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Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti–programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti–PD-1 Ab was examined in a P-glycoprotein–knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti–PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti–PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti–PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Legends for supplementary figures S1-S9

Supplementary Materials and Methods not included in the main manuscript

Eribulin is a microtubule dynamics inhibitor with tumor microenvironment modulation activity such as vascular remodeling activity. Here, we investigated antitumor and immunomodulatory activities of eribulin and its liposomal formulation (eribulin-LF) as monotherapies or in combination with anti–programmed death 1 (PD-1) Ab. The antitumor activity of eribulin or eribulin-LF as monotherapy or in combination with anti–PD-1 Ab was examined in a P-glycoprotein–knockout 4T1 model. Eribulin and eribulin-LF showed stronger antitumor activity in immunocompetent mice compared with immunodeficient mice, indicating that they have immunomodulatory activity that underlies its antitumor activity. Combination therapy of eribulin and eribulin-LF with anti–PD-1 Ab showed antitumor activity, and the combination activity of eribulin-LF with anti–PD-1 Ab was observed at a lower dose and longer interval of administration compared with that using eribulin. To examine the immunomodulatory activity of eribulin and eribulin-LF and its underlying mechanisms, we performed flow cytometry, IHC, and gene expression profiling. IHC and flow cytometry revealed that eribulin-LF increased microvessel density and intratumoral populations of cytotoxic T cells and natural killer cells rather than eribulin. Gene expression profiling demonstrated that eribulin-LF induces IFNγ signaling. Furthermore, IHC also showed that eribulin-LF increased infiltration of CD8-positive cells together with increased CD31-positive cells. Eribulin-LF also increased ICAM-1 expression, which is essential for lymphocyte adhesion to vascular endothelial cells. In conclusion, eribulin showed combination antitumor activity with anti–PD-1 Ab via immunomodulation due to its vascular remodeling activity, and the liposomal formulation showed improved antitumor activity over the standard formulation.

Antibody lists used in the flow cytometric analysis

Fig S1 shows isotype control has no antitumor activity. Fig S2 shows gating strategy for flow cytometric analysis of TILs. Fig S3 shows induction of ICAM-1 by eribulin. Fig S4 shows establishment of P-gp KO cell lines and ICD activity of eribulin. Fig S5 shows dose-dependent antitumor activity of eribulin-LF + anti-PD-1 Ab. Fig S6 shows PK analysis of eribulin and eribulin-LF in plasma and tumor. Fig S7 shows immunomodulatory activity of eribulin in different 3 regimens. Fig S8 shows TIL population changes not included in the main figures. Fig S9 shows activation of IFN signature by eribulin-LF.

Background: Aberrant activation of Wnt/β-catenin signaling plays an important role in both carcinogenesis and modulation of the tumor microenvironment. E7386, an investigational anti-cancer agent, inhibits the binding of β-catenin to its transcriptional co-activator, CREB binding protein (CBP), thereby modulating Wnt/β-catenin signaling. We previously reported that E7386 suppressed tumor growth in preclinical mouse models with aberrant activation of Wnt/β-catenin signaling. Moreover, the combination of E7386 plus lenvatinib, a multiple receptor tyrosine kinase inhibitor that mainly targets vascular endothelial growth factor receptors and fibroblast growth factor receptors, showed stronger antitumor and antiangiogenic activities than each treatment alone. Here, we investigated the molecular mechanism of the combination activities of E7386 plus lenvatinib. Methods: We examined antitumor activity of E7386 plus lenvatinib in multiple human tumor xenograft and mouse syngeneic models. Tumor samples underwent RNA sequencing (RNA-seq) followed by weighted gene correlation network analysis. Furthermore quantitative PCR was used to assess changes in the expression of some hypoxia-related genes. The protein interaction between β-catenin and HIF1 was analyzed by immunoprecipitation-western blotting. Results: RNA-seq analysis of the effects of E7386, lenvatinib, and E7386 plus lenvatinib on the tumor microenvironment in the Hepa1-6 hepatocellular carcinoma syngeneic tumor model in mice showed that lenvatinib suppressed the expression of angiogenesis-related genes and that E7386 plus lenvatinib enhanced this suppression. Although lenvatinib increased the expression of hypoxia-related genes, E7386 plus lenvatinib attenuated this phenomenon. According to these results, we hypothesized that E7386 inhibits the formation of the β-catenin/HIF-1 complex. To confirm the effect of E7386 on the hypoxic response, we conducted an in vitro quantitative PCR assay under hypoxic conditions in Hepa1-6 cells and the human hepatocellular carcinoma cell lines, HepG2 and Hep3B. In accordance with the RNA-seq result, E7386 significantly suppressed the expression levels of hypoxia-related genes in the HepG2 and Hep3B cells. Finally, data from immunoprecipitation-western blotting demonstrated that β-catenin formed a complex with HIF-1 and that E7386 clearly inhibited this complex formation in HepG2 cells. Conclusion: These results suggest that E7386 enhances angiogenesis inhibition when used in combination with lenvatinib and that E7386 suppresses the hypoxic response induced by angiogenesis inhibition. Further analysis is required to clarify the precise mechanisms of actions of the combination of E7386 plus lenvatinib. Citation Format: Mizuki Kimura, Yusaku Hori, Megumi Kuronishi, Takayuki Kimura, Ryoga Ishida, Kenji Ichikawa, Yoichi Ozawa, Yu Kato. E7386, a CREB binding protein (CBP)/β-catenin interaction inhibitor, suppresses the hypoxic response induced by angiogenesis inhibition in hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1437.

Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.