[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is a malignant disorder of post-germinal center B cells, characterized by the clonal proliferation of malignant plasma cells (PCs) within the bone marrow (BM). The reciprocal and complex interactions that take place between the different compartments of BM and the MM cells result in tumor growth, angiogenesis, bone disease, and drug resistance. Given the importance of the BM microenvironment in MM pathogenesis, we investigated the possible involvement of Hypoxia-Inducible transcription Factor-1 alpha (HIF-1α) in the PCs-bone marrow stromal cells interplay. To test this hypothesis, we used EZN-2968, a 3rd generation antisense oligonucleotide against HIF-1α, to inhibit HIF-1α functions. Herein, we provide evidence that the interaction between MM cells and BM stromal cells is drastically reduced upon HIF-1α down-modulation. Notably, we showed that upon exposure to HIF-1α inhibitor, neither the incubation with IL-6 nor the co-culture with BM stromal cells were able to revert the anti-proliferative effect induced by EZN-2968. Moreover, we observed a down-modulation of cytokine-induced signaling cascades and a reduction of MM cells adhesion capability to the extracellular matrix proteins in EZN-2968-treated samples. Taken together, these results strongly support the concept that HIF-1α plays a critical role in the interactions between bone BM cells and PCs in Multiple Myeloma.
Experimental Cell Research 09/2014; · 3.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The increasing importance of hypoxia-inducible factor-1α (HIF-1α) in tumorigenesis raises the possibility that agents which specifically inhibit this transcription factor, would provide significant therapeutic benefit. The constitutive expression of HIF-1α in about 35% of Multiple Myeloma (MM) patients suggests HIF-1α suppression might be part of a therapeutic strategy. Accordingly, we explored the effect of EZN-2968, a small 3rd generation antisense oligonucleotide against HIF-1α, in a panel of MM cell lines and primary patients samples. Here, we demonstrated that EZN-2968 is highly specific for HIF-1α mRNA and that exposure of MM cells to EZN-2968 resulted in an efficient and homogeneous loading of the cells showing a long lasting low HIF-1α protein level. In MM cells, HIF-1α suppression induced a permanent cell cycle arrest by prolonging S-phase through cyclin A modulation and in addition it induced a mild apoptotic cell death. Moreover, HIF-1α suppression caused a metabolic shift that leaded to increased production of ATP by oxidative phosphorylation (i.e. Warburg effect reversion), that was confirmed by the observed mitochondrial membrane potential decrease. These results show that HIF-1α is an important player in MM homeostasis and that its inhibition by small antisense oligonucleotides provides a rationale for novel therapeutic strategy to improving MM treatment.
[Show abstract][Hide abstract] ABSTRACT: Bortezomib (bort)-dexamethasone (dex) is an effective therapy for relapsed/refractory (R/R) multiple myeloma (MM). This retrospective study investigated the combination of bort (1.3 mg/m(2) on days 1, 4, 8, and 11 every 3 weeks) and dex (20 mg on the day of and the day after bort) as salvage treatment in 85 patients with R/R MM after prior autologous stem cell transplantation or conventional chemotherapy. The median number of prior lines of therapy was 2. Eighty-seven percent of the patients had received immunomodulatory drugs included in some line of therapy before bort-dex. The median number of bort-dex cycles was 6, up to a maximum of 12 cycles. On an intention-to-treat basis, 55 % of the patients achieved at least partial response, including 19 % CR and 35 % achieved at least very good partial response. Median durations of response, time to next therapy and treatment-free interval were 8, 11.2, and 5.1 months, respectively. The most relevant adverse event was peripheral neuropathy, which occurred in 78 % of the patients (grade II, 38 %; grade III, 21 %) and led to treatment discontinuation in 6 %. With a median follow up of 22 months, median time to progression, progression-free survival (PFS) and overall survival (OS) were 8.9, 8.7, and 22 months, respectively. Prolonged PFS and OS were observed in patients achieving CR and receiving bort-dex a single line of prior therapy. Bort-dex was an effective salvage treatment for MM patients, particularly for those in first relapse.
