Abstract 3729
The serine/threonine kinase Akt plays a critical signaling role downstream of phosphatidylinositol-3-kinase (PI3K) and is important in promoting cell survival and inhibiting apoptosis. ...Indeed, Akt activation and overexpression is often associated with resistance to chemotherapy or radiotherapy. Previous studies demonstrated the potential therapeutic value of targeting the PI3K pathway in lymphoma, as both the selective PI3Kδ inhibitor CAL-101, and everolimus and temsirolimus, which target PI3K and mTOR, produce clinical responses in a variety of lymphomas. We evaluated the effect of the novel allosteric Akt inhibitor, MK-2206, in a panel of lymphoma cell lines and primary lymphoma cells. We found that Akt, and activated pAkt, are highly expressed in lymphoma cells. After 72 hours of incubation, the Akt inhibitor MK-2206 demonstrated antiproliferative activity in a variety of lymphoma cell lines, with an IC50 ranging between 0.1 and 5μM. There was no correlation between pre-treatment levels of pAKT, PI3K isoforms, or PTEN protein expression and sensitivity to MK-2206. Within the diffuse large B cell lymphoma cell lines, those of GCB cell of origin were more sensitive to MK-2206, compared with the ABC-derived cell lines. Resistant cell lines tended to had weak or absent expression of p-GSK3 and p-4EBPI. Mechanistically, MK-2206 treatment decreased the level of p-Akt (Ser473), and p-Akt (Thr308), irrespective of drug sensitivity. Furthermore, MK-2206 decreased the phosphorylation level of Akt downstream targets, including p-GSK3 beta and p-PRAS40, upregulated p27. and dephosphorylated p70S6K. Moreover, MK-2206 treatment decreased HIF-1 alpha and VEGF expression. Depending on the cell of origin, the antiproliferative effect resulted from cycle arrest at the G0/G1 phase, autophagy, orapoptosis. MK-2206 showed synergistic effect in combination with the HDAC inhibitor, Vorinostat. Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, the combination of MK-2206 and Vorinostat effectively altered p53 and p27 levels, which were associated with increased PARP cleavage and induction of apoptosis. Our data demonstrate that AKT is a promising target for the treatment of lymphoma, and provide a rationale for an ongoing trial, evaluating MK-2206 for the treatment of patients with relapsed lymphoma.
No relevant conflicts of interest to declare.
Abstract 2852
TGF-b-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, plays a key role in regulating inflammation, immunity, metabolism, and ...cell death in a variety of cell types. It is activated in response to a variety of cytokines, including tumor necrosis factor (TNF), TGF-b, and interleukin 1 (IL-1). Upon receptor binding, TAK1 binds to adaptor proteins, and subsequently phosphorylate downstream molecules leading to activation of p38MAPK, JNK, and NF-kB. In this study, we examined the expression pattern of TAK1 and its potential therapeutic role as a target for lymphoma. First, we examined TAK1 expression in a panel of lymphoid cell lines by western blot, and found it to be highly expressed in mantle cell lymphoma cell lines (Mino, SP53, and Jeko-1). In contrast, PBL from healthy donors had no expression of TAK1 protein. TAK1 was also highly expressed in primary lymph node sections of MCL compared with benign reactive lymph nodes. Subsequently, we investigated the in vitro activity of the novel TAK1 small molecule inhibitor AZ-Tak1 in these cell lines. AZ-Tak1 is a potent and a relatively selective inhibitor of TAK1 kinase activity, with an IC50 of 0.009 mM. AZ-Tak1 treatment decreased the level of p38 and ERK in mantle cell lymphoma cells, and induced apoptosis in a dose and time dependent manner, with an IC50 of 0.1–0.5 mM. Using the annexin-V and PI staining and FACS analysis, After 48 hours of incubation, AZ-Tak1 (0.1 mM) induced apoptosis in 28%, 34% and 86% of Mino, SP53, and Jeko cells, respectively, which was increased to 32%, 42%, and 86% when 0.5 mM concentration was used. Similar activity was also observed when TAK1 expression in MCL cells was downregulated by TAK1- specific SiRNA and when primary mantle cell lymphoma specimens were examined after treatment with AZ-Tak1 for 24h (300nM). Using pathway-specific protein arrays focusing on apoptosis, kinases, and transcription factors, AZ-Tak1 (0.5 mM) altered the level of several proteins that regulate cell growth and survival, especially members of the inhibitors of apoptosis (IAP) family. Specifically, nuclear NF-κB p65 levels were decreased, cytosolic levels of SMAC/DIABLO and cytochrome-C were increased in AZ-Tak1 treated cells, which were associated with a decrease in the level of the anti-apoptotic protein X-linked IAP (XIAP) and activation of the intrinsic apoptotic pathway as evident by activation of caspase 9, cleavage of caspase 3, and consequent cells apoptosis. Collectively, our data demonstrate that TAK1 is essential for MAPK and NF-κB activation. Inhibition of TAK1 by the small molecule inhibitor AZ-Tak1 or TAK1-SiRNA induces cell death in mantle cell lymphoma by activating the intrinsic apoptosis pathway, suggesting that targeting TAK1 may have a therapeutic value for the treatment of mantle cell lymphoma.
