ALBERT

Description: 8-Aminoadenosine (8-NH2-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-NH2-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-NH2-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-NH2-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-NH2-Ado was highly associated with intracellular accumulation of 8-NH2-adenosine triphosphate (ATP) and loss of endogenous ATP. Formation of 8-NH2-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-NH2-ATP but maintained their viability with 8-NH2-Ado treatment, primary lymphoma cells accumulated higher concentrations of 8-NH2-ATP, had increased loss of ATP, and underwent apoptosis. We conclude that 8-NH2-Ado is efficacious in preclinical models of MCL and inhibits signaling of Akt/mTOR and Erk pathways.

Description: We have examined the cytotoxic effects of cyclic adenosine-3′,5′-monophosphate (cAMP) derivatives on multiple myeloma cells lines and determined that the 8-Chloro substituted derivative (8Cl-cAMP) is one of the most potent. We report here that 8Cl-cAMP is cytotoxic to both steroid sensitive and insensitive myeloma cells with a half maximal concentration of approximately 3 μmol/L. 8Cl-cAMP toxicity in myeloma cells is dependent on phosphodiesterase activity in the serum of cell culture medium. A metabolite of 8Cl-cAMP, 8-Chloro-adenosine (8Cl-AD), kills myeloma cells as effectively as 8Cl-cAMP. Adenosine deaminase (ADA) converts 8Cl-AD into 8Cl-inosine and abrogates the cytotoxic effects of 8Cl-cAMP, 8Cl-AMP, and 8Cl-AD, as does 5-(p-Nitrobenzyl)-6-Thio-Inosine (NBTI), an inhibitor of nucleoside uptake. These data suggest that 8Cl-cAMP must be converted to 8Cl-AD and that 8Cl-AD is the compound that enters the cell. Contrary to glucocorticoid-mediated cell death in myeloma cells, the pathway of 8Cl-AD–mediated cell death appears to be independent of interleukin-6 (IL-6) actions. Although the exact mode of action for this agent is currently unknown, its ability to kill steroid sensitive and insensitive multiple myeloma cells in an IL-6 independent fashion may offer exciting new therapeutic options. © 1998 by The American Society of Hematology.

Description: 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.

Description: Cutaneous T-Cell Lymphomas (CTCL) represent a heterogeneous group of hematopoietic malignancies that account for 5-10% of Non-Hodgkin Lymphomas. The initial disease presentation is characterized by the infiltration of the skin by malignant clonal CD4+ lymphocytes that possess a mature memory helper T-cell phenotype. CTCL patients present in a spectrum of defined disease phenotypes, Mycosis Fungoides (MF) and Sézary Syndrome (SS) being the most common. Thus, it is not surprising that no underlying molecular basis for disease has been identified. Standard treatment protocols are designed to provide palliation, as no chemotherapeutic compound(s) has demonstrated increased long-term or disease-free survival. Currently, no curative therapy for CTCL exists. Our long-term goal is to gain a better understanding of the pathways governing CTCL cell survival and proliferation while identifying druggable targets for the development of new and more effective therapeutics. The principles of chemical biology, to discover and elucidate molecular pathways fundamental in cellular, developmental, and disease biology through synthetic organic chemistry, are readily applied to the field drug discovery. Using these methods, we have previously observed that inhibition of protein kinase C (PKC) β with the small molecule Enzastaurin (Enz), combined with inhibition of glycogen synthase kinase 3 (GSK3) with AR-A014418 (ARA), causes synergistic apoptosis in CTCL cell lines (Hut78 and H9) and patient cells. Further investigations have shown combined treatment with Enz and ARA increased β-catenin protein levels and that β-catenin downstream transcription activation negatively impacts CTCL viability. However, β-catenin alone is not sufficient to induce CTCL apoptosis. These data indicate there are additional mechanisms of cell death stimulated by the combined inhibition of PKC and GSK3. To identify the β-catenin independent targets of combined PKC and GSK3 inhibition, we have assayed drug-treatment induced changes in global gene expression through a microarray approach. We have identified transcripts that are significantly regulated after combined inhibition of PKC and GSK3 with Enz, ARA, and a combination of both small molecules. We are focusing our efforts on those targets and signaling pathways that are minimally regulated by each drug alone (or no change at all), but are dramatically impacted by the combination treatment to identify mechanisms of synergy. Using Gene Set Enrichment Analysis (GSEA), we noted enrichment in two pathways that are key to T-cell viability, namely the T-cell receptor (TCR) pathway and interferon response pathway. To elucidate candidate genes that may have therapeutic importance and to further explore the clinical relevance of our data set, in a pilot study, we interrogated the gene expression profile of a set of CTCL patients normalized to normal CD4+ T-cells. We then selected those genes whose expression is negatively correlated between CTCL patients and drug treated CTCL cell lines. From this pilot study, we have identified members of the TCR pathway that are over-expressed in CTCL patients and strikingly down-regulated by combined inhibition of PKC and GSK3. Taken together, these observations prompted us to further explore genes of the TCR signaling cascade as potential therapeutic targets. These data will provide the basis for downstream target identification for further drug development. Disclosures: No relevant conflicts of interest to declare.

Description: We have examined the cytotoxic effects of cyclic adenosine-3′,5′-monophosphate (cAMP) derivatives on multiple myeloma cells lines and determined that the 8-Chloro substituted derivative (8Cl-cAMP) is one of the most potent. We report here that 8Cl-cAMP is cytotoxic to both steroid sensitive and insensitive myeloma cells with a half maximal concentration of approximately 3 μmol/L. 8Cl-cAMP toxicity in myeloma cells is dependent on phosphodiesterase activity in the serum of cell culture medium. A metabolite of 8Cl-cAMP, 8-Chloro-adenosine (8Cl-AD), kills myeloma cells as effectively as 8Cl-cAMP. Adenosine deaminase (ADA) converts 8Cl-AD into 8Cl-inosine and abrogates the cytotoxic effects of 8Cl-cAMP, 8Cl-AMP, and 8Cl-AD, as does 5-(p-Nitrobenzyl)-6-Thio-Inosine (NBTI), an inhibitor of nucleoside uptake. These data suggest that 8Cl-cAMP must be converted to 8Cl-AD and that 8Cl-AD is the compound that enters the cell. Contrary to glucocorticoid-mediated cell death in myeloma cells, the pathway of 8Cl-AD–mediated cell death appears to be independent of interleukin-6 (IL-6) actions. Although the exact mode of action for this agent is currently unknown, its ability to kill steroid sensitive and insensitive multiple myeloma cells in an IL-6 independent fashion may offer exciting new therapeutic options. © 1998 by The American Society of Hematology.