Immunomodulatory drug (IMiD) resistance is a key clinical challenge in myeloma treatment. Previous data suggests almost one third of myeloma patients acquire mutations in the key IMiD effector cereblon by the time they are pomalidomide refractory. Some events, including stop codons/frameshift mutations and copy loss, having clearly explicable effects on cereblon function. Missense mutations have also been reported throughout the length of cereblon but their functional impact has not been systematically studied. This study modelled selected missense mutations and examined their effect on cereblon function also analysing whether any mutations deleterious to IMiD action could be overcome using the novel cereblon binding agents (CELMoDs). Three patterns of response to missense mutations were apparent, mutations that led to complete loss of CRBN function for all agents, those that had no effect on CRBN function and those with agent-dependent effect on CRBN function. The latter group of 4 mutations were profiled in more detail with confirmatory experiments demonstrating an ability of the more potent CELMoDs to lead to neosubstrate degradation and cell death even though IMiDs were not active. Dynamic modelling based on a newly generated crystal structure of the DDB1/CRBN/lenalidomide complex, with greater resolution than those published to date, helped to understand the impact of these mutations. These results have important implications for the interpretation of CRBN sequencing results from patients for future therapy decisions, particularly differentiating those who may, despite relapsing on IMiDs with CRBN mutations, have the potential to still benefit from the use of CELMoD agents.

Introduction Immunomodulatory drugs (IMiDs) such as Lenalidomide (Len) have a direct impact on myeloma (MM) cells by functioning as a molecular glue, binding to a CRL4CRBN E3 ubiquitin ligase (cereblon) and leading to degradation of neo-substrates including ikaros (IKZF1) and aiolos (IKZF3). This results in downregulation of IRF4/MYC and MM cell death. IMiDs have also been shown to have direct action on the immune system, including T cells via upregulation of IL-2. We hypothesized that the enhanced degradation of neo-substrates in the tumour cell could be associated with a third mechanism of action, through altered antigen presentation of the neo-substrate degradation products as peptides via MHC class I (MHC-I), leading to upregulation of CD8+ T cell immunosurveillance. Additionally, we hypothesized that the novel cereblon binding agents (CELMoDs), such as Mezigdomide (Mezi), would lead to enhanced altered antigen presentation and a greater induced T cell response compared to IMiDs. Methods H929 cells were treated with either 10uM Len, 0.01uM Mezi or DMSO. IKZF1/IKZF3 degradation was measured via Western blot, after 2 and 24hrs. Global HLA levels were measure via flow cytometry after 24 hours of drug exposure with a pan HLA-ABC antibody. Subsequently, 1x10^8 H929 cells per condition were incubated with 10uM Len, 0.01uM Mezi or DMSO for 2 hours and 24hours. Cells were harvested and lysed in 1% CHAPS buffer. MHC-I peptide complexes were then isolated via incubation with bead/antibody complex, and the peptides then eluted and dried. MHC-peptides were labelled with tandem mass tags (TMT) to facilitate relative quantification via mass spectrometry (TMT-MS2). Predicted binding was analysed using NetMHC website. Normalised peptide abundance values were compared to their relative DMSO control using Log2 fold change (FC). Results Degradation of IKZF1 and IKZF3 was confirmed at 2 and 24hrs. There was greater degradation with Mezi compared to Len and at the longer time point. Global MHC-I expression did not change following incubation with Len and Mezi, as seen by flow. This indicates that any upregulated peptides subsequently identified by MS were not due to upregulation of MHC-I itself. After isolation of MHC-I bound peptides and MS identification, a total of 7809 peptides (between 8-15 amino acids in length) from 3901 protein sources were identified across all samples. Most of the peptides were 9-mers (53.7%), consistent with the literature for MHC-I isolated peptides. 82% of peptides had high predicted binding for all the MHC-I alleles for H929 (HLA-A03:01, HLA-A24:02, HLA-B07:02, HLA-B18:01, HLA-C07:02). All peptides with high predicted binding and consistently upregulated by a log2FC of at least 0.3 in all three replicates were considered further. Both Len and Mezi led to enhanced antigen presentation compared to DMSO, with more unique peptides presented at 24hrs vs 2hrs and with Mezi vs Len (Len 2hrs 2 peptides, Len 24hrs 24 peptides, Mezi 2hrs 10 peptides, Mezi 24hrs 40 peptides). 