ALBERT

Description: Multiple Myeloma (MM) is a malignancy of monoclonal plasma cell proliferation in the bone marrow, which accounts for 10% of all hematologic cancers. This slow proliferating B-cell malignancy accumulates apoptosis-resistant and replication-quiescent cell populations, posing a challenge for current chemotherapeutics, which target rapidly replicating cells. MM remains an incurable disease in need of new therapeutic approaches. We have previously shown that the nucleoside analog, 8-chloro-adenosine (8-Cl-Ado), has significant activity for MM and that its sister compound, 8-amino-adenosine (8-NH2-Ado), appears to be even more potent, exhibiting greater activity in pre-clinical studies. 8-NH2-Ado is cytotoxic to MM cell lines both sensitive and resistant to conventional chemotherapies. This cytotoxic effect requires phosphorylation of 8-NH2-Ado to its tri-phosphate form, 8-NH2-ATP, and accumulation of 8-NH2-ATP results in a concomitant loss of endogenous ATP levels. 8-NH2-Ado induces apoptosis in MM cells 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 subG1 DNA content. Here we demonstrate the novel effect of 8-NH2-Ado on the phosphorylation status of key cellular signaling molecules. MM cells treated with 8-NH2-Ado exhibit a dramatic loss of phosphorylation of several important signaling proteins, including ERK1/2, p38 MAPK and Akt kinase, within 30–120 minutes. Cells depleted of ATP by antimycin A or 2-deoxyglucose treatments to mimic 8-NH2-Ado-induced ATP depletion 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 be affecting the activity of protein phosphatases involved in the negative regulation of these signaling molecules. Based on studies using the phosphatase inhibitor okadaic acid, it appears that PP2A may play a role in the 8-NH2-Ado-induced decrease in phosphorylation of p38. The distinctive effect of 8-NH2-Ado on the phosphorylation status of cellular proteins is a novel phenomenon for a nucleoside analog drug and appears to be unique to 8-NH2-Ado among this class of drugs. The kinetics of 8-NH2-Ado-mediated changes in phosphorylation levels of critical pro-survival and apoptosis-regulating proteins suggest that the modulation of these proteins by de-phosphorylation at early time-points may be an important mechanistic step in 8-NH2-Ado-induced programmed cell death.

Description: Multiple Myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of terminally differentiated plasma cell in the bone morrow, which accounts for 10% of all hematological cancers. Despite major advance of last decade, MM remains an incurable disease in need of new therapeutic approaches. Leflunomide was originally developed as an immunosuppressive drug capable of alleviating the severity of autoimmune disease and preventing allograft and xenograft rejection and it has been FDA approved to use in the treatment of rheumatoid arthritis (RA) since 1998. It rapidly converted in gastrointestinal tract and plasma to open ring metabolite A771726. In current study, we examine the effects of A771726 to the growth of MM cell lines in vivo and show that A771726 inhibits the proliferation of four different well-characterized MM cell lines (MM.1, U266, RPMI8226 and MDR10V) in a dose- and time- dependent manner, regardless their sensitivity to the conventional chemotherapies. Cell cycle analyses further demonstrate that G1-S phase transition is blocked by A771726 in those cells. Moreover, we find that, at higher concentration, A771726 is able to induce apoptosis in at least two of the cell lines tested as measured by an increase of the cells with subG1 DNA content, an increasing in caspase activity and cleavage of caspase substrate. Previous studies suggested that A771726 exerts its anti-proliferate function mostly by inhibiting de novo pyrimidine synthesis in lymphocyte and supplementation of the cultures with exogenous uridine could prevent A771726-induced cell cycle block by restoring the intracellular UTP and CTP lever. However, we find that additions of exogenous uridine can prevent neither A771726-induced cell cycle block nor apoptosis in the MM cell lines tested, suggesting a novel mechanism. It shall be noted that pharmacologically, the highest concentration of A771726 (200uM) used in our study is readily achievable in the plasma through current drug regiment of Leflunomide in RA patients. Since its approval, Leflunomide has been proved to be an effective antirheumatic drug with mild side effects. Given the results from current study, we are tempting to suggest that Leflunomide might also provide a safe alternative in the treatment of MM. The vast knowledge gained from clinical use of Leflunomide in last six year will greatly help us greatly in testing this hypothesis in a clinical setting in a near future.

