Spatial and temporal intra-tumoral heterogeneity in advanced HGSOC: Implications for surgical and clinical outcomes.

Limited evidence exists on the impact of spatial and temporal heterogeneity of high-grade serous ovarian cancer (HGSOC) on tumor evolution, clinical outcomes, and surgical operability. We perform systematic multi-site tumor mapping at presentation and matched relapse from 49 high-tumor-burden patients, operated up front. From SNP array-derived copy-number data, we categorize dendrograms representing tumor clonal evolution as sympodial or dichotomous, noting most chemo-resistant patients favor simpler sympodial evolution. Three distinct tumor evolutionary patterns from primary to relapse are identified, demonstrating recurrent disease may emerge from pre-existing or newly detected clones. Crucially, we identify spatial heterogeneity for clinically actionable homologous recombination deficiency scores and for poor prognosis biomarkers CCNE1 and MYC. Copy-number signature, phenotypic, proteomic, and proliferative-index heterogeneity further highlight HGSOC complexity. This study explores HGSOC evolution and dissemination across space and time, its impact on optimal surgical cytoreductive effort and clinical outcomes, and its consequences for clinical decision-making.

CELLector: Genomics-Guided Selection of Cancer In Vitro Models.

Selecting appropriate cancer models is a key prerequisite for maximizing translational potential and clinical relevance of in vitro oncology studies. We developed CELLector: an R package and R Shiny application allowing researchers to select the most relevant cancer cell lines in a patient-genomic-guided fashion. CELLector leverages tumor genomics to identify recurrent subtypes with associated genomic signatures. It then evaluates these signatures in cancer cell lines to prioritize their selection. This enables users to choose appropriate in vitro models for inclusion or exclusion in retrospective analyses and future studies. Moreover, this allows bridging outcomes from cancer cell line screens to precisely defined sub-cohorts of primary tumors. Here, we demonstrate the usefulness and applicability of CELLector, showing how it can aid prioritization of in vitro models for future development and unveil patient-derived multivariate prognostic and therapeutic markers. CELLector is freely available at https://ot-cellector.shinyapps.io/CELLector_App/ (code at https://github.com/francescojm/CELLector and https://github.com/francescojm/CELLector_App).

Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.

Structural rearrangements generate cell-specific, gene-independent CRISPR-Cas9 loss of fitness effects.

BACKGROUND: CRISPR-Cas9 genome editing is widely used to study gene function, from basic biology to biomedical research. Structural rearrangements are a ubiquitous feature of cancer cells and their impact on the functional consequences of CRISPR-Cas9 gene-editing has not yet been assessed. RESULTS: Utilizing CRISPR-Cas9 knockout screens for 250 cancer cell lines, we demonstrate that targeting structurally rearranged regions, in particular tandem or interspersed amplifications, is highly detrimental to cellular fitness in a gene-independent manner. In contrast, amplifications caused by whole chromosomal duplication have little to no impact on fitness. This effect is cell line specific and dependent on the ploidy status. We devise a copy-number ratio metric that substantially improves the detection of gene-independent cell fitness effects in CRISPR-Cas9 screens. Furthermore, we develop a computational tool, called Crispy, to account for these effects on a single sample basis and provide corrected gene fitness effects. CONCLUSION: Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing in cancer cells.

Combined inhibition of the PI3K/mTOR/MEK pathway induces Bim/Mcl-1-regulated apoptosis in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) progression and chemotherapy insensitivity have been associated with aberrant PI3K/mTOR/MEK signalling. However, cell death responses activated by inhibitors of these pathways can differ - contextually varying with tumour genetic background. Here, we demonstrate that combining the dual PI3K/mTOR inhibitor PF5212384 (PF384) and MEK inhibitor PD325901 (PD901) more effectively induces apoptosis compared with either agent alone, independent of KRAS mutational status in PDAC cell lines. Additionally, a non-caspase dependent decrease in cell viability upon PF384 treatment was observed, and may be attributed to autophagy and G0/G1 cell cycle arrest. Using reverse phase protein arrays, we identify key molecular events associated with the conversion of cytostatic responses (elicited by single inhibitor treatments) into a complete cell death response when PF384 and PD901 are combined. This response was also independent of KRAS mutation, occurring in both BxPC3 (KRAS wildtype) and MIA-PaCa-2 (KRASG12C mutated) cells. In both cell lines, Bim expression increased in response to PF384/PD901 treatment (by 60% and 48%, respectively), while siRNA-mediated silencing of Bim attenuated the apoptosis induced by combination treatment. In parallel, Mcl-1 levels decreased by 36% in BxPC3, and 30% in MIA-PaCa-2 cells. This is consistent with a functional role for Mcl-1, and siRNA-mediated silencing enhanced apoptosis in PF384/PD901-treated MIA-PaCa-2 cells, whilst Mcl-1 overexpression decreased apoptosis induction by 24%. Moreover, a novel role was identified for PDCD4 loss in driving the apoptotic response to PF384/PD901 in BxPC3 and MIA-PaCa-2 cell lines. Overall, our data indicates PF384/PD901 co-treatment activates the same apoptotic mechanism in wild-type or KRAS mutant PDAC cells.

