Bromodomain inhibition overcomes treatment resistance in distinct molecular subtypes of melanoma.

Therapy of BRAF-mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF-mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS- and NF1-mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Niraparib Suppresses Cholangiocarcinoma Tumor Growth by Inducing Oxidative and Replication Stress.

Cholangiocarcinoma (CCA) is the second most common hepatobiliary cancer, an aggressive malignancy with limited therapeutic options. PARP (poly (ADP-ribose) polymerase) 1 and 2 are important for deoxyribonucleotide acid (DNA) repair and maintenance of genomic stability. PARP inhibitors (PARPi) such as niraparib have been approved for different malignancies with genomic alteration in germline BRCA and DNA damage response (DDR) pathway genes. Genomic alterations were analyzed in DDR genes in CCA samples employing The Cancer Genome Atlas (TCGA) database. Mutations were observed in various DDR genes, and 35.8% cases had alterations in at least one of three genes (ARID1A, BAP1 and ATM), suggesting their susceptibility to PARPi. Niraparib treatment suppressed cancer cell viability and survival, and also caused G2/M cell cycle arrest in patient-derived xenograft cells lines (PDXC) and established CCA cells harboring DDR gene mutations. PARPi treatment also induced apoptosis and caspase3/7 activity in PDXC and CCA cell lines, and substantially reduced expression of BCL2, BCL-XL and MCL1 proteins. Niraparib caused a significant increase in oxidative stress, and induced activation of DNA damage markers, phosphorylation of CHK2 and replication fork stalling. Importantly, niraparib, in combination with gemcitabine, produced sustained and robust inhibition of tumor growth in vivo in a patient-derived xenograft (PDX) model more effectively than either treatment alone. Furthermore, tissue samples from mice treated with niraparib and gemcitabine display significantly lower expression levels of pHH3 and Ki-67, which are a mitotic and proliferative marker, respectively. Taken together, our results indicate niraparib as a novel therapeutic agent alone or in combination with gemcitabine for CCA.