Panel Of Tumor Cell Lines Research Articles

Abstract 1862: CX5461 in combination with radiotherapy I effective against gynecological tumors

Abstract An increased rate of cellular proliferation is a hallmark of cancer and may be accompanied by increase in ribosome biogenesis and dysregulation in rRNA synthesis. In this regard CX-5461 has been developed as a novel RNA POL1 inhibitor and is currently in Phase I/II clinical trials. We have set out to explore the use of this agent in combination with radiation therapy and to understand the associated molecular mechanisms using this approach. A panel of human tumour cell lines treated with CX-5461 indicated a broad spectrum of sensitivity (MTT assay) with IC50 values varying from 25nM - >2µM. No clear relationship between p53 status and sensitivity to CX-5461 was indicated and the mode of cell death was cell context dependent - either via autophagy (LC3I→ LC3II conversion, with P62 sequestosome decrease), sometimes via apoptosis (annexinV staining, caspase-3 activation), also in the presence of increased senescence (β-galactosidase activity). Combination treatment experiments involving CX-5461 alongside radiation therapy (0-16Gy) showed marked synergistic effects for some cancer cell lines, particularly the cervical cancer CASKI line (HPV16+ve). Highly significant effects were seen when low dose radiation and low dose CX-5461 were used together in CASKI cells: combination index CI=< 0.2 with 6nM CX-5461 and 2Gy (isobologram analysis). Cell cycle analysis of CASKI cells treated in combination indicated cell cycle disruption with prolonged S-phase, giving some insight into some of the associated mechanisms involved. In addition, we have found that BRCA-mutant PEO1 ovarian cancer cells were sensitive to CX-5461, IC50 approx. 100nM, whereas carboplatin-resistant PEO1CarbR (with reversed BRCA mutant status) cells gave an IC50 value >3µM In addition, proteomic studies have shown that PEO1CarbR cells overexpress a number of nucleolar proteins and we are investigating the relevance of this in relation to CX-5461 sensitivity. Our further studies aim to identify potential biomarkers of response to CX-5461 and may also inform the design of clinical trials of this novel agent. Citation Format: Mohammed Ismael, Roger Webb, Mazhur Ajaz, Helen Coley. CX5461 in combination with radiotherapy I effective against gynecological tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1862.

Abstract 2507: Axl suppression inhibits cell migration, invasion and survival in breast and prostate cancer cell lines

Abstract Cancer is the second leading cause of death in the United States with approximately 90% of cancer-related deaths resulting from metastatic disease. In tumor cells, upregulation of certain signaling pathways promote metastatic phenotypes characterized by enhanced invasion, migration, survival, proliferation and induction of angiogenesis. One pathway of considerable interest involves the receptor tyrosine kinase Axl that is expressed in a variety of tumor types and is associated with poor prognosis and metastasis. Furthermore, Axl's ligand, Growth arrest specific 6 (Gas6) protein, can activate a number of downstream signaling pathways that promote metastatic phenotypes. The purpose of the current study was to characterize Axl and Gas6 in a number of human tumor cell lines and to determine the effect of Axl suppression on the metastatic phenotypes, specifically cell migration, invasion and survival. A panel of human tumor cell lines, including prostate, breast, colorectal, renal, and osteosarcoma, were characterized for Axl expression by Western blot and qPCR. Gas6 expression was assessed by qPCR and Gas6 secretion was determined in conditioned media by ELISA. Axl was genetically inactivated by shRNA in prostate (DU145) and breast (MDA-MB-231) cancer cell lines. The efficiency of knockdown was determined by Western blot. The effect of Axl knockdown on the migratory and invasive potentials of these cell lines was evaluated by transwell migration and invasion assays. A clonogenic cell survival assay was used to assess the impact of Axl knockdown on tumor cell viability. Axl-expressing tumor cells expressed and secreted its ligand Gas6, suggesting that Axl is co-expressed with Gas6, and may be mediated by autocrine regulation. Genetic inactivation of Axl (shRNA) inhibited cell migration, invasion and reduced clonogenic cell survival in both the MD-MB-231 breast and DU145 prostate cancer cell lines. These findings suggest that Axl may be a novel therapeutic target to inhibit the dissemination of tumor cells. Ongoing experiments are evaluating the impact of the Axl-targeting agent R428 on Axl signaling and metastatic phenotype. Citation Format: Mai Tanaka, Samantha S. Dykes, Dietmar W. Siemann. Axl suppression inhibits cell migration, invasion and survival in breast and prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2507.

