John Culpepper

Multiple myeloma (MM) cells have high rates of secretion of proteins rich in disulfide bonds and depend upon compartmentalized redox balance for accurate protein folding. The proteasome inhibitor bortezomib (Btz) is a successful frontline treatment for the disease, but its long-term efficacy is restricted by the acquisition of resistance. We found that MM cell lines resistant to Btz maintain high levels of oxidative stress and are cross resistant to endoplasmic reticulum (ER) stress-inducing agents thapsigargin (ThG), and tunicamycin (TuM). Moreover, cells expressing high/wild type levels of glutathione S-transferase P (GSTP) are more resistant than Gstp1/p2 knockout cells. In agreement, basal levels of S-glutathionylated proteins and redox regulation enzymes, including GSTP are elevated at mRNA and protein levels in resistant cells. GSTP mediated S-glutathionylation (SSG) regulates the activities of a number of redox active ER proteins. Here we demonstrated that the post-translational modification determines the balance between foldase and ATPase activities of the binding immunoglobulin protein (BiP), with Cys41-SSG important for ATPase, and Cys420-SSG for foldase. BiP expression and S-glutathionylation are increased in clinical specimens of bone marrow from MM patients compared to non-cancerous samples. Preventing S-glutathionylation in MM cells with a GSTP specific inhibitor restored BiP activities and reversed resistance to Btz. Therefore, S-glutathionylation of BiP confers pro-survival advantages and represents a novel mechanism of drug resistance in MM cells. We conclude that altered GSTP expression leads to S-glutathionylation of BiP, and contributes to acquired resistance to Btz in MM.

Recent advancements in the treatment of melanoma are encouraging, but there remains a need to identify additional therapeutic targets. We identify a role for microsomal glutathione transferase 1 (MGST1) in biosynthetic pathways for melanin, and as a determinant of tumor progression. Knockdown (KD) of MGST1 depleted midline-localized, pigmented melanocytes in zebrafish embryos, while in both mouse and human melanoma cells, loss of MGST1 resulted in a catalytically dependent, quantitative and linear depigmentation, associated with diminished conversion of L-dopa to dopachrome (eumelanin precursor). Melanin, especially eumelanin, has antioxidant properties and MGST1 KD melanoma cells are under higher oxidative stress, with increased reactive oxygen species, decreased antioxidant capacities, reduced energy metabolism and ATP production, and lower proliferation rates in 3D culture. In mice, when compared to non-target control, Mgst1 KD B16 cells had less melanin, more active CD8+ T cell infiltration, slower growing tumors, and enhanced animal survival. Thus, MGST1 is an integral enzyme in melanin synthesis and its inhibition adversely influences tumor growth.

Reductive stress is characterized by an excess of cellular electron donors and can be linked with various human pathologies including cancer. We developed melanoma cell lines resistant to reductive stress agents: rotenone (ROTR), n-acetyl-L-cysteine, (NACR), or dithiothreitol (DTTR). Resistant cells divided more rapidly and had intracellular homeostatic redox-couple ratios that were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROTR cells had significant changes in mitochondrial morphology with higher numbers that were more isolated, fragmented and swollen, with greater membrane depolarization and decreased numbers of networks. These changes were accompanied by lower basal oxygen consumption and maximal respiration rates. Whole cell flux analyses and mitochondrial function assays showed that NACR and DTTR preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROTR used ketone body substrates such as D, L-β-hydroxybutyric acid. NACR and DTTR cells had constitutively decreased levels of reactive oxygen species (ROS), although this was accompanied by activation of nuclear factor erythroid 2-related factor 2 (Nrf2), with concomitant increased expression of the downstream gene products such as glutathione S-transferase P (GSTP). Further adaptations included enhanced expression of endoplasmic reticulum proteins controlling the unfolded protein response (UPR). Although expression patterns of these UPR proteins were distinct between the resistant cells, a trend implied that resistance to reductive stress is accompanied by a constitutively increased UPR phenotype in each line. Overall, tumor cells, although tolerant of oxidative stress, can adapt their energy and survival mechanisms in lethal reductive stress conditions.

<b>Abstract ID 20639</b> <b>Poster Board 448</b> Reductive stress is characterized by an excess of cellular electron acceptors and can be linked with a number of human pathologies including increased cytotoxicity in cancer cells. In order to characterize cellular adaptations to reductive stress, we used stepwise, incremental selection to generate melanoma cell lines with acquired resistance to rotenone (ROT^R, 5-fold), n-acetyl cysteine, (NAC^R, 2-fold), or dithiothreitol (DTT^R, 23-fold) or rotenone (ROT^R, 5-fold). Cells divided more rapidly in resistant lines and intracellular homeostatic redox- couple ratios (e.g., glutathione, NADPH) were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROT^R cells had significant changes in mitochondrial morphology (higher numbers, and more swollen; isolated and more fragmented; decrease in networks; greater membrane depolarization). These changes were accompanied by lower basal oxygen consumption and maximal respiration rates and a more marked reliance on glycolysis for energy production. Whole cell flux analyses and mitochondrial function assays showed that NAC^Rand DTT^Rpreferentially utilized TCA cycle intermediates, while ROT^Rused ketone body substrates such as D, L, b-hydroxybutyric acid. While NAC^R and DTT^REach resistant line cells had constitutively elevated decreasedd levels of reactive oxygen species (ROS), in ROT^R these were increased. This was accompanied by and spuriously inhibited/activated Nrf2, with concomitant decreased/increased expression of downstream gene products, such as glutathione S-transferase P in ROT^R/ NAC^Rand DTT^R. Adaptations to reductive stress also included enhanced expressionalteredion of proteinsexpression of proteins controlling the unfolded protein response (UPR). These included BiP, PDI, CHOP, ATF4, ATF6 and PERK and although expression patterns of these UPR proteins were distinct between the resistant cells, there was an enhancement of expression implying that resistance to reductive stress accompanies a constitutively increased UPR phenotype in each line, but does not result in cell death. Overall, while most tumor cell lines adapt to survive conditions of high oxidative stress, they are also flexibly capable of adapting various pathways to regulate growth and survival in conditions concentrations of drugs that would cause cell death throughof reductive stress. This work was supported by grants from the National Institutes of Health (CA08660, CA117259, NCRR P20RR024485 - COBRE in Oxidants, Redox Balance and Stress Signaling) and support from the South Carolina Centers of Excellence program and was conducted in a facility constructed with the support from the National Institutes of Health, Grant Number C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources.

While recent targeted and immunotherapies in malignant melanoma are encouraging, most patients acquire resistance, implicating a need to identify additional drug targets to improve outcomes. Recently, attention has been given to pathways that regulate redox homeostasis, especially the lipid peroxidase pathway that protects cells against ferroptosis. Here we identify microsomal glutathione S-transferase 1 (MGST1), a non-selenium-dependent glutathione peroxidase, as highly expressed in malignant and drug resistant melanomas and as a specific determinant of metastatic spread and therapeutic sensitivity. Loss of MGST1 in mouse and human melanoma enhanced cellular oxidative stress, and diminished glycolysis, oxidative phosphorylation, and pentose phosphate pathway. Gp100 activated pmel-1 T cells killed more Mgst1 KD than control melanoma cells and KD cells were more sensitive to cytotoxic anticancer drugs and ferroptotic cell death. When compared to control, mice bearing Mgst1 KD B16 tumors had more CD8+ T cell infiltration with reduced expression of inhibitory receptors and increased cytokine response, large reduction of lung metastases and enhanced survival. Targeting MGST1 alters the redox balance and limits metastases in melanoma, enhancing the therapeutic index for chemo- and immunotherapies.