiotoxicity after follow-up. Meta-analysis was performed using frequentist’s approach to estimate odds ratio (OR). Results: Eighteen RCTs including 8,479 MM patients were included in this analysis. Eleven studies compared iMiDs and seven studies compared PIs with control. CTACE high-grade (≥ grade 3) cardiotoxic events were increased with iMiDs in comparison to their control counterpart (OR 2.05; 95% CI 1.30-3.26). Cardiotoxicity was also noted more frequently with PI use when compared to the control group (OR 1.47; 95% CI 1.19-1.82). Similar difference was observed between high-grade cardiotoxic complication between these groups. There was no evidence of publication bias among studies. Conclusions: Our results showed iMiDs and PIs are independently associated with cardiotoxicity, which may warrant increased awareness among treating physicians and close monitoring of patients. The adverse cardiac event profile with these drugs require further investigation in future studies.

Mitochondrial Electron Transport Chain Inhibition Promotes Resistance to Proteasome Inhibitors in Multiple Myeloma

INTRODUCTIONProteasome inhibitors (PI) such as bortezomib (Velcade), carfilzomib (Kyprolis) and ixazomib (Ninlaro)) have been shown to be efficacious in multiple myeloma (MM) therapy. However, despite being an effective first line therapy, resistance to PI usually develops, leading to relapse and refractory disease. Previous studies have implicated a role of OXPHOS, glycolysis, antioxidants and serine metabolism in PI resistance. Mitochondrial metabolism plays a central role in malignant progression not only by generating ATP but also by providing precursors for synthesis of several biomolecules such as proteins, nucleotides, fatty acids and antioxidants that can influence the efficacy of MM therapies. We previously determined reduced mitochondrial electron transport chain (ETC) activity promotes sensitivity to the BCL-2 antagonist, venetoclax. However, the relationship between the metabolic state of the mitochondria and proteasome inhibitor (PI) sensitivity is not fully understood. Unexpectedly, we found ETC inhibition or reduced ETC activity to promote resistance to PIs. Here, we investigate the mechanistic basis for divergent effects of mitochondrial stress on sensitivity to PIs and venetoclax.METHODSWe have used RNA-Seq and carbon isotope tracing using labeled U 13C-glucose or U 13C-glutamine, flow cytometry and western blot analysis in MM cell lines treated with mitochondrial Complex I inhibitor, IACS-010759 (IACS) +/- Bortezomib; and immunostaining of MM patient samples and interrogation of Multiple Myeloma Research Foundation's CoMMpass Study (NCT01454297, Interim Analysis 15).RESULTSWe find that mitochondrial ETC (complex I-V) inhibition antagonizes bortezomib (BTZ) and carfilzomib (CFZ) induced cell death in MM in contrast to promoting sensitivity to venetoclax. Additionally, cell lines exhibiting intrinsically reduced ETC activity were more resistant to PI in comparison to cell lines with higher ETC activity. Evaluation of CoMMpass MM trial (NCT0145429, IA15) and serial samples from 50 patients before PI treatment and after relapse, show pathways related to OXPHOS and TCA cycle to be downregulated in poor survival patients, corroborating our in vitro observations on reduced ETC activity promoting resistance to PI. To elucidate the mechanistic basis of ETC-inhibition induced PI resistance we performed RNA-Seq and U 13C-glucose and glutamine tracing in L363 cells treated with the ETC Complex I inhibitor IACS +/- BTZ. RNA-Seq analysis and further confirmation by western blot analysis reveals integrated stress response (ISR) upregulation, ATF4 induction, and suppression of protein translation and global protein ubiquitination levels, likely responsible for resistance to proteasome inhibition in cells co-treated with IACS and BTZ compared to BTZ alone. Stable isotope tracing reveals an upregulation of reductive carboxylation; while RNA-Seq data and flow cytometry demonstrate increase in the cystine/glutamate transporter SLC7A11. The ensuing metabolic rewiring in mitochondrially suppressed MM induces several metabolic vulnerabilities including sensitivity to the SLC7A11 inhibitor, erastin. We also show that knockdown of ATF4 re-sensitizes ETC-inhibited cells to BTZ while ablating sensitivity to venetoclax (previously reported). Furthermore, examination of patient samples demonstrates inter-cellular heterogeneity in ATF4 expression. Our results thus, support the role ATF4 as key determinants of PI and BCL-2 antagonist efficacy.CONCLUSIONWe show mitochondrial ETC inhibition induces ISR mediated resistance to PI. These ETC-inhibited cells are however sensitive to BCL-2 antagonists and afford additional metabolic vulnerabilities that can be capitalized upon to target metabolic heterogeneity in MM. Our study underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic heterogeneity-mediated resistance. DisclosuresLonial: Janssen: Consultancy, Honoraria, Research Funding; BMS/Celgene: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria. Boise: AstraZeneca: Consultancy, Research Funding; Abbvie: Consultancy. Jaye: Stemline Therapeutics: Honoraria. Nooka: Janssen Oncology: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Other: Travel expenses; Sanofi: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Oncopeptides: Consultancy; Adaptive technologies: Consultancy; Karyopharm Therapeutics: Consultancy.

