Accumulation Of Cells In G1 Research Articles

Abstract 3229: IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets

Abstract Block in differentiation and accumulation of undifferentiated blasts are hallmarks of Acute Myeloid Leukemia (AML) and epigenetic processes have been shown to play a critical role in hematological malignancies. Paralogs CREBBP and EP300, in association with co-factors, are hijacked during leukemogenesis by aberrant transcriptional factors, thus driving blast proliferation and maintaining the undifferentiated phenotype. Here, we introduce IACS-16559, a bromodomain inhibitor targeting CREBBP/EP300. IACS-16559 is highly selective for the bromodomains of CBP and EP300 versus BRD4, showing a dose-dependent inhibition of H3K27 acetylation residue in vivo and in vitro. In addition, IACS-16559 pharmacokinetic properties show low clearances, high oral exposures and predicted QD dosing in human. We screened AML cell lines in vitro for their response to IACS-16559 and identified specific subtypes of AML that exhibit heightened sensitivity to CBP/EP300 inhibition, specifically AML with AML1-ETO and MLL-AF9 rearrangements, and AML featuring mutations in the nucleophosmin (NPM1c) gene. IACS-16559 inhibited proliferation of AML responder cell lines in vitro, halted cell cycle progression, with an accumulation of cells in G1 and a decrease of cells in S phase. IACS-16559 did not induce apoptosis across a panel of AML cell lines, indicating that induction of apoptosis is not a major mechanism of action of CBP/EP300 inhibition. To augment the anti-tumor effects of IACS-16559, we prioritized the pharmacological combination of CBP/EP300 and MLL-Menin inhibitors. This combination synergistically impeded cell proliferation and potently triggered apoptosis in vitro and in vivo, whereas none of the individual agents alone had this effect. The changes in gene and surface marker expression produced by these treatments, as single agents and in combination with synergistic effects, were in line with an induced myeloid differentiation phenotype. Collectively, our findings provide a compelling basis for further exploring the use of a CBP/EP300 bromodomain inhibitor in conjunction with an inhibitor of the MLL-Menin interaction in a subset of AML patients with MLL rearrangements or NPM1 mutations. Citation Format: Ciro Zanca, Michael Soth, Guang Gao, Jason Gay, Ningping Feng, Christopher Bristow, Kang Le, Quanyun Xu, Phuong Nguyen, Yongying Jiang, Teresa Perry, Faika Mseeh, Paul Leonard, Jason Cross, Virginia Giuliani, Joe Marszalek, Philip Jones, Tim Heffernan. IACS-16559, a CBP/P300 bromodomain inhibitor for the treatment of specific AML subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3229.

Intracellular Potassium in Cell Growth and Proliferation of Human Mesenchymal Stem Cells and Blood Lymphocytes

Many data show that K+ ions are essential for cell proliferation. In this brief review, we summarize our own studies and literature data that characterize the relationship between modulations of intracellular K+ content and the intensity of cell proliferation. Using flame emission assay we compared the transport of monovalent cations in transformed cells and in human mesenchymal stem cells in growing cultures, as well as during stress-induced cell cycle arrest and transition from monolayer (2D) to three-dimensional (3D) spheroids. A decrease in the intracellular content of K+ (evaluated as the ratio of the content of K+ in cells to the mass of cellular protein) associated with the accumulation of G1 cells in the population and accompanied by a stop in proliferation was revealed. The relationship between intracellular K+ and initiation of cell proliferation was further analyzed in human blood lymphocytes (HBL) as a model for the transition of cells from quiescence to proliferation. In HBL stimulated to proliferate, the content of K+ in cells increases during the transition G0/G1/S, accompanying the growth of small lymphocytes into blasts. Cellular water content, calculated from buoyant cell density, is higher in proliferating HBLs and in Jurkat leukemic T cells than in resting HBL. Available data suggest that intracellular K+ is important for successful cell proliferation as the main intracellular ion involved in the regulation of cell volume during the transition from quiescence to proliferation, and high K+ content and associated high water content in the cell are a characteristic feature of cell proliferation and transformation.

