Jan Dominik Kuhlmann

Methylstat sensitizes ovarian cancer cells to PARP-inhibition by targeting the histone demethylases JMJD1B/C

Abstract PARP-inhibitors (PARPi) are an integral part of ovarian cancer treatment. However, overcoming acquired PARPi resistance or increasing the benefit of PARPi in patients without homologous recombination deficiency (HRD) remains an unmet clinical need. We sought to identify genetic modulators of PARPi response, guiding pharmacological PARPi sensitization. CRISPR-Cas9 mediated loss-of-function screen with a focused sgRNA library revealed that DNA-demethylases JMJD1B/JMJD1C, targetable by the small inhibitor methylstat, promote PARPi resistance. Methylstat synergistically interacted with olaparib, and (re-)sensitized ovarian cancer cells to PARPi treatment, surpassing the efficacy of common demethylase inhibitors. Genetic knockout of JMJD1B and/or JMJD1C phenocopied the effect of methylstat in an additive manner. Validation studies revealed methylstat to be a universal PARPi-sensitizing drug, effective, regardless of PARPi resistance status or BRCA1 mutational background. Methylstat modulated clonal cancer dynamics by mitigating positive selection of PARPi-resistant or BRCA1-proficient cells under olaparib treatment. Using a model of PARPi-induced cellular toxicity, we showed that methylstat impairs cellular DNA repair, indicated by an increased susceptibility of ovarian cancer cells to olaparib-induced DNA double strand breaks after methylstat exposure. This study proposes the histone demethylase inhibitor methylstat as an epigenetic drug for overcoming PARPi-resistance or for increasing efficacy of PARPi beyond HRD in ovarian cancer patients.

Diphenhydramine increases the therapeutic window for platinum drugs by simultaneously sensitizing tumor cells and protecting normal cells

Platinum‐based compounds remain a well‐established chemotherapy for cancer treatment despite their adverse effects which substantially restrict the therapeutic windows of the drugs. Both the cell type‐specific toxicity and the clinical responsiveness of tumors have been associated with mechanisms that alter drug entry and export. We sought to identify pharmacological agents that promote cisplatin (CP) efficacy by augmenting the levels of drug‐induced DNA lesions in malignant cells and simultaneously protecting normal tissues from accumulating such damage and from functional loss. Formation and persistence of platination products in the DNA of individual nuclei were measured in drug‐exposed cell lines, in primary human tumor cells and in tissue sections using an immunocytochemical method. Using a mouse model of CP‐induced toxicity, the antihistaminic drug diphenhydramine (DIPH) and two methylated derivatives decreased DNA platination in normal tissues and also ameliorated nephrotoxicity, ototoxicity, and neurotoxicity. In addition, DIPH sensitized multiple cancer cell types, particularly ovarian cancer cells, to CP by increasing intracellular uptake, DNA platination, and/or apoptosis in cell lines and in patient‐derived primary tumor cells. Mechanistically, DIPH diminished transport capacity of CP efflux pumps MRP2, MRP3, and MRP5 particularly in its C2+C6 bimethylated form. Overall, we demonstrate that DIPH reduces side effects of platinum‐based chemotherapy and simultaneously inhibits key mechanisms of platinum resistance. We propose that measuring DNA platination after ex vivo exposure may predict the responsiveness of individual tumors to DIPH‐like modulators.

Serum calretinin as an independent predictor for platinum resistance and prognosis in ovarian cancer

