CHEK1 Inhibitor Research Articles

Inhibition of checkpoint kinase prevents human oocyte apoptosis induced by chemotherapy and allows enhanced tumour chemotherapeutic efficacy.

Could inhibition of the checkpoint kinase (CHEK) pathway protect human oocytes and even enhance the anti-tumour effects, during chemotherapy? CHEK inhibitors prevented apoptosis of human oocytes induced by chemotherapy and even enhanced the anti-tumour effects. CHEK inhibitors showed ovarian protective effects in mice during chemotherapy, while their role in human oocytes is unclear. This experimental study evaluated the ovarian reserve of young patients (120 patients) with cancer, exposed or not exposed to taxane and platinum (TP)-combined chemotherapy. Single RNA-sequencing analysis of human primordial oocytes from 10 patients was performed to explore the mechanism of oocyte apoptosis induced by TP chemotherapy. The damaging effects of paclitaxel (PTX) and cisplatin on human oocytes were also evaluated by culturing human ovaries in vitro. A new mouse model that combines human ovarian xenotransplantation and patient-derived tumour xenografts was developed to explore adjuvant therapies for ovarian protection. The mice were randomly allocated to four groups (10 mice for each group): control, cisplatin, cisplatin + CK1 (CHEK1 inhibitor, SCH 900776), and cisplatin + CK2 (CHEK2 inhibitor, BML277). In the prospective cohort study, human ovarian follicles were counted and serum AMH levels were evaluated. RNA-sequencing analysis was conducted, and staining for follicular damage (phosphorylated H2AX histone; γH2AX), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) assays and assessments of apoptotic biomarkers (western blot and immunofluorescence) were conducted in human ovaries. After the treatments, histological analysis was performed on human ovarian samples to investigate follicular populations, and oocyte damage was measured by γH2AX staining, BAX staining, and TUNEL assays. At the same time, the tumours were evaluated for volume, weight, and apoptosis levels. Patients who received TP chemotherapy showed decreased ovarian reserves. Single RNA-sequencing analysis of human primordial oocytes indicated that TP chemotherapy induced apoptosis of human primordial oocytes by causing CHEK-mediated TAp63α phosphorylation. In vitro culture of human ovaries showed greater damaging effects on oocytes after cisplatin treatment compared with that after PTX treatment. Using the new animal model, CHEK1/2 inhibitors prevented the apoptosis of human oocytes induced by cisplatin and even enhanced its anti-tumour effects. This protective effect appeared to be mediated by inhibiting DNA damage via the CHEK-TAp63α pathway and by generation of anti-apoptotic signals in the oocytes. N/A. This was a preclinical study performed with human ovarian samples, and clinical research is required for validation. These findings highlight the therapeutic potential of CHEK1/2 inhibitors as a complementary strategy for preserving fertility in female cancer patients. This work was financially supported by the National Natural Science Foundation of China (nos. 82001514 and 81902669) and the Fundamental Research Funds for the Central Universities (2021yjsCXCY087). The authors declare no conflict of interest.

AMT-562, a Novel HER3-targeting Antibody-Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors.

HER3 is a unique member of the EGFR family of tyrosine kinases, which is broadly expressed in several cancers, including breast, lung, pancreatic, colorectal, gastric, prostate, and bladder cancers and is often associated with poor patient outcomes and therapeutic resistance. U3-1402/Patritumab-GGFG-DXd is the first successful HER3-targeting antibody-drug conjugate (ADC) with clinical efficacy in non-small cell lung cancer. However, over 60% of patients are nonresponsive to U3-1402 due to low target expression levels and responses tend to be in patients with higher target expression levels. U3-1402 is also ineffective in more challenging tumor types such as colorectal cancer. AMT-562 was generated by a novel anti-HER3 antibody Ab562 and a modified self-immolative PABC spacer (T800) to conjugate exatecan. Exatecan showed higher cytotoxic potency than its derivative DXd. Ab562 was selected because of its moderate affinity for minimizing potential toxicity and improving tumor penetration purposes. Both alone or in combination therapies, AMT-562 showed potent and durable antitumor response in low HER3 expression xenograft and heterogeneous patient-derived xenograft/organoid models, including digestive system and lung tumors representing of unmet needs. Combination therapies pairing AMT-562 with therapeutic antibodies, inhibitors of CHEK1, KRAS, and tyrosine kinase inhibitor showed higher synergistic efficacy than Patritumab-GGFG-DXd. Pharmacokinetic and safety profiles of AMT-562 were favorable and the highest dose lacking severe toxicity was 30 mg/kg in cynomolgus monkeys. AMT-562 has potential to be a superior HER3-targeting ADC with a higher therapeutic window that can overcome resistance to generate higher percentage and more durable responses in U3-1402-insensitive tumors.

