How to measure homologous recombination deficiency in ovarian cancer

Defective DNA repair via homologous recombination (HR) is common in ovarian high grade serous carcinomas, and homologous recombination deficiency (HRD) represents an important therapeutic target in epithelial ovarian cancers (EOCs). The development of poly(ADP ribose) polymerase (PARP) inhibitors (PARPi) has been an important advance in the treatment of HR-deficient EOCs with the potential to change daily clinical practice. However, while germline and somatic mutations in BRCA1 and BRCA2 are still the most important mechanisms of HRD, alterations in other DNA repair pathways might also contribute to defective HR. In this review, we focus on current and emerging approaches for identifying and targeting HR-deficient EOCs, and discuss the challenges associated with these approaches

Activation of MAPK signalling results in resistance to saracatinib (AZD0530) in ovarian cancer

SRC tyrosine kinase is frequently overexpressed and activated in late-stage, poor prognosis ovarian tumours, and preclinical studies have supported the use of targeted SRC inhibitors in the treatment of this disease. The SAPPROC trial investigated the addition of the SRC inhibitor saracatinib (AZD0530) to weekly paclitaxel for the treatment of platinum resistant ovarian cancer; however, this drug combination did not provide any benefit to progression free survival (PFS) of women with platinum resistant disease. In this study we aimed to identify mechanisms of resistance to SRC inhibitors in ovarian cancer cells. Using two complementary strategies; a targeted tumour suppressor gene siRNA screen, and a phospho-receptor tyrosine kinase array, we demonstrate that activation of MAPK signalling, via a reduction in NF1 (neurofibromin) expression or overexpression of HER2 and the insulin receptor, can drive resistance to AZD0530. Knockdown of NF1 in two ovarian cancer cell lines resulted in resistance to AZD0530, and was accompanied with activated MEK and ERK signalling. We also show that silencing of HER2 and the insulin receptor can partially resensitize AZD0530 resistant cells, which was associated with decreased phosphorylation of MEK and ERK. Furthermore, we demonstrate a synergistic effect of combining SRC and MEK inhibitors in both AZD0530 sensitive and resistant cells, and that MEK inhibition is sufficient to completely resensitize AZD0530 resistant cells. This work provides a preclinical rationale for the combination of SRC and MEK inhibitors in the treatment of ovarian cancer, and also highlights the need for biomarker driven patient selection for clinical trials

CD40 ligand induced cytotoxicity in carcinoma cells is enhanced by inhibition of metalloproteinase cleavage and delivery via a conditionally-replicating adenovirus

Background CD40 and its ligand (CD40L) play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death. The most profound induction of cell death in carcinoma cells is induced by membrane-bound rather than recombinant soluble CD40L, but like other TNF family ligands, it is cleaved from the membrane by matrix metalloproteinases. Results We have generated a replication-deficient adenovirus expressing a mutant CD40L that is resistant to metalloproteinase cleavage such that ligand expression is retained at the cell membrane. Here we show that the mutated, cleavage-resistant form of CD40L is a more potent inducer of apoptosis than wild-type ligand in CD40-positive carcinoma cell lines. Since transgene expression via replication-deficient adenovirus vectors in vivo is low, we have also engineered a conditionally replicating E1A-CR2 deleted adenovirus to express mutant CD40L, resulting in significant amplification of ligand expression and consequent enhancement of its therapeutic effect. Conclusions Combined with numerous studies demonstrating its immunotherapeutic potential, these data provide a strong rationale for the exploitation of the CD40-CD40L pathway for the treatment of solid tumours

The potential and challenges of targeting MTAP-negative cancers beyond synthetic lethality

Approximately 15% of cancers exhibit loss of the chromosomal locus 9p21.3 – the genomic location of the tumour suppressor gene CDKN2A and the methionine salvage gene methylthioadenosine phosphorylase (MTAP). A loss of MTAP increases the pool of its substrate methylthioadenosine (MTA), which binds to and inhibits activity of protein arginine methyltransferase 5 (PRMT5). PRMT5 utilises the universal methyl donor S-adenosylmethionine (SAM) to methylate arginine residues of protein substrates and regulate their activity, notably histones to regulate transcription. Recently, targeting PRMT5, or MAT2A that impacts PRMT5 activity by producing SAM, has shown promise as a therapeutic strategy in oncology, generating synthetic lethality in MTAP-negative cancers. However, clinical development of PRMT5 and MAT2A inhibitors has been challenging and highlights the need for further understanding of the downstream mediators of drug effects. Here, we discuss the rationale and methods for targeting the MAT2A/PRMT5 axis for cancer therapy. We evaluate the current limitations in our understanding of the mechanism of MAT2A/PRMT5 inhibitors and identify the challenges that must be addressed to maximise the potential of these drugs. In addition, we review the current literature defining downstream effectors of PRMT5 activity that could determine sensitivity to MAT2A/PRMT5 inhibition and therefore present a rationale for novel combination therapies that may not rely on synthetic lethality with MTAP loss

Activation of MAPK signalling results in resistance to saracatinib (AZD0530) in ovarian cancer

