Selective Cancer Cell Death Research Articles

Combined HAT/EZH2 modulation leads to cancer-selective cell death.

Epigenetic alterations have been associated with both pathogenesis and progression of cancer. By screening of library compounds, we identified a novel hybrid epi-drug MC2884, a HAT/EZH2 inhibitor, able to induce bona fide cancer-selective cell death in both solid and hematological cancers in vitro, ex vivo and in vivo xenograft models. Anticancer action was due to an epigenome modulation by H3K27me3, H3K27ac, H3K9/14ac decrease, and to caspase-dependent apoptosis induction. MC2884 triggered mitochondrial pathway apoptosis by up-regulation of cleaved-BID, and strong down-regulation of BCL2. Even aggressive models of cancer, such as p53–/– or TET2–/– cells, responded to MC2884, suggesting MC2884 therapeutic potential also for the therapy of TP53 or TET2-deficient human cancers. MC2884 induced massive apoptosis in ex vivo human primary leukemia blasts with poor prognosis in vivo, by targeting BCL2 expression. MC2884-treatment reduced acetylation of the BCL2 promoter at higher level than combined p300 and EZH2 inhibition. This suggests a key role for BCL-2 reduction in potentiating responsiveness, also in combination therapy with BCL2 inhibitors.Finally, we identified both the mechanism of MC2884 action as well as a potential therapeutic scheme of its use. Altogether, this provides proof of concept for the use of epi-drugs coupled with epigenome analyses to ‘personalize’ precision medicine.

Targeting Pyruvate Kinase M2, β Catenin Signaling by Juglone Silver Nano Framework for Selective Cancer Cell Death

AbstractThe modern biomedical nanotechnology necessitates functionalized nanoparticles for targeted action on diseased cells with excellent physico chemical properties. The present study revealed the potential of fabricating juglone silver nano framework for selective cancer cell death by altering the redox states in the cancer cell and selectively inhibiting its growth and proliferation. Conjugation of Juglone with silver nanoparticle reduced the toxicity against normal cells and specifically only inhibited the proliferation of cancer cells. The mechanism of the selective cytotoxic and apoptotic effect on cancer cell was due to down‐regulation of NADPH oxidase 4, Hypoxia‐inducible factor 1α and subdued Pyruvate kinase M2 by arresting Wnt/β catenin signaling through ROS dependent caspase‐3 pathway. Collectively, our findings epitomized that functionalization of nanoparticle gives up a new perspective for adding‐in and tuning the therapeutic values by altering the redox states in cancer cells. Implications of this study opens up a new horizon in modern biomedicine for tuning therapeutic entities to meet the needs of practical applications in complex biological systems.

Reduced HeLa cell viability in methionine‐containing cell culture medium irradiated with microwave‐excited atmospheric‐pressure plasma

The mechanism underlying the death of cancer cells incubated in plasma‐activated medium (PAM) prepared by irradiation of culture medium with atmospheric‐pressure plasma is revealed by analyzing changes in the culture medium composition using liquid chromatography‐tandem mass spectrometry and nuclear magnetic resonance spectroscopy. The concentration of the essential amino acid methionine declined in microwave‐excited plasma‐irradiated medium, whereas, that of methionine sulfoxide increased due to oxidation of methionine. The decrease in methionine concentration in the cell culture medium rather than the increase in methionine sulfoxide leads to the decline in the survival rate of HeLa cells incubated in PAM. These results provide useful insights into the mechanism of selective cancer cell death in PAM.

Concentration Does Matter: The Beneficial and Potentially Harmful Effects of Ascorbate in Humans and Plants.

Ascorbate (Asc) is an essential compound both in animals and plants, mostly due to its reducing properties, thereby playing a role in scavenging reactive oxygen species (ROS) and acting as a cofactor in various enzymatic reactions. Recent Advances: Growing number of evidence shows that excessive Asc accumulation may have negative effects on cellular functions both in humans and plants; inter alia it may negatively affect signaling mechanisms, cellular redox status, and contribute to the production of ROS via the Fenton reaction. Both plants and humans tightly control cellular Asc levels, possibly via biosynthesis, transport, and degradation, to maintain them in an optimum concentration range, which, among other factors, is essential to minimize the potentially harmful effects of Asc. On the contrary, the Fenton reaction induced by a high-dose Asc treatment in humans enables a potential cancer-selective cell death pathway. The elucidation of Asc induced cancer selective cell death mechanisms may give us a tool to apply Asc in cancer therapy. On the contrary, the regulatory mechanisms controlling cellular Asc levels are also to be considered, for example, when aiming at generating crops with elevated Asc levels.