No relevant conflicts of interest to declare.
Abstract 2850
Based on recent favorable in vitro and in vivo activity of several HDACi (histone deacetylase inhibitors) in HL (Hodgkin lymphoma), we investigated the in vitro activity of SNDX-275, an ...oral, class 1 isoform of selective HDACi in HL-derived cell lines.
Proliferation and cell death were examined by MTS assay, Annexin-V/PI and FACS analysis. For combination studies, cells were incubated with SNDX-275 (0.1-2 μM) and either ABT-737 (0.01-0.2 μM), Obatoclax (0.1-2 μM), Gemcitabine (1-20 nM) or Bortezomib (1-20 nM) for 72 hours. Gene and protein expression were measured by RT-PCR, Western blot, and immunohistochemistry. A multiplex assay was used to determine 30 cytokines and chemokines.
SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased histone-3 acetylation, up-regulated p21 expression, and activated the intrinsic apoptosis pathway by down-regulating the XIAP (X-linked inhibitor of apoptosis protein), which was associated with activation of Caspase 9 and 3. Similarly to other HDACis, SNDX-275 decreased the expression of anti-apoptotic Bcl-2 and Bcl-xL, while level of Mcl-1 and pro-apoptotic Bax remained the same level. Combination studies demonstrated that SNDX-275 had more synergistic effect when combined with Bcl-2 inhibitors ABT-737 or Obatoclax and less when combined with Gemcitabine or Bortezomib. Dysregulated cytokine/chemokine production has been shown previously to contribute to HL pathology, including immune tolerance of the cancer cells. Hence, we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Increased IL12 p40-70, IP10, and RANTES, and decreased IL13, IL4 and TARC levels were found, thus favoring Th1-type cytokines/chemokines. Recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of CTAs (cancer testis antigens), leading to a favorable immune response. SNDX-275 was able to induce CTA expression of SSX2 and NY-ESO only in one cell line whereas MAGE-A4 was induced in both HL cell lines.
Our studies demonstrate that SNDX-275 has a dual effect on apoptotic and immunomodulatory pathways in HL, which can be enhanced by the addition of agents targeting cell survival pathways. Phase II studies with SNDX-275 in HL are ongoing, future clinical studies should investigate combinations with SNDX-275.
No relevant conflicts of interest to declare.
Abstract 3959
Entinostat (ENT) is an oral, class 1 isoform selective HDACi. In vitro in HL cell lines, ENT decreases proliferation and induces cell death in a dose dependent manner with an IC50 of ...0.4 uM. As an epigenetic agent, ENT modulates immune pathways from a TH2 to a TH1 cytokine/chemokine profile and can upregulate the expression of cancer testis antigens, thus acting as an immunomodulator. This phase 2 study was initiated to define ENT single agent antitumor activity and safety and tolerability.
The trial is a Simon 2-stage design that has completed enrollment in 2 dosing schedules with 24 evaluable patients at 6 sites, 6 patients remain active; enrollment is continuing in an alternate dosing schedule. ENT was administered at a dose of 10 mg every 14 days (d), in a 28 d cycle (C) for the first stage of the study, for the second stage the dose was escalated to 15 mg every 14 d beginning C1d15. Subjects ongoing from the first stage were also allowed to escalate. CT/PET scans are conducted every 2 cycles. Blood samples for correlative study analysis are obtained pre-treatment, C1D8, C1D15, and C3D15.
