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Page 280 Schwarzenbach et al. Cancer Drug Resist 2019;2:271-96 I http://dx.doi.org/10.20517/cdr.2019.010

synergistic cytotoxicity and apoptosis in human ovarian cancer cells and reduced among others Bcl-2 and
inhibited both DNMT and histone deacetylase (HDAC) activities.

Applying reduced representation bisulfite sequencing, Lund et al. examined malignant ascites cells
[19]
from patients with high-grade serous ovarian cancer, the most common ovarian cancer type, to clarify
the molecular mechanisms of drug resistance in this cancer type. Cisplatin resistance was associated with
hypomethylation at several CpG sites, primarily localized in the intergenic regions of the genome. The
genes close to the differentially methylated sites were associated with canonical pathways, such as cAMP-
mediated signaling, G-protein coupled receptor (GPCR) signaling, WNT/beta-catenin signaling and human
embryonic stem cell pluripotency.

Carboplatin resistance
Several mechanisms associated with the development of acquired drug resistance in ovarian cancer have also been
reported for carboplatin. Fang et al. compared the response of patients with recurrent platinum-resistant ovarian
[35]
cancer who received carboplatin plus the DNMT inhibitor guadecitabine with a standard-of-care chemotherapy
regimen. Epigenomic and transcriptomic profiling were performed using the Infinium methylation bead
chips. They defined 94 gene promoters that were significantly hypomethylated by guadecitabine, with 1,659
genes differentially expressed in pretreatment versus post-treatment tumors resulting in altered immune
reactivation and DNA repair pathways. In functional analyses, upregulation of the tumor suppressors docking
protein 2 (DOK2), an adapter protein downstream of tyrosine kinase, and miR-193a silenced by promoter
methylation restored platinum drug sensitivity of ovarian cancer cells. Lum et al. also characterized and
[34]
functionally validated DOK2 among the genes identified in the epigenome screening using a tissue culture
carboplatin resistance assay. In the set of methylated candidate genes associated with platinum resistance,
the loss of DOK2 induced chemotherapy resistance by decreasing the level of apoptosis in response to the
treatment. de Leon et al. used an Illumina DNA methylation array and profiled carboplatin sensitive
[33]
and resistant ovarian cancer xenografts. In particular, they confirmed that the mRNA expression levels
of transmembrane protein 88 (TMEM88) were increased in resistant compared to control xenografts and
correlated with promoter hypomethylation. Its transcriptional regulation by promoter methylation was
supported by the administration of ovarian cancer cells with the DNMT inhibitor guadecitabine which
increased TMEM88 mRNA expression levels. TMEM88 knock-down re-sensitized cells to platinum and
induced upregulation of cyclin D1 and c-Myc, suggesting that TMEM88 inhibited the Wnt signaling pathway.

In approximately 11% of high-grade serous ovarian cancer, BRCA1 promoter methylation is an important
somatic driver . Using whole-genome sequencing of tumor and germline DNA samples from these patients,
[77]
Patch et al. observed several molecular events associated with acquired resistance to carboplatin-combination
[78]
treatment. They identified a patient who displayed extensive promoter methylation and low expression of BRCA1.
During the relapse, the patient lost BRCA1 methylation and the gene was expressed at comparable levels to
homologous-recombination-intact tumors. Comparison of the global methylation patterns of primary and
recurrence samples suggested a specific rather than a generalized altered methylation status at relapse in this
patient.

A phase 1 trial of low-dose decitabine combined with carboplatin in ten patients with recurrent, platinum-resistant
ovarian cancer was performed by Fang et al. The most common toxicities were nausea, allergic reactions,
[79]
neutropenia, fatigue, anorexia, vomiting and abdominal pain. LINE-1 hypomethylation in peripheral blood
mononuclear cells (PBMCs) and hypermethylation of HOXA11 and BRCA1 in plasma were detected. One
complete response was observed, and three additional patients had stable disease for about six months.
Furthermore, a phase 1b-2a clinical trial of a sequential combination of azacitidine and carboplatin was initiated
in patients with platinum-resistant or platinum-refractory epithelial ovarian cancer by Fu et al. Among 29
[80]
evaluable patients, this treatment produced one complete response, three partial responses and ten cases of