Constitutive Expression of CD40L on Bone Marrow Mast Cells Supports the Growth of Lymphoplasmacytic Cells in Patients with Waldenstrom’s Macroglobulinemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2350-2350 ◽  
Author(s):  
Olivier Tournilhac ◽  
Daniel Ditzel Santos ◽  
Lian Xu ◽  
Jeffery Kutok ◽  
Yu Tsu Tai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mast Cells ◽  
Cell Surface ◽  
Tumor Cells ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Potent Inducer ◽  
Dose Dependent

Abstract CD40 ligand (CD40L) is a potent inducer of normal and malignant B-cell proliferation through interaction with CD40. We and others have observed excess mast cells (MC) in bone marrow (BM) biopsies of WM patients, which are commonly found admixed with tumor aggregates. (Tournilhac et al, JCO 2004, 22:571S). We therefore sought to clarify the role of MC in WM. Co-culture of 0.5% paraformaldehyde fixed, or sublethally irradiated HMC-1, LAD, and KU mast or basophilic cell lines and sorted BM lymphoplasmacytic cells (LPC) from 10 WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies. As demonstrated by immunohistochemical, multicolor flow cytometric (CD117+FceRI+) and/or RT-PCR analysis, CD40L was expressed on BM MC from 29 of 31 (94%), 11 of 13 (85%), and 7 of 9 (78%) of WM patients, respectively. In contrast, cell surface CD40L expression was not detected by immunohistochemistry (p=0.00005) and flow cytometry (p=0.003) in 5 normal donors, and only faint expression for 1 of 5 normal donors by RT-PCR (p=0.09). Moreover, by multicolor flow cytometry, CD40 was expressed on BM tumor cells from 14/17 (83%) patients. CD40 functionality was confirmed either by the G28.5 CD40 agonistic antibody which induced dose dependent proliferation or by the rh-CD40L which partly prevented serum starvation-induced-apoptosis of WM LPC from 4/4 and 3/4 patients respectively. Importantly, expansion of tumor cells from 3 of 4 patients in mixed cultures with paraformaldehyde fixed MC was blocked in a dose dependent manner by use of a CD40L blocking protein (CD40:Fc). These studies demonstrate that CD40L is constitutively expressed on the cell surface of BM MC in WM and support the growth of WM tumor cells, and therefore provide the framework for therapeutic targeting of MC and MC-WM cell interactions in WM.

The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1844-1844 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Michelle Kuhne ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Mouse Models ◽  
Research Funding ◽  
Therapeutic Agents ◽  
Dependent Manner ◽  
Apoptotic Pathways ◽  
Dose Dependent

Abstract Abstract 1844 Background. We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods. Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results. MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion. These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures: Roccaro: Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

High Levels of Soluble Immunoregulatory Receptors in Patients with WaldenströM’s Macroglobulinemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4881-4881 ◽  
Author(s):  
Zachary R. Hunter ◽  
Andrew R. Branagan ◽  
Daniel Ditzel Santos ◽  
Olivier Tournilhac ◽  
Evdoxia Hatjiharissi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Mast Cells ◽  
Immune Responses ◽  
Pcr Analysis ◽  
P Value ◽  
Rt Pcr ◽  
T Cell Immune Responses ◽  
Immunoregulatory Receptors ◽  
Cell Disorder

