sub-G1 Analysis Research Articles

Fucoxanthin Inhibits the Proliferation of ABCC2-Over Expressing Cisplatin-Resistance Ovarian Cancer Cells via Inducing Apoptosis

Background: The development of multidrug resistance (MDR) is a major barrier to achieving effective chemotherapy in cancer. Studies have shown that epithelial ovarian cancer initially responds to platinum-based therapy, however, the recurrent type is often resistant to treatment and is associated with high mortality. Fucoxanthin, a natural component found in marine algae, possesses various pharmacologic properties. This study evaluated the cytotoxicity and resistance reversal activity of fucoxanthin on multidrug resistance-associated protein 2 (MRP2)- overexpressing, cisplatin-resistant ovarian cancer cells (A2780RCIS) and their parental cells (A2780). Methods: Cell viability was evaluated in the presence of different concentrations of fucoxanthin or cisplatin or fucoxanthin/cisplatin combination using the MTT assay. Propidium iodide staining and subG1 analysis were used to evaluate fucoxanthin potential for cell cycle modification and apoptosis induction in cancer cell lines. Results: The results showed that fucoxanthin was able to cause similar toxicity in both cell lines via apoptosis induction. Co-treatment of cells with cisplatin (3.125 to 100 µM) and nontoxic concentrations of fucoxanthin (1 and 2.5 µM) did not reverse resistance to cisplatin in A2780RCIS cells. Conclusion: Although fucoxanthin was not able to modify cisplatin resistance in ovarian cancer cells, it was equally effective in inducing apoptosis and death in both A2780 and A2780RCIS cells, indicating it is not an MRP2 substrate.

In Vitro and In Vivo Relevant Antineoplastic Activity of Platinum(II) Complexes toward Triple-Negative MDA-MB-231 Breast Cancer Cell Line.

Two platinum complexes [Pt(HL3)Cl]·H2O (3) and [Pt(HL4)Cl]·H2O (4) containing α- and β-naphthyl groups, respectively, were investigated in more detail in vitro and in vivo for antineoplastic activity. The cytotoxicity activity induced by these platinum(II) compounds against breast cancer (MDA-MB-231 and MCF-7), lung (A549), prostate (PC3), pancreas (BXPC-3), and normal peripheral blood mononuclear (PBMC) cells were evaluated by MTT assay. The cell viability MTT assay showed that complex (4) was more cytotoxic to all cancer cell lines tested and less cytotoxic against human PBMC. Therefore, complex (4) was selected to further investigate the mechanism of cytotoxic effects involved against MDA-MB-231 cell line (human triple-negative breast cancer). Sub-G1 analysis of the cell cycle showed that this complex induces cell death by apoptosis due to the cell loss of DNA content detected in flow cytometry. The cytotoxic effect induced by complex (4) was associated with the capability of the complex to induce mitochondrial membrane depolarization, as well as increase ROS levels and caspase activation, as a result of the activation of both extrinsic and intrinsic apoptosis pathways. Ultrastructural alterations were observed using scanning and transmission electron microscopy (SEM and TEM), such as membrane blebbing, filopodia reduction, empty mitochondrial matrix, and DNA fragmentation. Furthermore, complex (4) was tested in an MDA-MB-231 tumor nodule xenograft murine model and demonstrated a remarkable reduction in tumor size in BALB/c nude mice, when compared to the control animals.

Abstract 1044: ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma

Abstract Gastric adenocarcinoma is commonly asymptomatic early in its course, leading to late-stage diagnosis at a time when patients are inoperable. Currently available chemotherapy options are non-targeted and highly toxic, leading to a poor 5-year survival of only 31.5%. ONC201 is a small molecule imipridone anti-cancer therapy discovered by our lab that induces cell death in a variety of malignant tumors via a multitude of mechanisms including activation of the integrated stress response (ISR) leading to up-regulation of TNF-related apoptosis-inducing ligand (TRAIL), which induces apoptosis after binding to the death receptor 5 (DR5). We investigated the ability of ONC201 to induce cell death in gastric adenocarcinoma cells when used in combination with recombinant human TRAIL (rhTRAIL), and its affect on DR5 cell surface expression and expression of inhibitors of apoptosis (IAP) in order to further establish mechanism of action. AGS (caspase 8, KRAS, PIK3CA mutant, HER2 amplified), SNU-1 (KRAS and MLH1 mutant, microsatellite instable), SNU-5 (p53 mutant) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. All lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. All cell lines exhibited strong synergy in response to dual therapy with ONC201 and rhTRAIL, with combination indices <0.6 at doses that did not induce cell death in normal fibroblast cells. Synergy was confirmed via flow cytometry analysis with increased cells in the sub-G1 phase of the cell cycle with dual therapy in excess of what would be expected from an additive effect. On western blot analysis, apoptosis was confirmed with increased cleavage of PARP, caspase 8 and caspase 3 after dual treatment with ONC201 and rhTRAIL. Flow cytometry revealed increased cell surface expression of DR5 with ONC201 therapy, and western blot analysis showed that ONC201 lead to up-regulation of ATF-4 and CHOP, indicating activation of the ISR, and down-regulation of anti-apoptotic CIAP-2 and XIAP in all cell lines except AGS, as well as FLIP in all cell lines except SNU-16. These in vitro results suggest that ONC201 in combination with rhTRAIL may be an effective and non-toxic option for the treatment of gastric adenocarcinoma by inducing apoptosis via activation of the ISR, increased cell surface expression of DR5 and down-regulation of inhibitors of apoptosis. We are currently applying this regimen to an organoid model, as well as a murine sub-cutaneous xenograft model using AGS and SNU-1 cells. Citation Format: Cassandra Susan Parker, Lanlan Zhou, Varun Prabhu, Josh Allen, Wafik El-Deiry. ONC201 as a novel anti-cancer therapeutic via modulation of inhibitors of apoptosis and up-regulation of DR5 in gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1044.

