Synergistic Inhibition Of Cell Proliferation Research Articles

Abstract 4711: Evaluation of effect of autophagy inhibition by SAR405, a selective Vps34 inhibitor, on proliferation of pleural mesothelioma cells

Abstract Pleural mesothelioma is a poor prognostic malignancy because the efficacy of therapies remains limited; thus, there is an urgent need to elucidate the pathophysiology leading to newer treatment modalities. Macroautophagy (hereafter referred to as autophagy) is an intracellular degradation system in which cytoplasmic components are degraded by the lysosome. Autophagy plays pivotal roles in various physiological processes, including adaptation to starvation, preimplantation embryonic development, elimination of intracellular pathogens, and regulation of innate and adaptive immunity. In cancer cells, autophagy may have the potential to function protectively for the cells and, alternatively, to work suppressively. It can promote tumor growth in established cancers through autophagy-mediated intracellular recycling, providing substrates for metabolism and maintain that the functional pool of mitochondria. On the other hand, it may lead to a cytotoxic effect called autophagy-dependent cell death. Several reports have demonstrated that autophagy has cytoprotective effects on pleural mesothelioma cell lines. In this study, we assessed effects of inhibition of autophagy using SAR405, which specifically inhibits Vps34 in an early step of autophagy, on pleural mesothelioma cells. Additionally, we investigated whether SAR405 could enhance the growth suppression induced by cisplatin in these cells. Mesothelioma cell lines, including NCI-H28 (H28), NCI-H2452 (H2452), and MSTO-211H (211H), were cultured with 0.1-50 μM SAR405 or 1.0-5.0 μM cisplatin for cell viability analysis using water-soluble tetrazolium salt-8, and cell cycle analysis. For the assessment of autophagy, a plasmid, pMRX-IP-GFP-LC3-RFP-LC3ΔG, was stably transfected into mesothelioma cells. Transfected cells generate GFP-LC3-RFP-LC3ΔG, and it is cleaved into GFP-LC3 and RFP-LC3ΔG by inherent ATG4. GFP-LC3 levels are decreased by autophagy activation, while RFP-LC3ΔG serves as an internal control. SAR405 dose-dependently suppressed cell viability, accompanied by a significantly increase in the proportions of cells in the G2/M phase. The IC50 of SAR405 at 72 hours was 11.5 μM for H28, 16.7 μM for H2452, and 14.9 μM for 211H. SAR405 inhibited autophagy 24 hours after treatment at 2.5 μM for H28 and 211H, and at 5.0 μM for H2452. Furthermore, cisplatin induced autophagy at 5.0 μM for H28, 0.5 μM for H2452, 1.0 μM for 211H. The combination of 2.5-10 µM SAR405 and 0.5-5.0 µM cisplatin exhibited additive or synergistic effects on cell growth inhibition in all plural mesothelioma cell lines. In conclusion, Vps34 inhibition by SAR405 suppressed cell growth in pleural mesothelioma cells, accompanied by autophagy inhibition. The combination of SAR405 and cisplatin resulted in a synergistic inhibition of cell proliferation. Citation Format: Yoshiki Kuwabara, Kosuke Sakai, Shigeru Ishi, Shin Yokosuka, Masatoshi Abe, Tomoyuki Takahashi, Yuichiro Kawano, Hiroaki Nishimura, Maiko Toda-Sasaki, Yumiko Kobayashi-Ogawa, Satoshi Kikuchi, Yusuke Hirata, Hiroyuki Kyoyama, Gaku Moriyama, Nobuyuki Koyama, Kazutsugu Uematsu. Evaluation of effect of autophagy inhibition by SAR405, a selective Vps34 inhibitor, on proliferation of pleural mesothelioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4711.

Abstract B047: Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma

Abstract Medulloblastoma is the most common pediatric malignant brain tumor. Polyamines, critical intracellular polycations governing key cell processes such as DNA replication, translation, and cell proliferation, are frequently upregulated in cancer. We previously showed that the polyamine pathway is critical to the growth and survival of pediatric diffuse midline gliomas and MYC amplified neuroblastoma. Given the activity of polyamine inhibition in DMG, and given the critical role of MYC in medulloblastoma, we sought to investigate the potential of polyamine pathway inhibition as a therapeutic approach for medulloblastoma. We compared the expression of regulators of the polyamine pathway in a cohort of pediatric medulloblastoma tumors from patients enrolled in the Zero Childhood Cancer (ZERO) Personalized Medicine Program and found significant over-expression compared with normal fetal brain samples. Analysis of Cavalli dataset of 763 medulloblastoma samples, found that high expression of polyamine biosynthetic genes predicted poor overall survival, and low expression of catabolic flux genes significantly correlated with better outcomes. We next treated medulloblastoma cells treated with a polyamine synthesis inhibitor, DFMO, and found that led to a compensatory increase in polyamine transporter protein expression. Conversely, treatment with the polyamine transport inhibitor AMXT 1501 led to compensatory increase in ODC1, the key polyamine synthetic enzyme. Dual targeting of the polyamine pathway, (DFMO and AMXT 1501) showed a potent and synergistic inhibition of cell proliferation and reduced the clonogenic potential of medulloblastoma cells in soft agar clonogenic assays. Flow cytometric analysis of Annexin V-stained cells showed that treatment with DFMO and AMXT 1501 in combination led to induction of apoptosis. Furthermore, we found that dual polyamine inhibition potently sensitized medulloblastoma cells, particularly Group 3, to the chemotherapeutic agent SN-38, leading to complete eradication of medulloblastoma cells in both cytotoxicity assays and clonogenic assays at nanomolar chemotherapeutic concentrations. We are currently validating this treatment strategy in aggressive orthotopic medulloblastoma models. Our findings suggest that inhibiting both polyamine synthesis and uptake holds promise for sensitizing highly aggressive pediatric medulloblastoma tumors to chemotherapy. Currently, AMXT 1501 combined with DFMO is undergoing evaluation in a Phase I clinical trial for adults (NCT05500508). Building on these promising results, we are developing a Phase I/II clinical trial for children with aggressive brain tumors through the international CONNECT consortium. Citation Format: Aaminah Khan, Anjana Gopalakrishnan, Jie Liu, Mark R. Burns, Maria Tsoli, David S. Ziegler. Inhibition of the polyamine pathway: a novel therapeutic approach to treat aggressive pediatric medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B047.

