Lenushka Maharaj

Bortezomib Therapy in Patients With Relapsed or Refractory Lymphoma: Potential Correlation of In Vitro Sensitivity and Tumor Necrosis Factor Alpha Response With Clinical Activity

PURPOSE To determine the efficacy of bortezomib in patients with lymphoid malignancy, correlating clinical response with effect on plasma cytokines and in vitro activity in primary cultures. PATIENTS AND METHODS Patients received bortezomib (1.3 mg/m2) on days 1, 4, 8, and 11 of a 3-week cycle. Plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-6 were measured before each treatment, and bortezomib activity was examined in patient samples grown in primary culture. RESULTS Fifty-one patients received a total of 193 cycles of treatment. Twenty-four patients had mantle cell lymphoma (MCL), 13 had follicular lymphoma (FL), six had lymphoplasmacytic lymphoma, six had Hodgkins disease (HD), and one each had diffuse large B-cell lymphoma and adult T-cell leukemia/lymphoma. Patients were heavily pretreated with a median of four previous therapies. Significant grade 3 to 4 toxicities were thrombocytopenia (n = 22), fatigue (n = 10), and peripheral neuropathy (n = 3). Seven patients with MCL responded to treatment (one complete response, six partial responses [PRs]; overall response rate, 29%). Two patients with FL achieved a late PR 3 months after discontinuing therapy. Two patients with Waldenströms macroglobulinemia and one patient with HD achieved a PR. MCL primary cultures demonstrated greater sensitivity to bortezomib than FL (median 50% effective concentration for viability, 209 nmol/L v 1,311 nmol/L, respectively; P = .07), which correlated with clinical response. A median reduction in plasma TNF-alpha of 98% was observed in six patients with MCL who responded to bortezomib compared with a reduction of 38% in six nonresponders (P = .07). CONCLUSION Bortezomib demonstrates encouraging efficacy in MCL in heavily pretreated individuals. Response was associated with a reduction in plasma TNF-alpha and in vitro sensitivity in a small number of patients.

The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines.

Bortezomib is a proteasome inhibitor with proven efficacy in multiple myeloma and non-Hodgkins lymphoma. This study reports the effects of bortezomib in B-cell lymphoma cell lines with differing sensitivity to bortezomib to investigate factors that influence sensitivity. Bortezomib induced a time- and concentration-dependent reduction in cell viability in five lymphoma cell lines, with EC(50) values ranging from 6 nmol/L (DHL-7 cells) to 25 nmol/L (DHL-4 cells) after 72 h. Bortezomib cytotoxicity was independent of p53 function, as all cell lines exhibited mutations by sequence analysis. The difference in sensitivity was not explained by proteasome or nuclear factor-kappaB (NF-kappaB) inhibition as these were similar in the most and least sensitive cells. NF-kappaB inhibition was less marked than that of a specific NF-kappaB inhibitor, Bay 11-7082. Cell cycle analysis showed a marked G(2)-arrested population in the least sensitive DHL-4 line only, an effect that was not present with Bay 11-7082 treatment. Conversely, in DHL-7 cells, bortezomib treatment resulted in cells moving into an aberrant mitosis, indicative of mitotic catastrophe that may contribute to increased sensitivity to bortezomib. These studies show that although bortezomib treatment had similar effects on apoptotic and NF-kappaB signaling pathways in these cell lines, different cell cycle effects were observed and induction of a further mechanism of cell death, mitotic catastrophe, was observed in the more sensitive cell line, which may provide some pointers to the difference in sensitivity between cell lines. An improved understanding of how DHL-7 cells abrogate the G(2)-M cell cycle checkpoint may help identify targets to increase the efficacy of bortezomib.

P110α-mediated constitutive PI3K signaling limits the efficacy of p110δ-selective inhibition in mantle cell lymphoma, particularly with multiple relapse

Phosphoinositide-3 kinase (PI3K) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis, but early-phase studies of the PI3K p110δ inhibitor GS-1101 have reported inferior responses in MCL compared with other non-Hodgkin lymphomas. Because the relative importance of the class IA PI3K isoforms p110α, p110β, and p110δ in MCL is not clear, we studied expression of these isoforms and assessed their contribution to PI3K signaling in this disease. We found that although p110δ was highly expressed in MCL, p110α showed wide variation and expression increased significantly with relapse. Loss of phosphatase and tensin homolog expression was found in 16% (22/138) of cases, whereas PIK3CA and PIK3R1 mutations were absent. Although p110δ inhibition was sufficient to block B-cell receptor-mediated PI3K activation, combined p110α and p110δ inhibition was necessary to abolish constitutive PI3K activation. In addition, GDC-0941, a predominantly p110α/δ inhibitor, was significantly more active compared with GS-1101 against MCL cell lines and primary samples. We found that a high PIK3CA/PIK3CD ratio identified a subset of primary MCLs resistant to GS-1101 and this ratio increased significantly with relapse. These findings support the use of dual p110α/p110δ inhibitors in MCL and suggest a role for p110α in disease progression.