Annals of Hematology 07/2013; · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AIM: The objective of this study was to analyze the prognostic value of F-FDG PET/CT after therapy in patients with multiple myeloma (MM). PATIENTS AND METHODS: One hundred seven patients prospectively recruited with MM had FDG PET/CT at staging 3 months after therapy (autologous stem cell transplantation) and every 6 to 12 months during the follow-up (mean 41 months). Patients were divided into group 1 (relapsed) and group 2 (nonrelapsed). In group 1, PET results and SUVmax were compared to the time to relapse (TTR). In group 2, the presence of PET finding changes during follow-up was analyzed to identify typical patterns of disease behavior (ie, late responders or stabilized disease). Patients with a negative PET at staging were excluded from further evaluation. RESULTS: Forty-seven out of 107 (44%) patients relapsed: 10 were excluded because of a negative PET at staging. In group 1, 22 patients had a negative posttherapy PET (59%, mean TTR = 27.6 months) and 15 had a positive posttherapy PET (41%, mean TTR = 18 months). There was a significant difference between the TTR of the two subgroups (t test P = 0.05). In patients with a positive posttherapy PET, the SUVmax was inversely correlated to the TTR (correlation coefficient = -0.7; P < 0.01).Sixty out of 107 (56%) patients did not relapse. Twenty patients were excluded because of a negative PET at staging. In group 2, 27 patients had a negative posttherapy PET (68%) and 13 had a positive posttherapy PET (32%). None of nonrelapsed patients showed a progressive increase in SUVmax during the follow-up. There was no significant difference between relapsed and nonrelapsed patients in terms of SUVmax at posttherapy PET/CT (t test P = 0.7). CONCLUSION: In our series of MM patients, a negative posttherapy PET was predictive for nonrelapse or a long disease-free survival. In contrast, a persistent significantly increased SUVmax after therapy was correlated to a short TTR.
Clinical nuclear medicine 11/2012; · 3.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The p53 tumor suppressor pathway is tightly kept in check or completely silenced in cancer cells. A potent inhibitor of p53 is MDM4, which is located on chr.1q: it is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) and autologous stem cell transplantation (ASCT) in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. CD138+ plasma cells obtained at diagnosis from 61 pts, subsequently treated with VTD and ASCT were analysed by means of GEP and unpaired analysis of CNA (Affymetrix U133 Plus2.0 and 6.0 SNP arrays). Nineteen out of 61 pts (31%) carried a minimal amplification region on chr.1q, harboring MDM4. Eight out of 61 pts (13%) carried a 482 Kb minimal deletion region on chr.17 harboring TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 24 pts) or the absence of both these abnormalities (group B, 37 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≤ 3 bone lesions in pts of group A. The rate of best complete or near complete response was 40% in group A and 14% in group B (p = 0.04). With a median follow-up of 36 months, the risk of progression was 46% for pts in group A and 19% for those in group B (p = 0.03). The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03). A comparison of expression profiles of the two subgroups of pts highlighted a deregulated expression of genes involved in the mechanisms of response to genotoxic stress, i.e. of damage sensor genes (ATM, RAD9, RAD 50, ATRip), of damage signal mediator genes (H2AFX, 14-3-3), of genes involved in regulation of cell proliferation (CDK6, CDC25, CCND2) and of anti-apoptotic genes (BCL2, p73) (one-way ANOVA plus multiple-test correction with FDR <0,05). Finally, this group of pts significantly over-express the transcription factor YY1, which is known to interact with p53, thus inhibiting its transcriptional activity. In conclusion, pts carrying amplified MDM4 and/or deleted p53 showed a significantly higher number of CNAs and the significant over-expression of genes involved in the response mechanisms to genotoxic stress, as compared to pts lacking these chromosomal aberrations. This might account for the worse outcome of patients harboring del(17p) and/or amp(1q). The MDM4 locus amplification and the YY1 over-expression might contribute to maintain p53 in an OFF state by an indirect mechanism.