Phase IB Dose Escalation and Expansion Study of AKT Inhibitor Afuresertib with Carboplatin and Paclitaxel in Recurrent Platinum-resistant Ovarian Cancer.

PURPOSE: Preclinically, AKT kinase inhibition restores drug sensitivity in platinum-resistant tumors. Here the pan-AKT kinase inhibitor afuresertib was given in combination with paclitaxel and carboplatin (PC) in patients with recurrent platinum-resistant epithelial ovarian cancer (PROC) and primary platinum-refractory ovarian cancer (PPROC). PATIENTS AND METHODS: Part I was a combination 3+3 dose escalation study for recurrent ovarian cancer. Patients received daily continuous oral afuresertib at 50-150 mg/day with intravenous paclitaxel (175 mg/m2) and carboplatin (AUC5) every 3 weeks for six cycles followed by maintenance afuresertib at 125 mg/day until progression or toxicity. Part II was a single-arm evaluation of the clinical activity of this combination in recurrent PROC (Cohort A) or PPROC (Cohort B). Patients received oral afuresertib at the MTD defined in Part I in combination with PC for six cycles, followed by maintenance afuresertib. Primary endpoints were safety and tolerability of afuresertib in combination with PC (Part I, dose escalation), and investigator-assessed overall response rate (ORR) as per RECIST version 1.1 (Part II). RESULTS: Twenty-nine patients enrolled into Part I, and 30 into Part II. Three dose-limiting toxicities of grade 3 rash were observed, one at 125 mg and two at 150 mg afuresertib. The MTD of afuresertib in combination with PC was therefore identified as 125 mg/day. The most common (≥50%) drug-related adverse events observed in Part I of the study were nausea, diarrhea, vomiting, alopecia, fatigue, and neutropenia and, in Part II, were diarrhea, fatigue, nausea, and alopecia. The Part II ORR in the intention to treat patients was 32% [95% confidence interval (CI), 15.9-52.4] by RECIST 1.1 and 52% (95% CI, 31.3-72.2) by GCIG CA125 criteria. Median progression-free survival was 7.1 months (95% CI, 6.3-9.0 months). CONCLUSIONS: Afuresertib plus PC demonstrated efficacy in recurrent PROC with the MTD of afuresertib defined as 125 mg/day.

Unsupervised correction of gene-independent cell responses to CRISPR-Cas9 targeting.

BACKGROUND: Genome editing by CRISPR-Cas9 technology allows large-scale screening of gene essentiality in cancer. A confounding factor when interpreting CRISPR-Cas9 screens is the high false-positive rate in detecting essential genes within copy number amplified regions of the genome. We have developed the computational tool CRISPRcleanR which is capable of identifying and correcting gene-independent responses to CRISPR-Cas9 targeting. CRISPRcleanR uses an unsupervised approach based on the segmentation of single-guide RNA fold change values across the genome, without making any assumption about the copy number status of the targeted genes. RESULTS: Applying our method to existing and newly generated genome-wide essentiality profiles from 15 cancer cell lines, we demonstrate that CRISPRcleanR reduces false positives when calling essential genes, correcting biases within and outside of amplified regions, while maintaining true positive rates. Established cancer dependencies and essentiality signals of amplified cancer driver genes are detectable post-correction. CRISPRcleanR reports sgRNA fold changes and normalised read counts, is therefore compatible with downstream analysis tools, and works with multiple sgRNA libraries. CONCLUSIONS: CRISPRcleanR is a versatile open-source tool for the analysis of CRISPR-Cas9 knockout screens to identify essential genes.

Biomarker Assessment of HR Deficiency, Tumor BRCA1/2 Mutations, and CCNE1 Copy Number in Ovarian Cancer: Associations with Clinical Outcome Following Platinum Monotherapy.