Ipatasertib exhibits anti‑tumorigenic effects and enhances sensitivity to paclitaxel in endometrial cancer invitro and invivo.

Endometrial cancer is the most common gynecologic cancer and one of the only cancers for which incidence and mortality is steadily increasing. Although curable with surgery in the early stages, endometrial cancer presents a significant clinical challenge in the metastatic and recurrent setting with few novel treatment strategies emerging in the past fifty years. Ipatasertib (IPAT) is an orally bioavailable pan‑AKT inhibitor, which targets all three AKT isoforms and has demonstrated anti‑tumor activity in pre‑clinical models, with clinical trials emerging for many cancer types. In the present study, the MTT assay was employed to evaluate the therapeutic efficacy of IPAT or IPAT in combination with paclitaxel (PTX) in endometrial cancer cell lines and primary cultures of endometrial cancer. The effect of IPAT and PTX on the growth of endometrial tumors was evaluated in a transgenic mouse model of endometrial cancer. Apoptosis was assessed using cleaved caspase assays and cellular stress was assessed using ROS, JC1 and tetramethylrhodamine ethyl ester assays. The protein expression levels of markers of apoptosis and cellular stress, and DNA damage were evaluated using western blotting and immunohistochemistry. IPAT significantly inhibited cell proliferation, caused cell cycle G1 phase arrest, and induced cellular stress and mitochondrial apoptosis in a dose dependent manner in human endometrial cancer cell lines. Combined treatment with low doses of IPAT and PTX led to synergistic inhibition of cell proliferation and induction of cleaved caspase 3 activity in the human endometrial cancer cell lines and the primary cultures. Furthermore, IPAT effectively reduced tumor growth, accompanied by decreased protein expression levels of Ki67 and phosphorylation of S6 in the Lkb1fl/flp53fl/fl mouse model of endometrioid endometrial cancer. The combination of IPAT and PTX resulted in increased expression of phosphorylated‑H2AX and KIF14, markers of DNA damage and microtubule dysfunction respectively, as compared with IPAT alone, PTX alone or placebo‑treated mice. The results of the present study provide a biological rationale to evaluate IPAT and the combination of IPAT and PTX in future clinical trials for endometrial cancer.

Enhanced Sestrin expression through Tanshinone 2A treatment improves PI3K-dependent inhibition of glioma growth

Glioblastomas are a highly aggressive cancer type which respond poorly to current pharmaceutical treatments, thus novel therapeutic approaches need to be investigated. One such approach involves the use of the bioactive natural product Tanshinone IIA (T2A) derived from the Chinese herb Danshen, where mechanistic insight for this anti-cancer agent is needed to validate its use. Here, we employ a tractable model system, Dictyostelium discoideum, to provide this insight. T2A potently inhibits cellular proliferation of Dictyostelium, suggesting molecular targets in this model. We show that T2A rapidly reduces phosphoinositide 3 kinase (PI3K) and protein kinase B (PKB) activity, but surprisingly, the downstream complex mechanistic target of rapamycin complex 1 (mTORC1) is only inhibited following chronic treatment. Investigating regulators of mTORC1, including PKB, tuberous sclerosis complex (TSC), and AMP-activated protein kinase (AMPK), suggests these enzymes were not responsible for this effect, implicating an additional molecular mechanism of T2A. We identify this mechanism as the increased expression of sestrin, a negative regulator of mTORC1. We further show that combinatory treatment using a PI3K inhibitor and T2A gives rise to a synergistic inhibition of cell proliferation. We then translate our findings to human and mouse-derived glioblastoma cell lines, where both a PI3K inhibitor (Paxalisib) and T2A reduces glioblastoma proliferation in monolayer cultures and in spheroid expansion, with combinatory treatment significantly enhancing this effect. Thus, we propose a new approach for cancer treatment, including glioblastomas, through combinatory treatment with PI3K inhibitors and T2A.

