Selective Cell Growth Inhibition Research Articles

Abstract PR009: Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening

Abstract NRAS mutations play a crucial role as oncogenic drivers of tumor cell growth and chemo-resistance in melanoma and acute myeloid leukemia (AML). In melanoma, NRAS mutations occur in approximately 25% of cases, and are generally mutually exclusive with BRAF-mutations. NRAS mutant tumors often exhibit an aggressive phenotype, such as thicker lesions, increased mitotic activity, and higher rates of lymph node metastasis. Surgical resection of NRAS mutant melanoma has a poor prognosis with a lower median overall survival (OS) compared to non-NRAS-mutant melanoma (8.2 vs. 15.1 months for stage IV disease). Current treatment strategies for stage III/IV non-resectable metastatic NRAS mutant melanoma include immunotherapy and indirect targeting of NRAS associated signaling pathways, highlighting the urgent need to develop small molecule NRAS inhibitors. NRAS palmitoylation is essential for its oncogenic function by regulating its trafficking and anchoring to the plasma membrane. Unlike other RAS isoforms, NRAS relies on a single palmitoylation site (C181) for its plasma membrane localization and this unique dependency makes NRAS more sensitive to palmitoylation inhibitors. To identify selective NRAS inhibitors that covalently modify C181 and prevent its palmitoylation, we employed an imaging-based NRAS translocation high-throughput screen using a library of 23,000 covalent molecules. We identified a set of covalent inhibitors that selectively target NRAS translocation but do not affect HRAS localization. We further confirmed the direct binding of these compound hits to NRAS at its palmitoylation site (C181) using intact mass spectrometry and chemo-proteomic peptide mapping techniques. Moreover, we validated the functional impact of these compound hits by demonstrating their selective inhibition of cell growth in mutant NRAS-driven melanoma cell lines. In conclusion, our study presents the first direct inhibitor for NRAS, discovered through a high-throughput covalent screening approach. This finding holds promising implications for the development of novel targeted therapies for NRAS mutant tumors. Citation Format: Zhao Wang, Keith Moore, Simon Ng, Chao Wang, Ronald Tomlinson, Huay-Keng Loke, Jamie Rice, Ivan Cornella. Discovery of covalent NRAS inhibitors targeting the Palmitoylation site through imaging-based high-throughput screening [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR009.

Daphnanes diterpenes from the latex of Hura crepitans L. and their PKCζ-dependent anti-proliferative activity on colorectal cancer cells

Hura crepitans L. (Euphorbiaceae) is a thorn-covered tree widespread in South America, Africa and Asia which produces an irritating milky latex containing numerous secondary metabolites, notably daphnane-type diterpenes known as Protein Kinase C activators. Fractionation of a dichloromethane extract of the latex led to the isolation of five new daphnane diterpenes (1–5), along with two known analogs (6–7) including huratoxin. Huratoxin (6) and 4′,5′-epoxyhuratoxin (4) were found to exhibit significant and selective cell growth inhibition against colorectal cancer cell line Caco-2 and primary colorectal cancer cells cultured as colonoids. The underlying mechanism of 4 and 6 was further investigated revealing the involvement of PKCζ in the cytostatic activity.

Selectively Targeting STAT3 Using a Small Molecule Inhibitor is a Potential Therapeutic Strategy for Pancreatic Cancer.

Pancreatic cancer is the worst prognosis among all human cancers, and novel effective treatments are urgently needed. Signal transducer and activator of transcription 3 (STAT3) has been demonstrated as a promising target for pancreatic cancer. Meanwhile, selectively targeted STAT3 with small molecule remains been challenging. To specifically identify STAT3 inhibitors, more than 1.3 million compounds were screened by structure-based virtual screening and confirmed with the direct binding assay. The amino acid residues that WB436B bound to were verified by induced-fit molecular docking simulation, RosettaLigand computations, and site-directed mutagenesis. On-target effects of WB436B were examined by microscale thermophoresis, surface plasmon resonance, in vitro kinase assay, RNA sequencing, and selective cell growth inhibition assessment. In vivo studies were performed in four animal models to evaluate effects of WB436B on tumor growth and metastasis. Kaplan-Meier analyses were used to assess survival. WB436B selectively bound to STAT3 over other STAT families protein, and in vitro antitumor activities were improved by 10 to 1,000 fold than the representative STAT3 inhibitors. WB436B selectively inhibits STAT3-Tyr705 phosphorylation, STAT3 target gene expression, and the viability of STAT3-dependent pancreatic cancer cells. WB436B significantly suppresses tumor growth and metastasis in vivo and prolongs survival of tumor-bearing mice. Mechanistic studies showed that WB436B have unique binding sites located in STAT3 Src homology 2 domain. Our work presents the first-in-class selective STAT3 inhibitor WB436B as a potential therapeutic candidate for the treatment of pancreatic cancer.

