Research ArticleIMMUNOLOGY

Inhibition of Vps34 reprograms cold into hot inflamed tumors and improves anti–PD-1/PD-L1 immunotherapy

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Science Advances  29 Apr 2020:
Vol. 6, no. 18, eaax7881
DOI: 10.1126/sciadv.aax7881
  • Fig. 1 Targeting Vps34 decreases tumor growth and tumor weight and improves mice survival in multiple tumor types.

    (A and B) Growth curves, weight in grams, and mice survival curves of control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (A) and CT26 CRC (B) tumors. Each curve represents three independent experiments of four or five mice per group for B16-F10 and two independent experiments of five mice per group for CT26. (C to F) Growth curves, weight in grams, and mice survival curves of B16-F10 melanoma (C); CT26 colorectal (D), GEMM (YUMM) melanoma (E); or Renca renal carcinoma (F) tumors in mice treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). For B16-F10, each curve represents three independent experiments of eight or nine mice per group. For CT26, YUMM, or Renca, each curve represents two independent experiments of five mice per group. All results are shown as mean ± SEM (error bars). Statistically significant differences (indicated by asterisks) are calculated compared to control conditions using an unpaired two-tailed Student’s t test. Not significant (ns) = P > 0.05; *P < 0.05; **P < 0.005; and ***P < 0.0005. Mice survival curves (five mice per group for all tumor models) were generated from tumor-bearing mice. Lack of survival was defined as death or tumor size >1000 mm3. Mice survival percentage was defined using GraphPad Prism, and P values were calculated using the log-rank (Mantel-Cox) test (*P ≤ 0.05 and **P ≤ 0.01).

  • Fig. 2 Targeting Vps34 induces profound changes in the immune landscape of tumors.

    (A) Top: Flow cytometry quantification of CD45+ leukocytes (gated in live cells) infiltrating the following tumors: control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (left) and B16-F10 melanoma (middle) or CT26 colorectal (right) tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). Bottom: Representative images of immunohistochemical staining of CD45 performed on B16-F10 tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). Scale bars, 100 μm. (B) Flow cytometry quantification of NK cells (NK), CD8+ T cells (CD8), CD4+ effector T cells (CD4 eff), DCs, and type 1 macrophages (M1) infiltrating the following tumors: control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (top) and B16-F10 melanoma (middle) or CT26 colorectal (bottom) tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). Representative images of immunohistochemical staining of CD8+ performed on B16-F10 tumors treated with vehicle (vehicle) or Vps34i (SB02024 or SAR405) are included in the bottom of the middle panel. Scale bars, 100 μm. (C) Quantification of the percent of CD69+ activated NK cells, CD8+ T cells, and CD4+ effector T cells infiltrating the following tumors: control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (top) and B16-F10 melanoma (middle) or CT26 colorectal (bottom) tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). (D) Quantification of the percent of type 2 macrophages (M2), Treg, and MDSCs infiltrating the following tumors: control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (top) and B16-F10 melanoma (middle) or CT26 colorectal (bottom) tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). (E) CD8+–to–Treg cell ratio (CD8/Treg) in control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (left) and in B16-F10 melanoma tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405) (right). Experiments described in (A) to (E) were conducted on well-established tumors on day 15. The defined subpopulations were gated and quantified in live CD45+ cells. Each dot represents one tumor. The data are reported as the average of five mice per group (each dot represents one mouse). Results are shown as mean ± SEM (error bars). Statistically significant differences (indicated by asterisks) are calculated compared to control conditions using an unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).

  • Fig. 3 Tumor growth inhibition in Vps34-targeted tumors is dependent on NK and CD8+ T cells.

    (A) Growth curve (top) and weight in grams (bottom) in immunodeficient NSG mice of shCT and shVps34 B16-F10 tumors. (B) Growth curve (top) and weight in grams (bottom) of B16-F10 tumors in immunodeficient NSG mice treated with vehicle or Vps34i (SB02024 or SAR405). (C) Growth curves (top) and weight in grams (bottom) of shCT and shVps34 B16-F10 tumors in control (iso), NK-depleted (αNK1.1), and CD8-depleted (αCD8) mice. (D) Growth curves (top) and weight in grams (bottom) of B16-F10 tumors in control (iso) or NK-depleted (αNK1.1) mice treated with vehicle or Vps34i (SB02024 or SAR405). (E) Growth curves (top) and weight in grams (bottom) of B16-F10 tumors in control (iso) or CD8-depleted (αCD8) mice treated with vehicle or Vps34i (SB02024 or SAR405). Data described in (A) to (E) are reported as the average of 10 (A and B) and 5 (C to E) mice per group. Each dot represents one mouse. Results are shown as mean ± SEM (error bars). Tumor weights were assessed at day 17. Statistically significant differences (indicated by asterisks) are calculated compared to control conditions using an unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).

  • Fig. 4 Targeting of Vps34 increases pro-inflammatory CCL5 and CXCL10 chemokine secretion in tumors.

