IDO Pathway Analysis for Identifying Novel Biomarkers in Multiple Myeloma

Abstract Background: Indoleamine 2,3-dioxygenase (IDO) is an intracellular enzyme that catalyzes the initial rate-limiting step in Tryptophan (Trp) degradation along the Kynurenine pathway. Trp can be catabolized by tryptophan hydroxylase into serotonin (Ser) or alternatively, by IDO and tryptophan-2,3-dioxygenase (TDO) into Kynurenine (Kyn). A variety of human tumors have been shown to express an active IDO which leads to the induction of immune tolerance and malignant cell survival (Mellor et al, 1999). Elevated IDO activity within tumor microenvironment impairs the function of antigen specific T cells and suppresses the anti-tumor immune response, while in tumor-draining lymph nodes an active IDO inhibits the antigen presentation reducing the population of antigen-specific T cells and stimulates regulatory T cells (Treg) activation (Curti et al. 2009). IDO overexpression has been correlated with poor prognosis in a number of human solid tumors including ovarian, endometrial and colon carcinoma (Okamoto et al. 2005, Ino et al. 2006, Brandacher et al. 2006). In hematological malignancies, such as AML and DLBCL, IDO expression has also shown significant prognostic value (Folgiero et al. 2014, Ninomiya et al. 2011). Currently, our understanding of the role of IDO in multiple myeloma (MM) is limited to a few existing studies correlating the abundance of selected IDO pathway metabolites with the disease (Bonanno et al. 2012, Zdzisinska et al. 2010). We hypothesize that IDO activity correlates with the disease onset and progression in MM patients. Methods: We quantified the levels of Trp, Kyn, and Ser in human serum by mass spectrometry using the Biocrates AbsoluteIDQ p180 kit. Analysis was performed using MetaboAnalyst 3.0 and Graphpad prism software. The serum samples derived from healthy donors and MM patients were obtained commercially. Results: We found significantly reduced levels of Trp (n=13, p<0.001) and elevated levels of Kyn (n=13, p<0.05) in serum samples from MM patients compared to matching (sex and age paired) healthy donor samples, while Ser levels did not differ significantly. Similarly, samples obtained from Newly Diagnosed (ND) MM patients showed a significant reduction in Trp (n=16, p<0.01) and a trend towards increased Kyn. The overall results in Relapsed/Refractory (R/R) MM patients were similar to the ND setting, although we only detected a trend towards Trp reduction, and observed a significant increase in Kyn levels (n=26, p<0.01) when compared to healthy donors. These results were confirmed when analyzing the data using the widely employed Kyn/Trp ratio, a potential marker for assessing IDO activity. A significantly elevated Kyn/Trp ratio was observed in all of the MM malignant populations (paired: n=13, p<0.001; ND: n=16, p<0.01; R/R: n=26, p<0.01) as compared to healthy individuals. Conclusions: Our study suggests that increased activity of IDO, and/or the production of downstream metabolites is one of the features of MM, and could potentially define a valuable set of novel biomarkers. Here we show a consistent pattern of increased Kyn and decreased Trp levels as well as an elevated Kyn/Trp ratio, all of which are hallmarks of high IDO activity in MM subjects when compared to healthy individuals. We did not detect any significant differences in Ser levels between any of the groups which may suggest that the catabolic pathway is not dependent on tryptophan hydroxylase. Additional studies are being initiated to further investigate the role of IDO/Trp pathway in MM pathogenesis as well as better understand its potential prognostic and predictive impact in currently used MM therapies. Disclosures No relevant conflicts of interest to declare.

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Acetate correlates with disability and immune response in multiple sclerosis

PeerJ ◽

10.7717/peerj.10220 ◽

2020 ◽

Vol 8 ◽

pp. e10220 ◽

Cited By ~ 1

Author(s):

Silvia Pérez-Pérez ◽

María Inmaculada Domínguez-Mozo ◽

Aitana Alonso-Gómez ◽

Silvia Medina ◽

Noelia Villarrubia ◽

...