Targeting ribosomal G-quadruplexes with naphthalene-diimides as RNA polymerase I inhibitors for colorectal cancer treatment

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures commonly found in regulatory genomic regions. G4 targeting has emerged as a therapeutic approach in cancer. We have screened naphthalene-diimides (NDIs), a class of G4 ligands, in a cellular model of colorectal cancer (CRC). Here, we identify the leading compound T5 with a potent and selective inhibition of cell growth by high-affinity binding to G4s in ribosomal DNA, impairing RNA polymerase I (Pol I) elongation. Consequently, T5 induces a rapid inhibition of Pol I transcription, nucleolus disruption, proteasome-dependent Pol I catalytic subunit A degradation and autophagy. Moreover, we attribute the higher selectivity of carbohydrate-conjugated T5 for tumoral cells to its preferential uptake through the overexpressed glucose transporter 1. Finally, we succinctly demonstrate that T5 could be explored as a therapeutic agent in a patient cohort with CRC. Therefore, we report a mode of action for these NDIs involving ribosomal G4 targeting.

Daphnanes diterpenes from the latex of Hura crepitans L. And activity against human colorectal cancer cells Caco-2

Hura crepitans (Euphorbiaceae) is a tree from South America that produces an irritant latex used as a fish poison. A bio-guided fractionation of an ethanolic extract of the latex led to the isolation and structural identification of three known daphnane-type diterpenes (1–3) including huratoxin (1), together with two new analogs (4, 5). Compound 1 was found to exhibit significant and selective cell growth inhibition against the colorectal cancer cell line Caco-2, with morphological modifications suggesting formations mimicking the intestinal crypt architecture. The underlying mechanism of 1 was further investigated, in comparison with 12-O-tetradecanoylphorbol-13-acetate (TPA), revealing two different mechanisms.

Abstract 4757: Targeting the mevalonate pathway in HER2+breast cancer to overcome resistance and enhance anti-HER2 therapy efficacy

Abstract Background: Despite the advent of HER2-targeted therapies, including the monoclonal antibody trastuzumab (T) and the HER1/2 inhibitor lapatinib (L), for HER2+ breast cancer (BC), resistance still poses a major challenge. L and L+T resistant (R) HER2+ cells with inhibited HER2 signaling showed upregulation of RNA levels of the mevalonate pathway (MVA) enzymes (which were inhibited by short term treatment of parental (P) cells with L or L+T) and increased sensitivity to MVA inhibition by statins (e.g. simvastatin (Sim)), suggesting MVA’s role as an escape mechanism of resistance. Here we investigated the therapeutic potential of another MVA inhibitor Zoledronate (ZA) and the role of mTOR and YAP/TAZ (Y/T) in mediating this resistance. Lastly, we tested if co-blockade of the MVA or its downstream effectors further sensitizes HER2+ models to anti-HER2 therapies. Methods: SKBR3 and AU565 P cells and their LR and LTR derivatives were used. The effects of MVA perturbations on cell growth and HER2 or MVA signaling were studied by methylene blue staining and Western blot (WB), respectively. Y/T activity was tested by a luciferase reporter assay and functionally validated by siRNA knockdown and dominant-active (DomA) YAP overexpression. YAP target gene expression was assessed by RT PCR. SCID-Beige mice bearing HER2+ BCM-3963 PDX tumors were treated with vehicle, L, Sim, or L+Sim and monitored for time to complete response (CR). Results: ZA, like Sim, showed a selective inhibition of cell growth and mTOR signaling in R vs. P cells, which was rescued only by the downstream metabolite geranyl geranyl pyrophosphate (GGPP), but not by the upstream metabolite mevalonate, indicating the on-target effect of ZA. Increased Y/T activity in R models was confirmed, and both Sim and ZA inhibited TAZ levels and induced phospho-YAP levels, which were rescued by the corresponding downstream metabolites. Y/T knockdown inhibited growth and mTOR signaling in R vs. P cells, and DomA YAP negated the mTOR inhibition by Sim. Sim and ZA also significantly decreased levels of the Y/T target gene survivin in R vs. P cells, and the expression was rescued by the downstream metabolites. Inhibition of MVA by Sim or ZA or its downstream signaling effectors, Y/T (by siRNA) and mTOR (by everolimus), enhanced the L sensitivity in P cells. Conversely, DomA YAP reduced the sensitivity of P cells to L. In the presence of Sim or ZA, L treatment more strongly inhibited levels of phospho-S6, a downstream target of mTORC1, compared to L alone. Preliminary in vivo data showed that treatment with L+Sim vs. L alone shortened the median time to CR and numerically increased CR rates. Conclusions: The MVA pathway mediates anti-HER2 therapy resistance via Y/T, survivin, and mTOR, in some cell models and this resistance can be overcome by Sim and ZA. The potential of MVA pathway inhibition to enhance anti-HER2 therapy efficacy warrants further clinical studies. Citation Format: Vidyalakshmi Sethunath, Huizhong Hu, Carmine DeAngelis, Jamunarani Veeraraghavan, Lanfang Qin, Martin Shea, Tamika Mitchell, Sarmistha Nanda, Resel Pereira, Susan G. Hilsenbeck, Mothaffar F. Rimawi, Kent C. Osborne, Rachel Schiff. Targeting the mevalonate pathway in HER2+breast cancer to overcome resistance and enhance anti-HER2 therapy efficacy [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 4757.