Arsenic compound sensitizes homologous recombination proficient ovarian cancer to PARP inhibitors

The poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors show survival benefits in ovarian cancer patients with BRCA1/2 mutation or homologous recombination (HR) deficiency, but only limited efficacy in HR-proficient ones. Another drug, arsenic trioxide (ATO) or arsenic drug (RIF), exerts antitumor effects via inducing DNA damage. Here, we investigated the combined therapeutic effects of the PARP inhibitors and the arsenic compound in HR-proficient ovarian cancer. The combined treatment of niraparib, olaparib, or fluazolepali with ATO showed a significant suppression in tumor cell viability and colony formation. The drug treatment also induced synergistic inhibition of cell proliferation and DNA damage, and acceleration of cell apoptosis in two HR-proficient ovarian cancer cell lines SKOV3 and CAOV3, either by simultaneous or sequential administration. The mechanism underlying these synergistic effects were reflected by the significantly increased ratio of cleaved-PARP/total PARP and decreased ratio of p-AKT/total AKT. Consistently, the combination of olaparib with RIF synergistically reduced the tumor growth in mouse xenograft models. In conclusion, the arsenic compound greatly sensitizes HR-proficient ovarian cancer cells to the PARP inhibitors, and our findings provide an evidence for the clinical treatment development of this combination in HR-proficient ovarian cancer patients.

Andrographolide Induces Apoptosis in Gastric Cancer Cells through Reactivation of p53 and Inhibition of Mdm-2.

Andrographolide is a labdane diterpenoid isolated from Andrographis paniculata. The plant extract and andrographolide has long been used in traditional medicine practices mainly for gastrointestinal diseases and improving liver function. Andrographolide has shown various pharmacological properties including anti-inflammatory, antioxidant and anticancer activity. This study evaluated the effect of andrographolide on proliferation of human gastric carcinoma cells in relevance to p53 and Mdm-2 pathways. Andrographolide inhibited the proliferation of SGC7901 and AGS cells in a dose-dependent manner with estimated IC50 values 38 and 44 μM respectively. Effect of andrographolide on p53 activity was ascertained by using a p53 activator (RITA) which showed synergistic inhibition of cell proliferation. While andrographolide when used in combination with a p53 inhibitor (pifithrin-α) showed potent restriction over its response. Andrographolide caused decrease in mitochondrial membrane potential as an indicator of apoptotic activity. Andrographolide activated the expression of p53 protein and gene and downregulated the levels of Mdm-2 (negative regulator of p53). Andrographolide inhibited the colony formation abilities in SGC7901 in a p53-dependent manner followed by induction of mitochondrial intrinsic apoptosis through activation of caspases-9 and -3, cleavage of PARP, and inhibition of pro-apoptotic Bcl-2. Andrographolide induced p53 mediated apoptosis in gastric carcinoma cells which adds to a novel approach in anticancer therapies.

A kinome-centered CRISPR-Cas9 screen identifies activated BRAF to modulate enzalutamide resistance with potential therapeutic implications in BRAF-mutated prostate cancer

Resistance to drugs targeting the androgen receptor (AR) signaling axis remains an important challenge in the treatment of prostate cancer patients. Activation of alternative growth pathways is one mechanism used by cancer cells to proliferate despite treatment, conferring drug resistance. Through a kinome-centered CRISPR-Cas9 screen in CWR-R1 prostate cancer cells, we identified activated BRAF signaling as a determinant for enzalutamide resistance. Combined pharmaceutical targeting of AR and MAPK signaling resulted in strong synergistic inhibition of cell proliferation. The association between BRAF activation and enzalutamide resistance was confirmed in two metastatic prostate cancer patients harboring activating mutations in the BRAF gene, as both patients were unresponsive to enzalutamide. Our findings suggest that co-targeting of the MAPK and AR pathways may be effective in patients with an activated MAPK pathway, particularly in patients harboring oncogenic BRAF mutations. These results warrant further investigation of the response to AR inhibitors in BRAF-mutated prostate tumors in clinical settings.

Dual targeting of EZH2 and androgen receptor as a novel therapy for castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) emerges after androgen withdrawal therapy and remains incurable due to the lack of effective treatment protocols. Treatment with enzalutamide, a second generation androgen receptor (AR) antagonist, offers an initial response followed by drug resistance and tumor relapse. Enhancer of zeste homolog 2 (EZH2), a member of PRC2 complex, is an important target that acts as a coactivator of AR-mediated gene suppression whose oncogenic activity increases during castration. We hypothesize that dual targeting of EZH2 and AR could be highly effective in CRPC treatment. The present study aimed to examine the effectiveness of combination using EZH2 inhibitor GSK126 with antiandrogen enzalutamide in the treatment of CRPC cells. Treatment of 22Rv1 and C42B CRPC cells with a combination of GSK126 and enzalutamide led to synergistic inhibition of cell proliferation, cell cycle arrest and marked increase in cell death. Mechanistically, this combination treatment significantly reduced expression of AR and AR-v7, decrease in PSA and Akt activity, diminution of EZH2 and other members of PCR2 complex including SUZ12 and EED, with simultaneous loss of H3K27 trimethylation and dissociation between AR and PRC2 complex members compared to individual treatment. This study provides preclinical proof-of-concept that combined treatment of EZH2 inhibitor with AR antagonist results in synergistic anticancer effects opening new possibilities for treatment of CRPC tumors.