Abstract 5644: Activation of p53 in prolonged prometaphase

Abstract The tumor suppressor p53 plays a critical role in protecting cells from aberrant divisions in the face of DNA damage, and while its role in DNA damage checkpoints have been extensively studied, its role in the mitotic checkpoint is less well characterized. The mitotic checkpoint prevents completion of mitosis until chromosomes have made bipolar kinetochore attachments to microtubules. Previous studies have shown that cells that experience a prolonged prometaphase(>1.5 h) following treatment with microtubule destabilizing drugs will arrest in G1 following eventual exit from mitosis. Given its role in DNA damage checkpoints, p53 appears to be a likely candidate for involvement in this arrest. The goal of the current study is to investigate the role of p53 in this G1 arrest following prolonged prometaphase. Three cells lines were utilized:MCF10A, MCF10A/αp53 which overexpressed shRNA to p53, and MCF10A/OD which overexpresses the p53 oligomerization domain. Cells were treated with 9 μM RO-3306 for 18 h to synchronize cells at the G2/M transition. The RO-3306 was then removed and cells were treated with a microtubule destabilizing drug, nocodazole, at a concentration of 0.08 μM for 1.5 or 2.5 h to cause prometaphase arrest. The cells were then washed to remove nocodazole, and the cells were harvested at various time points and analyzed via flow cytometry and Western blot. Flow cytometry analysis in in all three cell lines showed an accumulation of cells in G1 at 12 hours in for both treatment times (1.5 and 2.5 h), with subsequent increases in sub-G1 populations by 24 hours. Western blot analysis revealed activation of p53 by phosphorylation of ser20 and activation of p21 in the MCF10A and MCF10A/OD, but not in the MCF10A/αp53 for both treatment times. Taken together, these results suggest that activation of p53 alone is not sufficient to elicit the G1 arrest, as the MCF10A/αp53 cells arrested in G1 despite the absence of p53 protein. Citation Format: Megan Anderson, Aime A. Levesque. Activation of p53 in prolonged prometaphase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5644.

The budding yeast transition to quiescence.

A subset of Saccharomyces cerevisiae cells in a stationary phase culture achieve a unique quiescent state characterized by increased cell density, stress tolerance, and longevity. Trehalose accumulation is necessary but not sufficient for conferring this state, and it is not recapitulated by abrupt starvation. The fraction of cells that achieve this state varies widely in haploids and diploids and can approach 100%, indicating that both mother and daughter cells can enter quiescence. The transition begins when about half the glucose has been taken up from the medium. The high affinity glucose transporters are turned on, glycogen storage begins, the Rim15 kinase enters the nucleus and the accumulation of cells in G1 is initiated. After the diauxic shift (DS), when glucose is exhausted from the medium, growth promoting genes are repressed by the recruitment of the histone deacetylase Rpd3 by quiescence-specific repressors. The final division that takes place post-DS is highly asymmetrical and G1 arrest is complete after 48 h. The timing of these events can vary considerably, but they are tightly correlated with total biomass of the culture, suggesting that the transition to quiescence is tightly linked to changes in external glucose levels. After 7 days in culture, there are massive morphological changes at the protein and organelle level. There are global changes in histone modification. An extensive array of condensin-dependent, long-range chromatin interactions lead to genome-wide chromatin compaction that is conserved in yeast and human cells. These interactions are required for the global transcriptional repression that occurs in quiescent yeast.

Abstract 1023: Combining all-trans retinoic acid therapy with androgen receptor N-terminal domain inhibitors for the treatment of castration-resistant prostate cancer

Abstract Androgen receptor (AR) signaling plays an essential role in all stages of prostate cancer. Androgen-deprivation therapy is generally effective for advanced prostate cancer until progression to lethal metastatic castration-resistant prostate cancer (mCRPC). Most CRPC continues to be driven by AR signaling. AR transcriptional activity requires a functional N-terminal domain (NTD). Transactivation of AR is mediated by ligand-independent activation by cross-talk with signal transduction pathways targeting the AR NTD, gain-of-function mutations in AR ligand-binding domain (LBD), or expression of truncated AR splice variants that lack LBD (e.g., AR-V7). The intrinsically disordered AR NTD is essential for its transcriptional activity. It harbors six putative binding sites for Pin1, a proline isomerase that regulates protein conformation at specific phosphorylated-Ser/Thr-Pro motifs. All-trans retinoic acid (ATRA) is a validated and potent Pin1 inhibitor. Since conformational changes within the AR NTD are required for transactivation, perturbation of its structure may be a promising approach to block its activity. The purpose of this study was to assess a therapy that combined ATRA with antagonists of AR NTD, ralaniten (EPI-002) and its analogues. We hypothesized that targeting Pin1 activity with ATRA would disrupt the AR NTD and enhance inhibition by EPI compounds which bind to Tau-5 in the NTD. Using reporter gene assays, proliferation assays, and cell cycle analysis by flow cytometry, we tested ATRA in isolation and in combination with EPI in androgen-sensitive (LNCaP) and androgen-independent (LN95) prostate cancer cell lines. We found that treatment with ATRA decreased the transcriptional activity of AR and androgen-induced expression of PSA. ATRA also attenuated the transcriptional activity of AR-V7 and androgen-independent growth of LN95 cells expressing both full-length AR and AR-V7. Co-immunoprecipitation studies confirmed interactions between Pin1 and specific regions of AR NTD. In combination, ATRA had synergistic interaction with EPI compounds and lowered the effective inhibitory concentrations for blocking AR transcriptional activity. Furthermore, combinations decreased cell cycle progression of LN95 cells through S-phase, leading to accumulation of cells in G1 and induction of senescence. These preclinical findings showed that ATRA enhanced the potency of AR NTD inhibitors and support a novel therapeutic strategy for CRPC. Future studies will determine the in vivo antitumor effect of ATRA in combination with EPI compounds on the growth of CRPC xenografts. Citation Format: Jacky K. Leung, Marianne D. Sadar. Combining all-trans retinoic acid therapy with androgen receptor N-terminal domain inhibitors for the treatment of castration-resistant prostate cancer [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 1023.