The current study evaluated three biomarkers [homologous recombination deficiency (HRD), tumor BRCA1/2 (tBRCA) mutations, and CCNE1 copy-number variation (CNV)] in ovarian tumors from patients enrolled on the SCOTROC4 clinical trial for associations with outcome following carboplatin monotherapy. Ovarian tumors (n = 250), with high-grade serous (HGSOC) subgroup analysis (n = 179) were classified as HRD positive (HRD score ≥42 or tBRCA mutation) and as CCNE1 amplification positive (CCNE1 CNV score >2.4). Seventy-four (30%) tumors were HRD positive, including 34 (14%) with tBRCA mutations. Forty-seven (19%) were CCNE1 amplification positive, all of which were tBRCA wild-type. HRD and tBRCA, but not CCNE1 amplification, were significantly associated with CA125 complete response in the entire cohort (HRD, P = 0.00015; tBRCA P = 0.0096), and the HGSOC subgroup (HRD, P = 0.0016; tBRCA P = 0.032). HRD and lack of CCNE1 amplification were associated with improved progression-free survival (PFS) and overall survival (OS) in the full cohort and HGSOC subgroup (HRD, P = 0.00021; CCNE1 status P = 0.038). HRD remained significant for OS and PFS after adjusting for clinical factors, while CCNE1 status only remained significant for PFS. Patients with HRD-positive tumors had greater PFS and OS benefit from platinum dose intensification than HRD-negative tumors (P = 0.049 and P = 0.035, respectively). An alternative exploratory HRD score threshold (≥33 or tBRCA mutation) was also significantly associated with both PFS and OS in the HGSOC subset.Implications: HRD, tumor BRCA1/2 mutations, and absence of CCNE1 amplification are associated with improved survival of ovarian cancer patients treated with platinum monotherapy and HRD-positive patients may benefit from platinum dose intensification. Mol Cancer Res; 16(7); 1103-11. ©2018 AACR.

Dietary geranylgeraniol can limit the activity of pitavastatin as a potential treatment for drug-resistant ovarian cancer.

Pre-clinical and retrospective studies of patients using statins to reduce plasma cholesterol have suggested that statins may be useful to treat cancer. However, prospective clinical trials have yet to demonstrate significant efficacy. We have previously shown that this is in part because a hydrophobic statin with a long half-life is necessary. Pitavastatin, the only statin with this profile, has not undergone clinical evaluation in oncology. The target of pitavastatin, hydroxymethylglutarate coenzyme-A reductase (HMGCR), was found to be over-expressed in all ovarian cancer cell lines examined and upregulated by mutated TP53, a gene commonly altered in ovarian cancer. Pitavastatin-induced apoptosis was blocked by geranylgeraniol and mevalonate, products of the HMGCR pathway, confirming that pitavastatin causes cell death through inhibition of HMGCR. Solvent extracts of human and mouse food were also able to block pitavastatin-induced apoptosis, suggesting diet might influence the outcome of clinical trials. When nude mice were maintained on a diet lacking geranylgeraniol, oral pitavastatin caused regression of Ovcar-4 tumour xenografts. However, when the animal diet was supplemented with geranylgeraniol, pitavastatin failed to prevent tumour growth. This suggests that a diet containing geranylgeraniol can limit the anti-tumour activity of pitavastatin and diet should be controlled in clinical trials of statins.

MicroRNAs associated with small bowel neuroendocrine tumours and their metastases.

Novel molecular analytes are needed in small bowel neuroendocrine tumours (SBNETs) to better determine disease aggressiveness and predict treatment response. In this study, we aimed to profile the global miRNome of SBNETs, and identify microRNAs (miRNAs) involved in tumour progression for use as potential biomarkers. Two independent miRNA profiling experiments were performed (n=90), including primary SBNETs (n=28), adjacent normal small bowel (NSB; n=14), matched lymph node (LN) metastases (n=24), normal LNs (n=7), normal liver (n=2) and liver metastases (n=15). We then evaluated potentially targeted genes by performing integrated computational analyses. We discovered 39 miRNAs significantly deregulated in SBNETs compared with adjacent NSB. The most upregulated (miR-204-5p, miR-7-5p and miR-375) were confirmed by qRT-PCR. Two miRNAs (miR-1 and miR-143-3p) were significantly downregulated in LN and liver metastases compared with primary tumours. Furthermore, we identified upregulated gene targets for miR-1 and miR-143-3p in an existing SBNET dataset, which could contribute to disease progression, and show that these miRNAs directly regulate FOSB and NUAK2 oncogenes. Our study represents the largest global miRNA profiling of SBNETs using matched primary tumour and metastatic samples. We revealed novel miRNAs deregulated during SBNET disease progression, and important miRNA-mRNA interactions. These miRNAs have the potential to act as biomarkers for patient stratification and may also be able to guide treatment decisions. Further experiments to define molecular mechanisms and validate these miRNAs in larger tissue cohorts and in biofluids are now warranted.