Research progress in proteasome inhibitor resistance to multiple myeloma.

Multiple myeloma (MM) is a highly heterogeneous malignant plasma cell disease. Proteasome inhibitors (PIs) are the first line of medicine for MM. Bortezomib, ixazomib, and carfilzomib are also widely used for MM. Marizomib, oprozomib, and KZR-616 are in clinical trials. However, the drug resistance of PIs in MM is still a problem. The mechanisms for PIs resistance to MM include ubiquitin-proteasome pathway, autophagy lysosome pathway, endoplasmic reticulum stress pathway, cell survival signal pathway, exosome-mediated resistance, and bone marrow microenvironment-mediated resistance.

S100 Calcium Binding Protein Family Members Associate With Poor Patient Outcome and Response to Proteasome Inhibition in Multiple Myeloma.

Despite several new therapeutic options, multiple myeloma (MM) patients experience multiple relapses and inevitably become refractory to treatment. Insights into drug resistance mechanisms may lead to the development of novel treatment strategies. The S100 family is comprised of 21 calcium binding protein members with 17 S100 genes located in the 1q21 region, which is commonly amplified in MM. Dysregulated expression of S100 family members is associated with tumor initiation, progression and inflammation. However, the relationship between the S100 family and MM pathogenesis and drug response is unknown. In this study, the roles of S100 members were systematically studied at the copy number, transcriptional and protein level with patients’ survival and drug response. Copy number analysis revealed a predominant pattern of gains occurring in S100 genes clustering in the 1q21 locus. In general, gains of genes encoding S100 family members associated with worse patient survival. However, S100 gene copy number and S100 gene expression did not necessarily correlate, and high expression of S100A4 associated with poor patient survival. Furthermore, integrated analysis of S100 gene expression and ex vivo drug sensitivity data showed significant negative correlation between expression of S100 family members (S100A8, S100A9, and S100A12) and sensitivity to some drugs used in current MM treatment, including proteasome inhibitors (bortezomib, carfilzomib, and ixazomib) and histone deacetylase inhibitor panobinostat. Combined proteomic and pharmacological data exhibited significant negative association of S100 members (S100A4, S100A8, and S100A9) with proteasome inhibitors and panobinostat. Clinically, the higher expression of S100A4 and S100A10 were significantly linked to shorter progression free survival in patients receiving carfilzomib-based therapy. The results indicate an association and highlight the potential functional importance of S100 members on chromosome 1q21 in the development of MM and resistance to established myeloma drugs, including proteasome inhibitors.