Bioassay-guided isolation of cytotoxic compounds from Chrysophthalmum montanum (DC.) Boiss

Bioassay-guided isolation of the 80% methanol extract of the aerial parts of Chrysophthalmum montanum (DC.) Boiss. (Asteraceae) led to the isolation of four known guaianolide-type sesquiterpene lactones, 6α-acetoxy-4α-hydroxy-1βH-guaia-9.11(13)-dien-12.8α-olide (1), 6α-acetoxy-4α-hydroxy-9β.10β-epoxy-1βH-guaia-11(13)-en-12.8α-olide (2), 4α,6α-dihydroxy-1β,5α,7αH-guaia-9(10),11(13)-dien-12,8α-olide (3), and (4α,5α,8β,10β)-4,10-dihydroxy-1,11(13)-guaidien-12,8-olide (4), along a steroidal glycoside mixture (5a and 5b). The structures of the compounds were identified on the basis of spectroscopic data. Among them, 2, 4 and a steroidal glycoside mixture were obtained from C. montanum for the first time. All isolates were also first time assayed for in vitro cytotoxicities against four human cancer cell lines, i.e. breast (MCF-7, MDA-MB 231), colon (HT-29), and lung (PC3). Among the isolates, 1–3 showed significant inhibitory effect on the proliferation of cancer cells with viability ranging from 6.86 to 26.51%, while steroidal glycoside mixture showed no cytotoxicity, except against HT-29 (viability 61.99%). Compound 4 exhibited strong and selective cell growth inhibition against HT-29 with viability 20.99% and was identified as a promising compound with high selectivity between cancer cells and normal human lung cells (BEAS-2B), especially against HT-29 (IC50 = 12.2 μg/mL) compared to that of cisplatin. These results suggested that 4 is worthy of further study to determine its cytotoxicity mechanisms.

Cytotoxic, proapoptotic and antioxidative potential of flavonoids isolated from propolis against colon (HCT-116) and breast (MDA-MB-231) cancer cell lines

Isolated and structurally confirmed, eleven flavonoids from propolis were examined for their cytotoxicity toward human colon cancer and human breast cancer cells. Their effect on induction of apoptosis and their antioxidative activities were also evaluated. Six flavonoids induced cytotoxic effects in both cell lines. Luteolin had a marked effect on both cell lines, especially on HCT-116 cells (IC50 72h, 66.86μM). Also, luteolin was observed to have the highest apoptotic potential after 72h treatment of examined cell lines (27.13% and 37.09%, respectively). Myricetin exhibited selective inhibition of cell growth (IC50 114.75μM) and induced apoptosis in MDA-MB-231 cells only. Luteolin and galangin exhibited prooxidative properties 24h after the treatment in HCT-116 cells, while myricetin induced prooxidative effects in MDA-MB-231 cells. On the other hand, selected flavonoids exhibited antioxidative properties 72h after the treatment, decreasing superoxide anion radical and nitrite levels in both cell lines. Cytotoxic and proapoptotic effects on colon and breast cancer cell lines and the influence on their redox status make tested flavonoids good candidates for developing new anticancer drugs.