Abstract 309: FAK blockade enhances antitumor effect of BTK inhibitor in esophageal squamous cell carcinoma via EGFR-ERK-Akt pathway inhibition

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is a fatal disease with poor prognosis and lack of effective targeted therapy. Recently, some researches have shown that BTK can be a novel therapeutic target for esophageal cancer, while the mechanism remains unknown. In addition, FAK is one of the important factors to promote the metastasis of esophageal cancer. In this study, we sought to explore the synergistic antitumor effect and underline mechanism of combining BTK inhibitor Ibrutinib with a novel FAK inhibitor APG-2449 for esophageal squamous cell carcinoma. Methods: Cell viability and growth inhibition were determined by CCK-8 assay and colony formation assay. Migration ability was detected by Transwell assay. EMT markers and ki67 were measured by RT-qPCR. Cell cycle analysis and apoptosis were confirmed by flow cytometry. Western Blot analysis and siRNA interference for mechanism exploration. ESCC xenograft models were established to evaluated the synergistic antitumor effect in vivo. Results: We found that ESCC cell lines harbor EGFR or c-myc-amplified exhibited more sensitive to Ibrutinib inhibition. Moreover, Ibrutinib combined with APG-2449 showed synergistic inhibition of cell proliferation in ESCC cell lines. By analyzing data from the Gene Expression Profiling Interactive Analysis database, we found that EGFR expression positively correlated with FAK expression in ESCC samples. The combination of Ibrutinib and APG-2449 exerted obviously synergistic apoptosis induction and colony formation inhibition. Furthermore, we found that Ibrutinib combined with APG-2449 could induce more cancer cells arrested in G1/S phase. In addition, the co-treatment of Ibrutinib and APG-2449 could significantly suppress ESCC cells migration by upregulating E-cadherin. Ibrutinib alone could down-regulate the protein expression of p-EGFR and p-ERK. Moreover, combination treatment significantly reduced the expression of p-Akt and p-c-myc, and upregulated P21 and PUMA expression. In ESCC xenotransplantation models, single therapy with either Ibrutinib or APG-2449 was equivalent in delaying tumor growth, while the combination therapy demonstrated more significantly tumor growth suppression. Conclusions: Taken together, our data strongly suggest that the combination of FAK inhibitor APG-2449 and Ibrutinib provide an effective and precise therapeutic strategy for treating esophageal squamous cell carcinoma. Citation Format: Qiu-Yun Luo, Su-Na Zhou, Wen-Tao Pan, Meng-xian Pan, Lu-ping Yuan, Lin Zhang, Xiang-Lei Yan, Yu-xin Zhang, Jian Sun, Miaozhen Qiu, Da-Jun Yang. FAK blockade enhances antitumor effect of BTK inhibitor in esophageal squamous cell carcinoma via EGFR-ERK-Akt pathway inhibition [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 309.

Impact of the Anticancer Drug NT157 on Tyrosine Kinase Signaling Networks.

The small-molecule drug NT157 has demonstrated promising efficacy in preclinical models of a number of different cancer types, reflecting activity against both cancer cells and the tumor microenvironment. Two known mechanisms of action are degradation of insulin receptor substrates (IRS)-1/2 and reduced Stat3 activation, although it is possible that others exist. To interrogate the effects of this drug on cell signaling pathways in an unbiased manner, we have undertaken mass spectrometry-based global tyrosine phosphorylation profiling of NT157-treated A375 melanoma cells. Bioinformatic analysis of the resulting dataset resolved 5 different clusters of tyrosine-phosphorylated peptides that differed in the directionality and timing of response to drug treatment over time. The receptor tyrosine kinase AXL exhibited a rapid decrease in phosphorylation in response to drug treatment, followed by proteasome-dependent degradation, identifying an additional potential target for NT157 action. However, NT157 treatment also resulted in increased activation of p38 MAPK α and γ, as well as the JNKs and specific Src family kinases. Importantly, cotreatment with the p38 MAPK inhibitor SB203580 attenuated the antiproliferative effect of NT157, while synergistic inhibition of cell proliferation was observed when NT157 was combined with a Src inhibitor. These findings provide novel insights into NT157 action on cancer cells and highlight how globally profiling the impact of a specific drug on cellular signaling networks can identify effective combination treatments. Mol Cancer Ther; 17(5); 931-42. ©2018 AACR.

Synergistic inhibition of cell proliferation by combined targeting with kinase inhibitors and dietary xanthone is a promising strategy for melanoma treatment.

α-Mangostin is a dietary xanthone that displays various biological activities, and numerous reports have shown its efficacy in cancer prevention and inhibition. As most agents have been shown to be ineffective as single-agent therapy for malignant melanoma (MM), the principle of targeted chemotherapy for MM is to use effective inhibitors and combination methods. In this study, we tested the cytotoxicity of several kinase inhibitors, including the glycogen synthase kinase (GSK)-3 inhibitor CHIR99021, and rapamycin, in combination with a dietary xanthone, α-mangostin, by screening from a kinase inhibitor library for melanogenesis in SK-MEL-2 MM cells, and verified these by clone formation efficiency, terminal dUTP nick end labelling, and expression of apoptosis-related proteins. We also explored the molecular mechanisms for the apoptosis-inducing effects reported. We found a marked synergistic effect of CHIR99021 or rapamycin in combination with α-mangostin, which we verified through apoptosis-related methods. These data provide a strong rationale for the use of α-mangostin as an adjunct to GSK-3 inhibitor or mammalian target of rapamycin inhibitor treatment. The intrinsic mechanism behind α-mangostin might be inhibition of phosphatidylinositol 3-kinase/AKT signalling and autophagy, and induction of reactive oxygen species generation.

Cholesterol esterification inhibition and imatinib treatment synergistically inhibit growth of BCR-ABL mutation-independent resistant chronic myelogenous leukemia.

