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.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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