3-Aryl/Heteroaryl-5-amino-1-(3′,4′,5′-trimethoxybenzoyl)-1,2,4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization

Many natural and synthetic substances are known to interfere with the dynamic assembly of tubulin, preventing the formation of microtubules. In our search for potent and selective antitumor agents, a novel series of 1-(3′,4′,5′-trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had different heterocycles, including thiophene, furan or the three isomeric pyridines, and they possessed a phenyl ring bearing electron-releasing or electron-withdrawing substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-two tested compounds showed moderate to potent antiproliferative activities against a panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing strong antiproliferative activity (IC50 < 1 μM) against selected cancer cells. Among them, several molecules preferentially inhibited the proliferation of leukemic cell lines, showing IC50 values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly inhibited tubulin assembly, with an IC50 value of 0.66 μM, half that obtained in simultaneous experiments with CA-4 (IC50 = 1.3 μM).

Homopiperazine-rhodamine B adducts of triterpenoic acids are strong mitocans

Parent pentacyclic triterpenoic acids such as ursolic-, oleanolic, glycyrrhetinic, betulinic and boswellic acid were converted into their acetylated piperazinyl amides that were coupled with rhodamine B. SRB assays to evaluate their cytotoxicity showed all of these triterpene-homopiperazinyl-rhodamine adducts 16–20 being highly cytotoxic for a panel of human tumor cell lines even in nanomolar concentrations while being significantly less cytotoxic for non-malignant cells. Interestingly enough, these compounds were even more cytotoxic than previously prepared piperazinyl analogs, thus making the homopiperazinyl spacer a very interesting scaffold for the development of biologically active compounds. Extra staining experiments showed that the cytostatic effect of compounds 18 and 20 onto A2780 cancer cells is due to their ability to act as a mitocan.

Anticancer Gold(III) Peptidomimetics: From Synthesis to in vitro and ex vivo Biological Evaluations

Five new AuIII -peptidodithiocarbamato complexes of the type [AuIII Br2 (dtc-AA1 -AA2 -OR] (in which AA1 =N-methylglycine (Sar), l/d-Pro; AA2 =l/d-Ala, α-aminoisobutyric acid (Aib); R=OtBu, triethylene glycol methyl ether), differing with regard to the amino acid sequence and/or the chiral amino acid configuration, were designed to enhance tumor selectivity and bioavailability. The gold(III)-based moiety was functionalized to exploit the targeting properties of the peptidomimetic ligand toward two peptide transporters (namely PEPT1 and PEPT2), which are upregulated in several tumor cells. The compounds were synthesized and fully characterized, mainly by means of elemental analysis, one- and two-dimensional NMR spectroscopy, FT-IR, and UV/Vis spectrophotometry. The crystal structures of three compounds were also solved by X-ray diffraction. In vitro cytotoxicity studies using a panel of human tumor cell lines (A549 [non-small-cell lung carcinoma], MCF-7 [breast cancer], A2780 [ovarian carcinoma], H1975 [non-small-cell lung carcinoma], H460 [large-cell lung carcinoma], and A431 [human epidermoid carcinoma]) showed the dtc-Pro-Aib-OtBu derivative to be very effective, with GI50 values much lower than those of cisplatin. This complex was thus selected for evaluating stability under physiological conditions and possible interactions with serum albumin, as well in PARP-1 enzyme inhibition assays and preliminary ex vivo toxicity experiments on healthy rat tissues.

