Some possible ways in which replication of plasmids containing the Epstein-Barr virus (EBV) plasmid maintenance origin, oriP, might be controlled were investigated. Virtually all plasmid molecules were found to replicate no more than once per cell cycle, whether replication was observed after stable introduction of the plasmids into cells by drug selection or during the first few cell divisions after introducing the DNA into cells. The presence in the cells of excess amounts of EBNA1, the only viral protein needed for oriP function, did not increase the number of oriP-replicated plasmids maintained by cells under selection. In the cell lines studied, EBNA1 and oriP seem to lack the capacity to override the cellular controls that limit DNA replication to one initiation event per DNA molecule per S phase. The multicopy status of EBV-derived, selectable plasmids appears to result from the initial uptake by cells of large numbers of plasmid molecules, the efficient maintenance of these plasmids, and the pressure of genetic selection against plasmid loss. Other unknown controls must be responsible for the amplification of EBV genomes soon after latent infection of cells.

AKT1E17K mutations occur at low frequency in a variety of solid tumors, including those of the breast and urinary bladder. Although this mutation has been shown to transform rodent cells in culture, it was found to be less oncogenic than PIK3CA mutations in breast epithelial cells. Moreover, the therapeutic potential of AKT inhibitors in human tumors with an endogenous AKT1 E17K mutation is not known. Expression of exogenous copies of AKT1 E17K in MCF10A breast epithelial cells increased phosphorylation of AKT and its substrates, induced colony formation in soft agar, and formation of lesions in the mammary fat pad of immunodeficient mice. These effects were inhibited by the allosteric and catalytic AKT inhibitors MK-2206 and AZD5363, respectively. Both AKT inhibitors caused highly significant growth inhibition of breast cancer explant models with AKT1 E17K mutation. Furthermore, in a phase I clinical study, the catalytic Akt inhibitor AZD5363 induced partial responses in patients with breast and ovarian cancer with tumors containing AKT1 E17K mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1 E17K and FGFR3 Y373C mutations, AZD5363 monotherapy did not significantly reduce tumor growth, but tumor regression was observed in combination with the FGFR inhibitor AZD4547. The data show that tumors with AKT1 E17K mutations are rational therapeutic targets for AKT inhibitors, although combinations with other targeted agents may be required where activating oncogenic mutations of other proteins are present in the same tumor.

Polymerase chain reaction has been applied to the amplification of long DNA fragments from a variety of sources, including genomic, mitochondrial, and viral DNAs. However, polymerase chain reaction amplification from cDNA templates produced by reverse transcription has generally been restricted to products of less than 10 kilobases. In this paper, we report a system to effectively amplify fragments up to 20 kilobases from human coronavirus 229E genomic RNA. We demonstrate that the integrity of the RNA template and the prevention of false priming events during reverse transcription are the critical parameters to achieve the synthesis of long cDNAs. The optimization of the polymerase chain reaction conditions enabled us to improve the specificity and yield of product but they were not definitive. Finally, we have shown that the same reverse transcription polymerase chain reaction technology can be used for the amplification of extended regions of the dystrophin mRNA, a cellular RNA of relatively low abundance.

Drug resistance is ultimately the cause of death for most cancer patients – even initially strong responses to treatment are usually followed by the emergence of resistance over time. This suggests the existence of residual or persistent cancer cells, creating a reservoir that ultimately gives rise to stable resistance. These drug tolerant persisters (‘the deadly survivors’) have been described for over a decade in numerous studies; they are often present as a minor fraction of the total tumour population and may exploit non-genetic (transcriptional) programs to allow the cells to survive drug treatment. 10-20% of lung adenocarcinoma patients harbour activating mutations in EGFR. Although treatment with the EGFR kinase inhibitor osimertinib has improved overall survival in such patients, almost all patients ultimately develop drug resistance. We carried out parallel genome-wide CRISPR gain and loss of function screens in EGFR mutant lung cancer cell lines treated with EGFR inhibitors, to identify the genes and pathways that may be important in enabling the survival of persister cells. We observed recurrent resistant genes in previously identified resistance pathways including PI3K (PTEN, TSC1, TSC2), MAPK (KRAS, NF1, MET), cell death (BCL2L11, BAX), the mediator complex (MED24, MED19) and ubiquitination (KCTD5, KEAP1). A secondary screen of 63 resistance genes that combined high content microscopy with CRISPR gene knockout demonstrated that 21% (13/63) of genes were associated with increased nuclear localisation of YAP1/WWTR1, key activators of the Hippo pathway. A closer review of the CRISPR screen data confirmed that many resistance hits are members of this pathway - upstream regulators (NF2, AMOTL2), core signalling genes (LATS1, LATS2), main effectors (WWTR1, YAP1), transcriptional co-effectors (TEAD3, FOSL1, VGLL4) and the SWI/SNF complex (ARID2, SMARCA4, SMARCB1, PBRM1). Hippo signalling is mediated through YAP1 and WWTR1 which bind to TEAD transcription factors and activate transcriptional programs affecting cell proliferation and apoptosis. We confirmed using CRISPR that knockout (NF2) or overexpression (YAP1, WWTR1) of key Hippo genes in the EGFR mutant lung cancer cell lines PC-9, HCC827 and HCC4006 resulted in up to 60-fold increased resistance to osimertinib and increased expression of canonical Hippo transcriptional targets. We therefore reasoned that the Hippo pathway might be involved in maintaining the survival of drug tolerant persister cells in this setting. Acute treatment of EGFR mutant cell lines with osimertinib was associated with increased nuclear localisation of YAP1 and WWTR1 and increased expression of canonical Hippo transcriptional targets. Furthermore, the combination of osimertinib and a TEAD inhibitor (K-975) almost completely abolished the survival of drug tolerant persister cells following treatment, indicating that this pathway is an important survival mechanism following drug treatment. Consequently, we propose Hippo signalling as an important target mechanism for the prevention of resistance to osimertinib. Citation Format: Matthias Pfeifer, Jonathan Brammeld, Stacey Price, Matthew Martin, Hannah Thorpe, Aurelie Bornot, Ercia Banks, Nin Guan, Shanade Dunn, Maria Lisa Guerriero, Daniel O'Neill, James Pilling, Davide Gianni, James Brownell, Paul Smith, Ultan McDermott. Gain and loss of function genome-wide CRISPR screens identify Hippo signalling as an important driver of resistance in EGFR mutant lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P066.

