Fig 2: NDNB1182 enhanced ICB efficacy in the PPS syngeneic prostate cancer model. (A) Growth curves of Myc-CaP tumors treated with isotype IgG or ICB (anti-PD1 plus anti-CTLA4) administered intraperitoneally (i.p.) at 10mg/kg, twice/week. N=9/group. (B) Growth curves of Myc-CaP tumors treated with vehicle, Ganetespib (25mg/kg, i.p. daily) or NDNB1182 (50mg/kg, i.p. daily). N=10 tumors/group at the start of the treatment. (C) Survival curves for mice bearing Myc-CaP tumors and treated with vehicle, Ganetespib or NDNB1182. N=5/group. (D) Growth curves of PPS tumors treated with vehicle control (N=6), NDNB1182 (50mg/kg, i.p. daily; N=6), ICB (anti-PD1 plus anti-CTLA4, 10mg/kg, i.p., twice/week; N=10), or concurrent NDNB1182 plus ICB (N=8). In (A, B, D), the randomized groups on day 0 of the treatment showed no significant difference based on statistical tests (for (A) two groups at day 0, P=0.8238 based on Mann Whitney test; for (B) three groups at day 0, P=0.4265 based on ANOVA test; for (D) four groups at day 0, P= 0.5561 based on ANOVA test). (E) Representative images and quantification results for CD8a immunohistochemistry on PPS tumors treated with the four regimens. The tumor regions (“T”) and the margin contours are denoted. N=5 for each condition. Image analysis and quantification was performed with ImageJ following a published protocol (59), where DAB signals were normalized to the number of nuclei present in the images. Scale bar 200µm. (F) Body weight changes of mice treated with vehicle, NDNB1182, or NDNB1182 plus ICB. (G) Representative H&E staining images of liver from vehicle-treated and NDNB1182-treated mice. Scale bar 50µm. (H) Quantification results for cleaved caspase 3 immunohistochemistry on liver and spleen tissues from vehicle-treated and NDNB1182-treated mice. In (A–H), data represent mean ± SEM. #P>0.05; *P<0.05, **P<0.01, ****P<0.0001, Student’s t-test. In (B, D–F), one-way ANOVA test result was shown in red (#P>0.05; ****P<0.0001). In (C), #P>0.05; *P<0.05, logrank test.

Fig 3: NDNB1182 enhanced ICB therapy in the PyMT-7160 syngeneic mammary tumor model. (A) Growth curves of PyMT-7160 tumors treated with vehicle control (N=8), NDNB1182 (50mg/kg, i.p. daily; N=10), ICB (anti-PD1 plus anti-CTLA4, 10mg/kg, i.p., twice/week; N=10), or concurrent NDNB1182 plus ICB (N=8). (B, C) Quantification results and gating strategy for tumor-infiltrating CD8+ T cells in treated PyMT-7160 tumors. (D, E) Quantification results and gating strategy for tumor-infiltrating CD11c+ MHC-II+ dendritic cells (DCs) in treated PyMT-7160 tumors. (F) qRT-PCR result of Ifit1/2/3 and two ERV elements for PyMT-7160 tumors treated with DMSO, NDNB1182, ICB, or NDNB1182 plus ICB (N=3/group).In (A, B, D, F), data represent mean ± SEM. #P>0.05; *P<0.05, **P<0.01, ****P<0.0001, Student’s t-test. In (A, B, D), one-way ANOVA test result was shown in red (*P<0.05, **P<0.01, ***P<0.001).

Fig 4: Hsp90ß-selective inhibitor NDNB1182. (A) Association of high HSP90AB1 expression with worse PSA progression free survival (PFS) and overall survival, with data extracted from the database of a phase 2 clinical trial of ipilimumab in metastatic castration-resistant prostate cancer *P<0.05, log rank test between the two cohorts (47). (B) Association of high HSP90AB1 expression with worse overall survival for anti-PD1 or anti-PD-L1 treatment with data from multiple clinical studies, drawn with KMplot. (C) The generic structure of NDNB1182 and IC50 values against Hsp90 isoforms determined using fluorescence polarization (FP) assay. (D) Western blot of Src in mouse prostate cancer cell line PPS treated with DMSO, NDNB1182 or luminespib for 12 hours, with the semi-quantitative result presented on the right. (E) HPLC result confirming the high purity of NDNB1182. (F) Dose-response curves and IC50 values of NDNB1182 on four cancer cell lines. Nonlinear regression modeling with log(inhibitor) and normalized response of variable slopes was conducted in Graphpad Prism. (G) Dose-response curves and IC50 values of NDNB1182 on primary prostate epithelial cells grown from wild type C57BL/6 mice and the mouse fibroblast cell line L cells. (H) HSP90AB1 transcript levels for prostate-lineage cell lines in the Depmap database portal with the plot generated by Depmap.

Fig 5: Blockade of PD-1/PD-L1 signaling enhances proinflammatory cytokine release in infected lung tissue. (A, B) Cytokine and chemokine concentrations in lung tissue homogenates from R. arrhizus-infected mice receiving ICIs or isotype antibodies were measured on day +4 (A) and day +7 (B) using a 19-plex Luminex assay. Individual and median concentrations of selected cytokines are displayed. Raw data are shown in Figure S1 (day +4) and Figure S2 (day +7). The two-sided Mann-Whitney U test with Benjamini-Hochberg adjustment for a false discovery rate of 0.2 was used for significance testing (comparisons between ICI-treated mice and isotype controls as well as between the two ICI-treated cohorts). (C) Comparison of median day-4 and day-7 lung concentrations of selected cytokines and chemokines in ICI-treated mice. Error bars represent inter-quartile ranges. The two-sided Mann-Whitney U test with Benjamini-Hochberg adjustment for a false discovery rate of 0.2 was used to compare the two sampling time points for each treatment arm. * p < 0.05, ** p < 0.01. C(X)CL, C(-X)-C motive chemokine receptor ligand; (G)M-CSF, (Granulocyte) Macrophage Colony-Stimulating Factor; IL, Interleukin; MCP, Monocyte Chemoattractant Protein; MIP, Macrophage Inflammatory Protein; TNF, Tumor Necrosis Factor.