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Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

Background We have previously demonstrated adaptive antibody responses targeting public tumor antigens in cancer patients. ATRC-101, a clinical stage, engineered version of an antibody identified in such a patient, displays robust single-agent activity in syngeneic tumor models requiring Fc receptors (FcRs) expressed by innate immune cells and the presence of CD8+ T cells. The novel target of ATRC-101 was found to be a tumor-restricted ribonucleoprotein (RNP) complex, and because RNP complexes drive T cell responses in infectious and autoimmune disease via innate immune cells, we further characterized the mechanism-of-action of ATRC-101. Here we describe changes in immune cell populations in a tumor model proximal to treatment initiation with ATRC-101. Methods Mice bearing EMT6 tumors received ATRC-101 beginning on day 7 post-tumor inoculation. Tissues were harvested between days 7 and 14 and analyzed by flow cytometry and immunohistochemistry. Transcriptome analysis was performed using RNA sequencing on whole tumors taken on days 7, 9, and 12. Results The earliest significant changes induced by ATRC-101, relative to vehicle, were noted just 24 hours after dosing: increased numbers of cDC1 cells in blood, and decreased numbers of cDC2 cells in blood and M-MDSCs in tumor. A significant increase of CD8+ T cells was observed in blood 48 hours after dosing and in tumor 96 hours after dosing. Increased numbers of NK cells were also observed in blood and tumor at this later time. Multiplex analysis of circulating cytokines demonstrated a very early increase in myeloid chemo-attractants, such as MCP1 and MIP1a.Whole exome sequencing of tumor samples showed that ATRC-101 dosing drives a significant increase, relative to vehicle, in the expression of interferon-stimulated genes. Co-culturing experiments demonstrated that induced, bone marrow-derived dendritic cells are activated by ATRC-101 and its target in a dose-dependent fashion. Conclusions Dosing with ATRC-101 in the EMT6 syngeneic tumor model, in which ATRC-101 displays notable single-agent activity, leads to changes in immune cell composition in the blood and tumor, with the earliest changes observed in myeloid or myeloid-derived cell populations, and to the early appearance of myeloid chemo-attractants. We believe these data indicate that ATRC-101 acts proximally on the myeloid cell populations in the tumor, leading to a remodeling of the tumor environment and an adaptive immune response that includes CD8+ T cells driving tumor regression. Our data demonstrate that ATRC-101, bound to its target which is an RNP complex, can activate myeloid cells and are consistent with this activation occurring via FcR and Toll-like receptor (TLR) pathways.

Background We have previously described ATRC-101, a fully human, engineered IgG1 antibody binding a tumor-restricted ribonucleoprotein (RNP) complex as its target. ATRC-101 is currently under evaluation in the clinic as a monotherapy for solid tumors. Following target engagement, ATRC-101 functions in an Fc-mediated fashion to deliver the target to the innate immune system, which modifies the tumor microenvironment and generates an adaptive immune response involving CD8+ T cells leading to anti-tumor activity in syngeneic mouse models. Binding of ATRC-101 appears restricted to malignant tissues in both mouse models and human, across a range of cancer histologic phenotypes, including carcinomas that are known candidates for anti-PD-1 treatment. In the EMT6 mouse model, representing a T cell-excluded phenotype in which anti-PD-1 agents display limited activity, ATRC-101 monotherapy was uniformly vigorous with persistent anti-tumor memory. When co-administered at a lower dose with anti-PD-1, the combination of therapy demonstrated a robust and heightened anti-tumor response relative to either agent dosed as monotherapy at similar concentrations. Methods To gain insight into the mechanisms that contribute to the anti-tumor effect with combination therapy, in vivo experiments in the EMT6 syngeneic mouse model were performed to determine temporal and spatial patterns of infiltrates and assessed tumors by using whole exome sequencing following administration of ATRC-101 vs. vehicle control. Within naive human tumor samples, coincident immunoreactivities of ATRC-101 and PD-L1 were also characterized. Results In mice treated with ATRC-101, analysis by immunofluorescence revealed a significant increase in the percentage of PD-1 reactive T cells within the tumor microenvironment. Elevated transcripts for PD-L1 also were detected in tumors from mice administered ATRC-101 vs baseline levels or vehicle control. When human tumor tissues were characterized for coincident expression of these targets, a high prevalence of ATRC-101 immunoreactivity was noted in both PD-L1 reactive and non-reactive tumor cores. Across multiple indications, ATRC-101 immunoreactivity was apparent in > 50% of PD-L1+ cores. Conclusions In situ studies suggest the target of ATRC-101 may co-locate with PD-L1, and in vivo studies indicate that ATRC-101 administration increases PD-L1 transcripts and PD-1-positive infiltrates in mouse tumor. Altogether, our data support studies to combine ATRC-101 with agents targeting PD-1 in the clinical treatment of solid tissue malignancies. Acknowledgements We acknowledge the significant effort and contributions of our colleagues from the clinical, in vivo pharmacology, translational sciences, in vitro pharmacology, and cell biology groups. This includes Mark Armanini, Erin Brosey, Chantia Carroll, Sean M. Carroll, Nicole Haaser, Benjamin Haugen, Dongkyoon Kim, Beatriz Millare, Yann Chong Tan, Danhui Zhang, and Patricia Zuno. Trial Registration NCT04244552 Ethics Approval The study was approved by WIRB (Western Institutional Review Board) on Jun 11, 2013. The WIRB study number is 20130121. Reference DeFalco J, Harbell M, Manning-Bog A, et al. Non-progressing cancer patients have persistent B cell responses expressing shared antibody paratopes that target public tumor antigens. Clinical Immunology 2018; 187:37–45.