Ion Homeostasis during the Growth of Human Mesenchymal Stem Culture. I. Density-Dependent Changes of Intracellular K+ and Na+ Content and K+ Influxes

In this study, we reported proliferation-related changes in cell K+ and Na+ content and K+ influxes in cultivated human mesenchymal stem cells (MSCs). The intracellular K+ content calculated per cell protein amount was found to decrease in the growing culture, whereas intracellular Na+ content was not significantly changed. It was also revealed that, at higher densities of an hMSC monolayer, the ouabain-sensitive K+ influx was decreased thus indicating a decline in Na+, K+ pump-mediated transport. We analyzed the cell cycle profiles of hMSC cultures and found that, under optimal culture conditions in high-density cultures, the decline in K+ content per cell protein was correlated with accumulation of G1 cells and slowing down cell proliferation in the population. It is concluded that cell K+ content per cell protein is an informative test for assessing the functional status of stem cells in vitro.

Proliferation-related changes in K+ content in human mesenchymal stem cells

Intracellular monovalent ions have been shown to be important for cell proliferation, however, mechanisms through which ions regulate cell proliferation is not well understood. Ion transporters may be implicated in the intracellular signaling: Na+ and Cl− participate in regulation of intracellular pH, transmembrane potential, Ca2+ homeostasis. Recently, it is has been suggested that K+ may be involved in “the pluripotency signaling network”. Our study has been focused on the relations between K+ transport and stem cell proliferation. We compared monovalent cation transport in human mesenchymal stem cells (hMSCs) at different passages and at low and high densities of culture as well as during stress-induced cell cycle arrest and revealed a decline in K+ content per cell protein which was associated with accumulation of G1 cells in population and accompanied cell proliferation slowing. It is suggested that cell K+ may be important for successful cell proliferation as the main intracellular ion that participates in regulation of cell volume during cell cycle progression. It is proposed that cell K+ content as related to cell protein is a physiological marker of stem cell proliferation and may be used as an informative test for assessing the functional status of stem cells in vitro.

Abstract 3509: The glutaminase inhibitor CB-839 synergizes with CDK4/6 and PARP inhibitors in pre-clinical tumor models

Abstract Many tumor cells utilize the amino acid glutamine to meet the elevated bioenergetic and biosynthetic demands of rapid cell growth. The enzyme glutaminase converts glutamine to glutamate, which is used to fuel the TCA cycle, synthesize amino acids and nucleotides, and balance cellular oxidative stress. We developed CB-839, a potent and orally bioavailable small molecule inhibitor of glutaminase, that blocks production of glutamate and generation of downstream metabolites glutathione, malate and aspartate. Mice treated with CB-839 had decreased levels of nucleotides in their tumors, likely due to glutamine-derived aspartate being required for nucleotide biosynthesis. Consistent with our finding that CB-839 decreases nucleotide pools, CB-839 treatment delayed cancer cells from either entry into S-phase or progression through S-phase. Based on this observation, the ability of CB-839 to synergize with therapies that block cell cycle progression was tested. CB-839 synergized with the CDK4/6 inhibitor palbociclib in colorectal carcinoma (CRC), triple negative breast cancer (TNBC) and ER+ breast cancer cell lines resulting in anti-proliferative activity. The combination of CB-839 with palbociclib also led to decreased cell cycle progression through S-phase and caused an accumulation of cells in G1 as measured by an EdU DNA incorporation assay. In vivo, the combination of CB-839 with palbociclib resulted in enhanced anti-tumor activity in both an ER+ breast cancer and CRC xenograft tumor model. We next investigated whether CB-839 treatment would enhance the anti-tumor effects of DNA repair inhibitors, given the ability of CB-839 treatment to decrease nucleotide pools. CB-839 treatment in combination with the PARP inhibitors niraparib and talazoparib led to synergistic anti-proliferative activity in TNBC, CRC, non-small cell lung carcinoma, ovarian and prostate cancer cells. In vivo, the combination of CB-839 with PARP inhibitors showed enhanced anti-tumor activity compared to single agent treatment in a CRC tumor xenograft model. CB-839 is currently undergoing evaluation for efficacy in the treatment of cancer in several phase I/II clinical trials. These encouraging pre-clinical results support the testing of CB-839 with CDK4/6 or PARP inhibitors in cancer patients. Citation Format: Ethan D. Emberley, Mark Bennett, Jason Chen, Matthew Gross, Tony Huang, Amani Makkouk, Gisele Marguier, Alison Pan, Sandra M. Spurlock, Susanne Steggerda, Francesco Parlati. The glutaminase inhibitor CB-839 synergizes with CDK4/6 and PARP inhibitors in pre-clinical tumor models [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 3509.