Preventing Damage Limitation: Targeting DNA-PKcs and DNA Double-Strand Break Repair Pathways for Ovarian Cancer Therapy.

Platinum-based chemotherapy is the cornerstone of ovarian cancer treatment, and its efficacy is dependent on the generation of DNA damage, with subsequent induction of apoptosis. Inappropriate or aberrant activation of the DNA damage response network is associated with resistance to platinum, and defects in DNA repair pathways play critical roles in determining patient response to chemotherapy. In ovarian cancer, tumor cell defects in homologous recombination - a repair pathway activated in response to double-strand DNA breaks (DSB) - are most commonly associated with platinum-sensitive disease. However, despite initial sensitivity, the emergence of resistance is frequent. Here, we review strategies for directly interfering with DNA repair pathways, with particular focus on direct inhibition of non-homologous end joining (NHEJ), another DSB repair pathway. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a core component of NHEJ and it has shown considerable promise as a chemosensitization target in numerous cancer types, including ovarian cancer where it functions to promote platinum-induced survival signaling, via AKT activation. The development of pharmacological inhibitors of DNA-PKcs is on-going, and clinic-ready agents offer real hope to patients with chemoresistant disease.

A putative biomarker signature for clinically effective AKT inhibition: correlation of in vitro, in vivo and clinical data identifies the importance of modulation of the mTORC1 pathway.

Our identification of dysregulation of the AKT pathway in ovarian cancer as a platinum resistance specific event led to a comprehensive analysis of in vitro, in vivo and clinical behaviour of the AKT inhibitor GSK2141795. Proteomic biomarker signatures correlating with effects of GSK2141795 were developed using in vitro and in vivo models, well characterised for related molecular, phenotypic and imaging endpoints. Signatures were validated in temporally paired biopsies from patients treated with GSK2141795 in a clinical study. GSK2141795 caused growth-arrest as single agent in vitro, enhanced cisplatin-induced apoptosis in vitro and reduced tumour volume in combination with platinum in vivo. GSK2141795 treatment in vitro and in vivo resulted in ~50-90% decrease in phospho-PRAS40 and 20-80% decrease in fluoro-deoxyglucose (FDG) uptake. Proteomic analysis of GSK2141795 in vitro and in vivo identified a signature of pathway inhibition including changes in AKT and p38 phosphorylation and total Bim, IGF1R, AR and YB1 levels. In patient biopsies, prior to treatment with GSK2141795 in a phase 1 clinical trial, this signature was predictive of post-treatment changes in the response marker CA125. Development of this signature represents an opportunity to demonstrate the clinical importance of AKT inhibition for re-sensitisation of platinum resistant ovarian cancer to platinum.

Rethinking ovarian cancer II: reducing mortality from high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSOC) accounts for 70-80% of ovarian cancer deaths, and overall survival has not changed significantly for several decades. In this Opinion article, we outline a set of research priorities that we believe will reduce incidence and improve outcomes for women with this disease. This 'roadmap' for HGSOC was determined after extensive discussions at an Ovarian Cancer Action meeting in January 2015.

Dose-Finding Quantitative 18F-FDG PET Imaging Study with the Oral Pan-AKT Inhibitor GSK2141795 in Patients with Gynecologic Malignancies.

