Crosstalk Between Notch and CD80/86 Signaling In Multiple Myeloma

Abstract 1111

Multiple myeloma is a neoplasm of bone marrow resident plasma cells characterized by its dependence on the bone marrow microenvironment (BME) for production of survival factors including IL-6, a prototypic cytokine in myeloma biology. However, little is known about the molecular and cellular components of the BME involved in IL-6 production. At the cellular level, we and others have previously shown that dendritic cells (DC)-expressing CD80/CD86 (ligands with short cytoplasmic tails and signaling partners of CD28 expressed on myeloma cells) - in the bone marrow microenvironment have been implicated as being an important component. At the molecular level, the CD28-CD80/86 and Notch1-Jagged2 pathways were separately implicated by us (in DC) and others (in stromal cells) in myeloma induced IL-6 production. Blocking either of the pathways causes significant decrease in IL-6 production suggesting crosstalk between the two pathways.

To test our hypothesis, DC were stimulated with CD28-Ig (a soluble form of CD28 which mimicks myeloma cell-bound CD28) in the presence or absence of an inhibitor of Notch signaling -DAPT. DC treated with CD28-Ig and DAPT significantly downregulated IL-6 production when compared to DC treated with CD28-Ig alone. This decrease was not due to the decrease in CD80/86 expression on DC. Our results suggest that CD28 mediated IL-6 production is dependent on Notch signaling and crosstalk between the Notch1-Jagged2 and CD28-CD80/86 pathways leads to IL-6 production by DC. Crosstalk between CD28-CD80/86 and Notch1-Jagged2 pathways was also observed in murine bone marrow derived dendritic cells (BMDC), where a significant down regulation of IL-6 was observed upon blocking Notch signaling.

One possible mechanism of crosstalk involves direct effect of CD80/86 crosslinking by CD28-Ig on Notch expression/signaling leading to increase in IL-6 production. We tested for this possibility in DC and found no significant change in Notch expression/signaling. We thus hypothesized that the mechanism of crosstalk involves molecules downstream of Notch and/or CD80/86. Notch signaling has been reported to be involved in the regulation of PTEN (a negative regulator of the PI3K/Akt pathway). Previous studies have also shown the importance of FoxO3a-a transcription factor tightly regulated by Akt- in regulating IL-6 production in BMDC upon CD80/86 crosslinking. We therefore tested the possible involvement of PTEN (molecule downstream of Notch signaling), PI3K-Akt-FoxO3a axis (downstream of CD80/86) in crosstalk between the two pathways aforementioned by testing the effect of GSI on their regulation at the protein level. Blocking PI3K causes significant decrease in IL-6 production by DC and also decreases phosphorylation of Akt and FoxO3a. Similarly results were observed with blocking Akt activation. Blocking Notch signaling downregulates activation of p-Akt and p-FoxO3a suggesting that crosstalk between Notch-CD80/86 signaling involves PI3K-Akt-FoxO3a axis. Additionally, Notch regulates PI3K pathway via inactivation of PTEN and activation of casein kinase II (a molecule known to phosphorylate PTEN). We propose a model of crosstalk between Notch and CD80/86 signaling involving negative regulation of PTEN (which in turn is regulated by casein kinase II) which drives IL-6 production by PI3K-Akt-FoxO3a upon crosslinking CD80/86 by CD28-Ig.

We have previously reported that in the myeloma BME, DC backsignaling via CD80/86 is also involved in production of indoleamine 2, 3 dioxygenase (IDO), an immunosuppressive enzyme which breaks down tryptophan to L-kynurenine rendering T cells inactive. To test if the model of crosstalk between Notch and CD80/86 signaling is similar in IDO activity, we treated DC with CTLA4-Ig with DAPT/PI3K inhibitor and found significant downregulation of IDO activity suggesting the involvement of PI3K pathway in crosstalk.

Though the median survival and progression-free survival of myeloma patients has doubled over the past decade, it remains incurable prompting the need for finding new targets. Our work helps decipher molecules involved in IL-6 and IDO (important cytokines in myeloma biology) production in the BME of myeloma thus providing novel therapeutic targets. Furthermore, expression of CD28 on T cells and long lived plasma cells (as shown by us previously) helps extend our model of crosstalk to understanding their biology as well.