CD56 Has a Critical Role in Regulating Multiple Myeloma Cell Growth and Response to Therapies

Multiple myeloma (MM) is a disease derived from genetically abnormal clonal plasma cells. MM cells aberrantly express several surface antigens compared with normal plasma cells. Among others, CD56/NCAM1 is present at variable levels in approximately 70% of MM patients. Very little is known about its role in MM; however, CD56 positivity in MM correlates with greater osteolytic burden and lower frequency of good prognostic features, such as the presence of t(11;14). We first analyzed 569 patients with MM diagnosed between 1/1/2005 and 12/31/2014 at the Ohio State University Wexner Medical Center, stratifying them based on the percentage of CD56-expressing clonal MM cells. The mean percentage of CD56-expressing clonal MM was 26.5%, with range from 0 to 100%; the Mean Fluorescent Intensity values varied, with a quarter of patients expressing CD56 at high intensity. We then evaluated patient outcomes based on the percentage of CD56-expressing clonal MM cells. We noticed that MM patients with more than 30 or 50 percent of CD56-expressing MM clonal cells have inferior clinical outcomes than patients with less than 30 or 50 percent of CD56-expressing MM clonal cells, with median overall survival of 9.61 versus 7.64 years (log-rank p-value: 0.004) or 9.30 versus 6.47 years (log-rank p-value: 0.0009), respectively. We then demonstrated by conventional and real-time PCR analyses that the predominately expressed CD56 isoform in MM has signaling potential with a transmembrane portion and cytosolic tail. Therefore, we evaluated the functional role of CD56 in MM. By gain-of function studies in U266 and MM.1S MM cell lines, we showed that overexpression of CD56 promotes MM growth and viability; the opposite effect occurred with CD56 silencing in H929, OPM-2, and RPMI-8226 MM cell lines, which leads to reduced MM growth and increased apoptotic cell death. Overexpression of CD56 resulted in the phosphorylation and hence activation of ribosomal protein S6 kinase A3 (RSK2) and of the transcription factor, cAMP responsive element binding protein 1 (CREB1). This induced CREB1 binding to DNA consensus CRE elements, and promoted transcription of CREB1 targets, the anti-apoptotic genes BCL2 and MCL1. CD56 silencing in H929 and OPM-2 MM cell lines had opposite effects, with reduction of phospho-RSK2, phospho-CREB1, MCL1, and BCL2 levels. We then used shRNAs targeting RSK2 and CREB1 or specific inhibitors (BI-D1870 that is a RSK2 inhibitor, and 666-15 that is a CREB1 inhibitor) at 0.1-1 microM concentration. We evaluated viability by MTT assay or Zombie dye staining on CD138 positive MM cells and apoptosis by Annexin V-PI staining. We demonstrated that CD56 positive MM cell lines (H929, OPM-2, and RPMI-8226) or patients with high CD56 expression (>30% of CD56-expressing clonal MM cells) are more sensitive to RSK2/CREB1 inhibition compared with CD56 negative MM cell lines (U266, L363, and MM.1S) or patients with low CD56 expression (<30% of CD56-expressing clonal MM cells). Using similar strategies, we also proved that CREB1 is essential to CD56-protumoral phenotype, since CREB1 inhibition reduces cellular growth and viability in CD56 overexpressing U266 cells. RSK2 and CREB1 inhibition mimic CD56 silencing with decrease of BCL2 and MCL1 mRNA and protein levels. Furthermore, we observed that CD56 signaling by CREB1 activation decreases CRBN expression, reducing responses to lenalidomide. Conversely, CREB1/RSK2 blockade rescued CRBN levels in CD56 positive MM cells and increased lenalidomide response. These results support the hypothesis that targeting CREB1 is hence a so far unexplored but potentially effective synthetic lethal strategy for CD56-expressing MM patients. In conclusion, our study defines an effective threshold for therapeutic intervention in CD56-expressing MM patients. Moreover, our data pioneer the use of CREB1/RSK2 inhibition in CD56-expressing MM cells, either as single agents or in combination with lenalidomide, suggesting that CD56 can be a prognostic and predictive factor of response in MM. Disclosures No relevant conflicts of interest to declare.