Since the advent of tyrosine kinase inhibitors (TKIs) such as imatinib, nilotinib, and dasatinib, chronic myelogenous leukemia (CML) prognosis has improved greatly. However, ~30–40% of patients develop resistance to imatinib therapy. Although most resistance is caused by mutations in the BCR-ABL kinase domain, 50–85% of these patients develop resistance in the absence of new mutations. In these cases, targeting other pathways may be needed to regain clinical response. Using label-free Raman spectromicroscopy, we evaluated a number of leukemia cell lines and discovered an aberrant accumulation of cholesteryl ester (CE) in CML, which was found to be a result of BCR-ABL kinase activity. CE accumulation in CML was found to be a cancer-specific phenomenon as untransformed cells did not accumulate CE. Blocking cholesterol esterification with avasimibe, a potent inhibitor of acyl-CoA cholesterol acyltransferase 1 (ACAT-1), significantly suppressed CML cell proliferation in Ba/F3 cells with the BCR-ABLT315I mutation and in K562 cells rendered imatinib resistant without mutations in the BCR-ABL kinase domain (K562R cells). Furthermore, the combination of avasimibe and imatinib caused a profound synergistic inhibition of cell proliferation in K562R cells, but not in Ba/F3T315I. This synergistic effect was confirmed in a K562R xenograft mouse model. Analysis of primary cells from a BCR-ABL mutation-independent imatinib resistant patient by mass cytometry suggested that the synergy may be due to downregulation of the MAPK pathway by avasimibe, which sensitized the CML cells to imatinib treatment. Collectively, these data demonstrate a novel strategy for overcoming BCR-ABL mutation-independent TKI resistance in CML.

Abstract 1089: Anti-tumor activity of selinexor is enhanced by palbociclib in preclinical models of HER2+ breast cancer

Abstract Introduction: XPO1 (exportin-1/ CRM1) inhibitor selinexor (KPT-330) is the first-in-class, orally bioavailable, clinical stage SINE (Selective Inhibitor of Nuclear Export) compound with marked anti-tumor activity towards solid and hematological malignancies. This activity can be further enhanced by other therapeutic agents. We have previously shown strong synergistic preclinical activity of selinexor plus trastuzumab against HER2+ breast cancer. In cancer models of acquired resistance to HER2-targeted therapies, G1/S phase cell cycle regulators Cyclin D1 and CDK4/6 are inappropriately activated. We therefore investigated the combinatorial effect of selinexor plus palbociclib, a CDK4/6 inhibitor, in HER2+ breast cancer models as a treatment option for recurrent and relapsed HER2+ breast cancers. Methods: The effects of selinexor or palbociclib single agents or in combination were tested in vitro with BT474 HER2+ breast cancer cell line. Total RNA and protein was extracted from cell lysates and analyzed by qPCR and immunoblots. In vivo, a subcutaneous BT474 xenograft mouse model was treated with selinexor (5 mg/kg or 15 mg/kg; qodx3) or palbociclib (50 mg/kg or 150 mg/kg; qd) single agents or in combination. Tumor growth and body weights were monitored for 60 days. Tumors were harvested and analyzed by immunohistochemistry (IHC). Results: Selinexor plus palbociclib was highly effective in vitro and in vivo in BT474 breast cancer cells. In in vitro assays, selinexor or palbociclib single agents demonstrated inhibitory effects on cell proliferation and showed strong synergy when combined. In vivo, the combination resulted in significant survival benefit and enhanced tumor growth inhibition compared to vehicle or either single agent. IHC analysis of xenograft tumors showed synergistic inhibition of cell proliferation by selinexor plus palbociclib. The Ki67 proliferation index determined by IHC was 25% for vehicle control, 20% for selinexor, 7% for palbociclib and 2% for the combination. Based on IHC analysis, the synergistic anti-tumor activity of selinexor plus palbociclib was achieved at multiple levels of the CDK4/6 pathway. Selinexor treatment increased p21, p27 and Rb nuclear staining. Both p21 and p27 are inhibitors of CDK4/6 while Rb is a negative regulator of cell cycle progression. CDK4/6 phosphorylates and inactivates Rb, which allows cell cycle progression. In selinexor as well as palbociclib treated samples, phosphorylated Rb in the nucleus decreased, indicating a down-regulation of the CDK4/6 pathway. Conclusion: Selinexor plus palbociclib shows synergistic inhibition of cell proliferation in vivo and in vitro in HER2+ breast cancer cell line BT474 by down-regulation of CDK4/6 pathway. This combination therapy warrants further investigation as an effective treatment option for recurrent and relapsed HER2+ breast cancer. Citation Format: Hua Chang, Sharon Friedlander, Trinayan Kashyap, Boris Klebanov, Christian Argueta, Oscar A. Gonzalez, Erkan Baloglu, Yosef Landesman, Sharon Shacham, Margaret Lee, William Senapedis. Anti-tumor activity of selinexor is enhanced by palbociclib in preclinical models of HER2+ breast cancer [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 1089. doi:10.1158/1538-7445.AM2017-1089

Abstract 3141: Combined cetuximab and pemetrexed therapy enhances cytotoxicity against crizotinib-resistant non-small cell lung cancer cells by downregulating thymidylate synthase