Inactivated Sendai virus particle upregulates cancer cell expression of intercellular adhesion molecule-1 and enhances natural killer cell sensitivity on cancer cells.

We have already reported that the inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ‐E) has multiple anticancer effects, including induction of cancer‐selective cell death and activation of anticancer immunity. The HVJ‐E stimulates dendritic cells to produce cytokines and chemokines such as β‐interferon, interleukin‐6, chemokine (C‐C motif) ligand 5, and chemokine (C‐X‐C motif) ligand 10, which activate both CD8+ T cells and natural killer (NK) cells and recruit them to the tumor microenvironment. However, the effect of HVJ‐E on modulating the sensitivity of cancer cells to immune cell attack has yet to be investigated. In this study, we found that HVJ‐E induced the production of intercellular adhesion molecule‐1 (ICAM‐1, CD54), a ligand of lymphocyte function‐associated antigen 1, in several cancer cell lines through the activation of nuclear factor‐κB downstream of retinoic acid‐inducible gene I and the mitochondrial antiviral signaling pathway. The upregulation of ICAM‐1 on the surface of cancer cells increased the sensitivity of cancer cells to NK cells. Knocking out expression of ICAM‐1 in MDA‐MB‐231 cells using the CRISPR/Cas9 method significantly reduced the killing effect of NK cells on ICAM‐1‐depleted MDA‐MB‐231 cells. In addition, HVJ‐E suppressed tumor growth in MDA‐MB‐231 tumor‐bearing SCID mice, and the HVJ‐E antitumor effect was impaired when NK cells were depleted by treatment with the anti‐asialo GM1 antibody. Our findings suggest that HVJ‐E enhances NK cell sensitivity against cancer cells by increasing ICAM‐1 expression on the cancer cell surface.

Real-time monitoring of microdistribution of antibody-photon absorber conjugates during photoimmunotherapy in vivo

Photoimmunotherapy (PIT) is an emerging low side effect cancer therapy based on a monoclonal antibody (mAb) conjugated with a near-infrared (NIR) phthalocyanine dye IRDye 700DX. IR700 is fluorescent, can be used as an imaging agent, and also is phototoxic. It induces rapid cell death after exposure to NIR light. PIT induces highly selective cancer cell death, while leaving most of tumor blood vessels unharmed, leading to an effect called super-enhanced permeability and retention (SUPR). SUPR significantly improves the effectiveness of the anticancer drug. Currently, the therapeutic effects of PIT are monitored using the IR700 fluorescent signal based on macroscopic fluorescence reflectance imagery. This technique, however, lacks the resolution and depth information to reveal the intratumor heterogeneity of mAb-IR700 distribution. We applied a minimally invasive two-channel fluorescence fiber imaging system by combining the traditional fluorescence imaging microscope with two imaging fiber bundles (~0.85mm). This method monitored mAb-IR700 distribution and therapeutic effects during PIT at different intratumor locations (e.g., tumor surface vs. deep tumor) in situ and in real time simultaneously. This enabled evaluation of the therapeutic effects in vivo and treatment regimens. The average IR700 fluorescence intensity recovery after PIT to the tumor surface is 91.50%, while it is 100.63% in deep tumors. To verify the results, two-photon microscopy combined with a microprism was also used to record the mAb-IR700 distribution and fluorescence intensity of green fluorescent protein (GFP) at different tumor depths during PIT. After PIT treatment, there was significantly higher IR700 fluorescence recovery in deep tumor than in the tumor surface. This phenomenon can be explained by increased vascular permeability immediately after NIR-PIT. Fluorescence intensity of GFP at the tumor surface decreased significantly more compared to that of deep tumor and in controls (no PIT).

Abstract B12: Effect of Ganoderma lucidum on DNA damage and DNA repair in colorectal cancer cell lines