3 peptides (derived from proteins IKZF3, TMEM147 and ITGB7) were seen to have an increased expression across more than one treatment condition, and3 additional peptide sequences (derived from proteins APOL6 and ITGB7) corresponded to the same protein source across different treatment conditions. All peptides derived from the IKZF3 and IKZF1 proteins were explored in more detail. Across the dataset 4 distinct peptides from IKZF1 (x1) and IKZF3 (x3) were identified. Their pulldown was enriched after incubation with Len (IKZF1: mean Log2 FC 2hrs -0.04, 24 hrs 0.44, IKZF3 mean Log2 FC 2hrs -0.12, 0.07, 0.34, 24 hrs -0.09, 0.16, 0.40) and Mezi (IKZF1: mean Log 2 FC 2hrs 0.3, 24hrs 0.43, IKZF3 mean Log2 FC 2hr 0.17, 0.49, 0.53, 24 hrs 0.18, 0.43, 0.63) compared to DMSO control, most marked for the 24hour time point and with Mezi. These, and the additional peptides of interest from protein sources TMEM147, ITGB7 and APOL6 will be analysed in immunogenicity assay to confirm stimulation of CD8+ T cells. Discussion Our data suggest that myeloma cells can present peptides from the known neo-substrates IKZF1 and IKZF3, along with additional peptides, after IMiD/CELMoD therapy via MHC-I. This occurred to a greater extent with the CELMoD Mezi compared to the IMiD Len. Experiments to assess the immunogenicity of the presented peptides are ongoing. These data highlight a potential novel mechanism by which IMiDs and CELMoDs may stimulate the immune system.

2017 to 2022. Treatment drop-out decreased in 2L to 3L and 3L to 4L+, with a drop-out rate of 17% and 19% per switch subgroup, respectively, in 2022. Main 1L regimens in SCT-E (VCd and VTd) and SCT-NE subgroups (VMP and VCd) remained stable, with daratumumab-based regimens increase in 2022 in all subgroups.Patient share in maintenance therapy increased from 5% to 16% in the overall treated population, dominated by lenalidomide in 2022.The 2L and 3L treatment pattern changed considerably, with daratumumab-based regimens dominating patient share in 2022 (2L-43%; 3L-35%).In 4L+ a heterogenous treatment pattern emerged, with no standard of care identified, being carfilzomib-and daratumumab-based regimens the most relevant in 2022.Overall median TD increased in SCT-E subgroup from 5 to 19 months between time series, driven by maintenance therapy, and remained stable for SCT-NE patients (10 months).Regarding TTNT, median time increased between 3L to 4L+ in SCT-E patients from 5.5 to 9 months.Conclusions: MM treatment landscape changed during a 5-year period, characterized by an increase in treated patients, patients achieving advanced treatment lines, and improved access to therapeutic innovation in earlier treatment stages of the disease.

Introduction Immunomodulatory agents (IMiDs) are effective therapies for multiple myeloma (MM), approved for use at all stages of disease. IMiDs function as molecular glues at cereblon (CRBN), part of the CRL4 CRBN E3 ubiquitin ligase complex, designating neo-substrates for proteasomal degradation. In myeloma cells the B cell transcription factors Ikaros and Aiolos are degraded, leading to downregulation of IRF4 which is critical for myeloma cell survival. Novel CRBN ligase modulators (CELMoDs) are currently in early phase clinical trials and have demonstrated activity in patients refractory to IMiDs, but resistance inevitably develops over time. EZH2 is a histone methyltransferase which acts at histone 3 lysine 27 (H3K27) leading to trimethylation (H3K27me3). The aberrant expression of EZH2 leads to abnormal gene repression, contributing to tumour development and progression. EZH2 inhibitors (EZH2i) have been shown to overcome drug resistance in several cancers and we hypothesized that combining EZH2i with IMiDs/CELMoDS would overcome resistance to these agents in MM cell lines. Methods MM cell lines KMS11, RPMI and MOLP8 were used as models of IMiD/CELMoD resistance and MM.1S as a control model of sensitivity. The isogenic pair of KMS11 with CRBN knockout (KMS11 CRBN KO) was established using CRISPR-Cas9. Following concentration/duration optimisation, cell lines were treated with EZH2i (Tazemetostat) 0.25-1µM or DMSO control for 5 days alone, and then in combination with IMiD (Lenalidomide, Len, 0-20 µM, Pomalidomide, Pom, 0-8 µM) or CELMoD (Iberdomide, CC-220, 0-2 µM and Mezigdomide, CC-92480, 0-0.1uM) for a further 5 days. Cell viability was measured using CellTiter Blue® and the level of synergy assessed using SynergyFinder (Bliss model). The effect on apoptosis and cell cycle was examined using flow cytometry after AnnexinV-PI and/or DAPI staining respectively. To elucidate the mechanism of synergy, the effect of the single agents and combinations on key CRBN pathway effectors (CRBN, Ikaros/Aiolos, IRF4) were examined using WB and qPCR. ChIP-PCR was employed to assess the changes in enrichment of H3K27me3 and key MM transcription factors (Ikaros/Aiolos) within the IRF4 promoter region upon EZH2i treatment. Results MM.1S cells were sensitive to IMiDs (Len/Pom) and CELMoDS (CC-220/CC-92480) with GI50s of 0.279 µM, 0.08 µM, 2 nM and 0.3 nM respectively. KMS11, MOLP8 and RPMI cell lines were resistant at more than 10x