Single Agent and Synergistic Combinatorial Efficacy of First-in-Class Small Molecule Imipridone ONC201 in Hematological Malignancies In Vitro and In Vivo through Apoptosis Via the Integrated Stress Response Pathway

▪ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies.ONC201 was evaluated in 20 human leukemia cell lines (Figure 1): 13 acute myeloid leukemia (AML), 5 acute lymphoblastic leukemia (ALL) and 2 chronic myelogenous leukemia (CML) cell lines. We observed a dose-dependent decrease in cell viability in AML, ALL and CML cells (GI50 1-8 µM). ONC201 also decreased cell viability in 2 primary chronic lymphocytic leukemia (CLL) patient samples with 48-72h treatment. Dose- and time-dependent induction of apoptosis (caspase-3 cleavage), inhibition of Akt and Foxo3a phosphorylation was observed in AML cells treated with ONC201 for 24-48 hr. Sub-G1 analysis confirmed that ONC201 induced apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. ONC201-mediated apoptosis in ALL cells involved PARP cleavage and caspase-9 activation. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim was upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells, which are known to confer a poor clinical prognosis.Low micromolar concentrations of ONC201 also reduced multiple myeloma (MM) cell viability in a time- and dose-dependent manner. ONC201 reduced cell viability and induced apoptosis in dexamethasone-resistant MM.1R cells with efficacy comparable to dexamethasone-sensitive MM.1S cells (Figure 2). ONC201 caused activation of the integrated stress response (ISR) pathway, as observed with induction of ATF/CHOP in MM cells. XIAP is highly up-regulated in MM cells and sownregulation of IAP family proteins induces apoptosis. ONC201 treatment of MM cells resulted in a dose-dependent decrease in the anti-apoptotic proteins XIAP, cIAP1 and cIAP2.Next, ONC201 was evaluated in 33 human lymphoma cell lines: 3 anaplastic large cell lymphoma (ALCL), 7 Burkitt's lymphoma, 2 cutaneous T-cell lymphoma (CTCL), 8 diffuse large B-cell lymphoma (DLBCL), 1 MCL, 2 nodular sclerosis (NS) and 10 other lymphoma cell lines. ONC201 demonstrated low micromolar range efficacy (GI50 1-6 µM) across all lymphoma subtypes. Low micromolar concentrations of ONC201 reduced cell viability of MCL and DLBCL cell lines and primary cells in a time- and dose-dependent manner (Figure 3). ONC201 caused activation of the ISR pathway, as observed with induction of ATF4 in Pfeiffer DLBCL cells. In patient-derived MCL cells, ONC201 reduced cyclin D1 expression, a diagnostic feature of MCL.We then tested the efficacy of ONC201 in combination with approved therapies in hematological malignancies in vitro and in vivo. ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine confirmed by combination indices in AML cell lines CMK, THP-1, HL60 and ALL Kasumi-2 cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model exhibited superior tumor growth inhibition compared to either agent alone (Figure 4). ONC201 demonstrated synergistic reduction in cell viability in combination with 5-azacytidine that was confirmed by combination indices in AML cell lines HL-60, MOLM14 and MV411. ONC201 synergistically reduced cell viability in combination with dexamethasone or ixazomib in RPMI-8226 MM cells. ONC201 in combination with bortezomib synergistically reduced cell viability in KARPAS 299 ALCL cells and RPMI-8226 MM cells and improved apoptosis induction in UPN2 MCL cells. ONC201 combined with bortezomib was evaluated in a Burkitt's lymphoma xenograft model. Histological evaluation of tumor xenografts revealed a reduced tumor cell density at 3-4 weeks and enhanced CHOP induction at 48 hours with combination treatment compared to either agent alone. The combination treatment did not result in body weight loss.These results serve as a rationale for ongoing ONC201 single agent trials in relapsed/refractory acute leukemia (NCT02392572), non-Hodgkin's lymphoma (NCT02420795), MM (NCT02609230) and combination trial with dexamethasone in MM (NCT02863991). Our findings provide pharmacodynamic biomarkers and identify synergistic combinatorial regimens that are well tolerated and can be explored in the clinic. [Display omitted] DisclosuresPrabhu:Oncoceutics: Employment, Equity Ownership. Grupp:Novartis Pharmaceuticals Corporation: Consultancy, Other: grant; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties; Jazz Pharmaceuticals: Consultancy. Oster:Oncoceutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Allen:Oncoceutics: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc: Equity Ownership, Patents & Royalties.

Abstract 3012: ONC201 induces apoptosis in gastric adenocarcinoma and exhibits synergy with rhTRAIL

Abstract Gastric adenocarcinoma is highly lethal with a 5-year survival of only 31.5%, largely due to late presentation of disease and ineffective chemotherapy options. ONC201 is a first-in-class small molecule anti-cancer therapy that induces cell death in many different types of malignant tumors via a multitude of mechanisms including up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) by Foxo3a and death receptor 5 (DR5) via activation of the integrated stress response. We investigated the efficacy of ONC201 in inducing cell death in gastric adenocarcinoma cells as monotherapy and in combination with recombinant human TRAIL (rhTRAIL). AGS (caspase 8 and PIK3CA mutant, HER2 amplified) and SNU-16 (p53 mutant) gastric adenocarcinoma cell lines were treated with ONC201 and rhTRAIL and viability assays were performed to determine cell line sensitivities. Both cell lines were then treated with combination therapy based on sensitivities and viability assays were performed in order to determine combination indices. Cell death was verified with sub-G1 analysis via flow cytometry and protein expression was established via western blotting. SNU-16 cells were sensitive to ONC201, while AGS cells were shown to be resistant to monotherapy. Both cell lines were sensitive to rhTRAIL and exhibited synergy when treated in combination with ONC201, resulting in combination indices of less than 0.5 at doses that did not induce cell death in fibroblasts. The proportion of cells in sub-G1 phase of the cell cycle increased in a dose-dependent manner with combination treatment. PARP cleavage increased in a dose-dependent manner after combination therapy, indicating apoptosis and ATF4 and CHOP expression also increased with increasing doses of combination therapy, indicating activation of the integrated stress response. These results suggest that ONC201 may be an effective therapeutic agent for gastric adenocarcinoma in combination with rhTRAIL by inducing apoptosis via the integrated stress response. We are currently expanding the in vitro model to include additional cell lines SNU-1(Ras mutant, microsatellite instability) and SNU-5 (p53 and CDH1 mutant). We are further exploring the role of ONC201 in priming cells via up-regulation of DR5 receptors such that they have increased sensitivity to rhTRAIL when pretreated with ONC201 as a potential source for the established synergy in gastric adenocarcinoma cells. We will also apply this study regimen to an organoid and in vivo model. Citation Format: Cassanda Susan Parker, Lanlan Zhou, Wafik S. El-Deiry. ONC201 induces apoptosis in gastric adenocarcinoma and exhibits synergy with rhTRAIL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3012.