Abstract Although non-small cell lung cancer (NSCLC) cells with anaplastic lymphoma kinase (ALK)-rearranged initially show a dramatic response to crizotinib, these cells eventually develop resistance to crizotinib. This resistance is caused by secondary mutations and copy number gain in the ALK gene. However, other resistance mechanisms through activation of the bypass tracts have yet to be clearly elucidated. To investigate the mechanisms of acquired resistance to ALK fusion-directed treatment in NSCLC, we generated crizotinib-resistant NSCLC cell lines in vitro through chronic exposure of an ALK fusion NSCLC line (SNU2292) to crizotinib. Interestingly, the resultant SNU2292-CR cells maintained activation of epidermal growth factor receptor (EGFR) and expressed increased levels of transforming growth factor alpha (TGF-α), an EGFR ligand. Additionally, we found that thymidylate synthase (TS) was overexpressed in SNU2292-CR cells compared with the parental cells. We showed reduction of TS enhanced synergistic inhibition of cell proliferation when cetuximab, an EGFR inhibitor, was combined with pemetrexed, a TS inhibitor. As a result, combined therapy was found to exhibit a synergistic growth inhibitory effect against crizotinib-resistant cells by downregulating TS expression. Taken together, these results support a potential role of activation of the bypass tracts in acquired resistance to ALK-directed treatment in ALK-rearranged NSCLC, and provide insights into strategies for preventing and/or overcoming this resistance in patients Citation Format: Hwang-Phill Kim, Sae-Won Han, Sang-Hyun Song, Tae-You Kim. Combined cetuximab and pemetrexed therapy enhances cytotoxicity against crizotinib-resistant non-small cell lung cancer cells by downregulating thymidylate synthase [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 3141. doi:10.1158/1538-7445.AM2017-3141

Enhanced efficacy of AKT and FAK kinase combined inhibition in squamous cell lung carcinomas with stable reduction in PTEN.

Squamous cell lung carcinoma (SCC) accounts for 30% of patients with NSCLC and to date, no molecular targeted agents are approved for this type of tumor. However, recent studies have revealed several oncogenic mutations in SCC patients, including an alteration of the PI3K/AKT pathway, i.e. PI3K point mutations and amplification, AKT mutations and loss or reduced PTEN expression. Prompted by our observation of a correlation between PTEN loss and FAK phosphorylation in a cohort of patients with stage IV SCC, we evaluated the relevance of PTEN loss in cancer progression as well as the efficacy of a new combined treatment with the pan PI3K inhibitor buparlisip and the FAK inhibitor defactinib. An increase in AKT and FAK phosphorylation, associated with increased proliferation and invasiveness, paralleled by the acquisition of mesenchymal markers, and overexpression of the oncomir miR-21 were observed in SKMES-1-derived cell clones with a stable reduction of PTEN. Notably, the combined treatment induced a synergistic inhibition of cell proliferation, and a significant reduction in cell migration and invasion only in cells with reduced PTEN. The molecular mechanisms underlying these findings were unraveled using a specific RTK array that showed a reduction in phosphorylation of key kinases such as JNK, GSK-3 α/β, and AMPK-α2, due to the concomitant decrease in AKT and FAK activation. In conclusion, the combination of buparlisib and defactinib was effective against cells with reduced PTEN and warrants further studies as a novel therapeutic strategy for stage IV SCC patients with loss of PTEN expression.

Abstract LB-011: PAK1 inhibitor FRAX1036 sensitizes ovarian cancer cells with amplified 11q13 to cytotoxic effect of rottlerin

Abstract p21-activated kinases (PAKs) are Cdc42/Rac-activated serine-threonine protein kinases that regulate several key cancer-relevant signaling pathways, such as the Mek/Erk, PI3K/Akt, and Wnt/b-catenin. Pak1 is frequently overexpressed and/or hyperactivated in different human cancers, including human breast, ovary, prostate, and brain cancer, due to amplification of the PAK1 gene in an 11q13 amplicon. In our previous in vitro and in vivo studies we have shown that ovarian cancer cells with amplified/overexpressed Pak1 were significantly more sensitive to pharmacologic or genetic inhibition of Pak1 compared to cells without 11q13 amplification. In the present study we examined the combination effect of the Pak1 inhibitor FRAX1036 in ovarian cancer cells using ICCB Known Bioactives Library (Enzo). We found that the cytotoxic effect of FRAX1036 was significantly higher when combined with the PKC delta inhibitor, Rottlerin. We tested FRAX1036 alone and in combination with Rottlerin on 11q13-amplified human ovarian cancer cell lines with respect to cell proliferation, migration and apoptosis. FRAX1036 alone inhibited cell proliferation and migration in 11q13-amplified ovarian cancer cell lines. Co-administration of FRAX1036 with Rottlerin further synergized the inhibition of cell proliferation in vitro and decreased tumor growth in vivo. To explain the synergistic inhibition of cell proliferation induced by the FRAX1036/Rottlerin combination we analyzed Pak1 and PKC delta down-stream signaling molecules. Western blot data indicated that FRAX1036 and Rottlerin synergistically inhibited phosphorylation of critical signaling molecules such as MEK, ERK1/2, b-Catenin, and 4EBP1. Our findings suggest that Pak1 small molecule inhibitors in combination with Rottlerin hold potential clinical value as chemotherapeutic drugs for the ∼25% of ovarian cancers characterized by PAK1 gene amplification. Citation Format: Tatiana Prudnikova, Jonathan Chernoff. PAK1 inhibitor FRAX1036 sensitizes ovarian cancer cells with amplified 11q13 to cytotoxic effect of rottlerin. [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 LB-011.