As of August 6, 2010, demographic data are available on 23 evaluable subjects, median age is 28 years (19-53), median prior regimens is 3 (2-10), 13 (56%) had prior autologous transplant, 3 (13%) had prior allogeneic and 3 (13%) had prior autologous and allogeneic transplant. 23 patients have >1 post baseline result available, 65% have disease control (CR+PR+SD), 35% had PD and 2 patients with SD discontinued due to AE (Pericarditis and thrombocytopenia). Two (9%) responses (PR) are reported with time on study 14.7 months and one >6 months. An additional 4 (17%) patients have tumor reduction (25-49%) for at least 4 cycles. Six (26%) patients completed ≥ 6 cycles. Common G1/2 AEs including all causalities were gastrointestinal, fatigue, and pyrexia. Of the 32 patients evaluated for safety, G 3/4 thrombocytopenia occurred in 19 (59.4%) patients, G 3/4 neutropenia in 9 (28.1%) patients, and G 3/4 anemia in 11 (34.4%) patients, including all causalities. Profiling of cytokine, chemokine and growth factor levels in patient serum samples provides support for the biological activity of entinostat as an immunomodulatory agent with ongoing evaluation demonstrating a reduction of IL-13 and TNFα levels within one week of therapy.
ENT as a single agent in HL was well tolerated and demonstrated encouraging activity in this heavily pretreated patient population. Further dose intensity is currently being tested; updated results on the immunomodulatory effects will be presented.
Off Label Use: Given the CD20 positivity of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) rituximab has been evaluated previously for relapsed NLPHL and was shown to be efficacious. Rituximab however is not FDA approved for NLPHL. This is a retrospective study that evaluates the use of R-CHOP and other therapies for NLPHL. Current NCCN guidelines support consideration of R-CHOP for NLPHL treatment, and given the rarity of the disease there is no one defined preferred chemotherapy regimen. This information will be disclosed to the audience.
Abstract 1562
Poster Board I-585
SNDX-275 is an oral, class 1 isoform selective HDACi. Phase 1 studies in leukemia demonstrated the agent has a long half-life and that weekly or every other week ...dosing is sufficient for antitumor activity. Based on recent favorable in vitro and in vivo activity of several HDAC inhibitors in HL, we investigated the in vitro activity of SNDX275 in HL-derived cell lines.
For apoptosis and gene expression analysis 05 × 106 cells were incubated with 0.1-2 μM of SNDX-275 for 24-72 hours before they were examined for proliferation and cell death by the MTS assay and the annexin-PI and FACS analysis. For combination studies, cells were incubated with 0.1-2 uM of SNDX-275 and 1-20 nM of either gemcitabine or bortezomib for 48-72 hours. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. SNDX-275 effects on a panel of 30 cytokines and chemokines was assayed on 05 × 106 cells after incubation of 48 hrs using a multiplex assay.
SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased H3 acetylation, up-regulated p21 protein expression, and activated the intrinsic apoptosis pathway by down-regulating the anti-apoptotic X-linked inhibitor or apoptosis (XIAP) protein, which was associated with activation of caspase 9 and 3. Combination studies demonstrated that SNDX-275 had synergistic effects when combined with gemcitabine and bortezomib. To further investigate the potential for SNDX-275 activity in HL we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Dysregulated cytokine/chemokine production has been shown to contribute to HL pathology, including immune tolerance of the cancer cells. SNDX-275 increased IL12 p40-70, IP10, and RANTES, and decreased the level of IL13 and IL4, thus favoring Th1-type cytokines/chemokines. In addition, recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of cancer testis tumor associated antigens, leading to a favorable immune response. None of the lines expressed the CTAs without induction. SNDX275 was able to induce CTA expression of SSX2 in L428 but not HDLM2 whereas MAGE-A was induced in both HL cell lines. NY-ESO expression was not induced.
Our studies demonstrate that SNDS-275 has dual effect on apoptotic and immunomodulatory pathways in HL. Furthermore, this data demonstrates that SNDX-275 may upregulate CTAs suggesting that this treatment may render the tumor more immunogeneic and susceptible to immune mediated killing with tumor-specific cytotoxic T lymphocytes. The selectivity profile of SNDX-275 also suggests that HDAC1 and 2 are the primary targets for HDAC inhibition in these cells. Phase 2 studies with SNDX-275 in HL are ongoing.
Younes:MethylGene: Honoraria, Research Funding.
