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Background/Aim: Although CDK4/6 inhibitors have been increasingly used in combination with hormonal agents to treat hormone-receptor positive and human epithelial growth factor receptor 2-negative breast cancer, the mechanism of CDK4/6 inhibitor resistance and its impact on established therapy for post-resistance, especially bevacizumab combined with chemotherapy, are unclear. Materials and Methods: Sensitivity of RB knockout MCF7 clones to CDK4/6 inhibitors was evaluated in vitro. One RB knockout clone was subcutaneously implanted in nude mice and the effects of bevacizumab on volume and microvessel density (MVD) of tumors were investigated. Results: Palbociclib did not exhibit antitumor efficacy against the RB knockout tumor, in contrast to the parental MCF7 xenograft model. Bevacizumab significantly exhibited antitumor efficacy and suppressed the MVD both in RB knockout and parental MCF7 xenograft models. Conclusion: Bevacizumab inhibited tumor growth by suppressing MVD in the CDK4/6 inhibitor-resistant tumor acquired due to RB loss, suggesting its efficacy also in patients after treatment with CDK4/6 inhibitors.

The efficacy of programmed cell death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) blockade therapy has been demonstrated but is limited in patients with PD-L1low or immune desert tumors. This limitation can be overcome by combination therapies that include anti-vascular endothelial growth factor (VEGF) therapy. Such combinations have been investigated in clinical trials for a number of cancer types; however, evidence on the mechanisms underlying their effects in these types of patients is still not sufficient. Therefore, the present study investigated the efficacy and effects on CD8+ T cell and C-X-C motif chemokine receptor 3 (CXCR3) ligand expression in tumors by combining anti-PD-L1 and anti-VEGF antibodies using an OV2944-HM-1 mouse model with PD-L1low and immune desert-like phenotypes. Although the model exhibited anti-PD-L1 insensitivity, anti-PD-L1 antibody treatment combined with anti-VEGF antibody inhibited tumor growth compared with anti-VEGF monotherapy, which itself inhibited tumor growth compared with the control treatment on Day 25. In combination-treated mice, a higher percentage of CD8+ T cells and higher levels of CXCR3 ligands were observed in tumor tissues compared with those in the anti-VEGF antibody treatment group, which was not significantly different from control treatment on Day 8. The increase in the intratumoral percentage of CD8+ T cells following the combination treatment was reversed by CXCR3 blocking to the same level as the control. In an anti-PD-L1 insensitive model with PD-L1low and immune desert-like phenotypes, although anti-PD-L1 antibody alone was not effective, anti-PD-L1 antibody in combination with anti-VEGF antibody exhibited antitumor combination efficacy with an increase of CD8+ T cell infiltration, which was suggested to be dependent on the increase of intratumoral CXCR3 ligands. This mechanism could explain the efficacy of anti-PD-L1 antibody and anti-VEGF antibody combination therapy in the clinical setting.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.

Background Bevacizumab in combination with chemotherapeutics has shown significant survival benefit in clinical studies in patients with non-small cell lung cancer (NSCLC). Since bevacizumab was administered as standard treatment until disease progression, the importance of bevacizumab during the maintenance phase was not prospectively investigated in these studies. Materials and methods Three human NSCLC cell line xenograft models were used to investigate antitumor effect of bevacizumab and tumor microvessel density (MVD) was analyzed. Results In A549 and NCI-H292 models, bevacizumab maintenance following combination with paclitaxel exhibited stronger tumor suppression than its absence. In an NCI-H292 model, bevacizumab maintenance continuously inhibited increase of MVD. In an NCI-H2228 model following induction treatment with pemetrexed and bevacizumab, maintenance with pemetrexed plus bevacizumab, had stronger efficacy than pemetrexed alone and led to lower MVD and level of thymidylate synthase. Conclusion Continuous suppression of angiogenesis by bevacizumab may contribute to the superior efficacy of maintenance treatment containing bevacizumab.