Abstract Colorectal carcinoma (CRC) is the third most common type of cancer in the world and second most common cause of cancer related deaths in Europe. Countries of Central Europe (Czech Republic, Slovakia and Hungary) have one of the highest rates both for incidence and mortality of CRC and this disease therefore possesses a serious health, social and economic problem. CRC is a complex disease that develops as consequence of environmental and health risk factors with particular involvement of suboptimal DNA repair, resulting in accumulation of DNA damage. Reactive oxygen species (ROS) represent a group of highly reactive molecules tightly controlled by cellular antioxidant system. Disturbance of the prooxidation–antioxidation homeostasis can lead to ROS accumulation and consequently to DNA damage and apoptosis. Many natural compounds possess anti-cancer activities with the generation of ROS. Cancer cells are more sensitive to oxidative DNA damage than non-malignant cells. Modulation of oxidative DNA damage and DNA repair pathways by natural compounds may lead to selective cancer cell-death and to sensitization of cancer cells to treatment. Ganoderma Lucidum (GLC) (Reishi, Ling-Zhi) is a mushroom used in Chinese medicine for thousands of years for prevention or therapy of many different disorders including cancer. The aim of our study is to define the effect of Ganoderma lucidum (GLC) extract on DNA damage and DNA repair machinery in colorectal cancer cell lines (HTC116, HCT116p53-/-, HT29, SW480). Our results showed that 6hrs GLC treatment in colorectal cancer cells (0.5µg/µl) inhibits activity of superoxid dismutase 1 (25%, p<0.01) and enzymatic activity of glutathione peroxidase (20%, p<0.01), followed by abnormal ROS accumulation (1,5-fold, p<0.05). Furthermore the specific oxidative DNA damage increased (10-fold, p<0.01) after GLC treatment, whereas the base excision DNA repair process was supressed (40%, p<0.001). Finally, above events resulted in cell cycle arrest (HCT116) or apoptosis (HCTp53-/-) and subsequent decrease of colorectal cancer cell survival (25%, p<0.05). Our results suggest that GLC extract decreases activity of the cellular antioxidant system, which leads to oxidative DNA damage and subsequently to genotoxic effects of GLC extract in colorectal cancer cell lines. These data indicate that specific oxidative DNA damage caused by natural compounds may become a potential tool for improvement of anti-cancer treatment. Acknowledgement: AMVIS LH13061 and GACR 15-14789S, AZV 15-27580A Citation Format: Alena Opattova, Andrea Cumova, Sona Vodenkova, Peter Macinga, Jozef Horak, Daniel Sliva, Pavel Vodicka. Effect of Ganoderma lucidum on DNA damage and DNA repair in colorectal cancer cell lines [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B12.

In Silico Design of Optimal Dissolution Kinetics of Fe-Doped ZnO Nanoparticles Results in Cancer-Specific Toxicity in a Preclinical Rodent Model.

Cancer cells have unique but widely varying characteristics that have proven them difficult to be treated by classical therapeutics and calls for novel and selective treatment options. Nanomaterials (NMs) have been shown to display biological effects as a function of their chemical composition, and the extent and exact nature of these effects can vary between different biological environments. Here, ZnO NMs are doped with increasing levels of Fe, which allows to finely tune their dissolution rate resulting in significant differences in their biological behavior on cancer or normal cells. Based on in silico analysis, 2% Fe-doped ZnO NMs are found to be optimal to cause selective cancer cell death, which is confirmed in both cultured cells and syngeneic tumor models, where they also reduce metastasis formation. These results show that upon tuning NM chemical composition, NMs can be designed as a targeted selective anticancer therapy.

Novel Oligopyrrole Carboxamide based Nickel(II) and Palladium(II) Salens, Their Targeting of Human G-Quadruplex DNA, and Selective Cancer Cell Toxicity.

DNA targeting by various metal complexes is a key strategy toward the restriction of cancer cell proliferation. Toward this end, we designed and synthesized novel salen-based Ni(II) and Pd(II) metal complexes with positively charged flanking side chains comprising N-methylpyrrole carboxamides of varying lengths. The compounds showed high specificity toward G-quadruplex DNA over duplex DNA. Sufficient inhibition of the telomerase activity was observed, which was ascertained by the prominent restriction of cancer cell proliferation in the long-term cell viability and telomerase inhibition assays. The compounds exhibited selective cancer cell death following an apoptotic pathway. Analysis of the binding mode showed partial stacking of the salen moiety over the G-tetrads and association of the pendant oligopyrrole carboxamide units with the grooves. The conjugation of the tetrad-binding metal salen core with groove-oriented flexible oligopyrrole moieties resulted in the high selectivity and stabilization of the human G-quadruplex DNA structures.

Inhibition OF USP7 Induces Selective Cancer Cell Death in Chronic Lymphocytic Leukemia

Inhibition OF USP7 Induces Selective Cancer Cell Death in Chronic Lymphocytic Leukemia

The novel anthraquinone derivative IMP1338 induces death of human cancer cells by p53-independent S and G2/M cell cycle arrest.