Keyword(s):

Multiple Sclerosis ◽

T Cells ◽

Mononuclear Cells ◽

Immune Cell ◽

Short Chain Fatty Acids ◽

Liquid Chromatography Mass Spectrometry ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Disability Status

Background Gut microbiota has been related to multiple sclerosis (MS) etiopathogenesis. Short-chain fatty acids (SCFA) are compounds derived from microbial metabolism that have a role in gut-brain axis. Objectives To analyse SCFA levels in plasma of MS patients and healthy donors (HD), and the possible link between these levels and both clinical data and immune cell populations. Methods Ninety-five MS patients and 54 HD were recruited. Patients were selected according to their score in the Expanded Disability Status Scale (EDSS) (49 EDSS ≤ 1.5, 46 EDSS ≥ 5.0). SCFA were studied in plasma samples by liquid chromatography-mass spectrometry. Peripheral blood mononuclear cells were studied by flow cytometry. Gender, age, treatments, EDSS and Multiple Sclerosis Severity Score (MSSS) were evaluated at the recruitment. Results Plasma acetate levels were higher in patients than in HD (p = 0.003). Patients with EDSS ≥ 5.0 had higher acetate levels than those with EDSS≤ 1.5 (p = 0.029), and HD (p = 2.97e–4). Acetate levels correlated with EDSS (r = 0.387; p = 1.08e–4) and MSSS (r = 0.265; p = 0.011). In untreated MS patients, acetate levels correlated inversely with CD4+ naïve T cells (r = − 0.550, p = 0.001) and directly with CD8+ IL-17+ cells (r = 0.557; p = 0.001). Conclusions Plasma acetate levels are higher in MS patients than in HD. In MS there exists a correlation between plasma acetate levels, EDSS and increased IL-17+ T cells. Future studies will elucidate the role of SCFA in the disease.

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Correlation of the commercial anti-SARS-CoV-2 receptor binding domain antibody test with the chemiluminescent reduction neutralizing test and possible detection of antibodies to emerging variants

10.1101/2021.05.25.21257828 ◽

2021 ◽

Author(s):

Yoshitomo Morinaga ◽

Hideki Tani ◽

Yasushi Terasaki ◽

Satoshi Nomura ◽

Hitoshi Kawasuji ◽

...

Keyword(s):

Receptor Binding ◽

Disease Onset ◽

Antibody Test ◽

Binding Domain ◽

Receptor Binding Domain ◽

Healthy Individuals ◽

Wild Type ◽

Serum Samples ◽

Serological Tests ◽

Convalescent Phase

Background Serological tests are beneficial for recognizing the immune response against SARS-CoV-2. To identify protective immunity, optimization of the chemiluminescent reduction neutralizing test (CRNT), using pseudotyped SARS-CoV-2, is critical. Whether commercial antibody tests are comparably accurate is unknown. Methods Serum samples collected before variants were locally found were obtained from confirmed COVID-19 patients (n = 74), confirmed non-COVID-19 individuals (n = 179), and unscreened individuals (suspected healthy individuals, n = 229). The convalescent phase was defined as the period after day 10 from disease onset. The CRNT against pseudotyped viruses displaying the wild-type spike protein and a commercially available anti-receptor binding domain (RBD) antibody test were assayed. The CRNT was also assayed, using South African (SA) and United Kingdom (UK)-derived variants. Results The CRNT (cut off value, 50% inhibition) and the anti-RBD antibody test (cut off value, 0.8 U/mL) concurred regarding symptomatic COVID-19 patients in the convalescent phase and clearly differentiated between patients and suspected healthy individuals (sensitivity; 95.8% and 100%, specificity; 99.1% and 100%, respectively). Anti-RBD antibody test results correlated with neutralizing titer (r = 0.47, 95% CI 0.20-0.68). Compared with the wild-type, CRNT reduction was observed for the SA and UK-derived variants. Of the samples with ≥100 U/mL by the anti-RBD antibody test, 77.8% and 88.9% showed ≥50% neutralization against the UK and the SA variants, respectively. Conclusion The CRNT and commercial anti-RBD antibody test effectively classified convalescent COVID-19 patients. The strong positive results using the commercial antibody test can reflect neutralizing activity against emerging variants.

