Vepafestinib is a pharmacologically advanced RET-selective inhibitor with high CNS penetration and inhibitory activity against RET solvent front mutations.

RET receptor tyrosine kinase is activated in various cancers (lung, thyroid, colon and pancreatic, among others) through oncogenic fusions or gain-of-function single-nucleotide variants. Small-molecule RET kinase inhibitors became standard-of-care therapy for advanced malignancies driven by RET. The therapeutic benefit of RET inhibitors is limited, however, by acquired mutations in the drug target as well as brain metastasis, presumably due to inadequate brain penetration. Here, we perform preclinical characterization of vepafestinib (TAS0953/HM06), a next-generation RET inhibitor with a unique binding mode. We demonstrate that vepafestinib has best-in-class selectivity against RET, while exerting activity against commonly reported on-target resistance mutations (variants in RETL730, RETV804 and RETG810), and shows superior pharmacokinetic properties in the brain when compared to currently approved RET drugs. We further show that these properties translate into improved tumor control in an intracranial model of RET-driven cancer. Our results underscore the clinical potential of vepafestinib in treating RET-driven cancers.

Modulation of tumor immune microenvironment by TAS-115, a multi-receptor tyrosine kinase inhibitor, promotes antitumor immunity and contributes anti-PD-1 antibody therapy.

TAS-115 is an oral multi-receptor tyrosine kinase inhibitor that strongly inhibits kinases implicated in antitumor immunity, such as colony stimulating factor 1 receptor and vascular endothelial growth factor receptor. Because these kinases are associated with the modulation of immune pathways, we investigated the immunomodulatory activity of TAS-115. An in vitro cytokine assay revealed that TAS-115 upregulated interferon γ (IFNγ) and interleukin-2 secretion by T cells, suggesting that TAS-115 activated T cells. Gene expression analysis suggested that TAS-115 promoted M1 macrophage differentiation. In in vivo experiments, although TAS-115 exerted a moderate antitumor effect in the MC38 mouse colorectal cancer model under immunodeficient conditions, this effect was enhanced under immunocompetent conditions. Furthermore, combination of TAS-115 and anti-PD-1 antibody exhibited greater antitumor activity than either treatment alone. Flow cytometry analysis showed the increase in IFNγ- and granzyme B (Gzmb)-secreting tumor-infiltrating T cells by TAS-115 treatment. The combination treatment further increased the percentage of Gzmb+CD8+ T cells and decreased the percentage of macrophages compared with either treatment alone. These results highlight the potential therapeutic effect of TAS-115 in combination with PD-1 blockade, mediated via activation of antitumor immunity by TAS-115.

TAS4464, a NEDD8-activating enzyme inhibitor, activates both intrinsic and extrinsic apoptotic pathways via c-Myc-mediated regulation in acute myeloid leukemia.

TAS4464, a potent, selective small molecule NEDD8-activating enzyme (NAE) inhibitor, leads to inactivation of cullin-RING E3 ubiquitin ligases (CRLs) and consequent accumulations of its substrate proteins. Here, we investigated the antitumor properties and action mechanism of TAS4464 in acute myeloid leukemia (AML). TAS4464 induced apoptotic cell death in various AML cell lines. TAS4464 treatments resulted in the activation of both the caspase-9-mediated intrinsic apoptotic pathway and caspase-8-mediated extrinsic apoptotic pathway in AML cells; combined treatment with inhibitors of these caspases markedly diminished TAS4464-induced apoptosis. In each apoptotic pathway, TAS4464 induced the mRNA transcription of the intrinsic proapoptotic factor NOXA and decreased that of the extrinsic antiapoptotic factor c-FLIP. RNA-sequencing analysis showed that the signaling pathway of the CRL substrate c-Myc was enriched after TAS4464 treatment. Chromatin immunoprecipitation (ChIP) assay revealed that TAS4464-induced c-Myc bound to the PMAIP1 (encoding NOXA) and CFLAR (encoding c-FLIP) promoter regions, and siRNA-mediated c-Myc knockdown neutralized both TAS4464-mediated NOXA induction and c-FLIP downregulation. TAS4464 activated both caspase-8 and caspase-9 along with an increase in NOXA and a decrease in c-FLIP, resulting in complete tumor remission in a human AML xenograft model. These findings suggest that NAE inhibition leads to anti-AML activity via a novel c-Myc-dependent apoptosis induction mechanism.

Crucial roles of thymidine kinase 1 and deoxyUTPase in incorporating the antineoplastic nucleosides trifluridine and 2'-deoxy-5-fluorouridine into DNA.