Abstract PD2-09: The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC)

Abstract Background: The CDK4/6 inhibitors have emerged as standard first- or second-line regimens in combination with an antiestrogen for patients with HR+/HER2- MBC. While these agents convey significant clinical benefit in many patients, intrinsic resistance can occur and, in patients who respond, acquired resistance is unfortunately inevitable. Despite their widespread use, we have limited insight into the molecular mechanisms governing response and resistance to these agents. Methods: Whole exome sequencing (WES) was performed on metastatic tumor biopsies from 58 patients (pts) with HR+/HER2- MBC who received a CDK4/6 inhibitor with or without an antiestrogen at the Dana-Farber Cancer Institute, including 7 pts with pre/post-exposure biopsy pairs. Among these biopsies, 69.5% were characterized as resistant (intrinsic or acquired) and 30.5% were characterized as sensitive. To validate putative resistance mediators identified in patient samples, HR+/HER2- breast cancer cells were modified via CRISPR knockout or lentiviral overexpression. Sensitivity of these cells to antiestrogens and CDK4/6i was interrogated via cell-titer-glo assay. In parallel, HR+/HER2- breast cancer cells were cultured to resistance in the presence of an escalating dose of CDK4/6i. Derivative cell lines were subjected to western blotting in an effort to interrogate the putative resistance mediators identified in pts. Novel dependencies were identified in these derivative cell lines via treatment with targeted therapeutic agents in vitro. Results: WES of tumors with CDK4/6i exposure revealed candidate mechanisms of resistance including biallelic RB1 disruption (n=4, 10%) and activating events in AKT1 (n=5, 12.5%), RAS (n=4, 10%), aurora kinase A (AURKA, n=11, 27.5%), and cyclin E2 (CCNE2, n=6, 15%). Convergent evolution toward biallelic RB1 disruption was identified in a single patient with one pre- and two post-exposure biopsies, while acquisition of AKT1 mutation and amplification was identified in two separate instances. Knockout of RB1 and overexpression of AKT1, KRAS G12D, AURKA, and CCNE2 provoked CDK4/6i and antiestrogen resistance in vitro. Breast cancer cells cultured to resistance in CDK4/6i demonstrated concordant acquisition of RB1 downregulation, RAS/ERK activation, AURKA overexpression, and CCNE2 overexpression. Derivative resistant cell lines with RB1 loss or AURKA gain demonstrated enhanced sensitivity to a novel AURKA inhibitor (LY3295668), while cells with RAS activation were highly sensitive to ERK inhibition (via LY3214996). CCNE2-overexpressing cells were highly sensitive to prexasertib, a CHEK1 inhibitor. Conclusions: The genomic landscape of resistance to CDK4/6i is heterogeneous with multiple potential mediators that play well-established roles in cell division and oncogenic signal transduction. We present novel mechanisms of clinical resistance including activation of AKT1 and RAS family oncogenes as well as amplification of AURKA and CCNE2. These drivers were able to provoke resistance to CDK4/6i in vitro. Finally, in each case, a novel dependency was identified which is readily translatable into the clinic. These results underscore the potential of next-generation sequencing as a critical tool to enable identification of resistance mediators, while also suggesting that the presence of specific genomic alterations may define new therapeutic opportunities in CDK4/6i-resistant HR+ MBC. Citation Format: Seth A. Wander, Ofir Cohen, Xueqian Gong, Gabriela N. Johnson, Jorge Buendia-Buendia, Maxwell Lloyd, Dewey Kim, Flora Luo, Pingping Mao, Karla Helvie, Kailey Kowalski, Utthara Nayar, Stephen Parsons, Ricardo Martinez, Lacey Litchfield, Xiang Ye, Chun Ping Yu, Valerie Jansen, Levi A. Garraway, Eric P. Winer, Sara M. Tolaney, Nancy U. Lin, Sean Buchanan, Nikhil Wagle. The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) in patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-09.

