Iberdomide increases innate and adaptive immune cell subsets in the bone marrow of patients with relapsed/refractory multiple myeloma.

Iberdomide is a potent cereblon E3 ligase modulator (CELMoD agent) with promising efficacy and safety as a monotherapy or in combination with other therapies in patients with relapsed/refractory multiple myeloma (RRMM). Using a custom mass cytometry panel designed for large-scale immunophenotyping of the bone marrow tumor microenvironment (TME), we demonstrate significant increases of effector T and natural killer (NK) cells in a cohort of 93 patients with multiple myeloma (MM) treated with iberdomide, correlating findings to disease characteristics, prior therapy, and a peripheral blood immune phenotype. Notably, changes are dose dependent, associated with objective response, and independent of prior refractoriness to MM therapies. This suggests that iberdomide broadly induces innate and adaptive immune activation in the TME, contributing to its antitumor efficacy. Our approach establishes a strategy to study treatment-induced changes in the TME of patients with MM and, more broadly, patients with cancer and establishes rational combination strategies for iberdomide with immune-enhancing therapies to treat MM.

Pharmacodynamic changes in tumor and immune cells drive iberdomide's clinical mechanisms of activity in relapsed and refractory multiple myeloma.

Iberdomide is a next-generation cereblon (CRBN)-modulating agent in the clinical development in multiple myeloma (MM). The analysis of biomarker samples from relapsed/refractory patients enrolled in CC-220-MM-001 (ClinicalTrials.gov: NCT02773030), a phase 1/2 study, shows that iberdomide treatment induces significant target substrate degradation in tumors, including in immunomodulatory agent (IMiD)-refractory patients or those with low CRBN levels. Additionally, some patients with CRBN genetic dysregulation who responded to iberdomide have a similar median progression-free survival (PFS) (10.9 months) and duration of response (DOR) (9.5 months) to those without CRBN dysregulation (11.2 month PFS, 9.4 month DOR). Iberdomide treatment promotes a cyclical pattern of immune stimulation without causing exhaustion, inducing a functional shift in T cells toward an activated/effector memory phenotype, including in triple-class refractory patients and those receiving IMiDs as a last line of therapy. This analysis demonstrates that iberdomide's clinical mechanisms of action are driven by both its cell-autonomous effects overcoming CRBN dysregulation in MM cells, and potent immune stimulation that augments anti-tumor immunity.

Extended exposure to low doses of azacitidine induces differentiation of leukemic stem cells through activation of myeloperoxidase.

Oral azacitidine (oral-Aza) treatment results in longer median overall survival (OS) (24.7 vs. 14.8 months in placebo) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy. The dosing schedule of oral-Aza (14 days/28-day cycle) allows for low exposure of Aza for an extended duration thereby facilitating a sustained therapeutic effect. However, the underlying mechanisms supporting the clinical impact of oral-Aza in maintenance therapy remain to be fully understood. In this preclinical work, we explore the mechanistic basis of oral-Aza/extended exposure to Aza through in vitro and in vivo modeling. In cell lines, extended exposure to Aza results in sustained DNMT1 loss, leading to durable hypomethylation, and gene expression changes. In mouse models, extended exposure to Aza, preferentially targets immature leukemic cells. In leukemic stem cell (LSC) models, the extended dose of Aza induces differentiation and depletes CD34+CD38- LSC. Mechanistically, LSC differentiation is driven in part by increased myeloperoxidase (MPO) expression. Inhibition of MPO activity either by using an MPO-specific inhibitor or blocking oxidative stress, a known mechanism of MPO, partly reverses the differentiation of LSC. Overall, our preclinical work reveals novel mechanistic insights into oral-Aza and its ability to target LSC.

ETV4-Dependent Transcriptional Plasticity Maintains MYC Expression and Results in IMiD Resistance in Multiple Myeloma.

Immunomodulatory drugs (IMiD) are a backbone therapy for multiple myeloma (MM). Despite their efficacy, most patients develop resistance, and the mechanisms are not fully defined. Here, we show that IMiD responses are directed by IMiD-dependent degradation of IKZF1 and IKZF3 that bind to enhancers necessary to sustain the expression of MYC and other myeloma oncogenes. IMiD treatment universally depleted chromatin-bound IKZF1, but eviction of P300 and BRD4 coactivators only occurred in IMiD-sensitive cells. IKZF1-bound enhancers overlapped other transcription factor binding motifs, including ETV4. Chromatin immunoprecipitation sequencing showed that ETV4 bound to the same enhancers as IKZF1, and ETV4 CRISPR/Cas9-mediated ablation resulted in sensitization of IMiD-resistant MM. ETV4 expression is associated with IMiD resistance in cell lines, poor prognosis in patients, and is upregulated at relapse. These data indicate that ETV4 alleviates IKZF1 and IKZF3 dependency in MM by maintaining oncogenic enhancer activity and identify transcriptional plasticity as a previously unrecognized mechanism of IMiD resistance. SIGNIFICANCE: We show that IKZF1-bound enhancers are critical for IMiD efficacy and that the factor ETV4 can bind the same enhancers and substitute for IKZF1 and mediate IMiD resistance by maintaining MYC and other oncogenes. These data implicate transcription factor redundancy as a previously unrecognized mode of IMiD resistance in MM. See related article by Welsh, Barwick, et al., p. 34. See related commentary by Yun and Cleveland, p. 5. This article is featured in Selected Articles from This Issue, p. 4.

Luspatercept stimulates erythropoiesis, increases iron utilization, and redistributes body iron in transfusion-dependent thalassemia.

Luspatercept, a ligand-trapping fusion protein, binds select TGF-ß superfamily ligands implicated in thalassemic erythropoiesis, promoting late-stage erythroid maturation. Luspatercept reduced transfusion burden in the BELIEVE trial (NCT02604433) of 336 adults with transfusion-dependent thalassemia (TDT). Analysis of biomarkers in BELIEVE offers novel physiological and clinical insights into benefits offered by luspatercept. Transfusion iron loading rates decreased 20% by 1.4 g (~7 blood units; median iron loading rate difference: -0.05 ± 0.07 mg Fe/kg/day, p< .0001) and serum ferritin (s-ferritin) decreased 19.2% by 269.3 ± 963.7 µg/L (p < .0001), indicating reduced macrophage iron. However, liver iron content (LIC) did not decrease but showed statistically nonsignificant increases from 5.3 to 6.7 mg/g dw. Erythropoietin, growth differentiation factor 15, soluble transferrin receptor 1 (sTfR1), and reticulocytes rose by 93%, 59%, 66%, and 112%, respectively; accordingly, erythroferrone increased by 51% and hepcidin decreased by 53% (all p < .0001). Decreased transfusion with luspatercept in patients with TDT was associated with increased erythropoietic markers and decreasing hepcidin. Furthermore, s-ferritin reduction associated with increased erythroid iron incorporation (marked by sTfR1) allowed increased erythrocyte marrow output, consequently reducing transfusion needs and enhancing rerouting of hemolysis (heme) iron and non-transferrin-bound iron to the liver. LIC increased in patients with intact spleens, consistent with iron redistribution given the hepcidin reduction. Thus, erythropoietic and hepcidin changes with luspatercept in TDT lower transfusion dependency and may redistribute iron from macrophages to hepatocytes, necessitating the use of concomitant chelator cover for effective iron management.

Immunomodulation of NK, NKT and B/T cell subtypes in relapsed/refractory multiple myeloma patients treated with pomalidomide along with velcade and dexamethasone and its association with improved progression-free survival.

Background: Multiple Myeloma (MM) patients exhibit dysregulated immune system, which is further weakened by chemotherapeutic agents. While cereblon-modulating agents, such as pomalidomide and lenalidomide, have been found to improve the immune profile, the efficacy of their impact in combination with other treatments is yet unknown. Methods: We conducted an immune-profiling of a longitudinal cohort of 366 peripheral blood samples from the CC4047-MM-007 (OPTIMISMM, NCT01734928) study. This study followed relapsed/refractory Multiple Myeloma (RRMM) patients who were treated with Velcade + dexamethasone (Vd), or Vd with pomalidomide (PVd). 366 blood samples from 186 patients were evaluated using multi-color flow cytometry at 3 timepoints: screening, day 8 of cycle 1, and cycle 3. Results: Among NK and NKT cell populations, adding pomalidomide showed no inhibition in the frequency of NK cells. When expression of double positivity for activation markers like, p46/NKG2D, on NK cells was higher than the median, PVd treated patients showed significantly better (p=0.05) progression-free survival (PFS) (additional 15 months) than patients with lower than the median expression of p46/NKG2D on NK cells. PVd treated patients who expressed CD158a/b below the median at cycle 1 demonstrated a significantly better PFS (more than 18months). Among B cell subtypes, PVd treatment significantly increased the abundance of B1b cells (p<0.05) and decreased Bregs (p<0.05) at day 8 of both cycle 1 and cycle 3 when compared to screening samples. Of all the B cell-markers evaluated among paired samples, a higher expression of MZB cells at day 8 of cycle 1 has resulted in enhanced PFS in PVd treated patients. Within T cells, pomalidomide treatment did not decrease the frequency of CD8 T cells when compared with screening samples. The higher the surface expression of OX-40 on CD8 T cells and the lower the expression of PD-1 and CD25 on CD4 T cells by PVd treatment resulted in improved PFS. Conclusion: The prognostic significance for the number of immune markers is only seen in the PVd arm and none of these immune markers exhibit prognostic values in the Vd arm. This study demonstrates the importance of the immunomodulatory effects and the therapeutic benefit of adding pomalidomide to Vd treatment.

Multiple myeloma and its treatment contribute to increased platelet reactivity.

Multiple myeloma (MM) and its precursor states, smoldering myeloma (SM) and monoclonal gammopathy of undetermined significance (MGUS) are associated with increased incidence of thrombosis, however the cause of this is unknown. Lenalidomide treatment of MM substantially improves patient survival, although significantly increases thrombotic risk by an unknown mechanism. This pilot study aimed to establish the impact of MM and its treatment with Lenalidomide on platelet function. We analyzed platelet function in MGUS, SM and MM compared to healthy controls. We report an increase in platelet reactivity in MGUS, SM, and MM where increases in fibrinogen binding, P-selectin exposure, altered receptor expression, elevated levels of aggregation and enhanced sensitivity to agonist stimulation were observed. We also demonstrate an increase in patient platelet reactivity post Lenalidomide treatment compared to pre-treatment. We show Lenalidomide treatment of platelets ex vivo increased reactivity that was associated with formation of larger thrombi at arterial shear rates but not venous shear rates. This study demonstrates a clear increase in platelet reactivity and prothrombotic potential in patients with MGUS, SM and MM which is elevated further upon treatment with Lenalidomide. Our observations suggest that more detailed studies are warranted to determine mechanisms of thrombotic complications to enable the development of new preventative strategies that specifically target platelets.

What is the context?Multiple myeloma is associated with increased risk of thrombosis, although the potential role of platelets in this has not been evaluated.What is new?We show in this pilot study that multiple myeloma and its precursor states of smoldering myeloma and monoclonal gammopathy of undetermined significance are associated with increased levels of platelet responses. This is further exacerbated by treatment with the immunomodulatory drug lenalidomide.What is the impact?This study suggests that more detailed studies are warranted to explore the mechanisms that cause these effects in a larger population of patients, since this may reveal new approaches to prevent myeloma-associated thrombotic complications.

Oral azacitidine modulates the bone marrow microenvironment in patients with acute myeloid leukaemia in remission: A subanalysis from the QUAZAR AML-001 trial.

Oral azacitidine (Oral-AZA) maintenance therapy improved relapse-free (RFS) and overall survival (OS) significantly versus placebo for AML patients in remission after intensive chemotherapy (IC) in the phase 3 QUAZAR AML-001 study. Immune profiling was performed on the bone marrow (BM) at remission and on-treatment in a subset of patients with the aim of identifying prognostic immune features and evaluating associations of on-treatment immune effects by Oral-AZA with clinical outcomes. Post-IC, increased levels of lymphocytes, monocytes, T cells and CD34 + CD117+ BM cells were prognostically favourable for RFS. CD3+ T-cell counts were significantly prognostic for RFS in both treatment arms. At baseline, high expression of the PD-L1 checkpoint marker was identified on a subset of CD34 + CD117+ BM cells; many of which were PD-L2+. High co-expression of T-cell exhaustion markers PD-1 and TIM-3 was associated with inferior outcomes. Oral-AZA augmented T-cell numbers during early treatment, increased CD4+:CD8+ ratios and reversed T-cell exhaustion. Unsupervised clustering analysis identified two patient subsets defined by T-cell content and expression of T-cell exhaustion markers that were enriched for MRD negativity. These results indicate that Oral-AZA modulates T-cell activity in the maintenance setting of AML, and these immune-mediated responses are associated with clinical outcomes.

High-dose melphalan treatment significantly increases mutational burden at relapse in multiple myeloma.

High-dose melphalan (HDM) improves progression-free survival in multiple myeloma (MM), yet melphalan is a DNA-damaging alkylating agent; therefore, we assessed its mutational effect on surviving myeloma cells by analyzing paired MM samples collected at diagnosis and relapse in the IFM 2009 study. We performed deep whole-genome sequencing on samples from 68 patients, 43 of whom were treated with RVD (lenalidomide, bortezomib, and dexamethasone) and 25 with RVD + HDM. Although the number of mutations was similar at diagnosis in both groups (7137 vs 7230; P = .67), the HDM group had significantly more mutations at relapse (9242 vs 13 383, P = .005). No change in the frequency of copy number alterations or structural variants was observed. The newly acquired mutations were typically associated with DNA damage and double-stranded breaks and were predominantly on the transcribed strand. A machine learning model, using this unique pattern, predicted patients who would receive HDM with high sensitivity, specificity, and positive prediction value. Clonal evolution analysis showed that all patients treated with HDM had clonal selection, whereas a static progression was observed with RVD. A significantly higher percentage of mutations were subclonal in the HDM cohort. Intriguingly, patients treated with HDM who achieved complete remission (CR) had significantly more mutations at relapse yet had similar survival rates as those treated with RVD who achieved CR. This similarity could have been due to HDM relapse samples having significantly more neoantigens. Overall, our study identifies increased genomic changes associated with HDM and provides rationale to further understand clonal complexity.

Prolyl-tRNA synthetase as a novel therapeutic target in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy characterised by aberrant production of immunoglobulins requiring survival mechanisms to adapt to proteotoxic stress. We here show that glutamyl-prolyl-tRNA synthetase (GluProRS) inhibition constitutes a novel therapeutic target. Genomic data suggest that GluProRS promotes disease progression and is associated with poor prognosis, while downregulation in MM cells triggers apoptosis. We developed NCP26, a novel ATP-competitive ProRS inhibitor that demonstrates significant anti-tumour activity in multiple in vitro and in vivo systems and overcomes metabolic adaptation observed with other inhibitor chemotypes. We demonstrate a complex phenotypic response involving protein quality control mechanisms that centers around the ribosome as an integrating hub. Using systems approaches, we identified multiple downregulated proline-rich motif-containing proteins as downstream effectors. These include CD138, transcription factors such as MYC, and transcription factor 3 (TCF3), which we establish as a novel determinant in MM pathobiology through functional and genomic validation. Our preclinical data therefore provide evidence that blockade of prolyl-aminoacylation evokes a complex pro-apoptotic response beyond the canonical integrated stress response and establish a framework for its evaluation in a clinical setting.

The location of the t(4;14) translocation breakpoint within the NSD2 gene identifies a subset of patients with high-risk NDMM.

Although translocation events between chromosome 4 (NSD2 gene) and chromosome 14 (immunoglobulin heavy chain [IgH] locus) (t(4;14)) is considered high risk in newly diagnosed multiple myeloma (NDMM), only ∼30% to 40% of t(4;14) patients are clinically high risk. We generated and compared a large whole genome sequencing (WGS) and transcriptome (RNA sequencing) from 258 t(4;14) (n = 153 discovery, n = 105 replication) and 183 non-t(4;14) NDMM patients with associated clinical data. A landmark survival analysis indicated only ∼25% of t(4;14) patients had an overall survival (OS) <24 months, and a comparative analysis of the patient subgroups identified biomarkers associated with this poor outcome, including translocation breakpoints located in the NSD2 gene and expression of IgH-NSD2 fusion transcripts. Three breakpoints were identified and are designated as: "no-disruption" (upstream of NSD2), "early-disruption" (in the 5' UTR), and "late-disruption" (within the NSD2 gene). Our results show a significant difference in OS based on the location of DNA breakpoints (median OS 28.6 "late-disruption" vs 59.2 "early disruption" vs 75.1 months "no disruption"). These findings have been replicated in an independent replication dataset. Also, univariate and multivariate analysis suggest high-risk markers such as del17p, 1p independently contribute to poor outcome in t(4;14) MM patients.

Whole-genome analysis identifies novel drivers and high-risk double-hit events in relapsed/refractory myeloma.

Large-scale analyses of genomic data from patients with newly diagnosed multiple myeloma (ndMM) have been undertaken, however, large-scale analysis of relapsed/refractory MM (rrMM) has not been performed. We hypothesize that somatic variants chronicle the therapeutic exposures and clonal structure of myeloma from ndMM to rrMM stages. We generated whole-genome sequencing (WGS) data from 418 tumors (386 patients) derived from 6 rrMM clinical trials and compared them with WGS from 198 unrelated patients with ndMM in a population-based case-control fashion. We identified significantly enriched events at the rrMM stage, including drivers (DUOX2, EZH2, TP53), biallelic inactivation (TP53), noncoding mutations in bona fide drivers (TP53BP1, BLM), copy number aberrations (CNAs; 1qGain, 17pLOH), and double-hit events (Amp1q-ISS3, 1qGain-17p loss-of-heterozygosity). Mutational signature analysis identified a subclonal defective mismatch repair signature enriched in rrMM and highly active in high mutation burden tumors, a likely feature of therapy-associated expanding subclones. Further analysis focused on the association of genomic aberrations enriched at different stages of resistance to immunomodulatory agent (IMiD)-based therapy. This analysis revealed that TP53, DUOX2, 1qGain, and 17p loss-of-heterozygosity increased in prevalence from ndMM to lenalidomide resistant (LENR) to pomalidomide resistant (POMR) stages, whereas enrichment of MAML3 along with immunoglobulin lambda (IGL) and MYC translocations distinguished POM from the LEN subgroup. Genomic drivers associated with rrMM are those that confer clonal selective advantage under therapeutic pressure. Their role in therapy evasion should be further evaluated in longitudinal patient samples, to confirm these associations with the evolution of clinical resistance and to identify molecular subsets of rrMM for the development of targeted therapies.

Prognostic impact of NPM1 and FLT3 mutations in patients with AML in first remission treated with oral azacitidine.

The randomized, placebo-controlled, phase 3 QUAZAR AML-001 trial (ClinicalTrials.gov identifier: NCT01757535) evaluated oral azacitidine (Oral-AZA) in patients with acute myeloid leukemia (AML) in first remission after intensive chemotherapy (IC) who were not candidates for hematopoietic stem cell transplantation. Eligible patients were randomized 1:1 to Oral-AZA 300 mg or placebo for 14 days per 28-day cycle. We evaluated relapse-free survival (RFS) and overall survival (OS) in patient subgroups defined by NPM1 and FLT3 mutational status at AML diagnosis and whether survival outcomes in these subgroups were influenced by presence of post-IC measurable residual disease (MRD). Gene mutations at diagnosis were collected from patient case report forms; MRD was determined centrally by multiparameter flow cytometry. Overall, 469 of 472 randomized patients (99.4%) had available mutational data; 137 patients (29.2%) had NPM1 mutations (NPM1mut), 66 patients (14.1%) had FLT3 mutations (FLT3mut; with internal tandem duplications [ITD], tyrosine kinase domain mutations [TKDmut], or both), and 30 patients (6.4%) had NPM1mut and FLT3-ITD at diagnosis. Among patients with NPM1mut, OS and RFS were improved with Oral-AZA by 37% (hazard ratio [HR], 0.63; 95% confidence interval [CI], 0.41-0.98) and 45% (HR, 0.55; 95% CI, 0.35-0.84), respectively, vs placebo. Median OS was improved numerically with Oral-AZA among patients with NPM1mut whether without MRD (48.6 months vs 31.4 months with placebo) or with MRD (46.1 months vs 10.0 months with placebo) post-IC. Among patients with FLT3mut, Oral-AZA improved OS and RFS by 37% (HR, 0.63; 95% CI, 0.35-1.12) and 49% (HR, 0.51; 95% CI, 0.27-0.95), respectively, vs placebo. Median OS with Oral-AZA vs placebo was 28.2 months vs 16.2 months, respectively, for patients with FLT3mut and without MRD and 24.0 months vs 8.0 months for patients with FLT3mut and MRD. In multivariate analyses, Oral-AZA significantly improved survival independent of NPM1 or FLT3 mutational status, cytogenetic risk, or post-IC MRD status.

Loss of COP9 signalosome genes at 2q37 is associated with IMiD resistance in multiple myeloma.

The acquisition of a multidrug refractory state is a major cause of mortality in myeloma. Myeloma drugs that target the cereblon (CRBN) protein include widely used immunomodulatory drugs (IMiDs), and newer CRBN E3 ligase modulator drugs (CELMoDs), in clinical trials. CRBN genetic disruption causes resistance and poor outcomes with IMiDs. Here, we investigate alternative genomic associations of IMiD resistance, using large whole-genome sequencing patient datasets (n = 522 cases) at newly diagnosed, lenalidomide (LEN)-refractory and lenalidomide-then-pomalidomide (LEN-then-POM)-refractory timepoints. Selecting gene targets reproducibly identified by published CRISPR/shRNA IMiD resistance screens, we found little evidence of genetic disruption by mutation associated with IMiD resistance. However, we identified a chromosome region, 2q37, containing COP9 signalosome members COPS7B and COPS8, copy loss of which significantly enriches between newly diagnosed (incidence 5.5%), LEN-refractory (10.0%), and LEN-then-POM-refractory states (16.4%), and may adversely affect outcomes when clonal fraction is high. In a separate dataset (50 patients) with sequential samples taken throughout treatment, we identified acquisition of 2q37 loss in 16% cases with IMiD exposure, but none in cases without IMiD exposure. The COP9 signalosome is essential for maintenance of the CUL4-DDB1-CRBN E3 ubiquitin ligase. This region may represent a novel marker of IMiD resistance with clinical utility.

Myeloma Genome Project Panel is a Comprehensive Targeted Genomics Panel for Molecular Profiling of Patients with Multiple Myeloma.

PURPOSE: We designed a comprehensive multiple myeloma targeted sequencing panel to identify common genomic abnormalities in a single assay and validated it against known standards. EXPERIMENTAL DESIGN: The panel comprised 228 genes/exons for mutations, 6 regions for translocations, and 56 regions for copy number abnormalities (CNA). Toward panel validation, targeted sequencing was conducted on 233 patient samples and further validated using clinical FISH (translocations), multiplex ligation probe analysis (MLPA; CNAs), whole-genome sequencing (WGS; CNAs, mutations, translocations), or droplet digital PCR (ddPCR) of known standards (mutations). RESULTS: Canonical immunoglobulin heavy chain translocations were detected in 43.2% of patients by sequencing, and aligned with FISH except for 1 patient. CNAs determined by sequencing and MLPA for 22 regions were comparable in 103 samples and concordance between platforms was R2 = 0.969. Variant allele frequency (VAF) for 74 mutations were compared between sequencing and ddPCR with concordance of R2 = 0.9849. CONCLUSIONS: In summary, we have developed a targeted sequencing panel that is as robust or superior to FISH and WGS. This molecular panel is cost-effective, comprehensive, clinically actionable, and can be routinely deployed to assist risk stratification at diagnosis or posttreatment to guide sequencing of therapies.

The Next Frontier: Translational Development of Ubiquitination, SUMOylation, and NEDDylation in Cancer.

Post-translational modifications of proteins ensure optimized cellular processes, including proteostasis, regulated signaling, cell survival, and stress adaptation to maintain a balanced homeostatic state. Abnormal post-translational modifications are associated with cellular dysfunction and the occurrence of life-threatening diseases, such as cancer and neurodegenerative diseases. Therefore, some of the frequently seen protein modifications have been used as disease markers, while others are targeted for developing specific therapies. The ubiquitin and ubiquitin-like post-translational modifiers, namely, small ubiquitin-like modifier (SUMO) and neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8), share several features, such as protein structures, enzymatic cascades mediating the conjugation process, and targeted amino acid residues. Alterations in the regulatory mechanisms lead to aberrations in biological processes during tumorigenesis, including the regulation of tumor metabolism, immunological modulation of the tumor microenvironment, and cancer stem cell stemness, besides many more. Novel insights into ubiquitin and ubiquitin-like pathways involved in cancer biology reveal a potential interplay between ubiquitination, SUMOylation, and NEDDylation. This review outlines the current understandings of the regulatory mechanisms and assay capabilities of ubiquitination, SUMOylation, and NEDDylation. It will further highlight the role of ubiquitination, SUMOylation, and NEDDylation in tumorigenesis.

Multiple Myeloma Patient Tumors With High Levels of Cereblon Exon-10 Deletion Splice Variant Upregulate Clinically Targetable Pro-Inflammatory Cytokine Pathways.

Immunomodulatory drugs (IMiDs), including lenalidomide and pomalidomide, are used in the routine treatment for multiple myeloma (MM) patients. Cereblon (CRBN) is the direct molecular target of IMiDs. While CRBN is not an essential gene for MM cell proliferation, the frequency of CRBN genetic aberrations, including mutation, copy number loss, and exon-10 (which includes a portion of the IMiD-binding domain) splicing, have been reported to incrementally increase in later-line patients. CRBN exon-10 splicing has also been shown to be associated with decreased progression-free survival in both newly diagnosed and relapsed refractory MM patients. Although we did not find significant general splicing defects among patients with CRBN exon-10 splice variant when compared to those expressing the full-length transcript, we identified upregulated TNFA signaling via NFKB, inflammatory response, and IL-10 signaling pathways in patients with exon-10 splice variant across various data sets-all potentially promoting tumor growth via chronic growth signals. We examined master regulators that mediate transcriptional programs in CRBN exon-10 splice variant patients and identified BATF, EZH2, and IKZF1 as the key candidates across the four data sets. Upregulated downstream targets of BATF, EZH2, and IKZF1 are components of TNFA signaling via NFKB, IL2/STAT5 signaling pathways, and IFNG response pathways. Previously, BATF-mediated transcriptional regulation was associated with venetoclax sensitivity in MM. Interestingly, we found that an EZH2 sensitivity gene expression signature also correlated with high BATF or venetoclax sensitivity scores in these tumors. Together, these data provide a rationale for investigating EZH2 inhibitors or venetoclax in combination with the next generation CRBN-targeting agents, such as CELMoDs, for patients overexpressing the CRBN exon-10 splice variant.

Overcoming IMiD resistance in T-cell lymphomas through potent degradation of ZFP91 and IKZF1.

Immunomodulatory (IMiD) agents like lenalidomide and pomalidomide induce the recruitment of IKZF1 and other targets to the CRL4CRBN E3 ubiquitin ligase, resulting in their ubiquitination and degradation. These agents are highly active in B-cell lymphomas and a subset of myeloid diseases but have compromised effects in T-cell lymphomas (TCLs). Here, we show that 2 factors determine resistance to IMiDs among TCLs. First, limited CRBN expression reduces IMiD activity in TCLs but can be overcome by newer-generation degrader CC-92480. Using mass spectrometry, we show that CC-92480 selectively degrades IKZF1 and ZFP91 in TCL cells with greater potency than pomalidomide. As a result, CC-92480 is highly active against multiple TCL subtypes and showed greater efficacy than pomalidomide across 4 in vivo TCL models. Second, we demonstrate that ZFP91 functions as a bona fide transcription factor that coregulates cell survival with IKZF1 in IMiD-resistant TCLs. By activating keynote genes from WNT, NF-kB, and MAP kinase signaling, ZFP91 directly promotes resistance to IKZF1 loss. Moreover, lenalidomide-sensitive TCLs can acquire stable resistance via ZFP91 rewiring, which involves casein kinase 2-mediated c-Jun inactivation. Overall, these findings identify a critical transcription factor network within TCLs and provide clinical proof of concept for the novel therapy using next-generation degraders.

Integrative multi-omics identifies high risk multiple myeloma subgroup associated with significant DNA loss and dysregulated DNA repair and cell cycle pathways.

BACKGROUND: Despite significant therapeutic advances in improving lives of multiple myeloma (MM) patients, it remains mostly incurable, with patients ultimately becoming refractory to therapies. MM is a genetically heterogeneous disease and therapeutic resistance is driven by a complex interplay of disease pathobiology and mechanisms of drug resistance. We applied a multi-omics strategy using tumor-derived gene expression, single nucleotide variant, copy number variant, and structural variant profiles to investigate molecular subgroups in 514 newly diagnosed MM (NDMM) samples and identified 12 molecularly defined MM subgroups (MDMS1-12) with distinct genomic and transcriptomic features. RESULTS: Our integrative approach let us identify NDMM subgroups with transversal profiles to previously described ones, based on single data types, which shows the impact of this approach for disease stratification. One key novel subgroup is our MDMS8, associated with poor clinical outcome [median overall survival, 38 months (global log-rank p-value < 1 × 10-6)], which uniquely presents a broad genomic loss (> 9% of entire genome, t-test p value < 1e-5) driving dysregulation of various transcriptional programs affecting DNA repair and cell cycle/mitotic processes. This subgroup was validated on multiple independent datasets, and a master regulator analyses identified transcription factors controlling MDMS8 transcriptomic profile, including CKS1B and PRKDC among others, which are regulators of the DNA repair and cell cycle pathways. CONCLUSION: Using multi-omics unsupervised clustering we were able to discover a new high-risk multiple myeloma patient segment. This high-risk group presents diverse previously known genetic markers, but also a new characteristic defined by accumulation of genomic loss which seems to drive transcriptional dysregulation of cell cycle, DNA repair and DNA damage. Finally, our work identified various master regulators, including E2F2 and CKS1B as the genes controlling these key biological pathways.