UNLABELLED: AKT (a serine/threonine-specific protein kinase) regulates many cellular processes contributing to cytotoxic drug resistance. This study's primary objective examined the relationship between GSK2141795, an oral, pan-AKT inhibitor, and (18)F-FDG PET markers of glucose metabolism in tumor tissue to determine whether (18)F-FDG PET could be used to guide personalized dosing of GSK2141795. Biomarker analysis of biopsies was also undertaken. METHODS: Twelve patients were enrolled in 3 cohorts; all underwent dynamic (18)F-FDG PET scans and serial pharmacokinetic sampling at baseline, week 2, and week 4 with tumor biopsies before treatment and at week 4. Response was evaluated by RECIST v1.1 and Gynecologic Cancer Intergroup criteria. Biopsy samples were analyzed for mutations and protein expression. RESULTS: GSK2141795 did not significantly influence blood glucose levels. No dose-response relationship was observed between GSK2141795 pharmacokinetics and (18)F-FDG PET pharmacodynamic measures; however, an exposure-response relationship was seen between maximum drug concentrations and maximal decrease in (18)F-FDG uptake in the best-responding tumor. This relationship also held for pharmacokinetic parameters of exposure and 1,5-anhydroglucitol (a systemic measure of glucose metabolism). Phospho-AKT upregulation at week 4 in biopsies confirmed AKT inhibition by GSK2141795. Single-agent activity was observed with a clinical benefit rate of 27% (3/11) and 30% (3/10) CA125 response in the study's platinum-resistant ovarian patients. AKT pathway activation by PIK3CA/PIK3R1 mutation did not correlate with clinical activity, whereas RAS/RAF pathway mutations did segregate with resistance to AKT inhibition. CONCLUSION: GSK2141795 demonstrated an exposure-response relationship with decreased (18)F-FDG uptake and is active and tolerable. This study's design integrating (18)F-FDG PET, pharmacokinetics, and biomarker analyses demonstrates the potential for clinical development for personalized treatment.

The role of interleukin-8 (IL-8) and IL-8 receptors in platinum response in high grade serous ovarian carcinoma.

Platinum based drugs are the cornerstone of chemotherapy for ovarian cancer, however the development of chemoresistance hinders its success. IL-8 is involved in regulating several pro-survival pathways in cancer. We studied the expression of IL-8 and IL-8 receptors in platinum sensitive and resistant cell lines. Using qRT-PCR and immunohistochemistry, both platinum sensitive (PEA1, PEO14) and resistant (PEA2, PEO23) show increased expression of IL-8 and IL-8 receptors. IL-8RA shows nuclear and cytoplasmic expression, whilst IL-8RB is present solely in the cytoplasm. Knockdown of IL-8 increased sensitivity to cisplatin in platinum sensitive and reversed platinum resistance in resistant cell lines, decreased the expression of anti-apoptotic Bcl-2 and decreased inhibitory phosphorylation of pro-apoptotic Bad. IL-8 receptor antagonist treatment also enhanced platinum sensitivity. Nuclear localisation of IL-8RA was only detected in platinum resistant tumours. Inhibition of IL-8 signalling can enhance response in platinum sensitive and resistant disease. Nuclear IL-8RA may have potential as a biomarker of resistant disease.

Modeling platinum sensitive and resistant high-grade serous ovarian cancer: development and applications of experimental systems.

High-grade serous ovarian cancer remains the most common sub-type of ovarian cancer and, characterized by high degrees of genomic instability and heterogeneity, is typified by a transition from early response to acquired resistance to platinum-based chemotherapy. Conventional models for the study of ovarian cancer have been largely limited to a set of relatively poorly characterized immortalized cell lines and recent studies have called into question the validity of some of these as reliable models. Here, we review new approaches and models systems that take into account advances in our understanding of ovarian cancer biology and advances in the technology available for their generation and study. We discuss primary cell models, 2D, 3D, and organotypic models, and "paired" sample approaches that capture the evolution of chemotherapy failure within single cases. We also overview new methods for non-invasive collection of representative tumor material from blood samples. Adoption of such methods and models will improve the quality and clinical relevance of ovarian cancer research.

Opportunities for translation: targeting DNA repair pathways in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the poorest prognosis neoplasms. It is typified by high levels of genomic aberrations and copy-number variation, intra-tumoural heterogeneity and resistance to conventional chemotherapy. Improved therapeutic options, ideally targeted against cancer-specific biological mechanisms, are urgently needed. Although induction of DNA damage and/or modulation of DNA damage response pathways are associated with the activity of a number of conventional PDAC chemotherapies, the effectiveness of this approach in the treatment of PDAC has not been comprehensively reviewed. Here, we review chemotherapeutic agents that have shown anti-cancer activity in PDAC and whose mechanisms of action involve modulation of DNA repair pathways. In addition, we highlight novel potential targets within these pathways based on the emerging understanding of PDAC biology and their exploitation as targets in other cancers.

Molecular subtypes of serous borderline ovarian tumor show distinct expression patterns of benign tumor and malignant tumor-associated signatures.