the GI50 in MM.1S. In these models of resistance, the cells express functional CRBN protein, demonstrated by the degradation of neo-substrates Ikaros/Aiolos. However, whilst their degradation results in reduced IRF4 expression and cell death in sensitive cells (e.g. MM.1S), this does not occur in the resistant models suggesting an uncoupling of Ikaros/Aiolos control of IRF4 expression in the resistant setting. Combining EZH2i with IMiDs/CELMoDs led to a reduction in cell viability (e.g. CC-220 shown in Fig.1A) and demonstrated synergy across resistant cell lines (KMS11, MOLP8 and RPMI). An increase in apoptosis was seen using AnnexinV/PI staining, with no demonstrable change in cell cycle. WB and qPCR results showed a significant reduction in IRF4 mRNA and protein with the combination treatment. The combination of EZH2i and IMiD/CELMoDs did not reduce cell viability or IRF4 expression in the KMS11 CRBN KO cell line ( Fig.1A), indicating that the observed synergistic effect is CRBN-dependent. Compared to DMSO control, EZH2i increased IRF4 mRNA, with a reduction of H3K27me3 enrichment observed within the IRF4 promoter region by ChIP-PCR in KMS11 ( Fig.1B). In addition, EZH2i also enhanced Ikaros and Aiolos enrichment within the IRF4 promoter region ( Fig.1B). Conclusions Our results suggest that combining EZH2i with IMiDs/CELMoDs can overcome resistance to these agents in MM cell line models, with synergy that is CRBN-dependent. By examining the key components of the CRBN pathway we identified that EZH2i reduced H3K27me3 and increased Ikaros and Aiolos association at the IRF4 promoter, suggesting a possible re-coupling of Ikaros/Aiolos to IRF4 expression, which may be responsible for reinstating IMiD/CELMoD activity, driving the synergistic effect seen.

Immunomodulatory agents (IMiDs) and cereblon E3 ligase modulators (CELMoDs) are a cornerstone of Multiple Myeloma (MM) treatment. They bind to the cereblon (CRBN) component of the CRL4 E3 ubiquitin ligase complex and designate a new set of substrates for proteasomal degradation. Most patients initially respond well to IMiD/CELMoD therapy but over time will become resistant. Understanding resistance mechanisms will enable the development of new targeted treatment strategies to overcome this pressing clinical challenge. Acquired IMiD/CELMoD resistant human MM cell lines were generated by treating IMiD/CELMoD sensitive MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Cell lines were characterized by whole exome sequencing, RNA-Seq and proteomics. All resistant lines had reduced CRBN expression and some had CRBN mutations, reflecting patient data. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s. Gene effect scores were calculated with the Chronos algorithm and compared to parental MM1s using data from the Broad Institute DepMap portal. Potential new dependencies in the resistant setting (defined as gene effect score ←1 in resistant cells and >-0.5 in parental cells) were identified in 47 genes including SETD2, a histone 3 lysine 36 methyltransferase (H3K36me2 → H3K36me3). The specific SETD2 inhibitor EPZ-719 reduced viability to a greater extent in the IMiD/CELMoD resistant lines compared to their controls in a 14-day trypan blue exclusion assay. This was most pronounced in the Pom- and Iber-resistant H929 lines. For example in Iber-resistant H929 compared to its control line the viability with EPZ-719 was 13% vs 66% at 1µM, 5% vs 46% at 5µM and 2% vs 45% at 10µM, p<0.0001 (% viability compared to DMSO control at day 14). To investigate whether this novel dependency was related to reduced CRBN expression a CRBN knockout MM1s cell line was generated using CRISPR/Cas9. These cells showed a markedly greater reduction in viability with EPZ-719 compared to control MM1s (viability 20% vs 32% at 1µM, 8% vs 25% at 5µM and 2% vs 18% at 10µM, p<0.001). A reduction in H3K36me3 was seen on immunoblotting after EPZ-719 incubation across all cell lines suggesting an on-target effect. ChIP-Seq and RNA-Seq experiments are underway to identify downstream mechanisms. In conclusion, SETD2 inhibition is more active in IMiD/CELMoD-resistant cell lines compared to sensitive counterparts. Rapid clinical translation of these findings would be possible as a SETD2 inhibitor is currently in a phase 1 trial which includes MM patients. Previous pre-clinical data suggests the t(4;14) MM subgroup may be more sensitive to SETD2 inhibition but our data highlights a novel vulnerability in the IMiD/CELMoD resistant setting that should be explored further. Citation Format: Sarah Anne Bird, Marco Licciardello, Yakinthi Chrisochoidou, James Smith, Amy Barber, Jack Cheung, Yura Grabovska, Shannon Martin, Fernando Sialana, Harvey Che, Habib Bouguenina, Benjamin Bellenie, Brian Walker, Paul Clarke, Charlotte Pawlyn. SETD2 is a novel and druggable dependency in IMiD/CELMoD resistant multiple myeloma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3879.

Introduction Immunomodulatory drugs (IMiDs) have revolutionized the treatment of myeloma (MM), with lenalidomide (Len) and pomalidomide (Pom) approved and novel cereblon E3 ligase modulators (CELMoDs, e.g. iberdomide, Iber and mezigdomide, Mezi) in clinical trials. IMiDs and CELMoDs bind to cereblon (CRBN), which is part of the CRL4 CRBN Ubiquitin E3 ligase complex, leading to the ubiquitination and subsequent degradation of neo-substrates including the transcription factors Ikaros and Aiolos which are critical for MM cell survival. Previous data suggests almost one third of myeloma patients acquire CRBN mutations by the time they are Pom refractory. Some events, including stop codons/frameshift mutations and copy loss, have clearly explicable effects on cereblon function. However, many are point mutations with some occurring outside of the thalidomide binding domain and at variable cancer clonal fractions. We sought to understand which mutations were deleterious to cereblon function and whether any mutations were associated with a differential response between IMiDs and CELMoDs. Methods From the 36 CRBN mutations previously reported in patient datasets, 12 missense mutations were selected for functional investigation (fig.1a). All mutations were mapped on to the publicly available crystallographic 3D structures of CRBN bound to IMiDs and CELMoDs to aid in predicting the potential functional effect of each mutation. CRBN knock out (KO) MM1.s cell lines were generated using CRISPR-Cas9 and confirmed to have homozygous loss at the first few exons of the CRBN sequence. Once validated, two independent MM1.s CRBNKO cell line clones were transduced with plasmids expressing either CRBN wild type control (CRBN WT) or mutated CRBN (generated using site directed mutagenesis) leading to stable expression. All cell lines were assessed for response to IMiDs and CELMoDs using cell viability assays (CellTitre Blue, 5 days, Len max. concentration 20uM, Pom 8uM, Iber 2uM and Mezi 1uM) and their ability to degrade neo-substrates assessed using Western blotting for Aiolos and downstream effects using RT-qPCR for IRF4, at 24 hrs. Results Knock out of CRBN in MM1.s cells led to complete resistance to IMiDs/CELMoDs. The re-expression of wild type CRBN re-sensitised MM1.s CRBNKO cells to IMiDs/CELMoDs with degradation of Aiolos, downregulation of IRF4 and a similar effect on cell viability to that seen in parental MM1.s. In contrast, the “positive control” mutation p.W386A located within the tri-tryptophan IMiD/CELMoD binding pocket appeared to completely inactivate CRBN with no change in Aiolos/IRF4 expression levels and no effect on cell viability after incubation with all IMiDs/CELMoDs. A second mutation, also in very close proximity to the tri-tryptophan binding pocket and within the neo-substrate binding area (p.H397Y), had the same effect suggesting mutations here entirely prevent all IMiD and CELMoD activity (fig.1b). In contrast, some CRBN mutations appeared to have no deleterious effect on CRBN function with response to all IMiDs and CELMoDs regained on re-expression at the same magnitude as seen with wild type CRBN re-expression. These mutations included all those located in the Lon protease-like domain (p.D50H, p.A143V, p.L190F, p.R283K) and two within the thalidomide binding domain but not close to the tri-tryptophan binding pocket nor neo-substrate binding area (p.A347V and p.W415G)(fig.1b). The remainder of the mutations studied appeared to abrogate the activity of the IMiDs lenalidomide and pomalidomide but retained some response (both degradation and viability) to iberdomide (p.C326G), and to a greater extent mezigdomide (p.C326G, p.P352S, p.C366Y and p.F381S), suggesting these compounds were able to partially compensate the effect of the mutation (fig.1b). This may be due to the more potent CELMoD compounds being able to overcome structural changes induced by some mutations but not others. Discussion These data highlight key differences in the functional impact of different CRBN missense mutations that have been previously identified in patients. These results may have important implications for the interpretation of CRBN sequencing results from patients for future therapy decisions, particularly differentiating those who may, despite relapsing on IMiDs with CRBN mutations, have the potential to still benefit from the use of CELMoD agents.

