Myeloid Derived Suppressor Cell mediated AMPK activation regulates Multiple Myeloma cell survival

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that accumulate in different cancer types, including Multiple Myeloma (MM). Besides immune regulation, MDSC promote tumor angiogenesis and tumor growth by secretion of cytokines and growth factors. The presence and activation of MDSC in MM patients has been well-documented, however direct actions of MDSC on cancer cells have been poorly defined. In this study, we investigated MDSC distribution in the immunocompetent 5T33MM murine model and the effects of in vivo MDSC targeting by 5-Fluorouracil (5FU) in the presence of bortezomib. In addition, we determined direct effects of MDSC on MM cells in vitro in the presence of currently used anti-MM drugs (Bortezomib and Melphalan) and analyzed downstream pathways.
In a first part, we determined the effects of the MM microenvironment on the total MDSC population using the 5T33MM model. The number of MDSC (defined as CD11b+ cells) was only increased the first week after MM cell inoculation, while circulating myeloid cells increased at end-stage of disease. In addition, we evaluated the effect of 5FU, an MDSC depleting chemotherapy agent, on tumor progression. 5T33MM mice (n=7/group) were treated with 50mg/kg 5FU on day 4, with bortezomib (0.7mg/kg, 2 times a week), with the combination of both or with vehicle and tumor load was assessed at day 17. We observed a significant reduction in serum M-spike and idiotype positive cells in the BM and spleen (30% reduction compared to vehicle, pBesides immune suppression, MDSC may exert direct effects on tumor growth. To investigate direct actions on MM cells, we performed co-culture experiments of 5T33MM derived MDSC with 5T33MM cells in vitro, resulting in increased survival and proliferation of MM cells. Also in the presence of Bortezomib (2.5-5nM) and Melphalan (15-30µM) we could observe a protection against induced apoptosis. In addition, transwell experiments demonstrated that both cell-cell contact and soluble factors are involved in the observed MDSC-mediated increased survival of MM cells. To investigate underlying pathways we performed a Pathscan® Intracellular Signaling Array (Cell Signaling) demonstrating an upregulation of pAMPK (phospho Adenosine Monophosphate-activated Protein Kinase) in 5T33MM cells after co-culture with MDSC. AMPK plays a key role in energy homeostasis and has been described to be expressed and activated in MM (Baumann et al., 2007). AMPK activation was confirmed by western blot and we could observe an increase in anti-apoptotic factors Mcl-1 and Bcl-2. We subsequently targeted AMPK by BML-275 (compound C) and observed a decrease of AMPK phosphorylation, Mcl-1 and Bcl-2 expression, and an increase in PARP cleavage and apoptosis of MM cells. Using distinct human myeloma cell lines (LP-1, RPMI-8226, U266) we confirmed the effect of BML-275 to reduce MM viability, indicating a role of AMPK in MM cell survival.
In conclusion, our data clearly demonstrate that MDSC directly increase the survival of MM cells, even in the presence of bortezomib or melphalan, mediated by AMPK phosphorylation. In addition, targeting of MDSC in combination with bortezomib showed promising preclinical results for treatment of MM patients.