BackgroundWe previously described ATRC-101, a fully human, engineered IgG1 antibody which is currently under evaluation in the clinic as a monotherapy for solid tumors. A chimeric version of this antibody expressed on a mouse IgG2a (mATRC-101) has shown robust anti-tumor activity as a monotherapy in the EMT6 syngeneic tumor model. In order to assess the potential utility of ATRC-101 in combination with chemotherapeutic agents, non-clinical studies were performed to assess the efficacy of mATRC-101 in combination with chemotherapeutic agents, including Doxorubicin and pegylated liposomal Doxorubicin (PLD), and the impact of these anti-tumor small molecules on mATRC-101 immunoreactivity in mouse tumor and normal tissues.MethodsFemale BALB/c mice with established EMT6 tumors were dosed with mATRC-101 (1 or 3 mg/kg) or vehicle intraperitoneally (IP) twice weekly plus Doxorubicin (2 or 5 mg/Kg) or vehicle (saline) IV once weekly following randomization on Day 6. Statistical analyses of tumor volumes were performed using the normalized area above the curve and the normalized growth rate metrics developed at Atreca. One-sided log-rank (Mantel-Cox) test was used to assess survival advantage relative to the indicated reference group. P-values ≤0.05 were considered significant. In monotherapy studies with Doxorubicin and PLD, EMT6 tumor and non-tumor bearing mice were dosed with vehicle or Doxorubicin (2, 10 mg/kg) or PLD (1, 2, 5,10 mg/kg). Normal mouse tissues were collected at 24 hours and 2 weeks after the last dose. Reactivity for mATRC-101 in EMT6 tumor and normal mouse tissues was evaluated by immunohistochemistryResultsThe combination of 3 mg/kg mATRC-101 and 5 mg/kg Doxorubicin demonstrated significant tumor growth inhibition compared to either monotherapy, or vehicle (pConclusionsThis study provides non-clinical evidence that administration of mATRC-101 in combination with Doxorubicin increases anti-tumor activity in the EMT6 model. Exposure to Doxorubicin or PLD in EMT6 tumor-bearing mice increased mATRC-101 immunoreactivity in the tumor, in a dose-dependent manner Moreover, mATRC-101 immunoreactivity in normal tissues was not influenced by Doxorubicin or PLD. Taken together, these findings support clinical evaluation of the combination of ATRC-101 and doxorubicin.

While clinical benefit of the proteasome inhibitor (PI) bortezomib (BTZ) for multiple myeloma (MM) patients remains unchallenged, dose-limiting toxicities and drug resistance limit the long-term utility. The E3 ubiquitin ligase Skp1–Cullin-1–Skp2 (SCFSkp2) promotes proteasomal degradation of the cell cycle inhibitor p27 to enhance tumor growth. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance. SCFSkp2 activity is increased by the Cullin-1-binding protein Commd1 and the Skp2-binding protein Cks1B. Here we observed higher CUL1, COMMD1 and SKP2 mRNA levels in CD138+ cells isolated from BTZ-resistant MM patients. Higher CUL1, COMMD1, SKP2 and CKS1B mRNA levels in patient CD138+ cells correlated with decreased progression-free and overall survival. Genetic knockdown of CUL1, COMMD1 or SKP2 disrupted the SCFSkp2 complex, stabilized p27 and increased the number of annexin-V-positive cells after BTZ treatment. Chemical library screens identified a novel compound, designated DT204, that reduced Skp2 binding to Cullin-1 and Commd1, and synergistically enhanced BTZ-induced apoptosis. DT204 co-treatment with BTZ overcame drug resistance and reduced the in vivo growth of myeloma tumors in murine models with survival benefit. Taken together, the results provide proof of concept for rationally designed drug combinations that incorporate SCFSkp2 inhibitors to treat BTZ resistant disease.

Several epidemiological studies show that aspirin can act as a chemopreventive agent and decrease the incidences of various cancers including melanoma. In this work, we investigated the in vitro and in vivo efficacy of acetylsalicylic acid (ASA) as an antimelanoma agent in B16-F0 cells and skin B16-F0 melanoma tumor mouse model. Our findings indicate that the IC50 (48 h) for ASA in B16-F0 melanoma cells was 100 μM and that ASA caused a dose- and time-dependent GSH depletion and increase in reactive oxygen species (ROS) formation in B16-F0 melanoma cells. Male C57BL/6 mice were inoculated s.c. with 1 × 106 B16-F0 melanoma cells. ASA (80, 100, and 150 mg/kg) was initiated on day 1 or day 7, or day 9 after cell inoculation and continued daily for 13, 7, and 5 days, respectively. Animals were weighed daily and sacrificed on day 13. The tumors were excised and weighed. The animals receiving 13 days of ASA therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 1 ± 12 %, 19 ± 22 %, and 50 ± 29 %, respectively. Animals receiving 7 days of therapy at 80, 100, and 150 mg/kg demonstrated tumor growth inhibition by 12 ± 14 %, 27 ± 14 %, and 40 ± 14 %, respectively. No significant tumor growth inhibition was observed with 5 days of therapy. ASA at 100 and 150 mg/kg caused significant tumor growth inhibition in C57BL/6 mice when administered for 13 and 7 days, respectively. The results obtained in this study are consistent with the recent epidemiologically based report that aspirin is associated with lower melanoma risk in humans.

In the current work, we investigated the in vitro biochemical mechanism of Caffeic Acid Phenylethyl Ester (CAPE) toxicity and eight hydroxycinnamic/caffeic acid derivatives in vitro, using tyrosinase enzyme as a molecular target in human SK-MEL-28 melanoma cells. Enzymatic reaction models using tyrosinase/O(2) and HRP/H(2)O(2) were used to delineate the role of one- and two-electron oxidation. Ascorbic acid (AA), NADH and GSH depletion were used as markers of quinone formation and oxidative stress in CAPE induced toxicity in melanoma cells. Ethylenediamine, an o-quinone trap, prevented the formation of o-quinone and oxidations of AA and NADH mediated by tyrosinase bioactivation of CAPE. The IC(50) of CAPE towards SK-MEL-28 melanoma cells was 15muM. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased CAPE's toxicity towards SK-MEL-28 cells indicating quinone formation played an important role in CAPE induced cell toxicity. Cyclosporin-A and trifluoperazine, inhibitors of the mitochondrial membrane permeability transition pore (PTP), prevented CAPE toxicity towards melanoma cells. We further investigated the role of tyrosinase in CAPE toxicity in the presence of a shRNA plasmid, targeting tyrosinase mRNA. Results from tyrosinase shRNA experiments showed that CAPE led to negligible anti-proliferative effect, apoptotic cell death and ROS formation in shRNA plasmid treated cells. Furthermore, it was also found that CAPE selectively caused escalation in the ROS formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 cells which express functional tyrosinase. In contrast, CAPE did not lead to ROS formation and ICG depletion in amelanotic C32 melanoma cells, which do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in CAPE's selective toxicity towards melanocytic melanoma cell lines. Our findings suggest that the mechanisms of CAPE toxicity in SK-MEL-28 melanoma cells mediated by tyrosinase bioactivation of CAPE included quinone formation, ROS formation, intracellular GSH depletion and induced mitochondrial toxicity.

Introduction: We have reported that fenretinide (4-HPR) is active against high-risk neuroblastoma (NB) in vitro and in vivo; our Phase I trials of novel 4-HPR formulations evidenced clinical activity. Mechanisms of activity include increase of cytotoxic dihydroceramides. Fenretinide activity in vitro is enhanced by safingol (S), the L-threo diastereomer of sphinganine. Here, we delineate stress pathways activated in response to 4-HPR±S and identify ixazomib, a proteasomal inhibitor, and antimalarial, mefloquine, a disruptor of autophagy, as new potential synergizing agents for 4-HPR±S. Methods: Sphingolipids were assessed by LC/MS/MS; cytotoxicity by fluorescence-based plate assay in 2% and 5% oxygen; apoptosis by TUNEL assay. ER stress markers, unfolded protein response (UPR), and autophagy, were assessed using immunoblotting, immunoprecipitation, and electron/confocal microscopy. Target validation was by gene silencing. Results: 4-HPR rapidly increased D-erythro-dihydroceramides; safingol was catabolized to L-threo-dihydroceramides. Safingol (2-3 µM) caused multi-log cytotoxic synergy of 4-HPR in eight of ten GBM and five NB cell lines (CI Citation Format: Nikhil Vad, Dong Wang, Hwangeui Cho, Dattesh Verlekar, Charlie Linch, C Patrick Reynolds, Min Kang, Barry J. Maurer. Dihydroceramide increase precedes golgi dispersal, pro-survival autophagy, ER stress, and UPR in fenretinide + safingol treated neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2885.

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.

Although the therapeutic benefit of proteasome inhibition in multiple myeloma remains unchallenged, drug resistance inevitably emerges through mechanisms that remain elusive. Bortezomib provokes unwanted protein accumulation and the endoplasmic reticulum stress to activate the unfolded protein response (UPR) and autophagy as compensatory mechanisms that restore protein homeostasis. High-throughput screens to detect pharmacologics that modulated autophagy to enhance the anti-myeloma effect of bortezomib revealed metformin, a widely used antidiabetic agent with proven efficacy and limited adverse effects. Metformin co-treatment with bortezomib suppressed induction of the critical UPR effector glucose-regulated protein 78 (GRP78) to impair autophagosome formation and enhance apoptosis. Gene expression profiling of newly diagnosed myeloma patient tumors further correlated the hyperexpression of GRP78-encoding HSPA5 with reduced clinical response to bortezomib. The effect of bortezomib was enhanced with metformin co-treatment using myeloma patient tumor cells and the chemoresistant, stem cell-like side population that may contribute to disease recurrence. The relevance of the findings was confirmed in vivo as shown by metformin co-treatment with bortezomib that delayed the growth of myeloma xenotransplants. Taken together, our results suggest that metformin suppresses GRP78, a key driver of bortezomib-induced autophagy, and support the pharmacologic repositioning of metformin to enhance the anti-myeloma benefit of bortezomib.

Cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased reactive oxygen species and/or de novo synthesized D-erythro-dihydroceramides. Co-treatment with L-threo-sphinganine (safingol, S), synergistically increased fenretinide cytotoxicity in cell lines of many cancer types coincident with increase of safingol-derived, L-threo-dihydroceramides. Previously, we showed that 4-HPR+S induced ER stress, unfolded protein response (UPR), pro-survival autophagy, and mixed non-apoptotic and apoptotic cell death in glioblastoma multiforme (GBM) cells in vitro. However, the mechanisms regulating such activity remained unclear, including the role of ceramide-activated signal transducers, such as ASK1. We now report that 4-HPR+S activated stress kinase ASK1 via phosphorylation at Thr845 (+1-6 hrs), an event known to induce p38 MAPK phosphorylation. Furthermore, 4-HPR+S induced an increase in the nuclear localization of transcription factor FOXO3a coincident with decreased Akt signaling and increased phosphorylated levels of JNK1/2, p38 MAPK, ERK1/2, and autophagy regulator, AMPK (+1-6 hrs). 4-HPR+S caused a time-dependent (+6-24 hrs) increase of GRP78 protein levels, a key regulator of ER stress response and UPR, and increased levels of pro-apoptotic transcription factor, CHOP. GRP78 increase was p38 MAPK-dependent, as p38 MAPK inhibitor, SB203580, greatly reduced GRP78 increase. Interestingly, 4-HPR+S induced cytotoxicity was unchanged in the presence of an inhibitor of ERK 1/2 (PD98059) but significantly decreased when cells were concurrently treated with p38 MAPK inhibitor, SB203580, or JNK1/2 inhibitor, SP600125 (+24-72 hrs, p ≤ 0.05). Significantly, co-treatment with dorsomorphin, an AMPK inhibitor, increased and accelerated 4-HPR+S cytotoxicity (+24-72 hrs, p ≤ 0.05). Moreover, 4-HPR+S treatment induced FOXO3a Ser7 phosphorylation (+1-6 hrs) and a corresponding decrease in levels of nuclear E3-Ub protein ligase, Skp2. The decrease of SKp2 protein was coincident with increased levels of CARM1 protein, a known transcriptional activator of autophagy-related genes. Activation of ASK1, JNK1/2, p38 MAPK and ERK1/2 by 4-HPR+S occurred earlier, and to a greater extent, than single agent 4-HPR and S exposures at all time points (+1-24 hrs). Together, these results support that 4-HPR+S induced both pro-survival autophagy and cell death at least partly through activation of FOXO3a and ASK1-dependent activation of p38 MAPK, possibly in response to the known progressive increase of de novo synthesized D-erythro and L-threo dihydroceramides. A Phase I trial of intravenous 4-HPR+S in adult solid tumors (ClinicalTrials.gov Identifier:NCT0155307) is currently open in the Texas State-supported, South Plains Oncology Consortium (SPONC.org). Citation Format: Nikhil Vad, Dong Wang, Charlie Linch, Barry J. Maurer. Co-treatment with fenretinide and safingol induced p38 MAPK and/or FOXO3a activated pro-survival autophagy in glioblastoma multiforme cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2017-1057

Malignant gliomas are highly aggressive primary brain tumors with few treatment options and invariably fatal outcomes. The receptor tyrosine kinase epidermal growth factor receptor (EGFR) is hyperactivated through gene amplification or mutation in many cancers. EGFR activity is associated with increased cell proliferation, disease progression and therapeutic resistance. Intratumoral heterogeneity of EGFR arises as a signature pathogenetic events in the highly aggressive and invariably fatal brain tumor glioblastoma multiforme (GBM). Functional blockade of the EGFR kinase activity alone does not achieve maximal therapeutic benefit to support the role for EGFR kinase-independent functions. We performed a high-throughput, pharmacologic screening to identify EGFR-targeted therapeutics in glioma cells engineered to overexpress either a high or low copy number of EGFR wildtype (WT), a constitutively active mutant EGFR-vIII or an EGFR kinase-dead (KD) mutant. We discovered that the heat shock protein (HSP)90 inhibitor ganetespib selectively and potently killed glioma cells that overexpressed either the WT, vIII or KD forms of EGFR (IC50 ∼20-40nM). Real-time measurements demonstrated that ganetespib reduced proliferation, induced caspase-dependent apoptosis and inhibited glioma migration. Overexpression of the EGFR-WT, vIII or KD forms significantly increased intracellular ATP levels and WT, vIII and KI EGFR all translocalized to the mitochondria. Ganetespib addition rapidly reduced ATP in cells that overexpressed either kinase-active or kinase-inactive forms. Ganetespib reduced the proliferation of human patient GBM patient-derived tumor stem cells and effectively impaired the growth of subcutaneously placed glioma implants in nude mouse models. Ganetespib impairs the EGFR kinase-(in)dependent activities with the potential benefit to overcome intratumoral heterogeneity benefit and improve GBM patient outcome.

Small molecules that modulate cancer stem cell (CSC) fate and function offer therapeutic potential to improve patient outcome. Malignant gliomas are highly aggressive primary brain tumors with few treatment options and an invariably fatal outcome. High-throughput screening and structure-guided design identified a novel, dual phosphatidylinositol-3-kinase (PI3K)/mammaliam target of rapamycin (mTOR) inhibitor that potently induced apoptosis in glioma cells and glioma patient-derived CSCs. The compound, designated DT61, inhibited PI3K, mTORC1 and mTORC2 kinase activities at nanomolar concentrations in a dose-dependent manner. DT61 dramatically reduced the level of the PI3-K α and β isoforms and led to a reduction in the site-specific phosphorylation and activity of the pro-survival serine-threonine kinase Akt/Protein Kinase B (PKB). DT61 inhibition of PI3K activity prevented the inactivation of glycogen synthase kinase (GSK-3beta) to enhance site-specific phosphorylation and proteasomal degradation of the c-myc oncoprotein that drives gliomagenesis. The apoptotic effect of DT61 on CSCs was synergistically enhanced when combined with radiation treatment, a standard-of-care modality for glioma treatment. Taken together, the results demonstrate that DT61 is a novel, highly effective dual PI3K/mTOR inhibitor that is cytotoxic to brain CSC under normoxia and hypoxia with potential benefit for glioma patients.

The cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased dihydroceramide and reactive oxygen species (ROS) levels and enhanced by co-treatment with safingol (S), an L-threo-dihydroceramide precursor. The ER stress response displays dichotomic effects wherein mild short-term stressors activate responses to neutralize or adapt to stress, but severe or long-lasting stressors activate cell death cascades. Here, we report that ER stress and autophagy are pro-survival responses in glioblastoma multiforme (GBM) cells to unfolded proteins-related cytotoxicity induced by 4-HPR+S in vitro. In T98G and A172 cells, 4-HPR+S induced mixed cell death associated with time-dependent increase of TUNEL-positivity (+12-36 h) and cleavage of caspase-3 (+12-36 h), but pan- caspase inhibitor, boc-D-fmk, did not rescue cell death. Cytotoxicity (+6-24 h) was associated with minimal mitochondrial depolarization or dual-staining of Annexin V/PI. Assays for known non-apoptotic mechanisms were negative. There was time-dependent (+6-24 h) increase of GRP78 protein levels, a key regulator of ER stress response; ER stress sensors, PERK, IRE1α, and ATF6, were activated (+6-24 h); levels of pro-apoptotic transcription factor, CHOP, increased (+6-24 h). Cytotoxicity was increased by PERK pathway inhibitors, salubrinal (+24-72 h, p≤0.05), GSK 2606414 (+24-72 h, p≤0.05), and GSK 2656157 (+48-72 h, p≤0.05), indicating that PERK activation was pro-survival. Treatment increased levels of CHIP, a key co-chaperone targeting unfolded proteins for degradation (+6-24 h). There was strong activation of the ubiquitin-proteasome system (UPS) and autophagy preceding or concurrent with the accumulation of poly-ubiquitinated proteins (+12-24 h). Autophagy was evidenced by increased conversion of LC3B-I to LC3B-II (+6-24 h). LC3B-II levels were increased by lysosomal degradation inhibitor, bafilomycin A1, indicating increased autophagic flux. Cytosolic levels of HDAC6, a coordinator of protein turnover via UPS and autophagy through trafficking of protein aggregates into pre-lysosomal structures (aggresomes) prior to autophagosome engulfment, increased (+6-24 h). siRNA knockdown of CHIP enhanced cytotoxicity (+24-72 h, p≤0.05). siRNA ablation of autophagy (ATG7, BECN1)(+24-36 h, p≤0.05), or pharmacological disruption by mefloquine, increased cytotoxicity (+24-72 h, p≤0.05), indicating that autophagy was pro-survival. Cytotoxicity was increased by siRNA knockdown of HDAC6 (+24-72 h, p≤0.05) and selective HDAC6 inhibitor, ACY1215, (+24-72 h, p≤0.05). Together, these results support that ER stress/unfolded protein response (UPR) is pro-survival in GBM cells exposed to 4-HPR+S. This suggests that co-agents that reduce ER stress response, UPR, or autophagy processes may further sensitize GBM cells to 4-HPR+S. Citation Format: Nikhil M. Vad, Dong Wang, Barry J. Maurer. ER stress, unfolded protein response, and autophagy are pro-survival responses to fenretinide + safingol treatment in GBM cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4656.

The aim of this study was to identify a phenolic prodrug compound that is minimally metabolized by rat liver microsomes, but yet could form quinone reactive intermediates in melanoma cells as a result of its bioactivation by tyrosinase. In current work, we investigated 24 phenolic compounds for their metabolism by tyrosinase, rat liver microsomes and their toxicity towards murine B16-F0 and human SK-MEL-28 melanoma cells. A linear correlation was found between toxicities of phenolic analogs towards SK-MEL-28 and B16-F0 melanoma cells, suggesting similar mechanisms of toxicity in both cell lines. 4-HEB was identified as the lead compound. 4-HEB (IC(50) 48h, 75muM) showed selective toxicity towards five melanocytic melanoma cell lines SK-MEL-28, SK-MEL-5, MeWo, B16-F0 and B16-F10, which express functional tyrosinase, compared to four non-melanoma cells lines SW-620, Saos-2, PC3 and BJ cells and two amelanotic SK-MEL-24, C32 cells, which do not express functional tyrosinase. 4-HEB caused significant intracellular GSH depletion, ROS formation, and showed significantly less toxicity to tyrosinase specific shRNA transfected SK-MEL-28 cells. Our findings suggest that presence of a phenolic group in 4-HEB is critical for its selective toxicity towards melanoma cells.

In current work, we investigated the in-vitro efficacy of Caffeic acid Phenethyl Ester (CAPE) as an anti-melanoma agent in five melanoma cell lines B16-F0, B16F10, SK-MEL-28, SK-MEL-5, and MeWo and in-vivo efficacy study in skin B16-F0 melanoma tumor model in C57BL/6 mice. The IC(50) (48 h) of CAPE in above five melanoma cell lines was 15 µM. CAPE (20-200 µM) led to intracellular GSH depletion of 16-54%, and 10-25 fold increase in Reactive Oxygen Species (ROS) formation in B16-F0 cells. CAPE (15-30 µM) caused 5-7 fold increase in apoptosis in B16-F0 cells. CAPE (10, 20 and 30 mg/Kg/day) led to tumor size growth inhibition by 39 ± 33%, 54 ± 36%, and 57 ± 18%, respectively. The respective therapies led to plasma Alanine Amino Transferase (ALT) levels corresponding to 85 ± 18, 107 ± 26, 154 ± 35 IU/L in comparison to controls 66 ± 14 IU/L. At corresponding doses, the lipid peroxidation levels as measured by malondialdehyde (MDA) formation in liver homogenates were 255 ± 8 μM, 304 ± 21 μM, and 342 ± 14 μM in comparison to 208 ± 6 μM in controls. The level of MDA in kidney homogenates was 263 ± 21 μM, 282 ± 18 μM, and 350 ± 28 μM, respectively, in comparison to 212 ± 8 μM in controls. Administration of CAPE (10, 20, 30 mg/Kg/day) diminished free thiol contents in liver for 21 ± 15%, 40 ± 17%, and 44 ± 19% and in kidney homogenates for 25 ± 15%, 37 ± 18%, and 40 ± 22%, respectively, as compared to controls. Our study suggests that CAPE at 10 mg/Kg/day has significant anti-melanoma efficacy with minimal toxicity.

Previously, we reported that acetaminophen (APAP) showed selective toxicity towards melanoma cell lines. In the current study, we investigated further the role of tyrosinase in APAP toxicity in SK-MEL-28 melanoma cells in the presence of a short hairpin RNA (shRNA) plasmid, silencing tyrosinase gene. Results from tyrosinase shRNA experiments showed that APAP led to negligible toxicity in shRNA plasmid-treated cells. It was also found that APAP selectively caused escalation in reactive oxygen species (ROS) formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 and murine B16-F0 melanoma cells that express functional tyrosinase whereas it lacked significant effects on ROS formation and ICG in amelanotic C32 melanoma cells that do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in APAP selective induced toxicity in melanocytic melanoma cell lines. Furthermore, the in vivo efficacy and toxicity of APAP in the skin melanoma tumor model in mice was investigated. Mice receiving APAP at 60, 80, 100 and 300 mg/kg/day, day 7 through 13 post melanoma cell inoculation demonstrated tumor size growth inhibition by 7+/-14, 30+/-17, 45+/-11 and 57+/-3%, respectively. Mice receiving APAP day 1 through 13 post melanoma cell inoculation showed tumor size growth inhibition by 11+/-7, 33+/-9, 36+/-20 and 44+/-28%, respectively.

In the current work, we investigated the biochemical toxicity of acetylsalicylic acid (ASA; Aspirin) in human melanoma cell lines using tyrosinase enzyme as a molecular cancer therapeutic target. At 2 h, ASA was oxidized 88% by tyrosinase. Ascorbic acid and NADH, quinone reducing agents, were significantly depleted during the enzymatic oxidation of ASA by tyrosinase to quinone. The 50% inhibitory concentration (48 h) of ASA and salicylic acid toward SK-MEL-28 cells were 100 micromol/l and 5.2 mmol/l, respectively. ASA at 100 micromol/l was selectively toxic toward human melanocytic SK-MEL-28, MeWo, and SK-MEL-5 and murine melanocytic B16-F0 and B16-F10 melanoma cell lines. However, ASA was not significantly toxic to human amelanotic C32 melanoma cell line, which does not express tyrosinase enzyme, and human nonmelanoma BJ, SW-620, Saos, and PC-3 cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased ASA toxicity toward SK-MEL-28 cells indicating quinone formation and intracellular GSH depletion played important mechanistic roles in ASA-induced melanoma toxicity. Ascorbic acid, a quinone reducing agent, and GSH, an antioxidant and quinone trap substrate, prevented ASA cell toxicity. Trifluoperazine, inhibitor of permeability transition pore in mitochondria, prevented ASA toxicity. ASA led to significant intracellular GSH depletion in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ASA also led to significant reactive oxygen species (ROS) formation in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ROS formation was exacerbated by dicoumarol and 1-bromoheptane in SK-MEL-28. Our investigation suggests that quinone species, intracellular GSH depletion, ROS formation, and mitochondrial toxicity significantly contributed toward ASA selective toxicity in melanocytic SK-MEL-28 melanoma cells.

In this work, we investigated the biochemical mechanism of acetaminophen (APAP) induced toxicity in SK-MEL-28 melanoma cells using tyrosinase enzyme as a molecular cancer therapeutic target. Our results showed that APAP was metabolized 87% by tyrosinase at 2 h incubation. AA and NADH, quinone reducing agents, were significantly depleted during APAP oxidation by tyrosinase. The IC(50) (48 h) of APAP towards SK-MEL-28, MeWo, SK-MEL-5, B16-F0, and B16-F10 melanoma cells was 100 microM whereas it showed no significant toxicity towards BJ, Saos-2, SW-620, and PC-3 nonmelanoma cells, demonstrating selective toxicity towards melanoma cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, enhanced APAP toxicity towards SK-MEL-28 cells. AA and GSH were effective in preventing APAP induced melanoma cell toxicity. Trifluoperazine and cyclosporin A, inhibitors of permeability transition pore in mitochondria, significantly prevented APAP melanoma cell toxicity. APAP caused time and dose-dependent decline in intracellular GSH content in SK-MEL-28, which preceded cell toxicity. APAP led to ROS formation in SK-MEL-28 cells which was exacerbated by dicoumarol and 1-bromoheptane whereas cyslosporin A and trifluoperazine prevented it. Our investigation suggests that APAP is a tyrosinase substrate, and that intracellular GSH depletion, ROS formation and induced mitochondrial toxicity contributed towards APAP's selective toxicity in SK-MEL-28 cells.

Multiple Myeloma (MM) is a complex disease that frequently leads to fatal outcome and accurate risk classification to optimize the choice of therapy may have impact on clinical outcomes. MM stratification based upon cytogenetic abnormalities and protein-coding gene expression does not adequately correlate with the depth or durability of therapeutic response. Therefore, the new class of molecular effectors, non-coding RNAs (ncRNAs), may enhance the basic understanding of myelomagenesis, drug resistance and provide better stratification of myeloma subtypes. NcRNAs include long ncRNAs (lncRNAs) and microRNAs (miRNAs) that may act as positive or negative regulators of gene expression to control cell proliferation, apoptosis and drug resistance. We hypothesized that lncRNAs commonly deregulated in the 3 resistant cell lines would have significant overlap to the deregulated lncRNA in MM patients. To investigate the role of lncRNAs in resistance to proteasome inhibitors (PIs), we compared global lncRNA profiling in drug-naïve cells to cells with acquired resistance to the PIs bortezomib, carfilzomib and ixazomib. RPMI 8226 cells resistant to each of the three PIs were generated through successive exposure to bortezomib, carfilzomib or ixazomib. Total RNA was isolated and genome-wide ncRNA expression profiling was performed using Affymetrix 3.0 microarray chips LncRNA expression profiles from drug-resistant cells were compared to that of drug-naïve cells treated with vehicle alone. MM patients’ bone marrow aspirates were obtained after UCCOM IRB-approval. Bioinformatic analysis identified a panel of 18 lncRNAs that were significantly (>100-fold) deregulated in all three drug-resistant cell lines relative to drug-naïve cells. Strikingly, the majority of the deregulated lncRNAs exhibited a similar expression pattern in all 3 PI-resistant cell lines (Figure 2a). RPMI 8226 cells carry a t(14,16) and none of the deregulated lncRNAs detected localized to chromosomes 14 or 16, suggestive of a cytogenetic-independent mechanism of drug resistance. We also identified lncRNAs deregulated in MM patient samples relative to plasma cells from healthy age-matched individuals. We found a significant overlap (>90%) between deregulated lncRNAs in PI-resistant cells and MM cells. The lncRNA COL4A-2A was upregulated >5,000-fold in resistant cells and displayed extensive sequence complementarity to miRNA-29 that was downregulated in resistant cells. Taken together, we identified a curated panel of deregulated lncRNAs in common within myeloma cells generated with acquired resistant to three different clinically-relevant proteasome inhibitors and MM patients. Further investigation is warranted to shed light on the role of these lncRNAs in the development of MM, to identify their targets and to define their role in drug resistance. Note: This abstract was not presented at the meeting. Citation Format: Ehsan Malek, Rebekah Karns, Anil G. Jegga, Sajjeev Jagannathan, Nikhil Vad, Mohamed A.Y Abdel Malek, James J. Driscoll. Long non-coding RNAs deregulated in Multiple Myeloma impact therapeutic response to proteasome inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2015-153

Multiple myeloma (MM) patients invariably relapse through mechanisms that remain elusive. Disease recurrence after treatment suggests that rare, tumor-initiating subpopulations with chemoresistant phenotypes persist or emerge to propagate and mediate tumor regrowth. Stem cell-like side population (SP) cells were isolated from myeloma cell lines and patient samples and exhibited resistance to proteasome inhibitors relative to the main population (MP). SP cell-based high-throughput screening detected pharmacologics that enhanced the anti-proliferative effect of bortezomib and revealed the anti-diabetic agent metformin. Bortezomib treatment increased levels of the molecular chaperone glucose-regulated protein GRP78 but metformin co-treatment suppressed bortezomib-induced GRP78 upregulation. Bortezomib treatment also promoted GRP78 co-localization with the signal transducer phosphatidylinositol-3-kinase (PI3K) that activates cell growth and survival pathways. SP cells displayed greater levels of the PI3K product phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and enhanced PI3K-mediated phosphorylation of protein kinase B/AKT compared to MP cells. Metformin treatment or knockdown of GRP78-encoding HSPA5 enhanced sensitivity to bortezomib preferentially in SP cells. Tumor growth was significantly delayed and overall survival prolonged after metformin co-treatment with bortezomib in mice injected with parental or SP cells. Taken together, the results support the pharmacologic repositioning of metformin to inhibit GRP78 induction in therapy-resistant cells and enhance the anti-myeloma benefit of bortezomib. Note: This abstract was not presented at the meeting. Citation Format: James J. Driscoll, Sajjeev Jagannathan, Mohamed AY Abdel Malek, Nikhil Vad, Ehsan Malek. Metformin suppresses GRP78-dependent PI3-Kinase activity in clonogenic side population to enhance the anti-myeloma benefit of bortezomib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4242. doi:10.1158/1538-7445.AM2015-4242

Background: Multiple myeloma (MM) is a malignancy of post-germinal center B-cells whose pathogenesis remains poorly understood and that remains invariably fatal. Evading apoptosis is a cancer hallmark that remains a serious obstacle in current treatment approaches and contributes to drug resistance. Despite the therapeutic benefit of the proteasome inhibitor bortezomib, drug resistance still accounts for the majority of tumor relapses and cancer-related deaths in MM patients. A precise understanding of the molecular mechanism(s) of resistance to bortezomib in myeloma is lacking. The goal of the present study was to identify and functionally characterize novel microRNAs (miRNAs) that inhibit the ubiquitin (Ub)+proteasome system (UPS) and also disrupt the aggresome-autophagy pathway for anti-myeloma benefit. Functional blockade of the UPS using targeted therapeutics has translated into clinical success and transformed the management of MM. However, drug resistance emerges through induction of the aggresome-autophagy pathway as a compensatory protein clearance mechanism leading to treatment failure, disease relapse and ultimately fatal outcome. Methods: Myeloma cells were separately exposed to the proteasome inhibitors bortezomib, carfilzomib or ixazomib to generate cells with acquired drug resistance. Genome-wide, microarray-based profiling was then performed to identify miRNAs significantly deregulated in the drug-resistant myeloma cells relative to parental drug-naïve cells. Based upon the miRNA profiling, we identified a curated panel of miRNAs that were significantly deregulated in the drug-resistant cells. To determine the effect of individual miRNAs on myeloma cells, synthetic miRNA replacements or antagomirs were transfected into myeloma cells. The effect of the single miRNAs on the growth and drug resistance was then quantified using cell viability assays. Individual miRNA replacements or antagomirs were also transfected into human embryonic kidney (HEK) cells that expressed a short-lived green fluorescent protein that is degraded by the proteasome. The effect of individual miRNAs on the UPS was then quantitated in myeloma or HEK cells by confocal microscopy and fluorescent imaging. Results: MiRNA-29b was significantly downregulated in the myeloma cells generated with acquired resistance to bortezomib, carfilzomib and ixazomib relative to the parental cells. MiRNA-29b was also downregulated in MM patient bone marrow-derived tumor cells relative to plasma cells obtained from healthy individuals. MiRNA-29b targets PSME4which encodes the proteasome activator PA200. Transfection of synthetically-engineered miRNA-29b replacements reduced the viability of myeloma cells and patient tumor cells and synergistically enhanced the cytotoxic effect of proteasome inhibitors. The miRNA-29b replacement also reduced the growth of myeloma xenografts in mouse models in vivo. MiRNA-29b replacements decreased expression of PSME4and the protein product PA200, a proteasome activator. The reduction of PA200 levels inhibited the proteasome’s chymotrypsin-like peptidase activity and the turnover of ornithine decarboxylase, a proteasome substrate degraded through Ub-independent mechanisms. Following proteasome inhibition, perinuclear aggregates of insoluble Ub-conjugated proteins, termed aggresomes, accumulate and are degraded by the autophagy pathway. Importantly, in contrast to bortezomib, miRNA-29b replacement inhibited proteasome activity but did not induce the formation of perinuclear aggresomes or autophagosomes. Rather, immunofluorescence studies indicated that miRNA-29b replacement promoted the accumulation of small, ubiquitinated protein aggregates that were dispersed throughout the cytoplasm but were not transported by microtubules and were not coalesced into mature aggresomes. Ub-conjugated proteins were also not detected in autophagosomes. Conclusions: MiRNA-29b replacement represents the first-in-class miRNA-based agent to inhibit proteasomes and to disrupt the efficient delivery of Ub-conjugate-containing aggresomes to the autophagy pathway. Taken together, our study highlights the potential of miRNA replacement therapy to synergistically enhance the anti-myeloma benefit of proteasome inhibitors. Disclosures No relevant conflicts of interest to declare.

Background: Functional blockade of the ubiquitin (Ub)+proteasome system (UPS) using the small molecule bortezomib represents a remarkable bench-to-bedside success that has improved the outcome of patients diagnosed with multiple myeloma (MM). However, certain patients do not respond to bortezomib and those that do inevitably develop resistance through mechanisms that remain elusive and despite all currently available therapies, MM remains invariably fatal. While bortezomib disrupts protein homeostasis leading to tumor cell death but also activates pro-survival aggresome and autophagosome formation as compensatory protein clearance mechanisms to promote the emergence of drug resistance, tumor survival and disease relapse. While the therapeutic benefit of targeting the proteasome is unchallenged, more precise modalities that do not induce drug resistance are urgently needed. These studies were performed to identify pharmacologics that inhibited the autophagy pathway and to uncover their mechanisms of action as a means to synergistically enhance the cytotoxic effect of bortezomib for myeloma treatment. Methods: High-throughput screening (HTS) was performed to identify FDA-approved pharmacologic agents and bioactive molecules that prevented bortezomib-mediated induction of autophagosomes. Confocal microscopy combined with novel, dye-based fluorescent methods were used to visualize and quantitate aggresomes and autophagosomes. The effect of pharmacologics on aggresome and autophagosome formation was then determined. The effects of metformin, alone or combined with bortezomib were evaluated using MM cell lines, myeloma patient bone marrow-derived tumor cells and myeloma cells xenotransplanted into mouse models. Results: Bortezomib inhibits the UPS to promote the unwanted intracellular accumulation of ubiquitinated proteins that form aggregates dispersed throughout the cytoplasm. These aggregates are then coalesced into perinuclear aggresomes and subsequently disposed through autophagosome formation and the lysosomal pathway. Bortezomib treatment of myeloma cells increased levels of the endoplasmic reticulum (ER) molecular chaperone Glucose-Regulated Protein 78 (GRP78), a critical regulator of the unfolded protein response (UPR) and autophagosome formation. HTS revealed that the anti-diabetic biguanide metformin inhibited bortezomib-mediated autophagosome formation. Co-treatment with metformin and bortezomib increased the level of proteotoxic aggresomes but suppressed GRP78 and autophagosome formation. Metformin synergistically enhanced the anti-proliferative effect of bortezomib in myeloma cells and bone marrow-derived patient PCs. Co-treatment with metformin and proteasome inhibitors also overcame drug-resistance. shRNA-mediated knockdown of HSPA5, which encodes GRP78, similarly enhanced bortezomib-induced aggresome formation and blocked autophagosome formation. A GRP78 mutant that lacked the carboxy-terminal KDEL ER-retention motif also led to bortezomib-induced aggresomes without autophagosomes. Metformin and bortezomib together reduced myeloma xenotransplant growth in mice more effectively than either agent administered as single-agent. Conclusions: The ER chaperone GRP78 facilitates aggresome clearance by promoting autophagosome formation. Although bortezomib alone promotes GRP78 induction and aggresome formation, metformin suppresses GRP78 to uncouple the cytotoxic aggresomes from the autophagy pathway and to enhance the anti-myeloma effect of bortezomib. The results support the pharmacologic repositioning of metformin in combination with bortezomib as an effective anti-myeloma strategy. The molecular chaperone GRP78 holds promise as a target for cancer treatment as well as for neurodegenerative diseases in which intracellular protein aggregates are central to disease pathogenesis Disclosures No relevant conflicts of interest to declare.