Modeling cell survival and change in amount of DNA during protracted irradiation

Hyper-radiosensitivity (HRS) is a well-known bioresponse under low-dose or low-dose-rate exposures. Although disorder of the DNA repair function, non-targeted effects and accumulation of cells in G2 have been experimentally observed, the mechanism for inducing HRS by long-term irradiation is still unclear. On the basis of biological experiments and a theoretical study, we have shown that change in the amount of DNA associated with accumulation of cells in G2 enhances radiosensitivity. To demonstrate continuous irradiation with 250 kVp X-rays, we adopted a fractionated regimen of 0.186 or 1.00 Gy per fraction at intervals of 1 h (i.e. 0.186 Gy/h, 1.00 Gy/h on average) to Chinese Hamster Ovary (CHO)-K1 cells. The change in the amount of DNA during irradiation was quantified by flow cytometric analysis with propidium iodide (PI). Concurrently, we attempted a theoretical evaluation of the DNA damage by using a microdosimetric-kinetic (MK) model that was modified to incorporate the change in the amount of DNA. Our experimental results showed that the fraction of the cells in G2/M phase increased by 6.7% with 0.186 Gy/h and by 22.1% with 1.00 Gy/h after the 12th irradiation. The MK model considering the change in amount of DNA during the irradiation exhibited a higher radiosensitivity at a high dose range, which could account for the experimental clonogenic survival. The theoretical results suggest that HRS in the high dose range is associated with an increase in the total amount of DNA during irradiation.

Abstract 393: NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma

Abstract Recently published whole exome sequencing studies in head and neck squamous cell carcinoma (HNSCC) tumors revealed that few had therapeutically targetable alterations using current strategies. This finding defines translational gap between genomics and HNSCC treatment. One potential targetable alteration is PIK3CA mutations. However, clinical trials testing PI3K/mTOR pathway inhibitors have had limited success and these inhibitors only lead to cell cycle arrest in PIK3CA mutant HNSCC cell lines. Thus, there is a critical need to identify therapeutic vulnerabilities for common mutation groups, including tumor suppressors, in HNSCC. One of these molecular subgroups is NOTCH1 which is the second most frequently mutated gene in HNSCC, with a 10-15% prevalence of inactivating mutations. Although there are several studies underscoring the importance of NOTCH1 as a tumor suppressor in HNSCC, none has identified a therapy that targets NOTCH1 mutant (mut) HNSCC. Our objective was to identify predictive biomarkers of sensitivity to PI3K/mTOR inhibitors by integrating drug and multiple-omics data. Cell viability with six PI3K/mTOR inhibitors in 68 HNSCC lines was measured by the CellTiter Glo assay. The peak plasma concentration of each drug was used as the cut-off to determine sensitivity. We observed a striking correlation between NOTCH1mut and sensitivity to PI3K/mTOR pathway inhibitors. When fisher's exact test was performed, NOTCH1mut lines were more sensitive to GSK2126458 (P<0.027), BYL719 (P<0.004) and PQR309 (P<0.014) than NOTCH1 wild type cell lines. NOTCH1 was also identified as an upstream regulator in sensitive cell lines by Ingenuity® Pathway Analysis. Basal NOTCH1 protein expression was higher in HNSCC lines resistant to PI3K/mTOR inhibition using unsupervised hierarchical clustering of Reverse Phase Protein Array data. NOTCH1mut lines underwent more apoptosis after GSK2126458 treatment compared to NOTCH1wt lines (PCI15B- 48.1 fold; P<0.05, HN31- 46.9 fold; P<0.05). There was also increased accumulation of cells in G1 after GSK2126458 treatment in NOTCH1mut lines (PCI15B-1.3 fold, P<0.05; HN31- 1.4 fold, P<0.05). To check if inhibition of NOTCH1 pathway inhibition sensitizes NOTCH1wt lines to PI3K/mTOR inhibition, resistant NOTCH1wt lines were treated with Gamma secretase inhibitors and GSK2126458. The combination led to significantly decreased cell viability (DAPT- 1.5 fold and YO010227- 1.7 fold). The combination studies will be further expanded to 38 NOTCH1wt lines. On-going studies include assessment of drug sensitivity in vivo, mechanistic studies and the effect of genetic manipulation of NOTCH1 signaling on sensitivity to PI3K/mTOR inhibitors. Our data suggests that loss of active NOTCH1 signaling confers sensitivity to PI3K/mTOR inhibition. If the combination of NOTCH1 and PI3K/mTOR inhibition leads to apoptosis, this combination could be translated into the clinic. Citation Format: Faye M. Johnson, Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Pan Tong, Tuhina Mazumdar, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick. NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393.