Structure dependent selective efficacy of pyridine and pyrrole based Cu(II) Schiff base complexes towards in vitro cytotoxicity, apoptosis and DNA-bases binding in ground and excited state

This work highlights a systematic and comparative study of the structure-dependent influence of a series of biologically active Cu(II) Schiff base complexes (CSCs) on their in vitro cytotoxicity, apoptosis and binding with polymeric DNA-bases in ground and photo-excited states. The structure-activity relationship of the closely resembled CSCs towards in vitro cytotoxicity and apoptosis against cervical cancerous HeLa and normal human diploid WI-38 cell lines has been investigated by MTT assay and FACS techniques respectively. The steady-state and time-resolved spectroscopic studies have also been carried out to explore the selective binding affinities of the potential complexes towards different polymeric nucleic acid bases (poly d(A), poly d(T), poly d(G), poly d(C), Poly d(G)-Poly d(C)), which enlighten the knowledge regarding their ability in controlling the structure and medium dependent interactions in ‘ground’ and ‘excited’ states. The pyridine containing water soluble complexes (CuL1 and CuL3) are much more cytotoxic than the corresponding pyrrole counterparts (CuL2 and CuL4). Moreover the acidic hydrogens in CuL1 increase its cytotoxicity much more than methyl substitution as in CuL3. The results of MTT assay and double staining FACS experiments indicate selective inhibition of cell growth (cell viability 39% (HeLa) versus 85% (WI-38)) and occurrence of apoptosis rather than necrosis. The ground state binding of CuL1 with polymeric DNA bases, especially with guanine rich DNA (Kb=6.41±0.122×105), that enhances its cytotoxic activity, is further confirmed from its binding isotherms. On the other hand the pyrrole substituted CuL4 complex exhibits the structure and medium dependent selective electron-transfer in triplet state as observed in laser flash photolysis studies followed by magnetic field (MF) effect.

Selective in vitro anticancer effect of superparamagnetic iron oxide nanoparticles loaded in hyaluronan polymeric micelles.

Due to its native origin, excellent biocompatibility and biodegradability, hyaluronan (HA) represents an attractive polymer for superparamagnetic iron oxide nanoparticles (SPION) coating. Herein, we report HA polymeric micelles encapsulating oleic acid coated SPIONs, having a hydrodynamic size of about 100 nm and SPION loading capacity of 1-2 wt %. The HA-SPION polymeric micelles were found to be selectively cytotoxic toward a number of human cancer cell lines, mainly those of colon adenocarcinoma (HT-29). The selective inhibition of cell growth was even observed when the SPION loaded HA polymeric micelles were incubated with a mixture of control and cancer cells. The selective in vitro inhibition could not be connected with an enhanced CD44 uptake or radical oxygen species formation and was rather connected with a different way of SPION intracellular release. While aggregated iron particles were visualized in control cells, nonaggregated solubilized iron oxide particles were detected in cancer cells. In vivo SPION accumulation in intramuscular tumor following an intravenous micelle administration was confirmed by magnetic resonance (MR) imaging and histological analysis. Having a suitable hydrodynamic size, high magnetic relaxivity, and being cancer specific and able to accumulate in vivo in tumors, SPION-loaded HA micelles represent a promising platform for theranostic applications.

OP449, a Novel SET Antagonist, Is Cytotoxic To Leukemia Cells and Enhances Efficacy Of Tyrosine Kinase Inhibitors In Drug-Resistant Myeloid Leukemias

