The epigenetic modifier HDAC2 and the checkpoint kinase ATM determine the responses of microsatellite instable colorectal cancer cells to 5-fluorouracil.

The epigenetic modifier histone deacetylase-2 (HDAC2) is frequently dysregulated in colon cancer cells. Microsatellite instability (MSI), an unfaithful replication of DNA at nucleotide repeats, occurs in about 15% of human colon tumors. MSI promotes a genetic frameshift and consequently a loss of HDAC2 in up to 43% of these tumors. We show that long-term and short-term cultures of colorectal cancers with MSI contain subpopulations of cells lacking HDAC2. These can be isolated as single cell-derived, proliferating populations. Xenografted patient-derived colon cancer tissues with MSI also show variable patterns of HDAC2 expression in mice. HDAC2-positive and HDAC2-negative RKO cells respond similarly to pharmacological inhibitors of the class I HDACs HDAC1/HDAC2/HDAC3. In contrast to this similarity, HDAC2-negative and HDAC2-positive RKO cells undergo differential cell cycle arrest and apoptosis induction in response to the frequently used chemotherapeutic 5-fluorouracil, which becomes incorporated into and damages RNA and DNA. 5-fluorouracil causes an enrichment of HDAC2-negative RKO cells in vitro and in a subset of primary colorectal tumors in mice. 5-fluorouracil induces the phosphorylation of KAP1, a target of the checkpoint kinase ataxia-telangiectasia mutated (ATM), stronger in HDAC2-negative cells than in their HDAC2-positive counterparts. Pharmacological inhibition of ATM sensitizes RKO cells to cytotoxic effects of 5-fluorouracil. These findings demonstrate that HDAC2 and ATM modulate the responses of colorectal cancer cells towards 5-FU.

Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3.

Acute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

Cytotoxic and Senolytic Effects of Methadone in Combination with Temozolomide in Glioblastoma Cells.

Methadone is an analgesic drug used for pain treatment and heroin substitution. Recently, methadone has been proposed to be useful also for cancer therapy, including glioblastoma multiforme (GBM), the most severe form of brain cancer, because experiments on cultured glioma cells treated with doxorubicin showed promising results. Doxorubicin, however, is not used first-line in GBM therapy. Therefore, we analyzed the cytotoxic effect of methadone alone and in combination with temozolomide, a DNA-alkylating drug that is first-line used in GBM treatment, utilizing GBM-derived cell lines and a human fibroblast cell line. We show that methadone is cytotoxic on its own, inducing apoptosis and necrosis, which was observed at a concentration above 20 µg/mL. Methadone was similar toxic in isogenic MGMT expressing and non-expressing cells, and in LN229 glioblastoma and VH10T human fibroblasts. The apoptosis-inducing activity of methadone is not bound on the opioid receptor (OR), since naloxone, a competitive inhibitor of OR, did not attenuate methadone-induced apoptosis/necrosis. Administrating methadone and temozolomide together, temozolomide had no impact on methadone-induced apoptosis (which occurred 3 days after treatment), while temozolomide-induced apoptosis (which occurred 5 days after treatment) was unaffected at low (non-toxic) methadone concentration (5 µg/mL), and at high (toxic) methadone concentration (20 µg/mL) the cytotoxic effects of methadone and temozolomide were additive. Methadone is not genotoxic, as revealed by comet and γH2AX assay, and did not ameliorate the genotoxic effect of temozolomide. Further, methadone did not induce cellular senescence and had no effect on temozolomide-induced senescence. Although methadone was toxic on senescent cells, it cannot be considered a senolytic drug since cytotoxicity was not specific for senescent cells. Finally, we show that methadone had no impact on the MGMT promoter methylation. Overall, the data show that methadone on glioblastoma cells in vitro is cytotoxic and induces apoptosis/necrosis at doses that are above the level that can be achieved in vivo. It is not genotoxic, and does not ameliorate the cell killing or the senescence-inducing effect of temozolomide (no synergistic effect), indicating it has no impact on temozolomide-induced signaling pathways. The data do not support the notion that concomitant methadone treatment supports temozolomide-based chemotherapy.

Strong apoptotic response of testis tumor cells following cisplatin treatment.