103rd Annual Meeting of the American Association for Cancer Research; 06/2012
[Show abstract][Hide abstract] ABSTRACT: In a randomized, phase 3 study, superior complete/near-complete response (CR/nCR) rates and extended progression-free survival were demonstrated with bortezomib-thalidomide-dexamethasone (VTD) versus thalidomide-dexamethasone (TD) as induction therapy before, and consolidation after, double autologous stem cell transplantation for newly diagnosed myeloma patients (intention-to-treat analysis; VTD, n = 236; TD, n = 238). This per-protocol analysis (VTD, n = 160; TD, n = 161) specifically assessed the efficacy and safety of consolidation with VTD or TD. Before starting consolidation, CR/nCR rates were not significantly different in the VTD (63.1%) and TD arms (54.7%). After consolidation, CR (60.6% vs 46.6%) and CR/nCR (73.1% vs 60.9%) rates were significantly higher for VTD-treated versus TD-treated patients. VTD consolidation significantly increased CR and CR/nCR rates, but TD did not (McNemar test). With a median follow-up of 30.4 months from start of consolidation, 3-year progression-free survival was significantly longer for the VTD group (60% vs 48% for TD). Grade 2 or 3 peripheral neuropathy (8.1% vs 2.4%) was more frequent with VTD (grade 3, 0.6%) versus TD consolidation. The superior efficacy of VTD versus TD as induction was retained despite readministration as consolidation therapy after double autologous transplantation. VTD consolidation therapy significantly contributed to improved clinical outcomes observed for patients randomly assigned to the VTD arm of the study. The study is registered at www.clinicaltrials.gov as #NCT01134484.
[Show abstract][Hide abstract] ABSTRACT: Multiple Myeloma (MM) is an incurable hematologic malignancy characterized by the accumulation of malignant plasma cells. Dysregulation of MYC by rearrangement or translocation are common somatic events described either in early or late stage of the disease, and transcriptional profiling of MYC pathway activation is observed in more than 60% of MM cell lines. Hypoxia Inducible Factor-1α (HIF-1α) overexpression has been described in several MM cell lines and in about 30% of MM patients samples. In solid tumours, deregulation of c-MYC has been associated with HIF-1α up-regulation.
In the present study we explored the interaction between c-MYC and HIF-1α in a panel of MM cell lines. We had previously shown that treatment with EZN2968, an antisense oligonucleotide against HIF-1α, resulted in a significantly reduction of HIF-1α protein level as soon as 24h and lasted over 96h. To confirm the inhibition of HIF-1α activity, MM1S cells were treated with EZN2968 for 24h, lysed, co-precipitated with p300, and incubated with anti-HIF-1α antibody. We showed that HIF-1α was no longer associated p300 in EZN-treated compared to untreated samples, suggesting an inhibitory effect of HIF-1α activity. We next observed that treatment with EZN2968 induced a progressive accumulation of cells in S-phase with concomitant reduction of G2/M phase. By western blot analysis, we observed that p21, cell cycle check point negatively regulated by c-MYC, was up-regulated in treated samples. We further verified the effect of HIF-1α inhibition on c-MYC protein level by western blotting. After treatment with EZN2968, c-MYC protein expression was reduced in a time dependent manner, suggesting that c-MYC protein level is associated with inhibition of HIF-1α. To examine whether HIF-1α and c-MYC regulate each other promoter activity, we performed Chromatin Immunoprecipitation (ChIP) assays with HIF-1α or c-MYC antibodies. HIF-1A and MYC promoter amplification signals, were present in the controls samples, and decreased after EZN2968 exposure.