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: Glucocorticoid (GC) is an effective anti-myeloma agent by directly inducing apoptosis and growth inhibition in myeloma cells. The anti-myeloma action of GC is mediated by its intracellular receptor, the glucocorticoid receptor (GR). The response to GC is variable among myeloma patients and resistance to glucocorticoid treatment develops almost invariably. The mechanism regulating glucocorticoid-sensitivity in not well understood, although it has been hypothesized that the regulation might occur at both the receptor (GR) and post-receptor level. In MM.1 myeloma cell lines, we observed significant down-regulation of GR expression as the cells develop GC-resistance, suggesting that GR expression level is an important determinant of GC-response. Here we present evidence that GR expression level directly correlates with the clinical outcome in myeloma patients who were treated with GC-containing regimens. First, using a quantitative real-time PCR analysis, we performed a retrospective analysis of GR gene expression on bone marrow plasma cell samples from 35 patients with newly diagnosed myeloma who were then treated on the ECOG study E1A00. This is a phase 3 trial which randomized patients to dexamethasone (dex) alone vs. dex in combination with thalidomide. Pre-treatment expression levels of GR-alpha, GR-beta and GR-P splicing isoforms were measured for 35 patients, of whom, 10 had post-treatment samples available. Interestingly, a large variation of the GR expression levels was observed across the patient samples, with the GR-alpha being the predominant isoform. However, a clear correlation between GR expression and treatment response was not seen, likely due to the small sample size. To access a larger patient pool, we retrospectively analyzed the GR expression levels using the cDNA microarray data from the Arkansas group on 351 newly diagnosed myeloma patients treated with Total Therapy 2, of which dexamethasone was an essential component throughout the treatment course. Of the 351 patients, both event-free (EFS) and overall survivals (OS) were compared between 49 patients (14%) with low levels of GR expression and the rest (86%) with high levels of GR expression. Strikingly, the cases with low levels of GR expression were associated with statistically significantly worse EFS and OS compared to those with high levels of GR expression, with the 5-year EFS of 20% vs. 52% and OS of 34% vs. 68%, P 〈 0.01. In summary, we have shown for the first time that the baseline level of glucocorticoid receptor expression in newly diagnosed myeloma patients predicts their clinical response to GC-containing regimen and the overall clinical outcome.

Description: Abstract 2755 Poster Board II-731 Cutaneous T cell lymphomas (CTCL) represent a spectrum of several distinct extranodal non-Hodgkin's lymphomas and are characterized by an invasion of the skin by malignant, clonal CD4+ lymphocytes. Although current treatments may achieve remission, early chemotherapeutic treatment of CTCL has not demonstrated survival benefits and no treatments have proven to be curative. Our lab has previously demonstrated that treatment with the Protein Kinase C (PKC) inhibitor Enzastaurin increases apoptosis in CTCL which directly led to the clinical trial of Enzastaurin for CTCL. PKCs phosphorylate a number of substrates important in modulating cell survival, including Bcl-2 family members. To investigate the effects of PKC signaling on Bcl-2 family member levels in CTCL we treated cells with Enzastaurin and measured downstream effects. Inhibition of PKC signaling by Enzastaurin led to a decrease of Bcl-xL protein levels but not Bcl-2 levels. Apoptosis was assessed by flow cytometry and cells cultured in Enzastaurin showed an increase in cell death which correlated with the decrease in anti-apoptotic Bcl-2 family member levels. Enzastaurin treatment also resulted in a decrease of both Bad and Bcl-2 phosphorylation as well as a decrease in Bcl-xL but not Mcl-1 or Bcl-2 mRNA levels. To determine if PKC modulates Mcl-1 levels through phosphorylation and inactivation of GSK3β in CTCL, cells were exposed to Enzastaurin for 24 hours and immunoblots were performed for Mcl-1, total GSK3β and phosphor-Ser9 GSK3β. Enzastaurin blocked basal phosphorylation as well as TPA-induced phosphorylation of GSK3β. However, Mcl-1 levels were not affected by Enzastaurin. This suggests that PKC modulates GSK3β activity but that this is independent of Mcl-1. To determine if PKC negative regulation of GSK3β inhibits apoptosis we treated cells with Enzastaurin and the GSK3β inhibitor ARA014418. Surprisingly, the addition of the GSK3β inhibitor did not reverse the effects of Enzastaurin but instead increased apoptosis. This suggests that PKC does not deliver survival signals in CTCL by negatively regulating GSK3β. Treatment with Enzastaurin alone as well as treatment with both Enzastaurin and GSK3β inhibitors resulted in an upregulation of β catenin. However, a higher percentage of β catenin was unphosphorylated when both Enzastaurin and the GSK3β inhibitor were added. Interestingly, the combination of both inhibitors resulted in a dramatic increase in β catenin / TCF transcription as measured by TOPGLOW luciferase assay. The downregulation of Bcl-2 family member levels by PKC inhibition strongly suggests that PKC modulation of Bcl-2 family members is important for CTCL survival. PKC may be affecting Bcl-2 family members by modulating phosphorylation or by indirectly activating or repressing transcription. Additionally, β catenin upregulation may play a role in apoptosis mediated by inhibition of PKC. β catenin TCF/LEF complexes can activate transcription of several genes linked to apoptosis, including c-Jun, fra-1, p53 and Myc. β catenin may also be functioning to repress transcription of Bcl-2 family members such as Bcl-xL. Elucidating the underlying mechanism will help to identify new drug targets for CTCL which may be applicable to a broad spectrum of malignancies. Disclosures: Rosen: Eli Lilly: Consultancy.

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.