Most solid metastatic cancers are resistant to chemotherapy. However, metastatic testicular germ cell tumors (TGCT) are cured in over 80% of patients using cisplatin-based combination therapy. Published data suggest that TGCTs are sensitive to cisplatin due to limited DNA repair and presumably also to a propensity to undergo apoptosis. To further investigate this aspect, cisplatin-induced activation of apoptotic pathways was investigated in cisplatin-sensitive testis tumor cells (TTC) and compared to cisplatin-resistant bladder cancer cells. Apoptosis induction was investigated using flow cytometry, caspase activation and PARP-1 cleavage. Immunoblotting and RT-PCR were applied to investigate pro- and anti-apoptotic proteins. Transfections were performed to target p53- and Fas/FasL-mediated apoptotic signaling. Immunoblotting experiments revealed p53 to be induced in TTC, but not bladder cancer cells following cisplatin. Higher levels of pro-apoptotic Bax and Noxa were observed in TTC, anti-apoptotic Bcl-2 was solely expressed in bladder cancer cells. Cisplatin led to translocation of Bax to the mitochondrial membrane in TTC, resulting in cytochrome C release. Cisplatin increased the expression of FasR mRNA and FasL protein in all tumor cell lines. Targeting the apoptotic pathway via siRNA-mediated knockdown of p53 and FAS reduced death receptor-mediated apoptosis and increased cisplatin resistance in TTC, indicating the involvement of FAS-mediated apoptosis in the cisplatin TTC response. In conclusion, both the death receptor and the mitochondrial apoptotic pathway become strongly activated in TTC following cisplatin treatment, explaining, together with attenuated DNA repair, their unique sensitivity toward platinum-based anticancer drugs.

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells.

INTRODUCTION: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers. OBJECTIVES: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells. METHODS: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells. RESULTS: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62. CONCLUSION: These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.

Mechanistic insights into p53-regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells.

Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.

Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression.

BACKGROUND: Cisplatin based chemotherapy cures over 80% of metastatic testicular germ cell tumours (TGCT). In contrast, almost all other solid cancers in adults are incurable once they have spread beyond the primary site. Cell lines derived from TGCTs are hypersensitive to cisplatin reflecting the clinical response. Earlier findings suggested that a reduced repair capacity might contribute to the cisplatin hypersensitivity of testis tumour cells (TTC), but the critical DNA damage has not been defined. This study was aimed at investigating the formation and repair of intrastrand and interstrand crosslinks (ICLs) induced by cisplatin in TTC and their contribution to TTC hypersensitivity. RESULTS: We observed that repair of intrastrand crosslinks is similar in cisplatin sensitive TTC and resistant bladder cancer cells, whereas repair of ICLs was significantly reduced in TTC. γH2AX formation, which serves as a marker of DNA breaks formed in response to ICLs, persisted in cisplatin-treated TTC and correlated with sustained phosphorylation of Chk2 and enhanced PARP-1 cleavage. Expression of the nucleotide excision repair factor ERCC1-XPF, which is implicated in the processing of ICLs, is reduced in TTC. To analyse the causal role of ERCC1-XPF for ICL repair and cisplatin sensitivity, we over-expressed ERCC1-XPF in TTC by transient transfection. Over-expression increased ICL repair and rendered TTC more resistant to cisplatin, which suggests that ERCC1-XPF is rate-limiting for repair of ICLs resulting in the observed cisplatin hypersensitivity of TTC. CONCLUSION: Our data indicate for the first time that the exceptional sensitivity of TTC and, therefore, very likely the curability of TGCT rests on their limited ICL repair due to low level of expression of ERCC1-XPF.

Synthesis of 131I-labeled glucose-conjugated inhibitors of O6-methylguanine-DNA methyltransferase (MGMT) and comparison with nonconjugated inhibitors as potential tools for in vivo MGMT imaging.

O(6)-Substituted guanine derivatives are powerful agents used for tumor cell sensitization by inhibition of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). To provide targeted accumulation of MGMT inhibitors in tumor tissue as well as tools for in vivo imaging, we synthesized iodinated C(8)-alkyl-linked glucose conjugates of 2-amino-6-(5-iodothenyl)-9H-purine (O(6)-(5-iodothenyl) guanine, ITG) and 2-amino-6-(3-iodobenzyloxy)-9H-purine (O(6)-(5-iodobenzyl) guanine, IBG). These compounds have MGMT inhibitor constants (IC(50) values) of 0.8 and 0.45 microM for ITGG and IBGG, respectively, as determined in HeLa S3 cells after 2-h incubation with inhibitor. To substantiate that the (131)I-(hetero)arylmethylene group at the O(6)-position of guanine is transferred to MGMT, both the glucose conjugated inhibitors ITGG and IBGG and the corresponding nonglucose conjugated compounds ITG and IBG were labeled with iodine-131. The radioiodinations of all compounds with [(131)I]I(-) were performed with radiochemical yields of >70% for the destannylation of the corresponding tri-n-butylstannylated precursors. The binding ability of [(131)I]ITGG, [(131)]IBGG, [(131)I]ITG, and [(131)I]IBG to purified MGMT was tested. All radioactive compounds were substrates for MGMT, as demonstrated using a competitive repair assay. The newly synthesized radioactive inhibitors were utilized to study ex vivo biodistribution in mice, and the tumor-to-blood ratio of tissue uptake of [(131)I]IBG and [(131)I]IBGG was determined to be 0.24 and 0.76 after 0.5 h, respectively.

