Introduction In a recent phase I/II clinical trial in hepatocellular carcinoma (HCC), (NCT02400788), the combination of HDAC-inhibitor resminostat (YHI1001) with a multi-kinase inhibitor sorafenib was clinically beneficial compared to sorafenib mono-treatment in patients with platelet counts ≥1.5 × 105/µl, but not below that threshold. In HCC, platelets have been linked to bad prognosis, tumour growth and metastasis as well as to resistance against the standard treatment with sorafenib. This begs the question how platelets affect HCC cells and modulate their drug response. Material and methods Several HCC cell lines were used to compare phenotypical features, susceptibility to platelets and drug-mediated effects. To determine effects on cancer cell features, in vitro cell growth and transwell invasion assays were performed. Furthermore, the underlying mechanism of a platelet-modulated drug response on the molecular level was explored. Results and discussions In HCC cells, the anti-proliferative potency of sorafenib was counteracted by platelet factors and the mesenchymal phenotype. However, resminostat alone and in combination with sorafenib effectively triggered an anti-proliferative response independently of platelets or the mesenchymal phenotype. Therefore, resminostat determined the anti-proliferative response of the drug combination. Moreover, recent reports highlight HCC cell subpopulations which express cancer stem cell genes and harbour clonogenic growth and cell invasive capacities as critical for metastasis. Intriguingly, we found that platelets induced the cell-invasive capacity in HCC cells with detectable levels of several cancer stem cell markers and which featured a mixed epithelial-mesenchymal phenotype. Importantly, only the combination of resminostat with sorafenib, but not the mono-treatments, significantly reversed the platelet-induced cell invasion. Conclusion Our pre-clinical data provide evidence on how platelets mediate pro-tumorigenic effects and modulate the therapeutic response to the resminostat/sorafenib drug combination. Platelet factors negatively modulated the drug response to sorafenib. This was overcome by the anti-proliferative activities of resminostat. Importantly, providing an explanation for the clinical benefit of the combination therapy, the platelet-induced invasive capacity was reversed only by the combination of resminostat with sorafenib, but not the mono-treatments.

Background: Various HDAC inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors have been investigated in syngeneic tumor models, the mode of anti-tumoral action is not yet fully understood. Here, we analyzed the anti-tumoral mode-of-action of 4SC-202, an orally available clinical stage epigenetic small molecule inhibitor targeting histone deacetylases (HDAC) class I. We used a clinically equivalent dosage regimen to ensure that the results would be relevant for the clinical situation.

Introduction: The optimal management of patients aged ≥ 65 years with newly diagnosed glioblastoma (GB) is evolving. In the UK, hypofractionated radiotherapy (HRT: 30 Gy in 6 fractions over 2 weeks) has been the standard of care for individuals aged >70 years with PS=0-1, any age with PS≥2, and/or multifocal tumours. Occasionally, patients aged >70 years with PS 0-1 and no medical comorbidities may be considered for standard radiotherapy (SRT - 60 Gy in 30 fractions over 6 weeks) with or without temozolomide (TMZ). Materials and methods: In this retrospective study we analysed the outcome of radiotherapy for patients aged ≥ 65 years with glioblastoma treated between 2009 and 2015. Survival was calculated the Kaplan -Meier method. Differences in patient- and disease-related factors between the HRT and SRT groups were compared using the Fisher exact test. The Cox-proportional hazard regression analysis was used to correlate these factors with survival. Results: Patients received HRT (n=45), SRT (n= 8) or SRT with TMZ (n=15). The median age was 71.5 years (range: 65 to 83 years). 30 patients were aged 65-70 years and 38 were >70 years. 35 patients underwent debulking surgery and the remaining had either a biopsy (n=29) or a radiological diagnosis (n=4). 21 patients had multifocal disease. Patients receiving HRT were significantly older (median age 75 years vs. 69 years, p=0.003), had worse PS (≥2) (p=0.000), and the majority had only a biopsy or radiological diagnosis (p=0.021), compared with those receiving SRT with or without temozolomide Patients who received SRT+/-TMZ had a better median overall (11 vs. 5 months; p=0.000) and progression-free survival (9 vs. 3 months, p=0.040) compared with patients who received HRT. The corresponding 1-year OS for both groups were 43.5% and 6.7% respectively. On univariate analysis, overall survival correlated with PS (HR=0.42, 95% CI:0.24-0.74), multifocality (HR=2.07, 95% CI 1.21-3.55), and age (HR=0.96, 95% CI: 0.99-1.09), but not with the extent of resection (HR=1.44, 95% CI 0.95-2.18). MGMT methylation status was not available for all patients and therefore, not analysed. CONCLUSION: SRT with or without TMZ may improve survival in selected patients aged ≥ 65 years with newly diagnosed glioblastoma. The survival of patients treated with SRT+/-TMZ in our cohort is better than that of patients treated with chemo-radiotherapy in the recently published EORTC 26062-22061 study. An individualised-approach should be favoured over a one-fits-all strategy in the management of elderly patients with glioblastoma.