To identify novel small molecules that induce selective cancer cell death, we screened a chemical library containing 1040 compounds in HT29 colon cancer and CCD18-Co normal colon cells, using a phenotypic cell-based viability assay system with the Cell Counting Kit-8 (CCK-8). We discovered a novel anthraquinone derivative, N-(4-[{(9,10-dioxo-9,10-dihydro-1-anthracenyl)sulfonyl}amino]phenyl)-N-methylacetamide (IMP1338), which was cytotoxic against the human colon cancer cells tested. The MTT cell viability assay showed that treatment with IMP1338 selectively inhibited HCT116, HCT116 p53(-/-), HT29, and A549 cancer cell proliferation compared to that of Beas2B normal epithelial cells. To elucidate the cellular mechanism underlying the cytotoxicity of IMP1338, we examined the effect of IMP1338 on the cell cycle distribution and death of cancer cells. IMP1338 treatment significantly arrested the cell cycle at S and G2/M phases by DNA damage and led to apoptotic cell death, which was determined using FACS analysis with Annexin V/PI double staining. Furthermore, IMP1338 increased caspase-3 cleavage in wild-type p53, p53 knockout HCT116, and HT29 cells as determined using immunoblotting. In addition, IMP1338 markedly induced the phosphorylation of histone H2AX and Chk1 in both cell lines while the combination of 5-fluorouracil (5-FU) and radiation inhibited the viability of HCT116, HCT116 p53(-/-), and HT29 cells compared to 5-FU or radiation alone. Our findings indicated that IMP1338 induced p53-independent cell death through S and G2/M phase arrest as well as DNA damage. These results provide a basis for future investigations assessing the promising anticancer properties of IMP1338.

Oxidative Stress and JNK Activation cause Mitochondrial Dysfunction and Cell Death in Hepatocarcinoma after VDAC-Tubulin Antagonists

Background: Flux of metabolites into mitochondria sustains membrane potential (ΔΨ) and reactive oxygen species (ROS) formation. Free α,β-tubulin dimers close VDAC in vitro and high free tubulin in cancer cells closes VDAC. The small molecule erastin antagonizes the inhibition of tubulin on VDAC. Here, we hypothesized that VDAC-tubulin antagonists (VTA) open VDAC, increase mitochondrial metabolism, decrease glycolysis and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of hepatocarcinoma (HCC) cells. Our AIM was to evaluate the effects of VTAs on mitochondrial ΔΨ, NAD(P)H+ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 HCC cells.Methods: Confocal/multiphoton fluorescence microscopy assessed ΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry.Results: Erastin and small molecules X1-2 identified in a high-throughput screen increased ΔΨ, NAD(P)H and cmDCF and MitoSOX fluorescence. The mitochondrial antioxidant MitoQ blocked ROS increases. Mitochondrial hyperpolarization was followed by depolarization. Additionally, erastin and X1 activated JNK. JNK inhibitors (JNK VIII and SP600125, 30 µM) blocked X1-induced hyperpolarization/depolarization and decreased ROS formation. Lactate generation after X1 decreased by 60%. Both X1 and X2 killed cancer cells (∼93% and ∼76% respectively), which the antioxidant N-acetylcysteine (100 µM) blocked and JNKVIII and SP600125 decreased. By contrast, X1-2 caused <25% cell death in primary rat hepatocytes. X1 (5 mg/kg, i.p. daily) decreased tumor growth in a Huh7 xenograft model.Conclusion: AVTs are anti-Warburg compounds that promote mitochondrial metabolism, inhibit glycolysis and cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants and JNK inhibitors.

