Mari Masuda

Pimitespib–induced reduction in FOXP3 expression depends on STAT5 degradation in MJ cells. (A). Immunoprecipitation of HSP90 and STAT5. MJ cell lysates were incubated with normal rabbit IgG, anti-HSP90 mAb, or anti-STAT5 mAb, and immunocomplexes were purified, separated, and blotted. (B, C). The effect of STAT5B knockdown on FOXP3 expression. siRNA was used for knockdown of STAT5B (B). MJ cells after knockdown of STAT5B were cultured with the indicated concentrations of pimitespib for 48 hours. FOXP3 expression was examined in the MJ cells (C). (D). MJ cells were mock transduced or transduced with STAT5B to develop an MJ cells with STAT5b overexpression (OE). MJ cells with or without STAT5b overexpression were cultured in the presence or absence of pimitespib (1 μM) for 48 hours, and the mean fluorescence intensity (MFI) of FOXP3 was evaluated. (E). MJ cells were cultured with the indicated concentrations of pimitespib for 48 hours. Protein expression of the TCR-related signaling molecules LCK was examined. GAPDH was used as the internal control. All in vivo experiments were performed at least twice. **, P < 0.01; *, P < 0.05; **, P < 0.01; n.s., not significant.

Pimitespib treatment does not significantly alter the infiltration of myeloid cell populations in gastric cancers. (A, B). Representative multiple IHC images of T cell subsets (A), and myeloid subsets (B) before pimitespib treatment with or without nivolumab. (C). A summary of the frequencies of CD14+ cells (monocyte/macrophage), HLA-DR+ cells, CD14+CD206+ cells, and CD14+HLA-DR+ cells. The data are presented as a total of six fields per biopsy specimen from five patients. Scale bars, 50 μm.

Antigen-specific CD8+ T cells are significantly increased after pimitespib treatment. (A–C). Antigen-specific CD8+ T-cell induction in the presence or absence of pimitespib. Representative flow cytometry staining of antigen-specific CD8+ T cells (left) and summaries of the frequency of antigen-specific CD8+ T cells (right). For in vitro sensitization of antigen-specific CD8+ T cells, 1.5–2.0 × 106 cells were cultured with antigen-presenting cells pulsed with various peptides (Melan-A/MART-1, A; influenza, B; CMV, C) with or without pimitespib (1 μM). Seven to ten days later, pre-sensitized CD8+ T cells were stained with PE-labeled peptide antigen/HLA-A*0201 dextramers and analyzed with flow cytometry. The data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

Pimitespib augments the antitumor efficacy of anti–PD-1 mAb. (A–C). Antitumor efficacy of combined treatment with anti–PD-1 mAb and pimitespib against CMT-93. A total of 2 × 106 CMT-93 cells were inoculated subcutaneously, and the cancer size was monitored twice weekly (n = 6). The mice were randomized, and some mice received intraperitoneal administration of anti–PD-1 mAb (0.5 mg/body) on that day (day 0) and/or oral administration of pimitespib (14 mg/kg/body) five times weekly from day 0. Cancer growth (A for summary and B for each mouse) and PFS (C) are shown. PFS was defined as the time from day 0 until the cancer was twice as large as when treatment was initiated. All in vivo experiments were performed at least twice. *, P < 0.05; **, P < 0.01.

MHC/peptide multimers used for antigen-specific T-cell induction and detection.

Antibodies Used in RPPA Analysis.

A schematic summary of the reduction in Treg cells by pimitespib. HSP90 inhibition by pimitespib impairs Treg cells via STAT5 degradation. The IL-2/STAT5 signaling pathway is essential for the development and maintenance of Treg cells (left). STAT5, a client protein of HSP90, is inhibited by pimitespib treatment, resulting in the inhibition of FOXP3 transcription. Decreased FOXP3 expression inhibits the development, maintenance, and immunosuppressive function of Treg cells (right). Treg cell impairment, therefore, is mainly caused by pimitespib–induced STAT5 degradation.

Antibodies used for flow cytometry.

Antitumor efficacy of combined treatment with anti–PD-1 mAb and pimitespib is not observed in CD8+ T cell–depleted mice. (A). Representative flow cytometry staining of PBMCs from C57BL/6 mice. For CD4+ T-cell and CD8+ T-cell depletion, anti-CD4 mAb (0.5 mg/body) or anti-CD8β mAb (0.2 mg/body), respectively, was administered intraperitoneally once weekly. CD4+ T-cell or CD8+ T-cell depletion was confirmed by flow cytometry. (B). Antitumor efficacy of combined treatment with anti–PD-1 mAb and pimitespib in CD4+ or CD8+ T cell–depleted mice. A total of 2 × 106 MC-38 cells were injected subcutaneously, and the cancer size was monitored twice weekly (n = 6). After the cancer volumes reached 100–150 mm3, the mice were randomized, and some mice received intraperitoneal administration of anti–PD-1 mAb (0.05 mg/body) on that day (day 0) and/or oral administration of pimitespib (14 mg/kg/body) five times weekly from day 0. Anti-CD4 mAb or anti-CD8β mAb was administered intraperitoneally every 7 days. All in vivo experiments were performed at least twice. *, P < 0.05; **, P < 0.01; n.s., not significant.

Primary antibodies and associated Opal fluorophore applications used for multiplex immunohistochemistry.