Recently, it has been shown that SIRT1, a transcription factor involved in a development and cellular stress responses, can modulate HIF-1α and c-MYC activity. By Immunoblotting assay, we observed that SIRT1 physically interacts with both proteins and that, after 24h of exposure to EZN2968, c-MYC and HIF-1α were strongly associated to SIRT1. These results were also confirmed at the transcriptional level, by ChIP assay using an anti-SIRT1 antibody. After 24h of treatment with EZN2968, we observed a significant increase of HIF-1A and MYC promoter amplification signals in treated compared to untreated samples, suggesting that SIRT1 recruitment at both promoters is dependent on HIF inhibition.
We showed that in MM cells the expression of HIF-1α and c-MYC are linked and mediated by SIRT1 deacetylase protein. The data suggests a new regulatory mechanism for controlling c-MYC and HIF-1α activity by SIRT1.
104th Annual Meeting of the American Association for Cancer Research; 01/2012
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that plays a critical role in survival and angiogenesis. In solid tumors, elevated expression of HIF-1α, in response to hypoxia or activation of growth factor pathways, is associated with tumor proliferation, metastasis, and drug resistance and correlated with poor prognosis. In contrast to solid tumours, the role of HIF-1α in hematological malignancies is not completely known. In particular in multiple myeloma (MM) HIF-1α has been suggested to be constitutively expressed and HIF-1α knockdown cell lines have shown higher sensitivity to standard chemotherapy, suggesting a role in the pathophysiology of MM. In the present study, we explored the effect of EZN2968, an antisense oligonucleotide against HIF-1α, as a molecular target in MM.
We showed that the expression of HIF-1α in several MM cell lines is detectable in either normoxia or hypoxia conditions and is increased in the presence of growth stimuli (IL-6 and stroma cells). In vitro, EZN2968 induced selective and stable down-modulation of HIF-1α mRNA and protein expression, either in normoxia or hypoxia conditions. Immunofluorescence analysis confirmed the reduction of the protein after 24 hours of treatment, as well as a lower expression of VEGF.
Clinically achievable doses of EZN2968 induced cell cycle arrest and cell death (10% to 60% of apoptotic cells compared to the controls after 24 to 96 hours, respectively) in MM cell lines. The activation of the apoptotic pathway was confirmed by western blot analysis at different time points. Moreover we observed an irreversible commitment to cell death after 48 hours of exposure. To evaluate the effect of microenvironment, MM cells treated with EZN2968 were exposed to IL-6 and stroma cells for additional 24 hours. EZN2968 overcame the proliferative effect induced by cytokines. EZN2968 showed moderate increase in efficacy in combination with conventional agents such as Melphalan, and immunomodulatory agents (Thalidomide and Lenalidomide).
No significant effect on cell viability was observed on peripheral blood mononuclear cells and CD34+ cells from healthy donors treated with increasing doses of EZN2968.
Evaluation of gene expression profiling modulation induced by EZN 2968 is ongoing.
Our data suggest that HIF-1α inhibitor is an attractive therapeutic target for MM patients and provide the rationale for clinical evaluation of HIF inhibitor in combination with currently used MM agents.
Second AACR International Conference on Frontiers in Basic Cancer Research; 12/2011
[Show abstract][Hide abstract] ABSTRACT: Thal-dex (TD) is an effective therapy for advanced MM. We evaluated TD as salvage treatment of MM patients at first relapse. Thal was given at a daily dose of 100 or 200 mg until progression. Dex was administered 160 mg/month. One hundred patients were enrolled. First line therapy included ASCT (72%) and conventional CHT (28%). Fifty-nine percent received a fixed thal dose of 100 mg/day. The most frequent adverse events were constipation (42%), peripheral neuropathy (58%, 5% grade 3), bradycardia (20%), skin rash (11%), and VTE (7%). Discontinuation of thal due to adverse events was recorded in eight patients. On ITT, 46% of patients achieved at least a PR. Median DOR was 28 months, median time to next therapy was 15.5 months. Median OS, TTP, and PFS were 43, 22, and 21 months, respectively. TTP and PFS were significantly longer for patients with at least PR to TD. TD was an effective salvage treatment for MM patients at first relapse, as demonstrated by durable disease control and prolonged OS. TD was well tolerated, as reflected by the long stay on treatment without disease progression (median 25 months) and a low discontinuation rate due to toxicity (8%).