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Role of Antilipopolysaccharide Antibodies in Serum Bactericidal Activity against Salmonella enterica Serovar Typhimurium in Healthy Adults and Children in the United States

Clinical and Vaccine Immunology ◽

10.1128/cvi.00289-13 ◽

2013 ◽

Vol 20 (10) ◽

pp. 1491-1498 ◽

Cited By ~ 36

Author(s):

Estela Trebicka ◽

Susan Jacob ◽

Waheed Pirzai ◽

Bryan P. Hurley ◽

Bobby J. Cherayil

Keyword(s):

United States ◽

Bactericidal Activity ◽

Salmonella Enterica ◽

The United States ◽

Healthy Children ◽

Healthy Individuals ◽

Serum Samples ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTRecent observations from Africa have rekindled interest in the role of serum bactericidal antibodies in protecting against systemic infection withSalmonella entericaserovar Typhimurium. To determine whether the findings are applicable to other populations, we analyzed serum samples collected from healthy individuals in the United States. We found that all but 1 of the 49 adult samples tested had robust bactericidal activity againstS. Typhimurium in a standardin vitroassay. The activity was dependent on complement and could be reproduced by immunoglobulin G (IgG) purified from the sera. The bactericidal activity was inhibited by competition with soluble lipopolysaccharide (LPS) fromS. Typhimurium but not fromEscherichia coli, consistent with recognition of a determinant in the O-antigen polysaccharide. Sera from healthy children aged 10 to 48 months also had bactericidal activity, although it was significantly less than in the adults, correlating with lower levels of LPS-specific IgM and IgG. The lone sample in our collection that lacked bactericidal activity was able to inhibit killing ofS. Typhimurium by the other sera. The inhibition correlated with the presence of an LPS-specific IgM and was associated with decreased complement deposition on the bacterial surface. Our results indicate that healthy individuals can have circulating antibodies to LPS that either mediate or inhibit killing ofS. Typhimurium. The findings contrast with the observations from Africa, which linked bactericidal activity to antibodies against anS. Typhimurium outer membrane protein and correlated the presence of inhibitory anti-LPS antibodies with human immunodeficiency virus infection.

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Targeting Deubiquitylating Enzyme USP1 in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.1813.1813 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1813-1813

Author(s):

