Christopher Groves

EZH2 Is Required for Breast and Pancreatic Cancer Stem Cell Maintenance and Can Be Used as a Functional Cancer Stem Cell Reporter

Although cancer is largely seen as a disease stemming from genetic mutations, evidence has implicated epigenetic regulation of gene expression as a driving force for tumorigenesis. Epigenetic regulation by histone modification, specifically through polycomb group (PcG) proteins such as EZH2 and BMI‐1, is a major driver in stem cell biology and is found to be correlated with poor prognosis in many tumor types. This suggests a role for PcG proteins in cancer stem cells (CSCs). We hypothesized that epigenetic modification by EZH2, specifically, helps maintain the CSC phenotype and that in turn this epigenetic modifier can be used as a reporter for CSC activity in an in vitro high‐throughput screening assay. CSCs isolated from pancreatic and breast cancer lines had elevated EZH2 levels over non‐CSCs. Moreover, EZH2 knockdown by RNA interference significantly reduced the frequency of CSCs in all models tested, confirming the role of EZH2 in maintenance of the CSC population. Interestingly, genes affected by EZH2 loss, and therefore CSC loss, were inversely correlated with genes identified by CSC enrichment, further supporting the function of EZH2 CSC regulation. We translated these results into a novel assay whereby elevated EZH2 staining was used as a reporter for CSCs. Data confirmed that this assay could effectively measure changes, both inhibition and enrichment, in the CSC population, providing a novel approach to look at CSC activity. This assay provides a unique, rapid way to facilitate CSC screening across several tumor types to aid in further CSC‐related research.

Mycophenolic Acid Differentially Impacts B Cell Function Depending on the Stage of Differentiation

Production of pathogenic Abs contributes to disease progression in many autoimmune disorders. The immunosuppressant agent mycophenolic acid (MPA) has shown clinical efficacy for patients with autoimmunity. The goal of these studies was to elucidate the mechanisms of action of MPA on B cells isolated from healthy individuals and autoimmune patients. In this study, we show that MPA significantly inhibited both proliferation and differentiation of primary human B cells stimulated under various conditions. Importantly, MPA did not globally suppress B cell responsiveness or simply induce cell death, but rather selectively inhibited early activation events and arrested cells in the G0/G1 phase of the cell cycle. Furthermore, MPA blocked expansion of both naive and memory B cells and prevented plasma cell (PC) differentiation and Ab production from healthy controls and individuals with rheumatoid arthritis. Finally, whereas MPA potently suppressed Ig secretion from activated primary B cells, terminally differentiated PCs were not susceptible to inhibition by MPA. The target of MPA, IMPDH2, was found to be downregulated in PCs, likely explaining the resistance of these cells to MPA. These results suggest that MPA provides benefit in settings of autoimmunity by directly preventing activation and PC differentiation of B cells; however, MPA is unlikely to impact autoantibody production by preexisting, long-lived PCs.

The Plasma Cell Signature in Autoimmune Disease

Production of pathogenic autoantibodies by self‐reactive plasma cells (PCs) is a hallmark of autoimmune diseases. We undertook this study to investigate the prevalence of PCs and their relationship to known pathogenic pathways to increase our understanding of the role of PCs in disease progression and treatment response.

Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging

Regulatory authorities require that cell lines used in commercial production of recombinant proteins must be derived from a single cell progenitor or clone. The limiting dilution method of cell cloning required multiple rounds of low‐density cell plating and microscopic observation of a single cell in order to provide evidence of monoclonality. Other cloning methods rely on calculating statistical probability of monoclonality rather than visual microscopic observation of cells. We have combined the single cell deposition capability of the Becton Dickinson Influx™ cell sorter with the microscopic imaging capability of the SynenTec Cellavista to create a system for producing clonal production cell lines. The efficiency of single cell deposition by the Influx™ was determined to be 98% using fluorescently labeled cells. The centrifugal force required to settle the deposited cells to the bottom of the microplate well was established to be 1,126g providing a 98.1% probability that all cells will be in the focal plane of the Cellavista imaging system. The probability that a single cell was deposited by the cell sorter combined with the probability of every cell settling into the focal plane of the imager yield a combined >99% probability of documented monoclonality.

Type I IFNs Regulate Inflammation, Vasculopathy, and Fibrosis in Chronic Cutaneous Graft-versus-Host Disease.

Type I IFNs play a critical role in the immune response to viral infection and may also drive autoimmunity through modulation of monocyte maturation and promotion of autoreactive lymphocyte survival. Recent demonstrations of type I IFN gene signatures in autoimmune diseases, including scleroderma, led us to investigate the pathological role of IFNs in a preclinical model of sclerodermatous graft-versus-host disease. Using a neutralizing Ab against the type I IFN receptor IFNAR1, we observed a marked reduction in dermal inflammation, vasculopathy, and fibrosis compared with that seen in the presence of intact IFNAR1 signaling. The ameliorative effects of IFNAR1 blockade were restricted to the skin and were highly associated with inhibition of chronic vascular injury responses and not due to the inhibition of the T or B cell alloresponse. Inhibition of IFNAR1 normalized the overexpression of IFN-inducible genes in graft-versus-host disease skin and markedly reduced dermal IFN-α levels. Depletion of plasmacytoid dendritic cells, a major cellular source of type I IFNs, did not reduce the severity of fibrosis or type I IFN gene signature in the skin. Taken together, these studies demonstrate an important role for type I IFN in skin fibrosis, and they provide a rationale for IFNAR1 inhibition in scleroderma.