Background Acquired resistance to immunomodulatory drugs (IMiDs)/cereblon (CRBN) E3 ligase modulators (CELMoDs) is a major challenge in multiple myeloma (MM) treatment. These drugs bind to the CRBN component of the CRL4CRBN E3 ubiquitin ligase and designate a new set of substrates for degradation via the proteasome. Generation of resistance is frequently associated with decreased CRBN expression and this is due to genetic alteration in ~1/3rd of patients. Alternative drivers of the low CRBN state, and other mechanisms of resistance, need to be elucidated. To tackle this complex problem, MM models with acquired IMiD/CELMoD resistance were generated and multiomics analysis performed. Methods IMiD/CELMoD resistant human MM cell lines were generated by treating MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Resistant cell lines and controls were characterised by whole exome sequencing (WES), RNA-Seq and proteomics. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s; gene effect scores were calculated with the Chronos model and compared to parental MM1s using data from the Broad Institute DepMap portal. Pathway analysis was performed using g:Profiler. The Membrane Bound Transcription Factor Peptidase, Site 1 (MBTPS1) inhibitor PF-429242 was used to inhibit activation of the Sterol Regulatory-Element Binding Protein (SREBP) pathway and the effect on cell viability measured using CellTiter-Blue®. The Multiple Myeloma Research Foundation CoMMpass database was used to explore the correlation between mRNA expression of SREBP pathway genes in newly diagnosed patients and progression-free survival (PFS). Results All models were resistant to the IMiD/CELMoD with which they were generated (to 20-100x the GI50 concentration) and exhibited cross-resistance to other IMiDs/CELMoDs. Functional assays showed that well-characterised effects of IMiD/CELMoD treatment, e.g. degradation of Ikaros/Aiolos, were abrogated. WES showed Pom-resistant MM1S and Pom- and Iber-resistant H929 cells had acquired mutations in CRBN predicted to have a high impact on function. Len-resistant H929 cells had new copy number loss at the CRBN locus but Len- and Iber-resistant MM1s cells had no genetic changes in CRBN. CRBN protein expression was reduced in all resistant lines compared to control (log2 fold changes (FCs) by proteomics ranging from -0.25 to -1.95, adj p <0.05). Together these models display diverse resistance mechanisms, reflecting the clinical picture. Proteomic analysis of the resistant lines identified key changes in the SREBP pathway. The proteomes of the resistant lines were heterogeneous and the only pathway with common significant enrichment was SREBP/fatty acid metabolism (on analysis performed per cell line of proteins with significantly decreased expression). Stearoyl-CoA Desaturase (SCD), a key effector of the SREBP pathway, was one of only 5 proteins with significantly altered expression in all 6 cell lines compared to control. SCD had log2FCs ranging from -0.37 to -1.50 (adj p <0.05). Other key pathway members are shown in Figure 1. A genome-wide CRISPR screen using Iber-resistant MM1s cells identified potential new dependencies (gene effect score <-1 in resistant cells and >-0.5 in parental cells) in 47 genes including SCD and MBTPS1. MBTPS1 is critical for activation of the SREBP pathway and demonstrated one of the largest changes in gene effect (-1.4 vs -0.3). The activity of PF-429242, an inhibitor of MBTPS1, was explored in the resistant lines. A significant difference in GI50 between Iber-resistant H929 cells and control was found (1uM and >10uM respectively) with greater activity in the resistant cells. The same pattern was observed with the other resistant H929 cell lines, but not the resistant MM1s. Other pathway inhibitors are being explored. The expression of genes encoding SREBP pathway components was explored in the CoMMpass dataset. High SCD or MBTPS1 mRNA expression was associated with significantly worse PFS (logrank p<0.01), providing a further rationale for targeting the pathway. Conclusions Models representing multiple different IMiD/CELMoD resistance mechanisms have unified alterations in the SREBP pathway, highlighting a role in resistance biology and potential novel and targetable vulnerabilities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal