Multiple myeloma exhibits novel dependence on GLUT4, GLUT8, and GLUT11: implications for glucose transporter-directed therapy.

Multiple myeloma is one of numerous malignancies characterized by increased glucose consumption, a phenomenon with significant prognostic implications in this disease. Few studies have focused on elucidating the molecular underpinnings of glucose transporter (GLUT) activation in cancer, knowledge that could facilitate identification of promising therapeutic targets. To address this issue, we performed gene expression profiling studies involving myeloma cell lines and primary cells as well as normal lymphocytes to uncover deregulated GLUT family members in myeloma. Our data demonstrate that myeloma cells exhibit reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption, maintenance of Mcl-1 expression, growth, and survival. We also establish that the activities of the enigmatic transporters GLUT8 and GLUT11 are required for proliferation and viability in myeloma, albeit because of functionalities probably distinct from whole-cell glucose supply. As proof of principle regarding the therapeutic potential of GLUT-targeted compounds, we include evidence of the antimyeloma effects elicited against both cell lines and primary cells by the FDA-approved HIV protease inhibitor ritonavir, which exerts a selective off-target inhibitory effect on GLUT4. Our work reveals critical roles for novel GLUT family members and highlights a therapeutic strategy entailing selective GLUT inhibition to specifically target aberrant glucose metabolism in cancer.

Clinical perspective on chemo-resistance and the role of RNA processing.

Pre-messenger RNA splicing is significantly changed in cancer cells leading to the expression of cancer-specific transcripts. These transcripts have the potential to be used as cancer biomarkers and also as targets for new therapeutic approaches. In addition, the cancer-specific transcripts have the potential to alter the drug response of the cancer cells creating a chemo-resistant state. This later property of alternative splicing presents a challenge to clinicians in the design of effective therapeutic regimens. When a patient's cancer relapses it is frequently refractory to standard chemotherapies resulting in a poor clinical outcome. Therefore, understanding the mechanisms of how alternative splicing can lead to chemo-resistance is critical to the effective delivery of treatment. Here, we will discuss the impact of alternative splicing variants on drug metabolism and activation; on drug interactions with cell signaling pathways; and on cell death pathways in cancer therapeutics. In addition to the initial characterization of splicing variants, the mechanisms leading to alterations in splicing are being studied in the setting of chemo-resistance and will be discussed here. The promise of therapeutic intervention to obviate the impact of these splicing variants will significantly enhance treatment options for cancer patients.

8-Aminoadenosine inhibits Akt/mTOR and Erk signaling in mantle cell lymphoma.

8-Aminoadenosine (8-NH(2)-Ado), a ribosyl nucleoside analog, in preclinical models of multiple myeloma inhibits phosphorylation of proteins in multiple growth and survival pathways, including Akt. Given that Akt controls the activity of mammalian target of rapamycin (mTOR), we hypothesized that 8-NH(2)-Ado would be active in mantle cell lymphoma (MCL), a hematological malignancy clinically responsive to mTOR inhibitors. In the current study, the preclinical efficacy of 8-NH(2)-Ado and its resulting effects on Akt/mTOR and extracellular-signal-regulated kinase signaling were evaluated using 4 MCL cell lines, primary MCL cells, and normal lymphocytes from healthy donors. For all MCL cell lines, 8-NH(2)-Ado inhibited growth and promoted cell death as shown by reduction of thymidine incorporation, loss of mitochondrial membrane potential, and poly (adenosine diphosphate-ribose) polymerase cleavage. The efficacy of 8-NH(2)-Ado was highly associated with intracellular accumulation of 8-NH(2)-adenosine triphosphate (ATP) and loss of endogenous ATP. Formation of 8-NH(2)-ATP was also associated with inhibition of transcription and translation accompanied by loss of phosphorylated (p-)Akt, p-mTOR, p-Erk1/2, p-phosphoprotein (p)38, p-S6, and p-4E-binding protein 1. While normal lymphocytes accumulated 8-NH(2)-ATP but maintained their viability with 8-NH(2)-Ado treatment, primary lymphoma cells accumulated higher concentrations of 8-NH(2)-ATP, had increased loss of ATP, and underwent apoptosis. We conclude that 8-NH(2)-Ado is efficacious in preclinical models of MCL and inhibits signaling of Akt/mTOR and Erk pathways.

Targeting glucose consumption and autophagy in myeloma with the novel nucleoside analogue 8-aminoadenosine.

Multiple myeloma, an incurable plasma cell malignancy, is characterized by altered cellular metabolism and resistance to apoptosis. Recent connections between glucose metabolism and resistance to apoptosis provide a compelling rationale for targeting metabolic changes in cancer. In this study, we have examined the ability of the purine analogue 8-aminoadenosine to acutely reduce glucose consumption by regulating localization and expression of key glucose transporters. Myeloma cells counteracted the metabolic stress by activating autophagy. Co-treatment with inhibitors of autophagy results in marked enhancement of cell death. Glucose consumption by drug-resistant myeloma cells was unaffected by 8-aminoadenosine, and accordingly, no activation of autophagy was observed. However, these cells can be sensitized to 8-aminoadenosine under glucose-limiting conditions. The prosurvival autophagic response of myeloma to nutrient deprivation or to nucleoside analogue treatment has not been described previously. This study establishes the potential of metabolic targeting as a broader means to kill and sensitize myeloma and identifies a compound that can achieve this goal.

Steroid receptor and microRNA regulation in cancer.

PURPOSE OF REVIEW: Steroid hormone receptors (SHR) are crucial regulators of disease and the basis for clinical intervention in cancers. Recent evidence confirms that microRNAs (miRNAs) impact the pathobiology of hormone-regulated malignancies. Therefore, elucidating miRNA regulation of SHR expression and modulation of miRNAs by SHRs may provide diagnostic biomarkers or therapeutic targets. RECENT FINDINGS: Estrogen receptor status has been established as a key factor in breast cancer prognosis and treatment. Recent studies detail the interactions between estrogen receptor and miRNAs in cancers. New evidence indicates involvement of miRNAs in the regulation of androgen receptor, progesterone receptor, glucocorticoid receptor in hormone responsive cancers. Several miRNAs regulate the expression of the SHRs, while other miRNAs are themselves regulated by SHR signaling in cancer. SUMMARY: Cancers have distinct miRNA expression profiles that contribute to the pathobiology of the disease. In hormone-responsive cancers, the regulatory interactions between the SHR and miRNA may contribute to disease progression. The miRNA regulation of estrogen receptor in cancer has been established in estrogen-dependent cancers. The role of miRNAs in regulating progesterone receptor, androgen receptor and glucocorticoid receptor is under investigation with new insights emerging. These interactions can provide prognostic utility as well as the potential for therapeutic intervention in the future.

Nuclear translocation of Atox1 potentiates activin A-induced cell migration and colony formation in colon cancer.

BACKGROUND: Colorectal cancer remains a deadly cancer due to metastatic disease. To understand the molecular mechanisms of metastasis in colon cancer, we investigated whether the copper chaperone antioxidant-1 (Atox1) protein plays a role in this process. Recent findings indicate that Atox1 protein has transcription factor activities and plays a vital role in cell proliferation in cancer cells. However, the role of Atox1 in metastasis has not been examined. METHODS: Atox1 expression was determined by immunofluorescence in a tissue microarray generated from a spectrum of CRC patients. Subcellular fractionation of colon cancer cell lines SW480 and SW620 cells was used to examine the cellular location of Atox1 in the face of activin A, a cytokine that stimulates colon cancer metastasis. Atox1 expression was genetically manipulated and cellular migration measured through trans-well assay and proliferation measured by colony formation assays. RESULTS: Here we demonstrate that in patients with metastatic colon cancer, there is a significant increase in the expression of nuclear Atox1. Interestingly, the metastatic CRC cell line SW620 has increased nuclear localization of Atox1 compared to its related non-metastatic cell line SW480. Further, inhibition of endogenous Atox1 by siRNA in SW620 decreased colony formation and reactive oxygen species generation via decreased expression of Atox1 targets cyclin D1 and NADPH oxidase subunit p47 phox, respectively. Additionally, overexpression of nuclear-targeted but not copper binding domain-mutated Atox1 in SW480 cells increased colony formation and cell migration that was further augmented by activin A stimulation, a known enhancer of colon cancer metastasis. CONCLUSIONS: Our findings suggest that nuclear Atox1 might be a new therapeutic target as well as a new biomarker for metastatic colorectal cancer.

Activin A Modulates Inflammation in Acute Pancreatitis and Strongly Predicts Severe Disease Independent of Body Mass Index.

INTRODUCTION: Acute pancreatitis (AP) is a healthcare challenge with considerable mortality. Treatment is limited to supportive care, highlighting the need to investigate disease drivers and prognostic markers. Activin A is an established mediator of inflammatory responses, and its serum levels correlate with AP severity. We hypothesized that activin A is independent of body mass index (BMI) and is a targetable promoter of the AP inflammatory response. METHODS: We assessed whether BMI and serum activin A levels are independent markers to determine disease severity in a cohort of patients with AP. To evaluate activin A inhibition as a therapeutic, we used a cerulein-induced murine model of AP and treated mice with activin A-specific neutralizing antibody or immunoglobulin G control, both before and during the development of AP. We measured the production and release of activin A by pancreas and macrophage cell lines and observed the activation of macrophages after activin A treatment. RESULTS: BMI and activin A independently predicted severe AP in patients. Inhibiting activin A in AP mice reduced disease severity and local immune cell infiltration. Inflammatory stimulation led to activin A production and release by pancreas cells but not by macrophages. Macrophages were activated by activin A, suggesting activin A might promote inflammation in the pancreas in response to injury. DISCUSSION: Activin A provides a promising therapeutic target to interrupt the cycle of inflammation and tissue damage in AP progression. Moreover, assessing activin A and BMI in patients on hospital admission could provide important predictive measures for screening patients likely to develop severe disease.

TGFß and activin A in the tumor microenvironment in colorectal cancer.

Although overall survival in colorectal cancer (CRC) is increasing steadily due to progress in screening, therapeutic options and precise diagnostic tools remain scarce. As the understanding of CRC as a complex and multifactorial condition moves forward, the tumor microenvironment has come into focus as a source of diagnostic markers and potential therapeutic targets. The role of TGFß in shifting the epithelial cancer compartment towards invasiveness and a pro-migratory phenotype via stromal signaling has been widely investigated. Accordingly, recent studies have proposed that CRC patients could be stratified into distinct subtypes and have identified one poor prognosis subset of CRC that is characterized by high stromal activity and elevated levels of TGFß. The TGFß superfamily member activin A is crucial for the pro-metastatic properties of the TGFß pathway, yet it has been under-researched in CRC carcinogenesis. In this review, we will elucidate the signaling network and interdependency of both ligands in the context of the tumor microenvironment in CRC.

Protein kinase A-independent inhibition of proliferation and induction of apoptosis in human thyroid cancer cells by 8-Cl-adenosine.

PURPOSE: Protein kinase A (PKA) affects cell proliferation in many cell types and is a potential target for cancer treatment. PKA activity is stimulated by cAMP and cAMP analogs. One such substance, 8-Cl-cAMP, and its metabolite 8-Cl-adenosine (8-Cl-ADO) are known inhibitors of cancer cell proliferation; however, their mechanism of action is controversial. We have investigated the antiproliferative effects of 8-Cl-cAMP and 8-CL-ADO on human thyroid cancer cells and determined PKA's involvement. EXPERIMENTAL DESIGN: We employed proliferation and apoptosis assays and PKA activity and cell cycle analysis to understand the effect of 8-Cl-ADO and 8-Cl-cAMP on human thyroid cancer and HeLa cell lines. RESULTS: 8-Cl-ADO inhibited proliferation of all cells, an effect that lasted for at least 4 d. Proliferation was also inhibited by 8-Cl-cAMP, but this inhibition was reduced by 3-isobutyl-1-methylxanthine; both drugs stimulated apoptosis, and 3-isobutyl-1-methylxanthine drastically reduced 8-Cl-cAMP-induced cell death. 8-Cl-ADO induced cell accumulation in G1/S or G2/M cell cycle phases and differentially altered PKA activity and subunit levels. PKA stimulation or inhibition and adenosine receptor agonists or antagonists did not significantly affect proliferation. CONCLUSIONS: 8-Cl-ADO and 8-Cl-cAMP inhibit proliferation, induce cell cycle phase accumulation, and stimulate apoptosis in thyroid cancer cells. The effect of 8-Cl-cAMP is likely due to its metabolite 8-Cl-ADO, and PKA does not appear to have direct involvement in the inhibition of proliferation by 8-Cl-ADO. 8-Cl-ADO may be a useful therapeutic agent to be explored in aggressive thyroid cancer.

Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma.

Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.

Enzastaurin (LY317615), a protein kinase Cbeta inhibitor, inhibits the AKT pathway and induces apoptosis in multiple myeloma cell lines.

Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is an oral inhibitor of the protein kinase Cbeta isozyme. The objective of this study was to assess the efficacy of enzastaurin in inducing apoptosis in multiple myeloma (MM) cell lines and to investigate possible mechanisms of apoptosis. Cell proliferation assays were done on a variety of MM cell lines with unique characteristics (dexamethasone sensitive, dexamethasone resistant, chemotherapy sensitive, and melphalan resistant). The dexamethasone-sensitive MM.1S cell line was used to further assess the effect of enzastaurin in the presence of dexamethasone, insulin-like growth factor-I (IGF-I), interleukin-6, and the pan-specific caspase inhibitor ZVAD-fmk. Enzastaurin increased cell death in all cell lines at clinically significant low micromolar concentrations (1-3 micromol/L) after 72 hours of treatment. Dexamethasone and enzastaurin were shown to have an additive effect on MM.1S cell death. Although IGF-I blocked the effect of 1 micromol/L enzastaurin, IGF-I did not abrogate cell death induced with 3 mumol/L enzastaurin. Moreover, enzastaurin-induced cell death was not affected by interleukin-6 or ZVAD-fmk. GSK3beta phosphorylation, a reliable pharmacodynamic marker for enzastaurin activity, and AKT phosphorylation were both decreased with enzastaurin treatment. These data indicate that enzastaurin induces apoptosis in MM cell lines in a caspase-independent manner and that enzastaurin exerts its antimyeloma effect by inhibiting signaling through the AKT pathway.

NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway.

Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB (TGFß) signaling are growth suppressive pathways that exert non-canonical pro-metastatic effects late in CRC carcinogenesis. We have recently shown that activin downregulates p21 via ubiquitination and degradation associated with enhanced cellular migration independent of SMADs. To investigate the mechanism of metastatic activin signaling, we examined activated NFkB signaling and activin ligand expression in CRC patient samples and found a strong correlation. We hypothesize that activation of the E3 ubiquitin ligase MDM2 by NFkB leads to p21 degradation in response to activin treatment. To dissect the link between activin and pro-carcinogenic NFkB signaling and downstream targets, we found that activin but not TGFB induced activation of NFkB leading to increased MDM2 ubiquitin ligase via PI3K. Further, overexpression of wild type p65 NFkB increased MDM2 expression while the NFkB inhibitors NEMO-binding domain (NBD) and Bay11-7082 blocked the activin-induced increase in MDM2. In conclusion, in colon cancer cell migration, activin utilizes NFkB to induce MDM2 activity leading to the degradation of p21 in a PI3K dependent mechanism. This provides new mechanistic knowledge linking activin and NFkB signaling in advanced colon cancer which is applicable to targeted therapeutic interventions.

The selective protein kinase C beta inhibitor enzastaurin induces apoptosis in cutaneous T-cell lymphoma cell lines through the AKT pathway.

Enzastaurin displays pro-apoptotic properties against a spectrum of malignancies and is currently being investigated in clinical trials. We have investigated the effects of enzastaurin on the viability of the cutaneous T-cell lymphoma cell lines HuT-78 and HH by using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, cell cycle analysis, propidium iodide and annexin-V staining, and caspase-3-mediated proteolytic activation. Enzastaurin-treatment decreased cell viability, increased annexin V-FITC-positive cells, and increased the proportion of sub-G1 populations in both cell lines that was not reversed by the T-cell growth stimulating cytokines IL-2, IL-7, IL-15. Enzastaurin-induced cell death involved caspase-3-activated cleavage of poly(ADP-ribose) polymerase that was inhibited by the pan-caspase inhibitor ZVAD-fmk, whereas the increase in sub-G1 population was only partially inhibited by ZVAD-fmk. Furthermore, enzastaurin downregulated AKT activity and its downstream effectors GSK3beta and ribosomal protein S6. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the growth and survival of hematologic malignancies and inhibition of this pathway is considered as a therapeutic target. Protein kinase C activation contributes to PI3K/AKT activation, but it is unknown how enzastaurin may interfere with signaling through this pathway. These results demonstrate that enzastaurin, at clinically achievable concentrations, induces apoptosis and affects AKT signaling, and provide a rationale for further in vivo studies addressing the therapeutic efficacy in cutaneous T-cell lymphoma patients.

8-Amino-adenosine induces loss of phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, and Akt kinase: role in induction of apoptosis in multiple myeloma.

Multiple myeloma is a slowly proliferating B-cell malignancy that accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics that target rapidly replicating cells. Multiple myeloma remains an incurable disease in need of new therapeutic approaches. The purine nucleoside analogue, 8-amino-adenosine (8-NH2-Ado), exhibits potent activity in preclinical studies, inducing apoptosis in several multiple myeloma cell lines. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its triphosphate form, 8-amino-ATP, and results in a concomitant loss of endogenous ATP levels. Here, we show the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. Multiple myeloma cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and Akt kinase. Cells depleted of ATP independent of 8-NH2-Ado do not exhibit the same decrease in phosphorylation of vital cellular proteins. Therefore, the significant shifts in endogenous ATP pools caused by 8-NH2-Ado treatment cannot account for the changes in phosphorylation levels. Instead, 8-NH2-Ado may influence the activity of select regulatory protein kinases and/or phosphatases, with preliminary data suggesting that protein phophatase 2A activity is affected by 8-NH2-Ado. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analogue drug and is unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical prosurvival and apoptosis-regulating proteins suggests that the modulation of these proteins by dephosphorylation at early time points may be an important mechanistic step in 8-NH2-Ado-induced apoptosis.

Mechanisms of glucocorticoid-mediated apoptosis in hematological malignancies.

Although glucocorticoids (GCs) have been used for their immunosuppressive, anti-inflammatory, and cytotoxic effects for many years, their precise mechanism of action has not been fully elucidated. Evidence indicates that GCs induce apoptosis in hematological cells, thus supporting their use as chemotherapeutic agents for leukemias, lymphomas, and myeloma. Although much research has been focused on investigating the mechanism of action responsible for GC-mediated cell death, the signaling pathways remain unclear. Two schools of thought have developed to account for GC-induced apoptosis. One supports the hypothesis that apoptosis is achieved via activation of death-inducing genes. The second theory states that GCs induce apoptosis via repression of transcription factor activity, thereby inhibiting the transcription of growth/survival genes. This review will attempt to clarify the complex signaling pathway responsible for mediating GC-induced apoptosis of hematological cells and to summarize the most current research in this field.

Characterization of the MM.1 human multiple myeloma (MM) cell lines: a model system to elucidate the characteristics, behavior, and signaling of steroid-sensitive and -resistant MM cells.

Multiple myeloma (MM) is a clonal B-lymphocyte malignancy, which is characterized by the accumulation of terminally differentiated antibody-producing cells in the bone marrow. Because current treatments offer only a median survival of 3 years, investigators continue to search for novel therapeutic strategies to combat the disease. Rational drug design is enhanced by understanding MM cell proliferation and key signaling pathways employed. In addition, a model system for preclinical evaluation of novel therapeutics is critical. Our laboratory has developed MM cell lines to study drug action and resistance, cell proliferation, and apoptosis. These cell lines are widely used in MM research. From a single MM patient, three separate cell lines were established that parallel the progression of the disease. These three cell lines, designated MM1.S, MM1.R(E), and MM1.R(L), can be distinguished on the basis of their sensitivity to steroid hormones such as glucocorticoids (GCs). Utilization of these cell lines to study the etiology of MM, effects of chemotherapeutic agents, and development of clinical resistance, will provide us with vital information for the evolution of new and more efficacious therapeutics. The aim of this review is to summarize the morphological, biochemical, and growth characteristics of these cells, and to review the results from investigations of the MM.1 signaling pathways. This information will enhance the study, treatment, and eventual eradication of MM.

Caspase activation is required for gemcitabine activity in multiple myeloma cell lines.

The objective of this study was to determine potential mechanisms of apoptotic activity of gemcitabine, a pyrimidine nucleoside analogue, in the MM1.S multiple myeloma (MM) cell line. A MM cell line that is sensitive to glucocorticoids (MM1.S) was used for this study. Immunoblotting analysis, cell cycle assays, and annexin V staining were performed to determine whether gemcitabine induced apoptosis in this model. Furthermore, we attempted to delineate the apoptotic pathway by measuring caspase-8 and -9 activity using fluorometric assays. Loss of mitochondrial membrane potential was measured by flow cytometry. Gemcitabine treatment caused apoptosis in MM cell lines as measured by an increase in DNA cleavage, an increase in annexin V binding, a decrease in the mitochondrial membrane potential, and activation of caspase activity. Furthermore, cleavage of the caspase substrate poly(ADP-ribose) polymerase and caspase-3 activation were documented as early as 8 h after treatment with gemcitabine. Caspase-8 and -9 were activated by gemcitabine treatment in this cell line, suggesting several mechanisms of action including death receptor pathway and mitochondrial damage. The addition of interleukin 6 to MM1.S cells treated with gemcitabine offered no protection against gemcitabine-induced cell death. Gemcitabine induced apoptosis in the MM1.S cell line, and its activity required caspase activation. There is a suggestion that mitochondrial integrity is being affected with gemcitabine in this system. Gemcitabine acts independently of interleukin 6, suggesting potential important therapeutic implications in MM patients.

8-amino-adenosine is a potential therapeutic agent for multiple myeloma.

UNLABELLED: Multiple myeloma (MM) is a malignancy of clonal B-cells that accounts for 10% of all hematologic malignancies. We have shown previously that a novel purine analogue, 8-chloro-adenosine, has significant activity for MM in preclinical studies. OBJECTIVE: Using MM cell lines, we investigated the molecular mechanism of related congener of adenosine, 8-amino-adenosine (8-NH2-Ado). METHODS: We employed biological and biochemical assays in MM cell lines to evaluate the clinical potential of 8-NH2-Ado. RESULTS: In MM cell lines both sensitive and resistant to conventional chemotherapies, 8-NH2-Ado is cytotoxic, with IC50 ranging from 300 nmol/L to 3 micromol/L. A mouse leukemic cell line lacking adenosine kinase activity was resistant to 8-NH2-Ado, indicating that phosphorylation of 8-NH2-Ado to its triphosphate form is required for cytotoxicity. A 4-hour incubation of MM cells with 10 micromol/L analogue resulted in an accumulation of >7 mmol/L 8-NH2-ATP with a parallel decline in the endogenous ATP levels. Accumulation of 8-NH2-ATP was dependent on both exogenous concentration of 8-NH2-Ado and incubation time. The accumulation of 8-NH2-ATP was accompanied by a decrease in both RNA and DNA synthesis. The mechanism of 8-NH2-Ado-mediated cytotoxicity was due to apoptosis as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a sub-G1 DNA content. CONCLUSION: Based on these results, we conclude that 8-NH2-Ado may hold great potential as a therapeutic agent for the treatment of MM.

Identification of p38ß as a therapeutic target for the treatment of Sézary syndrome.

Cutaneous T-cell lymphomas (CTCLs) represent a group of hematopoietic malignancies that home to the skin and have no known molecular basis for disease pathogenesis. Sézary syndrome (SS) is the leukemic variant of CTCL. Currently, CTCL is incurable, highlighting the need for new therapeutic modalities. We have previously observed that combined small-molecule inhibition of protein kinase C-ß (PKCß) and glycogen synthase kinase 3 (GSK3) causes synergistic apoptosis in CTCL cell lines and patient cells. Through microarray analysis of a SS cell line, we surveyed global gene expression following combined PKCß-GSK3 treatment to elucidate therapeutic targets responsible for cell death. Clinically relevant targets were defined as genes differentially expressed in SS patients that were modulated by combination-drug treatment of SS cells. Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways. Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment. This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells. These data establish p38 as a SS biomarker and a potential therapeutic target for the treatment of CTCL.

Identification of potential glucocorticoid receptor therapeutic targets in multiple myeloma.

Glucocorticoids (GC) are a cornerstone of combination therapies for multiple myeloma. However, patients ultimately develop resistance to GCs frequently based on decreased glucocorticoid receptor (GR) expression. An understanding of the direct targets of GC actions, which induce cell death, is expected to culminate in potential therapeutic strategies for inducing cell death by regulating downstream targets in the absence of a functional GR. The specific goal of our research is to identify primary GR targets that contribute to GC-induced cell death, with the ultimate goal of developing novel therapeutics around these targets that can be used to overcome resistance to GCs in the absence of GR. Using the MM.1S glucocorticoid-sensitive human myeloma cell line, we began with the broad platform of gene expression profiling to identify glucocorticoid-regulated genes further refined by combination treatment with phosphatidylinositol-3'-kinase inhibition (PI3Ki). To further refine the search to distinguish direct and indirect targets of GR that respond to the combination GC and PI3Ki treatment of MM.1S cells, we integrated 1) gene expression profiles of combination GC treatment with PI3Ki, which induces synergistic cell death; 2) negative correlation between genes inhibited by combination treatment in MM.1S cells and genes over-expressed in myeloma patients to establish clinical relevance and 3) GR chromatin immunoprecipitation with massively parallel sequencing (ChIP-Seq) in myeloma cells to identify global chromatin binding for the glucocorticoid receptor (GR). Using established bioinformatics platforms, we have integrated these data sets to identify a subset of candidate genes that may form the basis for a comprehensive picture of glucocorticoid actions in multiple myeloma. As a proof of principle, we have verified two targets, namely RRM2 and BCL2L1, as primary functional targets of GR involved in GC-induced cell death.