Calretinin (CRT) is a calcium‐binding protein that controls intracellular calcium signaling. Besides its prominent expression in neurons, serum CRT (sCRT) has recently been suggested as blood‐based biomarker for prediagnostic mesothelioma detection. CRT is expressed in ovarian cancer tissues in up to 40% of cases; however, its clinical relevance as blood‐based biomarker for ovarian cancer is unknown. sCRT was determined by calretinin enzyme‐linked immunoabsorbent assay (Calretinin‐ELISA, DLD Diagnostika GmbH, Hamburg, Germany) in a total of 515 serum samples from 116 healthy controls and 134 ovarian cancer patients (thereof 86% with Fédération Internationale de Gynécologie et d'Obstétrique [FIGO] III/IV), including samples at primary diagnosis and at four longitudinal follow‐up time points in the course of treatment and at recurrence. sCRT level was significantly increased in ovarian cancer patients compared to healthy controls (estimated difference = 0.3 ng/ml, p < 0.001), was mostly independent from CA125 (r ≤ 0.388) and enabled accurate discrimination between cases and controls (area under the curve = 0.85). Higher sCRT level at primary diagnosis predicted suboptimal debulking (p < 0.001) and was associated with advanced FIGO‐stage (p < 0.001) and increased amount of ascites (p < 0.001). sCRT levels at primary diagnosis and its dynamics in the course of chemotherapy were independent predictors for poor progression‐free survival (hazard ratio [HR] = 1.99, confidence interval [CI] = [1.13–3.52], p = 0.0181) and overall survival (HR = 15.4, CI = [1.92–124], p = 0.0099). Furthermore, sCRT at primary diagnosis or a relative sCRT increase in the time interval between surgery and the onset of chemotherapy were both independent predictors of platinum resistance (OR = 4.99, CI = [3.50–16,001], p = 0.0016; OR = 2.41, CI = [1.37–6,026], p = 0.0271, respectively). This is the first study that suggests sCRT as liquid biopsy marker for independent prediction of platinum resistance and prognosis.

Evaluation of circulating Dickkopf-1 as a prognostic biomarker in ovarian cancer patients

Abstract Objectives Dickkopf-1 (DKK1) is a secreted protein, known for suppressing the differentiation and activity of bone-building osteoblasts by acting as an inhibitor of Wnt-signalling. Soluble DKK1 (sDKK1) has been proposed as prognostic biomarker for a wide range of malignancies, however, clinical relevance of sDKK1 as potential blood-based marker for ovarian cancer is unknown. Methods sDKK1 levels were quantified in a cohort of 150 clinically documented ovarian cancer patients by a commercially available DKK1 ELISA (Biomedica, Vienna, Austria). Results Median sDKK1 level was significantly elevated at primary diagnosis of ovarian cancer compared to healthy controls (estimated difference (ED) of 7.75 ng/mL (95% CI: 3.01–12.30 ng/mL, p=0.001)). Higher levels of sDKK1 at diagnosis indicated an increased volume of intraoperative malignant ascites (ED 7.08 pmol/L, 95% CI: 1.46–13.05, p=0.02) and predicted suboptimal debulking surgery (ED 6.88 pmol/L, 95% CI: 1.73–11.87, p=0.01). sDKK1 did not correlate with CA125 and higher sDKK1 levels predicted a higher risk of recurrence and poor survival (PFS: HR=0.507, 95% CI: 0.317–0.809; p=0.004; OS: HR=0.561, 95% CI: 0.320–0.986; p=0.044). Prognostic relevance of sDKK1 was partly sustained in wtBRCA patients (PFS: HR=0.507, 95% CI: 0.317–0.809; p=0.004). Conclusions This is the first study demonstrating the prognostic relevance of sDKK1 in ovarian cancer patients, including those with wtBRCA 1/2 status. Our data encourage further evaluation of sDKK1 in ovarian cancer patients, possibly in terms of a therapy monitoring marker or a response predictor for sDKK1-directed targeted therapies.

Prognostic relevance of longitudinal HGF levels in serum of patients with ovarian cancer

The pleiotropic protein hepatocyte growth factor (HGF) is the only known ligand of the tyrosine kinase mesenchymal–epithelial transition (cMET) receptor. The HGF/cMET pathway mediates invasion and migration of ovarian cancer cells, and upregulation of HGF/cMET pathway components has been associated with poor prognosis. This study investigated the clinical relevance of circulating HGF in serum of patients with ovarian cancer. Serum HGF (sHGF) was determined by enzyme‐linked immunosorbent assay in a total of 471 serum samples from 82 healthy controls and 113 patients with ovarian cancer (88.5% with ≥ FIGO III). Patient samples were collected at primary diagnosis and at four follow‐up time points throughout treatment and at disease recurrence. Patients with ovarian cancer showed elevated median sHGF levels at primary diagnosis, and sHGF levels transiently increased after surgery and normalized in the course of chemotherapy, even dropping below initial baseline. Higher levels of sHGF were an independent predictor for shorter overall survival (OS) (a) at primary diagnosis (HR = 0.41, 95% CI: 0.22–0.78, P = 0.006), (b) at longitudinal follow‐up time points (after surgery and before/during/after chemotherapy), (c) along the patients’ individual dynamics (HR = 0.21, 95% CI: 0.07–0.63, P = 0.005), and (d) among a subgroup analysis of patients with BRCA1/2 wild‐type ovarian cancer. This is the first study proposing sHGF as an independent prognostic biomarker for ovarian cancer at primary diagnosis and in the course of platinum‐based chemotherapy, irrespective of the postoperative residual disease after surgical debulking. sHGF could be implemented into clinical diagnostics as a CA125 auxiliary tumor marker for individualized prognosis stratification and sHGF‐guided therapy monitoring.

