Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a particularly dismal prognosis. Histone deacetylases (HDAC) are epigenetic modulators whose activity is frequently deregulated in various cancers including PDAC. In particular, class-I HDACs (HDAC 1, 2, 3 and 8) have been shown to play an important role in PDAC. In this study, we investigated the effects of the class I-specific HDAC inhibitor (HDACi) 4SC-202 in multiple PDAC cell lines in promoting tumor cell differentiation. We show that 4SC-202 negatively affects TGFβ signaling and inhibits TGFβ-induced epithelial-to-mesenchymal transition (EMT). Moreover, 4SC-202 markedly induced p21 (CDKN1A) expression and significantly attenuated cell proliferation. Mechanistically, genome-wide studies revealed that 4SC-202-induced genes were enriched for Bromodomain-containing Protein-4 (BRD4) and MYC occupancy. BRD4, a well-characterized acetyllysine reader, has been shown to play a major role in regulating transcription of selected subsets of genes. Importantly, BRD4 and MYC are essential for the expression of a subgroup of genes induced by class-I HDACi. Taken together, our study uncovers a previously unknown role of BRD4 and MYC in eliciting the HDACi-mediated induction of a subset of genes and provides molecular insight into the mechanisms of HDACi action in PDAC.

Background: The efficacy of PD-(L)1 blockade depends on the composition of the tumor immune microenvironment (TIME) and is generally higher in tumors with pre-existing cytotoxic T cells (CTL) than in those with low CTL numbers. Nonetheless, a significant proportion of patients with pre-existing immunity fail to respond, indicating a therapeutic potential for combining PD-(L)1 blockade with additional immunomodulatory agents in both CTL-high and-low immune phenotypes. Here, we evaluated domatinostat (4SC-202), a class I-selective histone deacetylase (HDAC) inhibitor, for its effect on the TIME and its antitumoral efficacy using syngeneic mouse models with CTL-high or CTL-low tumors. Methods: Domatinostat was evaluated in PD-1 blockade-insensitive CTL-low (CT26) and CTL-high (C38) syngeneic models alone and in combination with different immune-inhibitory and-stimulatory approaches. Effects on the immunophenotype were assessed via flow cytometry and RNA-seq analyses. The changes in RNA-seq-based immune signatures determined in a murine setting were investigated in patient samples from the first-dose cohort of the SENSITIZE trial (NCT03278665) evaluating domatinostat combined with pembrolizumab in advanced-stage melanoma patients refractory/nonresponding to PD-1 blockade. Results: Domatinostat increased the expression of antigen-presenting machinery (APM) genes and MHC class I and II molecules, along with CTL infiltration, in tumors of both immune phenotypes. In combination with PD-(L)1 blockade, domatinostat augmented antitumor effects substantially above the effects of single-agent therapies, displaying greater benefit in tumors with pre-existing CTLs. In this setting, the combination of domatinostat with agonistic anti-4-1BB or both PD-1 and LAG3 blockade further increased the antitumor efficacy. In CTL-low tumors, domatinostat enhanced the expression of genes known to reinforce immune responses against tumors. Specifically, domatinostat increased the expression of Ifng and genes associated with responses to pembrolizumab and nivolumab. Clinically, these findings were confirmed in patients with advanced melanoma treated with domatinostat for 14 days, who demonstrated elevated expression of APM and MHC genes, the IFNG gene, and the IFN-γ and pembrolizumab response signatures in individual tumor samples. Conclusion: In summary, these data suggest a promising potential of domatinostat in combination with immunotherapy to improve the outcome of refractory cancer patients.

Oncolytic viruses (OV) constitute highly promising innovative biological anticancer agents. However, like every other antitumoral compound, OV are also faced with both primary and secondary mechanisms of resistance. To overcome those barriers and moreover amplify the therapeutic potential of OV, we evaluated a novel combined approach composed of the oral histone deacetylase inhibitor resminostat and an oncolytic measles vaccine virus (MeV) for a future epi‑virotherapy of pancreatic ductal adenocarcinoma. Cytotoxicity assays revealed that combined epi-virotherapeutic treatment of four well-characterized human pancreatic cancer cell lines resulted in a beneficial tumor cell killing as compared to either monotherapeutic approach. Notably, epi-virotherapeutic treatment of MIA PaCa-2 and partly also of PANC‑1 pancreatic cancer cells resulted in a tumor cell mass reduction being significantly more pronounced than it would be expected in case of an additive effect only, indicating a synergistic mode of action when combining resminostat with MeV. We further found that the epigenetic compound resminostat neither impaired MeV growth kinetics nor prevented the activation of the interferon signaling pathway which plays an important role in mediating primary and secondary resistances to OV. Moreover, we yielded information that the pharma-codynamic function of resminostat was presumably not altered in the course of pancreatic cancer cell infections with MeV. Taken together, these promising results favor the onset of epi-viro-thera-peutic clinical trials in patients suffering from advanced pancreatic ductal adenocarcinoma.