Transforming growth factor β-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of proinflammatory and stress signaling. It was shown previously that inhibition of TAK1 via the use of siRNA or small-molecule kinase inhibitors can inactivate NF-κB, down-regulate p38, and activate the intrinsic caspase pathway, resulting in profound induction of apoptosis. Herein we report the discovery of 5-fluoro-4-(imidazo[1,2-b]pyridazin-3-yl)-N-phenylpyrimidin-2-amines as TAK1 ATP competitive small molecule inhibitors. These compounds inhibit the kinase activity of TAK1 in vitro with low nanomolar potency. Evidence is presented that supports a mechanism of action consistent with inhibition of TAK1 kinase activity within the NF-κB pathway. Optimization of potency and selectivity for this series from hit to lead will be discussed, including the structure-based design and crystallographic determination of the binding mode. The outcome of efforts aimed at improving the in vivo pharmacokinetics of compounds is described. Results of kinase selectivity profiling for both an in vitro probe compound - AZ TAK1 - and an in vivo probe compound will be presented. Furthermore, we report low nanomolar antiproliferative activities in cell lines derived from Haematological Malignancies for these compounds. This cellular activity profile is suggesting that TAK1 inhibition presents therapeutic potential in Haematological Cancers. Citation Format: Dorin Toader, Jamal C. Saeh, Nin Guan, Francoise Powell, Raymond Chen, Corinne Reimer, Kate Byth. Discovery of small molecule TAK1 inhibitors with antiproliferative activity in cell lines derived from hematological malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2459. doi:10.1158/1538-7445.AM2013-2459

ATAD2 is a bromodomain (BD)-containing protein binding to chromatin at sites enriched in acetylated histones to modulate chromatin-related processes including transcription, DNA replication, and repair. ATAD2 is frequently amplified and/or overexpressed in breast cancer and is associated with aggressive disease, tumor metastasis, and poor outcome. Given the importance of the BD in targeting ATAD2 to relevant sites on the chromatin, we and others have hypothesized that the BD is essential for its mechanism of action and its targeting would have antiproliferative effects in cancer cells. The ATAD2 BD is one of the least tractable and to date reported inhibitors have been weak, nonselective, or exhibit poor cell permeability. Here we describe a potent, selective, and cell-permeable inhibitor of ATAD2 AZ4374 that modulates ATAD2 interaction with chromatin in cells and induces growth inhibition in breast cancer cell lines. AZ4374 has a potency (IC50) of 6.3 nM in the biochemical assay with >100-fold selectivity against other bromodomain-containing proteins. Chemoproteomic analysis of AZ4374 confirmed its selectivity. ATAD2 and BRD4 inhibition in cells was determined using displacement assays (NanoBRETTM) against the bromodomains. The IC50 for AZ4374 against ATAD2 was 80 nM and no inhibition of BRD4 was observed. The recently published GSK8814 was equipotent to AZ4374 in biochemical assays (IC50 ~6 nM) but less potent in the cell-based assay (IC50 ~600 nM). AZ4374 (10 µM) modulated the mRNA levels of CDK1, CDK2, and CCNE1 in EVSA-T cells (ATAD2 amplified). CRISPR/Cas9-based knockout of ATAD2 resulted in significant decrease in colony formation in breast cancer cell lines. Exposure of these cell lines to AZ4374 also reduced colony formation in a dose-dependent manner with IC50s ranging from 100 nM in SK-BR-3 to ~1µM in EVSA-T, so consistent with the potency observed in the cell-based assay. In addition, ATAD2 protacs caused a similar reduction of colony formation as AZ4374. Considering the role of chromatin modifiers in DNA damage response (DDR), we evaluated the impact of AZ4374 on the repair pathways HR and NHEJ using a CRISPR-reporter assay (traffic light reporter). A reduction in homology-directed repair was observed with 1 uM AZ4374, consistent with its biologic effect. Studies are ongoing to probe further the impact of ATAD2 inhibition on DNA repair. This is the first demonstration of highly potent and selective ATAD2 inhibition showing consistent cellular activity and biologic effect in cancer cell lines. Citation Format: Meghana M. Kulkarni, Jon Winter, David Whalley, ian Dale, Christopher Denz, Nin Guan, Deepa Bhavsar, Andrew Zhang, Timothy Rasmusson, Adrian Fretland, Sylvie M. Guichard. Discovery of a potent and selective ATAD2 inhibitor with antiproliferative activity in breast cancer 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 A170.

