Differential Targeting of Gr-MDSCs, T Cells and Prostate Cancer Cells by Dactolisib and Dasatinib

Abstract

Granulocytic myeloid-derived suppressor cells (Gr-MDSCs) promote immune evasion and resistance to immunotherapeutics in a variety of malignancies. Our previous study showed that dual PI3K/mTOR inhibitor Dactolisib impaired the viability and immunosuppressive function of Gr-MDSCs, and significantly synergized with immune checkpoint blockade (ICB) antibodies targeting PD1 and CTLA4 to eradicate metastatic castration-resistant prostate cancer (CRPC) in a preclinical transgenic mouse model. On the contrary, tyrosine kinase inhibitor Dasatinib diminished tumor-infiltrating T lymphocytes and showed no synergic activity with ICB. The understanding of the distinct effects of Dactolisib and Dasatinib on Gr-MDSCs, T cells and prostate neoplastic cells is inadequate, limiting the clinical translation of the combination immunotherapy. To address this question, we applied Reverse Phase Protein Array (RPPA) to profile 297 proteins and protein phosphorylation sites of Gr-MDSCs, T cells and prostate cancer cells isolated from the CRPC model. We found cell type-specific protein expression patterns and highly selective targets by the two drugs, including preferential inhibition of phospho-4E-BP1 in Gr-MDSCs by Dactolisib and preferential suppression of phospho-Src and phospho-p38 MAPK in T cells. Furthermore, transcriptomic profiling of Gr-MDSCs treated with the two inhibitors revealed downregulation of mitochondrial respiration pathways by Dactolisib but not Dasatinib. Overall, these results provide important mechanistic insight into the efficacious combination of Dactolisib and ICB as well as the detrimental effect of Dasatinib on anti-tumor immunity.

Keywords: Dactolisib; Dasatinib; PI3K/mTOR; Src; castration-resistant prostate cancer; combination immunotherapy; granulocytic myeloid-derived suppressor cells; immune checkpoint blockade; mitochondrial respiration; reverse phase protein array.

Figure 1

RPPA profiling of PCa cells, Gr-MDSCs and T cells of the CPPSML model of CRPC. (A) Workflow of cell isolation and RPPA. The figure was created with BioRender.com. (B) Unsupervised hierarchical clustering of PCa cells, T cells and Gr-MDSCs (untreated or DMSO-treated) with normalized RPPA signals. Each row represents a biological replicate sample and each column corresponds to an antibody in the RPPA array. (C) Volcano plots showing the pairwise fold change comparisons of the normalized protein or PTM levels among the three cell types. p values were calculated by Student’s t-test. Arrows help to pinpoint proteins of interest of whose labels were partly overlapped with adjacent labels.

Figure 2

Heatmaps showing the normalized protein expression signals under no treatment or 2 h treatment of DMSO, Dactolisib or Dasatinib (at indicated concentration) for Gr-MDSCs (A), PCa cells (B), and T cells (C). Proteins with significant changes under either inhibitor treatment are labeled with arrows. Dact = Dactolisib, Dasa = Dasatinib. Arrows highlight the differentially expressed proteins or PTMs by the inhibitor treatment.

Figure 3

Cell type-specific downregulation of phosphorylation levels of S6, 4E-BP1, Src and p38 MAPK by Dactolisib and Dasatinib. Phosphorylation level of the specified proteins was normalized to total protein level, and relative percentage was calculated with no drug treatment (0 μM) as 100%. (A) The phosphorylation of S6 at Ser235/236 and Ser240/244 dramatically decreased in PCa cells by Dactolisib, while Dasatinib had little effect. (B) The phosphorylation of 4E-BP1 at Thr37/46 in Gr-MDSCs was dramatically decreased by Dactolisib. The phosphorylation of 4E-BP1 at Ser65 in Gr-MDSCs, T cells and PCa cells was mildly decreased by Dactolisib. Dasatinib had little effect. (C) The phosphorylation of Src at Tyr416 and Tyr527 and p38 MAPK at Thr180/182 was dramatically decreased in T cells by Dasatinib, but not Dactolisib. In each plot, data represent mean ± standard error of the mean.

Figure 4

Validation of cell type-specific effect on S6, 4E-BP1, Src and p38 MAPK by Dactolisib and Dasatinib. Cell lysates used for RPPA were used to detect indicated total proteins and their phosphorylated forms by western blot for (A) Gr-MDSCs, (B) PCa cells and (C) T cells. The sign + and – denote the presence and absence of DMSO, Dactolisib or Dasatinib, respectively.

Figure 5

Dactolisib, but not Dasatinib, downregulated mitochondrial respiration genes in Gr-MDSCs based on microarray analysis. (A) p value distributions for differentially expressed probes between Dactolisib and DMSO control, or between Dasatinib and DMSO control based on unpaired t-test. (B) Volcano plots showing differentially expressed genes by Dactolisib or Dasatinib treatment on Gr-MDSCs. Some of the most significantly altered genes are labeled. Blue/Red: downregulated/upregulated genes by inhibitor treatment. (C) GO term over-representation analysis of downregulated genes by Dactolisib treatment, highlighting the enrichment of genes involved in mitochondrial respiration activity. BP: biological process; CC: cellular component; MF: molecular function.