Background: Multiple myeloma (MM) is a heterogeneous diseaseand there is an increased need for more accurate risk classification methods to improve treatment decision-making because of its high impact on clinical outcomes. Here, we demonstrate evidence to support the prognostic value of non-coding RNAs (ncRNAs) as newly discovered genetic biomarkers of drug-resistant and/or high-risk forms of MM. NcRNAs, e.g., long ncRNAs (lncRNAs) and microRNAs (miRNAs), act as positive or negative regulators of gene expression to control cell proliferation, apoptosis and drug resistance. NcRNAs have been shown to play a role in both solid and hematological tumors. Stratification of MM based upon cytogenetic abnormalities and protein-coding gene signatures does not adequately correlate with the depth and durability of response to novel agents such as bortezomib. Therefore, ncRNAs as new class of molecular effectors may enhance the basic understanding of myelomagenesis and provide better stratification of myeloma subtypes. To investigate the role of ncRNAs in resistance to proteasome inhibitors (PIs), we compared global ncRNA profiling in drug-naïve cells to cells with acquired resistance to the PIs bortezomib, carfilzomib and ixazomib. We hypothesized that ncRNAs commonly deregulated in the 3 resistant cell lines would yield a ncRNA signature and novel therapeutic targets. Experimental Procedures: RPMI 8226 cells resistant to PIs were generated through successive exposure to bortezomib, carfilzomib or ixazomib over a period of 6 months. Total RNA was isolated and genome-wide ncRNA expression profiling was performed using Affymetrix3.0 microarray chips that contained nearly 40,000 miRNA and 13,300 lncRNA probes. NcRNA expression profiles from drug-resistant cells were compared to that of drug- naïve parental cells treated with vehicle alone using the same treatment algorithm. Housekeeping genes were used for log expression normalization. MM patients' bone marrow aspirates were obtained from patients after University of Cincinnati Institutional Review Board approval. Results: Bioinformatic analysis of the ncRNA profiles identified a panel of 87 lncRNAs and ~40 miRNAs that were significantly (>100-fold) deregulated in all three drug-resistant cell lines relative to drug- naïve parental cells. Strikingly, ~90% of the deregulated lncRNAs exhibited a similar expression pattern in all 3 PI-resistant cell lines. Twenty lncRNAs were deregulated > 1000-fold in all 3 resistant cell lines (Figure 1). RPMI 8226 cells carry a chromosomal (14,16) translocation. Interestingly, none of the deregulated lncRNAs detected here localized to chromosome 14 or 16, suggestive of a cytogenetic-independent mechanism of drug resistance. The lncRNA COL4A-2A was upregulated >5,000-fold in resistant cells and displayed extensive sequence complementarity to miRNA-29 that was downregulated in resistant cells. Also, our microarray-based studies have identified ncRNAs deregulated in MM patient tumor samples relative to normal plasma cells from healthy age-matched individuals. A significant number of the deregulated ncRNAs between drug- naïve and drug resistant cells were also deregulated in normal plasma cells relative to myeloma cells. Studies are correlating the ncRNA patterns seen in drug-sensitive and drug-resistant cell lines with ncRNA patterns obtained from malignant plasma cells of patients currently receiving bortezomib-based therapy. Updated results to correlate ncRNA expression with myeloma patient response to bortezomib will be presented.Conclusions: Taken together, we have identified a curated panel of ncRNAs deregulated in common within myeloma cells generated with acquired resistant to three different clinically-relevant proteasome inhibitors. Ongoing studies will correlate ncRNA expression patterns from resistant cells with patterns generated from patients with monoclonal gammopathy of unknown significance (MGUS), Smoldering MM, newly diagnosed MM, refractory disease and plasma cell leukemia. In addition, ncRNA patterns will be generated based upon MM patient response to bortezomib. Further investigation is warranted to shed light on the role of these ncRNAs in the development of MM, to identify their targets and to define their role in drug resistance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Study aim was to characterize dihydroceramide-related cell death in the glioblastoma multiforme cell lines, A-172 and T98G. Methods: Cytotoxicity was evaluated using fluorescence digital image microscopy (DIMSCAN). Apoptosis was analyzed by Annexin V-FITC and TUNEL assays. Mitochondrial depolarization and Reactive Oxygen Species (ROS) were assayed by JC1 staining and DCFDA staining and flow cytometry. Autophagy marker, LC3BII, and caspase-3 cleavage were assayed by immunoblotting. Autophagy inhibition was by bafilomycin A1 and siRNA against ATG7 and BECN1. Sphingolipid levels were assayed by radiolabeling and mass spectrometry. Results: Fenretinide (4-HPR) increased dihydroceramides coincident with cytotoxicity. Artificial L-threo-sphinganine analogs, safingol (S) and C20-base safingol (C20-S), were incorporated into L-threo variant dihydroceramides coincident with synergistic enhancement of 4-HPR cytotoxicity. 4-HPR, alone or in combination with S or C20-S, resulted in a significant (p Citation Format: Nikhil Vad, Dong Wang, Barry J. Maurer. Molecular inhibition of autophagy enhances dihydroceramides-induced cell death in glioblastoma multiforme cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1709. doi:10.1158/1538-7445.AM2013-1709

Glutathione S-transferase (GST) play significant role in the metabolism and detoxification of drugs used in treatment of melanoma, resulting in a decrease in their efficacy against melanoma. In the present study, we investigated: i) the selective inhibition of GST by luteolin in the presence of tyrosinase, an abundant enzyme found in melanoma, and ii) the induction of apoptosis, change in mitochondrial permeability, cell viability, cell migration and GSH depletion by luteolin in human melanoma SK-MEL-28 cell culture. The inhibition of GST was investigated using CDNB method. DTNB method was used to measure GSH depletion. MTT assay, Annexin V apoptosis assay and TMRM fluorescence were used in the investigation of biochemical mechanism of luteolin toxicity in melanoma cells. Luteolin (40 µM) induced toxicity and death in 50% of cells after 48 h incubation. Cell treatment with luteolin (40 µM) resulted in apoptotic cell death in 70% of cells and caused 45% decrease in TMRM fluorescence indicating change in mitochondrial membrane permeability. At 1 h incubation, luteolin was bioactivated by tyrosinase to luteoline-quinone for 71% as measured by GSH depletion. The luteolin is effective in preventing metastasis of cancerous cells as evident by cell migration assay. At 48 h incubation, luteolin as low as 5 µM effectively prevented the cell migration. In the presence of tyrosinase, luteolin (10 µM) showed about 87% GST inhibition; whereas in the absence of tyrosinase, luteolin led to negligible GST inhibition. Both luteolin-SG conjugate and luteolin-quinone (10 µM) inhibited ≥90% of GST activity via reversible and irreversible competitive mechanisms with Ki of 0.9 and 6.4 µM with respect to GSH, respectively. The luteolin-SG conjugate inhibited GST activity reversibly and non-competitively with Ki of 1.8, whereas luteolin-quinone showed irreversible competitive inhibition of GST activity with Ki of 1.0 µM with respect to CDNB. While luteolin is not a substrate for GST, Luteolin (25 µM) non-competitively inhibited GST with Ki of 39 µM with respect to GSH and competitively with Ki of 61 µM with respect to CDNB. Luteolin, at the concentration range of 5-80 µM, exhibited 78-99% GST inhibition in human SK-MEL-28 cell homogenate. In summary, our results suggest that luteolin was bioactivated by tyrosinase to form a quinone and luteolin-glutathione conjugate, which played major role in the inhibition of GST. For the first time, we demonstrate that luteolin acts as a selective inhibitor of GST in the presence of tyrosinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1345. doi:10.1158/1538-7445.AM2011-1345