Abstract CD25, CD27, CD30 and CD40 are receptors for IL-2, CD70, CD153 and CD154, respectively, which provide important signaling for both B- and T-cell immune responses. We therefore examined the sera of patients with Waldenstrom’s macroglobulinemia (WM), a B-cell disorder characterized by excess IgM secreting bone marrow lymphoplasmacytic cells (LPC) for the presence of soluble CD25, CD27, CD30, CD40, IL-2, CD153, and CD154. Sera were from patients with active disease and off-therapy. Sera from healthy age matched donors (HD) was used for controls. The results of these studies were as follows: WM ELISA Results HD ELISA Results p-value n= median range n= median range sCD25 pg/ml 2.647x10−6 41 3418.99 3–19756.2 20 573.6 184.96–891.03 IL-2 pg/ml 0.72385 41 22.96 3–76.83 20 11.07 5.64–64.7 sCD27 U/ml 2.4727x10−7 26 7.45 0–19.42 10 0 0–2.78 sCD70 ND ND ND ND ND ND ND sCD30 U/ml 0.00088 25 89.25 0–550.81 15 39.12 0–135.25 sCD153 ng/ml 0.68388 25 8.18 0–43.8 15 5.65 0–24.82 sCD40 pg/ml 0.02484 25 101.39 11.76–584.99 11 50.73 0–150.5 sCD154 pg/ml 0.81317 55 949.58 92–4973.16 17 1078.03 351.88–2323.38 Considering our recent studies demonstrating a role for mast cells (MC) in supporting WM cell growth (JCO 2004 22:571S), we examined bone marrow LPC along with MC from WM patients for expression of CD25, CD27 and CD40 and/or their ligands by flow cytometry and RT-PCR analysis. The results of these studies are as follows: Flow Cytometry RT-PCR WM MC WM MC CD25 4/10 (40%) 2/7 (29%) 7/9 (78%) 7/15 (47%) IL-2 ND ND ND ND CD27 5/12 (42%) 2/8 (25%) 7/7 (100%) 4/7 (57%) CD70 6/6 (100%) 10/11 (91%) 7/7 (100%) 2/7 (29%) CD30 1/21 (4.7%) ND ND ND CD153 3/7 (43%) 12/13 (92%) 2/2 (100%) 9/11 (82%) CD40 25/30 (83%) ND ND ND CD154 2/3 (67%) 9/13 (69%) 7/9 (78%) ND The above studies demonstrate high levels of circulating immunoregulatory receptors, and expression of these receptors and/or their ligands on WM tumor and mast cells. Studies addressing their role in immune dysregulation in WM are underway.

Regulation of the New CXCR7 Receptor in Plasma Cell Dyscrasias.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3527-3527
Author(s):  
Anne-Sophie Moreau ◽  
Xavier Leleu ◽  
Xiaoying Jia ◽  
Judith Runnels ◽  
Costas Pitsillides ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Tumor Cells ◽  
Cell Lines ◽  
The Other ◽  
Pcr Analysis ◽  
Boyden Chamber ◽  
Embryonic Cells ◽  
Rt Pcr ◽  
Migration Assay

Abstract Background: Multiple Myeloma (MM) and Waldenstrom Macroglobulinemia (WM) are characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of tumor cells at that site. The receptor for SDF-1, CXCR4 has already been implicated by us and others in the migration and homing of tumor cells to the bone marrow. Recently, a new receptor for the chemokine SDF-1 has been described, namely CXCR7. To date, it has been implicated in the migration of embryonic cells during neurogenesis in zebrafish. It has been reported to be expressed in cancer cells but its function remains unknown. We have previously shown that disruption of the CXCR4/SDF-1 axis interferes with homing of MM cells to the BM. To better understand the homing and migration of MM and WM tumor cells, we sought to study the expression and function of CXCR7. Methods: MM (U266, MM1.S, RPMI, OPM2, OPM1, KMS12BM) and WM (BCWM.1 and WSU-WM) cell lines were used. CD19+ and CD138+ cells were extracted from patient bone marrow samples by microbeads selection after informed consent. To determine the expression of CXCR7 on MM and WM cell lines as well as primary samples, we used flow cytometry and RT-PCR analysis. The migration of tumor cells towards SDF-1 was studied using the transwell boyden chamber migration assay. The adhesion of tumor cells to fibronectin using a fluorometric assay. The CXCR7 inhibitor CCX-754 (ChemoCentryx Inc., Mountain View, CA) was used. Results: CXCR7 was expressed in all cell lines tested except WSU-WM by flow cytometry and RT-PCR. Interestingly, U266 cell line did not express surface CXCR4 and expressed CXCR7 and therefore was used in further experiments to determine the differential role of CXCR7 in SDF-1 related function. Primary WM (n=14) and MM (n=7) cells expressed low intensity CXCR7 by flow cytometry. Use of the above cells in experiments utilizing small molecule antagonists of both CXCR4 and CXCR7 suggested that inhibiting the binding of SDF-1 to one receptor could impact the signaling functions of the other. Experiments are ongoing to clarify the nature of the interaction between these two SDF receptors. Furthermore, adhesion of U266 cells to fibronectin was increased in presence of SDF-1, and inhibited in the presence of CCX-7754. Further analysis to examine CXCR7 and CXCR4 antagonism in adhesion are ongoing. Conclusion: we showed for the first time CXCR7 expression on MM and WM tumor cells. Our results lead us to conclude that through binding of a shared ligand, CXCR7 and CXCR4 may modulate the biological activity of the other.