BackgroundThe SET oncoprotein, an inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia cells, preventing PP2A from performing its regulatory role in deactivating signaling proteins by dephosphorylation. Restoration of PP2A activity in both chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cells to normal levels through shRNA-mediated knockdown of SET results in reduced leukemogenesis. Given the central role of PP2A and SET in regulating various kinase-dependent and -independent downstream signaling pathways, we evaluated the efficacy of SET antagonism in CML and AML cell lines as well as primary patient cells using OP449, a novel, specific, cell-penetrating SET antagonist. ResultsTreatment of human and murine CML cells with OP449 resulted in dose-dependent increase in PP2A activity and selective inhibition of cell growth (IC50: 0.60 to 1.11 μM), while parental Ba/F3 cells exhibited no measurable cytotoxicity. OP449-mediated decrease in the viability of leukemia cells was significantly rescued by co-treatment with okadaic acid, a PP2A inhibitor, confirming efficacy is mediated through PP2A activation. OP449 was also 3 to 8-fold more potent than FTY720 (a known activator of PP2A) and induced dephosphorylation/degradation of BCR-ABL1, AKT, and STAT5. Importantly, OP449 demonstrated activity against the ABL1 tyrosine kinase inhibitor-resistant BCR-ABL1T315I mutant and the BCR-ABL1E255V/T315I compound mutant (IC50: 1.62 and 1.97 μM, respectively). Consistent with cell line findings, OP449 also inhibited growth of primary cells from CML blastic phase patients harboring either wildtype BCR-ABL1 or BCR-ABL1T315I while normal CD34+ cells exhibited minimal effect. Further, treatment of CML cell lines and primary CD34+ CML cells with OP449 in combination with the ABL1 tyrosine kinase inhibitors showed significantly increased cytotoxicity as compared to each compound alone. For example, treatment of primary CD34+ CML cells with 2.5 μM OP449 or 200 nM nilotinib alone each resulted in a 50% reduction in colony formation, while combination of OP449 and nilotinib at these concentrations reduced colony formation by approximately 87%, suggesting synergistic reduction of clonogenicity (combination index: 0.195). Similar to our findings in CML cells, OP449 increased PP2A activity and suppressed growth in a dose-dependent manner in AML cell lines and primary patient samples harboring various different genetic lesions including FLT3-ITD, CSF1R overexpression, NRASQ61L, and JAK3A572V. Additionally, synergistic inhibition of these cells was observed when OP449 was combined with relevant tyrosine kinase inhibitors and chemotherapy. For example, treatment of MOLM-14 cells (FLT3-ITD) with 2.5 μM OP449 or 1 nM AC220 alone reduced cell viability by 58% and 75%, respectively; combined treatment reduced cell growth 96% (combination index: 0.723). Similarly, treatment of HL-60 cells (NRASQ61L) with 1 μM OP449 or 250 nM cytarabine alone reduced cell viability by 40% and 60%, respectively, whereas combined treatment led to a 94% reduction in viability (combination index: 0.630). Mechanistically, AML patient samples showed significantly increased SET expression compared to normal CD34+ cells, and treatment of AML cells with OP449 reduced phosphorylation of downstream ERK, STAT5, AKT and S6 ribosomal protein signaling. Finally, to evaluate OP449 antitumor efficacy in vivo, we tested OP449 (5 mg/kg intraperitoneally every 3 days) in xenograft mice bearing human HL-60 cell derived tumors. OP449 significantly inhibited tumor growth measured over time and resulted in a >2-fold reduction in tumor burden at the end of the experiment compared to vehicle-treated controls (Day 18: 1.14±0.06 g vs. 0.45±0.08 g, respectively; p<0.001). These results demonstrate the in vivo efficacy of OP449 in a murine leukemia model. ConclusionsSET antagonism is selectively cytotoxic to CML and AML cells harboring various genetic lesions and drug-resistant mutations. Our results demonstrate that combined targeting of SET and tyrosine kinases provides more efficient and selective inhibition of leukemia cell growth for a broad range of oncogenic lesions as compared to normal cells. Taken together, our findings suggest a novel therapeutic paradigm of SET antagonism in combination with tyrosine kinase inhibitors for the treatment of CML and AML patients with drug resistance. Disclosures:Agarwal:Oncotide Pharmaceuticals: Research Funding. Tyner:Incyte Corporation: Research Funding. Vitek:Oncotide Pharmaceuticals: Employment. Christensen:Oncotide Pharmaceuticals: Employment. Druker:Ambit Biosciences: Consultancy, PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb/Novartis: Currently PI or co-I on Novartis & Bristol-Myers Squibb clinical trials. His institution has contracts with these companies to pay for patient costs, nurse and data manager salaries, and institutional overhead. He does not derive salary, nor does his lab Other; Oncotide Pharmaceuticals: Research Funding, Subaward from NIH STTR, Subaward from NIH STTR Other.

