Proteasome Inhibitor Treatment Research Articles

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.

Myeloma Cell Addiction to the Transcription Factor TCF11

BACKGROUND: TCF11/NRF1 is a transcription factor transiently and post-translationally activated in response to metabolic challenges and endoplasmic reticulum (ER) stress. However, the genome-wide compendium of genes regulated by TCF11 has not been defined and its role in tumour biology has not been explored. Since malignant plasma cells (PC) in multiple myeloma are under a constant state of ER stress due to immunoglobulin secretion, we hypothesised that the expression and functional role of TCF11 would be altered in myeloma compared to normal cells.METHODS: We employed qPCR for mRNA quantitation, western blotting for protein expression analysis, constitutive and dox-inducible lentiviral shRNA-based knock-down of TCF11, DAPI staining and flow-cytometry for cell cycle analysis, RNA-seq for transcriptome analysis after TCF11knock-down, ChIP-seq against Flagx3-TCF11 in H929 myeloma cells, and a subcutaneous myeloma xenograft model in NSG mice following inducible TCF11 knock-down. Computational analysis of RNA-seq and ChIP-seq was performed using standard work-flows while cistrome-transcriptome integration was performed using the Binding and Expression Target Analysis package. Pathway enrichment analysis was performed using EnrichR and GSEA.RESULTS: We found that at the protein level TCF11 is undetectable in erythroblasts, monocytes, B and T cells but expressed at low level in normal PC and at higher levels in myeloma cell lines (n=5) as well as primary myeloma PC (n=10). Therefore, myeloma cells and to a lesser extent normal PC constitutively express TCF11. Depletion of TCF11 using 2 validated shRNAs (either constitutive or inducible) was toxic to 5/5 and 2/2 myeloma cell lines tested in vitro and in vivo respectively, revealing an absolute requirement for TCF11 expression for myeloma cell survival.Next we performed transcriptome analysis upon TCF11 depletion in 2 myeloma cell lines (H929 and U266) with divergent primary and secondary oncogenic events (CCND1 vs MMSET and MYC vs MYCL respectively). As well as its previously described role in the regulation of several metabolic processes, pathway enrichment analysis revealed in both cell lines a hitherto unknown function of TCF11 in cell cycle regulation. Indeed, inducible TCF11 depletion in myeloma cells resulted in a significant cell cycle arrest in G0/1 (n=5; p<0.01).To investigate whether TCF11 directly regulates genes involved in cell cycle and proliferation, we generated and analysed the first genome-wide maps of TCF11 binding in H929 cells (n=2). Significantly enriched peaks (n=3829) bind in roughly equal proportions to promoters, introns and enhancers and are highly enriched for the previously described TCF11-preferred ARE motif.Integrated transcriptome-cistrome maps revealed that 608 and 390 genes are directly activated or repressed by TCF11. Again, pathway enrichment analysis of TCF11 direct targets, as well as positive regulation of various metabolic processes (glycolysis, adipogenesis, unfolded protein response, oxidative phosphorylation), it also confirmed a direct role of TCF11 in the regulation of cell cycle and of the transcriptional programmes of MYC and E2F1/2. Low throughput ChIP-qPCR analysis validated TCF11 binding to the promoters of MYC and reciprocally, binding of MYC to the promoter of TCF11, thus establishing TCF11 as a novel co-operative factor of oncogenic MYC. Further, we confirmed that TCF11 binds to its own promoter and since TCF11 mRNA levels increase following proteasome inhibitor treatment, we conclude that TCF11 activates its own transcription.Finally, we detected constitutively high protein levels of TCF11 in several other haematological cancer cell lines, i.e., (ALL: BV173; AML: HL60 and KG1 and Burkitt lymphoma: Ramos) and solid tumour cell lines (breast: MCF7 and MCF7TR; prostate: DU145 and PC3; ovarian: SKOV3 and OVC433). In all cases, depletion of TCF11 was toxic to the cells.CONCLUSIONS: Myeloma cells are addicted to the constitutive expression of TCF11. As well as essential metabolic processes, TCF11 regulates cell cycle progression. At the transcriptional level, TCF11 regulated expression of MYC, E2F1/2 and of their respective transcriptional programmes in a direct co-operative fashion. These data establish a multifaceted role of TCF11 in tumour biology and link its function to cell cycle regulation in co-operation with oncogenic MYC in myeloma cells. DisclosuresAuner:Amgen: Honoraria, Research Funding. Karadimitris:GlaxoSmithKline: Research Funding.

Therapeutic Targeting of Interferon Regulatory Factor 4 with Next Generation Antisense Oligonucleotides Produces Robust In Vivo Antitumor Activity in Preclinical Models of Multiple Myeloma

Despite significant recent advances in therapeutic options, multiple myeloma (MM) remains an incurable disease, and novel agents targeting novel molecular pathways with activity in advanced patients are urgently needed. Interferon Regulatory Factor 4 (IRF4) is a transcription factor involved in immune responses in normal B and T cells, and is strongly implicated in the development of hematological malignancies, especially MM. Although recent publications have pointed to IRF4 as a driver oncogene in the progression of MM, it is considered an intractable target by conventional therapeutic approaches.Here we describe an antisense oligonucleotide (ASO) approach to selectively target IRF4. Administration of next generation human IRF4 ASOs via ‘free-uptake’ (without transfection) to a large panel of human MM cell lines resulted in a dose-dependent IRF4 reduction with concomitant induction of apoptosis. ASO-mediated IRF4 depletion also decreased the levels of c-Myc, another key oncogene in MM. MM cells were sensitive to even modest depletion of IRF4 (<50%), suggesting the strong dependence of MM cells on IRF4 for their survival. IRF4 ASOs also enhanced the sensitivity of the tumor cells to the treatment of IMiD (lenalidomide), proteasome inhibitor (Bortezomib) and BTK inhibitor (Ibrutinib) in MM cell, including KMS-11, JJN3 and MM1.R. In addition, Bortezomib-resistant clones of KMS-11 cells established from a long-term exposure to the drug remained sensitive to IRF4 inhibition. Importantly, in two different subcutaneously implanted MM tumor models, systemic administration of 2 independent human IRF4 ASOs depleted IRF4 protein levels in tumors and inhibited the growth of tumors compared to control treatment. Moreover, in a clinically relevant MM mouse model where human MM cells accumulate in the mouse bone marrow, treatment with human IRF4 ASOs substantially prolonged the survival of the tumor-bearing animals along with specific downregulation of human IRF4 and its downstream gene c-Myc. Finally, systematic treatment of mice with mouse-selective IRF4 ASOs at 50 mg/kg, twice a week for 4 weeks was well tolerated in normal mice, which is consistent with the lack of phenotypes observed in IRF4 heterozygous animals. Taken together, these results suggest that MM cells rely heavily on the survival pathway mediated by IRF4 and that selective knockdown of IRF4 by next generation ASOs is an attractive therapeutic strategy for the treatment of patients with advanced treatment-refractory MM. DisclosuresZhou:Ionis Pharmaceuticals: Employment, Equity Ownership. Schmidt:Ionis Pharmaceuticals: Employment. Kim:Ionis Pharmaceuticals: Employment. MacLeod:Ionis Pharmaceuticals: Employment.