e14090 Background: 4SC-207 is a novel small molecule with strong anti-mitotic activity. In this study the potency on in vitro cell proliferation, including chemotherapeutically resistant P-gp-expressing cells, was investigated. These observations were confirmed in in vivo xenograft tumor models. METHODS In vitro proliferation assay: 4SC-207 was tested on a panel of 50 ATCC cell lines. After a 24 h pre-growth period cells were incubated with 4SC-207 at different concentrations for 72 hours. After treatment cells were precipitated and stained with 0.4% wt/v sulforhodamine B solution in 1% acetic acid. Measurement of optical density was performed at 520 nm. Proliferation inhibition was determined as growth inhibition of 50% (GI50). In vivo xenograft model: 4SC-207 was tested both i.v. and p.o. in a xenograft NMRI mouse model using human colon adenocarcinoma cell line RKOp27. In the i.v. study 4SC-207 was administered at a dose of 40 mg/kg BID on days 1-7 and SID on days 8 and 11-14. In the p.o. study 4SC-207 was administered at a dose of 80 mg/kg SID on days 1, 2, 6, 7, 11, and 12. Endpoints consisted of tumor volume and body weight and hematologic parameters. RESULTS In vitro proliferation assay: 4SC-207 effectively inhibited the proliferation of most tested tumor cell lines with average GI50 values between 4 nM and 12 nM. 4SC-207 was also active on cell lines such as human colon cell lines HCT-15 and DLD-1 which display P-gp up-regulation and are known to be resistant to a large set of conventional anti-cancer agents (e.g. taxanes). In vivo xenograft model: 4SC-207 displayed a strong anti-tumor activity. Treatment with 4SC-207 induced complete tumor stasis (i.v.: Treated/Control = 0.09; p.o.: T/C = 0.1). Effects on body weight were mild and other signs of overt toxicity were not observed. CONCLUSIONS 4SC-207 is a highly potent, novel anti-mitotic compound with strong in vitro and in vivo anti-tumor activity. 4SC-207 is also active on P-gp expressing tumor cells and offers the opportunity for treatment of hematological and solid tumor types which are resistant to standard anti-cancer agents.

The biological activities of several previously synthesized [J. A. Montgomery et al., J. med. Chem.17, 1197 (1974)] adenine-substituted analogs of 5′-deoxy-5′-methylthio- or -deoxy-5′-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo[3,4-d]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5t-́deoxy-5′-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deflcient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5′-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5′-deoxy-5′-ethylthio-4-aminopyrazolo[3,4-d]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo[3,4-d]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.

A single amino acid substitution, from glutamic acid to lysine at position 627 of the PB2 protein, converts a nonlethal H5N1 influenza A virus isolated from a human to a lethal virus in mice. In contrast to the nonlethal virus, which replicates only in respiratory organs, the lethal isolate replicates in a variety of organs, producing systemic infection. Despite a clear difference in virulence and organ tropism between the two viruses, it remains unknown whether the dissimilarity is a result of differences in cell tropism or the reduced replicative ability of the nonlethal virus in mouse cells in general. To determine how this single amino acid change affects virulence and organ tropism in mice, we investigated the growth kinetics of the two H5N1 viruses both in vitro and in vivo. The identity of the PB2 amino acid at position 627 did not appreciably affect viral replicative efficiency in chicken embryo fibroblasts and a quail cell line; however, viruses with lysine at this position instead of glutamic acid grew better in the different mouse cells tested. When the effect of this substitution was investigated in mice, all of the test viruses showed the same cell tropism, but infection by viruses containing lysine at position 627 spread more rapidly than those viruses containing glutamic acid at this position. Further analysis showed a difference in local immune responses: neutrophil infiltration in lungs infected with viruses containing lysine at position 627 persisted longer than that associated with viruses lacking a glutamic acid substitution. Our data indicate that the amino acid at position 627 of the PB2 protein determines the efficiency of viral replication in mouse (not avian) cells, but not tropism among cells in different mouse organs. The presence of lysine leads to more aggressive viral replication, overwhelming the host's defense mechanisms and resulting in high mortality rates in mice.