Deepika Sharma Das ◽

Yan Song ◽

Arghya Ray ◽

Paul Richardson ◽

Dharminder Chauhan ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Repair ◽

Cell Lines ◽

Cancer Cell ◽

Therapeutic Index ◽

Cysteine Proteases ◽

Dependent Manner ◽

Healthy Donors ◽

Reporter Assays

Abstract Introduction Proteasome inhibitor Bortezomib is effective therapy of relapsed/refractory and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Importantly, the ability of bortezomib to overcome resistance to conventional therapies has validated therapeutically targeting the Ubiquitin Proteasome System (UPS), and suggested potential utility of inhibitors of other components of the UPS including deubiquitylating enzymes (DUBs). Therapeutic strategies directed against DUBs may allow for more specific targeting of the UPS, and therefore be less likely to have off-target activities with associated toxicities. Our prior studies have identified a role of USP7, USP14, and UCHL5 in MM pathogenesis, and provided the rationale for targeting these DUBs in MM (Chauhan et al., Cancer Cell 2012, 11:345-358; Tian et al., Blood 2014, 123:706-716). Among DUBs, USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner UAF1, and through its deubiquitylation of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. Here we examined the role of USP1 DUB in MM using both biochemical and RNA interference strategies. Methods We utilized MM cell lines, patient cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. Cell viability was assessed using WST and CellTiter-Glo assays. MM.1S MM cells were transiently transfected with control short interfering RNA (siRNA), USP1 ON TARGET plus SMART pool siRNA using the cell line Nucleofector Kit V. A biochemical inhibitor of USP1 SJB3-019A (SJB) was purchased from Medchem Express, USA. In vitro DUB enzymatic activity was assessed using Ubiquitin-AMC and Ubiquitin-Rhodamine assay kits, as well as Ub-CHOP-reporter and K48-linked Ubiquitin tetramers. Competitive Ub-VS probe labeling was performed, as previously described (Chauhan et al., Cancer Cell 2012, 11:345-358). Signal transduction pathways were evaluated using immunoblotting. Statistical significance of data was determined using a Student's t test. Results Immunoblot analyses show higher USP1 levels in MM cell lines and patient cells than normal cells.USP1-siRNA inhibited MM cell proliferation, which was rescued by transfection of USP1 (WT). Using Ub-Rhodamine, Ub-AMC, and Ub-EKL reporter assays, we found higher USP1 deubiquitylating activity in patient MM cells versus normal cells, suggesting a favorable therapeutic index for targeting USP1. Importantly, siRNA-knockdown of USP1 both promoted degradation of tumorigenic ID1 protein, and inhibited proliferation of bortezomib-resistant (ANBL-6.BR) MM cells, suggesting that novel agents targeting USP1 may overcome bortezomib resistance. We next examined the effects of USP1 inhibitor SJB3 on MM cell growth and survival in our models of MM. Analysis using Ub-Rhodamine, Ub-AMC, and Ub-EKL reporter assays in a panel of MM cell lines showed that SJB is a potent, specific, and selective inhibitor of USP1 DUB activity (EC50 = 50 ± 5.7 nM), which does not inhibit other DUBs (USP2/USP5/USP7/USP14) or other families of cysteine proteases (EC50>100 μM). SJB blocks labeling of USP1 with HA-Ub-VS probe in a concentration-dependent manner, but did not alter labeling of other DUBs with HA-Ub-VS. SJB inhibits USP1-mediated cleavage of K48 linked polyubiquitin chains, but not that mediated by USP2 or USP7. Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, ARP1, KMS11, U266, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreases their viability (IC50 range 100nM to 500nM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for SJB. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Mechanistic studies show that SJB-triggered apoptosis is associated with degradation of USP1 and Id1 protein. Finally, combination of SJB with lenalidomide, pomalidomide, HDACi ACY-1215, or bortezomib both induces synergistic anti-MM activity and overcomes drug resistance. Conclusion Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential MM therapy. Disclosures Chauhan: Stemline Therapeutics: Consultancy.

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Functional leukemia-associated antigen-specific memory CD8+ T cells exist in healthy individuals and in patients with chronic myelogenous leukemia before and after stem cell transplantation

Blood ◽

10.1182/blood-2003-01-0150 ◽

2003 ◽

Vol 102 (8) ◽

pp. 2892-2900 ◽

Cited By ~ 165

Author(s):

Katayoun Rezvani ◽

Matthias Grube ◽

Jason M. Brenchley ◽

Giuseppe Sconocchia ◽

Hiroshi Fujiwara ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Cd8 T Cells ◽

Healthy Individuals ◽

Low Frequencies ◽

Healthy Donors ◽

Before And After ◽

Ifn Γ

Abstract Antigens implicated in the graft-versus-leukemia (GVL) effect in chronic myeloid leukemia (CML) include WT1, PR1, and BCR-ABL. To detect very low frequencies of these antigen-specific CD8+ T cells, we used quantitative polymerase chain reaction (qPCR) to measure interferon-γ (IFN-γ) mRNA production by peptide-pulsed CD8+ T cells from HLA-A*0201+ healthy volunteers and from patients with CML before and after allogeneic stem cell transplantation (SCT). Parallel assays using cytomegalovirus (CMV) pp65 tetramers demonstrated the IFN-γ copy number to be linearly related to the frequency of tetramer-binding T cells, sensitive to frequencies of 1 responding CD8+ T cell/100 000 CD8+ T cells. Responses to WT1 and PR1 but not BCR-ABL were detected in 10 of 18 healthy donors. Responses to WT1, PR1, or BCR-ABL were observed in 9 of 14 patients with CML before SCT and 5 of 6 after SCT, often to multiple epitopes. Responses were higher in patients with CML compared with healthy donors and highest after SCT. These antigen-specific CD8+ T cells comprised central memory (CD45RO+CD27+CD57–) and effector memory (CD45RO–CD27–CD57+) T cells. In conclusion, leukemia-reactive CD8+ T cells derive from memory T cells and occur at low frequencies in healthy individuals and at higher frequencies in patients with CML. The increased response in patients after SCT suggests a quantitative explanation for the greater effect of allogeneic SCT.

