ALBERT

Description: We have previously shown that elevated homologous recombination (HR) activity mediates genomic instability and progression in myeloma. Moreover, elevated HR also plays critical role in tumor growth by contributing to telomere maintenance and other survival mechanisms. We have now investigated molecular mechanisms driving dysregulated HR in MM. We observe that elevated apurinic apyrimidic endonuclease 1 (APE1) significantly contributes to dysregulation of HR, directly through transcriptional control of RAD51 as well as indirectly through its ability to induce DNA breaks. The transgenic suppression using APE1-specifc shRNA inhibits RAD51 expression, HR activity, and genomic instability as measured by SNP array profile in MM cells; whereas its induction leads to increased RAD51 expression, HR activity, genomic instability and oncogenic transformation in normal human cells. We have further investigated how APE1, a base excision repair protein, regulates RAD51, the key component of HR in myeloma and evaluated a novel small molecule inhibitor of APE1 for its impact on HR and associated genomic instability. Using an antibody array we observed that APE1 physically interacts with p73, a known transcriptional regulator of RAD51. To demonstrate that APE1 and P73 interact with RAD51 promoter in MM cells, we conducted chromatin immunoprecipitation (chip) assays and observed both P73 and APE1 binding to adjacent loci on RAD51 promoter. Taken together, these data suggest that elevated APE1 induces RAD51 expression through its interaction with P73. We next evaluated effect of a small molecule inhibitor specifically targeting nuclease function of APE1 in MM cells, and observed that it inhibits RAD51 expression, RAD51 foci, HR activity and reduces DNA breaks as assessed by g-H2AX levels on western blotting. The suppression of APE1 by this small molecule was associated with significant loss of RAD51 promoter activity, as assessed by a RAD51-promoter driven luciferase construct, as well as reduced RAD51 transcript levels. As APE1 is required for DNA repair which plays a critical part in development of drug resistance, we evaluated if APE1 inhibitor can help sensitize MM cells to DNA damaging agents. To investigate this we pretreated RPMI8226 and LR5 MM cells with the small molecule inhibitor of APE1 and then exposed them to various concentrations of melphalan for 48 hrs and cell viability and growth assessed. Pretreatment with APE1 inhibitor not only sensitized RPMI8226 cells to melphalan but also resistant LR5 cell line. These observations suggest that elevated APE1 is a critical target to induce DNA damage or overcome certain type of resistance possibly driven by repair mechanisms. In summary, we conclude that elevated APE1 is a critical intermediate for dysregulated HR and associated genomic instability, and small molecule inhibitor of APE1 has potential to reduce genomic instability, prevent/delay progression and improve clinical outcome in MM. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2967 Activin-A is a member of the transforming-growth factor-beta superfamily most commonly associated with embryogenesis and gonadal hormone signaling. Activin-A is also involved in bone homeostasis having growth stimulatory effects on osteoclasts, but unclear effects on osteoblast function. A recent study showed that, in multiple myeloma (MM) microenvironment, activin-A is a stromally derived osteoblast inhibitor induced by myeloma cells and thus it seems to be implicated into the pathogenesis of myeloma bone disease (Vallet et al, PNAS 2010;107:5124). The aim of this study was to evaluate the circulating levels of activin-A in newly-diagnosed and relapsed patients with MM and explore possible correlations with clinical and laboratory data, including lytic bone disease and survival. We studied 113 MM patients (63M/50F, median age 69 years, range 31–92 years): 98 newly-diagnosed patients (13 with asymptomatic and 85 with symptomatic MM) and 15 patients with relapsed MM after previous response to front-line therapy, who received the combination of lenalidomide and high-dose dexamethasone (RD). All symptomatic patients received frontline treatment with novel agent-based regimens. For newly-diagnosed patients, serum was stored at -80°C at the time of diagnosis, while for relapsed patients serum was stored on day 1 of the first RD cycle and then on day 28 of the 4th cycle. Serum activin-A was measured using ELISA methodology (R&D Systems, Minneapolis, MN, USA) along with a series of markers of bone resorption (C-terminal cross-linking telopeptide of collagen type-1, CTX and tartate-resistant acid phosphatase isoform-5b, TRACP-5b) and bone formation (bone-specific alkaline phosphatase and osteocalcin). Evidence of bone involvement was documented using plain radiographs in patients at diagnosis and at the time of relapse. Activin-A and the above bone markers were also measured in 10 MGUS patients and in 17, gender- and age-matched, healthy controls. Circulating levels of activin-A of newly-diagnosed patients with symptomatic MM (median: 555 pg/mL, range: 129–2336 pg/mL) and of relapsed patients (677 pg/mL, 272–2088 pg/mL) were increased compared to controls (393 pg/mL, 204–899 pg/mL; p