Abstract 2999: Cytotoxicity with hydroxamic acid HDACis in combination with antimetabolites is highly sequence dependent in solid tumor cells

Abstract Used alone or in combination with other chemotherapeutics, the antimetabolites 2’, 2’-difluoro-2’-deoxycytidine (gemcitabine, dFdCyd), and 5-fluorouracil (5-FU)/fluorodeoxyuridine (FdUrd) comprise the mainstay of treatment for gastrointestinal malignancies, such as pancreatic and colorectal cancer. Gemcitabine with cisplatin is also first-line therapy for non-small cell lung cancer (NSCLC). While these treatments can prolong survival, many patients still relapse and there is need for improvement to current therapies. Cytotoxicity elicited by dFdCyd and 5-FU/FdUrd is influenced by drug metabolism/activation, DNA repair proteins, cell cycle progression, and apoptosis. Histone deacetylase inhibitors (HDACi) target histone deacetylases, which regulate gene expression through deacetylation of lysine residues on histones and some non-histone proteins. HDACis have been shown to increase expression of p21 and increase apoptosis which could enhance cytotoxicity with antimetabolites. The success of HDACis, including vorinostat and panobinostat, in the treatment of hematologic malignancies has encouraged their use in solid tumors. However, results from clinical trials in patients treated with HDACis alone or with other drugs, including antimetabolites have been mixed and the mechanism(s) underlying successful therapy with HDACis is largely unknown. In the present studies, we used solid tumor cell lines that represent tumors commonly treated with antimetabolites; A549 (NSCLC) and PANC1 (pancreatic cancer) cells, and HT29 (colon cancer) cells to investigate the effect of an HDACi with either dFdCyd or FdUrd, respectively, and we measured clonogenic survival. The data demonstrate that cytotoxicity with an HDACi and an antimetabolite is highly sequence dependent; vorinostat added concurrently with dFdCyd or FdUrd or prior to either antimetabolite resulted in antagonistic cytotoxicity, whereas the addition of the antimetabolite followed by HDACi was additive/synergistic with dFdCyd. Cytotoxicity with panobinostat and antimetabolites was also sequence dependent. Cell cycle distribution studies revealed the accumulation of cells in G1 and G2/M following HDACi addition (≥90% of cells), thus rendering S-phase specific dFdCyd less cytotoxic upon its subsequent addition. Similarly, concurrent addition of HDACi and dFdCyd produced less S-phase accumulation than observed with dFdCyd alone. These data demonstrate that administration of the HDACis and antimetabolites must be sequenced appropriately so that cell cycle effects complement rather than antagonize drug mechanisms. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Aaron Kramer, Jennifer Feigin, Donna S. Shewach. Cytotoxicity with hydroxamic acid HDACis in combination with antimetabolites is highly sequence dependent in solid tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2999.

Differential Effects of PI3K and Dual PI3K/mTOR Inhibition in Rat Prolactin-Secreting Pituitary Tumors.

Aggressive pituitary tumors are rare but difficult to manage, as there is no effective chemotherapy to restrict their growth and cause their shrinkage. Within these tumors, growth-promoting cascades, like the PI3K/mTOR pathway, appear to be activated. We tested the efficacy of two inhibitors of this pathway, NVP-BKM120 (Buparlisib; pan-PI3K) and NVP-BEZ235 (dual PI3K/mTOR), both in vitro on immortalized pituitary tumor cells (GH3) and on primary cell cultures of human pituitary tumors and in vivo on a rat model of prolactin (PRL) tumors (SMtTW3). In vitro, NVP-BEZ235 had a potent apoptotic and cytostatic effect that was characterized by decreased cyclin D/E and Cdk4/2 protein levels and subsequent accumulation of cells in G1 In vivo, the effect was transient, with a decrease in mitotic index and increase in apoptosis; long-term treatment had no significant inhibitory effect on tumor growth. In contrast, while NVP-BKM120 had little effect in vitro, it dramatically limited tumor growth in vivo Increased Akt phosphorylation observed only in the NVP-BEZ235-treated tumors may explain the differential response to the two inhibitors. Primary cell cultures of human PRL pituitary tumors responded to NVP-BEZ235 with reduced cell viability and decreased hormone secretion, whereas NVP-BKM120 had little effect. Altogether, these results show a potential for PI3K inhibitors in the management of aggressive pituitary tumors. Mol Cancer Ther; 15(6); 1261-70. ©2016 AACR.