Abstract LB-308: Protein kinase C beta selective inhibition induces apoptosis and arrests cell cycle in acute lymphoblastic leukemia cell lines

Abstract Acute lymphoblastic leukemia (ALL) is the most common leukemia in children and accounts for 20% of acute leukemia in adults. The intensive induction-consolidation-maintenance therapeutic regimens used currently have improved the 5-year disease free survival to around 80% in children and to 25%-40% in adults. The poorer response in adults is basically due to the inability to tolerate the intensive chemotherapy, and to the biology of adult disease which is associated with poor-risk prognostic factors. In the present era of target-specific therapy, PKCß targeting arose as a new, promising, and well tolerated treatment strategy in a variety of neoplasms, especially in B-cell malignancies. PKCß plays a major role in B-cell receptor signaling, but studies describing the role of PKCß in B-cell ALL are lacking. In the present study, we measured the sensitivity of a variety of B-cell ALL cell lines to PKC selective inhibition. Five cell lines were studied: RS4;11 and SEM-K2 [both Pro-B ALL with t(4;11)(q21;q23)], HB-1119 [Pre-B ALL with t(11;19)(q23;p13)] TOM-1 [Ph-positive Pro-B ALL with t(9;22)], and Reh [Pre-B ALL with t(12;21)(p13;q22)]. Cells were tested for PKCß1 and PKCß2 expression by immunoblot. Cell viability was measured in the presence of PKCß-selective inhibitor at concentrations of 1, 2.5, 5, 10, 20 and 30 μM for 48 hours with 10% fetal bovine serum (FBS). MTS assay was performed to quantify cell viability, and TUNEL assay with propidium iodide staining was used to detect apoptotic induction and effect on cell cycle. Results showed that all five cell lines express PKCß1 and PKCß2. Treatment with PKCß-selective inhibitor resulted in a dose-dependent inhibition of cell proliferation. RS4;11 and SEM-K2 cell lines showed the greatest sensitivity with statistically significant cell growth inhibition at inhibitor concentrations as low as 1 μM and an IC50 of 5 μM. Other cell lines required relatively higher inhibitor concentrations, with cell growth inhibition starting at 5 μM and an IC50 of 14 μM. The mechanism of PKC -selective cell growth inhibition was shown by flow cytometric TUNEL assay to involve apoptotic induction. No effect on cell cycle progression was observed, except in the Reh cell line. Apoptotic induction in RS4;11 and SEM-K2 cell lines was linked to the inhibition of GSK-3ß phosphorylation; flow cytometric analysis showed a 25% decrease in phosphorylated GSK-3ß after 24 hours incubation with the PKCß-selective inhibitor. These results indicate that PKCß plays an important role in the malignant process in B-cell ALL, and suggest that PKCß targeting should be considered as a potential treatment, whether in combination with the current regimens used or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCß inhibition and uncover possible relationships between PKCß signaling and t(4;11) (q21;q23). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-308.

Human telomeric G‐quadruplex: The current status of telomeric G‐quadruplexes as therapeutic targets in human cancer

The 3'-ends of human chromosomal DNA terminate in short single-stranded guanine-rich tandem-repeat sequences. In cancer cells, these are associated with the telomere-maintenance enzyme telomerase together with the end-binding protein hPOT1. Small molecules that can compete with these proteins and induce the single-stranded DNA to form quadruplex-ligand complexes are, in effect, able to expose these 3'-ends, which results in the activation of a DNA damage response and selective inhibition of cell growth. Several of these G-quadruplex binding molecules have shown promising anticancer activity in tumour xenograft models, which indicate that the approach may be applicable to the treatment of a wide range of human cancers. This minireview summarizes the available data on these compounds and the challenges posed for drug discovery.