Description: Abstract 1815 Poster Board I-841 Bortezomib is the first-in-class proteasome inhibitor with established activity in multiple myeloma (MM), which also increases osteoblast function both in vitro and in vivo. The addition of bortezomib to the combination of lenalidomide and dexamethasone (RD) regimen seems to increase the RD efficacy. There are very limited data in the literature for the effect of RD on bone metabolism while there are no reports for the effect of BRD on myeloma bone disease. The aim of this study was to evaluate the effect of BRD and RD on bone remodeling of patients with relapsed/refractory MM. We studied 91 patients who participated in a prospective study in which patients with pre-existing peripheral neuropathy (PN) grade

Description: Abstract 2963 Dickkopf-1 (Dkk-1) and sclerostin are inhibitors of the Wingless-type and integrase 1 (Wnt) signaling and they are implicated in the pathogenesis of multiple myeloma (MM) bone disease through inhibition of osteoblast function. There is very limited information for the circulating levels of Dkk-1 and sclerostin in different phases of MM and their alterations post therapies with novel agents. Therefore, we studied 284 MM patients (153M/131F, median age 66 years): 167 consecutive patients were newly-diagnosed (20 had asymptomatic MM and 147 symptomatic MM), 29 patients were at the plateau phase of MM and 88 patients had relapsed/refractory MM and received therapy with the combination of lenalidomide plus dexamethasone with or without bortezomib (VRD or RD; Dimopoulos et al, Leukemia 2010). For newly diagnosed patients, serum was stored at the time of diagnosis, while for patients at the plateau phase serum was collected at the time of confirmation of the plateau (at least 6 months with stable M-protein without criteria confirming progression) and for relapsed/refractory patients on day 1 of cycles 1, 4 and 7 of VRD or RD administration. Circulating levels of Dkk-1 and sclerostin were measured using ELISA methodology (R&D Systems, Minneapolis, MN, USA and Biomedica Medizinprodukte, Vienna, Austria, respectively) in all patients and in 20 gender- and age- matched healthy controls. Circulating Dkk-1 and sclerostin concentrations of newly diagnosed symptomatic patients (median: 1383 pg/mL, range:274-32, 862 pg/mL and 415 pg/mL, 0–3,340 pg/mL respectively) were increased compared to controls (1069 pg/mL, 540-2, 709 pg/mL; p

Description: Epigenomic changes have become an important component of cellular regulation and ultimately, of our understanding of oncogenomics in Multiple Myeloma (MM) as well as in other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we have evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM. First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (p

Description: Abstract 299 The aim of this study was to evaluate epigenetic modifications and alterations in cellular DNA damage response pathways that may be implicated in the multistep transformation of myelomagenesis. Peripheral blood mononuclear cells (PBMCs) and plasma cells from bone marrow aspirates were collected from 15 patients with MGUS (8M/7F), 22 with asymptomatic MM (AMM; 10M/12F), 41 patients with symptomatic MM (16M/25F) who underwent autologous stem cell transplantation as part of their first line therapy, and 12 healthy volunteers (7M/5F; only PBMCs). Epigenetics (chromatin condensation, transcription activity) and DNA damage response pathways (melphalan-induced DNA damage formation/repair in four genomic loci including beta-actin, p53, N-ras and delta-globin genes, accumulation of p53 protein and induction of apoptosis) were evaluated. In both PBMCs and plasma cells and in all genomic regions analyzed, significant differences in the local chromatin looseness between the different groups of patients were observed: healthy volunteers