The Interaction of NEK2 with USP7 Causes Resistance to Proteasome Inhibitor in Multiple Myeloma

Background: Drug resistance is the major reason for treatment failure in multiple myeloma (MM). Our previous work has demonstrated that high expression of chromosomal instability (CIN) genes results in increased cell survival and drug resistance and is associated with poor outcome in MM and other cancers. NEK2 was the most significant CIN gene to induce drug resistance and poor outcome in MM. Though we have demonstrated that NEK2 induces drug resistance through activation of efflux drug pumps, the detail mechanisms remain unknown.Materials and Methods: Multiple MM cell lines, xenograft MM NOD-Rag1nullmice with both subcutaneous and by tail vein injection, and primary MM samples were included in this study to test NEK2 inhibitors and gene expression profiles (GEP). The tandem affinity purification - mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were applied for the identification of NEK2 interacting partners. The dual-luciferase reporter assay and GEPs were performed to determine whether the interaction of NEK2 with USP7 activates the canonical NF-kB signaling pathway. Chromatin immunoprecipitation (ChIP)-qPCR and western blots were carried out to determine the direct target genes of the axis NEK2/USP7/NF-kB.Results: We demonstrate that NEK2 is stabilized by the de-ubiquitinating enzyme USP7, leading to accumulation of NEK2 within the cancer cells. Accumulation of NEK2 activates the canonical NF-kB through the AKT/IKK/IKB-a/p65 axis and, using this pathway as framework, we uncovered a novel bona fide NF-kB target, heparanase, a protein that is known to promote bone destruction in MM patients. A NF-kB or USP7 inhibitor could overcome NEK2-induced bortezomib resistance. The NEK2 inhibitor, INH1, showed great therapeutic effects on MM in vitro and in vivo . To corroborate our findings, we subcutaneously injected NEK2-OE ARP1 MM cells into NOD.Cg-Rag1 mice and targeted NEK2 with the small molecule drugs INH1 or P5091 (USP7 inhibitor) alone or in combination with bortezomib. As expected bortezomib had very little effect in reducing tumor burden since NEK2-OE cells are highly resistant to this drug. Inhibition of NEK2 with P5091 or INH1 alone had a strong anti-tumor effect and a synergistic effect was observed when combined with bortezomib.Conclusion: Our data present a novel mechanism for the interaction of NEK2 with USP7 driving drug resistance in MM. We conclude that (1) USP7 in MM cells leads to the de-ubiquitination and escape of proteasomal degradation of NEK2, leading to its accumulation in cancer cells. (2) Accumulated NEK2 binds and phosphorylates PP1a, suppressing its phosphatase activity thus promoting AKT overactivation. (3) AKT then phosphorylates the IKKa/b complex, triggering the canonical NF-kB cascade which phosphorylates and targets IKBa for proteasomal degradation, releasing the sequestered p65 to be phosphorylated at S536 and translocation to the nucleus where it drives bortezomib resistance by activating its target genes. Our data also show that INH1 and P5091 can deplete NEK2 protein in vitro and in vivo and suppress NEK2 downstream targets resulting in overcoming bortezomib resistance. Our findings should facilitate novel targeted approaches to the therapy and prevention of myeloma progression and relapse. DisclosuresNo relevant conflicts of interest to declare.

Defining the Most Effective Proteasome Inhibition in Multiple Myeloma: Head-to-Head Comparison of Currently Available Proteasome Inhibitors