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Enrichment of functional CD8 memory T cells specific for MUC1 in bone marrow of patients with multiple myeloma

Blood ◽

10.1182/blood-2004-01-0366 ◽

2005 ◽

Vol 105 (5) ◽

pp. 2132-2134 ◽

Cited By ~ 86

Author(s):

Carmen Choi ◽

Mathias Witzens ◽

Marianna Bucur ◽

Markus Feuerer ◽

Nora Sommerfeldt ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

T Cells ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Hematologic Malignancies ◽

Myeloma Cell ◽

Interferon Γ ◽

Multiple Myeloma Cell ◽

Healthy Donors

AbstractMultiple myeloma (MM) is one of the most common hematologic malignancies. Despite extensive therapeutical approaches, cures remain rare exceptions. An important issue for future immunologic treatments is the characterization of appropriate tumor-associated antigens. Recently, a highly glycosylated mucin MUC1 was detected on a majority of multiple myeloma cell lines. We analyzed bone marrow and peripheral blood of 68 patients with HLA-A2–positive myeloma for the presence and functional activity of CD8 T cells specific for the MUC1-derived peptide LLLLTVLTV. Forty-four percent of the patients with MM contained elevated frequencies of MUC1-specific CD8 T cells in freshly isolated samples from peripheral blood (PB) or bone marrow (BM) compared with corresponding samples from healthy donors. BM-residing T cells possessed a higher functional capacity upon specific reactivation than PB-derived T cells with regard to interferon γ (IFN-γ) secretion, perforin production, and cytotoxicity.

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Spatiotemporal Assessment of Immunogenomic Heterogeneity in Multiple Myeloma

Blood ◽

10.1182/blood-2020-142622 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 14-15 ◽

Cited By ~ 1

Author(s):

Maximilian Merz ◽

Almuth Maria Anni Merz ◽

Jie Wang ◽

Lei Wei ◽

Ahmed Belal ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Clonal Evolution ◽