Borderline ovarian tumors show heterogeneity in clinical behavior. Most have excellent prognosis, although a small percentage show recurrence or progressive disease, usually to low-grade serous carcinoma. The aim of this study was to understand the molecular relationship between these entities and identify potential markers of tumor progression and therapeutic targets. We studied gene expression using Affymetrix HGU133plus2 GeneChip microarrays in 3 low-grade serous carcinomas, 13 serous borderline tumors and 8 serous cystadenomas. An independent data set of 18 serous borderline tumors and 3 low-grade serous carcinomas was used for validation. Unsupervised clustering revealed clear separation of benign and malignant tumors, whereas borderline tumors showed two distinct groups, one clustering with benign and the other with malignant tumors. The segregation into benign- and malignant-like borderline molecular subtypes was reproducible on applying the same analysis to an independent publicly available data set. We identified 50 genes that separate borderline tumors into their subgroups. Functional enrichment analysis of genes that separate borderline tumors to the two subgroups highlights a cell adhesion signature for the malignant-like subset, with Claudins particularly prominent. This is the first report of molecular subtypes of borderline tumors based on gene expression profiling. Our results provide the basis for identification of biomarkers for the malignant potential of borderline ovarian tumor and potential therapeutic targets for low-grade serous carcinoma.

Evaluation of 2-deoxy-2-[18F]fluoro-D-glucose- and 3'-deoxy-3'-[18F]fluorothymidine-positron emission tomography as biomarkers of therapy response in platinum-resistant ovarian cancer.

PURPOSE: We evaluated whether 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) and 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) positron emission tomography (PET) could be used as imaging biomarkers of platinum resensitization in ovarian cancer. PROCEDURES: Paired platinum-sensitive and platinum-resistant ovarian cancer cells from the same patient, PEO1 and PEO4, grown as tumor xenografts in nude mice, were assessed by PET. RESULTS: The AKT inhibitor, API-2, resensitized platinum-resistant PEO4 tumors to cisplatin, leading to a markedly lower Ki67 labeling index (p ≤ 0.006, n = 6 per group). [(18)F]FDG-PET and [(18)F]FLT-PET imaging variables were lower after combination treatment compared with vehicle treatment (p ≤ 0.006, n = 6 per group). No changes were seen with either drug alone. PRAS40 phosphorylation status was a sensitive biochemical marker of pathway inhibition, whereas reductions thymidine kinase 1 expression defined the [(18)F]FLT response. CONCLUSIONS: Therapeutic inhibition of AKT activation in acquired platinum-resistant disease can be imaged noninvasively by [(18)F]FDG-PET and [(18)F]FLT-PET warranting further assessment.

DNA-PK mediates AKT activation and apoptosis inhibition in clinically acquired platinum resistance.

Clinical resistance to chemotherapy is a frequent event in cancer treatment and is closely linked to poor outcome. High-grade serous (HGS) ovarian cancer is characterized by p53 mutation and high levels of genomic instability. Treatment includes platinum-based chemotherapy and initial response rates are high; however, resistance is frequently acquired, at which point treatment options are largely palliative. Recent data indicate that platinum-resistant clones exist within the sensitive primary tumor at presentation, implying resistant cell selection after treatment with platinum chemotherapy. The AKT pathway is central to cell survival and has been implicated in platinum resistance. Here, we show that platinum exposure induces an AKT-dependent, prosurvival, DNA damage response in clinically platinum-resistant but not platinum-sensitive cells. AKT relocates to the nucleus of resistant cells where it is phosphorylated specifically on S473 by DNA-dependent protein kinase (DNA-PK), and this activation inhibits cisplatin-mediated apoptosis. Inhibition of DNA-PK or AKT, but not mTORC2, restores platinum sensitivity in a panel of clinically resistant HGS ovarian cancer cell lines: we also demonstrate these effects in other tumor types. Resensitization is associated with prevention of AKT-mediated BAD phosphorylation. Strikingly, in patient-matched sensitive cells, we do not see enhanced apoptosis on combining cisplatin with AKT or DNA-PK inhibition. Insulin-mediated activation of AKT is unaffected by DNA-PK inhibitor treatment, suggesting that this effect is restricted to DNA damage-mediated activation of AKT and that, clinically, DNA-PK inhibition might prevent platinum-induced AKT activation without interfering with normal glucose homeostasis, an unwanted toxicity of direct AKT inhibitors.