Mmprofiler with SKY92 Combined with ISS Identifies High and Low Risk Multiple Myeloma in the VTD Arm of Gimema-MMY-3006

SKY92 combined with ISS identifies high and low risk multiple myeloma in GIMEMA-MMY-3006 VTD armErik H. Van Beers*, Carolina Terragna*, Rowan Kuiper*, Marina Martello*, Elena Zamagni*, Giovanni Martinelli, Martin H. van Vliet*, Michele Cavo*.Introduction: Prior to this cohort, SKY92 demonstrated significance in several bortezomib-containing treatments (HOVON-65/GMMG-HD4, APEX, TT3, TT6) and thalidomide-containing treatments (HOVON-65/GMMG-HD4, HOVON-87/NMSG-18, TT2, TT3, TT6, MRC-IX). It remains unknown how this GEP-signature predicts outcome in IMiD plus proteasome inhibitor (PI) treatment. GIMEMA-MMY-3006 is the first dataset in which the front-line VTD in NDMM patients has been evaluated by SKY92 and other prognostic markers.Methods: The GIMEMA-MMY-3006 trial (NCT01134484) compared bortezomib-thalidomide-dexamethasone (VTD) versus thalidomide-dexamethasone (TD) as induction therapy prior to the autologous stem cell transplantation (ASCT) in transplant eligible NDMM patients [1]. The current analysis compares prognostic value of t(4;14), del(13), del(17), GEP and clinical characteristics in the VTD arm (GSE58133; n=114). PFS and OS were evaluated at median follow up of 3.3yrs. GEP data (U133Plus2.0 CEL files) were batch processed and GEP classifiers were calculated as reported [2]. R-ISS was constructed as described in Palumbo et al. [3], except for the missing marker t(14;16) while SKY92-ISS was constructed as described in Kuiper et al. [4] except that four classes were reduced to three (high, low, and the rest).Results: This is the first report of SKY92 evaluated in a VTD setting. The prognostic values of three risk models (ISS, SKY92-ISS and R-ISS) were compared (Table 1). A total of 36 progressions and 18 deaths occurred in 114 cases. Univariate PFS (results will be presented at the meeting). For OS, univariate analysis showed SKY92, UAMS70, IFM15, del(17), ISS (IIIvsI) and SKY92-ISS were significant. Figure 1 shows Kaplan Meier analyses for SKY92, SKY92-ISS, and R-ISS. Multivariate analysis for OS - with SKY92, del(17) and R-ISS (high risk vs others) - significance was observerd for SKY92 (HR 3.48) but not for del(17) or R-ISS (Table 2). Among the 31 R-ISS-I there were no deaths and therefore no HR (between I and III) was calculated (Figure 1C). A comparison of Figure 1B and C shows that SKY92-ISS captured more high-risk patients than R-ISS (23 vs.6). Of the 19 t(4;14) cases, eleven were also SKY92 high-risk. Of the 8 del(17) cases, 5 were also SKY92 high-risk.Both SKY92-ISS and R-ISS identified low risk patients. SKY92-ISS identified 12 more low risk cases than R-ISS (43/114, 38%, 31/105, 30%, respectively). Figure 1D shows concordance between the risk scores and association with OS (top rows) at 2 and 4y after diagnosis. None of the signatures predicted all deaths at 2 or 4y. SKY92 predicted more deaths than other markers (Figure 1D). Similar to a previous study [1] the risk models were concordant for 2 high risk and 66 standard risk cases, while discordant for 46 remaining cases. SKY92 best identified hazard for a large proportion of cases (Table 1).Conclusions: In this upfront VTD setting we observe proportions of SKY92 high risk and low risk myeloma that are comparable with previous studies with other treatments containing either bortezomib or thalidomide. SKY92-ISS identifies high risk in 20% and low risk in 38% MM patients with HR = 4.0 between high and standard risk (Fig 1A) and HR = 10.7 between high and low risk (Fig 1B) compared with R-ISS the obtained HR = infinite as there were no deaths in low risk (Fig 1C). SKY92-ISS identified a larger faction of low risk cases (38%) compared with R-ISS (30%) yet retains a very large and significant hazard ratio of 10.7 reproducing earlier validation data [1].Figure 1. Prognostic marker performance in n=114 VTD treated multiple myeloma patients. Kaplan Meier analyses for OS were stratified by A) SKY92, B) SKY92-ISS or C) R-ISS. D) shows GEP signature predictions (rows) and their overlap across all 114 cases (columns). Standard Risk (blue), High Risk (yellow), Alive at follow up (Green), dead at follow up (Red), lost to follow up (Grey).

Comparative Analysis of Gene Expression Status to Predict Response to Initial Multiple Myeloma Treatment: Utility of the Mmrf Commpass Database in Validating Historical Findings