Detection of Clonal Plasma Cells in POEMS Syndrome Using Multiparameter Flow Cytometry

Introduction POEMS syndrome is a rare systemic disorder characterized by various symptoms, including polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy (M-protein), and skin changes, which is based on the presence of monoclonal plasma cells (PC). We previously reported the analysis of the clonal immunoglobulin λ light-chain variable region (IGLV) gene rearrangements in POEMS syndrome using next-generation sequencing (NGS), and the clone identification rate was 36.7% (Kawajiri-Manako C, Am J Hematol. 2018;93(9):1161). EuroFlow-based next-generation flow (EuroFlow-NGF) has also been standardized for detecting minimal residual disease in multiple myeloma (MM). However, multiparameter flow cytometry (MFC) utility in detecting clonal PC of POEMS syndrome remains unknown. Therefore, to clarify the feasibility of detecting the clonality with MFC, we analyzed PC of bone marrow samples in patients with POEMS syndrome by MFC and validated gating strategies of PC in POEMS syndrome. Methods Patients with newly diagnosed or relapsed POEMS syndrome (n=25) at Chiba University Hospital from April 2019 to July 2021 were included in this study. Primary bone marrow aspirations were performed after obtaining informed consent following the Declaration of Helsinki. The collected bone marrow samples were analyzed by the SRL-Flow protocols (single-tube 8-color: CD138/CD27/CD38/CD56/CD45/CD19/CyIgκ/CyIgλ), which is highly correlated with EuroFlow-NGF (Takamatsu H, Int J Hematol. 2019;109(4): 377). Furthermore, 21 cases for which data files were available from SRL Inc. were reanalyzed by gating PC with lower expression of CD45 regarding the previous study (Dao LN, Blood. 2011;117(24):6438). This study was approved by the ethics committee of Chiba University Graduate School of Medicine. Results Among 25 patients, the median age was 59 (25-77) years, and 13 (52.0%) were men. The types of M-proteins identified by immunofixation electrophoresis (IFE) were IgA-λ in 12 (48.0%), IgG-λ in 9 (36.0%), IgA-κ in 1 (4.0%), IgG-κ in 1 (4.0%), and negative in 2 patients (8.0%), respectively. The median serum VEGF level was 3,240 (747-9,060) pg/ml. SRL-Flow identified the clonal PC in 9 of 25 (36.0%, λ in 7 and κ in 2): 7 of 23 IFE-positive cases (30.4%) and 2 of 2 IFE-negative cases (100%). The median clone size was 0.03 (0.01-3.78)%, smaller than MM. Interestingly, the clone size was not correlated with the level of serum VEGF (r = -0.17). Aberrant expression of clonal PC in POEMS syndrome was similar to MM: CD19-56+ in 5 (55.6%), CD19-56- in 2 (22.2%), CD19dim56- in 1 (11.1%), and CD19-56dim in 1 (11.1%) patient, respectively. No clone was detected in PC with CD19+56-. The clone identification rate by single-tube 8-color SRL-Flow protocols was lower than expected. Therefore, we reanalyzed the data by gating PC with lower expression of CD45 and CD38. In the reanalysis, we gated broadly for CD38 dim-to-bright and CD138 dim-to-bright cells and narrowly for CD38 dim and CD45 negative-to-dim cells. As a result, we identified the clonal PC in 15 of 21 cases (71.4%, λ in 13 and κ in 2): 13 of 19 IFE-positive cases (68.4%) and 2 of 2 IFE-negative cases (100%)). Of the 15 cases in which monoclonal PC were identified by reanalysis, 8 were negative by SRL-Flow. Of these, 6 were identified by selective gating of CD45- and CD38 dim, and 2 were identified by selective gating of CD45- and CD38+. The median clone size was 0.008 (0.001-3.27)%, smaller than that of MM, and the clone size was not correlated with the level of serum VEGF (r = -0.15). The aberrant expression of the clonal PC in POEMS remained similar to MM for CD19 and CD56 expression: CD19-56+ in 3 (20.0%), CD19-56- in 7 (46.7%), CD19-56dim in 4 (26.7%), and CD19dim 56- in 1 (6.7%) patient, respectively. No clone was detected in PC with CD19+56-. Conclusions EuroFlow-based SRL-Flow protocols detected the clonal PC in about one-third of POEMS patients. Aberrant immunophenotype of clonal PC in POEMS syndrome was similar to MM for CD19 and CD56 expression; however, CD45 and CD38 expression of POEMS PC tended to be downregulated. Selective gating of PC with CD38dim and CD45 negative-to-dim detected clonal PC in 71.4% of cases. This novel gating strategy is more accessible and might improve the identification rate of clonal PC in POEMS syndrome. Disclosures Tsukamoto: Daiichi Sankyo: Honoraria. Yokote: Kowa Company, Ltd.: Honoraria, Other: Scholarship; MSD K.K.: Honoraria, Other: Scholarship, Courses endowed by company; Astellas Pharma Inc.: Honoraria, Other: Scholarship; Mitsubishi Tanabe Pharma Corporation: Honoraria, Other: Scholarship; Amgen K.K.: Honoraria; Taisho Pharmaceutical Co., Ltd.: Honoraria, Other: Scholarship, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Honoraria, Other: Scholarship; Janssen Pharmaceutical K.K.: Honoraria; Kao Corporation: Other: Schlarship; Novo Nordisk Pharma Ltd.: Honoraria, Other: Scholarship; Teijin Pharma Limited: Other: Scholarship; Pfizer Japan Inc.: Honoraria, Other: Scholarship; Kyowa Kirin Co., Ltd.: Honoraria; Eli Lilly Japan K.K.: Honoraria, Other: Scholarship; Takeda Pharmaceutical Company Limited: Honoraria, Other: Scholarship; Sanofi K.K.: Honoraria; Ono Pharmaceutical Co., Ltd: Honoraria, Other: Scholarship; AstraZeneca K.K.: Honoraria; Daiichi Sankyo Company, Limited: Honoraria, Other: Scholarship; Novartis Pharma K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria, Other: Scholarship; Shionogi Co., Ltd.: Other: Scholarship; Bayer Yakuhin, Ltd.: Other: Scholarship. Nakaseko: Novartis Pharma KK.: Honoraria. Takamatsu: Adaptive Biotechnologies, Eisai: Honoraria; SRL: Consultancy; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding. Sakaida: Bristol Myers Squibb: Research Funding; Chugai: Research Funding; Ono: Research Funding; Kyowa Kirin: Research Funding.