<div>Abstract<p>AKT1^E17K mutations occur at low frequency in a variety of solid tumors, including those of the breast and urinary bladder. Although this mutation has been shown to transform rodent cells in culture, it was found to be less oncogenic than PIK3CA mutations in breast epithelial cells. Moreover, the therapeutic potential of AKT inhibitors in human tumors with an endogenous AKT1^E17K mutation is not known. Expression of exogenous copies of AKT1^E17K in MCF10A breast epithelial cells increased phosphorylation of AKT and its substrates, induced colony formation in soft agar, and formation of lesions in the mammary fat pad of immunodeficient mice. These effects were inhibited by the allosteric and catalytic AKT inhibitors MK-2206 and AZD5363, respectively. Both AKT inhibitors caused highly significant growth inhibition of breast cancer explant models with AKT1^E17K mutation. Furthermore, in a phase I clinical study, the catalytic Akt inhibitor AZD5363 induced partial responses in patients with breast and ovarian cancer with tumors containing AKT1^E17K mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1^E17K and FGFR3^Y373C mutations, AZD5363 monotherapy did not significantly reduce tumor growth, but tumor regression was observed in combination with the FGFR inhibitor AZD4547. The data show that tumors with AKT1^E17K mutations are rational therapeutic targets for AKT inhibitors, although combinations with other targeted agents may be required where activating oncogenic mutations of other proteins are present in the same tumor. <i>Mol Cancer Ther; 14(11); 2441–51. ©2015 AACR</i>.</p></div>

Breast cancer is one of the most common cancer types, with greater than 450,000 deaths reported per year worldwide. Through genome wide sequencing efforts, multiple genetic alterations have been identified, including mutations and amplifications in genes such as v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), GATA binding protein 3 (GATA3), phosphatidylinositol 3-kinase alpha catalytic subunit (PIK3CA) as well as novel genomic rearrangements such as the recently identified MAGI3-AKT3 fusion. Now that breast cancer can be characterized to an unprecedented level, one of the key challenges remaining is to identify and distinguish critical ‘driver’ events responsible for tumor progression, from neutral ‘passenger’ lesions. In order to achieve this, we utilized high resolution aCGH analysis of 50 purified breast cancer samples (made up of Her2+, estrogen receptor positive (ER+) and triple negative tumors with variable responses to SOC regimens), in combination with a Gain-of-Function transformation screen to identify and validate novel breast targets. 158 genomic regions were found to be recurrently amplified, consisting of 759 genes in total. The top 32 focally amplified genes, along with 12 cancer-relevant mutant alleles were prioritized and a library generated utilizing the pTRIPZ-tetracycline regulated inducible lentiviral vector system. These 44 genes were subsequently combined into 16 different target pools (5-13 targets per pool, co-expressing genes that were co-amplified in the same clinical specimen) and evaluated for their ability to transform immortalized breast epithelial MCF10A cells (both wild-type and p53 -/- cells). Through this screening approach, p21-activated kinase 1 (PAK1) was identified, whose kinase activity was required to robustly transform MCF10A cells through regulating multiple signalling pathways including MAPK. Several other putative oncogenes were also identified and will be presented here, including the glycosyltransferse asparagine-linked glycosylation 8 (ALG8). Interestingly, PAK1 and ALG8 are co-amplified in both breast (8%) and ovarian cancers (11%). Our target validation studies have suggested that ALG8 can support PAK1-induced transformation, as dramatic suppression of soft-agar colony growth was seen in co-amplified breast cancer cell lines upon combined siRNA treatment to both targets. Thus, this combined high resolution aCGH profiling and functional screening approach has enabled the successful identification of novel oncogenic targets in breast cancer. Citation Format: Krishna Vasudevan, Axel Hernandez, Zhongwu Lai, Yonghong Xiao, Nin Guan, Carolyn Hardy, Robert Godin, Christopher Denz, Minwei Ye, Elizabeth Lenkiewicz, Stephanie Savage, Michael T. Barrett, Donna Prunkard, Peter Rabinovitch, Mark Basik, Ewa Przybytkowski, Kevin Webster, Michael Zinda, Emma-Louise Jenkins. Identification and functional validation of novel genetically-linked breast cancer targets through pooled gain-of-function screening. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3133. doi:10.1158/1538-7445.AM2013-3133