Introduction: Treatment of multiple myeloma (MM) has evolved tremendously, resulting in significant improvement of overall survival in patients. Frontline therapy can be broadly categorized into: proteasome inhibitor (PI)-based, immunomodulatory (IMiD)-based or PI+IMiD-based. Despite their widespread benefit, a considerable proportion of patients achieve suboptimal response to treatment. It would be clinically valuable to understand disease biology prior to initiating therapy and be able to predict the likelihood of response. Prior studies have attempted such a process in which response to PI and/or IMiD-based treatments were retrospectively examined in correlation to genomic testing from pre-treatment patient samples. The MMRF CoMMpass study provides a unique opportunity for interrogation of genes predictive of response to PI and/or IMiD-based regimens. Here, we performed a comparative analysis of genes previously reported to be associated with response to treatment or survival in MM patients with data recorded from the CoMMpass trial.Methods: Previous studies with reported relationship of gene expression and response/survival outcome to PI and/or IMiD-based regimens were identified (Table 1). Next, we interrogated the CoMMpass database and abstracted patients by Best Response to First Line Therapy. Therapy class (PI and/or IMiD-based) was simplified to match the comparator studies in Table 1. We analyzed the genes reported in studies from Table 1 against our PI-based, IMiD-based and PI+IMiD-based CoMMpass patient cohorts. Logistics regression modeling and Bonferroni correction methods were applied adjusting for patient age, gender and ISS stage and gene odds ratios were calculated. An alpha of <0.00029 was considered significant due to multiple comparison of genes between the groups. We also compared mRNA expression for the genes identified in these 3 different treatment cohorts that had an alpha of ≤0.05 between patients classified as Responders (≥PR) and Non-responders (<PR).Results: Of the 7 previous studies identified, 4 were PI-based, 1 was IMiD-based and 2 were PI+IMiD-based. From among these studies, a total of 173 genes (132 genes in the PI-based group; 25 in the IMiD-based group and 17 in the PI+IMiD-based group) were cross-analyzed in patients from the CoMMpass study. In the PI-based cohort, 10 genes with an alpha of ≤0.05 were associated with an increased likelihood of not responding to treatment, with OR ranging from 1.32-E5 (CNGA1) - 0.99 (CCND1). In the IMiDs-based treatment cohort, we identified 2 genes associated with an increased likelihood of response, with OR of 1.2 (RPL26L1 and SNRPD1) and an alpha of 0.02 and 0.03, respectively. In the PI+IMiD-based group, 22 genes (p≤0.05) with OR ranging from 2.28 (HIST1H3B) - 0.85 (WIPF2) were associated with the likelihood of response. Relative gene expression analysis in the different treatment CoMMpass cohorts showed that in PI-based treatment patients NMT1, PSMD4, MAP4K4, CNGA1, DR1 and UBFD1 were significantly decreased in responders (n=228) vs. non-responders (n=14) (p<0.05-<0.001). In the IMiD-based cohort, C14orf2 and RPL26L1 were significantly increased in responders (n=32) vs. non-responders (n=7, p<0.05). In the PI+IMiD treatment group, ACVC1C, CRYGS, FUNDC1, PSMB3, RPL26L1, HIST1H3B, PSMB10 and STUB1 were significantly increased whereas TMC8, MAPK4, CCNB1IP1, SAP18, ANK3, JAK1, RUNX3, IKZF1, CUL4B, HNRNPC and WIPF2 were significantly decreased in responders (n=361) vs. non-responders (n=18) (p<0.05-<0.001).Conclusion: Our analysis investigates utility of the CoMMpass database to interrogate molecular data previously reported and shown to be associated with outcome to therapy in MM patients. While useful in comparison of relative gene expression changes and mutation data associated with patients receiving PI, IMiD or PI+IMiD-based therapy, cross-comparison of data from prior studies should be performed with caution considering variability in methodology and patients who have received treatment at different stages of their disease vs. frontline only in the CoMMpass dataset. Overall our analysis highlights the potential of CoMMpass data to develop future prospective studies with biomarkers and/or predictive models for MM therapeutics.Acknowledgments: We would like to thank Dr. Daniel Auclaire and the MMRF CoMMpass Network. [Display omitted] DisclosuresSher:LAM Therapeutics, Inc: Research Funding. Ailawadhi:Pharmacyclics: Research Funding; Novartis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Nelfinavir Blocks Export of Newly Synthesized Protein from the ER and Interacts with ER-Resident and Mitochondrial Proteins in an Activity-Dependent Fashion

The majority of multiple myeloma (MM) patients initially respond to proteasome inhibitor (PI)-based therapy, but later develop PI resistance and ultimately dies from PI-refractory MM. PI sensitivity of MM cells correlates with the activation status of the unfolded protein response (UPR) and its major activating axis IRE1/XBP1. PI-refractory MM cells downregulate IRE1/XBP1, and high IRE1/XBP1 expression correlates with clinical sensitivity to PI treatment. The anti-HIV drug nelfinavir induces IRE1/XBP1 and overcomes PI-resistance of MM in vitro . Nelfinavir showed unprecedented clinical activity in PI-refractory, heavily pretreated MM with ORR 65% in combination with bortezomib and dexamethasone. The molecular target of nelfinavir and its mechanism of action in MM cells are unknown. We synthesized structurally modified nelfinavir analogues equipped with a covalently protein-binding, photo-reactive moiety and functional groups that allow affinity purification of the nelfinavir analogue bound to its interacting cellular protein (SC-441), together with appropriate controls where the active center of nelfinavir was sterically occupied (SC-451). Isolation and identification of nelfinavir-interacting proteins in AMO-1 MM cells and cells adapted to carfilzomib (AMO-CFZ) was achieved with these tools by photoactivation in living cells, affinity purification and tandem mass spectrometry. We identified a small set of proteins that specifically bound to active nelfinavir. The majority of these were endoplasmic reticulum (ER)-resident proteins involved in protein quality control and transport from ER to Golgi apparatus. In addition, individual proteins from mitochondria or the nucleus were identified. We thus hypothesized that nelfinavir interferes with intracellular protein transport, in line with its UPR-activating properties. Using a construct of ST-SBP-TNFa-EGFP engineered with an ER-retention signal (KDEL) that can be released upon biotin treatment of cells (RUSH-system), in conjunction with confocal microscopy and flow cytometry in U2OS cells, we show that nelfinavir, but not its analogue SC-451, which lacks proteasome inhibitor-sensitizing activity, retains TNFa-EGFP in the ER and prevents its transport to the Golgi and into the extracellular space. We confirmed in MM cells that active nelfinavir, but not SC-451, likewise retains newly synthesized IgA by preventing its secretion, and similarly decreases MHC class I export to the cell surface.Functionally depletion of the putative ER-targets of nelfinavir by siRNA in U2OS cells sensitized cells to nelfinavir and proteasome inhibitors. The mitochondrial targets of nelfinavir were identified as constituents of the mitochondria permeability transition pore (mPTP), a multimeric protein complex that regulates the permeability of mitochondrial membranes. The formation of reactive oxygen species (ROS) and inhibition of ABCB1 efflux, known for nelfinavir, can be functionally linked to mPTP modulation.In conclusion, we demonstrate that nelfinavir blocks export of newly synthetized protein from the ER, consistent with its activity-dependent binding to ER proteins. The block in protein export from the ER by nelfinavir is consistent with its ER stress-inducing, IRE1/XBP1-activating, and PI-sensitizing effect in the clinic. We further identify mPTP as an additional protein target of nelfinavir, consistent with its ROS-inducing and ABCB1-modulating activity. The block in ER protein export represents a novel mechanism of action to sensitize MM cells for proteasome inhibitor treatment. DisclosuresNo relevant conflicts of interest to declare.

High Glutathione and NAD(P)H Levels, and Increased Protein Folding Capacity Are Involved in Mediating Proteasome Inhibitor-Resistance in Multiple Myeloma

