ALBERT

Beschreibung: INTRODUCTION: The phosphatidylinositol 3-kinase (PI3K) pathway is consistently activated in relapsed/refractory Hodgkin lymphoma (HL), suggesting that TGR-1202, a novel inhibitor of the δ isoform of PI3K (PI3K-δ) in clinical development for a variety of hematologic malignancies, might represent an attractive therapeutic option. The anti-CD30 monoclonal antibody, Brentuximab Vedotin (BV), a conjugate of Brentuximab and the microtubule-disrupting agent, monomethyl auristatin E (MMAE), induced a 75% objective response rate with limited duration of response in relapsed/refractory HL. Combination therapies aimed at enhancing the anti-tumor activity of BV may have the potential for significant clinical impact in the treatment of relapsed/refractory HL. Therefore, the present study was aimed at investigating the activity and mechanism(s) of action of the PI3K-δ inhibitor TGR-1202 in combination with BV. METHODS: Three HL cell lines, including L-540, KM-H2 and L-428, were used to investigate in vitro cell growth and cell survival. The activity of TGR-1202 and BV, each as single agents and in combination, on tubulin polymerisation and microtubule distribution across cell membrane was investigated by means of a tubulin polymerisation assay and a three-dimensional volume rendering technique. The efficacy of TGR-1202/BV in combination was finally analyzed in NOD/SCID mice with HL cell line xenografts. RESULTS: As compared to single agents, exposure of L-540, KM-H2, and L-428 cell lines to the TGR-1202 (10 µM) and BV (10 ng/ml) combination resulted in a synergistic inhibition of mean (±SEM) cell growth (TGR-1202: 40 ± 4%; BV: 30 ± 2%; TGR-1202/BV: 85 ± 1%, P ≤.0001) and a marked increase of cells in G2/M phase (TGR-1202/BV: 72 ± 3%). This finding was paralleled by a 3-fold reduction of cells in S phase (TGR-1202: 25 ± 1%; BV: 23 ± 1%; TGR-1202/BV: 9 ± 1%) and a marked Cyclin B1 and p21 overexpression. Upon TGR-1202/BV exposure, HL cell lines showed a 3-fold increase in apoptosis over that observed with single agents (TGR-1202: 27 ± 2%; BV: 27 ± 2%; TGR-1202/BV: 75 ± 2%, P ≤.0001). Activation of caspase-8, -9, -3, and cleavage of PARP were reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-dependent apoptosis. Analysis of α-tubulin by immunofluorescence showed a synergistic microtubule disruption induced by TGR-1202/BV treatment with a strong α-tubulin inhibition (40%, P ≤.0001) and a low diffuse staining with irregular microtubule fragments throughout the cytosol. In addition, TGR-1202/BV in combination strongly inhibited tubulin polymerization in a time-dependent manner, suggesting that TGR-1202/BV treatment abrogates microtubule assembly and disrupts microtubules. In NOD/SCID mice bearing human HL xenografts, TGR-1202 (150 mg/Kg) and BV (0.5 mg/Kg) combined treatment significantly reduced the growth of L-540 and L-428 nodules, resulting in an average 50% tumor growth inhibition (P ≤.0001) compared to single agent treatments. No systemic toxicity was observed in mice receiving the combination therapy. Interestingly, a significant increase of microtubule disruption resulting in a marked tumor necrosis (5-fold increase, P ≤.0001) detected in mice receiving TGR-1202/BV combination as compared to mice receiving single agents. Finally, TGR-1202/BV was found to interfere with the mitotic spindle integrity, which may suggest that the cytotoxicity of the combined TGR-1202/BV treatment primarily arises from the inhibition of tubulin polymerization. CONCLUSIONS: The novel PI3K-δ inhibitor TGR-1202 synergistically enhances the anti-tumor activity of BV by increasing drug-induced cell death and tubulin disruption in HL cell line xenografts. These data provide a strong rationale for clinical studies using TGR-1202/BV in combination in refractory/relapsed HL patients. A Phase I study of the combination of TGR-1202 and BV is ongoing in patients with relapsed/refractory HL. Disclosures Viswanadha: Incozen: Employment. Sportelli:TG Therapeutics: Employment, Equity Ownership. Vakkalanka:Rhizen: Employment, Equity Ownership.