Primary Waldesntrom Macroglobulinemia Cells Harbor Constitutive Activation of Akt, mTOR, Rictor and Raptor: Rational for Testing a Dual Inhibitor of the PI3K/Akt and mTOR Pathways in This Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3843-3843
Author(s):  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Emanuel N. Husu ◽  
Costas M Pitsillides ◽  
Steven Vesole ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Dna Synthesis ◽  
Cell Lines ◽  
Thymidine Uptake ◽  
Low Grade ◽  
Dependent Manner ◽  
Dual Inhibitor

Abstract Abstract 3843 Poster Board III-779 Background The PI3K/Akt and mTOR pathways play a pivotal role in the initiation and progression of malignancies, enhancing cell survival by stimulating cell proliferation and inhibiting apoptosis. Therefore, it is critical to examine therapeutic agents that explicitly target this pathway, specifically in tumors that harbor activation of the PI3K/Akt pathway, such as Waldenstrom macroglobulinemia (WM). Methods Primary-CD19+ bone marrow-derived WM cells, -bone marrow stromal cells, WM and IgM secreting low-grade lymphoma cell lines (BCWM.1, MEC1, RL), and primary normal CD19+ peripheral blood-derived (CD19+ PB)cells were used. Gene-expression and microRNA profiling have been performed on primary WM cells, as compared to CD19+ PB cells. Cytotoxicity, DNA synthesis, cell cycle and apoptosis were measured by thymidine uptake, MTT, PI staining, and Apo2.7/flow cytometry analysis, respectively. Cell signaling and apoptotic pathways were delineated by Western Blot and immunofluorescence analysis. In vivo homing has been assessed by in vivo flow cytometry. Results Primary bone-marrow derived WM cells are characterized by lower expression of PTEN gene and protein; higher expression of pospho(p)-Akt, p-mTOR, rictor and raptor, as compared to their normal cellular counterpart (CD19+ PB cells). We also observed that microRNA-542-3p and -494 are more highly expressed in primary WM cells as compared to normal CD19+ PB cells (P<.01); and they both target PTEN, as predicted using TargetScan, PicTar, and miRanda algorithms, suggesting their role in inhibiting PTEN expression. We next assessed the effect of the dual PI3K/Akt and mTOR inhibitor NVP-BEZ235 (Novartis, Basel, Switzerland). This agent induced cytotoxicity and inhibited DNA synthesis (IC50 20-25nM) in BCWM.1 at 48 hours. Similar effects were demonstrated in all IgM secreting cell lines and in primary CD19+ WM cells (IC50 20-50nM). No cytotoxicity was observed on CD19+ PB cells, indicating selective toxicity of the compound on the malignant lymphoplasmacytic clone. NVP-BEZ235 inhibited p-Akt and p-mTOR, as well as the downstream Akt- targeted proteins GSK3a/b, p-S6R and p-p70S6, in a dose-dependent manner. Akt and mTOR in vitro kinase activity was also inhibited by NVP-BEZ235 treatment. In addition, NVP-BEZ235 inhibited both rictor and raptor, thus abrogating the rictor-induced Akt phosphorylation in WM cells. NVP-BEZ235 also induced significant cytotoxicity in WM cells in a caspase-dependent and -independent manner, through targeting the forkhead box transcription factors. Finally, NVP-BEZ235 targeted WM cells in the context of bone marrow microenvironment evidenced by significant inhibition of migration, adhesion in vitro and homing in vivo. Conclusion These studies therefore show that dual targeting of the PI3K/mTOR pathway represents a promising therapy for tumors that harbor activation of the PI3K/mTOR signaling cascade such as WM. Disclosures: Ghobrial: Millennium : Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Disseminated tumor cells in bone marrow of breast cancer patients–Comparison of immunocytochemistry and RT-PCR

2006 ◽  
Vol 66 (S 01) ◽  
Author(s):  
T Fehm ◽  
S Becker ◽  
MJ Banys ◽  
G Becker-Pergola ◽  
S Duerr-Stoerzer ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Cancer Patients ◽  
Tumor Cells ◽  
Disseminated Tumor Cells ◽  
Breast Cancer Patients ◽  
Rt Pcr

Parathyroid Ca2+-conducting currents are modulated by muscarinic receptor agonists and antagonists

1997 ◽  
Vol 273 (5) ◽  
pp. E880-E890 ◽  
Author(s):  
Wenhan Chang ◽  
Tsui-Hua Chen ◽  
Stacy A. Pratt ◽  
Benedict Yen ◽  
Michael Fu ◽  
...  
Keyword(s):  
Muscarinic Receptors ◽  
Inositol Phosphate ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Pipette Solution ◽  
Pcr Products ◽  
Inositol Phosphate Accumulation ◽  
Receptor Cdna ◽  
Dose Dependent