Background. Proteasome inhibitors (PI) are a backbone for the treatment of multiple myeloma (MM). However, most MM patients ultimately acquire PI-resistance and die from MM. The biology of PI resistance is poorly understood and established treatment options for PI-refractory MM are lacking. We have recently generated a next-generation in vitro model for PI-refractory MM, based on single MM cell-derived subclones with specific (50-100x) resistance to either bortezomib (AMO-BTZ) or carfilzomib (AMO-CFZ). These cells consistently show downregulation of IRE1/XBP1, a hallmark of BTZ-refractory primary MM cells, and do or do not carry the PSMB5 A310G mutation. Our recent quantitative proteomic analysis suggested that the PI-resistant phenotype of AMO-BTZ and AMO-CFZ is in particular characterized by massive metabolic changes that render MM cells largely independent from proteasome activity. To verify such metabolic adaptation as a hallmark of proteasome inhibitor resistance of MM, and to identify therapeutic targets, we performed whole metabolome profiling of proteasome inhibitor-sensitive AMO-1 cells in comparison to BTZ or CFZ-resistant clones from the same cell line.Methods. Metabolomic profiling was performed using Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS); data were quantified and normalized by Metabolon, Inc. Metaboanalyst v3.0 software was used for Metabolite Set Enrichment Analysis (MSEA). Reactive oxygen species (ROS) formation was determined by flow cytometry after staining the cells with H2DCFDA and exposure to 0.08% H2O2. Protein folding was determined by MERO-GFP construct, viability was determined by MTS assay.Results. Based on MSEA, PI-adapted cells showed substantial metabolic changes in glutathione metabolism, protein biosynthesis, malate/aspartate shuttle, metabolism of purines, pyrimidines, amino acids and tricarboxylic acid (TCA). Relative to AMO-1, in both AMO-BTZ and AMO-CFZ, glutathione (l-γ-Glutamyl-l-cysteinyl-glycine; GSH and GSSG) was consistently identified as the metabolite with the most significant quantitative increase (fold change (FC) relative to AMO-1 for GSH in AMO-BTZ=52.92; in AMO-CFZ=56.79; FC for GSSG in AMO-BTZ=36.92; in AMO-CFZ=54.17), while many other metabolites in glycine/serine and cysteine/methionine pathways that are associated with glutathione synthesis were decreased. Consistently, PI-adapted cells showed accumulation of nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) (FC for NAD+ in AMO-BTZ=55.81; in AMO-CFZ=2.16; FC for NADH in AMO-BTZ=19.10; in AMO-CFZ=1.65), presumably to maintain balance between reduced and oxidized glutathione, together with changes in TCA and malate/aspartate shuttle fueling the NAD(P)H production and transport, respectively. PI-resistant cells in addition showed altered carbohydrate metabolism and a shift in availability of membrane lipids, from lysolipids to sphingomyelins, in comparison to non-adapted cells.The adaptive changes in these cells correspond well to the proteomic changes previously described, confirming that the metabolic adaptive changes of PI-refractory MM cells are in particular in the regulation of redox homeostasis and protein folding. Functional experiments confirmed that the PI-adapted cells show larger capacity to buffer ROS production and that protein re-folding after DTT challenge is more effective in PI-refractory MM, compared to PI-sensitive cells. Consequently, co-treatment of PI-adapted cells with respective proteasome inhibitors (bortezomib and carfilzomib) in the presence of the inhibitor of protein-disulfide isomerase (PDI), 16F16, re-sensitized cells to proteasome inhibition (FC of IC50 for bortezomib or carfilzomib alone vs bortezomib or carfilzomib + 16F16 in AMO-BTZ=1.26 or 1.88, respectively; in AMO-CFZ=1.25 or 2.59 respectively; in AMO-1=1.1 or 0.95, respectively.Conclusion. PI refractory MM cells show massive metabolic changes in particular in glutathione and NAD(P)H production. This is associated with increased protein folding capacity and likely results in decreased proteasome load. PI-refractory MM cells can be re-sensitized to PI treatment by reducing glutathione levels or by affecting protein folding. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of p62-ZZ Domain-Mediated Signaling Overcomes Bortezomib Resistance in Multiple Myeloma Cells Independent of Their p53 Status

Multiple myeloma (MM) cells depend on autophagy as well as proteasome activity for their survival. In MM cells, autophagy is essential to reduce the extraordinary ubiquitinated protein system stress derived from high levels of immunoglobulin synthesis. Thus, proteasome inhibitors (PIs) decrease tumor burden in the majority of MM patients but eventually a PI resistant MM clone(s) arises. Bortezomib (BTZ) resistance has been reported to correlate with p53 status of MM cells. Previous studies showed that MM patients harboring p53 genetic abnormalities are commonly resistant to standard therapies, increasing the need for new druggable targets.A possible mechanism of BTZ resistance involves the cargo receptor for ubiquitin-mediated autophagy SQSTM1/p62, that enhances MM cells survival by increasing their autophagic flux. We found that MM cells with wild type p53, are more sensitive to PI treatment, while MM cells harbouring mutant p53 or lacking p53 are more resistant to BTZ-mediated cytotoxicity. BTZ treatment induced a modest level of apoptosis in MM1.S cells (p53 wild type) at high doses (>5nM) by triggering the cleavage of caspase 3 and PARP-1. As expected BTZ treatment induced a significant compensatory increase in SQSTM1/p62 at the mRNA and protein levels in these cells. Conversely, BTZ treatment of p53 mutant MM cells failed to induce apoptosis, exhibiting only a mild effect on viability. This occurred in spite of a similar increase in SQSTM1/p62 expression as seen in MM.1S cells.To determine the role of p62 in the BTZ resistance of p53 mutant MM cells, we tested a novel small-molecule p62-ZZ domain inhibitor, XRK3F2 we recently identified. XRK3F2 treatment of mice with established MM, dramatically increased bone formation in MM-bearing bones, although it did not affect tumour burden in vivo . In vitro studies showed that high concentrations (10μM) of XRK3F2 were required to decrease the viability of primary and MM cell lines and induce a time-dependent activation of caspases 3, 7 and 9. XRK3F2 treatment dose-dependently increased levels of the lipidated form of LC3, LC3II, suggesting activation of pro-survival autophagy. Surprisingly, the expression levels of SQSTM1/p62 were either unchanged or increased following XRK3F2 treatment despite the increase of autophagosome formation, indicating that XRK3F2 inhibited the autophagic flux in MM cells. Defective autophagy occurs in tumor cells and leads to accumulation of p62, further promoting tumorigenesis by altering NFκB regulation and gene expression. We found that XRK3F2 treatment also significantly inhibited TNFα-induced NFκB activation, by inhibiting p65 nuclear translocation. These data suggested that XRK3F2 may sensitize MM cells to BTZ-induced cytotoxicity and possibly overcome BTZ resistance.To test this hypothesis, human MM cell lines MM1.S (p53 wild type), U266 (p53 mutant) and KMS-11 (p53 null) were exposed to different doses of a BTZ-XRK3F2 combination comprised of varying concentrations of BTZ combined with XRK3F2 at 5μM. We found treatment with the BTZ-XRK3F2 combination significantly reduced cell viability compared with either drug alone at the same doses, and increased MM cells sensitivity to BTZ, independent of their p53 status (IC50 BTZ vs BTZ-XRK3F2 for MM1.S 3 vs 1.5nM of BTZ; for U266 10 vs 1.7nM of BTZ; for KMS-11 7 vs 1nM BTZ).Mechanistic studies revealed that in p53 wild type MM cells, the combination (Bortezomib 3nM - XRK3F2 5 μM) significantly increased annexin V positive cells after 6h of treatment. This was followed by a reduction in expression of the anti-apoptotic Bcl-2 protein (mRNA and protein levels) and extensive activation of caspase 3 and cleaved PARP1 after 24h, demonstrating apoptosis-mediated cell death. Importantly, p62 expression was significantly reduced by the BTZ-XRK3F2 combination. The decrease in p62 level may result as a consequence of its caspase 3-dependent degradation. In p53 mutant cells, the combination significantly induced PARP1 cleavage, although only mildly increased caspase 3 activation. These results imply that the BTZ-XRK3F2 combination activates multiple parallel death pathways to enhance both apoptosis and necrosis.Taken together, these data suggest that p62-ZZ antagonists, alone or in combination with PIs, may have both anti-tumor and bone anabolic effects, and may overcome PI resistance in MM cells, making p62 an attractive target for the treatment of MM. DisclosuresXie:Oxis Biotech: Consultancy. Roodman:Amgen Denosumab Trial: Membership on an entity's Board of Directors or advisory committees.