Beschreibung: Introduction The phosphatidylinositol 3-kinase (PI3K) pathway is consistently activated in relapsed/refractory Hodgkin lymphoma (HL), suggesting that TGR-1202, a novel inhibitor of the delta isoform of PI3K (PI3K-δ), in clinical development for patients with hematologic malignancies, might represent an attractive therapeutic option. The anti-CD30 monoclonal antibody Brentuximab Vedotin (BV) conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) has recently been reported to induce an overall response rate of 75% in relapsed/refractory HL, but is associated with limited response duration. Combination therapies aimed at enhancing the anti-tumor activity of BV and reducing its side effects may have significant clinical impact in the treatment of relapsed/refractory HL. The present study was aimed at investigating the activity and mechanism(s) of action of the PI3K-δ inhibitor TGR-1202, in combination with BV in non-clinical models of HL. Methods Three HL cell lines, including L-540, KM-H2 and L-428, were used to test the effects of TGR-1202 alone, BV alone, or the combination of TGR-1202 with BV. Cell cycle effects and cell survival were determined by flow cytometry and Western blotting (WB). Additionally, WB was used to assess modulating effects of TGR-1202 on the PI3K/AKT pathway as well as microtubule interacting proteins. Cyclin B1, p21, and α-tubulin were detected by indirect immunofluorescence microscopy. The activity of TGR-1202 and/or BV on microtubule distribution and polymerization were quantified using a three-dimensional volume rendering technique. Results TGR-1202 and BV used as single agents were able to induce a time- and dose-dependent inhibition of cell proliferation and induction of cell death in all cell lines. Compared to the single agent effects, the combination of TGR-1202 (10 µM) and BV (10 ng/ml) synergistically inhibited the mean (±SEM) growth of L-540, KM-H2, and L-428 cell lines (TGR-1202: 40 ± 4%; BV: 30 ± 2%; TGR-1202/BV: 85 ± 1%). Inhibition of cell proliferation induced by the 2-drug combination was associated with a dramatic G2/M cell cycle arrest. Upon TGR-1202/BV treatment, the mean (±SEM) percentages of cells in G2/M phases were increased by 4-fold (72 ± 3%) as compared to TGR-1202 (18 ± 1%) or BV (18 ± 1%) alone. This finding was paralleled by a 3-fold reduction of cells in S phase (TGR-1202: 25 ± 1%; BV: 23 ± 1%; TGR-1202/BV: 9 ± 1%, mean ± SEM) and a marked Cyclin B1 and p21 overexpression. In comparison to each drug as a single agent, the TGR-1202/BV combination led to a synergistic cell death induction. In fact, upon TGR-1202/BV treatment, mean (±SEM) cell death values detected in L-540, KM-H2, and L-428 cell lines were increased by 3-fold over TGR-1202 or BV alone (TGR-1202: 27 ± 2%; BV: 27 ± 2%; TGR-1202/BV: 75 ± 2%). Analysis of caspase-3 and PARP cleavage and blocking experiments with the pan-caspase inhibitor Z-VAD-FMK revealed a caspase-dependent cell death mechanism. In addition, the antiproliferative and cytotoxic effects of TGR-1202 were associated with a marked time-dependent inhibition of PI3K/Akt pathway and dephosphorylation of GSK-3β, Aurora kinases, and stathmin, suggesting that modulation of molecules associated with microtubule polymerization are critically involved in TGR-1202/BV-triggered cell death. To asses potential effects on microtubule dynamics, HL cells were treated with TGR-1202, BV, or the combination for 24 hours, and the effect on microtubules was determined by α-tubulin staining. Compared with controls, TGR-1202 and BV treatment alone led to a modest loss of microtubules (TGR-1202: 11%; BV: 9%), while the combined TGR-1202/BV treatment resulted in a potent synergistic microtubule disruption (mean values of α-tubulin inhibition of 40%, P ≤.0001), supported by a diffuse stain and irregular microtubule fragments throughout the cytosol. Additionally, TGR-1202/BV was found to interfere with the mitotic spindle integrity which may suggest that the G2/M arrest and cytotoxicity of the combined TGR-1202/BV treatment primarily arises from the inhibition of tubulin polymerization. Conclusions Novel PI3K-δ inhibitor TGR-1202 enhances the anti-tumor activity of BV by increasing drug-induced apoptosis and tubulin disruption in all HL cell lines analyzed in the present study. Our data provides a strong rationale for evaluating TGR-1202 in combination with BV in patients with relapsed/refractory HL. Disclosures: Sportelli: TG Therapeutics, Inc.: Employment, Equity Ownership.

