En la provincia de Córdoba

The recent advances in the molecular techniques to a genome sequencing level have incredibly broadened our understanding of molecular pathways leading to oncogenesis and are continuously constituting a necessary tool for identifying new candidate genes that can be used as targets for therapy or biomarkers of diagnosis and/or response to treatments. It has been widely recognised how a proper patients’ stratification based on molecular profiling is essential to obtain better responses. The identification of new genes to use in the future in clinical practice is an urgent necessity for gynaecological malignancies too.

In an extensive work from Ciriello et al. [21], genomic and epigenetic data were combined from the The Cancer Genome Atlas (TCGA) database analysing 3000 tumour samples to identify common patterns in several solid cancers. Regarding ovarian carcinoma, the tumour was classified in the C class, which was described by an early mutation at tumour suppressor p53 followed by a cascade of events that leads to a mosaic of genetic instability often seen in cancer patients. Class C tumours were further subdivided whether they were showing mutations at chromosome 8 (MYC) and class C13 included the typical pattern seen in HGSEOC with germline or somatic alterations at BRCA1 and

BRCA2. Mutations in the PI3K-AKT-mTOR pathway were observed as well as KRAS, CNNE1 (Cyclin

E1), E2F3 and Aurora Kinase A (AURKA) (Figure 1.5-4). Interestingly, in the article published by the Cancer Genome Atlas Research Network [22], HGSEOC was categorised into descriptive subtypes considering differential genes’ expression; one of them, the “mesenchymal” subtype, was expressing high levels of HOX genes and other markers related to an expansion of the stromal components.

Each emerging target will be further discussed and its potential pharmaceutical inhibitions and clinical trials, where available, explored in the next paragraph implementing the description of some of the already briefly mentioned genes in Section 1.5.

1.6.1. P53

Tumour protein 53, TP53 or simply p53, is a 53kDa protein with a pivotal role in the control of DNA damage status of a cell. Its protein level is tightly regulated by MDM2 in a negative feedback fashion with increased concentrations seen in case of DNA damage. In fact, P53 is able to “sense” the stress status of a cell and interact with key regulators proteins of the cell cycle progression as p21, GADD45 and 14-3-3α to pause the cell and let the DNA repair systems to efficiently repair the damage. In the case of not solvable repairing, it is again P53 that activates pro-apoptotic genes like

31 Figure 1.5-2: P53 Functions.

P53, the guardian of the genome, is capable of sensing a variety of external or internal cellular stress and converting the damage into a molecular cascade. The image shows a non-exhaustive list of p53 functions after stress, including the cell cycle arrest via p21, 14 - 3-3 and GADD45 to allow the activation of DNA repair systems. In the case of a persistent stress and un -resolvable DNA damage, it is still p53 that initiates apoptosis via BAX, NOXA and PUMA. MDM2 regulates p53 protein content in the cytoplasm. Image from [23] under CC-BY-4.0 license.

It is not surprising that P53 has been nominated the “guardian of the genome” and it is one of the early mutations with loss of function that occurs in cancer leading to genomic instability, accounting for 95% of the cases for HGS epithelial ovarian cancer. Due to its high rate of alteration in OC in the early onset of tumour development as well as in other cancer types, p53 is not an ideal biomarker for diagnosis, but as target for therapy, strategies aim to insert the defective p53 using Adenoviruses or small molecules mimicking its function such as PRIMA-met [24].

Other approaches prevent the accumulation of defective p53 protein, which have been shown to interact with aberrant pathways and being oncogenic [25] . Nutlins are a classic example of inhibitor of MDM2 that regulates levels of p53.

1.6.2. BRCA1 and BRCA2

Germline mutations at BRCA1 and BRCA2 have been observed in 15 - 20% of the ovarian carcinoma patients and their alteration linked to a 50% increase in the possibility to develop ovarian cancer HGS type II [22]. The screening for BRCA1 and BRCA2 is now becoming clinical practice to stratify patients that could respond to PARPi treatment. The indirect targeting of BRCA1 and BRCA2

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represents nowadays one of the most promising target therapies for HGSEOC cases as described in

Section 1.5.2.

Figure 1.5-3: PARP inhibitors synthetic lethality.

When administering PARP inhibitors to BRCA negative patients, tumour cells becomes unable to solve DBSs and undergo apoptosis. There are recent evidences of acquired resistance through the activation of RAD52 involved in the homologo us recombination (HR). The eradication of residual HR is achieved when inducing a dual synthetic lethality with RAD52 inhibitors. Normal cells are protected by a fully functional BRCA dependent repair system [26]. Image taken from [26] under Creative Commons licence CC BY-NC-ND 4.0

1.6.3. Other molecular targets

c-myc belongs to a family of transcription factors with the ability to bind DNA and activate gene

expression via a basic-helix-loop-helix structural motif. The protein plays a central role in several molecular pathways and to name a few it is involved in cell-cycle control, proliferation, angiogenesis and apoptosis [27]. In ovarian cancer, abnormalities at c-myc results in its gene amplifications up to 30% of the cases [21]. Its central role in several biological processes makes c- myc an unattractive marker for diagnosis, but it is indeed a valid prognosis marker for poor outcome in patients demonstrating c-myc amplification [28]. The therapy targets the gene with siRNA [29] in platinum resistant cell lines or using small molecules inhibiting the c-myc promoter or binding with its cofactor MAX [30], but the limited efficacy can be explained by the amplification of the number of alleles of the gene that translate into abundant level of oncogenic protein.