Parathyroid cells express Ca2+-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration ≈10−8 M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution. (+)-Muscarine enhanced the adenosine 3′,5′-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed >90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.

scholarly journals New Insights on NETosis Induced by Entamoeba histolytica: Dependence on ROS from Amoebas and Extracellular MPO Activity

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 974
Author(s):  
César Díaz-Godínez ◽  
Joshue Fabián Jorge-Rosas ◽  
Mario Néquiz ◽  
Santiago Martínez-Calvillo ◽  
Juan P. Laclette ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nadph Oxidase ◽  
Entamoeba Histolytica ◽  
Pathogen Detection ◽  
Dependent Manner ◽  
Antimicrobial Proteins ◽  
Nadph Oxidase Activity ◽  
Mitochondrial Ros ◽  
Pre Treatment ◽  
Dose Dependent

NETosis is a neutrophil process involving sequential steps from pathogen detection to the release of DNA harboring antimicrobial proteins, including the central generation of NADPH oxidase dependent or independent ROS. Previously, we reported that NETosis triggered by Entamoeba histolytica trophozoites is independent of NADPH oxidase activity in neutrophils, but dependent on the viability of the parasites and no ROS source was identified. Here, we explored the possibility that E. histolytica trophozoites serve as the ROS source for NETosis. NET quantitation was performed using SYTOX® Green assay in the presence of selective inhibitors and scavengers. We observed that respiratory burst in neutrophils was inhibited by trophozoites in a dose dependent manner. Mitochondrial ROS was not also necessary, as the mitochondrial scavenger mitoTEMPO did not affect the process. Surprisingly, ROS-deficient amoebas obtained by pre-treatment with pyrocatechol were less likely to induce NETs. Additionally, we detected the presence of MPO on the cell surface of trophozoites after the interaction with neutrophils and found that luminol and isoluminol, intracellular and extracellular scavengers for MPO derived ROS reduced the amount of NET triggered by amoebas. These data suggest that ROS generated by trophozoites and processed by the extracellular MPO during the contact with neutrophils are required for E. histolytica induced NETosis.

scholarly journals The antigen presentation function of bone marrow-derived mast cells is spatiotemporally restricted to a subset expressing high levels of cell surface FcεRI and MHC II

2010 ◽  
Vol 11 (1) ◽  
pp. 34 ◽  
Author(s):  
Jian Gong ◽  
Ning-Sun Yang ◽  
Michael Croft ◽  
I-Chun Weng ◽  
Liangwu Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Cell Surface ◽  
Antigen Presentation ◽  
Mhc Ii

scholarly journals Ascorbic acid attenuates activation and cytokine production in sepsis-like monocytes

2021 ◽  
Author(s):  
Tobias Schmidt ◽  
Robin Kahn ◽  
Fredrik Kahn
Keyword(s):  
Flow Cytometry ◽  
Ascorbic Acid ◽  
Cytokine Production ◽  
In Vitro Study ◽  
Surface Expression ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Dose Dependent

Objective To investigate the effects of high dose ascorbic acid (AA) on monocyte polarization and cytokine production in vitro Design Experimental in vitro study of cells from healthy subjects and patients with sepsis Setting University research laboratory and academic hospital Subjects Six healthy controls and three patients with sepsis Interventions Monocytes were isolated from whole blood of healthy donors (n=6) and polarized in vitro for 48hrs using LPS or LTA. Polarization was confirmed by surface marker expression using flow cytometry. As a comparison, monocytes were also isolated from septic patients (n=3) and analyzed for polarization markers. The effect of AA on monocyte polarization was evaluated. As a functional assay, AA-treated monocytes were analyzed for cytokine production of TNF and IL-8 by intracellular staining and flow cytometry following activation with LPS or LTA. Measurements and Main Results Both LPS and LTA induced polarization in healthy monocytes in vitro, with increased expression of both pro- (CD40 and PDL1, p<0.05) and anti-inflammatory (CD16 and CD163, p<0.05) polarization markers, with non-significant effects on CD86 and CD206. This pattern resembled, at least partly, that of monocytes from septic patients. Treatment with AA significantly inhibited the upregulation of surface expression of CD16 and CD163 (p<0.05) in a dose dependent manner, but not CD40 or PDL-1. Finally, AA attenuated LPS or LTA-induced cytokine production of IL-8 and TNF in a dose-dependent manner (both p<0.05). Conclusions AA inhibits upregulation of anti-, but not pro-inflammatory related markers in LPS or LTA polarized monocytes. Additionally, AA attenuates cytokine production from in vitro polarized monocytes, displaying functional involvement. This study provides important insight into the immunological effects of high dose AA on monocytes, and potential implications in sepsis.