Targeting Homologous Recombination by Pharmacological Inhibitors Enhances the Killing Response of Glioblastoma Cells Treated with Alkylating Drugs.

Malignant gliomas exhibit a high level of intrinsic and acquired drug resistance and have a dismal prognosis. First- and second-line therapeutics for glioblastomas are alkylating agents, including the chloroethylating nitrosoureas (CNU) lomustine, nimustine, fotemustine, and carmustine. These agents target the tumor DNA, forming O6-chloroethylguanine adducts and secondary DNA interstrand cross-links (ICL). These cross-links are supposed to be converted into DNA double-strand breaks, which trigger cell death pathways. Here, we show that lomustine (CCNU) with moderately toxic doses induces ICLs in glioblastoma cells, inhibits DNA replication fork movement, and provokes the formation of DSBs and chromosomal aberrations. Since homologous recombination (HR) is involved in the repair of DSBs formed in response to CNUs, we elucidated whether pharmacologic inhibitors of HR might have impact on these endpoints and enhance the killing effect. We show that the Rad51 inhibitors RI-1 and B02 greatly ameliorate DSBs, chromosomal changes, and the level of apoptosis and necrosis. We also show that an inhibitor of MRE11, mirin, which blocks the formation of the MRN complex and thus the recognition of DSBs, has a sensitizing effect on these endpoints as well. In a glioma xenograft model, the Rad51 inhibitor RI-1 clearly enhanced the effect of CCNU on tumor growth. The data suggest that pharmacologic inhibition of HR, for example by RI-1, is a reasonable strategy for enhancing the anticancer effect of CNUs. Mol Cancer Ther; 15(11); 2665-78. ©2016 AACR.

Inhibition of O6-methylguanine-DNA methyltransferase by glucose-conjugated inhibitors: comparison with nonconjugated inhibitors and effect on fotemustine and temozolomide-induced cell death.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is an important suicide enzyme involved in the defense against O(6)-alkylating mutagens. It also plays a role in the resistance of tumors to anticancer drugs targeting the O(6)-position of guanine, such as temozolomide and fotemustine. Several potent MGMT inhibitors have been developed sensitizing cells to O(6)-alkylating agents. Aimed at targeting MGMT inhibitors to tumor cells, we synthesized MGMT inhibitory compounds conjugated with glucose to improve uptake in tumor cells. Here, we compared O(6)-benzylguanine, O(6)-2-fluoropyridinylmethylguanine (O(6)FPG), O(6)-3-iodobenzylguanine, O(6)-4-bromothenylguanine, and O(6)-5-iodothenylguanine with the corresponding C8-linker beta-d-glucose derivatives. All glucose conjugated inhibitors were 3- to 5-fold less effective than the corresponding nonconjugated drugs as to MGMT inhibition that was measured in cell extracts (in vitro) and cultivated HeLaS3 cells (in vivo). Except for O(6)FPG, IC(50) values of the guanine derivatives applied in vitro and in vivo were correlated. A similar correlation was not obvious for the corresponding glucosides, indicating differences in cellular uptake. C8-alpha-d-glucosides were less effective than beta-glucosides. From the newly developed glucose-conjugated inhibitors tested, O(6)-4-bromothenylguanine-C8-beta-d-glucoside (O(6)BTG-C8-betaGlu) was most potent in inhibiting MGMT both in vitro and in vivo. At a concentration of 0.1 microM, it inhibited cellular MGMT to completion. It was not toxic, even when applied chronically to cells at high dose (up to 20 microM). O(6)BTG-C8-betaGlu strongly potentiated the killing effect of fotemustine and temozolomide, causing reversal from MGMT+ to MGMT- phenotype. Therefore, O(6)BTG-C8-betaGlu seems to be especially suitable for approaching MGMT inhibitor targeting in tumor therapy.