Abstract A88: Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells

Abstract Background: Warburg metabolism is characterized by enhanced aerobic glycolysis and suppressed mitochondrial metabolism. Voltage dependent anion channels (VDAC) located in the mitochondrial outer membrane control flux of metabolites into mitochondria. Free α,β-tubulin closes VDAC in planar lipid bilayers (Rostovtseva et al., PNAS 105:18746), and high free tubulin in cancer cells decreases mitochondrial membrane potential (ΔΨ) by limiting ingress of respiratory substrates and ATP that supportΔΨ formation (Maldonado et al., Cancer Res. 70:10192). The small molecule erastin opens VDAC by antagonizing the inhibitory effect of tubulin on VDAC (Maldonado et al., JBC 288:11920). Here, we hypothesized that erastin and erastin-like antagonists of VDAC-tubulin interaction increase mitochondrial redox state, decrease glycolysis, increase mitochondrial formation of reactive oxygen species (ROS) and activate c-jun N-terminal kinase (JNK), culminating in mitochondrial dysfunction and death of cancer cells. Our AIM was to evaluate the effects of erastin/erastin-like compounds on mitochondrialΔΨ, NAD(P)H+ and ROS, lactate generation, JNK activation and cell killing in HepG2 and Huh7 hepatocarcinoma cells. Methods: Confocal/multiphoton fluorescence microscopy assessedΔΨ (tetramethylrhodamine methylester), ROS (chloromethyldichlorofluorescein [cmDCF]; MitoSOX Red) and NAD(P)H (autofluorescence). JNK was assessed by Western blotting and cell killing by propidium iodide fluorometry. Results: In both HepG2 and Huh7, erastin and small molecules X1 and X2 identified in a high-throughput screen increasedΔΨ and NAD(P)H. The three compounds also prevented mitochondrial depolarization by cytosolic high free tubulin induced by nocodazole (microtubule depolymerizing agent). Initial increases ofΔΨ were followed by mitochondrial depolarization occurring 1-2 h after X1 and X2 and 3-4 h after erastin. Lactate generation tested after X1 decreased by 60%. cmDCF and MitoSOX fluorescence increased 30 min after X1 and 60 min after X2 and erastin. The mitochondrially targeted antioxidant MitoQ blocked this increase of ROS. Additionally, erastin caused JNK activation with maximal phosphorylation within 1 h. Both X1 and X2 caused killing of cancer cells (~93% and ~76% respectively), which the antioxidant N-acetylcysteine (100µM) blocked. By contrast, X1 and X2 caused &amp;lt;25% cell death in primary rat hepatocytes. Conclusion: Antagonists of VDAC-tubulin interaction promote mitochondrial metabolism and inhibit glycolysis. These anti-Warburg drugs also cause mitochondrial generation of ROS, which in turn leads to JNK activation, mitochondrial dysfunction and selective death of cancer cells that is prevented by antioxidants. Grants: T32DK083262 to DND; DK073336, DK037034 and 14.Z50.31.0028 to JJL; ACS 13-043-01 and COBRE Pilot Project GM103542 to ENM. Citation Format: David N. DeHart, Monika Gooz, John J. Lemasters, John J. Lemasters, Eduardo N. Maldonado. Openers of voltage dependent anion channels are anti-Warburg agents that enhance mitochondrial metabolism, decrease glycolysis, activate JNK and induce ROS-dependent killing of cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A88.

Selective imaging and cancer cell death via pH switchable near-infrared fluorescence and photothermal effects.

Accurately locating and eradicating sporadically distributed cancer cells whilst minimizing damage to adjacent normal tissues is vital in image-guided tumor ablation. In this work, we developed four heptamethine cyanine based theranostic probes, IR1-4, that demonstrated unique pH switchable near-infrared (NIR) fluorescence and photothermal efficiency. While their fluorescence quantum yields increased up to 1020-fold upon acidification from pH 7.4 to 2.4, their photothermal efficiencies decreased up to 7.1-fold concomitantly. Theoretical calculations showed that protonation of the probes in an acidic environment increased the orbital energy gaps and reduced the intramolecular charge transfer efficiency, resulting in the conversion of absorbed light energy to NIR fluorescence instead of hyperthermia. Substitutions at the terminal indole of the probes fine-tuned their pKafluo values to a narrow physiological pH range of 4.0-5.3. IR2, with a pKafluo of 4.6, not only specifically illuminated cancer cells by sensing their more acidic lysosomal lumen, but also selectively ablated cancer cells via its maximized photothermal effects in the alkaline mitochondrial matrix. As far as we are aware, these probes not only offer the highest physiological acidity triggered NIR fluorescence enhancement as small molecules, but are also the first to specifically visualize and eradicate cancer cells by sensing their altered pH values in cellular organelles. Considering that a disordered pH in organelle lumen is a common characteristic of cancer cells, these theranostic probes hold the promise to be applied in image-guided tumor ablation over a wide range of tumor subtypes.

Sulfiredoxin inhibitor induces preferential death of cancer cells through reactive oxygen species-mediated mitochondrial damage

Recent studies have shown that many types of cancer cells have increased levels of reactive oxygen species (ROS) and enhance antioxidant capacity as an adaptation to intrinsic oxidative stress, suggesting that cancer cells are more vulnerable to oxidative insults and are more dependent on antioxidant systems compared with normal cells. Thus, disruption of redox homeostasis caused by a decline in antioxidant capacity may provide a method for the selective death of cancer cells. Here we show that ROS-mediated selective death of tumor cells can be caused by inhibiting sulfiredoxin (Srx), which reduces hyperoxidized peroxiredoxins, leading to their reactivation. Srx inhibitor increased the accumulation of sulfinic peroxiredoxins and ROS, which led to oxidative mitochondrial damage and caspase activation, resulting in the death of A549 human lung adenocarcinoma cells. Srx depletion also inhibited the growth of A549 cells like Srx inhibition, and the cytotoxic effects of Srx inhibitor were considerably reversed by Srx overexpression or antioxidants such as N-acetyl cysteine and butylated hydroxyanisol. Moreover, Srx inhibitor rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells and significantly suppressed the growth of A549 xenografts without acute toxicity. Our results suggest that Srx might serve as a novel therapeutic target for cancer treatment based on ROS-mediated cell death.

