Zay Yar Oo

Aurora A Is Critical for Survival in HPV-Transformed Cervical Cancer

Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7–expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers. Mol Cancer Ther; 14(12); 2753–61. ©2015 AACR.

Cell Cycle Checkpoint and DNA Damage Response Defects as Anticancer Targets: From Molecular Mechanisms to Therapeutic Opportunities

Cells have evolved a DNA damage response (DDR) pathway to monitor the integrity of their genome, which is tightly associated with cell cycle checkpoint controls, arresting cells to allow for DNA repair before continuing through the cell cycle. Defects in the DDR and checkpoint mechanisms frequently occur in human cancers, with failure of the cell to repair the DNA damage leading to genomic instability, increased mutation load, and cellular transformation. The loss of a DNA damage checkpoint in a tumour should make it vulnerable to checkpoint override strategies, providing therapeutic opportunities to inhibit mechanisms that compensate for the defect. Here we review the DDR pathway and cell cycle checkpoint responses to DNA damage, and explain how defects in these mechanisms present a significant opportunity for therapeutic intervention. These defects can be exploited using a synthetic lethal approach to target tumours with these defects and having limited normal tissue toxicity.

Endogenous replication stress marks melanomas sensitive to CHK1 inhibitors in vivo

Purpose: Checkpoint kinase 1 inhibitors (CHEK1i) have single-agent activity in vitro and in vivo. Here, we have investigated the molecular basis of this activity. Experimental Design: We have assessed a panel of melanoma cell lines for their sensitivity to the CHEK1i GNE-323 and GDC-0575 in vitro and in vivo. The effects of these compounds on responses to DNA replication stress were analyzed in the hypersensitive cell lines. Results: A subset of melanoma cell lines is hypersensitive to CHEK1i-induced cell death in vitro, and the drug effectively inhibits tumor growth in vivo. In the hypersensitive cell lines, GNE-323 triggers cell death without cells entering mitosis. CHEK1i treatment triggers strong RPA2 hyperphosphorylation and increased DNA damage in only hypersensitive cells. The increased replication stress was associated with a defective S-phase cell-cycle checkpoint. The number and intensity of pRPA2 Ser4/8 foci in untreated tumors appeared to be a marker of elevated replication stress correlated with sensitivity to CHEK1i. Conclusions: CHEK1i have single-agent activity in a subset of melanomas with elevated endogenous replication stress. CHEK1i treatment strongly increased this replication stress and DNA damage, and this correlated with increased cell death. The level of endogenous replication is marked by the pRPA2Ser4/8 foci in the untreated tumors, and may be a useful marker of replication stress in vivo. Clin Cancer Res; 24(12); 2901–12. ©2018 AACR.

Abstract A33: Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo

CHK1 inhibitors are being investigated as chemosensitizing agents with agents that increase replication stress. Here we have investigated the molecular basis of sensitivity to CHK1 inhibitors as single agents in melanoma and lung cancer. We have found that sensitivity in vitro and in vivo to single agent CHK1 inhibitor is loss of the S phase cell cycle checkpoint response. This is through a number of mechanisms including the uncoupling of CHK1 activation with the destabilization of CDC25A. Loss of checkpoint by over-expressing components of the checkpoint or inhibition of Wee1, covert CHK1 inhibitor insensitive cells to sensitive, and similarly depletion of CDC25A reduces CHK1 inhibitor sensitivity in sensitive lines. Loss of the S phase checkpoint provides cells with an adaptive advantage through introduction of moderate levels of genomic instability. The increased DNA damage found with CHK1 inhibitor treatment is not sufficient to induce cell death, but also involves a mechanism that is dependent in part on DNA-PK activity. Loss of S phase checkpoint function is predicted for Note: This abstract was not presented at the conference. Citation Format: Zay Yar Oo, Alexander Stevenson, Catherine Lanagan, Loredana Spoerri, Jill Larsen, Brian Gabrielli. Defect in S phase cell cycle checkpoint renders tumours vulnerable to CHK1 inhibitor single-agent treatment in vitro and in vivo [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A33.

Abstract C189: Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma

The S phase cell cycle checkpoint is critical for maintaining genomic integrity. This checkpoint is triggered in response to DNA damage and replication stresses to control cell cycle progression, halting DNA replication and allowing the time to repair to maintain genomic integrity. If this checkpoint is defective, cells lose their ability to ensure the fidelity of replication and repair any damage, resulting in replicative stress and genomic instability which will subsequently increase the risk of cancer. We have discovered that a large proportion of melanoma cell lines lose the ability to halt replication when challenged with high dose hydroxyurea-induced replicative stress, despite normal cell cycle checkpoint signalling. We found that failure in the crosstalk between ATR-CHK1 checkpoint signalling and the cell cycle mechanism in destabilization of CDC25A. Although CHK1-dependent phosphorylation of CDC25A which triggers its degradation was detected, the S phase checkpoint defective melanoma cell lines fail to destabilize CDC25A, maintaining normal activation of CDK2/Cyclin E. The melanoma cells with this S phase checkpoint defect are also hyper-sensitive to killing by CHK1 inhibitor. We have previously shown that melanoma with high levels of replicative stress are very sensitive to CHK1 inhibitors. I will report our studies investigating whether CHK1 inhibitor sensitivity is a direct outcome of the replicative stress arising from the S phase checkpoint defect, or whether the loss of the ATR-CHK1-CDC25A dependent S phase checkpoint signalling mechanism is directly responsible for the hyper-sensitivity to CHK1 inhibitors. Citation Format: Zay Y. Oo, Sheena Daignault, James Chen, Brian Gabrielli. Defective S phase cell cycle checkpoint: A potential culprit and target in melanoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C189.

Abstract 945: synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA HPV has been identified as the definitive agent in cancers of the cervix, penis, vulva, vagina, anus, skin, eye, and head and neck, and is responsible for 5% of the total cancer burden worldwide. HPV oncogenes disable a number of tumour suppressor pathways, including p53 and Rb, contributing to the transformed phenotype. We have performed an siRNA screen using the kinome (779 genes) library to identify genes that when depleted are synthetically lethal with HPV transformation. The primary and validations screens have confirmed Aurora A kinase (AURKA) as a potential synthetic lethal target selective for HPV transformed cells. In vitro research using the investigational selective small molecule AURKA inhibitor alisertib found alisertib to be significantly more potent towards the HPV transformed cells, and selectively promoted apoptosis in the HPV cancers. The drug was shown to target the HPVE7 oncogene, the level of expression of this oncogene possibly influencing sensitivity. Apoptosis was sensitive to Mcl-1 but not Bcl-2 over expression, indicating that the mechanism is associated with the proteolytic destruction of Mcl-1 in the extended mitosis in the alisertib treated HPV cancer lines. Xenograft experiments with cervical cancer cell lines showed alisertib inhibited growth of HPV and non-HPV xenografts during treatment. The non-HPV cancer growth was delayed, but in two separate HPV cancers models, regression and no resumption of growth was detected at even 50 days post-treatment. A second transgenic model of premalignant disease driven solely by HPVE7 similarly demonstrated sensitivity to drug treatment. These findings provide preclinical evidnce that alisertib warrants evaluation as a potential targeted compound with activity in HPV-transformed cervical cancer and premalignant disease that may have application to other HPV driven cancers. Citation Format: Brian G. Gabrielli, Fawzi Bokhari, Max Ranall, Zay Yar Oo, Alex Stevenson, Weili Wang, Sara McKee, Graham Leggatt, Paul Leo, Thomas J. Gonda, Nigel AJ McMillan. Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer. [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 945. doi:10.1158/1538-7445.AM2015-945