Synthesis and Antitumor Evaluation of Novel N-substituted Norcantharidin Imidazolium Derivatives

Aims and Objectives: Cantharidin is a terpenoid with a high vesicant potency isolated from Mylabris caraganae and various other insects, which originates from the Chinese traditional medicine and has a long history of use as antiproliferative agent. Modified cantharidin derivatives are researched for retainable antitumor activities and lower toxicity. And imidazolium salt is an important building block in drug discovery with pharmacological activities. This study was undertaken to identify that N-substituted norcantharidin imidazolium derivatives possess potential bioactivity. Materials and Method: Using readily available furan, maleic anhydride and imidazoles as starting materials, a series of novel N-substituted norcantharidin imidazolium derivatives have been designed and synthesized. The cytotoxic potential of all newly synthesized N-substituted norcantharidin imidazolium derivatives was assessed in vitro against a panel of human tumor cell lines, Human epidermal carcinoma, human lung carcinoma, liver hepatocellular carcinoma, pheochromocytoma of the rat adrenal medulla. Results: The imidazolium derivatives 6a-6f and 6m-6o, bearing a 5,6-dibromohexahydro-4,7-epoxyisobenzofuran- 1,3-dione or 5-bromo-7-oxabicyclo[2.2.1]hepta- 2,5-diene-2,3-dicarboxylate and electron-donating group, carbonyl and propenyl substituent at position-1 of the imidazole ring, were found to be the most potent compounds as antitumor agents. Notably, compounds 6m and 6n exhibited cytotoxic activity selectively against Hela and A549 cell lines with IC50 values 1.38-fold, 5.04-fold, lower than DDP, while compound 6f showed powerful inhibitory activities selectively against Hela and PC12 cell lines. Conclusion: Steric and electronic effects have an important role in determining the cytotoxic activity of imidazolium salts. The norcantharidin-imidazole 6f, 6m, 6n and 6o can be considered to be promising leads for further structural modifications guided by the valuable information derivable.

Synthesis and cytotoxicity of 3-amino-glycyrrhetinic acid derivatives

The aim of this study was to prepare 3-hydroximino- and 3-amino derivatives of glycyrrhetinic acid and derivatives to evaluate their in vitro cytotoxicity for a panel of human tumor cell lines. Thus, commercially available glycyrrhetinic acid (1) was acetylated or oxidized at position C-3 and transformed into a variety of different esters and amides followed by their conversion to 3-oximes and amines. While the parent compound was not cytotoxic at all, the 3-amino esters are highly cytotoxic. Interestingly, 3-amino amides were significantly less cytotoxic than 3-amino esters. The (3b, 18b, 20b) Benzyl 3-amino-11-oxoolean-12-en-30-oate was the most cytotoxic compound of this series showing an EC50 = 1.3 mM for 518A2 melanoma cells.

Cytotoxic prenylated acylphloroglucinols from Hypericum annulatum

A phytochemical investigation of the aerial parts of Hypericum annulatum Moris led to the isolation of five new prenylated acylphloroglucinol derivatives hyperannulatins A-E (1–3, 5 and 7) in addition to the known hypercalyxone A (4) and 3-geranyl-1-(2′-methylpropanoyl)phloroglucinol (6). The structures were determined by 1D and 2D NMR and MS spectroscopic techniques. Compounds 1 and 2 have in their structure evgenyl group, a rare hydrocarbon side chain. The cytotoxicity of isolated compounds was established on a panel of tumor cell lines (HL-60, HL-60/DOX, MDA-MB, SKW-3 and K-562) and was determined using MTT based assays. The compounds 1 and 2 showed to be the most potent cytotoxic agents, whose IC50 values against the chemosensitive cell lines ranged 3.42–5.87 μM and 1.48–8.21 μM, respectively. Noteworthy, albeit all tested compounds were less potent than podophyllotoxin their IC50 values were comparable to that of the other reference drug etoposide. In some of the cell lines compounds 1 and 2 even outclassed the cytotoxicity of etoposide.

SKLB-23bb, A HDAC6-Selective Inhibitor, Exhibits Superior and Broad-Spectrum Antitumor Activity via Additionally Targeting Microtubules.