Targeting the Ubiquitin-Proteasome System for Cancer Therapeutics by Small-Molecule Inhibitors.

Simple SummaryThe ubiquitin-proteasome system regulates multiple facets of protein homeostasis to modulate signal transduction in numerous biological processes. Not surprisingly, dysregulation of this delicately balanced system is frequently observed in cancer progression. In the past two decades, researchers in both academia and industry have made significant progress in developing small-molecule inhibitors targeting various components in the ubiquitin-proteasome system for cancer therapy. Here, we aim to provide a comprehensive summary of these efforts. Additionally, we overview the advancements of targeted protein degradation, a recently emerging drug discovery concept in cancer therapy.The ubiquitin-proteasome system (UPS) is a critical regulator of cellular protein levels and activity. It is, therefore, not surprising that its dysregulation is implicated in numerous human diseases, including many types of cancer. Moreover, since cancer cells exhibit increased rates of protein turnover, their heightened dependence on the UPS makes it an attractive target for inhibition via targeted therapeutics. Indeed, the clinical application of proteasome inhibitors in treatment of multiple myeloma has been very successful, stimulating the development of small-molecule inhibitors targeting other UPS components. On the other hand, while the discovery of potent and selective chemical compounds can be both challenging and time consuming, the area of targeted protein degradation through utilization of the UPS machinery has seen promising developments in recent years. The repertoire of proteolysis-targeting chimeras (PROTACs), which employ E3 ligases for the degradation of cancer-related proteins via the proteasome, continues to grow. In this review, we will provide a thorough overview of small-molecule UPS inhibitors and highlight advancements in the development of targeted protein degradation strategies for cancer therapeutics.

REAL-WORLD OUTCOME IN THE PRE-CAR-T ERA OF MYELOMA PATIENTS QUALIFYING FOR CAR-T CELL THERAPY

BackgroundCAR-T cell therapy is likely to be introduced starting from 2021 in patients with relapsed/refractory myeloma (r/r MM) in Europe. In order to qualify for commercial CAR-T treatment, it is assumed that r/r MM patients will have to be exposed to at least three lines of previous treatments including lenalidomide, bortezomib and anti-CD38 treatment. However, the outcome of this particular subgroup of r/r MM patients is largely unknown whereas this knowledge is crucial to estimate the possible benefit of eventual CAR-T treatment.MethodsIn this non-interventional, retrospective single-center study, we analyzed all subsequent r/r MM patients treated between 01/2016 (when anti-CD38 treatment was commercially introduced in Switzerland) and 04/2020 at the University Hospital of Bern. Patients were eligible for the study if they had received at least three lines of treatment including one proteasome inhibitor (PI), one immunomodulatory drug (IMID) and one anti-CD38 antibody, and if they were in need of subsequent treatment and effectively received further lines of treatment.ResultsAmong 56 patients fulfilling the criteria of at least three lines of treatment including PI, IMID and anti-CD38 treatment, only 34 (60%) effectively received subsequent further therapy. This suggests that 40% of r/r MM patients never receive additional treatment after at least three lines of treatment including PI, IMID and anti-CD38 treatment. For patients receiving further treatment, the median number of previous lines of treatment was 4.5 (range 2–12), including autologous stem cell transplantation in 31 (91%) patients. 13 (37%) patients were penta-refractory. The most frequently used treatment options were IMID/dexamethasone treatment in 11 (32%) patients, followed by PI/dexamethasone in 10 (29%) patients. 21 (62%) patients received two or more additional lines of therapy. The median PFS was 6.6 months (range 0–36.6 months), the median TTNT was 7.5 months (range 1.4–24.5 months) and the median OS was 13.5 months, (range 0.1–38 months) for the first subsequent treatment. The overall response rate (ORR) to the first subsequent treatment was 41%, with a median duration of the response of 5 months (range 1–37 months). 12% of the patients achieved VGPR or better, with a median duration of response of 8 months (range 3–37 months).ConclusionsMyeloma patients refractory after at least three lines of anti-CD38/PI/IMID treatment have a poor prognosis with a PFS of 6.6 months and OS of 13.5 months. These data may serve as reference to compare the potential benefit of CAR-T treatment in this group of myeloma patients when available in the near future.

Modulating proteasome inhibitor tolerance in multiple myeloma: an alternative strategy to reverse inevitable resistance

BackgroundResistance to proteasome inhibitors (PIs) is a major obstacle to the successful treatment of multiple myeloma (MM). Many mechanisms have been proposed for PI resistance; however, our mechanistic understanding of how PI resistance is inevitably acquired and reversed remains incomplete.MethodsMM patients after bortezomib relapse, MM cell lines and mouse models were used to generate matched resistant and reversed cells. RNA sequencing and bioinformatics analyses were employed to assess dysregulated epigenetic regulators. In vitro and in vivo procedures were used to characterise PI-tolerant cells and therapeutic efficacy.ResultsUpon PI treatment, MM cells enter a slow-cycling and reversible drug-tolerant state. This reversible phenotype is associated with epigenetic plasticity, which involves tolerance rather than persistence in patients with relapsed MM. Combination treatment with histone deacetylase inhibitors and high-dosage intermittent therapy, as opposed to sustained PI monotherapy, can be more effective in treating MM by preventing the emergence of PI-tolerant cells. The therapeutic basis is the reversal of dysregulated epigenetic regulators in MM patients.ConclusionsWe propose an alternative non-mutational PI resistance mechanism that explains why PI relapse is inevitable and why patients regain sensitivity after a ‘drug holiday’. Our study also suggests strategies for epigenetic elimination of drug-tolerant cells.

MIF as a biomarker and therapeutic target for overcoming resistance to proteasome inhibitors in human myeloma

AbstractMultiple myeloma (MM) remains largely incurable despite significant advances in biotherapy and chemotherapy. The development of drug resistance is a major problem in MM management. Macrophage migration inhibitory factor (MIF) expression was significantly higher in purified MM cells from relapsed patients than those with sustained response, and MM patients with high MIF had significantly shorter progression-free survival (PFS) and overall survival (OS). MM cell lines also express high levels of MIF, and knocking out MIF made them more sensitive to proteasome inhibitor (PI)-induced apoptosis not observed with other chemotherapy drugs. Mechanistic studies showed that MIF protects MM cells from PI-induced apoptosis by maintaining mitochondrial function via suppression of superoxide production in response to PIs. Specifically, MIF, in the form of a homotrimer, acts as a chaperone for superoxide dismutase 1 (SOD1) to suppress PI-induced SOD1 misfolding and to maintain SOD1 activity. MIF inhibitor 4-iodo-6-phenylpyrimidine and homotrimer disrupter ebselen, which do not kill MM cells, enhanced PI-induced SOD1 misfolding and loss of function, resulting in significantly more cell death in both cell lines and primary MM cells. More importantly, inhibiting MIF activity in vivo displayed synergistic antitumor activity with PIs and resensitized PI-resistant MM cells to treatment. In support of these findings, gene-profiling data showed a significantly negative correlation between MIF and SOD1 expression and response to PI treatment in patients with MM. This study shows that MIF plays a crucial role in MM sensitivity to PIs and suggests that targeting MIF may be a promising strategy to (re)sensitize MM to the treatment.

High Rates of Varicella Zoster Virus Antibody Seroconversion and Persistence after Administration of the Adjuvanted, Recombinant Varicella Zoster Vaccine in Multiple Myeloma Patients Undergoing Active Treatment

Multiple myeloma (MM) patients are predisposed to high rate of infectious complications, with viral infections and in particular varicella zoster virus infection having a more than 10-fold increased incidence compared to the general population. This risk increases with use of autologous stem cell transplant (ASCT), multiple lines of therapy for relapsed disease, and proteasome inhibitor (PI) or daratumumab treatment. An adjuvanted, recombinant vaccine, consisting of the varicella-zoster virus glycoprotein E antigen (Shingrix, GlaxoSmithKline; Triangle Park, NC) is now available but studies in MM patients have been limited to the post-ASCT setting. We aimed to analyze the initial seroconversion rates as well as persistence of seropositivity in our MM cohort undergoing active anti-myeloma treatment. We evaluated 85 MM patients undergoing active anti-myeloma treatment who were tested at baseline for VZV antibody (Ab) status. Nine (10.6%) patients were found to have high risk cytogenetics at diagnosis. The median number of prior lines of therapy was 1 (1-10), with 80 (94.1%) patients receiving a prior or current PI-based regimen, 66 (86.8%) receiving ASCT, and 15 (19.7%) receiving a prior or current daratumumab-based regimen. At the time of first vaccination, 35 (41.1%) patients were receiving post-ASCT immunomodulator (IMiD) maintenance. Seven (8.2%) patients had a prior history of VZV infection. 67 (78.8%) patients were actively receiving antiviral prophylaxis with either acyclovir or valacyclovir. To determine vaccine immunogenicity, we used an anti-VZV ELISA assay. Using this test, VZV Ab seropositivity was measured at baseline and after each of the 2 vaccinations. In the entire cohort, 41 (48.2%) patients were found to be VZV Ab negative at baseline. Multivariate modified Poisson regression models with adjustment for prognostic and clinical factors were used to estimate relative risk (RR) and 95% confidence intervals for baseline VZV Ab seropositive status. We identified ISS stage 2 (RR 0.31, 95% CI 0.21-0.61; p&amp;lt;0.001) or 3 (RR 0.52, 95% CI 0.32-0.86); p=0.010) disease and prior/ current daratumumab (RR 0.35, 95% CI 0.13-0.95; p=0.04) as strong predictors of baseline VZV seronegativity; IMiD maintenance (RR 2.15, 95% CI 1.00-4.65; p=0.05) and prior shingles (RR 2.61, 95% CI 1.24-5.51; p=0.011) were associated with VZV seropositivity. Sixty-two (72.9%) patients received one vaccine dose, 49 (57.6%) received 2 doses, and 3 (3.5%) refused the vaccine. The median time between both doses was 84 (53-189) days. Overall rates of seropositivity increased after 1 (87.9%; p=0.0002) and 2 (92.6%; p=0.0001) doses. Seroconversion from a baseline negative to positive test was observed in 16 (76.2%) and 23 (95.8%) patients after 1 and 2 doses, respectively. One patient who received prior cytotoxic chemotherapy failed to seroconvert after 2 doses, and 4 patients lost VZV seropositivity after a median of 348.5 days from the second vaccination dose. In a multivariate model, we observed that progressive disease and daratumumab treatment during time from vaccination to follow up VZV testing predicted for loss of VZV seropositivity. Of 42 patients on acyclovir after receiving both doses, 18 (42.9%) discontinued acyclovir with all patients but one remaining seropositive at most recent follow up. We report the first real world experience of the use of the adjuvanted, recombinant zoster vaccine in MM patients receiving active treatment. We show a high rate of initial VZV seronegativity and were able to show significant seroconversion after a single vaccine dose. In fact, despite studies showing benefit in patients receiving a total of 4 doses, we observed high seroconversion rates using 2 doses. Finally, we report for the first time strong predictors of baseline VZV seronegativity and maintenance of seropositivity in MM patients, in particular treatment with daratumumab. On the other hand, PI treatment was not associated with baseline seronegativity or loss of seropositivity. This data confirms that post-ASCT vaccination would be the optimal time to vaccinate patients, in particular when patients are immunocompetent while in remission on maintenance. The benefit of administering the vaccine in patients undergoing daratumumab or other treatment for progressive disease was not shown here and continued antiviral treatment rather than vaccination is likely to be the ideal prophylaxis strategy. Disclosures Calip: Flatiron Health: Current Employment. Patel:Amgen: Consultancy; Janssen: Consultancy; Celgene: Consultancy.

Comparison of 140mg/m2 vs 200mg/m2 Dose of Melphalan in Myeloma Autograft - a Single Centre Experience from Chennai, India

Introduction: - Melphalan at a dose of 200mg/m2 is considered the standard autograft dose in patients with multiple myeloma without significant co-morbidity. This drug was formulated in the early years before the era of novel agent induction and maintenance. Considering the deeper remission of novel agents and prevailing refractory gram-negative sepsis in most Indian setups, we compared 140mg/m2 (MEL140)vs. 200mg/m2(MEL200) of Melphalan in Indian myeloma patients who achieved a complete remission before transplant without having any significant co-morbidity. Methods and results: - We analysed myeloma autograft patients from September 2014 to August 2017. 42 patients received MEL200, and 43 patients received MEL140. Diabetes on oral medications was randomised equally on both sides. The median age of patients in MEL200 was 52 years, and MEL140 was 53.6 years. There were 60% and 55% of males in MEL200 vs. MEL140, respectively. ISS-III patients was 48% and 49% in MEL200 vs. MEL140, respectively. The standard induction regimen was three to four cycles of RvD chemotherapy. Around 16% of patients in the MEL140 group had a creatinine clearance between 30-50ml/min. The mobilisation regimen followed was five days of GCSF based mobilisation. The stem cell cut-off was kept to a minimum of 2 x 106/CD34+ve cells/kg body weight. Overall, 12% of patients in both groups needed a second-day stem cell collection. Three-year overall survival, progression-free survival, the incidence of relapse, rate of refractory sepsis, the time difference in hematopoietic recovery, and secondary cancers were compared between two groups. In outcome analysis, there was no transplant-related mortality in both groups. The mean day of engraftment was around Day +9 in MEL140 vs. Day +11 in MEL200. The rate of Gram-Negative Bacilli sepsis was around 14% in MEL140 vs. 27% in Mel200 (p 0.02). These findings were reflected in the use of high-end antibiotics like Colistin and Fosfomycin in such patients. There was a delay in engraftment, which may have been confounded by sepsis. The median progression-free survival was 26.1 months in MEL200and 27.2 months in the MEL140 group. The cumulative incidence of relapse at three years was not significantly different between the Mel200 (33.3%) and Mel140 (34.9%) groups. The adjusted HR for relapse was 0.98. Subgroup analysis suggests that in overall survival, progression-free survival, and cumulative incidence of relapse, there was no significant advantage associated with Melphalan 200 mg/m2. Age, renal status, proteasome inhibitor treatment, gender, or Karnofsky score did not interact with overall/progression-free survival or relapse rate in the two melphalan dose groups. One patient in the MEL200 group developed -5q deletion Myelodysplastic syndrome after five years of transplant. Discussion: This study was conducted taking into account the changing scenario in myeloma induction and maintenance to novel agents and increasing rate of resistant gram-negative sepsis in Indian febrile neutropenic patients. As we can achieve a deeper remission before transplant, we formulated to compare lower dose melphalan to prevent a stormy post-transplant course and prevent patients succumbing to sepsis. This may not be an issue in western countries where gram-negative sepsis rates are lower. The biggest worry for us is not compromising the myeloma outcome due to lower doses of Melphalan in fit patients who achieve complete remission. This study concluded that there is not much of a difference in overall survival, Progression-free survival, and cumulative incidence of relapse at three years among MEL200 vs. MEL140. Conclusion: - We conclude that Melphalan autograft at a dose 140mg/m2 can achieve comparable responses to 200mg/m2 in Indian myeloma patients who are taken to transplant post complete remission. We need a more prominent multicentric study and a longer follow-up to recommend MEL140 as a standard of care in Indian settings.