Advisory Board ◽

Braf Inhibitors ◽

Healthy Individuals ◽

Advisory Committees ◽

Site Specific

Introduction: Therapy and immune mediated processes are associated with clonal evolution in multiple myeloma (MM). In this study, we performed whole-exome sequencing (WES) and single cell RNA sequencing (scRNA-seq) on plasma cells (PC) from bone marrow aspirates of the iliac crest (BM) and corresponding osteolytic lesions (OL) to investigate spatial heterogeneity in patients with newly diagnosed (NDMM) and relapsed/refractory MM (RRMM). Next generation flow (NGF) and T-cell receptor sequencing (TCRseq) were performed to investigate the immunogenomic landscape surrounding malignant PC. Methods: In a prospective trial, 18 patients (NDMM: n=10; RRMM: n=8) consented to an imaging-guided biopsy of an OL in addition to the regular BM sampling. At inclusion, 37 different locations were biopsied. Follow-up samples were obtained from 5 patients in remission after therapy. After CD138+ selection, PC were subjected to WES and scRNA-seq (Chromium, 10x genomics). TCRseq was performed using multiplex PCR (ImmunoSEQ, Adaptive biotechnologies) on the CD138- fraction. For scRNA-seq data analyses, Cell Ranger (v3.1.0) and the Seurat R toolkit (v3.1) were used. TCRseq data were analyzed with immunoSEQ ANALYZER (v3.0) and the immunarch R toolkit (v0.6.6.). NGF was performed to study subsets of T-, B-, NK- and dendritic cells (DC). Results: Median PC infiltration was higher in OL compared to random BM (50.0% vs 12.5%, p=0.041). WES revealed more mutations in RRMM compared to NDMM (median; range: 189;120-523 vs 71;23-136, p<0.001). Based on mutational profiles from WES, 4 of 18 patients showed a branching evolution in PC isolated from OL. Three of the 4 patients had RRMM and one patient with NDMM had a prior history of solitary plasmacytoma. PC were obtained from OL with adjacent extramedullary disease (EMD) in 3 of 4 patients with branching evolution. Among site-specific mutations, we found in one patient two distinct BRAF mutations: V600E in the BM and G469R in the OL. An additional NRAS mutation (G12D) was found in the OL. BRAF G469R and NRAS G12D cause resistance to BRAF inhibitors, although this patient was naïve to BRAF-inhibitors. Clonal evolution was also reflected by chromosomal aberrations, including site-specific chromothripsis of chromosome 1 in a patient with RRMM. Even in patients without spatially divergent clones as detected by WES, scRNA-seq of more than 150,000 PC from 10 patients and 21 different locations revealed multiple clones. Distinct PC clones were identified by differential expression of genes associated with homing to the BM (CXCR4), malignant transformation (Jun/Fos, CD27, CD79a), apoptosis (BCL-2) bone disease (DKK1) and LAMP-5. In a patient with NDMM in remission after induction therapy, scRNA-seq demonstrated the emergence of a PC clone characterized by the overexpression of Interferon-induced genes (ISG15, IFI27, IFI44L) compared to the initially predominant PC clones. Next, we investigated spatiotemporal differences of immune cells. Estimation of median TCR richness using an abundance-based estimator (Chao1) revealed significantly lower values in patients with RRMM (120444; 57706-212744) compared to NDMM (389341; 50318-525082, p<0.001) and nine healthy individuals (460278; 138326-696419, p<0.001). No significant differences were found for TCR clonality as indicated by Simpson's D. While longitudinal tracking of TCR clones at primary diagnosis showed no clonal expansion after treatment, induction therapy restored sample richness in patients with NDMM to levels of healthy individuals (p=0.61). Overlap analysis showed a high concordance of TCR repertoires from OL and random BM with Morisita indices ranging in 90% of patients from 0.80 to 0.95. Nevertheless, significant site-specific expansion of TCR clones was detected. In accordance with TCRseq, NGF showed in the BM of patients with RRMM more regulatory T-cells (p=0.048) and less myeloid DC (p=0.024), Th9 cells and CD8 effector memory T-cells compared to NDMM. Conclusion: We report the first prospective clinical trial to investigate spatiotemporal immunogenomic heterogeneity in multiple myeloma as assessed by WES and scRNA-seq of PC and NGF and TCRseq of the non-PC compartment. We demonstrate spatial evolution and reduced TCR diversity especially in patients with RRMM and/or EMD. ScRNA-seq adds another layer of complexity compared to WES and helps identifying how PC create an immune suppressive BM niche. Disclosures Merz: Amgen, BMS, Celgene, Takeda: Honoraria. Block:GlaxoSmithKline LLC: Current Employment. McCarthy:Karyopharm: Consultancy, Honoraria; Magenta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Juno Therapeutics, a Bristol-Myers Squibb Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board , Research Funding is to Roswell Park, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Starton: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Genentech: Consultancy, Honoraria. Hillengass:Adaptive, Amgen, BMS, Celgene, GSK, Janssen, Oncotracker, Takeda: Honoraria.

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Epigenetic Deregulation of Telomere-Related Genes in Newly Diagnosed Multiple Myeloma Patients

Cancers ◽

10.3390/cancers13246348 ◽

2021 ◽

Vol 13 (24) ◽

pp. 6348

Author(s):

Samrat Roy Choudhury ◽

Cody Ashby ◽

Fenghuang Zhan ◽

Frits van Rhee

Keyword(s):