148 POSTER Comparison of the cellular and biochemical properties of ansamycin and non-ansamycin based Hsp90 inhibitors

Background: Heat shock protein 90 (Hsp90) has emerged as an important target for the treatment of cancer due to its essential role in several key oncogenic signaling pathways. Several classes of Hsp90 inhibitors have recently advanced into clinical trials including ansamycin derivatives that are semi-synthetic derivatives of the natural product geldanamycin (e.g. 17-AAG, IPI-504, 17-DMAG) or synthetic small molecules (e.g. purine derivatives, isoxazoles, pyrazoles). Ansamycin derivatives are potent Hsp90 inhibitors that demonstrate selective cell growth inhibition toward cancer cells as compared to normal cells. We have determined the biochemical and cellular properties of a group of published Hsp90 inhibitors, including both natural product derived and synthetic compounds. Materials and methods: The biochemical affinity of inhibitors to Hsp90 was determined using a competition binding assay using radioactively labeled competitor compound and Hsp90 purified from Hela cells. The growth inhibition induced by Hsp90 inhibitors was evaluated in human normal and cancer cells by measuring cell growth in the presence of varying concentrations of compounds using the Alamar Blue assay after 72h of compound addition. Results: The biochemical affinities to purified Hsp90 for the inhibitors tested range from 0.1 to 500 nM. There is an approximate correlation between biochemical affinity and cell growth inhibition of cancer cells with a relative activity ranking of isoxazoles > ansamycins > purines. While ansamycins demonstrate selective growth inhibition of cancer cells compared to normal cells as previously described in the literature, the more potent isoxazole derivative also potently inhibits the growth of some normal cell types. Three of the most potent compounds on cancer cells were also tested in vivo for their ability to induce the degradation of a Hsp90 client protein (mutEGFR) in a xenograft model of human NSCLC. In this model, we observe similar suppression of mutant EGFR and similar induction of cleaved caspase 3 for all three compounds, independent of their biochemical affinity for purified Hsp90. One possible explanation for this observation is provided by our measurements of off-rates of inhibitors from Hsp90. Conclusion: The experiments presented above raise the question of whether synthetic Hsp90 inhibitors with high affinity for Hsp90 have lost some of the in vitro therapeutic window between cancer and normal cells that makes Hsp90 inhibitors such attractive candidates for cancer therapeutics. Inhibitor Ki (nM)

Diastereomeric Selective Effects of Double-Stranded Peptides Conjugated with &amp;ndash;Phe&amp;ndash;Phe&amp;ndash; Residue for Growth Inhibition and Permeability of Ca&lt;sup&gt;2+&lt;/sup&gt; on A431, &lt;i&gt;src&lt;/i&gt;&lt;sup&gt;ts&lt;/sup&gt;NRK, and A549 Cells Proliferation

Our aim in this study is to elucidate the correlations between inhibition and chirality, especially, diastereomer, against cell proliferation of double-stranded peptides. In previous studies, we reported on the design, synthesis, and chemical properties on a series of novel double-stranded peptides, (y-AA-x-AA)(2)-spacer(S) (AA=amino acid, S=spacer, symbols x and y represent L- or D-forms, and (y-, x-) as represent of the symbol) conjugated with -y-AA-x-AA- and -z-AA-y-AA-x-AA- sequences to a spacer of carbon number n. The inhibition of A431 and src(ts)NRK cells growth by four diastereomers of the N(1),N(12)-bis(y-Phe-x-Phe)dodecanediamines (n=12) increased in the following order: (L-, L-)<(D-, D-)<(L-, D-)<(D-, L-). A similar trend was seen in the order for the activity of (y-AA-x-AA)(2)-spacer(S) with a spacer of carbon number n=2, 3, 4, 5, 6, and 12 against the cell growth inhibition. To understand the mechanism of diasteromer selective cell growth inhibition, the correlations between chirality and cell growth inhibition were investigated from the measurement of the changes in cytosolic Ca(2+) concentration (=[Ca(2+)](c)) of A431 cells. Although less active N(1),N(12)-bis(L-Phe-L-Phe)dodecanediamine increases cytosolic [Ca(2+)](c), more active diasteromers, N(1),N(12)-bis(L-Phe-D-Phe)dodecanediamine and N(1),N(12)-bis(D-Phe-L-Phe)dodecanediamine, decrease cytosolic [Ca(2+)](c) in A431 cell. This study provides diastereomeric effected new insights - this controls the polarity of double-stranded peptides - into the control of tumor cell proliferation and design of the uptake by penetration through the cell membrane of drugs.