scholarly journals Myeloma Cell Associated Therapeutic Protein Discovery Using Single Cell RNA-Seq Data

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Lijun Yao ◽  
Reyka G Jayasinghe ◽  
Tianjiao Wang ◽  
Julie O'Neal ◽  
Ruiyang Liu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Surface ◽  
B Cell ◽  
Tumor Cells ◽  
Plasma Cell ◽  
Tissue Specificity ◽  
Plasma Cells ◽  
Expression Data ◽  
Intracellular Proteins

Multiple myeloma (MM) is a hematological cancer of the antibody-secreting plasma cells. Despite therapeutic advancements, MM remains incurable due to high incidence of drug-resistant relapse. In recent years, targeted immunotherapies, which take advantage of the immune system's cytotoxic defenses to specifically eliminate tumor cells expressing certain cell surface and intracellular proteins have shown promise in combating this and other B cell hematologic malignancies. A major limitation in the development of these therapies lies in the discovery of optimal candidate targets, which require both high expression in tumor cells as well as stringent tissue specificity. In an effort to identify potential myeloma-specific target antigens, we performed an unbiased search for genes with specific expression in plasma and/or B cells using single-cell RNA-sequencing (scRNAseq) of 53 bone marrow samples taken from 42 patients. By comparing &gt;40K plasma cells to &gt;97K immune cells across our cohort, we were able to identify a total of 181 plasma cell-associated genes, including 65 that encode cell-surface proteins and 116 encoding intracellular proteins. Of particular interest is that the plasma cells from each patient were shown to be transcriptionally distinct with unique sets of genes expressed defining each patient's malignant plasma cells. Using pathway enrichment analysis, we found significant overrepresentation of cellular processes related to B-Cell receptor (BCR) signaling, protein transport, and endoplasmic reticulum (ER) stress, involving genes such as DERL3, HERPUD1, PDIA4, PDIA6, RRBP1, SSR3, SSR4, TXNDC5, and UBE2J1. To note, our strategy successfully captured several of the most promising MM therapeutic targets currently under pre-clinical and clinical trials, including TNFRSF17(BCMA), SLAMF7, and SDC1 (CD138). Among these, TNFRSF17 showed very high plasma cell expression, with concomitant sharp exclusion of other immune cell types. To ascertain tissue specificity of candidate genes outside of the bone marrow, we analyzed gene and protein expression data from the Genotype-Tissue Expression (GTEx) portal and Human Protein Atlas (HPA). We found further support for several candidates (incl. TNFRSF17,SLAMF7, TNFRSF13B (TACI), and TNFRSF13C) as being both exclusively and highly expressed in lymphoid tissues. While several surface candidates were not found to be lymphocyte-restricted at the protein level, they remain relevant considerations as secondary targets for bi-specific immunotherapy approaches currently under development. To further investigate potential combinatorial targeting, we examine sample-level patterns of candidate co-expression and mutually-exclusive expression using correlation analysis. As the majority of our detected plasma cell-specific genes encode intracellular proteins, we investigated the potential utility of these epitopes as therapeutic targets via MHC presentation. Highly expressed candidates include MZB1, SEC11C, HLA-DOB, POU2AF1, and EAF2. We analyzed protein sequences using NetMHC and NETMHCII to predict high-affinity peptides for common class-I and class-II HLA alleles. To correlate MHC allelic preference with candidate expression in our cohort, we performed HLA-typing for 29 samples using Optitype. To support our scRNAseq-driven findings, we cross-referenced gene expression data with 907 bulk RNA-sequencing samples, including 15 from internal studies and 892 from the Multiple Myeloma Research Foundation (MMRF), as well as bulk global proteomics data from 4 MM cell lines (TIB.U266, RPMI8226, OPM2, MM1ST) and 4 patients. We see consistent trends across both cohorts, with high positive correlation (Pearson R ranging between 0.60 and 0.99) for a majority of genes when comparing scRNA and bulk RNA expression in the same samples. Our experimental design and analysis strategies enabled the efficient discovery of myeloma-associated therapeutic target candidates. In conclusion, this study identified a set of promising myeloma CAR-T targets, providing novel treatment options for myeloma patients. Disclosures Goldsmith: Wugen Inc.: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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