Exploring the Molecular Mechanism of Cinnamic Acid-Mediated Cytotoxicity in Triple Negative MDA-MB-231 Breast Cancer Cells

Cinnamic Acid (CA), also known as 3-phenyl-2-propenoic acid, is a naturally occurring aromatic fatty acid found commonly in cinnamon, grapes, tea, cocoa, spinach and celery. Various studies have identified CA to have anti-proliferative action on glioblastoma, melanoma, prostate and lung carcinoma cells. Our objective was to investigate the molecular mechanism underlying the cytotoxic effect of CA in killing MDA-MB-231 triple negative breast cancer cells. We performed MTT assay and trypan blue assay to determine cell viability and cell death, respectively. Comet analysis was carried out to investigate DNA damage of individual cells. Furthermore, AO/EtBr assay and sub-G1 analysis using flow cytometry were used to study apoptosis. Protein isolation followed by immunoblotting was used to observe protein abundance in treated and untreated cancer cells. Using MTT assay, we have determined CA to reduce cell viability in MDA-MB-231 breast cancer cells and tumorigenic HEK 293 cells but not in normal NIH3T3 fibroblast cells. Subsequently, trypan blue assay and comet assay showed CA to cause cell death and DNA damage, respectively, in the MDA-MB-231 cells. Using AO/EtBr staining and sub-G1 analysis, we further established CA to increase apoptosis. Additionally, immunoblotting showed the abundance of TNFA, TNF Receptor 1 (TNFR1) and cleaved caspase-8/-3 proapoptotic proteins to increase with CA treatment. Subsequently, blocking of TNFA-TNFR1 signalling by small molecule inhibitor, R-7050, reduced the expression of cleaved caspase-8 and caspase-3 at the protein level. Thus, from the above observations, we can conclude that CA is an effective anticancer agent that can induce apoptosis in breast cancer cells via TNFA-TNFR1 mediated extrinsic apoptotic pathway.

Parthenolide inhibits transforming growth factor β1-induced epithelial-mesenchymal transition in colorectal cancer cells.

Background/AimsTransforming growth factor-β1 (TGF-β1) induction of epithelial-mesenchymal transition (EMT) is one of the mechanisms by which colorectal cancer (CRC) cells acquire migratory and invasive capacities, and subsequently metastasize. Parthenolide (PT) expresses multiple anti-cancer and anti-inflammatory activities that inhibit nuclear factor κB by targeting the IκB kinase complex. In the present study, we aimed to investigate whether PT can inhibit TGF-β1-induced EMT in CRC cell lines.MethodsHT-29 and SW480 cell lines were used in the experiment. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sub-G1 analysis was measured by flow cytometry. The induction of EMT by TGF-β1 and inhibition of the process by PT was analyzed by phase contrast microscopy, wounding healing, cellular migration and invasion assays, and Western blotting.ResultsTGF-β1 inhibits HT-29 cell proliferation, but has no effect on SW480 cell proliferation; different concentrations of TGF-β1 did not induce apoptosis in HT-29 and SW480 cells. PT attenuates TGF-β1-induced elongated, fibroblast-like shape changing in cells. PT inhibits TGF-β1-induced cell migration and cell invasion. In addition, other EMT markers such as β-catenin, Vimentin, Snail, and Slug were suppressed by PT, while E-cadherin was increased by PT.ConclusionsOur findings show that PT inhibits TGF-β1-induced EMT by suppressing the expression of the mesenchymal protein and increasing expression of the epithelial protein. These findings suggest a novel approach for CRC treatment by suppression of TGF-β1-induced EMT.

Abstract 3878: Newly identified p53-pathway restoring small molecule, CB002 analog #4 induces apoptosis and appears non-toxic in vivo

Abstract The transcription factor encoding TP53 tumor suppressor gene is mutated in more than half of human cancers. TP53 accounts for therapy resistance and poor patient survival in colorectal and other cancers. Upon cellular stress, p53 protein determines cell fate through the regulation of cell senescence, cell cycle arrest, and apoptosis pathways. Distinct from other tumor-suppressors, mutated p53 can acquire a gain-of-function activity, leading to chemotherapy resistance and a more aggressive tumor phenotype. Direct therapeutic targeting of transcription factors is challenging especially when reactivation is needed; thus, to date, there are no FDA approved drugs for p53 functional restoration in tumors harboring p53 mutations. To overcome this, we have screened for small molecules capable of restoring wild-type p53 pathway signaling rather than direct physical restoration of its function. We identified a small molecule, CB002 analog #4 capable of restoring the p53 pathway in HCT116, SW480 and DLD-1 colorectal cancer cell lines as indicated by the increased expression of p53-target genes NOXA, p21 and DR5, and apoptotic markers upon treatment. We found that analog #4 enhances ethidium homodimer staining of dead cells and decreases the number of positive Ki67 staining cells in colorectal cancer patient organoids. Sub-G1 analysis demonstrated that analog #4 does not induce apoptosis in normal human fibroblasts and preliminary in vivo studies indicate that it can be safely administered to mice. We are investigating the in vivo efficacy of analog #4 in addition to its mechanism of action through micro-array experiments. Our data supports the concept that small molecules can restore the p53-pathway as a therapeutic strategy that can be translated into a clinical setting. Citation Format: Liz J. Hernandez Borrero, Avital Lev, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Newly identified p53-pathway restoring small molecule, CB002 analog #4 induces apoptosis and appears non-toxic in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3878.

Abstract 262: Anti-tumorigenic effect of ONC201 is enhanced by combination treatment with TRAIL or a DR5 agonist in endometrial cancer in vitro

Abstract ONC201 is a well-tolerated, orally active small molecule in the novel imipridone class that has anti-tumorigenic properties in a number of solid tumors, but not in non-neoplastic cells. ONC201 has demonstrated promising activity as a single agent in patients with advanced endometrial cancer in the first-in-human clinical trial with Phase II trials in endometrial cancer patients now underway. Although the mechanism of action in endometrial cell pathology has not been well studied, ONC201’s anti-tumor effect has been shown in other model systems to be p53-independent and mediated through activation of the integrated stress response (ISR) leading to DR5 activation and through induction of cell death or growth arrest. ONC201 inhibits ERK and AKT leading to upregulation of TRAIL in many systems. We hypothesized that ONC201 upregulation of DR5 could sensitize tumor cells to TRAIL and that TRAIL could convert growth arrest to cell death in ONC201-treated cells. Three endometrial cancer cell lines AN3CA, HEC1A and KLE were treated with ONC201 alone or in combination with TRAIL, or a DR5 agonist. Effects on cell viability were assessed by the Cell Titer-Glo cell viability assay, colony formation assays and cell cycle analysis by propidium iodide staining. QPCR and Western blot analysis were used to evaluate mRNA and protein expression, respectively. Assessment of TRAIL and DR5 cell surface expression was evaluated by surface staining using flow cytometry and FlowJo data analysis. ONC201 decreased the cell viability of all three endometrial cancer cell lines at clinically achievable low micro-molar concentrations. ONC201 activated the ISR and an anti-proliferative effect involving a G1 phase arrest and little cell death as indicated by increased Sub-G1 analysis and PARP cleavage. DR5 mRNA and protein expression at the cell surface were induced by ONC201. All three endometrial cancer cell lines were resistant to TRAIL alone, however, pre-treatment with ONC201 sensitized the AN3CA and KLE to TRAIL and a DR5 agonist, leading to potent cell death induction. The combination of ONC201 plus TRAIL did not cause appreciable cell death in normal human fibroblast. ONC201 decreases cell viability in endometrial cancer cells lines primarily through growth arrest while the combination of ONC201 and TRAIL or a DR5 agonist promotes cell death in AN3CA and KLE cells but not in HEC1A endometrial cancer cells. Our results suggest a novel cancer therapeutic strategy that can be exploited in the clinic. Citation Format: Jocelyn Ray, Marie Ralff, David Dicker, Wafik El-Deiry. Anti-tumorigenic effect of ONC201 is enhanced by combination treatment with TRAIL or a DR5 agonist in endometrial cancer in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 262.

Anti-cancer activities of Bharangin against breast cancer: Evidence for the role of NF-κB and lncRNAs

Breast cancer remains one of the leading causes of cancer related deaths in women worldwide. Bharangin is a diterpenoid quinonemethide that has demonstrated therapeutic potential against leukemia, lymphoma, and multiple myeloma cells. Whether this diterpenoid exhibit activities against breast cancer cells and the underlying mechanism is largely unknown. Herein, we provide evidence that bharangin suppresses the proliferation of MCF-7, MDA-MB-231, MDA-MB-453, MDA-MB-468 and T-47D breast cancer cells. As examined by AO/PI staining, DAPI staining, sub-G1 analysis, phosphatidylserine externalization, caspase activation, DNA laddering, and poly-ADP ribose polymerase cleavage, the diterpenoid induced apoptosis in breast cancer cells. The growth inhibitory effect of bharangin on breast cancer cells was further confirmed from colony-formation assay. Furthermore, the cancer cell migration was also suppressed by the diterpenoid. Mechanistically, bharangin was found to modulate multiple cancer related cell signalling pathways in breast cancer cells. Bharangin suppressed the expression of cell survival and invasive proteins, and induced Bax and mitochondrial depolarization in breast cancer cells. The diterpenoid also suppressed the activation of pro-inflammatory transcription factor, nuclear factor (NF)-κB induced by okadaic acid. Finally, the diterpenoid induced the expression of tumor suppressor lncRNAs (MEG-3, GAS-5), while down-regulating oncogenic H19 expression. Overall, these results suggest that bharangin exhibits anti-carcinogenic, anti-proliferative and anti-inflammatory activities against breast cancer cells. The modulation of lncRNA expression and inhibition of NF-κB activation by bharangin may contribute to its anti-carcinogenic activities.

Abstract 4516: Dual inhibition of CDK1 and HSP90 destabilizes HIF1α and synergistically induces cancer cell death

Abstract Solid tumors are often characterized by intratumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) has been implicated in cell proliferation, survival, angiogenesis, invasion and migration. The expression of HIF-1α is increased in a variety of cancers but this is not restricted to hypoxic regions. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., Cell Cycle, 2013). Heat shock protein 90 (HSP90) is also an acknowledged VHL-independent HIF1α stabilizer. We sought to explore the link between CDK1-mediated and HSP90-mediated HIF1α stabilization. Administration of the CDK1 inhibitor, Ro-3306, reverses the heat shock (40°C)-increased level of HIF1α in normoxia suggesting that CDK1 activity may contribute to HSP90-mediated HIF1α stabilization. Under hypoxia, combination treatment with HSP90 inhibitor geldanamycin and CDK1 inhibition/knockdown decreases HIF1α levels more robustly than either treatment alone. This led us to investigate whether combined CDK1 and HSP90 inhibition results in synergistic anti-cancer effects. Indeed, dual inhibition of CDK1 and HSP90 synergistically decreases HCT116 colon cancer cell viability. PARP cleavage and sub-G1 analysis indicate the combinational treatment triggers cancer cell death through apoptosis. Consistent with these findings, we observed that inhibition of cell viability by the combination is impaired in HCT116 Bax-/- cells. Furthermore, the dual inhibition also suppresses cell migration in vitro. We further validated our results with CDK1 and HSP90 inhibitors that have entered clinical trials. Second-generation HSP90 inhibitor ganetespib and Ro3306 synergistically inhibit cell viability in HCT116 cells. We have previously demonstrated that CDK4 also contributes to the stabilization of HIF1α under hypoxia (Warfel et al., Cell Cycle, 2013). Interestingly, the combination of CDK4 and HSP90 inhibitors is also able to improve downregulation of HIF1α as compared to either agent alone in various cancer cell lines (eg. colon cancer, gliobastoma, etc). The FDA-approved CDK4 inhibitor palbociclib along with ganetespib synergistically inhibits cell viability in HCT116 cells under hypoxia. Such effect is not observed in WI38 normal cells under normoxia at similar concentrations. Our findings demonstrate that CDK1 inhibition disrupts the interaction between HSP90 and HIF1α providing a rationale for therapeutic targeting of HIF1α through the combination of CDK1/4 and HSP90 inhibitors in cancer. Ongoing studies are examining the effects of CDK1/4 plus HSP90 dual inhibition in other tumor types and in vivo. Citation Format: Shuai Zhao, David T. Dicker, Wafik S. El-Deiry. Dual inhibition of CDK1 and HSP90 destabilizes HIF1α and synergistically induces cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4516. doi:10.1158/1538-7445.AM2017-4516

Single Agent and Combinatorial Efficacy of First-in-Class Small Molecule ONC201 in Acute Leukemia and Multiple Myeloma

ONC201 is the founding member of the imipridone class of anti-cancer small molecules that possess a unique core chemical structure. ONC201 is currently being evaluated in several Phase I/II clinical trials for advanced cancers. In the current study, we evaluated the single agent and combinatorial efficacy of ONC201 in preclinical models of acute leukemia and multiple myeloma (MM).In acute leukemia, we evaluated ONC201 anti-cancer effects in acute myeloid leukemia (AML) (Kasumi-1, HL60) and acute lymphoblastic leukemia (ALL) (Reh, Jurkat and MOLT-4) cell lines. We observed a time- and dose-dependent decrease in cell viability for every cell line in the panel (EC50 1-5 µM). Vincristine-resistant cells HL60/VCR were also sensitive to single agent ONC201 with EC50 values on par with corresponding vincristine-sensitive parental cells. Dose- and time-dependent induction of apoptosis was noted in Western blot analysis of caspase-3 cleavage in AML cell lines treated with 2.5 µM or 5 µM of ONC201 for 48 hr. Western Blot analysis further demonstrated inhibition of Akt and Foxo3a phosphorylation in Kasumi-1 cells, in line with the previously reported late-stage signaling effects of ONC201 in solid tumor cells (Allen et al, 2013). Sub-G1 analysis indicated that ONC201 induces apoptosis in ALL cells and a pan-caspase inhibitor reduced ONC201-mediated apoptosis. Western blot analysis revealed ONC201-mediated apoptosis involves PARP cleavage and caspase-9 activation in ALL cells. Anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xl were downregulated while the pro-apoptotic Bcl-2 family member Bim is upregulated in response to ONC201 treatment in ALL cells. ONC201 also downregulates the inhibitor of apoptosis (IAP) family proteins cIAP1 and cIAP2 in ALL cells. We observed inhibition of Akt phosphorylation upon ONC201 treatment of ALL cells. Fresh AML patient cells were also found to be sensitive to ONC201 in cell viability and caspase 3/7 activity assays at 5µM.We observed that independent clones of cancer cells with acquired resistance to ONC201 were more sensitive to cytarabine compared to parental ONC201-sensitive cancer cells. In addition, ONC201 demonstrated synergistic reduction in cell viability in combination with cytarabine in AML cell lines. Determination of combination indices (CI) revealed synergy at several concentrations (CI 0.336-0.75 in CMK cells). Also, ONC201 combined additively with midostaurin in CMK cells and vincristine in HL60/VCR cells. Thus, ONC201 is a promising combinatorial partner for AML therapies based on these preclinical sensitization results.In accordance with ONC201-mediated activation of the integrated stress response that B cells are highly sensitive to (Kline et al and Ishizawa et al, 2016), MM was identified as one of the most ONC201-sensitive tumor types in the Genomics of Drug Sensitivity in Cancer collection of cell lines. Three human MM cell lines were used for validation (KMS18, MM.1S and RPMI-8226), which revealed a time- and dose-dependent decrease in cell viability (EC50 1-2.5 µM). Bortezomib-resistant cells MM.1S 33X were sensitive to ONC201 as a single agent with EC50 values comparable to bortezomib-sensitive parental cells. We observed an average of 10-fold induction of ONC201-mediated apoptosis using Sub-G1 analyses in MM cells at 5 µM, 48 hrs post-treatment. Rescue of ONC201-mediated apoptosis was demonstrated using the pan-caspase inhibitor (Z-VAD-FMK). In addition, Western blot analysis in MM cells indicated a dose-dependent decrease in the anti-apoptotic protein XIAP which is a key mediator of apoptosis inhibition and is reported to be highly up-regulated in MM cells. Furthermore, ONC201 demonstrated synergistic reduction in cell viability at various concentrations in combination with either ixazomib or dexamethasone, which are used in the clinical treatment of MM, in RPMI8226 cells (CI 0.228-0.75). Also, ONC201 combined additively with bortezomib in RPMI8226 and MM.1S 33X cells.In summary, these preclinical studies support the ongoing ONC201 single agent trials in acute leukemias and MM. Our findings suggest that ONC201 may be an important therapeutic option for patients with hematological malignancies who have developed resistance to approved therapies. Additionally, our results point to specific standard-of-care therapies that may be combined with ONC201 to exert durable responses without adding to the burden of toxicity. DisclosuresPrabhu:Oncoceutics: Employment. Tarapore:Oncoceutics: Employment, Equity Ownership. Oster:Oncoceutics: Employment, Equity Ownership. Allen:Oncoceutics: Employment, Equity Ownership. El-Deiry:Oncoceutics: Equity Ownership.

Abstract 3772: SHP-1 determines the radiosensitivity of liver cancer cell and dovitinib acts as a novel radiosensitizer in hepatocellular carcinoma via targeting SHP-1/STAT3 signaling

Abstract Background Hepatocellular carcinoma (HCC) is one of the most lethal human malignancies and curative therapy is not an option for most patients. There is growing interest in the potential benefit of radiotherapy (RT)-integrated therapy. This study aimed to investigate the biological impacts of a novel tumor suppressor Src homology 2 (SH2) domain-containing phosphatase 1 (SHP-1) and its downstream effecter, STAT3, in regulating the radiosensitivity of HCC cells. Furthermore, we explored the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. Material and methods To understand the impacts of SHP-1/STAT3 signaling affects radiosensitivity, HCC cells with ectopic expression of STAT3, SHP-1 and a catalytic mutant SHP-1 were treated with or without radiotherapy and analyzed by flow cytometry and colony formation assay. Furthermore, five HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T and Huh-7) were treated with dovitnib, RT or both. The in vitro and in vivo effects of above-mentioned treatments were analyzed. Results By sub-G1 analysis and colony formation, we found that HCC cell with ectopic expression of SHP-1 was much more sensitive to RT-induced apoptotic effects, while overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Next, we investigated the effects of dovitinib, which showed that dovitinib treatment resulted in SHP-1-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1 diminished the effects of dovitinib on HCC cells. Furthermore, by ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Importantly, we found that dovitinib potentiated the in vitor and in vivo effects of RT in HCC cells through affecting the SHP-1/STAT3 signaling. Conclusions SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. A combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced in vitro and in vivo anti-HCC effects. Citation Format: Man-Hsin Hung, Chao-Yuan Huang, Wei-Tien Tai, Ming-Hsien Tsai, Chih-Ting Shin, Szu-Yuan Wu, Chung-Wai Shiau, Kuen-Feng Chen. SHP-1 determines the radiosensitivity of liver cancer cell and dovitinib acts as a novel radiosensitizer in hepatocellular carcinoma via targeting SHP-1/STAT3 signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3772.

RHAMM splice variants confer radiosensitivity in human breast cancer cell lines.

