Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/β/δ treatment

Combining immune checkpoint therapy (ICT) and targeted therapy holds great promises for broad and long-lasting anti-cancer therapies. However, combining ICT with anti-PI3K inhibitors have been challenging because the multifaceted effects of PI3K on both cancer cells and immune cells within the tumor microenvironment. Here we find that intermittent but not daily dosing of a PI3Kα/β/δ inhibitor, BAY1082439, on Pten-null prostate cancer models could overcome ICT resistance and unleash CD8+ T cell-dependent anti-tumor immunity in vivo. Mechanistically, BAY1082439 converts cancer cell-intrinsic immune-suppression to immune-stimulation by promoting IFNα/IFNγ pathway activation, β2-microglubin expression and CXCL10/CCL5 secretion. With its preferential regulatory T cell inhibition activity, BAY1082439 promotes clonal expansion of tumor-associated CD8+ T cells, most likely via tertiary lymphoid structures. Once primed, tumors remain T cell-inflamed, become responsive to anti-PD-1 therapy and have durable therapeutic effect. Our data suggest that intermittent PI3K inhibition can alleviate Pten-null cancer cell-intrinsic immunosuppressive activity and turn “cold” tumors into T cell-inflamed ones, paving the way for successful ICT.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Source data are provided with this paper. All patient RNAseq data analyzed was available at TCGA database and from cBioPortal (https://www.cbioportal.org/). No restrictions on data availability. All RNAseq raw data used in Figure1, 2, 4, 6, 7, S2, S6, S9 and Supplementary data 1 was available at Gene Expression Omnibus under accession number GSE159660 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE159660). The remaining data are available within the Article, Supplementary Information or Source Data file.
Statistical methods were not used to predetermine sample size. Key findings were repeated in multiple independent experiments, as noted in figure legends. All sample size was at least three independent replicates. Animal and patient sample size was determined by experimental feasibility and sample availability to demonstrate certain results.
No data were excluded from the analyses.
Biological replications (three biological replicates at least) and statistics were indicated in the legends. All attempts at replication were successful based on replications on different days showing comparable significance level for biological comparison.
Samples were allocated into experimental groups by different drug treatments (Vehicle, or BAY1082439 treatment of different times) or by confirmed genetic modification of the cell line (doxycycline-induced protein expression). Mouse samples were randomly allocated into experimental groups after confirmed Pten genetic deletion.
The researchers were blinded during animals research data collection, experiments apart from animal studies, and data analysis. All western blot and flow cytometry antibodies in the manuscript had been validated. Each primary antibody data provided in the manuscript has been validated for the species and application on the manufacturer's website.
anti-Mouse B220 for FACS Biolegend RA3-6B2, #103244 was successfully stained in C57BL/6 mouse splenocytes.  Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Ultra-LEAF™ Purified anti-mouse CD28 Antibody for T cell culture Biolegend 37.51, #102115: 16 citations, reported applications include in vitro costimulation of T and NK cells.
Human prostate cancer cells (PC3 and LNCAP) were ordered from ATCC. Mouse prostate cancer cells (CAP2 and CAP8) were established by our lab (Jing Jiao. et al. Cancer research, 2007). PC3 WT/PTEN-inducible cells were established by our lab (David J Mulholland. et al. Cancer cell, 2011).
PC3 and LNCAP cell line have not been authenticated after purchasing from vendors. CAP2 and CAP8 cell were authenticated by PCR-based genotyping analysis. PC3 WT/PTEN-inducible cells were authenticated by immunoblotting of targeted proteins and Sanger sequencing of modification region of targeted genes.
All cells used have been tested negative for mycoplasma contamination.
No such cell lines were used.
The generation of the Pb-Cre+;PtenloxP/loxP prostate cancer model (male, 10 weeks) has been described previously (Wang, S. et al. Cancer cell, 2003). Cd8atm1Mak mice (CD8KO mice) was purchased from the Jackson lab (002665) then crossed with the Pten-null mice to generate Pb-Cre+;PtenL/L;Cd8-/-(Pten-null,Cd8-Ko mice. male, 10 weeks) mice. The generation of the Pb-Cre+;PtenL/L;K-rasG12D/W prostate cancer model (male, 12 weeks) has been described previously (Mulholland DJ. et al. Cancer research, 2012). Animal housing, breeding, and surgical procedures were approved by the Ethics Committee under ID LSC-WuH-1 and conducted in accordance with the regulations of the Division of Laboratory Animal Medicine at Peking University. Animals were housed at 22°C, with humidity of 40-70%, and dark/light cycle of 12/12 hours. For all animal experiments, the animals were monitored carefully, and no body-weight loss exceeds 20% in all treatment cohorts.
The study did not involve wild animals.
The study did not involve field-collected samples Animal housing, breeding, and surgical procedures were approved by the Ethics Committee under ID LSC-WuH-1 and conducted in accordance with the regulations of the Division of Laboratory Animal Medicine at Peking University.
Cell were digested by 0.25% trypin, mouse prostates were minced in sterile tissue culture dishes, and subjected to collagenase A (1.5 mg/ml; Roche) and DNase I (0.1 mg/ml; Roche) digestion for 1 h at 37°C with constant agitation. single cell were re-suspended in PBS containing 1% FBS.
For cell sorting, Cell were sorted on BD FACSAria™ III Cell Sorter. For cell population ratio/protein expression analysis, cell was analyzed in BD LSRFortessa™ Flow Cytometer.