In hepatocellular carcinoma (HCC), blood platelets have been linked to tumor growth, epithelial-to-mesenchymal transition (EMT), extrahepatic metastasis and a limited therapeutic response to the multikinase inhibitor (MKi) sorafenib, the standard of care in advanced HCC for the last decade. Recent clinical data indicated an improved overall survival for sorafenib in combination with the HDAC inhibitor resminostat in a platelet count dependent manner. Here, the impact of platelets on the sorafenib and resminostat drug effects in HCC cells was explored. In contrast to sorafenib, resminostat triggered an anti-proliferative response in HCC cell lines independent of platelets. As previously described, platelets induced invasive capabilities of HCC cells, a prerequisite for extravasation and metastasis. Importantly, the resminostat/sorafenib drug combination, but not the individual drugs, effectively blocked platelet-induced HCC cell invasion. Exploration of the molecular mechanism revealed that the combined drug action led to a reduction of platelet-induced CD44 expression and to the deregulation of several other epithelial and mesenchymal genes, suggesting interference with cell invasion via EMT. In addition, the drug combination decreased phosphorylated ERK level, indicating inhibition of the mitogenic signaling pathway MEK/ERK. Taken together, the resminostat plus sorafenib combination counteracts platelet-mediated cancer promoting effects in HCC cells.

Various histone deacetylases (HDAC) inhibitors were described as beneficially affecting anti-tumoral immune response. Although different HDAC inhibitors were investigated in syngeneic tumor models, their mode of anti-tumoral action (MOA) is not yet fully understood. Here, we analyzed the anti-tumoral MOA of 4SC-202, an orally available clinical stage small molecule inhibitor targeting HDAC class I. To ensure the relevance for the clinical situation we used a clinically equivalent dosage regimen. 4SC-202's effect on expression of tumor-associated antigen (TAA) and MHC molecules was analyzed in vitro and in vivo. Anti-tumoral efficacy and the impact on tumor microenvironment (TME) were analyzed in syngeneic CT26 and C38 models. Transcriptome analysis was performed by RNA-Seq, and the composition of immune cell subpopulations was determined by flow cytometry and immunohistochemistry. 4SC-202 increased expression of TAA and MHC molecules on tumor cells in vitro and in vivo suggesting a beneficial effect on immunogenicity of tumor cells. 4SC-202 significantly inhibited growth of syngeneic tumors at a dose that was inefficacious in immunocompromised mice. IFN-γ and chemokine expression was increased, and pro-inflammatory IL-1β and IL-23 decreased in the TME of CT26 tumors following 4SC-202 treatment. Detailed analysis revealed that 4SC-202 increased the number of cytotoxic CD8+ T cells (CTLs) in the tumor core without affecting their number in blood. Since the abundance of T cells in the tumor is pre-requisite for the efficacy of immune checkpoint blockade as well as the agonistic 4-1BB antibody, combinations of 4SC-202 with anti-PD-1, and anti-4-1BB antibodies were tested in the C38 model. The response rate to the antibodies alone was low in this model reflecting the refractory clinical situation. 4SC-202 was able to control the tumor growth, but did not induce tumor regression, whereas combination therapies resulted in significantly longer survival and durable complete responses in up to 83% of animals for the combination with the anti-PD-1 antibody. 4SC-202 already demonstrated a favorable safety profile with a low rate of > grade 3 treatment-related adverse events (17%) in a phase I study in heavily pretreated hematological cancer patients. With the daily dosing for 14 consecutive days a disease control rate of 83%, 1 partial and 1 complete response could be achieved. Complimentary to these clinical data, 4SC-202's immune priming capacity offers further options for development in combination with various cancer immunotherapy approaches. Combination of 4SC-202 with PD-1 blockade is now under evaluation in a phase Ib/II clinical trial in advanced cutaneous melanoma patients refractory/non-responding to treatment with anti-PD-1 antibodies (‘SENSITIZE', NCT03278665). Citation Format: Svetlana Hamm, Tanja Wulff, Kerstin Kronthaler, Sabine Schrepfer, Ulrike Parnitzke, Anne Catherine Bretz, René Bartz. 4SC-202 increases immunogenicity of tumor cells, induces infiltration of tumor microenvironment with cytotoxic T cells, and primes tumors for combinations with different cancer immunotherapy approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4722.