RAR Gamma Activation Sensitizes Human Myeloma Cells to Carfilzomib Treatment through OAS-RNase L Innate Immune Pathway

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) kill MM cells by disrupting the degradation of misfolded proteins, presumably derived from high-level immunoglobulin production, which provides an appealing explanation for why MM cells are so uniquely sensitive to PIs. However, relapses are frequent and acquired resistance to PI treatment emerges in most patients. Therefore, identifying novel and safe drugs overcoming PI resistance in MM will aid in chemo-(re)sensitization, reducing PI-induced side effects, and maximizing the outcomes of PI therapy. Here we performed a high-throughput screen of 1855 FDA-approved drugs (Figure 1) and identified all-trans retinoic acid (ATRA), a classic pan-retinoic acid receptor (RAR) agonist used successfully to treat acute promyelocytic leukemia (APL), as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and re-sensitized Cfz-resistant MM cells to Cfz in vitro. To determine which RARs are important for ATRA enhancement of Cfz-induced apoptosis in MM, esiRNAs of RARs for knocking down RARs and selective agonists of RARs were used in Cfz-treated MM cells. We identified that RARγ activation is important for ATRA sensitizing MM cells to Cfz treatment. To determine which signaling pathways are involved in ATRA-treated MM cells, gene-profiling data analysis of Cfz- versus ATRA+Cfz-treated MM cells was performed and real-time PCR was used to validate the microarray results. We found that ATRA treatment activated IFN-β response pathway (Figure 2), leading to upregulated expression of IRF1 and OAS1-3. Interestingly, similar to ATRA, IFN-β, which alone did not induce MM apoptosis, enhanced Cfz-induced MM cell apoptosis. Furthermore, using RNA integrity assay and dsRNA detection assay, we identified that ATRA treatment elevated the expression of OASs, which synthesized 2-5A upon binding to dsRNA induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death (Figure 3). By knocking down each gene of OAS1-3, we demonstrated that OAS1 is essential for ATRA to sensitize MM cells to Cfz-induced apoptosis. Furthermore, we determined the impact and significance of RARγ and OAS1 in human MM pathogenesis and drug response by analyzing the gene-profiling data of 264 MM patients from Mulligan et al. datasets and 1,143 MM patients from MMRF coMMpass study IA13. In support of these findings, analyses of the large patient's gene-profiling datasets showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment (Figure 4). Finally, BMS961, a selective RARγ agonist, similar to ATRA, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo (Figure 5). Thus, this study highlights the potential for RARγ agonists to sensitize MM and overcome MM resistance to Cfz treatment in patients. Disclosures No relevant conflicts of interest to declare.

Effect of Prior Treatment with Proteasome Inhibitors on the Efficacy and Safety of Once-Weekly Selinexor, Bortezomib, and Dexamethasone in Comparison with Twice-Weekly Bortezomib and Dexamethasone in Relapsed or Refractory Multiple Myeloma: Subgroup Analysis from the Boston Study

Effect of Prior Treatment with Proteasome Inhibitors on the Efficacy and Safety of Once-Weekly Selinexor, Bortezomib, and Dexamethasone in Comparison with Twice-Weekly Bortezomib and Dexamethasone in Relapsed or Refractory Multiple Myeloma: Subgroup Analysis from the Boston Study

In-Class Transition (iCT) from Parenteral Bortezomib to Oral Ixazomib Proteasome Inhibitor (PI) Therapy Increases the Feasibility of Long-Term PI Treatment and Benefit for Newly Diagnosed Multiple Myeloma (NDMM) Patients in an Outpatient Setting: Updated Real-World Results from the Community-Based United States (US) MM-6 Study

In-Class Transition (iCT) from Parenteral Bortezomib to Oral Ixazomib Proteasome Inhibitor (PI) Therapy Increases the Feasibility of Long-Term PI Treatment and Benefit for Newly Diagnosed Multiple Myeloma (NDMM) Patients in an Outpatient Setting: Updated Real-World Results from the Community-Based United States (US) MM-6 Study

Abstract 6426: Novel mechanism of PIM2 inhibition leads to significant effect on solid tumors

Abstract Background: PIM2 is overexpressed in hematopoietic and solid cancers, and is involved in multiple signaling pathways. In hematologic malignancies, PIM2 has been shown to be a pro-survival factor and demonstrated to be a possible therapeutic target. However, the role of PIM2 as a therapeutic target in solid cancers has not been fully elucidated. Methods: PIM2 expression was compared between normal and cancer tissues in The Cancer Genome Atlas (TCGA) datasets. Pan-PIM inhibitor treatment, as well as PIM2 overexpression/knockdown technique was utilized to investigate the PIM2 role in solid cancers. Multiple cancer models were utilized for in vitro and in vivo experiments to investigate if PIM2 as a therapeutic target and examine mechanism of anti-tumor activity by JP11646. Results: PIM2 was overexpressed in several types of cancer tissues, including breast, esophageal, head-neck, kidney, liver and endometrial cancers in TCGA. PIM2 overexpression promoted tumor growth in in vivo breast cancer mouse model. PIM2 inhibition by siRNA as well as pan-PIM inhibitor JP11646 activated apoptosis as seen by increased cleaved PARP. JP11646 treatment inhibited cancer cell proliferation in a dose-dependent manner in vitro in various types of solid cancers, including head-neck, ovarian, breast, prostate, liver, pancreatic, colorectal and non-small cell lung cancers. Interestingly, treatment with pan-PIM inhibitor JP11646 resulted in selective downregulation of PIM2 protein expression, but not PIM1 or PIM3 in breast cancer cell lines, BT549 and MDA-MB-231. The treatment also resulted in downregulation of phosphorylated downstream targets, 4EBP1 and TSC2 in breast cancer cells. However, additional proteasome inhibitor treatment blocked PIM2 downregulation and apoptosis activation was not seen. The result suggests that proteasome complex is required for PIM2 degradation which is necessary to induce apoptosis. JP11646 treatment inhibited tumor growth in breast, liver, pancreas, and non-small cell lung cancer in vivo xenograft models and was compared to multiple standard of care treatments showing advantage in lung tumors. Conclusion: We found that PIM2 promoted cancer progression in solid tumors. We show novel mechanism of JP11646 targeting PIM2 for degradation and induction of apoptosis. We show that PIM2 may serve as a novel therapeutic target for multiple types of solid cancers with enhanced therapeutic effect through protein degradation rather than kinase inhibition alone. Citation Format: Eriko Katsuta, Kazuaki Takabe, Alex Adjei, Mateusz Opyrchal. Novel mechanism of PIM2 inhibition leads to significant effect on solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6426.