Although bevacizumab maintenance following bevacizumab in combination with chemotherapy has demonstrated significant prolongation of progression-free survival in clinical studies in patients with ovarian cancer, the majority of the cancer cases in the study were of the serous histotype; therefore, data regarding clear cell carcinoma is limited. Furthermore, the efficacy of bevacizumab beyond progression has not yet been demonstrated in ovarian cancer. A xenograft model using the human ovarian clear cell carcinoma cell line RMG-I was used to investigate the antitumor effects and the mechanisms of bevacizumab in maintenance treatment and bevacizumab when administered beyond disease progression. In the RMG-I model, bevacizumab maintenance following bevacizumab in combination with paclitaxel exhibited increased tumor suppression, compared with its absence, and inhibited the increase of microvessel density (MVD) in tumors. Following disease progression during bevacizumab maintenance, continued bevacizumab treatment in combination with PEGylated liposomal doxorubicin as a secondary chemotherapeutic agent had increased efficacy, compared with PEGylated liposomal doxorubicin alone, and resulted in lower MVD accompanied with lower levels of insulin-like growth factor binding protein-3, which is reported to have angiogenic activity. Continuous suppression of angiogenesis by bevacizumab may contribute to the superior efficacy of bevacizumab maintenance and bevacizumab beyond progression in ovarian cancer.

Resistance of prostate cancer to castration is currently an unavoidable problem. The major mechanisms underlying such resistance are androgen receptor (AR) overexpression, androgen-independent activation of AR, and AR mutation. To address this problem, we developed an AR pure antagonist, CH5137291, with AR nuclear translocation-inhibiting activity, and compared its activity and characteristics with that of bicalutamide. Cell lines corresponding to the mechanisms of castration resistance were used: LNCaP-BC2 having AR overexpression and LNCaP-CS10 having androgen-independent AR activation. VCaP and LNCaP were used as hormone-sensitive prostate cancer cells. In vitro functional assay clearly showed that CH5137291 inhibited the nuclear translocation of wild-type ARs as well as W741C- and T877A-mutant ARs. In addition, it acted as a pure antagonist on the transcriptional activity of these types of ARs. In contrast, bicalutamide did not inhibit the nuclear translocation of these ARs, and showed a partial/full agonistic effect on the transcriptional activity. CH5137291 inhibited cell growth more strongly than bicalutamide in VCaP and LNCaP cells as well as in LNCaP-BC2 and LNCaP-CS10 cells in vitro. In xenograft models, CH5137291 strongly inhibited the tumor growth of LNCaP, LNCaP-BC2, and LNCaP-CS10, whereas bicalutamide showed a weaker effect in LNCaP and almost no effect in LNCaP-BC2 and LNCaP-CS10 xenografts. Levels of prostate-specific antigen (PSA) in plasma correlated well with the antitumor effect of both agents. CH5137291 inhibited the growth of LNCaP tumors that had become resistant to bicalutamide treatment. A docking model suggested that CH5137291 intensively collided with the M895 residue of helix 12, and therefore strongly inhibited the folding of helix 12, a cause of AR agonist activity, in wild-type and W741C-mutant ARs. In cynomolgus monkeys, the serum concentration of CH5137291 increased dose-dependently and PSA level decreased 80% at 100 mg/kg. CH5137291 is expected to offer a novel therapeutic approach against major types of castration-resistant prostate cancers.