Cooperation of Nutlin-3a and a Wip1 inhibitor to induce p53 activity.

Targeting the Mdm2 oncoprotein by drugs has the potential of re-establishing p53 function and tumor suppression. However, Mdm2-antagonizing drug candidates, e. g. Nutlin-3a, often fail to abolish cancer cell growth sustainably. To overcome these limitations, we inhibited Mdm2 and simultaneously a second negative regulator of p53, the phosphatase Wip1/PPM1D. When combining Nutlin-3a with the Wip1 inhibitor GSK2830371 in the treatment of p53-proficient but not p53-deficient cells, we observed enhanced phosphorylation (Ser 15) and acetylation (Lys 382) of p53, increased expression of p53 target gene products, and synergistic inhibition of cell proliferation. Surprisingly, when testing the two compounds individually, largely distinct sets of genes were induced, as revealed by deep sequencing analysis of RNA. In contrast, the combination of both drugs led to an expression signature that largely comprised that of Nutlin-3a alone. Moreover, the combination of drugs, or the combination of Nutlin-3a with Wip1-depletion by siRNA, activated p53-responsive genes to a greater extent than either of the compounds alone. Simultaneous inhibition of Mdm2 and Wip1 enhanced cell senescence and G2/M accumulation. Taken together, the inhibition of Wip1 might fortify p53-mediated tumor suppression by Mdm2 antagonists.

Abstract 2578: Synthetic lethal approaches to aurora inhibition in aggressive B-cell non-Hodgkin lymphoma

Abstract We explored synthetic lethality in the context of aurora inhibition with novel combination therapies for aggressive B-NHL. Aurora A and B are serine/threonine kinases and when amplified dysregulate the mitotic phase of the cell cycle. Pre-clinical and clinical evidence demonstrate that aurora inhibition has therapeutic benefit in aggressive B-NHL. Alisertib, an aurora inhibitor has single agent activity in B-NHL mouse xenograft tumors and clinical trials. Pre-clinical studies demonstrate Bruton's tyrosine kinase (BTK) is persistently expressed despite alisertib therapy. TMAs for DLBCL (S0313, S0515, N = 60) and MCL (S0213, S0601, N = 45) were generated to evaluate aurora A, B and BTK expression by IHC. We developed B-NHL cell lines resistant to alisertib to evaluate mechanisms of acute and chronic resistance and knock down of aurora A and B to evaluate Myc, Bcl2 and BTK expression. Mouse B-NHL xenograft models were conducted to demonstrate synthetic lethality of aurora plus BTK inhibition. We demonstrate synergistic inhibition of cell proliferation associated with apoptosis, particularly in ABC and double hit DLBCL and mantle cell lymphoma (MCL) cells. Ibrutinib completely inhibited BTK and AKT activity at 3.0μM without affecting BTK or AKT protein level in B-NHL cells that co-over-express Myc and Bcl-2. Aurora A and B knockdown show compromised oncogene expression. IHC indicated in DLBCL and MCL aurora B to be differentially over-expressed versus aurora A and BTK. Alisertib plus ibrutinib was highly synergistic in inhibiting cell proliferation and inducing apoptosis in B-NHL cells. Moreover, this effect was amplified with the addition of rituximab. Acute and chronic resistance to alisertib was due to ERK1/2 activation. Significantly greater apoptosis was induced in the triplet combination (alisertib + ibrutinib + rituximab) compared to doublet combinations (ibrutinib + rituximab or alisertib + ibrutinib or alisertib + rituximab). Mouse xenograft models of MCL (Granta-519) and double hit DLBCL (U2932) showed ibrutinib alone had no anti-B-NHL activity. Ibrutinib + alisertib lead to tumor growth inhibition (TGI) of ∼50% in MCL and ∼30% in DLBCL (p = 0.005). Interestingly, ibrutinib + rituximab lead to a TGI of ∼60% (p = 0.005) in both B-NHL mouse models. In contrast, triplet therapy with alisertib plus ibrutinib plus rituximab demonstrated a statistically significant tumor TGI of ∼70-90% (p = 0.005) in both B-NHL mouse models with an associated enhanced overall survival (>70 days). Harvested tumors at the end of treatment(s) demonstrated target inhibition and increased apoptosis with triple therapy. Inhibition of BTK plus rituximab in the presence of aurora inhibition is synthetic lethal in B-NHL and warrant clinical trial evaluation. Citation Format: Daruka Mahadevan, Carla Morales, Laurence Cooke, Manjari Pandey, Catherine Spier, Wenqing Qi. Synthetic lethal approaches to aurora inhibition in aggressive B-cell non-Hodgkin lymphoma. [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 2578. doi:10.1158/1538-7445.AM2015-2578

Abstract 3213: Rosehip (Rosa canina) extracts prevent MAPK and AKT-mediated cell proliferation in African American triple-negative breast cancer cells

