Abstract 5413: Multiplexed phosphoprotein cell signaling analysis in multiple myeloma reveals a pro-survival pathway elicited by amino-acid starvation

Background: Substantial advances in our understanding of the biology of the incurable plasma cell (PC) malignancy multiple myeloma (MM) came from the study of the bone marrow (BM) microenvironment (BMME). Our previous work disclosed an essential role for autophagy in sustaining MM cell proliferation and survival. However, the control exerted on PC autophagy by the BMME and its pathophysiological significance are virtually unknown. Experimental design: We integrated ex vivo multiplexed phosphoprotein cell signaling analyses of primary MM and BMME cells, metabolomic profiling of patient-derived BM and peripheral plasma, and in vitro studies on human MM cell lines. Primary CD138+ MM and BMME CD138- cells were isolated from BM aspirates obtained from 35 clinically characterized MM patients and 60 proteins - representative of autophagy, cell survival, proliferation, protein degradation and translation pathways - were analyzed by Reverse Phase Protein Arrays (RPPA).Comprehensive metabolomics profiling was achieved by ultra-high performance liquid and gas chromatography followed by mass spectrometry (UHPLC/GC-MS) on ad hoc-collected B and peripheral plasma samples from patients at different disease stages (30 MGUS, 17 smoldering MM, 16 MM) and age-matched healthy donors (29). For in vitro studies, human MM lines were exposed to amino acid (AA) depletion and selected responses evaluated by quantitative RT-PCR and immunoblotting analyses. Results: First, RPPA revealed that patients with the most aggressive clinical presentation (refractoriness, short time to progression and active bone disease) displayed higher activity of the PI3K/AKT/mTOR pathway associated to higher expression of the autophagic proteins ATG5, LC3B and p62. In search for extrinsic stimuli capable of raising both mTOR and autophagic activity, we recapitulated such expression pattern in MM cells through selective starvation of tryptophan (Trp) and arginine (Arg), two AA endowed with distinctive immune regulatory activity. When exposed to Trp-free or Arg-free medium, human MM cell lines activated GCN2-mediated responses, increasing mTOR, p-S6RP, CHOP, p62 and Blimp-1 and immunoglobulin secretion; conversely, stable lentiviral p62 silencing reduced Blimp-1 and caused the in vitro extinction of MM cell lines within 10 days of culture. UHPLC/GC-MS metabolomics analysis of plasma samples revealed a progressively and significantly lower concentration of Trp and Arg associated with disease evolution and s