Abstract 2074: Selective inhibitor of nuclear export (SINE) compounds show synergistic anti-tumor activity in combination with dexamethasone in multiple myeloma

Abstract Background: The primary nuclear export protein, Exportin 1 (XPO1/CRM1), is overexpressed in most cancers, which is frequently correlated with poor prognosis. SINE compounds are small-molecule bioavailable drugs that bind covalently to XPO1, which leads to nuclear retention of major tumor suppressor proteins, such as p53, FOXO and IκB, resulting in selective cancer cell death. Selinexor is the most advanced SINE with &amp;gt;500 hematological and solid tumor cancer patients treated to date in Phase I/II clinical trials. Glucocorticoid Receptor (GR) is an XPO1 cargo and dexamethasone (Dex) acts through GR agonism and inhibition of NF-κB activity. The combination of selinexor with Dex seems likely to have enhanced anti-tumor potency and this hypothesis was tested in multiple myeloma models. Methods: Total RNA and whole protein cell lysates from multiple myeloma cell line (MM1S) treated with selinexor with or without dexamethasone were analyzed by quantitative PCR and by immunoblots. Localization of GR was evaluated by immunofluorescence. GR and NF-κB transcriptional activity was analyzed using ELISA assays. MM1S xenograft model in NOD-SCID mice were treated with selinexor with or without dexamethasone to evaluate the effect on tumor growth. Results: In MM.1S cells, we found Dex, but not selinexor, induced phosphorylation of GR resulting in GR nuclear localization. Selinexor prevented nuclear export of phosphorylated GR, leading to the synergistic induction of GR-dependent transcriptional activity. RNA levels of GR regulated genes such as MNK2 were induced by this combination treatment. Interestingly, between the two MNK2 isomers, the combination treatment increased the expression of MNK2α, which is a tumor suppressor protein but not the MNK2β isoform. NF-κB transcriptional activity was inhibited additively by this treatment in MM1S cells. The combination treatment of suboptimal doses of selinexor (5 mg/kg; 60% TGI) and Dex (1 mg/kg; 47% TGI) led to nearly complete growth suppression (91% TGI) in the MM1.S multiple myeloma xenografts. Conclusions: We confirmed the hypothesized synergistic anti-tumor activity for the combination of selinexor with dexamethasone and provided evidence that this synergy is mediated through enhanced GR transcriptional activity, inhibition NF-κB and upregulation of the tumor suppressor MNK2α. This work provides a rationale basis for conducting clinical trials of combinations of selinexor with dexamethasone against cancers known to be sensitive to dexamethasone, such as multiple myeloma. Promising anti tumor activity for the combination of selinexor and dexamethasone in patients with relapsed/refractory MM was reported in the ongoing Phase 1 study (NCT01607892). Citation Format: Yosef Landesman, Trinayan Kashyap, Boris Klebanov, Sivan Elloul, Marsha Crochiere, Sharon Friedlander, William Senapedis, Robert Carlson, Michael Kauffman, Sharon Shacham. Selective inhibitor of nuclear export (SINE) compounds show synergistic anti-tumor activity in combination with dexamethasone in multiple myeloma. [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 2074. doi:10.1158/1538-7445.AM2015-2074

Abstract 4451: Novel small molecule AG311 induces tumor cell death through inhibition of mitochondrial electron transport