Mechanistic Distinctions between CHK1 and WEE1 Inhibition Guide the Scheduling of Triple Therapy with Gemcitabine.

Combination of cytotoxic therapy with emerging DNA damage response inhibitors (DDRi) has been limited by tolerability issues. However, the goal of most combination trials has been to administer DDRi with standard-of-care doses of chemotherapy. We hypothesized that mechanism-guided treatment scheduling could reduce the incidence of dose-limiting toxicities and enable tolerable multitherapeutic regimens. Integrative analyses of mathematical modeling and single-cell assays distinguished the synergy kinetics of WEE1 inhibitor (WEE1i) from CHEK1 inhibitor (CHK1i) by potency, spatiotemporal perturbation, and mitotic effects when combined with gemcitabine. These divergent properties collectively supported a triple-agent strategy, whereby a pulse of gemcitabine and CHK1i followed by WEE1i durably suppressed tumor cell growth. In xenografts, CHK1i exaggerated replication stress without mitotic CDK hyperactivation, enriching a geminin-positive subpopulation and intratumoral gemcitabine metabolite. Without overt toxicity, addition of WEE1i to low-dose gemcitabine and CHK1i was most effective in tumor control compared with single and double agents. Overall, our work provides quantitative insights into the mechanisms of DDRi chemosensitization, leading to the rational development of a tolerable multitherapeutic regimen.Significance: Multiple lines of mechanistic insight regarding DNA damage response inhibitors rationally guide the preclinical development of a tolerable multitherapeutic regimen.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/11/3054/F1.large.jpg Cancer Res; 78(11); 3054-66. ©2018 AACR.

RNF126 as a Biomarker of a Poor Prognosis in Invasive Breast Cancer and CHEK1 Inhibitor Efficacy in Breast Cancer Cells.

Purpose: (i) To investigate the expression of the E3 ligase, RNF126, in human invasive breast cancer and its links with breast cancer outcomes; and (ii) to test the hypothesis that RNF126 determines the efficacy of inhibitors targeting the cell-cycle checkpoint kinase, CHEK1.Experimental Design: A retrospective analysis by immunohistochemistry (IHC) compared RNF126 staining in 110 invasive breast cancer and 78 paired adjacent normal tissues with clinicopathologic data. Whether RNF126 controls CHEK1 expression was determined by chromatin immunoprecipitation and a CHEK1 promoter driven luciferase reporter. Staining for these two proteins by IHC using tissue microarrays was also conducted. Cell killing/replication stress induced by CHEK1 inhibition was evaluated in cells, with or without RNF126 knockdown, by MTT/colony formation, replication stress biomarker immunostaining and DNA fiber assays.Results: RNF126 protein expression was elevated in breast cancer tissue samples. RNF126 was associated with a poor clinical outcome after multivariate analysis and was an independent predictor. RNF126 promotes CHEK1 transcript expression. Critically, a strong correlation between RNF126 and CHEK1 proteins was identified in breast cancer tissue and cell lines. The inhibition of CHEK1 induced a greater cell killing and a higher level of replication stress in breast cancer cells expressing RNF126 compared to RNF126 depleted cells.Conclusions: RNF126 protein is highly expressed in invasive breast cancer tissue. The high expression of RNF126 is an independent predictor of a poor prognosis in invasive breast cancer and is considered a potential biomarker of a cancer's responsiveness to CHEK1 inhibitors. CHEK1 inhibition targets breast cancer cells expressing higher levels of RNF126 by enhancing replication stress. Clin Cancer Res; 24(7); 1629-43. ©2018 AACR.