Introduction: Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (β1, β2 and β5 of the constitutive (c) and the immunoproteasome (i), respectively). They differ in substrate preferences and affinity for PIs. β5c was identified as the rate-limiting proteolytic proteasome activity, while β2 and β1 activity received little attention. Consecutively, all proteasome-inhibiting drug candidates (bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, marizomib) by design target β5c, but differ in co-inhibition of non-β5 activities. Current chemical biology tools allow highly selective inhibition and visualization of individual proteasome subunits in living cells. We lack data that identify the most effective pattern of β5 and non-β5 type of proteasome inhibition in MM cells, as well as a head to head comparison of PIs and drug candidates, and their proteasome subunit inhibition profile in relation to pharmacodynamic activity. Such data shall allow rational selection and positioning of proteasome-inhibiting drugs.Methods: Head to head comparison of bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib and marizomib was performed in MM cells. Non-commercial, highly subunit-selective PIs and activity-based fluorescent chemical probes (ABP) were used to assess the most effective pattern of inhibition of proteasome subunits, in conjunction with a fluorescent ubiquitin Ub-G67V-GFP construct, and related to the viability of MM.Results: All commercial PI tested target primarily β5 activity and in higher concentrations co-targeted β2 and/or β1 (Table 1). In equimolar comparison, most effective PI was marizomib, followed by bortezomib, delanzomib, carfilzomib, ixazomib and oprozomib. MM cell viability inversely correlated with functional proteasome inhibition. Surprisingly, during short term, 1h pulse exposure mimicking the pharmacokinetics of i.v. proteasome inhibiting drugs, β5-selective proteasome inhibition was not sufficient to functionally inhibit protein degradation or induce cytotoxicity, even in PI-sensitive MM cells. Only co-inhibition β5 with either β1 or β2 resulted in accumulation of polyubiquitinated protein and decreased MM cell viability. Long time (48h) continuous β5-selective inhibition likewise induced cell death in PI-sensitive MM, however, only co-inhibition of β5 and β2 activity induced cytotoxicity in PI-resistant MM.Conclusion: All commercial PIs by design target β5c activity and differ in their co-targeting of non-β5 activities. Co-inhibition of β1 and β2 determines cytotoxicity. Selective inhibition of β5 by pulse exposure is not sufficient, but co-inhibition of β5 and β2 is the most effective combination to induce cytotoxicity, in particularly in PI resistant cells. This inhibition pattern is provided only by marizomib and high dose carfilzomib. By contrast, low dose of carfilzomib does not deliver co-inhibition of β2 and therefore has no advantage over bortezomib. Together, these results give a rational basis to select individual proteasome inhibitors for the treatment of MM: cytotoxicity in PI-sensitive MM is achieved by co-targeting of β5 together with β1 or β2 with a pulse treatment, or by prolonged β5 inhibition (s.c. bortezomib, orally administered ixazomib). In PI-resistant MM only dual β2/β5 inhibition is able to achieve meaningful cytotoxicity, which is provided by carfilzomib or marizomib. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Unbiased Phosphoproteomics Reveals a Therapeutically Relevant Connection between Pre-mRNA Splicing and Proteasome Inhibition in Multiple Myeloma