Abstract P5-03-04: Identification of compounds from natural sources with selective activity against triple-negative breast cancer molecular subtypes

Abstract Triple negative breast cancers (TNBCs) lack expression of the estrogen and progesterone receptors (ER/PR) and do not have amplified HER2. While targeted therapies for ER+/PR+ and HER2-amplified breast cancers have greatly improved patient survival, there are no targeted therapies for TNBCs and no effective therapies to treat metastatic disease. There is a need to identify new therapeutic agents and molecular targets for treating TNBCs, but efforts have been limited by a lack of understanding of the subtypes of these heterogeneous diseases. However, gene expression profiling of TNBC patients recently identified 6 molecular subtypes of TNBC and representative cell lines, providing the first opportunity to identify subtype-specific leads for TNBC. We performed high-content screening to evaluate novel libraries of extracts from Texas plants and diverse fungal cultures for antiproliferative and/or cytotoxic activity in a panel of cell lines modeling five different TNBC molecular subtypes. The aim was to identify extracts with selective activity in a single cell line. We hypothesized that extracts found to have selective activity in one of these cell lines may target a protein or cellular process critical to the growth of that subtype. We identified 11 extracts with selective activity against cell lines representing four different TNBC molecular subtypes. From a fungal culture we identified a new compound called maximiscin, which was found to have selective cytotoxic efficacy against the MDA-MB-468 cell line of the basal-like 1 subtype. From a plant extract we isolated deguelin, which had selective activity in the MDA-MB-453 cell line, a model of the luminal androgen receptor (LAR) subtype. The molecular mechanisms of action of each compound were investigated in cell line models. Initial cell cycle studies using flow cytometry showed that maximiscin caused an accumulation of cells in G1 after 18h of treatment. Protein microarray studies indicated that maximiscin increased levels of phospho-p53, which was consistent with the observed G1 accumulation. Based on these findings, we hypothesized that maximiscin induces DNA damage and investigated the effects of maximiscin on the phosphorylation of several DNA damage response proteins. Maximiscin increased phosphorylation of Chk1, Chk2, p53 and H2A.X as soon as 2h after treatment, indicating an accumulation of DNA damage. Previous studies have shown that LAR TNBC cells are particularly sensitive to PI3K inhibitors in vitro compared to other TNBC subtypes. The effects of deguelin on PI3K-Akt-mTORC1 signaling were evaluated in both MDA-MB-453 and MDA-MB-231 cells. Phosphorylation of both ribosomal protein S6 and 4E-BP1 were dramatically reduced in MDA-MB-453 cells 2h after deguelin treatment. Interestingly this was not observed in MDA-MB-231 cells, suggesting inhibition of mTORC1 signaling may be involved in the selective activity of deguelin in MDA-MB-453 cells. Preliminary studies suggest deguelin may also decrease androgen receptor abundance in MDA-MB-453 cells, indicating multiple molecular mechanisms may be involved in its selective effects. These results demonstrate that compounds with selective activity against TNBC subtypes can be identified from nature. Citation Format: Robles AJ, Du L, Cai S, Risinger AL, Cichewicz RH, Mooberry SL. Identification of compounds from natural sources with selective activity against triple-negative breast cancer molecular subtypes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-04.

WTC-01, a novel synthetic oxime-flavone compound, destabilizes microtubules in human nasopharyngeal carcinoma cells in vitro and in vivo.

Dynamic polymerization of microtubules is essential for cancer cell growth and metastasis, and microtubule-disrupting agents have become the most successful anti-cancer agents in clinical use. Besides their antioxidant properties, flavonoids also exhibit strong microtubule-disrupting activity and inhibit tumour growth. We have designed, synthesized and tested a series of oxime/amide-containing flavone derivatives. Here we report the evaluation of one compound, WTC-01 for its anti-proliferative effects in human cancer cells. We used a range of cancer cell lines including two human nasopharyngeal carcinoma (NPC) cell lines, measuring proliferation, cell cycle and apoptosis, along with caspase levels and mitochondrial membrane potentials. Assays of tubulin polymerisation in vitro and computer modelling of the colchicine binding site in tubulin were also used. In mice, pharmacokinetics and growth of NPC-derived tumours were studied. WTC-01 was most potent against proliferation of NPC cells (IC50 = 0.45 μM), inducing accumulation of cells in G2 /M and increasing apoptosis, time- and concentration-dependently. The colchicine competition-binding experiments and computer modelling results suggested that WTC-01 causes microtubule disruption via binding to the colchicine-binding site of tubulin resulting in mitochondrial membrane damage and cell apoptosis via activation of caspase-9/-3 without noticeable activation of the caspase-8. Notably, our in vivo studies demonstrated that at doses of 25 and 50 mg·kg(-1) , WTC-01 exhibited good pharmacokinetic properties and completely inhibited the growth of NPC-TW01 cells in a xenograft nude mouse model. WTC-01, a new synthetic oxime-containing flavone, exhibited potent anti-tumour activity against NPC cells and merits further investigation.