Abstract 1750: Analysis of non-metastatic HCC patient tumors revealed the significance of cell cycle regulation and tumor immunity in association with overall survival and identified clinically relevant druggable targets

Abstract The lack of efficacious therapies have hindered the progress in improving the survival of HCC patients worldwide. As a disease that associates with the underlying liver injuries caused by various etiologies and remains asymptomatic during most of its decades long progression, HCC presents challenges to accurately depict its biological landscape and determine suitable therapeutic intervention. An approach to tackle this issue is to define significant biological variants that impact patient survival in each disease stage, then attempt to chart paths of potential therapies. To identify significant biological determinants associated with the overall survival (OS) of early stage, non-metastatic HCC patients, we constructed focused Nanostring panels that are represented by 1,164 genes of validated components and transcriptional outputs of multiple signaling pathways, cellular machineries, and tumor immunity. An unbiased analysis of patient survival and gene transcript levels revealed four distinct groups of genes that are significantly prognostic. The first two (Group 1 & 2) are associated with poorer prognosis when expression is high (hazard ratio > 2), whereas the remaining two (Group 3 & 4) correspond to significantly better OS with elevated transcript levels (hazard ratio<0.5). Of note, Gene Set Enrichment Analyses (GSEA) clearly indicated that while Group 2 was substantially enriched in genes positively regulating cell cycle and cell division machinery, Group 4 was overrepresented by genes participating in the positive modulation and feedback mechanisms of adaptive immune response, implicating the survival benefit of acquiring higher immunogenic tumor environment in early stage HCC. Searching for druggable targets associated with poorer prognosis, we identified both EZH2 and CHEK1 as candidates from Group 2. In vitro treatment of three HCC cell lines with EPZ-6438 (EZH2 inhibitor) or GDC-0425 (CHEK1 inhibitor) resulted in significant modulation of H3K27me3 or pCHEK1 levels (s345) respectively. The growth of these cell lines was also dose-dependently suppressed by each inhibitor in colony formation assays . Furthermore, the combination of both inhibitors was able to achieve substantial cell growth inhibition at lower concentrations, suggesting a combinatorial effect of targeting both EZH2 and CHEK1 in HCC cell lines. Xenograft studies are ongoing to assess in vivo efficacy of this combination. In conclusion, utilizing well constructed Nanostring gene panels, we identified significant association of cell cycle machinery and adaptive tumor immunity with HCC patient survival. Our findings not only provided potential rationale for expand testing of cancer immunotherapies to non-metastatic, early stage HCC, but also revealed the potential utility of combining EZH2 and CHEK1 inhibitors to treat HCC. Citation Format: Pei-Zhen Miao, Jeff Cheng, Kwame Okrah, Bonnie Liu, Charlie Sun, Grace Cao, Tony Guo, Shijing Fu, Marie-Claire Wagle, Elizabeth Blackwood, David Shames, Garret Hampton, Lukas Amler, Astrid Kiermaier, Yevgeniya Fridlyand, Mark Lackner, Shih-Min Huang, Jian Zhou, Zhi Dai. Analysis of non-metastatic HCC patient tumors revealed the significance of cell cycle regulation and tumor immunity in association with overall survival and identified clinically relevant druggable targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1750. doi:10.1158/1538-7445.AM2017-1750

Abstract B62: Evaluation of scheduling for triple therapy gemcitabine/CHEK1 inhibitor/WEE1 inhibitor in pancreatic cancer models