    (A) Enzyme-linked immunosorbent assay (ELISA) quantification of the CCL5, CXCL10, and IFNγ secreted in the microenvironment of the following tumors: control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (top) and B16-F10 melanoma (middle) and CT26 colorectal (bottom) tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). Data are reported in pg/ml standardized to excised tumor weight (in grams) and represented as an average of 5 or 10 tumors per group (each dot represents one mouse). (B) ELISA quantification of CCL5 and CXCL10 protein levels in the blood of mice bearing the following tumors: control (shCT) or Vps34-targeted (shVps34) B16-F10 melanoma (top) and B16-F10 melanoma (middle) and CT26 colorectal (bottom) tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405). Data are reported in pg/ml and represented as an average of 10 or 15 blood samples per group collected from different mice (each dot represents one mouse). (C and D) Growth curves (top), weight in grams (middle), and flow cytometry quantification of NK cells and CD8+ T cells (bottom) infiltrating B16-F10 melanoma (C) and CT26 colorectal (D) tumors treated with control vehicle (vehicle) or Vps34i (SB02024 or SAR405) and injected with either control (iso) or CCL5-neutralizing antibody (αCCL5). Results are reported as the average of five mice per group. All results are shown as mean ± SEM (error bars). Statistically significant differences (indicated by asterisks) are calculated compared to control conditions using an unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).

  • Fig. 5 STAT1/IRF7 axis is involved in the up-regulation of CCL5 and CXCL10 in Vps34i (SB02024 or SAR405)–treated tumor cells.

    (A) Expression of pSTAT1 (Tyr701) and total STAT1 protein levels by Western blot in control (si-CT) or Vps34-targeted (si-Vps34) B16-F10 melanoma cells (left) and B16-F10 melanoma cells (middle) and CT26 CRC cells (right) treated with control dimethyl sulfoxide (DMSO) (control) or Vps34i (SB02024 or SAR405). β-Actin was used as a loading control. (B) Expression of STAT1 mRNA by real-time quantitative polymerase chain reaction (RT-qPCR) in control (si-CT) or Vps34-targeted (si-Vps34) B16-F10 melanoma cells (left) and B16-F10 melanoma cells (middle) and CT26 CRC cells (right) treated with control DMSO (control) or Vps34i (SB02024 or SAR405). Results are reported as fold change (FC) compared to control conditions. (C) The expression of Irf1, Irf3, Irf4, Irf7, and Irf8 mRNA by RT-qPCR in control (si-CT) or Vps34-targeted (si-Vps34) B16-F10 melanoma cells (left) and B16-F10 melanoma cells (middle) and CT26 CRC cells (right) treated with control DMSO (control) or Vps34i (SB02024 or SAR405). Results are reported as fold change compared to control conditions. (D) The expression of IRF7 and IRF1 protein levels by Western blot in control (si-CT) or Vps34-targeted (si-Vps34) B16-F10 melanoma cells (left) and B16-F10 melanoma cells (middle) and CT26 CRC cells (right) treated with control DMSO (control) or Vps34i (SB02024 or SAR405). β-Actin was used as a loading control. (E) Expression of CCL5 and CXCL10 mRNA by RT-qPCR (left) and ELISA quantification of CCL5 and CXCL10 protein levels in the supernatants (right) of B16-F10 melanoma cells and CT26 CRC cells pretransfected with control siRNA (si-CT) or two different siRNA sequences (1 or 2) targeting either STAT1 (si-STAT1) or IRF7 (si-IRF7) and then treated with control DMSO (C), SB02024 (SB), or SAR405 (SAR). Results in left panels are reported as fold change compared to control conditions. Results in right panels are reported in pg/ml. (F and G) Expression of Irf7 and Stat1 mRNA by RT-qPCR in B16-F10 melanoma (F) and CT26 CRC (G) cells described in (E). C, control DMSO; SB, SB02024; SAR, SAR405. Results represent the average of three independent experiments (A to D) and two independent experiments (E to G). All results are shown as mean ± SEM (error bars). Statistically significant differences (indicated by asterisks) are calculated compared to control conditions using an unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.005, and ***P < 0.0005).

  • Fig. 6 Treatment of tumor-bearing mice with Vps34i (SB02024 or SAR405) improves the therapeutic benefit of anti–PD-1/PD-L1 immunotherapy.

    (A and B) Tumor growth curves of B16-F10 melanoma (A) (top) or CT26 CRC (B) (top) in mice treated with control (vehicle) or Vps34i (SB02024 or SAR405) combined with either control isotype, anti–PD-L1 (αPD-L1), or anti–PD-1 (αPD-1). Tumor weight (in grams) of B16-F10 (A) (middle) on day 17 and CT26 (B) (middle) on day 15. Results are reported for B16-F10 as the average of 8 or 10 mice per group from two independent experiments conducted with four to five mice per group and for CT26 as the average of five mice per group. Results are shown as mean ± SEM (error bars). Statistically significant differences (indicated by asterisks) are calculated using an unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.005, and ***P < 0.0005). Mice survival curves (five mice per group) were generated from B16-F10 (A) (bottom) or CT26 (B) (bottom) tumor–bearing mice treated with control (vehicle) or Vps34i (SB02024 or SAR405) alone or in combination with either control isotype, anti–PD-L1 (αPD-L1), or anti–PD-1 (αPD-1). Lack of survival was defined as death or tumor size >1000 mm3. Mice survival percentage was defined using GraphPad Prism, and P values were calculated using the log-rank (Mantel-Cox) test (*P ≤ 0.05 and **P ≤ 0.01).

Supplementary Materials

  • Supplementary Materials

    Inhibition of Vps34 reprograms cold into hot inflamed tumors and improves anti–PD-1/PD-L1 immunotherapy

    Muhammad Zaeem Noman, Santiago Parpal, Kris Van Moer, Malina Xiao, Yasmin Yu, Tsolere Arakelian, Jenny Viklund, Angelo De Milito, Meriem Hasmim, Martin Andersson, Ravi K. Amaravadi, Jessica Martinsson, Guy Berchem, Bassam Janji

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