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The PI3K-AKT-mTOR pathway is another central molecular hub to regulate responses to growth factors and cytokines, therefore it is implicated in cell cycle regulation, control of apoptosis and cell proliferation. The ligand of the tyrosine kinase receptors activates a cascade of phosphorylation that switch on the PI3K protein to generate a second messenger PIP3 that in turn recruits AKT.

Conformational changes in AKT trigger the protein to phosphorylate its several targets as mTOR for survival, inhibition of BAD (apoptosis) and activation of MDM2 which inhibits P53 [31]. Alterations in the PI3K-AKT-mTOR pathway counts for up to 46% of cases in ovarian carcinoma, in particular the catalytic subunit of PI3K (PI3KCA) is amplified in 38% of the cases prevalently in clear cell and endometrioid cancer tumours. PTEN deletions, a protein that inhibits the phosphorylation of AKT by PI3K, are seen in 7% of the cases and generally associated with type I tumours (see Figure 1.3-2) [6, 21, 22]. Therefore, this pathway remains an attractive target to small molecules inhibitors and researcher have explored several compounds either directing PI3K, AKT or mTOR. BKM120 (Buparlisib) is a pan-PI3K inhibitor that blocks the catalytic unit p100α (PI3KCA) and prevents the formation of the second messenger PIP3.

The GINECO group run a phase II clinical trial exploring dosage and clinical efficacy of BKM120 as monotherapy in stratified patients according to type I or II OC classification. A total of 64 patients were enrolled, but the study was stopped before the end of recruitment due to the high toxicity of the drug observed and low clinical efficacy [32].

KRAS amplifications signature for mucinous ovarian carcinoma [22], have been observed in 15% of

OC cases in a study by Ciriello et al. [21]. KRAS belongs to a family of G-coupled proteins with an intrinsic GTP-ase activity. The hydrolysis from GTP into GDP induces conformational changes into an active KRAS able to phosphorylate RAF and PI3K creating a connection between the two pathways. Intermediate and final targets of this cascade of activation are MEK1/2 and ERK1 respectively. The RAS/RAF/ERK pathway is involved in survival and proliferation.

Several small molecules have been developed to target RAF or MEK1/2 just to name few and

Sorafenib and Selumetinib reached phase II clinical trials. Sorafenib was developed by Bayer and

approved by the FDA for treatment of liver and kidney cancer.

Amplifications at CCNE1 (cyclin E) and EF23 have been registered in 20% and 13% respectively of OC cases [21]. High levels of cyclin E impairs the inhibitory regulation of RB (retinoblastoma) on the E2F family, therefore the cells are able to enter G1/S phase. A strategy under investigation to stop the aberrant activity of the RB signalling is the use of small molecules targeting the CDK2 protein.

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To date there are no clinical trials testing the efficacy of CDK2 inhibitors in ovarian cancer patients, but in vitro pre-clinical studies are confirming that cells overexpressing CCNE1 are susceptible to CDK2 inhibitors and possible mechanisms of resistance to the drug are due to overexpression in CDK2 [33].

20% of amplification of Aurora Kinase A (AURKA) have also been observed for OC cases. AURKA is a key regulator kinase for the transition from late G2 phase into M (mitosis).

Danuseritib is the most encouraging small molecule compound with a phase II clinical trial

conducted by Schöffski et al. in 2015 [34]. Out of the 34 OC patients enrolled, only 4 had PFD after 4 months treatment out of the at least 10 required to carry on with the second stage of the trial suggesting the compound only had a partial and weak response when used as monotherapy in heavily pre-treated patients.

Figure 1.5-4: Molecular pathwa ys generally altered in ovarian carcinoma.

P53 mutation occurs in more than 90% of the new cases at early disease onset , in particular in HGS-EOC. Mutations at P53 results in a non -functional protein unable to correctly control the cell cycle (through p21 and GADD45) and DNA repair systems. The homologous recombination pathway is also mutated in 20% of OC with defects at BRCA1 and BRCA2. Overexpression of the oncogene c -MYC is probably associated with P53 mutations. Alterations in the RAS and PIK3A (a subu nit of PI3K) lead to an aberrant signalling cascade in response to cytokines and growth factors resulting in increased survival. PTEN mutations has been registered as well with a decreased ability to inhibit and regulate the PI3K -AKT- mTOR pathway. An incre ased expression in the level of messenger cyclin E is often seen in type I tumours resulting in the transition to G1 S phase of the cell cycle through the retinoblastoma Rb/E2F complex. Modified from [35].

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