Abstract Sensitization of cancer cells to gemcitabine (Gem) has been shown with checkpoint kinase CHEK1 (CHK1) and WEE1 inhibitors. Our aim is to determine, using in vitro and in vivo models, the optimal regime for application of the triple combination in pancreatic cancer. First, we have investigated the dual combinations of Gem + either CHEK1 or WEE1 inhibitors in growth inhibition assays in vitro. Synergistic growth inhibition was identified using both Bliss Independence and Loewe models, in MIA PaCa-2 cells at 10 – 30 nM Gem + 300 – 1000 nM CHEK1 inhibitor MK8776; or 10 – 30 nM Gem + 30 – 100 nM WEE1 inhibitor MK1775. The synergistic concentrations were submaximal, i.e. below the single agent GI50 concentrations, which were 30 nM for Gem, 6 μM for MK8776, and 500 nM for MK1775. The Panc-1 cell line is more resistant to Gem as a single agent, but synergy was evident: in colony forming assays the dual combination of either 30 nM gem + 300 nM MK1775 or 30 nM Gem + 1 μM MK8776 inhibited colony formation by 97 +/- 1.6 % and 91 +/- 2% respectively. Single agent Gem inhibited colony formation by only 23 +/- 4 % and MK1775 or MK8776 did not inhibit colony formation at these concentrations. Next, we investigated scheduling of the Gem/CHEK1i/Wee1i triple combination, at the synergistic concentrations, with kinetic live-cell imaging assays. MIA PaCa-2 cells treated continuously with 10 nM Gem plus 1 μM MK8776 showed durable growth inhibition over 72 hours. However, if both drugs were washed off after 24 hours the Gem + CHEK1i-treated cells recovered and began to proliferate within 24 hours. Different schedules of the trio were tested, and the most durable growth inhibition (> 5 days) was obtained when Gem + MK8776 were washed off at 24 hours and replaced with 300 nM MK1775, whereas 300 nM MK1775 in the absence of Gem + MK8776 pretreatment did not significantly inhibit growth, compared to control. Different scheduling options were also tested in Panc-1 cells in colony forming assays, and again the most effective schedule was Gem + MK8776 for 24 hours, followed by MK1775. MIA PaCa-2 xenograft studies are now underway, initially with the dual combination, to be followed with the drug trio of simultaneous Gem + MK8776, followed by MK1775. To reduce the likelihood of toxicity, Gem doses lower than the typical “full” dose (100 mg/kg IP twice per week) were tested. We found that 25 mg/kg Gem twice in a day (8h apart), twice per week was not well tolerated even as a single agent, but both 25 and 50 mg/kg Gem once in a day, twice per week was tolerated when co-dosed with 25 mg/kg MK8776, and when this combination was followed 8 hours later by 60 mg/kg MK1775 (OG). A pharmacokinetic study revealed that plasma Gem (dFdC) concentrations were not altered by co-dosing Gem with MK8776, but intratumor dFdCTP (the active intracellular metabolite of Gem) was elevated at 1 – 8 hours after the 25 mg/kg Gem + MK8776 dose (AUC0 –last 49 +/- 7.9 hr.pmoles/mg tissue) compared to mice with 25 mg/kg Gem alone (AUC0-last 21 +/- 2.6 hr.pmoles/mg tissue). CHEK1 target inhibition was demonstrated in vivo, with abrogation of Gem-induced CHEK1 S296 autophosphorylation for at least 4h post Gem + MK8776 dosing, using Western blot analyses of tumor lysate. There was also increased γH2AX and pRPA32 S4/8 at 4 - 8 hours post-dose with Gem + MK8776 compared to Gem alone, indicating enhanced DNA damage. Pharmacodynamic and efficacy studies with the triple combination, will determine whether enhanced efficacy can be observed, when compared to full dose, single agent gemcitabine (100 mg/kg IP twice per week). Citation Format: Siang-Boon Koh, Frances M. Richards, Yann Wallez, Duncan I. Jodrell.{Authors}. Evaluation of scheduling for triple therapy gemcitabine/CHEK1 inhibitor/WEE1 inhibitor in pancreatic cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B62.

Abstract 3490: Topotecan synergizes with CHEK1 inhibitor to induce apoptosis in ovarian cancer cells

Abstract Background Topotecan is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of topotecan is myelosuppression. This led us to seek a combination treatment to augment topotecan anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1, a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers. Methods We evaluated the cellular effects of topotecan in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of topotecan in the absence and presence of suboptimal concentrations of CHEK1 inhibitor, PF477736. Synergism between topotecan and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2A.X. Results Non-HGS ovarian cancer cells were generally more sensitive to topotecan treatment compared to HGS ovarian cancer cells. When combined with suboptimal concentrations of CHEK1 inhibitor, topotecan potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis. Conclusions Our findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to topotecan. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining topotecan with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit. Citation Format: Marianne K. H. Kim, Jana James, Christina Annunziata. Topotecan synergizes with CHEK1 inhibitor to induce apoptosis in ovarian cancer cells. [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 3490. doi:10.1158/1538-7445.AM2015-3490

Topotecan synergizes with CHEK1 (CHK1) inhibitor to induce apoptosis in ovarian cancer cells.

