ALBERT

Description: We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot- reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti- glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non- stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.

Description: We found that a monoclonal antibody (MoAb) to CD9 antigen, PMA2, induced a rise in cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded platelets, and we examined whether this response was due to direct action of PMA2 on CD9 antigen. The rise in [Ca2+]i was dependent on the PMA2 concentration, irrespective of the presence or absence of extracellular Ca2+. The role of secreted adenosine diphosphate (ADP) and thromboxane in the [Ca2+]i response to PMA2 was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. Combined treatment with CP/CPK and aspirin abolished the rise in [Ca2+]i, although either CP/CPK or aspirin alone produced only partial inhibition. Inhibition of adenosine triphosphate (ATP) secretion and thromboxane B2 synthesis by an MoAb to the glycoprotein IIb-IIIa complex, PMA1, resulted in little [Ca2+]i response to PMA2. In contrast, thrombasthenic platelets, in which ATP secretion and thromboxane B2 synthesis were normal, showed a normal [Ca2+]i response. When PMA2 was added to CD9+ mononuclear cells, no rise in [Ca2+]i was observed. Thus, we conclude that binding of monoclonal immunoglobulin G molecules to the CD9 antigen raises [Ca2+]i through the effect of secreted ADP and thromboxane on platelets, and that CD9 antigen is not directly involved in induction of Ca2+ influx and mobilization.

Description: We found that a monoclonal antibody to CD9 antigen, PMA2, induces fibrinogen binding to platelets and examined the mechanism for this. That PMA2 recognized the CD9 antigen was confirmed by its immunoblot- reactivity with a 24,000-dalton protein, reactivity with platelets and common acute lymphoblastic leukemia (ALL) cells, and competitive binding with the ALB6 antibody known as the CD9 antibody. At saturation, PMA2 bound to approximately 46,000 sites per platelet. The binding of 125I-fibrinogen to platelets occurred in a PMA2 concentration-dependent manner and was blocked by EDTA or an anti- glycoprotein (GP)IIb-IIIa monoclonal antibody. PMA2-stimulated platelets caused ATP secretion and thromboxane B2 synthesis under non- stirred conditions. The role of secreted ADP and thromboxane in fibrinogen-binding and subsequent platelet aggregation was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. CP/CPK or aspirin alone reduced fibrinogen binding to 20% to 30%; however, this binding was sufficient to support full platelet aggregation. Combined treatment with CP/CPK and aspirin abolished fibrinogen binding and aggregation. These results demonstrate that the binding of IgG molecules to the CD9 antigen exposes fibrinogen receptors through both secreted ADP and thromboxane and that either one of both can expose the receptors to an extent sufficient to aggregate platelets.

Description: Abstract 3527 Increasing evidence compiled from our lab and several others is illuminating the importance of epigenetic mechanisms in the pathogenesis of multiple myeloma (MM). The biochemical modifications that govern epigenetics are DNA methylation, and post-translational modifications of histone proteins on arginine and lysine residues. Modifications are generally associated with activation or repression of gene transcription depending upon the specific position of the modification. Histone methylation, catalyzed by histone methyltransferases (HMT) and histone demethylases (HDMT) are crucial for the proper programming of the genome during development. Deregulation of the methylation machinery can alter chromatin configuration and disrupt normal transcriptional programs, both features of cancer cells. Misregulated methylation may result from several genetic alterations in chromatin-modifying enzymes, which can include mutations, over-expression or chromosomal abnormalities. Indeed, genomic alterations of MLL, NSD1, CREBBP(CBP) MMSET and UTX, all encoding proteins involved in post-translational histone modifications, have been implicated in MM. The purpose of our study was to interrogate existing genomics datasets to identify additional HMT or HDMT genes that may be important in the pathogenesis of MM and, thus could become potential therapeutic targets. To identify novel HMT or HDMT in MM we mined existing expression, copy number and whole genome sequencing data generated as part of the Multiple Myeloma Research Consortium Genomics Initiative. Collectively, the datasets converged on numerous alterations involving histone methylation on lysine 9 (H3K9). One of these genes, a HDMT called JMJD1C, was over-expressed in approximately 15% of MM samples examined. We validated over-expression of JMJD1C expression by RT-PCR in over 50 clinical MM samples and 10 human myeloma cell lines (HMCLs). We also confirmed over-expression of JMJD1C by immunohistochemistry (IHC) of a tissue microarray (TMA) consisting of over 60 MM samples. Next we examined the levels of JMJD1C expression and H3K9 methylation by IHC, RT-PCR and western blot to determine the extent to which JMJD1C expression is associated with a decrease in H3K9 methylation (or vice versa). We demonstrated this negative correlation in 6/10 HMCL including OCIMY7 and KMS11 and in over 60% of clinical samples examined on the TMA. Furthermore, we demonstrated that H3K9 methylation, a repressive mark associated with heterochromatin, is low in a large percentage of samples. In addition, we generated double knockout isogenic cell line pairs using zinc finger nuclease technology to evaluate the direct role of JMJD1C on H3K9 methylation, cell function and chromatin and expression level changes using ChIP-seq and RNA-seq analyses. In summary our preliminary results demonstrate that JMJD1C is highly expressed in a significant percentage of MM patients. Our results demonstrate a noticeable inverse relationship of JMJD1C expression to H3K9. This reflects the importance of JMJD1C in modifying the H3K9 repressive mark and suggests that loss of repression due to JMJD1C-mediated histone demethylation disrupts the chromatin machinery and leads to neoplastic processes. Further studies are ongoing to realize JMJD1C as a potential and novel therapeutic target in MM. Disclosures: No relevant conflicts of interest to declare.

Description: We found that a monoclonal antibody (MoAb) to CD9 antigen, PMA2, induced a rise in cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded platelets, and we examined whether this response was due to direct action of PMA2 on CD9 antigen. The rise in [Ca2+]i was dependent on the PMA2 concentration, irrespective of the presence or absence of extracellular Ca2+. The role of secreted adenosine diphosphate (ADP) and thromboxane in the [Ca2+]i response to PMA2 was studied using creatine phosphate/creatine phosphokinase (CP/CPK) and aspirin. Combined treatment with CP/CPK and aspirin abolished the rise in [Ca2+]i, although either CP/CPK or aspirin alone produced only partial inhibition. Inhibition of adenosine triphosphate (ATP) secretion and thromboxane B2 synthesis by an MoAb to the glycoprotein IIb-IIIa complex, PMA1, resulted in little [Ca2+]i response to PMA2. In contrast, thrombasthenic platelets, in which ATP secretion and thromboxane B2 synthesis were normal, showed a normal [Ca2+]i response. When PMA2 was added to CD9+ mononuclear cells, no rise in [Ca2+]i was observed. Thus, we conclude that binding of monoclonal immunoglobulin G molecules to the CD9 antigen raises [Ca2+]i through the effect of secreted ADP and thromboxane on platelets, and that CD9 antigen is not directly involved in induction of Ca2+ influx and mobilization.