Increased Expression of CD56 on Plasmacytoid Dendritic Cells in Myeloid Leukemia Associated with Down Syndrome (ML-DS) Is Normal Regeneration and Not Measurable Residual Disease

Introduction: It has long been known that Down syndrome is associated with an increased risk for hematologic malignancies. One such disease is myeloid leukemia associated with Down syndrome (ML-DS), a disease that almost always occurs during the first 5 years of life. As research on ML-DS has progressed, understanding has grown that after the initiation of therapy, the non-neoplastic myeloid progenitor cells in ML-DS patients have a characteristic immunophenotype with the expression of CD56 on a subset of the CD34+ myeloid progenitor cells being one of the most notable features. The discovery that this immunophenotype is normal in ML-DS patients post-therapy has been of the utmost importance as it has led to such patients being properly classified as being negative for measurable residual disease. Plasmacytoid dendritic cells (pDCs) are another cell type in which CD56 expression is often part of the neoplastic immunophenotype, and we hypothesized that CD56 may also be differentially expressed in ML-DS pDCs post-therapy. Herein, we investigated the immunophenotype of pDCs in ML-DS patients found to be negative for measurable residual disease. Methods: A total of 10 bone marrow specimens from ML-DS patients post-treatment initiation and 7 bone marrow specimens from patients that did not have DS, were aged 0-4 years (matching the age range of ML-DS), had a myeloid neoplasm and were post-treatment initiation (non-DS) were included in this study. All specimens were found to be negative for measurable residual disease by difference from normal (ΔN) flow cytometry (the gold standard for the determination of residual disease in the Children's Oncology Group 1531 study on ML-DS) and were evaluated for CD56 and CD303 expression on pDCs. pDCs were defined as HLA-DR+/CD123++ (high intensity). Results: As expected, the ML-DS patients had a significantly greater percentage of CD34+CD56+ myeloid progenitor cells than the non-DS group, both in terms of percent total non-erythroid cells (0.9% vs 0.006%, P<0.001) and percent total myeloid progenitors (38% vs 0.57%, P<0.001). There was not a significant difference between groups in terms of pDC percentage (ML-DS 0.63% of total non-erythroid cells vs non-DS 0.53%, P=0.9%). There were also no significant differences in CD303 expression between the groups, both in terms of percent positive (ML-DS 89% vs non-DS 92%, P=0.5) and mean fluorescence intensity (MFI, in PE) (ML-DS 190 vs non-DS 246, P=0.3). On the other hand, the ML-DS group had significantly greater CD56 expression than the non-DS group, both in percent positive (74% vs 25%, P=0.005) and MFI (PE) (122 vs 5.9, P=0.005). Nine of the 10 ML-DS specimens had CD56 expression on greater than 50% of pDCs, and 3 showed a CD56 MFI of over 200. Conclusions: The data from this preliminary study indicate that much like the myeloid progenitor cells, the pDCs in ML-DS patients after the initiation of therapy have an immunophenotype that could be mistaken as abnormal. Importantly, they show that the setting of cutoff values for the determination of abnormal pDC CD56 expression, even relatively high ones, could lead to false positive results in ML-DS specimens post-treatment initiation. The dissemination of this knowledge is of increased importance as more flow cytometry laboratories begin to increase their investigation of pDCs. Further studies are needed to delineate common mechanisms between the expression of CD56 in myeloid progenitor cells and pDCs in ML-DS patients post-treatment initiation. Disclosures Pardo: Hematologics, Inc.: Current Employment. Lott: Hematologics, Inc.: Current Employment. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership.

