PIM2-dependent modulation of mitochondrial mass and function in multiple myeloma

Abstract

Abstract Multiple myeloma (MM) is a uncurable hematological malignancy of bone marrow resident plasma cells. The primary cause of treatment failure is adaptive upregulation of pro-survival mechanisms that lead to progressively greater resistance to therapy resulting in unrelenting relapsing disease, even with the newest immunotherapies. MM progression relies on mitochondria metabolic adaptation to high demands of antibody making concurrently with proliferation demands. This is consistent with a recent study demonstrating that mitochondria DNA copy number (mtDNA-CN) in normal plasma cells (PC) significantly increases in disease progression from normal PC to MGUS to SMM to MM to relapsed/refractory MM. We showed that oncogenic serine/threonine kinase PIM2 is overexpressed in MM and has significant role in maintaining survival, conferring chemotherapy resistance to these cells and associates with disease progression and poor prognosis. We hypothesize that PIM2 has an unexplored role in modulating mitochondrial biogenesis to maintain survival of MM cells. We employed PIM2 molecular manipulation: overexpression (OE) of wild type (WT) or kinase dead (K61A) and knockdown (with siRNA or doxycycline-inducible shRNA-ishPIM2) as well as pharmacological inhibition of PIM2 protein level and activity, in MM cell lines. The following new specific small molecules inhibitors were used: 1) JP11646 a PIM2-selective non-ATP competitive inhibitor associated with downregulation of PIM2 protein; 2)Cpd A with no kinase inhibition effect but inducer of PIM2 protein degradation and 3) Cpd B and AZD1208 potent PIM2 kinase inhibitors but ineffective on PIM2 protein level. We identified a kinase-independent (KI) modulation of MM cell's viability in vitro and in vivo. Lowering PIM2 protein expression (JP11646; 30 mg/kg by oral gavage) in Vk*myc tail vein injected mouse myeloma model significantly decreased disease progression in as little as 21 days, while AZD1208 treatment was not significantly changing mouse IgG levels when compared with vehicle treatment. MM disease progression has been associated with a heavy dependency on oxidative phosphorylation for energy production and GSEA analysis revealed that high expression of PIM2 associates with gene signature of mitochondrial metabolism and strongly correlates with c-Myc, a critical driver of MM progression. PIM2 WT or K61A MM cells favored a significant increase of mtDNA-CN, mitochondrial mass and function (OCR), regardless of the kinase functionality. ishPIM2 MM cells showed a significant decrease of mtDNA-CN, mitochondrial mass and function (OCR and ATP levels) as well as PGC1a and NRF1 levels in the nuclear fraction. Lowering PIM2 protein expression (JP11646, Cpd A) but not kinase only inhibition (Cpd B, AZD1208), significantly reduced mtDNA-CN, mitochondrial mass and function (OCR and ATP levels), the expression of key nuclear mitochondrial biogenesis genes, targets of c-Myc target genes (TFAM, PGC1a and LonP1), as well as PGC1a and NRF1 levels in the nuclear fraction. PIM2 OE inMM.1S(WT and K61A)provides a KI survival advantage to these cells after PGC1a inhibition with SR-18292, when compared with vector control counterparts. Together, these results suggest a KI role of PIM2, in mitochondria biogenesis. Moreover, the anti-survival effects resulted from PIM2 protein degradation in MM cells, might be partially attributed to inhibition of mitochondrial biogenesis. Thus, what sustains the functionality and metabolic integrity of MM mitochondria may represent novel therapeutic target to specifically disrupt the energy supply needed for disease progression and treatment resistance.