Dna Methylation ◽

Multiple Myeloma ◽

Dna Sequences ◽

Critical Length ◽

Newly Diagnosed ◽

Genome Wide ◽

Healthy Donors ◽

Binding Factor ◽

G Quadruplex

High-risk Multiple Myeloma (MM) patients were found to maintain telomere length (TL), below the margin of short critical length, consistent with proactive overexpression of telomerase. Previously, DNA methylation has been shown as a determinant of telomere-related gene (TRG) expression and TL to assess risk in different types of cancer. We mapped genome-wide DNA methylation in a cohort of newly diagnosed MM (NDMM; n = 53) patients of major molecular subgroups, compared to age-matched healthy donors (n = 4). Differential methylation and expression at TRG-loci were analyzed in combination with overlapping chromatin marks and underlying DNA-sequences. We observed a strong correlation (R2 ≥ 0.5) between DNA methylation and expression amongst selective TRGs, such that demethylation at the promoters of DDX1 and TERF1 were associated to their oncogenic upregulation, while demethylation at the bodies of two key tumor suppressors ZNF208 and RAP1A led to downregulation of the genes. We demonstrated that TRG expression may be controlled by DNA methylation alone or in cooperation with chromatin modifications or CCCTC-binding factor at the regulatory regions. Additionally, we showed that hypomethylated DMRs of TRGs in NDMM are stabilized with G-quadruplex forming sequences, suggesting a crucial role of these epigenetically vulnerable loci in MM pathogenesis. We have identified a panel of five TRGs, which are epigenetically deregulated in NDMM patients and may serve as early detection biomarkers or therapeutic targets in the disease.

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Biological correlates to support a clinical role for tagraxofusp, a novel targeted therapy directed to CD123, in combination with pomalidomide and dexamethasone, to target plasmacytoid dendritic cells in poor-risk patients with multiple myeloma.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e20537 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e20537-e20537

Author(s):

Dharminder Chauhan ◽

Arghya Ray ◽

Yan Song ◽

Arturo Olguin ◽

Janice Chen ◽

...

Keyword(s):

Multiple Myeloma ◽

Dendritic Cells ◽

Targeted Therapy ◽

Cell Growth ◽

Phase 1 ◽

Plasmacytoid Dendritic Cells ◽

Study Cohort ◽

Serum Samples ◽

Clinical Role

e20537 Background: We have previously described the pivotal pathogenetic role of plasmacytoid dendritic cells (pDCs; CD123/IL-3Ra+) in multiple myeloma (MM) (Chauhan et al Cancer Cell, 2009,16:309). In preclinical MM models, we demonstrated that tagraxofusp, a novel targeted therapy directed to CD123, triggers anti-MM activity by reducing the viability of MM-promoting pDCs. These observations led to an ongoing phase 1/2 clinical trial of tagraxofusp in MM patients (NCT02661022). The treatment regimen demonstrated safety and efficacy, with 5 of 9 heavily pretreated patients achieving durable partial response (PR). Here, we report the initial results of our translational exploratory studies using bone marrow (BM), peripheral blood (PB), and serum from the study cohort. Methods: pDCs and MM tumor cells were purified from BM/PB samples and quantified using FACS, as described ( Ray et al Leukemia, 2018,32:843). A high-throughput seroproteomics platform SOMAscan was utilized to analyze 1,310 protein analytes in serum samples from MM patients (N = 9). SOMAscan data were subjected to meta-analysis to generate heatmaps, followed by hierarchical cluster analysis. SOMAscan results were validated with ELISA using supernatants from MM patient pDCs cultured with or without tagraxofusp. Results: Analysis of BM/PB samples from MM patients receiving tagraxofusp therapy showed a marked reduction in the frequency of viable pDCs [average 2% at screen vs 0.75% post-tagraxofusp; N = 6; p = 0.036]. pDCs isolated from tagraxofusp-treated patients showed decreased ability to trigger MM cell growth. Seroproteomics analysis of MM patient serum before and after tagraxofusp therapy showed alterations in the levels of 100 proteins [Median Fold Change in expression: 0.39 to 4.5; n = 6; 3 each; p < 0.05]. Importantly, we found that tagraxofusp treatment reduced pDC-related soluble proteins, in particular, IFN-a and IL-3Rα. Additionally, our earlier study showed that pDC-MM interactions triggered secretion of MM cell growth including IL-3, which serves a dual role of promoting pDC survival and MM cell growth. Importantly, tagraxofusp decreased serum IL-3, suggesting that tagraxofusp attenuates survival mechanisms for tumor-promoting pDCs. Conclusions: Our current correlative studies validate target specificity of tagraxofusp and support further evaluation for this novel therapeutic to improve the clinical outcome of patients with MM.