GCS-100, a novel galectin-3 antagonist, modulates MCL-1, NOXA, and cell cycle to induce myeloma cell death

GCS-100 is a galectin-3 antagonist with an acceptable human safety profile that has been demonstrated to have an antimyeloma effect in the context of bortezomib resistance. In the present study, the mechanisms of action of GCS-100 are elucidated in myeloma cell lines and primary tumor cells. GCS-100 induced inhibition of proliferation, accumulation of cells in sub-G(1) and G(1) phases, and apoptosis with activation of both caspase-8 and -9 pathways. Dose- and time-dependent decreases in MCL-1 and BCL-X(L) levels also occurred, accompanied by a rapid induction of NOXA protein, whereas BCL-2, BAX, BAK, BIM, BAD, BID, and PUMA remained unchanged. The cell-cycle inhibitor p21(Cip1) was up-regulated by GCS-100, whereas the procycling proteins CYCLIN E2, CYCLIN D2, and CDK6 were all reduced. Reduction in signal transduction was associated with lower levels of activated IkappaBalpha, IkappaB kinase, and AKT as well as lack of IkappaBalpha and AKT activation after appropriate cytokine stimulation (insulin-like growth factor-1, tumor necrosis factor-alpha). Primary myeloma cells showed a direct reduction in proliferation and viability. These data demonstrate that the novel therapeutic molecule, GCS-100, is a potent modifier of myeloma cell biology targeting apoptosis, cell cycle, and intracellular signaling and has potential for myeloma therapy.

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.

Methylseleninic acid inhibits HDAC activity in diffuse large B-cell lymphoma cell lines

PurposeSelenium is a trace element that is fundamental to human health. Research has mainly focussed on its role in cancer prevention, but recent evidence supports its role in established cancer, with high concentrations inducing tumour cell death and non-toxic concentrations sensitising cells to chemotherapy. However, the precise mechanism of selenium action is not clear. The effect of methylseleninic acid (MSA), an organic selenium compound, on histone deacetylase (HDAC) activity in diffuse large B-cell lymphoma cell lines is reported here.MethodsLymphoma cell lines were exposed to MSA under normoxic and hypoxic conditions. Protein expression was determined by western blotting, HDAC activity and VEGF concentration by fluorimetric and electrochemiluminescence assays, respectively, and intracellular selenium metabolites quantified by mass spectrometry.ResultsMSA inhibited HDAC activity, which resulted in the acetylation of histone H3 and α-tubulin. However, cellular metabolism of MSA to methylselenol was required for this effect. Dimethylselenide, the methylation product of methylselenol, was found to be the major intracellular metabolite. MSA also inhibited HIF-1α expression and VEGF secretion, a possible consequence of HDAC inhibition.ConclusionThe ability of methylselenol to inhibit HDAC activity has not been previously reported, thus providing a novel mechanism of selenium action.

Schedule dependent cytotoxicity of bortezomib and melphalan in multiple myeloma

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The histone deacetylase inhibitor UCL67022 has potent activity in multiple myeloma and non-Hodgkin lymphoma pre-clinical models.

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Abstract 4489: The activity of novel, potent, selective PI3K/mTOR pathway inhibitors in B-cell malignancies