BackgroundTopotecan (TPT) is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of TPT is myelosuppression. This led us to seek a combination treatment to augment TPT anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1 (CHK1), a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers.MethodsWe evaluated the cellular effects of TPT in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of TPT in the absence and presence of CHEK1 inhibitor, PF477736. Synergism between TPT and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2AX.ResultsNon-HGS ovarian cancer cells were generally more sensitive to TPT treatment compared to HGS ovarian cancer cells. When combined with CHEK1 inhibitor, TPT potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis.ConclusionsOur findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to TPT. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining TPT with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1231-z) contains supplementary material, which is available to authorized users.

Abstract 5179: Context-specific dependence of ovarian cancer on IKKϵ and CHEK1

Abstract Background: The IκB kinase, IKKϵ, was previously identified as an oncogene in breast cancer and was associated with poor clinical outcome in ovarian cancer. Recently, we demonstrated that IKKϵ is a key regulator of invasion and metastasis in ovarian cancer. A highly specific inhibitor of IKKϵ is not readily available for use as a tool compound to study pathway interactions in the preclinical setting. Therefore, we utilized dual shRNA technique to create IKKϵ-matched cell line pairs and performed sensitization screens to identify the key molecular targets influenced by the loss of IKKϵ expression. Results: We screened a previously established shRNA library targeting the human kinome, and identified 65 genes that further inhibited the survival of ovarian cancer cells in combination with IKKϵ depletion. In order to evaluate potential clinical significance associated with their overexpression, we examined the expression levels of these 65 genes in TCGA ovarian serous cystadenocarcinoma dataset. Strikingly, CHEK1 was overexpressed in 100% of TCGA ovarian cancers with a cut-off of 1.5 fold change compared to normal ovarian surface epithelium. Interestingly, CHEK1 loss also sensitized IKKϵ depleted A2780, a wild-type p53 containing ovarian cancer cell line. Next, we investigated whether chemical inhibitors could recapitulate the effects of shRNAs, although they are less specific than RNAi and are unable to interrogate kinase-independent functions of cellular proteins. Consistent with the initial screening methodology, inhibition of IKKϵ and CHEK1 activities by BX795 and PF00477736, respectively, in 6 ovarian cancer cell lines was critical to the cooperative decrease in cell viability. Thus, the co-inhibition of IKKϵ and CHEK1 activities, regardless of p53 status, was more effective in killing ovarian cancer cells than single treatment. The inhibition of IKKϵ activity alone induced dramatic G2/M arrest in all 6 cell lines without inducing a DNA damage response, while CHEK1 inhibition resulted in significant DNA damage response with abnormal S phase profile in cell cycle analysis and an increase in gamma-H2A.X level. This combined inhibition dramatically decreased CHEK1 level and further increased apoptosis compared to CHEK1 inhibitor alone. Conclusion: Our dual shRNA technique efficiently identified CHEK1 as an IKKϵ synthetic lethal gene in ovarian cancer. CHEK1 inhibitor as a single agent has not been successful in clinic, and this may be due to increased pro-survival signaling mediated by IKKϵ, which may contribute to resistance and subsequent relapse. Identification of IKKϵ dependent signaling will lead to development of context-specific therapeutics in the poor prognostic group of patients with a high level of IKKϵ expression. Citation Format: Marianne K. H. Kim, Dong Joon Min, George Wright, Christina M. Annunziata. Context-specific dependence of ovarian cancer on IKKϵ and CHEK1. [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 5179. doi:10.1158/1538-7445.AM2014-5179

Aging alters tissue resident mesenchymal stem cell properties

Tissue resident mesenchymal stem cells (MSCs) are known to participate in tissue regeneration that follows cell turnover, apoptosis, or necrosis. It has been long known that aging impedes an organism's repair/regeneration capabilities. In order to study the age associated changes, the molecular characteristics of adipose tissue derived MSCs (ASCs) from three age groups of healthy volunteers i.e., young, middle aged, and aged were investigated. The number and multilineage differentiation potential of ASCs declined with age. Aging reduces the proliferative capacity along with increases in cellular senescence. A significant increase in quiescence of G2 and S phase was observed in ASCs from aged donors. The expression of genes related to senescence such as CHEK1 and cyclin-dependent kinase inhibitor p16 ink4a was increased with age, however genes of apoptosis were downregulated. Further, an age-dependent abnormality in the expression of DNA break repair genes was observed. Global microRNA analysis revealed an abnormal expression of mir-27b, mir-106a, mir-199a, and let-7. In ubiquitously distributed adipose tissue (and ASCs), aging brings about important alterations, which might be critical for tissue regeneration and homeostasis. Our findings therefore provide a better understanding of the mechanism(s) involved in stem cell aging and regenerative potential, and this in turn may affect tissue repair that declines with aging.