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Accumulation of Inherited and Acquired Mutations in Hyaluronan Synthase1 Gene May Contribute Oncogenesis in Multiple Myeloma and Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v108.11.3432.3432 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3432-3432

Author(s):

Sophia Adamia ◽

Jennifer Hodges ◽

Patrick M. Pilarski ◽

Steven Treon ◽

Michael J. Mant ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

B Cells ◽

Hematopoietic Cells ◽

Genetic Variations ◽

Waldenström’S Macroglobulinemia ◽

Aberrant Splicing ◽

Healthy Donors ◽

Waldenstrom's Macroglobulinemia ◽

Waldenström's Macroglobulinemia

Abstract In multiple myeloma (MM) and Waldenstrom’s macroglobulinemia (WM), we identified three alternatively and/or aberrantly spliced HAS1 transcripts—HAS1Va, HAS1Vb and HAS1Vc. Statistical analysis of samples taken from 172 untreated MM patients showed that expression of HAS1Vb, an intronic splice variant, strongly correlates with poor survival (P=0.005). We investigated the molecular basis of intron retention during HAS1 splicing in MM and WM patients. We speculated that aberrant HAS1 splicing and the associated reduced survival of MM patients, resulted from an accumulation of mutations in aberrantly spliced regions of HAS1. Exons and introns 3 and 4 of the HAS1 gene were sequenced, because they are hotspots for splicing aberrations. Sequencing of HAS1 was performed for a total 11 patients with WM and MM and 2 healthy donors. HAS1 gene templates for sequencing were isolated from a multiple sorted cell subpopulations, including malignant B and plasma cells (PC), non-malignant T cells and buccal epithelial cells (BECs), as well as hematopoietic progenitor cells (HPCs) from mobilized blood of MM patients. We detected sets of inherited and acquired genetic variations in HAS1 that were recurrent within 5–11 of the MM and WM patients analyzed, but absent from healthy donors. We also identified genetic variations that were unique to individual patients. Those HAS1 mutations found in all cell types tested, including BECs and from the hematopoietic cells (B, PC, T and HPCS) were classified as germline mutations. Those mutations found in hematopoietic cells but absent from BECs were classified as hematopoietic origin which acquired during the lifetime of the individual. Mutations identified only in malignant MM and WM B cells and PCs (absent from T cells, HPCs and BECs) were classified as acquired tumor specific mutations. Recurrent HAS1 mutations were found among both inherited and acquired sets of mutations. Some recurrent HAS1 mutations were common to both MM and WM. The high frequency of inherited HAS1 mutations suggests that they confer predisposition for developing MM or WM. Our sequencing analysis suggests that in MM and WM, sequential accumulation of genetic variations occurs as hematopoietic cells differentiate. Our data also suggest that hematopoietic origin mutations are necessary but by no means sufficient to drive HAS1 gene splicing. Effects of hematopoietic origin mutations on HAS1 splicing are manifested in malignant MM cells in context of additional tumor specific mutations, which are acquired by circulating B cells and passed to their plasma cell progeny. This suggests that mutations which lead to aberrant splicing of HAS1 pre-mRNA undergo mutational selection events, and leave a mutational “trace” throughout the hematopoietic cell lineage, including tumor cells. Existence of same mutational events detected in HAS1 gene from MM and WM supports the speculation that the precursors of both diseases may undergo a series of shared genetic events, diverging only when tumor specific mutations accumulate in distinct subsets of B lineage cells. In silico comparison of splicesomal assembly between wild type and mutated HAS1 gene gave a pattern that precisely predicts partial retention of intron and aberrant splicing of the HAS1 gene.

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