Molecular Pathways That Determine the Activation State of Macrophages within the Myeloma Niche

Abstract 443

Benefit from cytotoxic therapy in myeloma may be limited by persistence of residual tumor cells nested within favorable niches. However, the contribution of macrophages to the regulation of myeloma niches is still incompletely understood. We have previously shown that macrophages provide growth and anti-apoptotic signals to myeloma cells when grown in co-culture. These results prompted us to investigate the regulation of primary monocytes/macrophages that reside within myeloma niches in the bone marrow.

Unmanipulated CD14+ monocytic cells, freshly explanted from myeloma bone marrows, displayed a pre-dominantly pro-inflammatory transcriptomic profile when compared to normal monocytes. We found enhanced transcription of genes encoding pro-inflammatory mediators, known to support growth and survival of myeloma cells, such as TNFalpha, IL-1beta, IL-6, IL-8 and TACI. Downregulation of TGFbeta was also consistent with a pro-inflammatory (M1) signature. Interestingly, we also found concurrent transcription of some genes characteristic of “alternative macrophage activation” (M2 phenotype) such as IL-10 and IL1-receptor antagonist (IL-1RN). These results suggest that myeloma-associated macrophages, while being predominantly pro-inflammatory, display significant plasticity between the M1-M2 phenotypic extremes.

To obtain insights into the underlying mechanisms, we examined the role of TPL2 (Cot, MAP3K8), a serine/threonine kinase with central and non-redundant roles in regulating innate immune responses in macrophages following activation by Toll receptor (TLR) ligands and members of the TNF ligand superfamily. In myeloma-associated macrophages, we found constitutive activation of a TPL2 kinase-dependent, ERK-mediated pathway that promotes synthesis and processing of pro-inflammatory cytokines, including TNFalpha and IL-1beta. We also discovered constitutive activation of AKT at Ser473, a site dependent for its phosphorylation on TPL2 activity in macrophages responding to TLR signaling. Notably, the Akt/mTOR pathway limits the magnitude and duration of macrophage activation, in part through synthesis of the anti-inflammatory cytokine IL-10. These events involve signaling through STAT3. Accordingly, we discovered constitutive phosphorylation of STAT3 at a site regulated by TPL2 in activated macrophages.

In addition to non-tumor cell autonomous roles in regulating myeloma through macrophages, we showed a tumor cell-autonomous, growth-regulatory role of TPL2 kinase. Treatment of myeloma cells with a TPL2 small molecule inhibitor resulted in apoptosis that was not rescued by the presence of patient-derived stromal cells. We postulate that TPL2 inhibition interferes with growth signaling in myeloma cells because TPL2 has been shown to substitute for RAF proteins in growth signal transduction. Interestingly, we found that TPL2 was activated by phosphorylation as cells entered G2/M. Treatment with nocodazole increased the proportion of cells that co-expressed phosphorylated TPL2 and phosphorylated histone H3. Moreover, we found that TPL2 activity was required for MAPK pathway signal transduction in response to TNF receptor stimulation in myeloma cells.

Taken together, our results provide important novel insights into the regulation of macrophages within primary myeloma niches in the bone marrow. Plasticity between M1 and M2 phenotypes may correlate tightly with the actions of TPL2 kinase. In the myeloma niche, TPL2 activity helps to fine-tune macrophage activation by promoting synthesis and release of pro-inflammatory cytokines required by the myeloma tumor cell while engaging counter-regulatory mechanisms to prevent tissue destruction mediated by activated macrophages. Additionally, we described a growth regulatory role of TPL2 in the tumor cell itself. Thus, TPL2 blockade may disrupt crucial macrophage-tumor cell interactions within myeloma niches.