Background: Efficacy of combination therapy with atezolizumab plus bevacizumab was shown in the IMpower150 trial. In this study, we use the anti-PD-L1 sensitive Colon 38 mouse tumor model and the anti-PD-L1 insensitive OV2944-HM-1 (HM-1) mouse tumor model to investigate the efficacy and the mechanisms of this combination. Methods: Colon 38 and HM-1 tumor cells were subcutaneously inoculated into the right flank of C57BL/6J and B6C3F1 mice, respectively. Mice with established tumors were randomized into treatment groups (day1). Anti-PD-L1 mAb (anti-PD-L1, clone 6E11) or mouse IgG was administrated intraperitoneally to the mice at a dose of 5 mg/kg twice a week from day 1. Anti-VEGF mAb (anti-VEGF, clone B20-4.1.1) or mouse IgG was administrated intraperitoneally at a dose of 10 mg/kg weekly from day 1. Anti-CD8 mAb (anti-CD8, clone 116-13.1) was intraperitoneally administered at a dose of 100 µg/head twice a week from day -10. Anti-CXCR3 mAb (anti-CXCR3, clone CXCR3-173) was intraperitoneally administered at a dose of 100 µg/head twice a week from day 1. Tumor volume was measured twice a week. The tumors were sampled on day 1 and were subjected to immunohistochemical staining using the anti-CD8 mAb (anti-CD8, clone KT15) and anti-B7-H1/PD-L1 polyclonal antibody. The tumors were sampled on day 4 or 8 and were subjected to flow cytometry analysis using the antibody for CD8 and Granzyme B, and ELISA analysis for CXCL9. Results: HM-1 tumors showed lower PD-L1 expression under conditions where Colon 38 tumors were partially PD-L1 positive. CD8+ T cells were hardly observed in HM-1 tumors, while CD8+ T cells were prominently observed in Colon 38 tumors. In the Colon 38 model, combination treatment with anti-PD-L1 plus anti-VEGF significantly inhibited the tumor growth compared with the single agent treatments which themselves significantly inhibited the tumor growth compared with control treatment. In the HM-1 model, combination treatment with anti-PD-L1 plus anti-VEGF significantly inhibited the tumor growth compared with the anti-VEGF treatment which itself significantly inhibited the tumor growth compared with control treatment. In the HM-l model, significantly larger number of CD8+ T cells and Granzyme B+CD8+ T cells and a significantly higher level of CXCL9 were observed in tumor tissue with anti-PD-L1 treatment compared with control treatment on day 4. However, these statuses were not maintained on day 8. On the other hand, these statuses were observed in tumor tissue with combination treatment both on day 4 and day 8. Anti-CD8 canceled the potentiation of the antitumor effect in combination treatment to the level of anti-VEGF treatment. Anti-CXCR3 canceled the increased number of intratumoral CD8+ T cells in combination treatment to the level of control treatment. Conclusions: This is the first study to show that the combination treatment of anti-PD-L1 plus anti-VEGF exhibits more potent anti-tumor activity compared with single agent treatments, not only in an anti-PD-L1 sensitive model but also in an anti-PD-L1 insensitive model with PD-L1 low and immune desert like properties. At least in the anti-PD-L1 insensitive model, this can be mainly explained by the maintained increase of intratumoral CXCL9 leading to the increased infiltration of activated effector CD8+ T cells into tumor tissue. Conflict of interest: The authors are employees of Chugai Pharmaceutical Co., Ltd. Citation Format: Nobuyuki Ishikura, Keigo Yorozu, Mitsue Kurasawa, Masamichi Sugimoto, Osamu Kondoh. Addition of anti-VEGF overcomes the anti-PD-L1 refractoriness in a OV2944-HM-1 tumor model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B046. doi:10.1158/1535-7163.TARG-19-B046

We designed and fabricated porous silicon-based broadband rugate filters by combining up to seven rugate structures. A near-infrared stop-band filter having a reflection band width of 1926 nm with no higher-order harmonics and very small sidelobes were realized. The suppression of higher-order harmonics and the reduction of sidelobes also allowed us to produce a high quality pass-band filter having three high transmittance regions consisting of broad stop-bands.