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer, characterized by its lack of the human epidermal growth factor receptor-2 (HER-2), the estrogen receptor (ER), and the progesterone receptor (PR). The high prevalence of triple negative tumors in young women is more commonly observed in young African American (AA) women. Currently, the existing targeted therapy is of minimal benefit in TNBCs. Akt and MAPK have been shown to promote cell proliferation in triple negative breast cancer and MAPK expression may be an underlying mechanism contributing to the generation of chemo-resistance in triple-negative breast cancer. Due to major concerns involving the occurrence of side effects and the emergence of drug-resistant cancer cells, there has been growing interest in the use of naturally occurring molecules with chemo-preventive and chemo- therapeutic properties in cancer treatment. Rosehip extracts have been used as dietary supplements to relieve symptoms associated with diarrhea, gastritis, and rheumatoid arthritis and have been shown to prevent cell proliferation in glioblastomas. This study investigated the efficacy of rosehip extracts in preventing cell proliferation in African American triple negative (HCC70, HCC1806) and luminal (HCC1500) breast cancer cell lines. Each of the breast cancer cell lines were treated with rosehip extracts (1mg/mL -25ng/mL) demonstrated a significant decrease in cell proliferation. The rosehip extract-mediated decrease in cell proliferation was equal to or better than the decrease of cell proliferation observed when known inhibitors of the MAPK (U0126, 10 μM) or AKT (LY294002, 20 μM) signaling pathways were utilized. Additionally, pretreatment of these cell lines with these Rosehip extracts (1mg/mL -25ng/mL) selectively decreased AKT, MAPK, p70S6K, and S6 phosphorylation suggesting these extracts prevent AA TNBC cell proliferation by blocking both the MAPK and AKT signaling mechanisms. Results from cell cycle analysis by flow cytometry, western blot analysis, as well as apoptosis studies demonstrate that rosehip extracts inhibit cell proliferation but do not promote apoptosis. Rosehip extracts also have a synergistic effect with Doxorubicin (20µM), a chemotherapeutic agent used to treat patients with breast cancer, in preventing triple negative breast cancer cell proliferation. Taken together these data suggest that rosehip extracts are capable of decreasing cell proliferation in African American triple negative breast cancer cells without promoting apoptosis and demonstrate a synergistic inhibition of cell proliferation with Doxorubicin, and rosehip extracts may serve as an alternative or compliment to current chemotherapeutic regimens for triple negative breast cancer. Citation Format: Patrice Cagle, Tonisha Coburn, Patrick M. Martin. Rosehip (Rosa canina) extracts prevent MAPK and AKT-mediated cell proliferation in African American triple-negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2014-3213

Abstract CT405: A phase 1 study of neoadjuvant chemotherapy with gemcitabine plus nab-paclitaxel, followed by concurrent chemoradiation with gemcitabine, sorafenib, and vorinostat in locally advanced pancreatic cancer

Abstract Background: A third of pancreatic tumors initially deemed borderline or unresectable at diagnosis are ultimately able to undergo resection after neoadjuvant therapy. Survival rates improve significantly when curative resection with negative margins is achieved. Gemcitabine-based chemoradiation following combination chemotherapy is a common neoadjuvant regimen. Combination of gemcitabine and nab-paclitaxel is shown to be beneficial in metastatic disease, and is undergoing evaluation for use in neoadjuvant setting. Sorafenib is shown to reduce activation of c-kit, ERK, and VEGFR2 while gemcitabine inhibits Akt phosphorylation, leading to the synergistic inhibition of cell proliferation and the induction of apoptosis. Vorinostat has also been shown to sensitize pancreatic cancer cell lines to gemcitabine by enhancing the pro-apoptotic actions of gemcitabine, as evidenced by increased levels of the cleaved form of PARP and caspase 3 activities. Sorafenib and vorinostat kill carcinoma cells synergistically by activating CD95. Vorinostat appears to enhance tyrosine-phosphorylation of CD95 in a Src-dependent fashion, which also increases PDGFRβ inhibition by sorafenib. The combination also potently radiosensitizes pancreatic cancer cells in vivo by inducing cytosolic calcium, increasing reactive oxygen species and promoting CD95 activation. Full-dose radiation therapy with both agents has not been investigated in the clinical setting to date, and this represents a novel treatment modality appropriate for phase 1 study in a disease with limited treatment options. Methods: This is a phase 1 study using a traditional 3+3 dose-escalation design for the treatment combination dose escalation to determine appropriate phase 2 doses of sorafenib, vorinostat, and gemcitabine with radiation. Patients are treated with two cycles of combination chemotherapy with gemcitabine and nab-paclitaxel initially, followed by chemoradiation with the investigational agents with 50.4Gy of radiation. Patients will have locally advanced, non-metastatic pancreatic cancer with borderline resectable, unresectable, or node-positive disease, with ECOG performance status of 0 or 1, and preserved hepatic and endocrine pancreatic function with adequate bone marrow reserve by laboratory values. Patients who had prior radiation therapy, known intolerance to the agents, or other significant comorbidities such as poor cardiac function and prolonged QT interval are excluded. In correlative portion of the study, peripheral blood will be analyzed for circulating tumor cells, CD95 surface density and the degree of HDAC inhibition. Pretreatment biopsies obtained for diagnosis will be evaluated for CD95 expression, stromal density along with MCL1, SPARC, and PDGFR expression in tumor and desmoplastic stroma. Citation Format: Haeseong Park, Andrew Poklepovic. A phase 1 study of neoadjuvant chemotherapy with gemcitabine plus nab-paclitaxel, followed by concurrent chemoradiation with gemcitabine, sorafenib, and vorinostat in locally advanced pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT405. doi:10.1158/1538-7445.AM2014-CT405