Description: Abstract 3035 Data from patients with multiple myeloma (MM) treated with autologous transplantation indicate that a short remission period after high dose therapy, usually less than 12 months, is associated with a poor outcome and limited efficacy of salvage regimens. Also, failure to respond to upfront thalidomide or lenalidomide based-regimens is associated with poor outcome. However, such data are limited for the general, unselected population of elderly or non-transplant treated MM patients, especially after the introduction of novel agents in the upfront treatment of myeloma. Thus, we analyzed the outcome of 115 unselected patients, who were older than 65 years and who were treated upfront with novel agent-based regimens in a single center in Athens, Greece. Many of these patients had been included in clinical trials; however, several patients who were ineligible because of poor performance status, significant renal impairment or comorbidities were also treated with novel agent-based regimens. Thus, these patients are more representative of the general myeloma population. IMWG criteria were used for the assessment of response, progression-free (PFS) and overall survival (OS). Forty three percent of patients were 〉75 years. Nine percent died within 3 months from initiation of treatment. Among 106 patients who survived for at least 3 months, 77% achieved an objective response: 20% achieved a CR, 26% a VGPR and 33% a PR. On an intention to treat, 58% of patients have progressed so far. Among patients who responded to initial treatment, 19% who had achieved a CR, 27% who had achieved a VGPR and 37% who had achieved a PR, relapsed(p=0.012). At the time of relapse or progression, patients were treated again with novel agents. Median PFS for all patients was 21 months (95% CI: 18–23 months), while median PFS for patients who achieved CR/VGPR or PR as best response was 20 months and 22 months, respectively (p=0.9). Subsequently, we analyzed 94 patients who started treatment at least 12 months before this analysis. Among these patients, 32% had a PFS shorter than 12 months including 26% of patients who initially responded. Depth of response was predictive of probability for early relapse: CR 5%, VGPR 29% and PR 38% (p=0.04). In the univariate analysis, LDH ≥300 IU/L (upper limit of normal 225 IU/L) was the only factor associated with shorter PFS while other factors such as ISS stage, age 〉75 years, Hb

Description: Abstract 2969 LDH is elevated in approximately 10% of patients with newly-diagnosed, symptomatic, multiple myeloma (MM). In patients treated with conventional chemotherapy, elevated LDH is associated with poor outcome (Dimopoulos et al, Ann Intern Med 1991). In selected patients treated with combinations of novel agents and tandem transplantation, LDH is an independent prognostic factor and is associated with short event free and overall survival even when cytogenetic abnormalities and gene expression signature are considered (Barlogie et al, J Clin Oncol 2010). An analysis from our group has also suggested that LDH improves the prognostic value of the International Scoring System (ISS) for MM (Terpos et al, Eur J Hematol 2010). However, there is limited data about the prognostic importance of elevated LDH in unselected MM patients who have been treated upfront with novel agent-based regimens. To address this issue, we analyzed 180 consecutive, unselected patients from a single center in Athens, Greece, who were treated upfront with novel agent (thalidomide, bortezomib or lenalidomide)-based therapies. Many of these patients had been included in clinical trials; however, several patients who were ineligible because of poor performance status, significant renal impairment or comorbidities were also treated with novel agent-based regimens. Thus, these patients are more representative of the general myeloma population. Fifty-one per cent of our patients were older than 70 years of age. LDH was elevated (≥300 IU/L, upper limit of normal 225 IU/L) in 8% patients before initiation of treatment. Elevated LDH was associated with features of advanced disease such as ISS-3 stage (p=0.063) and impaired renal function (p=0.034). The objective response rate in patients with elevated LDH was 47% and in patients without elevated LDH was 79% (p=0.011). The median overall survival for all patients was 54 months. Elevated LDH was associated with poor outcome (median survival 21 months vs. 56 months for patients with LDH 70 years (p