The 3-(4-cyano-3-trifluoromethylphenyl)-5,5-dimethylthiohydantoin derivatives which have carboxy-terminal side chains were synthesized and their agonistic/antagonistic activities against androgen receptor (AR) measured. Among them, compound 13b showed antagonistic activity (IC50=130 nM) with no agonistic activity even at 10000 nM. This compound exhibited significant metabolic stability and oral antiandrogenic activity (ED50=7 mg/kg).

Previous observations that vitamin D hormone induces the expression of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL), thereby stimulating osteoclastogenesis in vitro, led to the widespread belief that 1alpha,25-dihydroxyvitamin D3 [1a,25(OH)2D3] is a bone-resorbing hormone. Here, we show that alfacalcidol, a prodrug metabolized to 1alpha,25(OH)2D3, suppresses bone resorption at pharmacologic doses that maintain normocalcemia in an ovariectomized (OVX) mouse model of osteoporosis. Treatment of OVX mice with pharmacologic doses of alfacalcidol does not increase RANKL expression, whereas toxic doses that cause hypercalcemia markedly reduce the expression of RANKL. When bone marrow (BM) cells from OVX mice were cultured with sufficient amounts of macrophage colony-stimulating factor (M-CSF) and RANKL, osteoclastogenic activity was higher than in sham mice. Marrow cultures from alfacalcidol- or estrogen-treated OVX mice showed significantly less osteoclastogenic potential compared with those from vehicle-treated OVX mice, suggesting that the pool of osteoclast progenitors in the marrow of vitamin D-treated mice as well as estrogen-treated mice was decreased. Frequency analysis showed that the number of osteoclast progenitors in bone marrow was increased by OVX and decreased by in vivo treatment with alfacalcidol or estrogen. We conclude that the pharmacologic action of active vitamin D in vivo is to decrease the pool of osteoclast progenitors in BM, thereby inhibiting bone resorption. Because of its unusual activity of maintaining bone formation while suppressing bone resorption, in contrast to estrogens that depress both processes, vitamin D hormone and its bone-selective analogs may be useful for the management of osteoporosis.

Background: Prostate cancer resistant to total androgen depletion therapies is termed castration-resistant prostate cancer and novel therapeutic approaches are in demand. The mechanisms of castration resistance in prostate cancer are reported to be: 1) hypersensitivity to androgen derived from overexpression of androgen receptor (AR), 2) androgen-independent activation of AR, termed “outlaw”, and 3) loss of ligand specificity derived from mutation of AR. To address this demand, we screened and developed an AR antagonist without agonist activity, a so-called AR pure antagonist, CH5137291. Methods: We compared the inhibitory effects of CH5137291 with bicalutamide on the growth of hormone-sensitive and castration-resistant prostate cancer cells. To confirm the in vivo efficacy of CH5137291, we used xenograft models. The antagonist/agonist effect of CH5137291 on AR transcriptional activity was analyzed using reporter gene assays. To elucidate the mechanisms of CH5137291, we examined the effect of CH5137291 on the protein level of nuclear AR in LNCaP-CS10 and the effect of CH5137291 on the subcellular localization of AR in LNCaP cells. Finally, the exposure of CH5137291 and its effect on serum prostate-specific antigen (PSA) level were investigated in cynomolgus monkey. Results: CH5137291 showed in vitro inhibitory effects superior to bicalutamide in all models tested including hormone-sensitive VCaP, LNCaP and castration-resistant, LNCaP-BC2 (overexpression model) and LNCaP-CS10 (outlaw model). CH5137291 treatment (10, 100 mg/kg) inhibited the tumor growth to initial levels and PSA production to lower than initial levels in castration-resistant LuCaP35V, LNCaP-BC2 and LNCaP-CS10 xenograft models. In contrast, bicalutamide (10, 100 mg/kg) did not inhibit tumor growth or PSA production in any of the xenograft models. In addition, CH5137291 exhibited prolongation of the time to progression compared with bicalutamide in the LNCaP xenograft model. In vitro functional assays clarified that CH5137291 showed pure antagonist activity against wild type, bicalutamide-resistant type (W741C), and flutamide-resistant type (T877A) AR in transcription and inhibited the nuclear translocation of all types of AR tested. CH5137291 also exhibited a dose-dependent increase in serum concentration and inhibited PSA production in cynomolgus monkey. Conclusion: Our novel AR pure antagonist CH5137291 inhibited AR nuclear translocation and showed not only superior efficacy in prostate cancers with castration-resistance acquired from AR overexpression, mutation and outlaw mechanisms but also prolonged time to progression in hormone-sensitive prostate cancer compared with bicalutamide. CH5137291 is expected to contribute to novel therapeutic approaches against a broad spectrum of prostate cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A219.