THU0005 Investigating the Plasma Cell Signature in Autoimmune Disease

Background Production of pathogenic autoantibodies by self-reactive plasma cells (PC) is a hallmark of autoimmune diseases; thus, PC levels could be associated with efficacy of B-cell depleting therapies. Additionally, investigating the prevalence of PC in autoimmune disease and their relationship with known pathogenic pathways may increase our understanding of the role of PC in disease progression and treatment response. Objectives Flow cytometry methods that are commonly used to enumerate and describe PC are difficult to implement routinely in the clinic and almost impossible in large clinical trials because of the fragility of PC. This difficulty has hampered thorough assessment of the effect of therapeutics on PC. For this reason, we first developed a highly sensitive and specific gene expression signature that can be easily implemented in the clinic, and then applied this signature to assess the impact of a novel therapeutic on PC and the prevalence of PC in different autoimmune diseases. Methods Whole genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA, IGJ, IGKC, IGKV, and TNFRSF17, whose expression is highly significantly enriched in PC. We combined expression levels of these genes to create a signature score that estimates PC counts in blood and tissue. The sensitivity of this PC signature score was assessed through ex vivo experiments with sorted cells. The PC signature was used to monitor changes in PC levels following anti-CD19 therapy; evaluate the relationship between PC and other autoimmune disease-related genes; and estimate PC levels in affected blood and tissue from multiple autoimmune diseases. Results Results indicated that the PC signature was highly sensitive and capable of detecting as few as 300 PCs. In scleroderma patients enrolled in a Phase I trial with MEDI-551, an anti-CD19 antibody with enhanced effector function, the PC signature was reduced over 90% following MEDI-551 treatment and this reduction was highly correlated (r=0.72, p=0.002) between blood and skin. Comparing the distribution of the PC signature in tissue and whole blood of patients with various autoimmune diseases revealed 30-35% of systemic lupus erythematosus, rheumatoid arthritis, and scleroderma patients with increased PC levels. Conclusions Our results demonstrate the utility of this newly developed gene expression-based PC signature. In addition to providing a robust and straightforward way to accurately measure PC levels in the clinic, the signature may be especially useful to tailor the treatment of autoimmune diseases by identifying those patients who may most benefit from PC-targeted therapies. Disclosure of Interest K. Streicher Shareholder of: MedImmune; AstraZeneca, C. Morehouse Shareholder of: MedImmune; AstraZeneca, C. Groves Shareholder of: MedImmune; AstraZeneca, B. Rajan Shareholder of: MedImmune; AstraZeneca, F. Pilataxi Shareholder of: MedImmune; AstraZeneca, K. Lehmann Shareholder of: MedImmune; AstraZeneca, P. Brohawn Shareholder of: MedImmune; AstraZeneca, B. Higgs Shareholder of: MedImmune; AstraZeneca, K. McKeever Shareholder of: MedImmune; AstraZeneca, S. Greenberg: None Declared, D. Fiorentino: None Declared, L. Richman: None Declared, B. Jallal Shareholder of: MedImmune; AstraZeneca, R. Herbst Shareholder of: MedImmune; AstraZeneca, Y. Yao Shareholder of: MedImmune; AstraZeneca, K. Ranade Shareholder of: MedImmune; AstraZeneca

Baseline Plasma Cell Gene Signature Predicts Improvement in Systemic Sclerosis Skin Scores Following Treatment With Inebilizumab (MEDI-551) and Correlates With Disease Activity in Systemic Lupus Erythematosus and Chronic Obstructive Pulmonary Disease

B cells impact the progression of systemic sclerosis (SSc; scleroderma) through multiple pathogenic mechanisms. CD19 inhibition in mice reduced skin thickness, collagen production, and autoantibody levels, consistent with CD19 expression on plasma cells (PCs), the source of antibody production. PC depletion could effectively reduce collagen deposition and inflammation in SSc; therefore, we investigated the effects of PC depletion on SSc disease activity.