SRC tyrosine kinase is frequently overexpressed and activated in late-stage, poor prognosis ovarian tumours, and preclinical studies have supported the use of targeted SRC inhibitors in the treatment of this disease. The SAPPROC trial investigated the addition of the SRC inhibitor saracatinib (AZD0530) to weekly paclitaxel for the treatment of platinum resistant ovarian cancer; however, this drug combination did not provide any benefit to progression free survival (PFS) of women with platinum resistant disease. In this study we aimed to identify mechanisms of resistance to SRC inhibitors in ovarian cancer cells. Using two complementary strategies; a targeted tumour suppressor gene siRNA screen, and a phospho-receptor tyrosine kinase array, we demonstrate that activation of MAPK signalling, via a reduction in NF1 (neurofibromin) expression or overexpression of HER2 and the insulin receptor, can drive resistance to AZD0530. Knockdown of NF1 in two ovarian cancer cell lines resulted in resistance to AZD0530, and was accompanied with activated MEK and ERK signalling. We also show that silencing of HER2 and the insulin receptor can partially resensitize AZD0530 resistant cells, which was associated with decreased phosphorylation of MEK and ERK. Furthermore, we demonstrate a synergistic effect of combining SRC and MEK inhibitors in both AZD0530 sensitive and resistant cells, and that MEK inhibition is sufficient to completely resensitize AZD0530 resistant cells. This work provides a preclinical rationale for the combination of SRC and MEK inhibitors in the treatment of ovarian cancer, and also highlights the need for biomarker driven patient selection for clinical trials

Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans) and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX), which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs). These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad) have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon), pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR) substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies), can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX), or alternatively, through the use of polymer-coated “stealth” vectors which avoid these interactions. Simultaneous retargeting and detargeting can be achieved by combining multiple genetic and/or chemical modifications

Angiotensin II promotes KV7.4 channels degradation through reduced interaction with HSP90 (heat shock protein 90)

Voltage-gated Kv 7.4 channels have been implicated in vascular smooth muscle cells’ activity because they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are downregulated in several arterial beds in different models of hypertension. Angiotensin II (Ang II) is a key player in hypertension that affects the expression of several classes of ion channels. In this study, we evaluated the effects of Ang II on the expression and function of vascular Kv 7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased Kv 7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (Kv 7)–sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the Kv 7 activator ML213. Ang II also reduced Kv 7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II–induced decrease of Kv 7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of Kv 7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of Kv 7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased Kv 7.4 ubiquitination. Similar alterations were found in mesenteric vascular smooth muscle cells isolated from Ang II–infused mice. The effect of Ang II was emulated by 17-AAG (17-demethoxy-17-(2-propenylamino) geldanamycin) that inhibits HSP90 interactions with client proteins. These results show that Ang II downregulates Kv 7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and Kv 7.4 ubiquitination and degradation via the proteasom

The Emerging Role of Interleukin 1β (IL-1β) in Cancer Cachexia

Treatment of cancer cachexia remains an unmet need. The host-tumour interface and the resulting sequestration of the pro-inflammatory cytokine Il-1β is critical in cachexia development. Neuroinflammation mediated via IL-1β through the hypothalamic pituitary axis results in increased muscle proteolysis and adipose lipolysis, thus creating a prolonged stress-like environment with loss of appetite and increased resting energy expenditure. Recent trials using a monoclonal antibody targeting IL-1β, canakinumab, have shown a potential role in lung cancer; however, a potential role of targeting IL-1β to treat cachexia in patients with lung cancer is unclear, yet the underlying pathophysiology provides a sound rationale that this may be a viable therapeutic approach

Repeatability of quantitative FDG-PET/CT and contrast-enhanced CT in recurrent ovarian carcinoma: test-retest measurements for tumor FDG uptake, diameter, and volume.

PURPOSE: Repeatability of baseline FDG-PET/CT measurements has not been tested in ovarian cancer. This dual-center, prospective study assessed variation in tumor 2[18F]fluoro-2-deoxy-D-glucose (FDG) uptake, tumor diameter, and tumor volume from sequential FDG-PET/CT and contrast-enhanced computed tomography (CECT) in patients with recurrent platinum-sensitive ovarian cancer. EXPERIMENTAL DESIGN: Patients underwent two pretreatment baseline FDG-PET/CT (n = 21) and CECT (n = 20) at two clinical sites with different PET/CT instruments. Patients were included if they had at least one target lesion in the abdomen with a standardized uptake value (SUV) maximum (SUVmax) of ≥ 2.5 and a long axis diameter of ≥ 15 mm. Two independent reading methods were used to evaluate repeatability of tumor diameter and SUV uptake: on site and at an imaging clinical research organization (CRO). Tumor volume reads were only performed by CRO. In each reading set, target lesions were independently measured on sequential imaging. RESULTS: Median time between FDG-PET/CT was two days (range 1-7). For site reads, concordance correlation coefficients (CCC) for SUVmean, SUVmax, and tumor diameter were 0.95, 0.94, and 0.99, respectively. Repeatability coefficients were 16.3%, 17.3%, and 8.8% for SUVmean, SUVmax, and tumor diameter, respectively. Similar results were observed for CRO reads. Tumor volume CCC was 0.99 with a repeatability coefficient of 28.1%. CONCLUSIONS: There was excellent test-retest repeatability for FDG-PET/CT quantitative measurements across two sites and two independent reading methods. Cutoff values for determining change in SUVmean, SUVmax, and tumor volume establish limits to determine metabolic and/or volumetric response to treatment in platinum-sensitive relapsed ovarian cancer.This study was funded by Merck and Co.This version is the author accepted manuscript. The OnlineFirst version of this article can be found on the publisher's website at: http://clincancerres.aacrjournals.org/content/20/10/2751.full.pdf+htm

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion