A distinct metabolic response characterizes sensitivity to EZH2 inhibition in multiple myeloma

AbstractMultiple myeloma (MM) is a heterogeneous haematological disease that remains clinically challenging. Increased activity of the epigenetic silencer EZH2 is a common feature in patients with poor prognosis. Previous findings have demonstrated that metabolic profiles can be sensitive markers for response to treatment in cancer. While EZH2 inhibition (EZH2i) has proven efficient in inducing cell death in a number of human MM cell lines, we hereby identified a subset of cell lines that despite a global loss of H3K27me3, remains viable after EZH2i. By coupling liquid chromatography-mass spectrometry with gene and miRNA expression profiling, we found that sensitivity to EZH2i correlated with distinct metabolic signatures resulting from a dysregulation of genes involved in methionine cycling. Specifically, EZH2i resulted in a miRNA-mediated downregulation of methionine cycling-associated genes in responsive cells. This induced metabolite accumulation and DNA damage, leading to G2 arrest and apoptosis. Altogether, we unveiled that sensitivity to EZH2i in human MM cell lines is associated with a specific metabolic and gene expression profile post-treatment.

Download Full-text

Differential Downregulation of Telomerase Activity by Bortezomib in Multiple Myeloma Cells- Regulatory Pathways and Clinical Implications.

Blood ◽

10.1182/blood.v114.22.4917.4917 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4917-4917

Author(s):

Chana Weiss ◽

Orit Uziel ◽

Ofir Wollach ◽

Shlomo Bulvick ◽

Meir Lahav

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Conflicts Of Interest ◽

Medical Center ◽

Telomerase Activity ◽

Response To Treatment ◽

Clinical Implications ◽

Myeloma Cells ◽

Regulatory Pathways ◽

Kinetics Of

Abstract Abstract 4917 Differential downregulation of telomerase activity by bortezomib in multiple myeloma cells- regulatory pathways and clinical implications C. Weiss1, O Uziel2, O. Wolach2, S. Bulvick1, and M. Lahav2 1Laniado Medical Center, Netania; 2Felsenstein Medical Research Center, Rabin Medical Center, Sackler School of Medicine, Tel-Aviv University, Israel. Background The proteasome inhibitor bortezomib is an effective drug in Multiple Myeloma (MM). However, its exact mechanism of action is not yet fully understood. The importance of telomerase in the biology and prognosis MM is well established, but its response to bortezomib has not been assessed yet. In light of common signaling pathways of connecting telomerase regulation and bortezomib known mechanisms of action, we surmised that the drug may affect the activity of telomerase in MM cells. Results Bortezomib downregulated telomerase activity in all MM cell lines. However, the kinetics of this downregulation varied between the two cell lines examined. Whereas in ARP-1 cells telomerase activity decreased 24 hours after bortezomib treatment, in CAG cells the effect was observed only after 48 hours. These finding imply different regulatory mechanisms between these lines. In both lines bortezomib transcriptionally downregulated telomerase activity by inhibiting hTERT expression. ChIP (Chromatin Immune Precipitation) analysis showed that SP-1 and C-Myc transcription factors mediate this transcriptional downregulation of telomerase. Interestingly, the binding of NFkB to hTERT promoter is enhanced in response to the drug, consistent with a recent study reporting upregulation of NFkB by bortezomib. We are currently looking in depth at the pathways leading to these phenomena. However, the two lines differed in the post translational modification of AKT, which phosphorylates telomerase post-translationally. Whereas in ARP-1 cells the phosphorylated form of AKT is downregulated in response to bortezomib, the drug did not affect AKT phosphorylation in CAG cells. These findings explain the different kinetics of telomerase downregulation and are in keeping with previous data showing different AKT response between these two cell lines. Conclusions and future studies Our results shed light on the effects and mechanism of bortezomib on telomerase activity in MM. Te differential response of pAKT pathway is in keeping with recent studies suggesting differential dependency of MM cells on pAKT. Telomerase inhibition by bortezomib may be of varying efficacy in subsets of MM patients in relation to their pAKT dependency. A correlation may also be found between the effect of bortezomib on telomerase activity and resistance to bortezomib in MM. As such, the ex vivo assessment of telomerase activity response to treatment may serve as a prognostic feature and add to therapeutic armamentarium by addition of telomerase inhibitors in defined subsets of patients. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Identification of a First-in-Class SETD2 Inhibitor That Shows Potent and Selective Anti-Proliferative Activity in t(4;14) Multiple Myeloma: T(4;14) Multiple Myeloma Cells Are Dependent on Both H3K36 Di and Tri-Methylation

Blood ◽

10.1182/blood-2018-99-110803 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3207-3207 ◽

Cited By ~ 1

Author(s):

Michael J Thomenius ◽

Jennifer Totman ◽

Kat Cosmopoulos ◽

Dorothy Brach ◽

Lei Ci ◽

...

Keyword(s):

Multiple Myeloma ◽

High Risk ◽

Cell Lines ◽

Cancer Cell ◽

Proliferative Activity ◽

Therapeutic Potential ◽

Response To Treatment ◽

Employment Equity ◽

Equity Ownership

Abstract t(4;14) chromosome translocations are found in 15% of newly diagnosed multiple myeloma (MM) cases and are associated with high risk. MM cells with t(4;14) over-express the histone methyltransferase (HMT), WHSC1/MMSET/NSD2, which leads to deregulation of gene expression due to increased di-methylation of Histone H3 at lysine 36 (H3K36me2). This activity has been shown to be essential for the survival of t(4;14) MM cells. In addition to WHSC1, another HMT, SETD2, has been shown to methylate H3K36. SETD2 is the only known enzyme capable of tri-methylation of H3K36 and has been reported to play a role in transcriptional elongation and alternative splicing. CRISPR pooled screening has shown that SETD2 activity is required for viability of a variety of cancer cell lines. This led Epizyme to develop small molecule inhibitors of SETD2 enzyme activity in order to understand the role of SETD2 in tumorigenesis. Through our drug discovery efforts, we identified EPZ-040414, a potent and selective inhibitor of SETD2 with low nM cell biochemical activity and broad selectivity against a panel of other HMTs. The proposed role of SETD2 in H3K36 methylation led us to test a panel of MM cells, including 6 t(4;14) cell lines with EPZ-040414. Inhibition of SETD2 resulted in reduced global tri-methylation of H3K36 in t(4;14) bearing MM cell lines. In contrast, there was no effect on global di-methyl H3K36 levels, indicating that WHSC1 activity is not affected by SETD2 inhibition. Moreover, 5/6 t(4;14) MM cell lines showed a cytotoxic response to treatment with EPZ-040414 with IC50s ranging between 60 and 200 nM, while all non-t(4;14) MM cell lines showed limited responses between 1 and 8 μM. Moreover, screening of a 280 cancer cell line panel with a SETD2 inhibitor showed minimal anti-proliferative activity in most cell lines tested. These findings show that t(4;14) MM cell lines require SETD2 activity for survival, suggesting that SETD2 inhibitors are strong candidates for the treatment of this high risk subgroup of MM. Efforts to further understand the interaction between SETD2 and WHSC1 in the molecular pathogenesis of t(4;14) myeloma will be presented. The current chemical series represented by EPZ-040414 is potent, selective, orally available, and currently under further evaluation for its therapeutic potential. Figure. Figure. Disclosures Thomenius: Epizyme Inc.: Employment, Equity Ownership. Totman:Epizyme Inc.: Employment, Equity Ownership. Cosmopoulos:Epizyme Inc.: Employment, Equity Ownership. Brach:Epizyme Inc.: Employment, Equity Ownership. Ci:Epizyme Inc.: Employment, Equity Ownership. Farrow:Epizyme Inc.: Employment, Equity Ownership. Smith:Epizyme Inc.: Employment, Equity Ownership. Chesworth:Epizyme Inc.: Employment, Equity Ownership. Duncan:Epizyme Inc.: Employment, Equity Ownership. Tang:Epizyme Inc.: Employment, Equity Ownership. Riera:Epizyme Inc.: Employment, Equity Ownership. Lampe:Epizyme Inc.: Employment, Equity Ownership.

Download Full-text

CRISPR/Cas9 Chemogenetic Profiling Identifies Candidate Biomarker Genes That Modulate Sensitivity to Selinexor

Blood ◽

10.1182/blood-2020-141785 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-14

Author(s):

Bert Kwanten ◽

Yosef Landesman ◽

Dirk Daelemans

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Nuclear Export ◽

B Cell Lymphoma ◽

Myeloma Cell ◽

Response To Treatment ◽

Loss Of Function ◽

License Agreement ◽

Potential Biomarker ◽

Increased Susceptibility

Introduction Selinexor (KPT-330) is a first-in-class selective inhibitor of XPO1-mediated nuclear export that has recently been approved by the US FDA for the treatment of relapsed/refractory multiple myeloma and relapsed diffuse large B-cell lymphoma. Additionally, the drug is being evaluated in ongoing clinical trials (phase I to III) on late-stage haematological and solid tumours. To improve outcomes and personalize therapeutics, specific biomarkers and underlying mechanisms of response to treatment need to be uncovered. Material and method We performed genome-wide CRISPR/Cas9-mediated loss-of-function genetic screening in two multiple myeloma (KMS-28-BM and SK-MM-1) and one chronic myeloid leukemia (HAP1) cell lines. After transduction with a knockout library, cells were divided and subsequently cultured into three parallel groups (control, IC20 and IC50) for at least 20 cell doublings. This setup allows monitoring of subtle relative changes between groups. After DNA extraction, sequencing of guide RNAs and statistical analysis, we identified genes that upon disruption confer either increased susceptibility or resistance to selinexor. Results and discussion For the multiple myeloma cell lines, the TGF-β/SMAD4 signalling pathway stood out as an important mediator of resistance to selinexor. Besides SMAD4 as a major hit in both cell lines, we identified five other genes from this pathway leading to resistance in KMS-28-BM cells, suggesting that downregulation of this pathway is a potential biomarker of response to SINE treatment in multiple myeloma. In addition, screens revealed significant variability across cell lines with hit genes involved in mRNA processing, proteasomal degradation and cell cycle regulation. Interestingly, we could simultaneously uncover resistance and sensitivity genes involved in the same pathway, suggesting that these have antagonistic effects. Notably, we identified knockout of the poorly characterized gene ASB8 as a strong, common sensitizer between the three cell lines. Conclusion Until now, most chemogenetic CRISPR/Cas9 knockout screens have focused on determining resistance genes, and few have employed the technique to screen for genes that enhance cell kill. We performed such screens with selinexor and found a number of general and cell type-specific hits. Genes identified to confer increased resistance are candidate biomarkers while genes inducing increased susceptibility represent new targets for drug combination therapies. Disclosures Kwanten: Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor). Landesman:Karyopharm Therapeutics Inc: Current Employment, Current equity holder in publicly-traded company. Daelemans:Karyopharm Therapeutics Inc.: Patents & Royalties: employees of KULeuven. KULeuven has a license agreement (royalties) with Karyopharm Therapeutics on XPO1 inhibitors (selinexor).

Download Full-text

Bone sialoprotein mRNA and protein expression in human multiple myeloma cell lines and patients

British Journal of Haematology ◽

10.1046/j.1365-2141.2000.02506.x ◽

2000 ◽

Vol 111 (4) ◽

pp. 1118-1121 ◽

Cited By ~ 5

Author(s):

A. Bellahcene ◽

I. Van Riet ◽

C. de Greef ◽

N. Antoine ◽

M. F. Young ◽

...

Keyword(s):

Multiple Myeloma ◽

Protein Expression ◽

Cell Lines ◽

Myeloma Cell ◽

Bone Sialoprotein ◽

Multiple Myeloma Cell ◽

Human Multiple Myeloma ◽

Mrna And Protein Expression

Download Full-text

Acetate metabolism in Multiple Myeloma identifies11C-Acetate PET as a novel strategy to image bone disease and response to treatment in preclinical models

Bone Abstracts ◽

10.1530/boneabs.5.p122 ◽

2016 ◽

Author(s):

Francesca Fontana ◽

Xia Ge ◽

Xinming Su ◽

Jingyu Xiang ◽

Simone Cenci ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Disease ◽

Response To Treatment ◽

Acetate Metabolism ◽

Preclinical Models ◽

Novel Strategy

Download Full-text

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines

Current Molecular Medicine ◽

10.2174/1566524019666190305134441 ◽

2019 ◽

Vol 19 (2) ◽

pp. 112-119 ◽

Cited By ~ 1

Author(s):

Mariana B. de Oliveira ◽

Luiz F.G. Sanson ◽

Angela I.P. Eugenio ◽

Rebecca S.S. Barbosa-Dantas ◽

Gisele W.B. Colleoni

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Viability ◽

Cell Lines ◽

Treatment Options ◽

Compensatory Mechanism ◽

Protein Homeostasis ◽

Protein Response ◽

Rpmi 8226

Introduction:Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR).Methods:We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors.Results:For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload.Conclusion:Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

Download Full-text

Diverse metabolic response of cancer cells treated with a 213Bi-anti-EGFR-immunoconjugate

Scientific Reports ◽

10.1038/s41598-021-84421-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Benedikt Feuerecker ◽

Philipp Biechl ◽

Christof Seidl ◽

Frank Bruchertseifer ◽

Alfred Morgenstern ◽

...

Keyword(s):

Amino Acids ◽

Cancer Cells ◽

Treatment Response ◽

Cell Lines ◽

Egf Receptor ◽

Metabolic Response ◽

Gas Chromatography Mass Spectrometry ◽

Alpha Emitter ◽

Early Treatment Response ◽

Bladder Cancer Cells

AbstractEvaluation of treatment response is among the major challenges in modern oncology. We herein used a monoclonal antibody targeting the EGF receptor (EGFR) labelled with the alpha emitter 213Bi (213Bi-anti-EGFR-MAb). EJ28Luc (bladder) and LN18 (glioma) cancer cells, both overexpressing EGFR, were incubated for 3 h with the radioimmunoconjugate. To assess the responses in the core carbon metabolism upon this treatment, these cancer cell lines were subsequently cultivated for 18 h in the presence of [U-13C6]glucose. 13C-enrichment and isotopologue profiles of key amino acids were monitored by gas chromatography–mass spectrometry (GC/MS), in order to monitor the impacts of the radionuclide-treatment upon glucose metabolism. In comparison to untreated controls, treatment of EJ28Luc cells with 213Bi-anti-EGFR-MAb resulted in a significantly decreased incorporation of 13C from [U-13C6]glucose into alanine, aspartate, glutamate, glycine, proline and serine. In sharp contrast, the same amino acids did not display less 13C-enrichments during treatment of the LN18 cells. The data indicate early treatment response of the bladder cancer cells, but not of the glioma cells though cell lines were killed following 213Bi-anti-EGFR-MAb treatment. The pilot study shows that the 13C-labelling approach is a valid tool to assess the responsiveness of cancer cells upon radionuclide-treatment in considerable metabolic detail.

Download Full-text

Telomere Architecture Correlates with Aggressiveness in Multiple Myeloma

Cancers ◽

10.3390/cancers13081969 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1969

Author(s):

Aline Rangel-Pozzo ◽

Pak Yu ◽

Sadhana LaL ◽

Yasmin Asbaghi ◽

Luiza Sisdelli ◽

...

Keyword(s):

Multiple Myeloma ◽

Disease Progression ◽

Genomic Instability ◽

Clinical Decision Making ◽

Three Dimensional ◽

Clinical Decision ◽

Response To Treatment ◽

Average Intensity ◽

Patient Response ◽

Smoldering Multiple Myeloma

The prognosis of multiple myeloma (MM), an incurable B-cell malignancy, has significantly improved through the introduction of novel therapeutic modalities. Myeloma prognosis is essentially determined by cytogenetics, both at diagnosis and at disease progression. However, for a large cohort of patients, cytogenetic analysis is not always available. In addition, myeloma patients with favorable cytogenetics can display an aggressive clinical course. Therefore, it is necessary to develop additional prognostic and predictive markers for this disease to allow for patient risk stratification and personalized clinical decision-making. Genomic instability is a prominent characteristic in MM, and we have previously shown that the three-dimensional (3D) nuclear organization of telomeres is a marker of both genomic instability and genetic heterogeneity in myeloma. In this study, we compared in a longitudinal prospective study blindly the 3D telomeric profiles from bone marrow samples of 214 initially treatment-naïve patients with either monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or MM, with a minimum follow-up of 5 years. Here, we report distinctive 3D telomeric profiles correlating with disease aggressiveness and patient response to treatment in MM patients, and also distinctive 3D telomeric profiles for disease progression in smoldering multiple myeloma patients. In particular, lower average intensity (telomere length, below 13,500 arbitrary units) and increased number of telomere aggregates are associated with shorter survival and could be used as a prognostic factor to identify high-risk SMM and MM patients.

Download Full-text

The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽

10.3390/cancers12092341 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2341

Author(s):

Normann Steiner ◽

Karin Jöhrer ◽

Selina Plewan ◽

Andrea Brunner-Véber ◽

Georg Göbel ◽

...

Keyword(s):

Multiple Myeloma ◽

Tyrosine Kinase ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Tyrosine Kinase Receptor ◽

Bone Marrow Biopsies ◽

Flt3 Inhibitors ◽

Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

Download Full-text