Our previous study reported that SKLB-23bb, an orally bioavailable HDAC6-selective inhibitor, exhibited superior antitumor efficiency both in vitro and in vivo in comparison with ACY1215, a HDAC6-selective inhibitor recently in phase II clinical trial. This study focused on the mechanism related to the activity of SKLB-23bb. We discovered that despite having HDAC6-selective inhibition equal to ACY1215, SKLB-23bb showed cytotoxic effects against a panel of solid and hematologic tumor cell lines at the low submicromolar level. Interestingly, in contrast to the reported HDAC6-selective inhibitors, SKLB-23bb was more efficient against solid tumor cells. Utilizing HDAC6 stably knockout cell lines constructed by CRISPR-Cas9 gene editing, we illustrated that SKLB-23bb could remain cytotoxic independent of HDAC6 status. Investigation of the mechanism confirmed that SKLB-23bb exerted its cytotoxic activity by additionally targeting microtubules. SKLB-23bb could bind to the colchicine site in β-tubulin and act as a microtubule polymerization inhibitor. Consistent with its microtubule-disrupting ability, SKLB-23bb also blocked tumor cell cycle at G2-M phase and triggered cellular apoptosis. In solid tumor xenografts, oral administration of SKLB-23bb efficiently inhibited tumor growth. These results suggested that SKLB-23bb was an orally bioavailable HDAC6 and microtubule dual targeting agent. The microtubule targeting profile enhanced the antitumor activity and expanded the antitumor spectrum of SKLB-23bb, thus breaking through the limitation of HDAC6 inhibitors. Mol Cancer Ther; 17(4); 763-75. ©2018 AACR.

Oleanane triterpenoids from the Algerian Salvia phlomoides

Three previously unreported triterpenoids 2α,3β,11α-trihydroxyolean-18-ene 1, 3β-acetoxy,2α,11α- dihydroxyolean-18-ene 2 and 2α-acetoxy,3β,11α-dihydroxyolean-18-ene 3 were isolated from the chloroform extract of aerial parts (leaves and flowers) of Salvia phlomoides collected from the Constantine region in the east of Algeria. Structures were determined using NMR spectroscopy and HR-ESI mass spectrometry. Compounds 1, 2 and 3 were tested against the NCI panel of human tumour cell lines at a single dose of 10 μM. Compound 2 was the most active showing a 38% growth inhibition against the leukaemia RPMI-8226 line.

Synthesis and investigations into the anticancer and antibacterial activity studies of β-carboline chalcones and their bromide salts

A series of sixteen β-carbolines, bearing chalcone moiety at C-1 position, were prepared from easily accessible 1-acetyl-β-carboline and various aldehydes under basic conditions followed by N2-alkylation using different alkyl bromides. The prepared compounds were evaluated for in vitro cytotoxicity against a panel of human tumor cell lines. N2-Alkylated-β-carboline chalcones 13a-i represented the interesting anticancer activities compared to N2-unsubstituted β-carboline chalcones 12a-g. Off the prepared β-carbolines, 13g exhibited broad spectrum of activity with IC50 values lower than 22.5 µM against all the tested cancer cell lines. Further, the N2-alkylated-β-carboline chalcone 13g markedly induced cell death in MDA-MB-231 cells by AO/EB staining assay. The most cytotoxic compound 13g possessed a relatively high drug score of 0.48. Additionally, the prepared β-carboline chalcones displayed moderate antibacterial activities against tested bacterial strains.

Novel 3‐substituted N‐methylcarbazole–imidazolium salt derivatives: Synthesis and cytotoxic activity

A series of novel 3-substituted N-methylcarbazole-imidazolium salt derivatives has been prepared and evaluated in vitro against a panel of tumor cell lines (Hep G-2, Hela and PC12). The results suggest that the presence of substituted 2-methyl-imidazole or imidazole ring and substitution of the imidazolyl-3-position with a naphthylacyl or 4-bromophenacyl group were important for improving cytotoxic activity. Compounds 17, 18, 27, and 28 with 4-bromophenacyl and naphthylacyl groups displayed good activities with IC50 values of 0.09-7.20μm against three tumor cell lines investigated and more active than DDP. Compound 35 exhibited cytotoxic activity selectively against Hela cell.