Trifluridine (FTD) and 2'-deoxy-5-fluorouridine (FdUrd), a derivative of 5-fluorouracil (5-FU), are antitumor agents that inhibit thymidylate synthase activity and their nucleotides are incorporated into DNA. However, it is evident that several differences occur in the underlying antitumor mechanisms associated with these nucleoside analogues. Recently, TAS-102 (composed of FTD and tipiracil hydrochloride, TPI) was shown to prolong the survival of patients with colorectal cancer who received a median of 2 prior therapies, including 5-FU. TAS-102 was recently approved for clinical use in Japan. These data suggest that the antitumor activities of TAS-102 and 5-FU proceed via different mechanisms. Thus, we analyzed their properties in terms of thymidine salvage pathway utilization, involving membrane transporters, a nucleoside kinase, a nucleotide-dephosphorylating enzyme, and DNA polymerase α. FTD incorporated into DNA with higher efficiency than FdUrd did. Both FTD and FdUrd were transported into cells by ENT1 and ENT2 and were phosphorylated by thymidine kinase 1, which showed a higher catalytic activity for FTD than for FdUrd. deoxyUTPase (DUT) did not recognize dTTP and FTD-triphosphate (F3dTTP), whereas deoxyuridine-triphosphate (dUTP) and FdUrd-triphosphate (FdUTP) were efficiently degraded by DUT. DNA polymerase α incorporated both F3dTTP and FdUTP into DNA at sites aligned with adenine on the opposite strand. FTD-treated cells showed differing nuclear morphologies compared to FdUrd-treated cells. These findings indicate that FTD and FdUrd are incorporated into DNA with different efficiencies due to differences in the substrate specificities of TK1 and DUT, causing abundant FTD incorporation into DNA.

Efficacy of combination chemotherapy using a novel oral chemotherapeutic agent, TAS-102, together with bevacizumab, cetuximab, or panitumumab on human colorectal cancer xenografts.

TAS-102 is a novel oral nucleoside antitumor agent that consists of trifluridine (FTD) and tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5, and was approved in Japan in March 2014 for the treatment of patients with unresectable advanced or recurrent colorectal cancer that is refractory to standard therapies. In the present study, we used colorectal cancer xenografts to assess whether the efficacy of TAS-102 could be improved by combining it with bevacizumab, cetuximab or panitumumab. TAS-102 was orally administered twice a day from day 1 to 14, and bevacizumab, cetuximab and panitumumab were administered intraperitoneally twice a week for 2 weeks. Growth inhibitory activity was evaluated based on the relative tumor volume (RTV) after 2 weeks of drug administration and time taken for the relative tumor volume to increase five-fold (RTV5). Tumor growth inhibition and RTV5 with TAS-102 and bevacizumab combination treatment were significantly better than those with TAS-102 or bevacizumab alone in the SW48 and HCT116 tumor models, and the concentration of phosphorylated FTD in tumors determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was higher in the TAS-102 and bevacizumab combination group than in the TAS-102 monotherapy group. The combination of TAS-102 and cetuximab or panitumumab was also significantly more effective than either monotherapy in the SW48 tumor model. There was no significant difference in the body weight between the mice treated with TAS-102 monotherapy and any of the combination therapies on day 29. Our preclinical findings indicate that the combination therapy of TAS-102, bevacizumab and cetuximab or panitumumab is a promising treatment option for colorectal cancer.

Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models.

TAS-102 is a novel oral nucleoside antitumor agent containing trifluridine (FTD) and tipiracil hydrochloride (TPI). The compound improves overall survival of colorectal cancer (CRC) patients who are insensitive to standard chemotherapies. FTD possesses direct antitumor activity since it inhibits thymidylate synthase (TS) and is itself incorporated into DNA. However, the precise mechanisms underlying the incorporation into DNA and the inhibition of TS remain unclear. We found that FTD-dependent inhibition of TS was similar to that elicited by fluorodeoxyuridine (FdUrd), another clinically used nucleoside analog. However, washout experiments revealed that FTD-dependent inhibition of TS declined rapidly, whereas FdUrd activity persisted. The incorporation of FTD into DNA was significantly higher than that of other antitumor nucleosides. Additionally, orally administered FTD had increased antitumor activity and was incorporated into DNA more effectively than continuously infused FTD. When TAS-102 was administered, FTD gradually accumulated in tumor cell DNA, in a TPI-independent manner, and significantly delayed tumor growth and prolonged survival, compared to treatment with 5-FU derivatives. TAS-102 reduced the Ki-67-positive cell fraction, and swollen nuclei were observed in treated tumor tissue. The amount of FTD incorporation in DNA and the antitumor activity of TAS-102 in xenograft models were positively and significantly correlated. These results suggest that TAS-102 exerts its antitumor activity predominantly due to its DNA incorporation, rather than as a result of TS inhibition. The persistence of FTD in the DNA of tumor cells treated with TAS-102 may underlie its ability to prolong survival in cancer patients.