Description: Abstract 2974 Multiple myeloma (MM) is a plasma cell malignancy which progresses from a “pre-malignant” precursor condition, the monoclonal gammopathy of undetermined significance (MGUS). Asymptomatic MM (AMM) is characterized by the absence of any myeloma related end-organ damage. The risk of progression of AMM to symptomatic MM is approximately 10% per year, while the risk of progression of MGUS is 1% per year. Previous work from our group showed that melphalan-induced DNA damage, formation/repair in the peripheral blood mononuclear cells (PBMCs) was associated with response and progression-free survival in MM patients who underwent high dose melphalan (HDM) with autologous stem cell transplantation (ASCT). The aim of this study was to compare DNA damage response pathways in the PBMCs of patients with MGUS, AMM, symptomatic MM and healthy controls. Three molecular end-points (chromatin condensation, transcription activity, melphalan-induced DNA damage formation/repair) were measured in PBMCs in four genomic loci (beta-actin, p53, N-ras and delta-globin genes). Furthermore, accumulation of p53 protein, recovery of both total RNA and poly(A) mRNA synthesis as well as induction of apoptosis were also studied in the PBMCs from 12 healthy volunteers (7M/5F; median age: 41 years), 10 patients with MGUS (5M/5F; 68.5 years), 10 with AMM (4M/5F; 64 years), and 32 patients with symptomatic MM (14M/18F; 59 years) who underwent HDM and ASCT as part of their first line therapy. In all subjects, beta-actin, p53 and N-ras genes were transcriptionally active. Importantly, delta-globin gene was silent in all healthy volunteers and MGUS patients, while an induction of the transcription activity of this gene was found in all MM patients and in 90% of MM patients (29/32). In all subjects, more relaxed chromatin structure and faster repair were observed in regions inside beta-actin, p53 and N-ras genes, compared to regions on both sides of the genes, while for delta-globin such a difference was observed only in AMM (10/10) and symptomatic MM patients (29/32). In all subjects, 5′- to 3′-end gradients of chromatin condensation and repair efficiency were observed along the active genes, with higher looseness of chromatin structure and faster repair at the 5′-end. Interestingly, inside all genes analyzed, repair was much slower in healthy volunteers relative to MM patients; the difference was greater and statistically significant at the 5′-end of the genes (p

Description: Abstract 2976 The alkylating agent melphalan is one of the most active chemotherapeutic agents in the treatment of patients with multiple myeloma (MM). It reacts with DNA, producing mostly N-alkylpurine monoadducts, a small proportion of which goes on to form interstrand cross-links, which play a major role in cytotoxicity. In this report, we investigated the molecular mechanisms of therapeutic action and drug resistance to alkylating drugs using melphalan as a model. We studied 12 healthy volunteers (7M/5F; median age 41 years) and 32 MM patients (14M/18F; median age 59 years) who underwent high-dose melphalan (HDM) therapy with autologous stem cells transplantation (ASCT) as part of their first line therapy. These patients had measurable monoclonal protein in serum and/or urine after induction treatment so that further response after HDM could be assessed. Twenty-three patients achieved a further reduction of monoclonal protein after ACST (responders) and 9 patients did not (non responders). Blood samples were obtained from the healthy volunteers and the patients, within 1 week prior to ASCT and at least 1 month after exposure to any anti-myeloma treatment. Peripheral blood mononuclear cells (PBMCs) were isolated and treated with 10 μ g/mL of melphalan for 1 h at 37°C. Three molecular end-points (chromatin condensation, transcription activity, melphalan-induced DNA damage formation/repair) were measured in four genomic loci (beta-actin, p53, N-ras and delta-globin genes). Furthermore, accumulation of p53 protein, recovery of both total RNA and poly(A) mRNA synthesis as well as induction of apoptosis were also studied. In all subjects, beta-actin, p53 and N-ras genes were transcriptionally active. Importantly, delta-globin gene was silent in all healthy volunteers, while an induction of the transcription activity of this gene was found in 90% of MM patients (29/32). In all subjects, more relaxed chromatin structure and faster repair were observed in regions inside beta-actin, p53 and N-ras genes, compared to regions on both sides of the genes, while for delta-globin such a difference was observed only in MM patients. In all subjects, 5′- to 3′-end gradients of chromatin condensation and repair efficiency were observed along the transcribed strand of the active genes, with higher looseness of chromatin structure and faster repair at the 5′-end. Interestingly, inside all genes analyzed, repair was slower in responders relative to non-responders, the difference being greater and statistically significant at the 5′-end (p