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Activation of the PI3K pathway is implicated in a number of tumour types, including haematological malignancies. Agents that target this pathway at different levels (e.g. RTK, PI3K, mTOR etc) have shown promising activity in preclinical models and in early phase trials. We have therefore investigated the activity of novel, potent, pan- and isoform-selective PI3K pathway inhibitors in lymphoma and myeloma models. Methods: Compounds studied included those with selectivity for mTOR (INK128, mTOR IC50 1.6 nM), PI3Kδ/γ (INK713, INK1048, IC50s 50 nM for others) and PI3Kδ/γ/β (INK1138, IC50s <10 nM for each isoform). IC87114 (δ- selective), GDC-0941 (α/δ selective), rapamycin and doxorubicin were included as comparators. Agents were studied in a panel of lymphoma and myeloma cell lines, in normal PBMCs and in primary tumours cultured with stromal cells. Effects on cell viability and proliferation were assessed using the Guava Viacount assay, on apoptosis induction by annexin V labelling, and on cell cycle distribution by flow cytometry. PI3K pathway activity and inhibition was determined by Western blotting as well as mass spectrometry. Results: In cell lines, inhibition of mTOR by INK128 resulted in potent inhibition of cell proliferation, in many at concentrations as low as 10 nM. Selective PI3K inhibitors were less potent anti-proliferative agents, although activity was seen in cells with activation of the PI3K pathway as determined by western blot analysis. For all compounds tested, the major impact was on cell proliferation rather than cell viability. PI3K inhibitors showed little effect on cell viability at concentrations < 1µM. The effect of these compounds in primary mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukaemia and myeloma samples was variable, both between and within tumour types, possibly due to differences in basal PI3K pathway activity. Effects in normal PBMCs were markedly different to those seen in tumour cells, with a concentration-dependent increase in cell number with either mTOR or PI3K inhibition. Conclusion: Selective PI3K pathway inhibition, particularly at the level of mTOR, results in cytostatic, and at higher concentrations cytotoxic, responses in haematological malignancies. The variable activity of these compounds between cell lines and primary samples suggests it will be important to characterise the activity of the PI3K pathway in individual tumours to identify patients likely to benefit from such agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4489.

Abstract 3651: Methylseleninic acid (MSA) is an HDAC inhibitor and suppresses VEGF production in lymphoma cell lines

Background: Serum selenium (Se) concentration at presentation is prognostic of outcome in patients with aggressive B-cell lymphoma (Last et al., JCO, 2003). We have shown that the Se compound MSA enhanced the efficacy of chemotherapeutic agents at non-cytotoxic concentrations in lymphoma cell lines (Juliger et al., Can Res, 2007). Others have reported that Se enhanced the anti-tumour activity of cytotoxic agents, but reduced toxicity to normal tissue in a xenograft model (Cao et al., Clin Can Res, 2004). In addition, Se has been reported to have antiangiogenic properties including the reduction of tumour microvessel density (Wang et al., Int J Can, 2008). We have therefore investigated the effect of MSA on histone deacetylases (HDACs) and HIF1α, the latter being in part regulated by HDAC activity. Methods: Lymphoma cell lines (RL, SUD4, DHL4) and peripheral blood mononuclear cells (PBMCs) were exposed to MSA in concentration and time-course experiments in normoxic and hypoxic conditions (1% O2). Changes in protein expression were determined by western blotting, HDAC activity measured using an HDAC fluorimetric assay (Biomol), and VEGF levels in cellular supernatants determined using an electrochemiluminescence assay (Meso Scale Discovery). Results: In lymphoma cell lines, exposure to MSA concentrations ≥5μM for 24 hours resulted in acetylation of histone H3 and induction of p21 protein. Acetylation of α-tubulin (mediated by HDAC6, a class II HDAC) occurred to a lesser extent. MSA did not inhibit HDAC activity in an isolated enzyme assay using HeLa cell nuclear extract. However, in whole cells pre-incubated with MSA HDAC activity was inhibited in a concentration-dependent manner. In DHL4 cells, exposure to 10μM and 30μM MSA for 2 hours inhibited HDAC activity by 34% (p=0.01 cf control) and 50% (p=0.02) respectively. 24 hour exposure to MSA concentrations ≥5μM markedly increased histone H3 acetylation in PBMCs. These results suggest that a cellular metabolite of MSA inhibits HDAC activity in intact cells. The effect of MSA on HIF1α expression and VEGF secretion was then investigated. In DHL4 cells, exposure to hypoxia for 24 hours induced HIF1α expression (1.8 fold), an effect not seen in the presence of 20µM MSA. In addition, hypoxia increased VEGF levels to 157% of basal levels, but this was reduced to 43% by 20μM MSA (p=0.007). Conclusions: At clinically relevant concentrations, a cellular metabolite of MSA inhibits HDAC activity and suppresses HIFα induction and VEGF secretion. This effect may account for some of the observed pre-clinical activity with Se species, particularly in animal models. The mechanism of HDAC inhibition is now being investigated. In addition, histone H3 acetylation in PBMCs may serve as a biomarker of Se activity in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3651.