Background: We hypothesized that the androgen receptor (AR) pure antagonists, which exhibit no agonist activities, would inhibit AR signaling completely and would be efficacious against castration-resistant prostate cancer (CRPC). Based on our understanding of estrogen receptor pure antagonists, we designed dihydrotestosterone (DHT) derivatives and nonsteroidal RU56187 derivatives that inhibit the folding of helix 12 of AR and screened AR pure antagonists. Although a non-steroidal derivative (CH4933468) showed improved metabolic stability compared with DHT derivatives, it metabolized into an extremely small amount of a dealkylated metabolite with strong agonist effect. Therefore, CH4933468 did not show antitumor activity against the CRPC xenograft model. Methods: For metabolic stability, we designed and synthesized sulfonamide-substituted aryl compounds without dealkylated agonist metabolites. The production of agonist metabolites was examined in rat, mouse, monkey and dog. The efficacy of our compounds was measured using an in vitro reporter assay, a cell growth assay and in vivo prostate cancer xenograft models. Results: As a result of our optimization, CH5137291 was discovered and found to be an orally-active androgen receptor pure antagonist without agonistic activities derived from dealkylated agonist metabolites in vivo in rat, mouse, monkey and dog. CH5137291 exhibited AR antagonistic activities of IC50 of 300 nM and no agonist activity even at 30,000 nM in the reporter gene assay. In LNCaP-BC2 cells (AR overexpression CRPC model), CH5137291 was more efficient than bicalutamide. CH5137291 completely inhibited cell growth, in contrast to the partial inhibition of bicalutamide. Furthermore, CH5137291 inhibited the tumor growth and PSA production in the LNCaP-BC2 xenograft model. CH5137291 also inhibited AR nuclear translocation in the presence of R1881. According to the binding models of CH5137291 and bicalutamide to wild type AR, the sulfonamide of CH5137291 directly collided with M895 and thus prevented the folding of helix 12 completely. On the other hand, bicalutamide did not collide with M895 from helix 12 and inhibited helix 12 folding indirectly through W741. Hence, CH5137291 might act as an AR pure antagonist, but bicalutamide apparently does not. Conclusions: CH5137291, a novel AR pure antagonist without agonistic activities derived from dealkylated agonist metabolites, has shown antitumor activities in both in vitro and in vivo CRPC models. Our experimental results for clinical candidate CH5137291 corroborate our hypothesis that pure antagonists inhibit the folding of helix 12 completely, leading to the complete inhibition of the transcription activity of AR. Based on the strong efficacy against CRPC xenograft model and the inhibition of AR nuclear translocation, CH5137291 may offer more benefit than bicalutamide. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A220.

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Rugate filters made of anisotropically nanostructured birefringent silicon have been fabricated and studied by polarization-resolved transmission measurements. Electrochemical etching of a (110) oriented Si wafer results in porous silicon layers which exhibit a strong in-plane birefringence. We demonstrate that a sinusoidal refractive index variation of birefringent porous silicon combined with index-matching layers and apodization results in a dichroic rugate filter having a stop-band dependent on the polarization direction of the incident light without higher-order harmonics and sidelobes. We also demonstrate that the combination of different dichroic rugate filters allow us to realize filters with more complex properties in a single preparation step.

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