Abstract 708: Androgen receptor activation inhibits papillary thyroid carcinoma cell cycle progression

Abstract The American Cancer Society predicts 62,980 new cases of thyroid cancer and 1,890 deaths due to the disease in 2014, making thyroid cancer the most prevalent endocrine malignancy. This is particularly concerning for women who have an approximately three-fold higher incidence of thyroid cancer than men. Significant research has been done to elucidate the role of estrogen as a driver of thyroid cancer. Taking the reverse approach, that men have a much lower incidence of thyroid cancer, led us to hypothesize that androgens and/or androgen receptors play a protective role and that PTC represents the escape from androgen-mediated cell regulation. Analysis of AR expression in 24 PTC patient tissue samples indicated a 2.7 fold reduction in AR expression compared to matched normal tissue (P<0.005). This suggests that the reduction of AR levels in the neoplastic process of thyroid tissue may be one mechanism of escape from androgen regulation. To more fully understand the function of androgen/AR in thyroid cells, the AR was stably transfected into 8505C anaplastic/PTC cells. Addition of DHT to the 8505C-transfected clone, 84E7, resulted in AR translocation into the nucleus and a 70% reduction in proliferation, as well as a shift in the cell cycle toward G1 arrest. Transcription profiling using RNA-Seq and gene ontology analysis revealed significant changes in genes associated with proliferation, cell cycle, and cell cycle regulation confirming the proliferation assay and cell cycle analysis above. Further, when we examined G1 associated cell cycle proteins, we found significant decreases in cell cycle progression proteins cdc25a, CDK6, CDK4, and CDK2 as well as significant increases in the inhibitors p27 and p21. Thus, protein, RNA and cell cycle analysis data are concordant and demonstrate an accumulation of cells in G1 when treated with DHT. Together these data demonstrate that expression of androgen/AR in normal thyroid cells may play a protective, anti-proliferative role that is lost in PTC resulting in dysregulation of genes normally under AR control. The differential expression of androgen/AR in males verses females may provide new insight into the different clinical presentations and outcomes between women and men. Citation Format: Melanie Elizabeth Jones, Timmy O'Connell, Anvita Gupta, Hong Zhao, Codrin Iacob, Augustine Moscatello, Edward Shin, Zbigniew Darzynkiewicz, Raj K. Tiwari, Jan Geliebter. Androgen receptor activation inhibits papillary thyroid carcinoma cell cycle progression. [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 708. doi:10.1158/1538-7445.AM2015-708

Identification of new therapeutic leads for triple negative breast cancer subtypes

High-content screening of plant and fungal extracts in a panel of cell lines modeling triple negative breast cancer subtypes identified 11 extracts with selective activity against particular cell lines. Maximiscin, (P/M) -1, was identified as having selective cytotoxic efficacy against the basal-like MDA-MB-468 cell line. Deguelin (2) was identified as having selective antiproliferative activity against the MDA-MB-453 cell line, representing the luminal androgen receptor subtype of TNBC. Mechanistic studies showed that maximiscin caused an accumulation of cells in G1. Immunoblotting of maximiscin-treated whole cell lysates showed that maximiscin increases levels of P-p53, P-Chk1, P-Chk2, and γ-H2A.X. These results are consistent with the observed G1 accumulation and collectively suggest that maximiscin induces DNA damage. Preliminary experiments suggest deguelin modulates AR localization and inhibits phosphorylation of Akt, ribosomal protein S6 and 4E-BP1 as soon as 2h after treatment. These results demonstrate that new compounds with potential therapeutic value for the treatment of TNBC subtypes can be identified from nature.