Introduction: Proteasome inhibitors (PIs) are first-line therapeutic agents in multiple myeloma. While these small molecules have revolutionized myeloma therapy, nearly all myeloma patients treated with PIs eventually experience drug resistance and relapse. We have previously examined the global effects of PIs on the transcriptional and translational landscape of multiple myeloma plasma cells (Wiita et al, eLife (2013) 2:01236). Here, we hypothesized that investigation into the intracellular signaling response to PI-induced stress would help elucidate additional PI mechanisms of action in this cancer.Methods: We applied an unbiased phosphoproteomic screen to MM.1S myeloma cells treated with the PI carfilzomib across a 24-hour time course. We used trypsin digestion, FeCl2 enrichment, and a single-shot 4-hour LC method on each sample harvested at each time point on a Thermo Q-Exactive Plus mass spectrometer, with phosphopeptide identification and label-free quantification performed in MaxQuant. Targeted mass spec was then performed for validation on a subset of upregulated and downregulated phosphosites by parallel reaction monitoring (PRM) on SILAC-labeled, carfilzomib treated MM.1S cells, as well as another multiple myeloma cell line, AMO1, at 24 hr of treatment. MM.1S cells were also treated with another multiple myeloma therapeutic agent, melphalan, to compare the response to a non-PI drug. Analysis was carried out in Skyline. For paired-end mRNA-seq, we constructed sequencing libraries from the SILAC samples and sequenced on an Illumina HiSeq4000 and analyzed with JuncBASE software to correlate changes in splicing factor phosphorylation with alternative splicing events. In vitro viability studies of myeloma cell lines were carried out with Cell-titer-glo assay. For in vivo studies, we used disseminated murine xenograft models of luciferase-labeled MM.1S implanted intravenously into NSG mice, with tumor burden measured by bioluminescence. Mice (n=6 per arm) were treated with 3 mg/kg E7107 or DMSO IV 5d/wk for 2 wks.Results: Among the more than 5,000 phosphopeptides quantified in the phosphoproteomic screen, the most profoundly upregulated phosphosites mapped to numerous serine-and-arginine rich (“SR”) core components of the pre-mRNA spliceosome (Fig. 1A: upregulated phosphosites across time course in gold; downregulated in cyan. Red arrows indicate SR protein phosphosites; green arrows are positive control phosphosites on EIF4EBP1 and RPS6, known to decrease after PI treatment.). These targets were confirmed by targeted mass spec, while simultaneous mRNA-seq confirmed no change in splicing factor abundance in response to PI (Fig. 1A). Intriguingly, intron retention appeared to be a dominant signature in pre-mRNA splicing after PI treatment based on JuncBASE analysis. This connection led us to investigate whether induction of intron retention may be sufficient to lead to myeloma tumor cell death. We therefore obtained the clinical candidate E7107, a specific inhibitor of the core spliceosome factor SF3B1, known to strongly induce intron retention across the transcriptome. We found that a panel of myeloma cell lines were highly sensitive to E7107 at low nanomolar concentrations, which are easily achievable in patients based on prior Phase I studies. We further found evidence of synergy in vitro between E7107 and PI treatment, and E7107 was equally efficacious in naïve and PI-resistant AMO1 cells. Furthermore, pre-clinical experiments in murine xenograft models with a brief two-week treatment with E7107 significantly decreased tumor burden and significantly increased lifespan (Fig 1B).Conclusion: Our approaches here reveal a novel connection between PI-induced stress and modulation of the core spliceosome machinery not previously observed in any other system. By capitalizing on this vulnerability, we further propose that targeting the spliceosome machinery may be a novel therapeutic option for multiple myeloma patients, either in combination with PIs or in the PI-refractory setting, addressing a major clinical need. [Display omitted] DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio, Inc.: Research Funding.

Targeting RFWD2 as an Effective Strategy to Inhibit Cellular Proliferation and Overcome Drug Resistance to Proteasome Inhibitor in Multiple Myeloma

The potential to overcome resistance to proteasome inhibitors is greatly related with ubiquitin-proteasome system during multiple myeloma (MM) treatment process. The constitutive photomorphogenic 1 (RFWD2), referred to an E3 ubiquitin ligase, has been identified as an oncogene in multiple cancers, yet important questions on the role of RFWD2 in MM biology and treatment remain unclear. Here we demonstrated that MM patients with elevated RFWD2 expression achieved adverse outcome and drug resistance by analyzing gene expression profiling. Moreover, we proved that RFWD2 participated in the process of cell cycle, cell growth and death in MM by mass spectrometry analysis. In vitro study indicated that inducible knockdown of RFWD2 hindered cellular growth and triggered apoptosis in MM cells. Mechanism study revealed that RFWD2 controlled MM cellular proliferation via regulating the degradation of P27 rather than P53. Further exploration unveiled that RFWD2 meditated P27 ubiquitination via interacting with RCHY1, which served as an E3 ubiquitin ligase of P27. Finally, in vivo study illustrated that blocking RFWD2 in BTZ-resistant MM cells overcame the drug resistance in a myeloma xenograft mouse model. Taken together, these findings provide compelling evidence for prompting that targeting RFWD2 may be an effective strategy to inhibit cellular proliferation and overcome drug resistance to proteasome inhibitor in MM.