Annals of Hematology 09/2011; 91(3):419-26. · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We prospectively analyzed the prognostic relevance of positron emission tomography-computed tomography (PET/CT) at diagnosis, after thalidomide-dexamethasone (TD) induction therapy and double autotransplantation (ASCT) in 192 newly diagnosed multiple myeloma (MM) patients. Presence at baseline of at least 3 focal lesions (FLs; 44% of cases), a standardized uptake value (SUV) > 4.2 (46%), and extramedullary disease (EMD; 6%) adversely affected 4-year estimates of progression-free survival (PFS; ≥ 3 FLs: 50%; SUV > 4.2: 43%; presence of EMD: 28%). SUV > 4.2 and EMD were also correlated with shorter overall survival (OS; 4-year rates: 77% and 66%, respectively). Persistence of SUV > 4.2 after TD induction was an early predictor for shorter PFS. Three months after ASCT, PET/CT was negative in 65% of patients whose 4-year rates of PFS and OS were superior to those of PET-positive patients (PFS: 66% and OS: 89%). In a multivariate analysis, both EMD and SUV > 4.2 at baseline and persistence of fluorodeoxyglucose (FDG) uptake after ASCT were independent variables adversely affecting PFS. PET/CT involvement at diagnosis, after novel agent-based induction and subsequent ASCT is a reliable predictor of prognosis in MM patients. This study is registered at www.clinicaltrials.gov as NTC01341262.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia-inducible factor-1 alpha (HIF-1a) is a transcription factor that
plays a critical role in survival and angiogenesis. In solid tumors, elevated
expression of HIF-1a, in response to hypoxia or activation of growth
factor pathways, is associated with tumor proliferation, metastasis, and
drug resistance and correlated with poor prognosis. In contrast to solid
tumours, the role of HIF-1a in hematological malignancies is not completely
known. In particular in multiple myeloma (MM) HIF-1a has been
suggested to be constitutively expressed and HIF-1a knockdown cell
lines have shown higher sensitivity to standard chemotherapy, suggesting
a role in the pathophysiology of MM. In the present study, we
explored the effect of EZN2968, an antisense oligonucleotide against
HIF-1a, as a molecular target in MM. We showed that the expression of
HIF-1a in several MM cell lines is detectable in either normoxia or hypoxia
conditions and is increased in the presence of growth stimuli (IL-6
and stroma cells). In vitro, EZN2968 induced selective and stable downmodulation
of HIF-1a mRNA and protein expression, either in normoxia
or hypoxia conditions. Immunofluorescence analysis confirmed the reduction of the protein after 24 hours of treatment, as well as a lower
expression of VEGF. Clinically achievable doses of EZN2968 induced cell
cycle arrest and cell death (10% to 60% of apoptotic cells compared to
the controls after 24 to 96 hours, respectively) in MM cell lines. The
activation of the apoptotic pathway was confirmed by western blot
analysis at different time points. Moreover we observed an irreversible
commitment to cell death after 48 hours of exposure. To evaluate the
effect of microenvironment, MM cells treated with EZN2968 were
exposed to IL-6 and stroma cells for additional 24 hours. EZN2968 overcame
the proliferative effect induced by cytokines. EZN2968 showed
moderate increase in efficacy in combination with conventional agents
such as Melphalan, and immunomodulatory agents (Thalidomide and
Lenalidomide). No significant effect on cell viability was observed on
peripheral blood mononuclear cells and CD34+ cells from healthy
donors treated with increasing doses of EZN2968. Our data suggest that
HIF-1a inhibitor is an attractive therapeutic target for MM patients and
provide the rationale for clinical evaluation of HIF inhibitor in combination
with currently used MM agents.