Abstract 3718
Based on preclinical experiments that demonstrated in vitro synergism between pan-deacetylase (DAC) inhibitor Panobinostat (LBH589) with mTOR inhibitor Everolimus (RAD001) in Hodgkin ...and non-Hodgkin lymphoma cell lines, we conducted a phase I/II study to determine the safety and efficacy of this novel regimen. Patients were eligible if they had relapsed or refractory Hodgkin or non-Hodgkin lymphoma regardless of the number of prior regimens, including autologous and allogeneic transplantation. Everolimus was self administered orally daily and Panobinostat three times weekly on 4 dose escalation levels Table 1. To date, a total of 30 patients have been treated. The histologic subtypes include small lymphocytic (n=1), follicular (n=2), mantle cell (n=3), hodgkin (n=12), diffuse large B-cell (n=7), T-cell (n=3), one discordant Hodgkin/marginal zone lymphoma and one discordant Hodgkin/large cell lymphoma. The median number of prior therapies is 3 with 11 patients receiving prior autologous transplantation. 30 patients received at least 1 dose and are evaluable for safety and 28 are evaluable for response. The DLT was thrombocytopenia observed in the 4th cohort with a dose of 30mg Panobinostat and 10mg of Everolimus. Therefore, 6 patients were treated at the lower dose level of 20mg Panobinostat and 10mg Everolimus which was determined to be the maximum tolerated dose (MTD) and starting dose in the phase II portion of the study. To date, 16 patients have been treated in the phase II portion of the study. Treatment side effects are manageable with the following grade 3/4 adverse events most commonly observed: thrombocytopenia 48%, neutropenia 48% and anemia 20%. One death occurred on study, possibly related to pulmonary embolus. 20 out of 28 evaluable patients (71%) had tumor reduction ranging from -12% to -72% of whom 50% had PR or CR. Our data demonstrate safety and promising clinical activity of this novel combination in a variety of lymphomas. The phase II portion continues to enroll patients to determine the efficacy of this novel regimen.
Dose LevelEverolimusPanobinostatN15mg10mg325mg20mg33 (MTD)10mg20mg6410mg30mg2Phase II10mg20mg16
Neelapu:celgene: Research Funding. Pro:Celgene: Consultancy, Honoraria.
Abstract 3964
mTOR and deacetylase (DAC) inhibitors have demonstrated single agent activity in patients with relapsed and refractory lymphoma. Synergy between DAC inhibitors and mTOR inhibitors has ...been observed in lymphoma cell lines in vitro. Thus we combined the pan-DACi panobinostat (LBH589) with mTOR inhibitor everolimus (RAD001) in a phase I/II study in patients with relapsed Hodgkin and non-Hodgkin lymphoma. The purpose of this study is to determine the maximum tolerated dose (MTD) and dose limiting toxicity (DLT) in addition to examining the clinical efficacy of this novel regimen. Patients were eligible if they had relapsed or refractory Hodgkin or non-Hodgkin lymphoma regardless of the number of prior regimens, including autologous and allogeneic transplantation. Everolimus was self administered orally daily and panobinostat three times weekly. To date, a total of 12 patients have been enrolled Table 1. The histologic subtypes include small lymphocytic (n=1), follicular (n=2), mantle cell (n=2), hodgkin (n=5), diffuse large B-cell (n=1) and one discordant Hodgkin/marginal zone lymphoma. The median number of prior therapies is 4 with 6 patients receiving prior autologous transplantation. 11 patients are evaluable for safety and response. The DLT was thrombocytopenia observed in the 4th cohort with a dose of 30mg panbobinostat and 10mg of everolimus. Therefore an additional 6 patients are being dosed at the 20mg panobinostat and 10mg everolimus cohort to confirm the safety of this combined dose. Treatment side effects were manageable with the following grade 3/4 adverse events most commonly observed: thrombocytopenia 45% (n=5), neutropenia 45% (n=5) and fatigue 18% (n=2). One death occurred on study, possibly related to pulmonary embolus. 10 out of 11 evaluable patients had tumor reduction ranging from -31% to -63% with 5 of these patients continuing on therapy. Changes in several cytokines and growth factors were observed at day 8 including VGEF, IL-10 and IL-13. This data demonstrates promising activity of this novel combination in a variety of lymphomas even at the lower dose levels. Once the MTD is identified, the phase II portion will enroll patients to confirm the efficacy of this novel regimen.
Dose LevelEverolimusPanobinostatN15mg10mg325mg20mg3310mg20mg4410mg30mg2
Younes:Novartis: Clinical Support, Honoraria; Seattle Genetics: Clinical Support, Honoraria; Syndax: Clinical Support, Honoraria; Roche: Honoraria; Biogen Idec: Honoraria; Sanofi Aventis: Clinical Support, Honoraria; SBIO: Clinical Support, Honoraria. Off Label Use: Panobinostat is an investigational agent currently being evaluated for the treatment of hematologic and solid malignancies. Fanale:Seattle Genetics: Research Funding; Novartis: Honoraria, Research Funding; Millenium: Research Funding; Genentech: Research Funding. Fayad:Genentech: Research Funding. Pro:Allos Therapeutics, Inc.: Research Funding.