Abstract P1-09-08: BCL2L1 (BCL-XL) expression and MYC super-enhancer positivity predict sensitivity to the covalent CDK7 inhibitor SY-1365 in triple negative breast cancer (TNBC) cell lines

Abstract Effective therapies for TNBC remain elusive. As such, TNBCs are associated with a high risk of relapse and short progression free- and overall-survival. Recent studies showed that TNBC cells are highly dependent on the transcriptional regulator CDK7, and suggest that the mitochondrial apoptosis pathway is important in mediating cell survival in CDK7-dependent cells. Further, TNBC has been shown to have a distinct epigenetic and transcriptional program, with super-enhancers (SE) mediating the expression of key oncogenic drivers such as MYC. SY-1365, a covalent and selective inhibitor of CDK7, was developed to exploit dysregulated programs thought to drive SE-mediated transcriptional-dependencies in TNBC and other cancers. To identify potential biomarkers predictive of sensitivity to SY-1365, we evaluated SY-1365 inhibitory activity in a large panel of human tumor cell lines, including TNBC lines, and correlated sensitivity with RNA expression and epigenetic profiles. SY-1365 dose-response curves were measured using the ATP-lite assay in a panel of 406 human tumor cell lines, including 19 TNBC cell lines. Clustering of growth-rate adjusted dose response curves of cell-lines treated with SY-1365 allowed the classification of cell-lines into low and high response groups. An unbiased genome wide approach was used to compare response classification to RNA expression data across all cell lines to identify gene expression markers predictive of sensitivity to SY-1365. Furthermore, a hypothesis driven approach was followed to interrogate whether the MYC SE predicted sensitivity to SY-1365. Twenty-five genes were differentially expressed between SY-1365-sensitive and -insensitive tumor lines (FDR&amp;lt;0.05). Lower expression of BCL2L1, which encodes the mitochondrial apoptosis regulator BCL-XL, was identified as the most predictive expression biomarker of sensitivity across all profiled cell lines, strongly separating the two classes of sensitivity (Accuracy=70%, FDR&amp;lt;0.005). Further, this predictive power of lower BCL2L1 expression was maintained in an analysis restricted to the subset of TNBC cell-lines (Accuracy=73%). Expanding beyond expression analysis, we also found that the strength of the MYC SE (as defined by H3K27Ac) was predictive of response to SY-1365 in TNBC (Accuracy=86%, FDR&amp;lt;0.05). In this study, we show for the first time that SY-1365 induced differential responses across a large panel of human tumor cell lines derived from multiple indications. We also show that in this panel of cell lines the response could be predicted in an “indication agnostic” manner by the level of expression of BCL2L1. Finally, in line with prior reports, in TNBC cell lines, MYC SE was significantly associated with sensitivity to SY-1365. These observations have generated strong hypotheses for selection strategies aimed at identifying patients with tumors particularly sensitive to CDK7 inhibition with SY-1365, and warrant further investigation with respect to predictive biomarkers of response in patients. SY-1365 is currently being assessed in a phase 1 trial in adult patients with advanced solid tumors, including a planned expansion cohort enriching for patients with TNBC (NCT03134638). Citation Format: Rajagopal N, Hodgson G, Hu S, McKeown M, Bush A, Fritz C, Orlando D, Olson E, di Tomaso E. BCL2L1 (BCL-XL) expression and MYC super-enhancer positivity predict sensitivity to the covalent CDK7 inhibitor SY-1365 in triple negative breast cancer (TNBC) cell lines [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-08.

Chagosendines A-C, New Metal Complexes of Imidazole Alkaloids from the Calcareous Sponge Leucetta chagosensis.

Chemical examination of the bright yellow sponge Leucetta chagosensis resulted in the isolation of three new imidazole-based alkaloid complexes namely chagosendines A-C (1-3), together with known analogues pyronaamidine, naamidine J, and naamine C. Their structures were determined on the basis of extensive spectroscopic (IR, MS, NMR, and single-crystal X-ray diffraction) analysis in association with the chemical conversion. The isolated alkaloids together with three synthesized new homodimer complexes were evaluated for the cytotoxic activities against a panel of tumor cell lines. The copper complexes of imidazole alkaloids 2 and 3, as found from nature for the first time, exerted selective and remarkable activities against the tumor cell lines K562, HepG2, and HeLa.

A Strategy for the Convergent and Stereoselective Assembly of Polycyclic Molecules.