Beschreibung: Introduction Disease relapse and resistance to chemotherapy represent challenging issues for Hodgkin Lymphoma (HL) patients. PI3K/AKT and RAF/MEK/ERK pathways are constitutively activated in the majority of HL patients, thus representing attractive therapeutic targets. Previous results from our phase II study indicate that combining the PI3K/AKT inhibitor perifosine with the RAF/MEK/ERK inhibitor sorafenib can achieve significant clinical responses in relapsed/refractory HL. The present study was therefore aimed at characterizing the in vitro and in vivo activity and mechanism(s) of action of a novel PI3K/ERK dual inhibitor AEZS-136 (Æterna Zentaris GmbH, Germany, EU). Methods Four HL cell lines (L-540, SUP-HD1, KM-H2 and L-428) were used to investigate the in vitro effects of AEZS-136 on cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Live cell imaging experiments were performed to asses the production of reactive oxygen species (ROS). Western blotting (WB) was used to assess the effects of AEZS-136 on MAPK and PI3K/AKT pathways as well as apoptosis. The antitumor efficacy of AEZS-136 was investigated in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results Exposure of L-540, SUP-HD1, KM-H2 and L-428 cell lines to AEZS-136 induced a marked, early and time-dependent dephosphorylation of PI3K/Akt and MAPK pathways that was associated with a significant time and dose-dependent cell growth inhibition [80 ± 3% (mean ±SEM) in the L-540 and SUP-HD1 responsive cell lines] and S phase cell cycle arrest. Indeed, upon AEZS-136 treatment the mean (±SEM) percentages of cells in S phase were reduced by 3-fold (13 ± 1%) as compared to control (33 ± 2%). Significant levels of cell death, as assessed by AnnexinV/PI staining, were only observed in L-540 (62 ± 9 vs 14 ± 3%, P ≤.0001) and SUP-HD1 (46 ± 2% vs 15 ± 2%, P ≤.0001) cell lines and were associated with severe mitochondrial dysfunction (up to 40%, P ≤.001). While no activation of caspase-3 and PARP cleavage were observed in L-540 and SUP-HD1 cells treated with AEZS-136, a potent generation of reactive oxygen species (ROS) was observed upon AEZS-136 treatment (up to 90%, P≤.0001). Pretreating cells with the ROS inhibitor YCG063 strongly inhibited AEZS-136-induced ROS generation, mitochondrial dysfunction and cell death, whereas the pan-caspase inhibitor Z-VADfmk did not. Since ROS generation has been implicated in mediating necroptosis, we tested if blocking programmed necrosis with Necrostatin-1 could prevent AEZS-136-induced cytotoxicity. When L-540 cells were treated with AEZS-136 in the presence of Necrostatin-1, cell death and ROS generation were completely prevented, suggesting that cell death was mechanistically related to necroptosis. Additionally, HL cells responsive to AEZS-136-induced cell death showed a pronounced JNK activation whose inhibition by the JNK inhibitor SP600125 reduced cell death and ROS generation. Furthermore, AEZS-136-increased JNK phosphorylation was inhibited by Necrostatin-1 or YCG063, suggesting that ROS-dependent necroptosis was linked to JNK. Interestingly, GEP analysis of L-540 and SUP-HD1 cell lines, but not KM-H2 and L-428 cells, indicated that AEZS-136 treatment induced upregulation of genes involved in positive regulation of cell death. In addition, in KM-H2 and L-428 cells, AEZS-136 strikingly induced the expression of the immediate early response 3 (IER3). Silencing of IER3 restored sensitivity of KM-H2 and L-428 cells to AEZS-136-induced necroptotic cell death, suggesting that IER3 acts as the signaling molecule that mediated AEZS-136-resistance to oxidative cell death. Finally, in vivo experiments were conducted to investigate the antitumor activity of AEZS-136. Treatment of NOD/SCID mice bearing L540 tumor nodules with increasing dose of AEZS-136 (30 – 60 mg/Kg body weight, PO, 5 days/2 weeks) resulted in a dose-dependent reduction of tumor growth (mean TGI of 70%, P ≤.0001) compared to vehicle-treated controls. No mice experienced any apparent treatment-related toxicity. Conclusions The PI3K/ERK dual inhibitor AEZS-136 demonstrates a potent antitumor activity against HL cell lines by targeting aberrant expression of MAPK and PI3K/Akt pathways. These data support further clinical evaluation of AEZS-136 in refractory/relapsed HL patients. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3711 INTRODUCTION: Patients with refractory or relapsed classical Hodgkin Lymphoma (cHL) represent an unmet medical need and would benefit from the development of new therapies. Histone deacetylases (HDACs) and the RAF/MEK/ERK pathway are aberrantly controlled in cHL and influence a broad repertoire of tumor processes, suggesting a rationale for therapeutically targeting these pathways. We targeted these pathways using the HDAC inhibitor Givinostat (Italfarmaco S.p.A., Milan, Italy), and the RAF/MEK/ERK inhibitor Sorafenib (Nexavar, Bayer, Germany, EU) in order to investigate in vitro and in vivo the activity and mechanism(s) of action of this two-drug combination. METHODS: Three cHL cell lines, including HDLM-2, L-540 and HD-MyZ, were used to investigate the effects of Givinostat and Sorafenib, used alone or in combination, by means of in vitro assays evaluating cell growth and cell survival. Additionally, live cell imaging was used to asses the production of reactive oxygen species (ROS), and Western blotting (WB) to assess modulating effects of the two-drug combination on MAPK, PI3K/AKT, HDACs as well as the apoptotic pathways. The efficacy of Givinostat/Sorafenib combination was finally confirmed in NOD/SCID mice with cHL cell line xenografts. RESULTS: While Givinostat and Sorafenib as single agents exerted a limited activity against cHL cells, the combined Givinostat/Sorafenib treatment was associated with potent dephosphorylation of MAPK and PI3K/Akt pathways and significantly increased H3 and H4 acetylation due to a nearly complete inhibition of class I and II HDACs. Furthermore, these events were associated with a time-dependent synergistic cell growth inhibition (70% to 90%) in all Givinostat/Sorafenib-treated cHL cells. Upon Givinostat/Sorafenib exposure, HDLM-2 and L-540 cell lines showed significantly (P ≤.0001) increased levels of apoptosis (90 ± 2% and 96 ± 1%, respectively) and mitochondrial dysfunction (up to 70%, P≤.0001), as compared with single agents. Apoptosis induced by Givinostat/Sorafenib combination failed to induce processing of caspase-8, −9, −3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, suggesting the occurrence of caspase-independent apoptosis. Besides downregulating the expression of the anti-apoptotic protein Mcl-1 and ERK1/2 phosphorylation, Givinostat/Sorafenib strongly increased expression of the BH-3 only protein Bim, compared to single treatments. These findings were dependent on a potent, early and time-dependent ROS generation (up to 60%, P≤.0001) that was synergistically induced by Givinostat/Sorafenib treatment. Additionally, pretreatment of cHL cells with the ROS inhibitor YCG063 prevented the generation of ROS as well as mitochondrial membrane depolarization along with cell death induced by the two-drug combination, suggesting that ROS generation is the triggering event in Givinostat/Sorafenib induced-cell death. In vivo Givinostat/Sorafenib treatment significantly reduced the growth of L-540 and HD-MyZ nodules, resulting in an average 35% to 65% tumor growth inhibition (P ≤.0001) compared to single treatments, in the absence of any toxicity. Interestingly, as compared to controls or treatment with single agents, the combined Givinostat/Sorafenib treatment significantly increased in vivo Bim expression (7- to 21-fold increase, P ≤.0001), resulting in a marked tumor necrosis (3- to 5-fold increase, P ≤.0001). CONCLUSIONS: The combined Givinostat/Sorafenib treatment demonstrates a potent preclinical in vitro and in vivo activity against cHL cell lines by targeting aberrant expression of HDACs and MAPK. Antitumor activity of this combination involves ROS generation and Bim upregulation and provides a rationale for clinical studies using this combination in refractory/relapsed cHL patients. Disclosures: No relevant conflicts of interest to declare.