Abstract 3735
Poster Board III-671
Recent clinical trials demonstrated a promising clinical activity of the HDAC inhibitor MGCD0103 in heavily pretreated patients with relapsed HL. However, the ...mechanisms underlying MGCD0103 activity in HL remains unclear. Here we show that MGCD0103 preferentially inhibited HDACs1, 2 with no effect on HDAC6. Using three HL-derived cell lines, the IC50 for MGCD0103 after 72 hour of incubation was in the submicromolar concentrations, and was more effective than vorinostat in inducing cell death. Using gene expression profiling (GEP) studies, pathway PCR array, and western blot analysis, we identified several pathways that are modulated by MGCD0103. MGCD0103 downregulated the X-linked inhibitor of apoptosis (XIAP) protein, activated the intrinsic caspase pathway, and synergized with TRAIL agonistic antibodies. MGCD0103 downregulated CD30 mRNA and surface protein expression in a dose dependent manner, but had no significant effect on B cell-associated proteins, including CD19 and CD20, or on the costimulatory receptors CD40 and CD80. MGCD0103 induced TNF-alfa expression and secretion, which was associated with NF-kB activation, upregulation of the silencer of cytokine signaling (SOCS)-3, downregulated STAT6, and upregulation of STAT1 and 2. Selective inhibition of TNF-alfa expression by short interfering mRNA enhanced MGCD0103-induced cell death. Similarly, inhibition of NF-kB by proteasome inhibitors also potentiated the effect of MGCD0103, thus demonstrating synergy independent of HDAC6 inhibition. These findings demonstrate that MGCD0103 has a potent anti lymphoma activity by modulating the expression of a variety of survival proteins, and provides mechanistic rationale for combining class-I HDAC inhibitors with proteasome inhibitors and TRAIL.
Mamidipudi:Celgene: Employment. Heise:Celgene Corporation: Employment, Equity Ownership. Besterman:Methylgene: Employment. Martell:Methylgene: Employment. MacBeth:Celgene Corporation: Employment, Equity Ownership. Younes:MethylGene: Honoraria, Research Funding.
Vorinostat (SAHA) Inhibits STAT6 Phosphorylation and Transcription, Downregulates Bcl-xL, and Induces Apoptosis in Hodgkin Lymphoma (HL) Cell Lines. Although the malignant Hodgkin and Reed Sternberg ...(HRS) cells of HL are of B-cell origin, they infrequently express B-cell antigens. Recent studies demonstrated that several B-cell specific genes are silenced in HRS cells by epigenetic mechanism, suggesting that this process may be reversible and could be explored therapeutically with deacetylase (DAC) inhibitors or hypomethylating agents. Pan-DAC and isotype-selective DAC inhibitors have shown promising activity in vitro and in vivo in a variety of lymphoid malignancies, including HL. However, the mechanisms of antiproliferative action of DAC inhibitors in HL remain unknown. In this study, we examined the antiproliferative effects of the pan-DAC inhibitor vorinostat (inhibits class I and class II DACs) on HL cell lines and determined its effect on signaling mechanisms that are known to promote HRS cell survival, including STAT3, STAT6, Akt, and ERK pathways. Vorinostat inhibited DACs as evident by the increase in histone-3 acetylation as early as 30 minutes of incubation. Furthermore, vorinostat induced the expression of the cell cycle regulatory protein p21 which was associated with an early increase in the G2M cell cycle fraction in HL cells. Vorinostat had no inhibitory effect on SATA3 or ERK, but inhibited STAT6 phosphorylation and transcription in a dose and time dependant manner. This effect was associated with a decrease in Akt phosphorylation on Ser473 residue. Because STAT6 has been reported to transcriptionally regulate Bcl-XL and Thymus and activation-regulated chemokines (TARC), we examined the effect of vorinostat on these two targets in HL cells. TARC has been shown to play an important role in attracting Th2-type T cells and T-regulatory (T-reg) cells, and to be elevated in sera from patients with HL. Vorinostat downregulated the mRNA expression of TARC in a dose dependent manner, suggesting that it may have a role in regulating chemotaxis of reactive T cells and T-reg cells to HL microenvironment in vivo. Moreover, vorinostat reduced the cellular level of the antiapoptotic protein Bcl-xL which was associated with activation of the caspase pathway, and induction of apoptosis in HL cells. Collectively, these data suggest that DAC inhibition in HL by vorinostat may induce cell death by inhibiting STAT6 and downregulating its antiapoptotic target bcl-xL protein. Furthermore, our data suggest that DAC inhibition may have an added effect in vivo on the cellular component in HL microenvironment by inhibiting TARC.
PDF