The stereoselective oxidative coupling of cyclic ketones via silyl bis-enol ethers followed by ring-closing metathesis is shown to be a general and powerful reaction sequence for the preparation of diverse polycyclic scaffolds from simple precursors. The modular strategy successfully constructs substructures prevalent in numerous bioactive natural product families, varying in substitution and carbocyclic composition. Several of the prepared compounds were shown to possess potent cytotoxic activity against a panel of tumor cell lines. The utility of this strategy was further demonstrated by a concise and highly convergent 17-step formal synthesis of the complex antimalarial marine diterpene, (+)-7,20-diisocyanoadociane.

Abstract B36: A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas

Abstract Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric sarcomas and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric sarcomas, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2 Gy IR and 1-10 nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations &amp;lt;10 nM in tumor cells expressing detectable levels of SLFN11. Conclusion: SLFN11 is present in select pediatric sarcomas and may induce a DNA repair defect that is best exploited by combining low doses of TAL and irinotecan with IR. Citation Format: Anang A. Shelat, Christopher L. Tinkle, Elizabeth Stewart, Sara Michele Federico, Brandon Bianski, Marcia Mellado-Lagarde. A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B36.

Abstract B109: ASN004, a novel 5T4-targeted Dolaflexin ADC, causes complete and durable tumor regression in a variety of tumor xenograft models

Abstract The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors, while showing very limited expression in normal adult tissues. This selective pattern of expression establishes 5T4 as an attractive therapeutic target for the Antibody-Drug Conjugate (ADC) strategy. ASN004 is an anti-5T4 ADC that incorporates a novel single-chain homo-dimer antibody, Fleximer® linker technology (Mersana Therapeutics), and cytotoxic dolastatin (auristatin) analog warheads, with a drug/antibody ratio of ~15:1. ASN004 shows high affinity for the 5T4 antigen (Kd &amp;lt; 30 pM), with selective binding and internalization to 5T4-expressing tumor cells. Potent, selective cytotoxicity has been determined for a diverse panel of tumor cell lines. ASN004 provides complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a range of 5T4 expression levels. Tumor-free survivors could be achieved in tumor models with a single dose of ASN004, as low as 1 mg/kg iv. The broad, superior activity of ASN004 was demonstrated in a head-to-head study against trastuzumab-DM1, in a low-5T4/high-HER2 expressing tumor model. Finally, efficacy of ASN004 in patient-derived tumor xenograft (PDX) models will be presented. In summary, ASN004 has been established as a promising ADC therapeutic for multiple tumor types, encompassing a wide range of 5T4-expression levels. IND-enabling studies are ongoing in preparation for advancement to clinical development. Citation Format: Roger A. Smith, David J. Zammit, Sanjeeva P. Reddy. ASN004, a novel 5T4-targeted Dolaflexin ADC, causes complete and durable tumor regression in a variety of tumor xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B109.

Abstract A014: Evaluation of the hollow fiber model as a highly predictive in vivo testing system for the selection of xenograft tumor models in cancer drug discovery

Abstract In cancer drug discovery, lead compounds found to possess the most antitumor efficacy in vitro need to be rapidly and economically prioritized for further analysis in conventional xenograft testing. To bridge this gap, Hollingshead et al. (1995) developed the in vivo hollow fiber model (HFM), a prescreen predicting xenograft efficacy. The screen is based on drug- (but not cell-) permeable fibers loaded with tumor cells that are implanted into mice. Following drug treatment, the number of viable cells in the fibers provides information about the drug's potential in vivo antitumor efficacy. Since one animal can receive up to three intraperitoneally and three subcutaneously implanted fibers, one lead can be simultaneously evaluated against up to three cell lines. To evaluate the predictability of the HFM, two target-specific kinase inhibitors were selected: crizotinib and VX-680. Crizotinib is an FDA-approved drug for NSCL that inhibits the kinases ALK, MET, and ROS1, whereas VX-680 inhibits the AURORA kinase family. Both drugs were run in a proliferation assay against a large panel of tumor cell lines to determine the IC50 value. Subsequently, 9 cell lines representing different inhibitor potencies (high, medium, low/none) were chosen for comparison of the proliferation, HFM, and xenograft tumor model results. For crizotinib the cell lines MKN-45, MiaPaca2, and KARPAS-299 were chosen with highly potent inhibition whereas for VX-680 the cell lines HCT-116, Molm-13, and Colo-205 are most potently inhibited in the proliferation assay. Cell lines SKOV-3, LN229, and HT-29 were chosen as noninhibited cell lines for both kinase inhibitors in the in vitro proliferation assay. For each of the 9 cell lines, a subcutaneous xenograft mouse model was established and the effect of both crizotinib and VX680 treatment on tumor growth determined. In parallel, all cell lines were tested in three separate HFMs with 3 cell lines simultaneously. The implanted hollow fibers were analyzed via MTT assay 14 days after therapy initiation. The correlation between the cell-based inhibitory activity and xenograft tumor model results and the correlation between HFM and xenograft tumor model results will be presented. Finally, the implementation of the HFM for the tumor model selection process in cancer drug discovery will be discussed. In case the HFM is a highly predictive in vivo prescreen for target-specific inhibitors, the HFM will reduce costs, time, and most importantly mice consumption according to reduction and refinement, criteria of the three rules (3Rs), guiding principles for more ethical use of animals in drug testing. Citation Format: Annika Sinz, Holger Weber, Cynthia Obodozie. Evaluation of the hollow fiber model as a highly predictive in vivo testing system for the selection of xenograft tumor models in cancer drug discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A014.