Biomarkers for prognosis in radiotherapy-treated breast cancer patients are urgently needed and important to stratify patients for adjuvant therapies. Recently, a role of the receptor of hyaluronan-mediated motility (RHAMM) has been suggested for tumor progression. Our aim was (i) to investigate the prognostic value of RHAMM in breast cancer and (ii) to unravel its potential function in the radiosusceptibility of breast cancer cells. We demonstrate that RHAMM mRNA expression in breast cancer biopsies is inversely correlated with tumor grade and overall survival. Radiosusceptibility in vitro was evaluated by sub-G1 analysis (apoptosis) and determination of the proliferation rate. The potential role of RHAMM was addressed by short interfering RNAs against RHAMM and its splice variants. High expression of RHAMMv1/v2 in p53 wild type cells (MCF-7) induced cellular apoptosis in response to ionizing radiation. In comparison, in p53 mutated cells (MDA-MB-231) RHAMMv1/v2 was expressed sparsely resulting in resistance towards irradiation induced apoptosis. Proliferation capacity was not altered by ionizing radiation in both cell lines. Importantly, pharmacological inhibition of the major ligand of RHAMM, hyaluronan, sensitized both cell lines towards radiation induced cell death. Based on the present data, we conclude that the detection of RHAMM splice variants in correlation with the p53 mutation status could help to predict the susceptibility of breast cancer cells to radiotherapy. Additionally, our studies raise the possibility that the response to radiotherapy in selected cohorts may be improved by pharmaceutical strategies against RHAMM and its ligand hyaluronan.

ONC201 Induces Apoptosis in Cutaneous T-Cell Lymphoma Cells through a Mechanism That Involves the Integrated Stress Response and Inactivation of Jak/Stat Signaling

Background: ONC201 (also called TIC10) is an orally active, first-in-class small molecule that is in phase II clinical trials for advanced cancers based on its ability to activate apoptosis in tumor cells, but not normal cells, in a p53-independent manner. Recent studies have implicated the integrated stress response as an early stage mechanism of ONC201 that may lead to the previously observed downstream anti-cancer effects. This study was conducted to evaluate the anti-tumor activity of ONC201 on Cutaneous T-Cell Lymphoma (CTCL) cell lines and Sézary cells.Methods: We treated 8 CTCL cell lines (H9, HH, Hut78, Mac2A, MJ, MyLA, Pb2b and SeAx), peripheral blood mononuclear cells (PBMC) from 5 Sézary syndrome (SS) patients and 6 healthy donors with ONC201. MTS viability Assay was used to assess the anti-proliferation effect. Apoptosis was assessed using Annexin V FITC/PI staining and sub-G1 analysis by flow cytometry. Protein expression by western blotting was analyzed in 3 CTCL cell lines (HH, Hut78, MJ) and in PBMCs from 2 SS patients treated for 72 hours with ONC201including Activating Transcription Factor 4 (ATF4), interferon regulatory factor 7 (IRF7/pIRF7), Signal transducer and activator of transcription 3 (STAT3), Janus Kinase 3 (JAK3/pJAK3) and NF-κB family members (p65, RelB, c-Rel and p105).Results: MTS viability assay showed pronounced cell growth inhibition, significantly increasing in a time-dependent manner in H9, HH, Hut78, Mac2A, MJ, MyLa, Pb2b, and SeAx lines after a 96-hour incubation within a very narrow efficacy threshold ranging from 1.25 to 10µM (n = 8, p < 0.05). To determine whether growth inhibition of ONC201 is due to cell-cycle arrest and/or to apoptosis in CTCL cell lines, sub-G1 analysis by flow cytometry was measured in 3 CTCL cell lines (HH, Hut78 and MJ). The percentages of HH, Hut78 and MJ cells with sub-G1 population increased, suggesting that cells are undergoing apoptosis (n = 3, p < 0.05). Induction of apoptosis was further confirmed by Annexin V FITC/PI staining, revealing dose and time dependent apoptosis induction in all 8 cell lines (Figure 1). To confirm cell line results in refractory ex vivo samples, we tested the pro-apoptotic effects of ONC201 on PBMCs from 5 SS patients who had high circulating CD4+CD26- malignant T-cells compared with PBMCs from 6 healthy donors. ONC201 induced significant levels of apoptosis in PBMCs from SS patients, but not in normal PBMCs (Figure 1, p < 0.001). Western blot analysis revealed that ONC201 increased the expression of ATF4, a hallmark of the integrated stress response and a negative regulator of IRF7. ONC201 decreased expression of IRF7/pIRF7 and down-regulated JAK3/pJAK3 and STAT3 expression in CTCL cell lines and PBMCs from SS patients. ONC201 also decreased the protein expression of NF-κB family members ( p65, RelB, c-Rel and p105) that can cause resistance to apoptosis in CTCL cells.Conclusions: ONC201 appears to be active as a single oral agent. It may impact key signaling pathways in CTCL preclinical models by inhibiting proliferation and inducing apoptosis through a mechanism that involves the integrated stress response, leading to inactivation of JAK/STAT signaling and down-regulation of the NFκB pathway. [Display omitted] DisclosuresDuvic:Eisai: Research Funding; MiRagen Therapeutics: Consultancy; Cell Medica Ltd: Consultancy; Tetralogics SHAPE: Research Funding; Soligenics: Research Funding; Array Biopharma: Consultancy; Innate Pharma: Research Funding; Allos (spectrum): Research Funding; Rhizen Pharma: Research Funding; Spatz Foundation: Research Funding; Oncoceutics: Research Funding; Huya Bioscience Int'l: Consultancy; Therakos: Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Most drugs for ctcl are non approved. This drug is a small molecule and is not approved for any cancer. Tarapore:Oncoceutics, Inc: Employment, Equity Ownership. Allen:Oncoceutics, Inc: Employment, Equity Ownership.