Abstract Mitochondria are emerging as an attractive target for new anticancer therapy. The inhibition of the mitochondrial electron transport chain (ETC) provides an alternate and selective mechanism for killing cancer cells, bypassing upstream apoptosis-inducing pathways. The mechanism is based on the fact that cancer cells have higher mitochondrial membrane potential rendering them inherently susceptible to increased reactive oxygen species (ROS) generation such as superoxide through the respiratory chain. In addition pharmacological inhibition of complex I and III at the ubiquinone-binding site could further increase superoxide production. Taken together, this mitochondrial sensitivity represents a promising approach to selectively kill cancer cells. The purpose of this study was to investigate the mitochondria-associated cell death of AG311, a novel anticancer compound. AG311 (5-[(4-Methylphenyl)thio]-9H-pyrimido[4,5-b]indole-2,4-diamine) is a small molecular weight compound designed by our group. AG311 has been shown to induce rapid mitochondrial depolarization resulting in necrotic cell death in a cancer cell-selective manner. In two mouse orthotopic breast cancer models (MDA-MB-435 and 4T1), AG311 significantly reduced tumor volume by 85% and 81%, respectively (n = 4 - 6), with no apparent systemic toxicity. In this study, we aimed to identify the mitochondrial protein target(s) of AG311 in cancer cells. First, upregulation of the mitochondrial ETC by culturing MDA-MB-435 cells in galactose media sensitized cells to AG311-induced cell death. Mitochondrial oxygen consumption (XFe96 extracellular flux analyzer) drastically decreased by 62.9% (± 12.9, n = 8) in response to AG311 (7.5 μM). The effect of AG311 on mitochondrial ETC complexes was determined by spectrophotometrically measuring NADH oxidation in the presence of antimycin A (complex I), ubiquinol reduction (complex III-IV) or cytochrome c oxidation (complex IV). It was found that AG311 inhibited complex I and III activity, but not complex IV. Further kinetic assays suggested that AG311 competitively inhibited ubiquinone binding and significantly induced generation of mitochondrial superoxide (estimated with MitoSOX Red). Finally, treatment with antioxidants (e.g. lipoic acid) partially prevented AG311-induced cell death. In summary, the present results indicate mitochondrial ubiquinone as a likely target for AG311-induced selective cancer cell death through generation of mitochondrial superoxide. This together with the previously demonstrated efficacy further substantiates the use of AG311 as an anticancer agent. Citation Format: Anja Bastian, Lora C. Bailey-Downs, Jessica E. Thorpe, Ravi Kumar Vyas Devambatla, Aleem Gangjee, Kenneth M. Humphries, Michael A. Ihnat. Novel small molecule AG311 induces tumor cell death through inhibition of mitochondrial electron transport. [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 4451. doi:10.1158/1538-7445.AM2015-4451

Abstract 4490: Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo

Abstract The primary nuclear export protein, Exportin 1 (XPO1/CRM1), is overexpressed in most cancers and this overexpression is frequently correlated with poor prognosis. Selective Inhibitors of Nuclear Export (SINE) compounds are a family of small-molecule bioavailable drugs that bind covalently to XPO1 to inhibit nuclear export. This results in nuclear retention of major tumor suppressor proteins, such as p53, pRB, FOXO3A and other critical proteins in cancer biology, which leads to selective cancer cell death. Selinexor is the most advanced SINE with &amp;gt;500 hematological and solid tumor cancer patients treated to date in a number of Phase I/II clinical trials. Blocking XPO1 function also blocks nuclear transport of key DNA damage repair (DDR) proteins and we therefore hypothesized that combination of selinexor with radiation therapy (RT) would lead to synergistic anti-tumor activity. Radiation-chemotherapy combination is an established therapeutic option for patients with medically inoperable non-small cell lung cancer (NSCLC), but its use is limited due to tolerability and adverse effects. Moreover, this treatment strategy typically has short-term benefits and the patients relapse with progressive disease. Here we report on studies designed to test whether selinexor could interfere with DDR and synergize with radiation to enhance anti-tumor potency relative to either treatment alone. Cell cycle analysis and clonogenic assays were conducted in the presence or absence of selinexor in combination with escalating doses of RT on the radiation-sensitive and radiation-insensitive H1299 and A549 cell lines, respectively. Interestingly, Selinexor-treatment induced G1 cell cycle arrest in the p53-deficient cell line H1299 and a prominent G2 cell cycle arrest in p53-wild type A549 cells 24 hrs post treatment. Furthermore, clonogenic assays revealed that selinexor + RT treatment act synergistically to produce a dose-dependent growth inhibition in both cell lines. In vivo combination treatment in the A549 NSCLC xenograft model with low doses of selinexor and RT displayed a strong synergistic effect for reduction of tumor volume. In addition, microscopic and immunohistochemical analysis of the resected tumors showed an overall reduction in tumor cell numbers, increased fibrosis and induction of apoptosis in the selinexor-RT treated tumors compared to controls. Finally, mechanistic studies revealed that selinexor does not induce DNA damage like RT, but downregulates CHK1, RAD51 and other DDR proteins. Together, our results suggest that selinexor treatment sensitizes cells to RT by preventing single-stranded DNA break repair via downregulation of DDR protein expression. In contrast, selinexor induces cell cycle arrest at G1 in p53 deficient H1299 cells, allowing DNA damage accumulation and induction of apoptosis. These results provide a rationale basis for combining selinexor with RT in clinical trials studies. Citation Format: Tami Rashal, Sivan Elloul, Marsha Crochiere, Trinayan Kashyap, William Senapedis, Ryan George, Sharon Friedlander, Maya Ilouze, Yosef Landesman, Robert Carlson, Nir Peled, Michael Kauffman, Sharon Shacham, Yaacov Lawrence. Selinexor (KPT-330) radio-sensitizes non-small cell lung cancer cells in vitro and in vivo. [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 4490. doi:10.1158/1538-7445.AM2015-4490

Targeting NEU Protein in Melanoma Cells with Non-Thermal Atmospheric Pressure Plasma and Gold Nanoparticles.