[Show abstract][Hide abstract] ABSTRACT: Background / Purpose:
The aim of the present study was to evaluate the clinical outcome of a large series of younger patients with symptomatic multiple myeloma (MM) who were enrolled in two subsequent clinical trials of thalidomide-dexamethasone (thal-dex) incorporated into double autologous stem-cell transplantation (ASCT) to support high-dose melphalan (200 mg/m2).
The analysis was performed on an intention-to-treat basis on a total of 593 patients who were followed for a median of 36 months. More than 80% of the patients were screened at diagnosis for the presence of cytogenetic abnormalities by FISH analysis. Forty-five percent of patients had del(13q), while t(4;14) and del(17p) were found in 16 % and 7 % of patients, respectively.In conclusion, incorporation of thal-dex into double autotransplantation failed to overcome the poor prognosis conferred by del(13 q), t(4;14) and del(17p). In a multivariate Cox regression analysis del(17p) and high levels of serum beta2-m at diagnosis were the strongest variables adversely influencing PFS and a. In comparison with the presence of t(4;14) but absence of del(17p), patients carrying del(17p) had a significantly shorter OS, possibly reflecting their worst outcome after relapse. Presence of del(13q) alone conferred a significantly shorter TTP, but did not have an adverse impact on OS due to the favourable role of effective salvage therapies incorporating either bortezomib or lenalidomide.
52nd American Society of Hematology Annual Meeting 2010; 12/2010
[Show abstract][Hide abstract] ABSTRACT: The bone marrow (BM) microenvironment consists of extracellular-matrix and the cellular compartment including immune cells. Multiple myeloma (MM) cell and BM accessory cell interaction promotes MM survival via both cell-cell contact and cytokines. Immunomodulatory agents (IMiDs) target not only MM cells, but also MM cell-immune cell interactions and cytokine signaling. Here we examined the in vitro effects of IMiDs on cytokine signaling triggered by interaction of effector cells with MM cells and BM stroma cells. IMiDs diminished interleukin-2, interferonγ, and IL-6 regulator suppressor of cytokine signaling (SOCS)1 expression in immune (CD4T, CD8T, natural-killer T, natural-killer) cells from both BM and PB of MM patients. In addition, coculture of MM cells with healthy PBMCs induced SOCS1 expression in effector cells; conversely, treatment with IMiDs down-regulated the SOCS1 expression. SOCS1 negatively regulates IL-6 signaling and is silenced by hypermethylation in MM cells. To define the mechanism of inhibitory-cytokine signaling in effector cells and MM cells, we next analyzed the interaction of immune cells with MM cells that were epigenetically modified to re-express SOCS1; IMiDs induced more potent CTL responses against SOCS1 re-expressing-MM cells than unmodified MM cells. These data therefore demonstrate that modulation of SOCS1 may enhance immune response and efficacy of IMiDs in MM.
[Show abstract][Hide abstract] ABSTRACT: In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110delta signaling in multiple myeloma (MM). Knockdown of p110delta by small interfering RNA caused significant inhibition of MM cell growth. Similarly, p110delta specific small molecule inhibitor CAL-101 triggered cytotoxicity against LB and INA-6 MM cell lines and patient MM cells, associated with inhibition of Akt phosphorylation. In contrast, CAL-101 did not inhibit survival of normal peripheral blood mononuclear cells. CAL-101 overcame MM cell growth conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cell coculture. Interestingly, inhibition of p110delta potently induced autophagy. The in vivo inhibition of p110delta with IC488743 was evaluated in 2 murine xenograft models of human MM: SCID mice bearing human MM cells subcutaneously and the SCID-hu model, in which human MM cells are injected within a human bone chip implanted subcutaneously in SCID mice. IC488743 significantly inhibited tumor growth and prolonged host survival in both models. Finally, combined CAL-101 with bortezomib induced synergistic cytotoxicity against MM cells. Our studies therefore show that PI3K/p110delta is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 to improve patient outcome in MM.
[Show abstract][Hide abstract] ABSTRACT: Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.