Abstract 3494: Combined MEK1/2 and PI3K inhibition induces synergistic caspase-dependent apoptosis in neuroblastoma

Abstract Background: Neuroblastoma is a pediatric cancer of the developing sympathetic nervous system that is often widely metastatic at diagnosis. Despite the intensive multimodal therapy patients receive including surgery, chemoradiotherapy, stem cell transplantation and immunotherapy, cure rates remain less than 40% for patients with high-risk disease. The identification of MAPK pathway genomic aberrations (NRAS mutations, loss of NF1) in primary and relapsed disease lends relevance to the use of MEK1/2 inhibitors in the treatment of neuroblastoma. Although PI3K and PTEN mutations are rarely detected in neuroblastoma, activation of the PI3K/Akt axis is described in primary tumors and is a well-defined mechanism of resistance to MEK inhibition in other solid tumors. We hypothesize that combined inhibition of MEK and PI3K will synergize to reduce neuroblastoma cell survival and proliferation. Methods: A panel of human-derived neuroblastoma cell lines (N = 15) was used to analyze the effect of combined binimetinib (MEK162, MEK1/2 inhibitor) and alpelisib (BYL719, alpha-specific PI3K inhibitor) treatment. Effects on cell survival were assayed with CellTiter-Glo® and pathway (MAPK, PI3K/Akt, mTOR) inhibition was analyzed following both single agent and combination treatments. Synergy was determined using the Chou-Talalay method. Additional cell death analysis included flow cytometry (sub-G1, Annexin V) and immunoblotting (BIM, cleaved-PARP). In vivo efficacy testing is ongoing in neuroblastoma xenograft models (N = 3) with defined MAPK aberrations. Results: Treatment with both binimetinib and alpelisib in 15 neuroblastoma cell line models induced a synergistic (N = 14; CI value range 0.098-0.637) or additive (N = 1; CI = 0.951) anti-proliferative response. The mechanism for the additive cytotoxicity was the conversion of binimetinib-induced cell cycle arrest to the induction of programmed cell death, as measured by morphology, sub-G1 analysis and PARP cleavage. Furthermore, the cell death response was rescued by treatment with the caspase inhibitory peptide, QVD-OPh. By immunoblot analysis, combination treatment decreased the levels of phospho-ERK, phospho-Akt, phospho-S6K, beyond that observed with ether single agent. Neuroblastoma xenograft studies are ongoing with models selected based on the presence of MAPK pathway aberrations, including NRAS mutations and copy number loss of NF1, and in vitro sensitivity to the combination. Conclusions: Synergistic activity of combined MEK1/2 and PI3K inhibition in human neuroblastoma cell lines via the induction of apoptosis was observed. Combined binimetinib and alpelisib treatment suppresses the activation of MAPK, PI3K/Akt and mTOR. The in vitro studies and preclinical in vivo models are expected to form the basis of testing binimetinib-alpelisib in neuroblastoma clinical trials designed to enrich for subjects with canonical MAPK activating lesions. Citation Format: Lori S. Hart, Lucy Chen, Vandana Batra, Matthew Tsang, Pichai Raman, Giordano Caponigro, Shiva Krupa, Markus Boehm, Malte Peters, John M. Maris. Combined MEK1/2 and PI3K inhibition induces synergistic caspase-dependent apoptosis in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3494. doi:10.1158/1538-7445.AM2015-3494

Abstract 108: Inhibition of demethylase, JMJD3 sensitizes diffuse large B-cell lymphoma (DLBCL) to chemotherapy

Abstract Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma (NHL) representing more than 30% of new cases diagnosed in the United States. DLBCL comprises multiple biologically and clinically distinct subtypes including germinal center B-cell-like (GCB) and activated B-cell like (ABC) DLBCL. Despite improvement in current therapies, nearly 40% of patients die from the disease. Recent studies of the epigenome and the methylome in particular, have revealed widespread epigenetic changes in DLBCL. Aberrant methylation of core histone tails occur with deregulation of methylase enzymes such as Ezh2 in DLBCL. In contrast, the role of JMJD3 and UTX, H3K27(me)3 demethylases in DLBCL remains unknown. In this study, we have evaluated the activity of the selective JMJD3 inhibitor GSK-J4 in DLBCL and investigated the mechanism of its action. Methods Panel of DLBCL cell lines: DB, KARPAS 422, Pfeiffer, Toledo, Sudhl2, Sudhl6, Sudhl8, TMD8, OCI-Ly1, OCI-Ly3, OCI-Ly7, OCI-Ly8, OCI-Ly4, OCI-Ly10, and primary DLBCL cells were analyzed for the expression of lysine demethylases (KDMs) using RT-PCR and Western blot analysis. Sub-G1 analysis was performed to analyze drug-induced apoptosis. Chemical inhibitor GSK-J4 and siRNAs were used for specific inhibition of the JMJD3. Results JMJD3 inhibitor, GSK-J4 induced the killing of DLBCL cell lines of both GC and ABC subtypes. Cell sensitivity to GSK-J4 could not be predicted from the expression of prognostic markers in DLBCL such as Bcl6, IRF4 and Ezh2. For further studies, we selected a cell line Sudhl6 (Bcl6 dependent), sensitive to the killing effect of GSK-J4. Treatment with GSK-J4 reduced the Bcl6 levels along with concomitant increase in IRF4 and FBXO11, negative regulators of Bcl6. Knockdown of JMJD3 by siRNAs also reduced the levels of Bcl6, implicating Bcl6 in the mechanism of action of GSK-J4. Additionally, GSK-J4 synergized in killing with vincristine, doxorubicin as well as proteasome inhibitors bortezomib and carfilzomib. Conclusion We demonstrate the potent effects of inhibition of H3K27(me)3 demethylase JMJD3 by GSK-J4 on proliferation and apoptosis of DLBCL cells. Our results clearly show synergistic effects of inhibition of demethylase with standard drugs in elimination of DLBCL cells. Note: This abstract was not presented at the meeting. Citation Format: Rohit Mathur, Lalit Sehgal, Zuzana Berkova, Sattva S. Neelapu, Felipe Samaniego. Inhibition of demethylase, JMJD3 sensitizes diffuse large B-cell lymphoma (DLBCL) to chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 108. doi:10.1158/1538-7445.AM2015-108

Abstract 4465: Sequential combination of docetaxel with a novel SHP-1 agonist enhanced anti-tumor effect in triple negative breast cancer cells