Abstract 5263: An RNAi screen of the protein kinome identifies CHEK1 as a therapeutic target in neuroblastoma

Abstract Background High-risk neuroblastoma is a pediatric cancer that is often fatal despite intense multimodality therapy associated with significant late effects for those who survive. Therefore, patients will benefit from treatment strategies that rationally exploit signaling pathways for which a tumor cell is selectively dependent. In an effort to identify novel therapeutic targets, we performed a comprehensive loss of function screen of the protein kinome in neuroblastoma cell models. Methods Using a validated siRNA library which targets the human protein kinome, we transiently transfected individual kinase siRNAs into four neuroblastoma cell lines and measured substrate adherent growth. We then ranked the top 100 most potent kinase siRNAs per cell line and integrated the lists to identify kinases with inhibition in multiple lines. Results Thirty targets emerged as potent in at least three of the four neuroblastoma cell lines. They were enriched for kinases involved in the cell cycle and mitosis. This select group was tested with an independent set of more specific siRNA pools in an expanded panel of eight neuroblastoma cell lines. The strikingly most potent to emerge from the secondary screen was the cell cycle checkpoint kinase CHEK1. CHEK1 knockdown appeared cytotoxic, often by 24 hours, coinciding with complete CHEK1 protein knockdown and an emergence of a substantial apoptotic fraction. As further validation of the siRNA results, pharmacologic inhibition with SB21807, a relatively specific CHEK1 inhibitor, showed cytotoxicity in 7 of 8 neuroblastoma cell lines with a median IC50 of 430 nM (range 62-677 nM), while the non-neuroblastoma lines RPE-1, DOAY and MCF7 cells were resistant with a median IC50 of 2609 nM (1066 - 2898 nM). The mechanism of selective inhibition in neuroblastoma is unclear as we did not identify mutations in the coding exons of CHEK1. Western blotting revealed, however, that CHEK1 is constitutively phosphorylated at the Ser 296 residue in 8/8 neuroblastoma cell lines and 10/16 primary tumors samples, but not in controls. Conclusion We have identified CHEK1 as a potential therapeutic target in neuroblastoma and are currently extending our work to understand the mechanism of this tumor's apparent selective sensitivity to CHEK1 inhibition. As CHEK1 inhibitors are currently in phase I/II clinical trials as chemosensitizers, we are also focused on determining the in vivo efficacy of combination CHEK1 inhibition with chemotherapy in neuroblastoma. 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 5263.

Synthetic Lethal RNAi Screen of the Human Kinome with Cytarabine in Myeloid Leukemias.