Abstract B002: CD47 expression predicts efficacy of macrophage-mediated phagocytosis of tumor cells

Abstract Most human cancers express CD47 to escape the macrophage surveillance. To reinstate macrophage functionality in clearing tumor cells, targeting CD47 has been an important strategy in immune-oncology therapy. We profiled a large panel of tumor cell lines of both solid and blood tumors that are frequently used in cancer drug research and in Mixeno® animal study in Crown Bioscience for CD47. Despite its being reported as a ubiquitously expressed immune-escape marker in cancer cells, the expression levels of CD47 vary among different tumor cell lines when the cells were examined by flow cytometry. It is of interest that a portion of tumor cells in fact do not present CD47 despite gene expression data, such as RNA-seq data, suggesting that they do. To establish a robust and reproducible in vitro assay platform to support anti-CD47 drug research, we applied a macrophage differentiation system using isolated CD14+ monocytes as starting material, and were able to achieve &amp;gt; 80% of M1 and M2 populations, respectively, through the 6-day differentiation. Differentiated macrophage was cocultured with target tumor cells labeled for flow cytometry analysis. In the presence of anti-CD47 antibody, steady phagocytosis effect was observed with aCD47 BRIC126 antibody and target tumor cells expressing high level of CD47 markers. BRIC126 antibody blocks the CD47-SIRPalpha interaction. To prove the finding, we constructed isogenic cell models from the same parental cells in that CD47 gene is knocked out in one sub-cell line and overexpressed in another sub-cell line. The isogenic systems are also subject to the CD47 biology studies and can be served as positive and negative controls in phagocytosis assays. Detailed data will be discussed. Citation Format: Huajun Yang, Zhongliang Li, Beibei Tang, Frank Xing, Qian Shi. CD47 expression predicts efficacy of macrophage-mediated phagocytosis of tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B002.

The cisplatin-based Pt(iv)-diclorofibrato multi-action anticancer prodrug exhibits excellent performances also under hypoxic conditions.

Multi-action cisplatin-based mono- (1) and di-clofibric acid (2) Pt(iv) "combo" derivatives were synthesized via both traditional and microwave assisted procedures. The two complexes offered very good performances (IC50 values in a nanomolar range) on a panel of human tumor cell lines, including the highly chemoresistant malignant pleural mesothelioma ones. Moreover, both 1 and 2 bypass the cisplatin resistance. Indeed, cisplatin and clofibric acid, the metabolites of the Pt(iv) → Pt(ii) intracellular reduction, proved to act synergistically. The adjuvant action of clofibric acid relies on the activation of peroxisome proliferator-activated receptor α (PPARα) that, in turn, decreases the level of Hypoxia-Inducible Factor-1α. Both compounds induced extensive apoptosis in tumor cells, also via oxidative stress. Finally, 2 exhibited excellent performances also under the hypoxic conditions typical of solid tumors, where cisplatin is less effective.