Abstract Background: Interfering the oncogenic STAT3 signaling is considered as promising anti-cancer strategy. STAT3 signaling can be negatively regulated by the protein tyrosine phosphatase SHP-1 through direct dephosphorylation of p-STAT3 (Tyr705). Our previous studies have confirmed SC-43 as a novel SHP-1 enhancer which down-regulate p-STAT3 and STAT3-related downstream effectors (Breast Cancer Research 2013). Here, we tested the efficacy of SC-43 in combination with docetaxel in triple negative breast cancer cells. Methods: TNBC cell lines (HCC-1937, MDA-MB-468, MDA-MB-231) were used for in vitro studies. Cell viability was examined by MTT assay. Combination index was determined using Calcusyn anaysis. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. In vivo efficacy of SC-43 in combination with docetaxel was tested in nude mice with breast cancer xenografts. Results: Sequential combination of docetaxel with SC-43 showed enhanced anti-proliferative activity in all tested TNBC cells. Synergistic effect was demonstrated when docetaxel combined with SC-43 at a dose ratio of 1 to 10, showing combination index below 1.0. Sub-G1 and western blot analysis showed that SC-43 enhanced docetaxel-induced apoptosis. In addition, combining SC-43 with docetaxel efficiently suppressed p-STAT3 and downstream effectors. Ectopic expression of STAT3 reduced the enhanced cytotoxicity by the combination therapy. Previous studies has shown SC-43 enhanced SHP-1 activity, here we showed that SC-43 in combination with docetaxel significantly enhanced SHP-1 activity. More importantly, intraperitoneal injection of SC-43 with tri-weekly docetaxel showed enhanced anti-tumor growth in xenografted tumor mice. Conclusion: Our results suggest that the novel SHP-1 agonist SC-43 enhanced docetaxel-induced cytotoxic killing by SHP-1 dependent STAT3 inhibition in human triple negative breast cancer cells. (Supported by Yen Tjing-Ling Medical Foundation CI-104-07; NSC-102-2325-B-075-006 and MOST 103-2325-B-075-002; and MOHW103-TD-B-111-02) Citation Format: Ling-Min Tseng, Chun-Yu Liu, Shiu-Ping Yang, Wan-Lun Wang, Jung-Chen Su, Chia-Yun Wu, Chung-Wai Shiau, Kuen-Feng Chen. Sequential combination of docetaxel with a novel SHP-1 agonist enhanced anti-tumor effect in triple negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4465. doi:10.1158/1538-7445.AM2015-4465

Abstract 2924: Suppressing SET reactivates PP2A function in EGFR wide-type NSCLC and synergizes with taxol to exert anti-cancer effects

Abstract Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-associated death in the world. Conventional chemotherapy is the standard-of-care for majority of patients with advance NSCLC and tyrosine kinase inhibitors of epithelial growth factor receptor (EGFR) only benefit patients with tumor containing specific mutation forms of EGFR. However, high disease recurrence rate after currently available therapeutics limited patient's survival. Therefore, there is a timely need to improve current treatment for patients with NSCLC, especially those with EGFR wide type tumors. Protein phosphatase 2A (PP2A) is an important tumor suppression whose function is inhibited in tumor tissue by the appearance of SET. In current study, we showed that inhibiting SET binding to PP2A by a novel small molecular compound significantly improved the chemosensitivity of EGFR wide-type NSCLC cells. Method: EGFR wide-type NSCLC cell lines, including A549, H358 and H460, were treated with taxol and a novel quinzoline derivative, PA, which was designed and developed to interrupt the binding of SET to PP2A. Apoptosis of cancer cell, signal transduction and phosphatase activity were analyzed after treatments. Combination index (CI) was applied to determine the synergistic effects of PA and taxol. Co-immunoprecipitation (co-IP) and proximity ligation assay (PLA) were used to identify in vivo binding between SET and PP2A. In vivo anti-tumor effects of treatment containing PA and taxol were also determined in A549 xenografted mice. Results: After treating the cancer cells with PA and taxol, apoptosis of cancer cells, in concordance with enhancing PP2A-dependent p-Akt downregulation, were observed in a dose-dependent manner in all EGFR wide-type NSCLC cell lines. CI determined by the results of sub-G1 analysis confirmed the synergy between PA and taxol. Overexpression of Akt and inhibition of PP2A diminished the therapeutic effects of this combination. By two different strategies, co-IP and PLA, we found that this combination treatment inhibited the association of SET and PP2A within lung cancer cells. When overexpression SET in treated cancer cells, the therapeutic effects of this combination were abolished. The anti-tumor effects of treatment combining PA and taxol were also validated in A549 xenografted mice. Our data suggested that PP2A inactivation determines the drug sensitivity of taxol in lung cancer cells, and by interfering the binding of SET and PP2A, PA significantly promote PP2A activity and increase the cytotoxic effects of taxol in EGFR wide-type NSCLC. Conclusion: By interfering the binding of SET to PP2A, the novel PP2A enhancer, PA, is shown to reactivate PP2A and work synergistically with taxol for the treatment of EGFR wide-type NSCLC. Citation Format: Man-Hsin Hung, Chung-Wai Shiau, Yung-Jen Hsiao, Hui-Chuan Yu, Wei-Tien Tai, Chun-Yu Liu, Cheng-Yi Wang, Kuen-Feng Chen. Suppressing SET reactivates PP2A function in EGFR wide-type NSCLC and synergizes with taxol to exert anti-cancer effects. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2924. doi:10.1158/1538-7445.AM2015-2924

Cationic Liposome- Multi-Walled Carbon Nanotubes Hybrids for Dual siPLK1 and Doxorubicin Delivery In Vitro.

PurposeTo formulate f-MWNTs-cationic liposome hybrids for the simultaneous delivery of siPLK1 and doxorubicin to cancer cells.Methodf-MWNTs-cationic liposome hybrids were prepared by the thin film hydration method where the lipid film was hydrated with 100 μg/ml or 1 mg/ml of ox-MWNTs-NH3+ or MWNTs-NH3+ in 5% dextrose. siRNA complexation and protection ability was determined by agarose gel electrophoresis. f-MWNTs and liposome interaction was evaluated using Nile Red (NR) fluorescence spectroscopy. Cellular uptake in A549 cells was assessed by flow cytometry. Silencing of target proteins was determined by Luciferase and MTT assays. Sub-G1 analysis was performed to evaluate apoptosis following co-delivery of siPLK1 and Doxorubicin (Dox).ResultsZeta potential and siRNA complexation profile obtained for all hybrids were comparable to those achieved with cationic liposomes. ox-MWNTs-NH3+ showed greater extent of interaction with cationic liposomes compared to MWNTs-NH3+. ox-MWNTs-NH3+ was able to protect siRNA from nuclease-mediated degradation. Enhanced cellular uptake of both the carrier and loaded siRNA in A549 cell, were observed for this hybrid compared to the liposomal carrier. A synergistic pro-apoptotic effect was obtained when siPLK1 silencing was combined with doxorubicin treatment for the hybrid:siRNA complexes compared to the lipoplexes, in A549 cells in vitro.Conclusionsf-MWNTs-cationic liposome hybrid designed in this study can serve as a potential vehicle for the co-delivery of siRNA and cytotoxic drugs to cancer cells in vitro.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-015-1707-1) contains supplementary material, which is available to authorized users.