Abstract 590 Background:To improve response and increase the number of patients responding to Ara-C, new genes/mechanisms/pathways need to be discovered that underlie and determine the sensitivity to Ara-C. We hypothesized that applying synthetic lethal RNAi screens of >570 kinases of the human kinome +/- Ara-C would be a fast and unbiased way to identify kinases that modulate sensitivity to Ara-C in myeloid cells. Methods:First, we developed a High-Throughput RNAi (HT-RNAi) platform for transient siRNA gene silencing in myeloid cells. Using lipid-based reverse transfection, TF1 and THP-1 myeloid cells were incubated with 2 different siRNA sequences per gene targeting 572 kinases (Tibes, EHA 2009). At 48 hrs, Ara-C at an IC30 dose was added and proliferation/viability was measured using a luminescence-based assay 48 hours after Ara-C treatment. Additionally, to assess high- vs. low dose Ara-C, a screen at an IC70 dose of Ara-C was performed in TF1 cells. Screens were performed in duplicate. The log2 ratio of each individual treatment of si+Ara-C over si alone was calculated [siRNA+drug : siRNA]. Hits were defined as the log2 ratio of individual siRNA >2 standard deviation (STD) below the median of all siRNA treatments (log2 ratios) on an individual plate or for the entire screen, respectively. Results:Transfection efficacy, determined by a universal lethal siRNA vs. non-silencing and buffer control, was between 75-80% for TF1 and >85% for THP-l. Correlation was high, i.e. for THP-1 (same Ara-C dose) with R2=0.67 for duplicate screens. Data was first analyzed to identify reproducible hits across independent duplicate RNAi screens for each cell line. Hits were defined of at least one same sequence siRNA reducing viability in combination with Ara-C in both (duplicate) screens to greater than 2 STD compared to siRNA or Ara-C alone (see also Methods). For TF1, 9 genes (1.6%) met these criteria. The top hit in TF1 was ATR for which all 4/4 siRNA sensitized to Ara-C; 4 genes each were positive for 3/4 and 2/4 siRNA. For THP-1, 8 genes were hits (7 genes with 2/4 siRNA and 1 gene with 3/4 siRNA) representing 1.4% of the entire screen. Strong hits in TF1 included ABL-1 and CHEK1 and for THP-1, ABL-2 and CHEK1. Apart from the top hit CHEK1, only one other gene reduced proliferation/viability in combination with Ara-C across both cell lines and thus, may be a novel universal sensitizer to Ara-C. This gene is centrally involved in regulating trafficking of membrane and receptor kinases of the MAPK family/pathways.Comparing high- vs. low-dose Ara-C, 4 genes exhibited sensitization to Ara-C at low concentrations only, and 10 genes at high Ara-C concentrations (each 2/2 si). Genes sensitizing at low Ara-C are involved in cell-cycle (3 of 4 genes); at high Ara-C, KEGG pathway analysis showed enrichment for purine, pyrimidine, IGF and MAPK pathways.In secondary functional validation screens using 4 siRNA sequences, CHEK1 has validated, whereas cell-cycle regulatory genes (i.e. AURKB) have not to date. In preliminary experiments, treatment of myeloid cells with CHEK1 inhibitors sensitized to Ara-C. A list of high-priority candidate targets is assembled and targets are being validated. Conclusions:Herein, we present the first large-scale kinase RNAi synthetic lethal screen in myeloid cells in combination with Ara-C. Only few kinases, 1-2% of the kinome, were identified whose silencing sensitizes to Ara-C. Two genes were potential universal sensitizers in AML: CHEK1, for which inhibitors are in early clinical development including in AML. We are currently conducting clinical trials with a novel, specific well tolerated CHEK inhibitor plus chemotherapy in solid tumors. Supported by our data we are encouraged to translating this concept into design of combination trials of CHEK1 inhibitor with Ara-C in AML. A potential novel sensitizer gene to Ara-C was identified that regulates kinase membrane trafficking and can activate the MAPK pathway. Validation of this gene is currently ongoing. The initial finding that distinct genes may modulate response at different Ara-C doses is of importance and could have clinical implications in the future.Functional genomics provide a fast and attractive approach to identify molecular targets and vulnerabilities in AML. RNAi synthetic lethal screens in leukemias hold promise for design of rational combination therapies that can be rapidly translated into the clinic. Disclosures:Tibes:Arizona Biomedical Research Commission: Peer reviewed grant funding for the presented project.

7-Hydroxystaurosporine (UCN-01) preferentially sensitizes cells with a disrupted TP53 to gamma radiation in lung cancer cell lines.

Mutations in TP53 occur in more than 50% of the lung cancer patients and are associated with an increased resistance to chemotherapy and radiotherapy. The human lung adenocarcinoma cell lines A549 and LXSN contain a wild-type TP53 and were growth arrested at both the G(1)- and G(2)-phase checkpoints after irradiation. However, a TP53-disrupted cell line, E6, was arrested only at the G(2)-phase checkpoint. UCN-01 (7-hydroxystaurosporine), a CHEK1 inhibitor that abrogates the G(2) block, has been reported to enhance radiation toxicity in human lymphoma and colon cancer cell lines. In this study, UCN-01 preferentially enhanced the radiosensitivity of the TP53-disrupted E6 cells compared to the TP53 wild-type cells. This effect was more pronounced in cells synchronized in early G(1) phase, where the E6 cells showed a higher resistance to radiation in the absence of drug. These results indicate that the combination of UCN-01 and radiation can more specifically target resistant TP53 mutated cancer cells and spare TP53 wild-type normal cells.