The levels of soluble cMET ectodomain in the blood of patients with ovarian cancer are an independent prognostic biomarker

The tyrosine kinase mesenchymal–epithelial transition (cMET) is typically overexpressed in up to 75% of patients with ovarian cancer, and cMET overexpression has been associated with poor prognosis. The proteolytic release of the soluble cMET (sMET) ectodomain by metalloproteases, a process called ectodomain shedding, reflects the malignant potential of tumour cells. sMET can be detected in the human circulation and has been proposed as biomarker in several cancers. However, the clinical relevance of sMET in ovarian cancer as blood‐based biomarker is unknown and was therefore investigated in this study. sMET levels were determined by enzyme‐linked immunosorbent assay in a set of 432 serum samples from 85 healthy controls and 86 patients with ovarian cancer (87% FIGO III/IV). Samples were collected at primary diagnosis, at four longitudinal follow‐up time points during the course of treatment and at disease recurrence. Although there was no significant difference between median sMET levels at primary diagnosis of ovarian cancervs. healthy controls, increased sMET levels at primary diagnosis were an independent predictor of shorter PFS (HR = 0.354, 95% CI: 0.130–0.968,P = 0.043) and shorter OS (HR = 0.217, 95% CI: 0.064–0.734,P = 0.014). In the follow‐up samples, sMET levels were prognostically most informative after the first three cycles of chemotherapy, with high sMET levels being an independent predictor of shorter PFS (HR = 0.245, 95% CI: 0.100–0.602,P = 0.002). This is the first study to suggest that sMET levels in the blood can be used as an independent prognostic biomarker for ovarian cancer. Patients at high risk of recurrence and with poor prognosis, as identified based on sMET levels in the blood, could potentially benefit from cMET‐directed therapies or other targeted regimes, such as PARP inhibitors or immunotherapy.

Bevacizumab May Differentially Improve Prognosis of Advanced Ovarian Cancer Patients with Low Expression of VEGF-A165b, an Antiangiogenic VEGF-A Splice Variant

Abstract Purpose: The identification of a robust IHC marker to predict the response to antiangiogenic bevacizumab in ovarian cancer is of high clinical interest. VEGF-A, the molecular target of bevacizumab, is expressed as multiple isoforms with pro- or antiangiogenic properties, of which VEGF-A165b is the most dominant antiangiogenic isoform. The balance of VEGF-A isoforms is closely related to the angiogenic capacity of a tumor and may define its vulnerability to antiangiogenic therapy. We investigated whether the expression of VEGF-A165b could be related to the effect of bevacizumab in advanced ovarian cancer patients. Experimental Design: Formalin-fixed paraffin-embedded tissues from 413 patients of the ICON7 multicenter phase III trial, treated with standard platinum-based chemotherapy with or without bevacizumab, were probed for VEGF-A165b expression by IHC. Results: In patients with low VEGF-A165b expression, the addition of bevacizumab to standard platinum-based chemotherapy significantly improved progression-free (HR: 0.727; 95% CI, 0.538–0.984; P = 0.039) and overall survival (HR: 0.662; 95% CI, 0.458–0.958; P = 0.029). Multivariate analysis showed that the addition of bevacizumab in low VEGF-A165b–expressing patients conferred significant improvements in progression-free survival (HR: 0.610; 95% CI, 0.446–0.834; P = 0.002) and overall survival (HR: 0.527; 95% CI, 0.359–0.775; P = 0.001), independently from established risk factors. Conclusions: We demonstrate for the first time that bevacizumab may differentially improve the prognosis of advanced ovarian cancer patients with low expression of VEGF-A165b, an antiangiogenic VEGF-A splice variant. We envision that this novel biomarker could be implemented into routine diagnostics and may have direct clinical implications for guiding bevacizumab-related treatment decisions in advanced ovarian cancer patients.