Non-thermal atmospheric pressure plasma effectively kills cancer cells, but it cannot selectively kill cancer cells. The authors targeted NEU (human epidermal growth factor receptor 2) protein, which is frequently over-expressed in the cell membrane of melanoma cells, using anti-NEU antibody-labeled gold nanoparticles. The labeled nanoparticles preferentially targeted melanoma cells rather than normal keratinocytes. After the addition of labeled gold nanoparticles to melanoma and normal keratinocyte cells, both cells were exposed to non-thermal atmospheric pressure plasma. The death rate of melanoma cells was significantly higher than that of normal keratinocyte cells; many vacuoles, indicative of cell death, were observed in melanoma cells treated with anti-NEU antibody labeled gold nanoparticles and plasma. This selective cancer cell death was attributed to the selective destruction of NEU protein and a downstream effector of NEU. Our study findings show that treatment with a combination of non-thermal atmospheric pressure plasma and anti-NEU antibody-labeled gold nanoparticles effectively and selectively kills melanoma cells.

Abstract P5-19-08: IMMU-132, a potential new antibody-drug conjugate (ADC) for the treatment of triple-negative breast cancer (TNBC): Preclinical and initial clinical results

Abstract Despite major advances in breast cancer treatment, TNBC, which comprises approximately 15% of all breast cancer cases, continues to have a poor prognosis, with an increased risk of recurrence and mortality. IMMU-132 is a new ADC targeting Trop-2, an antigen found in high prevalence in many epithelial cancers, including TNBC, and conjugated to SN-38, a topoisomerase inhibitor and active metabolite of irinotecan, at a high drug:antibody ratio (7.6:1). Studies in mice bearing human pancreatic tumor xenografts (Capan-1) have shown IMMU-132 remains intact in the serum until internalized within the tumor cell where SN-38 is released (pH dependent), resulting in selective cancer cell death. IMMU-132 was found to deliver &amp;gt;120-fold higher amount of SN-38 to xenografted tumors than irinotecan. In animals bearing established MDA-MB-468 TNBC xenografts, IMMU-132 at well-tolerated doses improved responses (more robust regression and delayed progression), compared to irinotecan at a maximum therapeutic dose regimen or to an irrelevant antibody conjugate control. Animals that progressed after receiving the control responded to IMMU-132 therapy, even after those tumors had more than doubled from their initial size. These encouraging pre-clinical data led to the development of the ongoing phase I/II clinical trial in patients (pts) with diverse relapsed/refractory epithelial cancers, including TNBC (NCT01631552). In dose escalation (8 to 18 mg/kg given on days 1 and 8 of a 3-week treatment regimen), dosing was limited primarily by neutropenia, with IMMU-132 being tolerated best for multiple cycles at 8 to 10 mg/kg. Since most (26/30) archival tumors from pts with TNBC expressed Trop-2 (30% ≥2+) and many cancer cell lines express abundant (i.e., &amp;gt;100,000) copies of Trop-2, no enrichment strategies for TNBC were employed to treat TNBC pts with IMMU-132. As of June 1, 2014, 10 pts with TNBC have completed their first response assessment by CT, with 6 having disease shrinkage as their best response; 2 pts had a partial response (-32 and -51% shrinkage of target lesions, with time to progression of 18+ and 30 wks, respectively), and 4 had stable disease (-3, -14 and -19 and -27% shrinkage for 18+, 14, 45, and 24 wks, respectively), according to RECIST. For all pts with diverse cancers treated to date at the phase II dose levels of 8 and 10 mg/kg, 6/25 (24%) had G3 neutropenia; G4 febrile neutropenia occurred in 1 pt. Alopecia, diarrhea, fatigue, nausea, and vomiting are the more common related non-hematological toxicities, but most occur at Grade ≤2. There has been no evidence of immunogenicity to the antibody or the drug, even over multiple cycles of treatment. These promising results in pts who failed multiple prior therapies (median, 4; range, 1-8) suggest IMMU-132 is a safe and potentially effective drug for pts with TNBC, justifying continued evaluation of IMMU-132 in less refractory pts and also in combination with other agents. Citation Format: David M Goldenberg, Linda T Vahdat, Alexander N Starodub, Aditya Bardia, Ellen Chuang, Rebecca L Moroose, Jennifer R Diamond, Robert M Sharkey, Pius P Maliakal, Steven A Hamburger, Allyson J Ocean. IMMU-132, a potential new antibody-drug conjugate (ADC) for the treatment of triple-negative breast cancer (TNBC): Preclinical and initial clinical results [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-19-08.