Abstract 800: Exploiting the proteasome to overcome RAS and WT1 mutation mediated resistance to FLT-3 inhibition in acute myeloid leukemia

Abstract FLT3 mutations (mFLT3) in acute myeloid leukemia (AML) carry a poor prognosis.The mandatory co-occurrence of other driver mutations with mFLT3 leads to suboptimal clinical responses with single agent FLT3 inhibitors (FLT3i). Pre-existing or emergent RAS mutations are one mechanism of primary and secondary resistance to single agent FLT3i. Therefore development of combinatorial strategies to overcome resistance conferring co-mutations is critical. Herein we report a novel in-vitro combination of the FLT3/SYK inhibitor (FSI) TAK-659 with Proteasome inhibitor (PI) ixazomib, to co-operatively inhibit proliferation of mFLT3 and RAS or WT1 co-mutated AML blasts, which are otherwise less sensitive to Flt3i and FSI. Peripheral blood or bone marrow derived cells from patients with AML were isolated and treated in vitro with TAK-659 and Ixazomib alone or in combination. Cytosolic/nuclear protein lysates were obtained at 12 hours of drug/control treatment and western blotting performed for proteins of interest. Tritiated thymidine proliferation assays were also performed with titrated doses of each agent alone, and combination of fixed dose of one agent with titration of the second. Patient samples were annotated for mutations by Next Generation Sequencing. Similar experiments were carried out on cell lines MV411 (Flt3ITD/MLL), THP-1 and OCI-AML3 (both NRAS). Proliferation assays on a series of primary AML samples revealed an inherent dichotomy in response patterns. mFLT-3 samples with co-mutations in DNMT3A, NPM1 or tMLL were very sensitive to TAK-659 (IC50 &amp;lt;75 nM) but much less sensitive to Ixazomib alone (IC50&amp;gt;100nM). The co-occurrence of RAS or WT1 mutations predicted for resistance to TAK-659 but interestingly sensitized cells to PI alone and in combination. In fact, presence of RAS mutations irrespective of FLT3 status predicted for sensitivity to PI (IC50&amp;lt;50nM). We also observed sensitivity to Ixazomib in THP-1 and OCI-AML3, while MV4-11 was sensitive to TAK-659 but relatively resistant to Ixazomib alone. Based on genome-wide CRISPR screen, overactivity of RAC-PAK signaling mediated by PREX1 is essential to RAS mutated AML (T Wang etal. Cell 168:890, 2017). Similarly, WT1 mutation in AML is known to ablate expression of a major nuclear chaperone for beta-catenin, TBL1X, and RAC has been found to be a substitute (Y Wang et al. Mol Cell. 57:662, 2015). We therefore hypothesized that PI decreased nuclear availability of RAC1 along with its chaperone partner beta-catenin - a determinant of leukemic stem cell activity, also regulated by RAC/PAK and the proteasomal system. Indeed, in primary RAS or WT1 mutated AML samples, we observed PI treatment led to significant reduction in nuclear RAC1 levels and decreased nuclear beta-catenin levels, specifically PAK phosphorylated p675 active beta-catenin. These findings suggest PI effects RAC1, PAK and beta-catenin simultaneously. Similar results were also noted in RAS mutant cell lines. siRNA knockdown experiments also linked p62 SQSTM1 in a pathway to RAS/RAC/PAK and beta-catenin. In summary, we delineate a RAS/RAC/PAK/Beta-catenin pathway to be linked to critical events in RAS or WT1 mutated AML progression and resistance. FLT3 mutated AML with these co-mutations are insensitive to single-agent activity of FLT3i or FSI alone and the addition of PI is critical to overcome them. Citation Format: Sravanti Rangaraju, Santosh Kumar Pasupuleti, Silvia Marino, Daniela Nicoleta Petrusca, Larry D. Cripe, Bhaskar Ramdas, Lakshmi Reddy Palam, Katie J. Sargent, Hamid Sayar, Heiko Konig, Reuben Kapur, David G. Roodman, Scott H. Boswell. Exploiting the proteasome to overcome RAS and WT1 mutation mediated resistance to FLT-3 inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 800.

Loss of NF-E2 expression contributes to the induction of profibrotic signaling in diabetic kidneys

AimsThis study aimed to examine the anti-fibrotic role of Nuclear Factor-Erythroid derived 2 (NF-E2) in human renal tubule (HK-11) cells and in type 1 and type 2 diabetic (T1D, T2D) mouse kidneys. Main methodsAnti-fibrotic effects of NF-E2 were examined in transforming growth factor-β (TGF-β) treated HK-11 cells by over-expressing/silencing NF-E2 expression and determining its effects on profibrotic signaling. NF-E2 proteasomal degradation was confirmed by proteasome inhibition in HK-11 cells and diabetic mice. Clinical relevance of changes in NF-E2 expression to fibrotic changes in the kidney were assessed in T1D and T2D mouse kidneys. Key findingsNF-E2 expression was significantly decreased in TGF-β treated HK-11 cells and in kidneys of diabetic mice with concurrent increase in expression of fibrotic proteins. TGF-β treatment of HK-11 cells did not inhibit NF-E2 mRNA expression, suggesting that the post-translational changes may contribute to NF-E2 protein degradation. The down-regulation of NF-E2 expression was attributed to its proteasomal degradation, as TGF-β- and diabetes-induced NF-E2 down regulation was prevented by proteasome inhibitor treatment. In HK-11 cells TGF-β treatment decreased E-cadherin expression and induced pSer82Hsp27/NF-E2 association, likely to promote NF-E2 degradation, as Hsp27 can target proteins to the proteasome. A critical role for NF-E2 in regulation of renal fibrosis was demonstrated as over-expression of NF-E2 or silencing NF-E2 expression, decreased or increased profibrotic proteins in TGF-β-treated HK-11 cells, respectively. SignificanceNF-E2, a novel anti-fibrotic protein, is down-regulated in diabetic kidneys. Preserving/inducing NF-E2 expression in diabetic kidneys may provide a therapeutic potential to combat DN.