Arylisothiocyanato selective androgen receptor modulators (SARMs) for prostate cancer

A new series of androgen receptor targeted agents (ARTA) was prepared and tested in androgen-dependent and -independent prostate cancer cell lines. These agents were bicalutamide analogs with isothiocyanato substituted B-rings. Also, the linker sulfone of R-bicalutamide was maintained or replaced with several alternative linkages including ether, amine, N-methylamine, thioether, and methylene (in this case the product was a racemic mixture) functional groups at the X-position. To expand the structure–activity relationship (SAR) of these arylisothiocyanato AR ligands, B-ring halogenated arylisothiocyanato ligands were also prepared and tested. The arylisothiocyanato AR ligands showed strong binding affinities to AR ranging from 0.6 to 54nM. Among them, thioether and ether linkages demonstrated high binding affinities (0.6 and 4.6nM, respectively) and selective cell growth inhibition (approximately 3- to 6-fold) for LNCaP, an androgen-dependent prostate cancer cell line, when compared to the androgen independent prostate cell lines (DU145, PC-3, and PPC-1) and a bladder cell line (TSU-Pr1). However, the ligands were inactive (IC50>100mM) in a normal monkey kidney cell line (CV-1) that was used as the control for non-specific toxicity.

Selective inhibition of cell growth by activin in SNU-16 cells

To investigate whether activin regulates the cell proliferation of human gastric cancer cell line SNU-16 through the mRNA changes in activin receptors, Smads and p21(CIP1/WAF1). The human gastric cancer cell lines were cultured, RNAs were purified, and RT-PCRs were carried out with specifically designed primer for each gene. Among them, the two cell lines SNU-5 and SNU-16 were cultured with activin A for 24, 48 and 72 h. The cell proliferation was measured by MTT assay. For SNU-16, changes in ActRIA, ActRIB, ActRIIA, ActRIIB, Smad2, Smad4, Smad7, and p21(CIP1/WAF1) mRNAs were detected with RT-PCR after the cells were cultured with activin A for 24, 48 and 72 h. The proliferation of SNU-16 cells was down regulated by activin A whereas other cells showed no change. Basal level of inhibin/activin subunits, activin receptors, Smads, and p21(CIP1/WAF1) except for activin betaB mRNAs was observed to have differential expression patterns in the human gastric cancer cell lines, AGS, KATO III, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-638, SNU-668, and SNU-719. Interestingly, significantly higher expressions of ActR IIA and IIB mRNAs were observed in SNU-16 cells when compared to other cells. After activin treatment, ActR IA, IB, and IIA mRNA levels were decreased whereas ActR IIB mRNA level increased in SNU-16 cells. Smad4 mRNA increased for up to 48 h whereas Smad7 mRNA increased sharply at 24 h and returned to the initial level at 48 h in SNU-16 cells. In addition, expression of the p21(CIP1/WAF1), the mitotic inhibitor, peaked at 72 h after activin treatment in SNU-16 cells. Our results suggest that inhibition of cell growth by activin is regulated by the negative feedback effect of Smad7 on the activin signaling pathway, and is mediated through p21(CIP1/WAF1) activation in SNU-16 cells.

Primary Antimitochondrial Activity of the Cancer Drug Methylglyoxal Bis(guanylhydrazone) in Yeast Cells

Primary action of methylglyoxal bis(guanylhydrazone) (MGBG) on the yeast mitochondrial system was demonstrated by (1) selective inhibition of cell growth in non-fermentable medium, (2) blockage of mitochondrial synthesis of cytochromes aa3 and b and (3) ultrastructural aberration. The drug caused extensive deletions in mitochondrial DNA detected by an increase in the frequency of the mitochondrial mutant petite but had little or no effect on cell viability. Growth inhibition by MGBG had any effect on growth inhibition by ethidium bromide. Strains showed no cross-correlation in their tolerance to MGBG and ethidium bromide.

Selective inhibition of cell growth and associated changes in glycolipid metabolism induced by monovalent antibodies to glycolipids

Monovalent antibodies directed against N -acetylhematoside are growth inhibitory for BALB/3T3 and NIL hamster fibroblasts but not their transformed counterparts. Within a similar dose range antibodies directed against globoside have no effect on cell growth. Inhibition of 3T3 cell growth by anti-hematoside correlates with a specific change in the metabolism of hematoside within the cell membrane. Following antibody treatment the radiolabeling of hematoside is elevated for cell in logarithmic growth but reduced relative to control at final saturation density. This effect is not observed for 3T3 cells transformed by Kirsten murine sarcoma virus. It is suggested that cell surface glycolipids may play a role in the control of normal cell growth in vitro.