OP0255 Inhibition of the Plasma Cell Signature Correlates with Reduced Collagen Expression in Systemic Sclerosis

Background Systemic sclerosis (SSc) is characterized by excessive extracellular matrix (ECM) deposition in the skin, as well as autoantibody production, release of various cytokines, and T-cell activation. Several lines of evidence indicate a potential role for B cells in the pathogenesis of SSc through effects on autoantibody production, T-cell activation, and fibrosis, among others. Accordingly, targeting B cells could effectively reduce ECM deposition and the inflammatory background of this disease. Objectives In animal models of SSc, decreased B-cell function following CD19 inhibition led to reduced skin thickness and a reduction in autoantibody production, which is consistent with the expression of CD19 on plasma cells (PC), the major source of antibody production. Therefore, our goal was to investigate the effect of CD19 inhibition on PC levels and collagen expression in the skin of SSc patients. Methods We evaluated skin biopsies obtained from a Phase I clinical trial of MEDI-551, an anti-CD19 antibody expected to deplete PC and B cells. Skin biopsies were collected pre-treatment (baseline) and at 29 days post-treatment with either MEDI-551 or placebo. To evaluate PC, we used whole genome microarray analysis of sorted cellular fractions to identify a panel of genes expressed predominantly in PC. To account for the multiple collagen types involved in skin fibrosis, a collagen score was calculated as the median expression of three collagen genes, COL1A1, COL3A1, and COL5A1. Results In patients with SSc, the collagen score was positively correlated with the modified Rodnan skin score (mRSS) disease activity measure at baseline (r=0.67, p=0.004), highlighting the role of collagen expression in SSc disease severity. Additionally, we identified a positive correlation between the PC signature and the collagen score at baseline (r=0.60, p=0.001). Following anti-CD19 treatment, the collagen score and the PC signature were both inhibited in skin as much as 90%, with a median inhibition of 35% and 50%, respectively. No change in the PC signature or collagen score was observed following placebo treatment. Interestingly, the inhibition of the PC signature was positively correlated with inhibition of the collagen score in all patients (r=0.80, p=0.001). Conclusions The positive correlation observed between inhibition of the PC signature and inhibition of this collagen score in scleroderma skin supports a role for PC in the pathogenic mechanism of this disease. These preliminary results need to be validated in a larger cohort of patients to determine if changes in PC signature and the collagen score could predict those who respond to treatment with PC-depleting therapeutics. Disclosure of Interest K. Streicher Shareholder of: MedImmune; AstraZeneca, C. Morehouse Shareholder of: MedImmune; AstraZeneca, C. Groves Shareholder of: MedImmune; AstraZeneca, B. Rajan Shareholder of: MedImmune; AstraZeneca, F. Pilataxi Shareholder of: MedImmune; AstraZeneca, K. Lehmann Shareholder of: MedImmune; AstraZeneca, P. Brohawn Shareholder of: MedImmune; AstraZeneca, B. Higgs Shareholder of: MedImmune; AstraZeneca, K. McKeever Shareholder of: MedImmune; AstraZeneca, L. Richman: None Declared, B. Jallal Shareholder of: MedImmune; AstraZeneca, R. Herbst Shareholder of: MedImmune; AstraZeneca, Y. Yao Shareholder of: MedImmune; AstraZeneca, K. Ranade Shareholder of: MedImmune; AstraZeneca

THU0034 CD19 Distinguishes Memory and Pre-Memory Antibody Secreting Cells

Background Plasma cells (PC), are differentiated B cells that play a major role in humoral immunity as they actively secrete antibodies which identify and neutralize foreign antigens. Normally, plasma cells develop in response to immune challenges such as infection or vaccination. In autoimmune diseases, however, PCs develop which secrete anti-self antibodies that may play a role in disease pathogenesis. Therefore, PCs are potential targets for therapeutic intervention in autoimmune disease. However, consideration must be given to the potential impact on protective immunity of PC targeted therapy. Objectives A recent report has identified long-lived PC in autoimmune patients which secrete Ig specific for self-antigen (1). These autoreactive cells represent a novel therapeutic target. The self-reactive PCs in the study were identified by commonly known cellular markers including the surface expression of CD19. CD19 is a molecule that is present on B cells from the earliest stages of development through the memory and plasma cell stage. Therefore, our goal was to characterize in detail the expression of CD19 on plasmablasts and plasma cells from human blood and tissues, and relate the phenotype of PCs from different tissues to production of antigen-specific immunoglobulin. Methods PCs were isolated from peripheral blood, tonsil, spleen, and bone marrow (BM) from human donors and characterized for surface antigen expression by flow cytometry. Samples of sorted cells were further characterized by mRNA expression profiling and by ELISPOT to determine total as well as antigen-specific production of immunoglobulin. Results Here we show that CD19 is expressed on normal PC found in human blood, tonsil, spleen, and bone marrow. However, we have identified a population of CD19- PC which exists in the spleen and bone marrow as a sub-population of PC in these tissues. We show that the bone marrow CD19- PC and CD19+ PC have nearly identical morphology, immunophenotype, mRNA expression, and function. However the CD19- PC fraction has a significantly increased frequency of cels which secrete antibodies specific for tetanus/diptheria toxin and influenza vaccine antigens when compared to CD19+ PC in human BM. Conclusions The results demonstrate that most PC from human blood and tissue are CD19+, although cell surface levels of CD19 can vary. Importantly, there exists a distinct CD19- population of PC in spleen and BM. These cells appear to be of a long-lived memory phenotype and secrete antibodies to vaccine antigens. The results have significant implications for the targeting of PC in autoimmune disease, especially with depleting antibodies against CD19, such as MEDI-551. References Mahévas M. et al (2013) B cell depletion in immune thrombocytopenia reveals splenic long-lived plasma cells. J Clin Invest 123(1):432-42. Disclosure of Interest None Declared