Abstract 3765: Indoleamine 2,3-dioxygenase mediates immune-independent human tumor cell resistance to olaparib, γ radiation, and cisplatin

Abstract Indoleamine 2,3-dioxygenase-1 (IDO) is an immunosuppressive molecule expressed by most human tumors. IDO levels correlate with poor prognosis in cancer patients and IDO inhibitors are under investigation to enhance endogenous anticancer immunosurveillance. Little is known of immune-independent functions of IDO relevant to cancer therapy. We show, for the first time, that IDO mediates human tumor cell resistance to a PARP inhibitor (olaparib), γ radiation, cisplatin, and combined treatment with olaparib and radiation, in the absence of immune cells. Antisense-mediated reduction of IDO, alone and (in a synthetic lethal approach) in combination with antisense to the DNA repair protein BRCA2 sensitizes human lung cancer cells to olaparib and cisplatin. Antisense reduction of IDO decreased NAD+ in human tumor cells. NAD+ is essential for PARP activity and these data suggest that IDO mediates treatment resistance independent of immunity and at least partially due to a previously unrecognized role for IDO in DNA repair. Furthermore, IDO levels correlated with accumulation of tumor cells in G1 and depletion of cells in G2/M of the cell cycle, suggesting that IDO effects on cell cycle may also modulate sensitivity to radiation and chemotherapeutic agents. IDO is a potentially valuable therapeutic target in cancer treatment, independent of immune function and in combination with other therapies. Supported by grants to JK and WM from the Canadian Institutes of Health Research (CIHR). Citation Format: Saman Maleki Vareki, Mateusz Rytelewski, Rene Figueredo, Di Chen, Peter J. Ferguson, Mark Vincent, Weiping Min, Xiufen Zheng, James Koropatnick. Indoleamine 2,3-dioxygenase mediates immune-independent human tumor cell resistance to olaparib, γ radiation, and cisplatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3765. doi:10.1158/1538-7445.AM2014-3765

Synthesis and Biological Evaluation of Imidazo[2,1‐b][1,3,4]thiadiazole‐Linked Oxindoles as Potent Tubulin Polymerization Inhibitors

A series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles composed of an A, B, C and D ring system were synthesized and investigated for anti-proliferative activity in various human cancer cell lines; test compounds were variously substituted at rings C and D. Among them, compounds 7 ((E)-5-fluoro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)-imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one), 11 ((E)-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one), and 15 ((E)-6-chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one) exhibited potent anti-proliferative activity. Treatment with these three compounds resulted in accumulation of cells in G2 /M phase, inhibition of tubulin assembly, and increased cyclin-B1 protein levels. Compound 7 displayed potent cytotoxicity, with an IC50 range of 1.1-1.6 μM, and inhibited tubulin polymerization with an IC50 value (0.15 μM) lower than that of combretastatin A-4 (1.16 μM). Docking studies reveal that compounds 7 and 11 bind with αAsn101, βThr179, and βCys241 in the colchicine binding site of tubulin.

Suppression of eukaryotic initiation factor 4E prevents chemotherapy-induced alopecia.

BackgroundChemotherapy-induced hair loss (alopecia) (CIA) is one of the most feared side effects of chemotherapy among cancer patients. There is currently no pharmacological approach to minimize CIA, although one strategy that has been proposed involves protecting normal cells from chemotherapy by transiently inducing cell cycle arrest. Proof-of-concept for this approach, known as cyclotherapy, has been demonstrated in cell culture settings.MethodsThe eukaryotic initiation factor (eIF) 4E is a cap binding protein that stimulates ribosome recruitment to mRNA templates during the initiation phase of translation. Suppression of eIF4E is known to induce cell cycle arrest. Using a novel inducible and reversible transgenic mouse model that enables RNAi-mediated suppression of eIF4E in vivo, we assessed the consequences of temporal eIF4E suppression on CIA.ResultsOur results demonstrate that transient inhibition of eIF4E protects against cyclophosphamide-induced alopecia at the organismal level. At the cellular level, this protection is associated with an accumulation of cells in G1, reduced apoptotic indices, and was phenocopied using small molecule inhibitors targeting the process of translation initiation.ConclusionsOur data provide a rationale for exploring suppression of translation initiation as an approach to prevent or minimize cyclophosphamide-induced alopecia.

Abstract A21: Autophagy: A potential target for colorectal cancer therapeutics.

Abstract Colorectal cancer is among the leading causes of death worldwide. Here, we investigate the molecular mechanism of crocin-induced apoptosis in colorectal cancer cells. Crocin was shown here to inhibit proliferation of both HCT wildtype and HCT p53-/- cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT wildtype cells in G1 compared to the control after 24 and 48 hours of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT p53-/- after 24 hours. Crocin induced apoptosis in both cell lines in a time dependent manner. Nevertheless, p53 deficient cells were not able to repair the damage induced by crocin. Crocin induced inefficient autophagy in HCT p53-/- cells where crocin induced the formation of LC3-II which was combined by a decrease in the protein levels of beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT p53-/- after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone, indicating an effective autophagic flux. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT wildtype cells. Baf-exposed-HCT p53-/- cells did not show any enhancement of apoptosis induction despite an increase in the DNA damage sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A21. Citation Format: Amr Amin, Khuloud Bajbouj, Adrian Koch, M. Gandesiri, Regine Schneider-Stock. Autophagy: A potential target for colorectal cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A21.