Defining the AHR regulated transcriptome in NK cells reveal gene expression program relevant to development and function

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular processes in cancer and immunity, including innate immune cell development and effector function. However, the transcriptional repertoire through which AHR mediates these effects remains largely unexplored. To elucidate the transcriptional elements directly regulated by AHR in NK cells, we performed RNA- and ChIP-sequencing on NK cells exposed to AHR agonist or antagonist. We show that mature peripheral blood NK cells lack AHR, but its expression is induced by Stat3 during IL-21-driven activation and proliferation, coincident with increased NCAM1 (CD56) expression resulting in a CD56bright phenotype. Compared to control conditions, NK cells expanded in the presence of the AHR antagonist, StemRegenin-1, were unaffected in proliferation or cytotoxicity, had no increase in NCAM1 transcription and maintained the CD56dim phenotype. However, it showed altered expression of 1,004 genes including those strongly associated with signaling pathways. In contrast, NK cells expanded in the presence of the AHR agonist, kynurenine, showed decreased cytotoxicity and altered expression of 97 genes including those strongly associated with oxidative stress and cellular metabolism. By overlaying these differentially expressed genes with AHR chromatin binding we identified 160 genes directly regulated by AHR, including hallmark AHR targets AHRR and CYP1B1, and known regulators of phenotype, development, metabolism, and function such as NCAM1, KIT, NQO1, and TXN. In summary, we define the AHR transcriptome in NK cells, propose a model of AHR and Stat3 coregulation, and identify potential pathways that may be targeted to overcome AHR-mediated immune suppression.

A Novel Mice Model for Studying the Efficacy and IRAEs of Anti-CTLA4 Targeted Immunotherapy

BackgroundPatient-derived orthotopic xenograft (PDOX) is a popular animal model for translational cancer research. Immunotherapy is a promising therapy against glioblastoma (GBM). However, the PDOX model is limited to evaluating immune-related events. Our study aims to establish GBM humanized PDOX (HPDOX) mice models to study the mechanism of anti-CTLA4 immunotherapy and immune-related adverse events (IRAEs).MethodsHPDOX models were established by culturing GBM tissues and intracranially implanting them in NSG mice. Meanwhile, peripheral blood mononuclear cells (PBMCs) were separated from peripheral blood and of GBM patients and administrated in corresponding mice. The population of CD45+, CD3+, CD4+, CD8+, and regulatory T (Treg) cells was estimated in the peripheral blood or tumor.ResultsT cells derived from GBM patients were detected in HPDOX mice models. The application of anti-CTLA4 antibodies (ipilimumab and tremelimumab) significantly inhibited the growth of GBM xenografts in mice. Moreover, residual patient T cells were detected in the tumor microenvironment and peripheral blood of HPDOX mice and were significantly elevated by ipilimumab and tremelimumab. Additionally, Treg cells were decreased in mice with IRAEs. Lastly, the proportion of CD4+/CD8+ T cells dramatically increased after the administration of ipilimumab. And the degree of IRAEs may be related to CD56+ expression in HPDOX.ConclusionsOur study established HPDOX mice models for investigating the mechanism and IRAEs of immunotherapies in GBM, which would offer a promising platform for evaluating the efficacy and IRAEs of novel therapies and exploring personalized therapeutic strategies.