Myeloma Cell Survival and Importance of Crosstalk Between Notch1-Jagged2 and CD28-B7 Pathways In Dendritic Cells

Abstract 1741

Multiple myeloma is a neoplasm of bone marrow resident plasma cells characterized by critical interactions between myeloma cells and bone marrow stromal cells. This interaction leads to production of IL-6, an important factor in myeloma cell biology. However, the molecular and cellular components involved in myeloma induced IL-6 production remain largely uncharacterized. While at the cellular level, dendritic cells (DC)-expressing CD80/CD86 (collectively called B7, 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-B7 and Notch1-Jagged2 pathways were separately implicated by us (in DC) and others in myeloma induced IL-6 production. Although Notch signaling leading to IL-6 production in DC is well understood, the mechanism of “backsignaling” via B7 is largely uncharacterized.

To better understand downstream B7 signaling leading to IL-6 production, 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, gamma secretase inhibitor (GSI). DC treated with CD28-Ig alone produced significantly (p< 0.001) higher levels of IL-6 when compared to DC treated with CD28-Ig and GSI. GSI specifically targeted Notch signaling as observed by decreased expression of Notch gene targets: Hes-1 (2 fold decrease) and Deltex-4 (4 fold decrease). Also, decreased IL-6 levels in presence of GSI were not due to the decrease in B7 expression on DC. To specifically implicate the importance of Notch1 and Jagged2, we blocked Notch1 signaling using blocking antibodies and observed a similar decrease in IL-6 production upon blocking Notch1 signaling. Our results suggest that CD28 mediated IL-6 production is dependent on Notch1 signaling and crosstalk between the Notch1-Jagged2 and CD28-B7 pathways leads to IL-6 production by DC. The model of crosstalk between CD28-B7 and Notch1-Jagged2 pathways was also observed in murine bone marrow derived dendritic cells (BMDC), where a significant (p<0.001) down regulation of IL-6 was observed upon blocking Notch signaling.

One possible mechanism of crosstalk involves direct effect of B7 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 to Notch and/or B7. 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 B7 crosslinking. We therefore tested the possible involvement of PTEN (molecule downstream of Notch signaling), Akt and FoxO3a (molecules downstream of B7) in crosstalk between the two pathways aforementioned by testing the effect of GSI on their regulation at the protein level. We observed an approximate 2 fold decrease in phospho-PTEN/PTEN ratio in DC treated with GSI and remained so even after B7 crosslinking at an early time point (15 min. post CD28-Ig treatment.) Further, phospho-Akt/Akt ratio decreased by 1.6 fold in DC treated with both GSI and CD28-Ig compared to CD28-Ig alone at 30 min. We therefore hypothesize a model of crosstalk involving Notch mediated regulation of PTEN leading to IL-6 production via regulation of Akt and possibly FoxO3a upon B7 crosslinking. Interestingly enough “backsignaling” via B7 in myeloma-induced IL-6 production seems to involve molecules well characterized in CD28 signaling of T-cells. Targeting IL-6 induced by crosstalk between these two pathways prompts not only clinical evaluation to improve MM patient outcome but also extends to advancing knowledge in T-cell and normal plasma cell biology as well.