Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer

Abstract

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.

Extended Data Figure 1. Impact of p110δ inactivation on CD4 and CD8 T cells in mice with 4T1 or EL4 tumours

a, Levels of CD44^highCD4⁺ and CD44^highCD8⁺ T cells in the indicated immune compartments of naive and 4T1 tumour-bearing on day 26 after inoculation in WT or δ^D910A mice. b, Distribution of cells on day 5 of culture of splenocytes, isolated from 4T1 tumour-bearing WT and δ^D910A mice 21 days after inoculation, in the presence of mitomycin-treated 4T1 cells. c, Gene expression in CTLs derived from splenocytes from WT and δ^D910A OT-I mice, cultured in the presence of SIINFEKL OVA peptide and IL2. GzmA, granzyme A; GzmB, granzyme B, Prf1, perforin and (FasL or CD95L) Fas ligand. Expression levels are presented relative to β2-microglobulin. a-b, Statistically significant differences are indicated by * (P < 0.05) or ** (P < 0.01), as determined by the non-parametric Mann-Whitney t test. Between brackets: number of mice used per experiment. Each dot represents an individual mouse.

Extended Data Figure 2. Impact of p110δ inactivation on myeloid cells in 4T1 tumours

a, Gating strategy used to identify myeloid cell subsets. Splenic cells were gated on CD11b^high cells followed by Ly6C and Ly6G gating. FSC, forward scatter; SSC, side scatter (top panel). Frequency of CD11b⁺ cells in the spleen of WT and δ^D910A naïve mice and in 4T1 tumour-bearing mice on day 21 after inoculation (bottom panel). b, [³H]-Thymidine incorporation in co-cultures of splenocytes and purified myeloid cells, in combinations as indicated, with or without stimulation with anti-CD3 antibodies. Cultures were made using cells derived from individual mice. Error bars represent standard deviation from the mean of biological replicates. Statistically significant differences are indicated by * (P < 0.05) or ** (P < 0.01), as determined by the non-parametric Mann-Whitney t test. Between brackets: number of mice used per experiment. Each dot represents an individual mouse.

Extended Data Figure 3. Characterisation of the p110δ-selective inhibitor PI-3065

a, PI-3065 structure and in vitro IC₅₀ on selected PI3K family members. No significant activity against 72 protein kinases was observed at ≤ 10 μM in a KinaseProfiler assay (Millipore). b, Pharmacokinetic parameters of PI-3065. Mean (±SD) plasma concentration profile of PI-3065 following a single oral dose (75 mg/kg) administred per os (po) to female BALB/c mice. AUCinf, area under the curve, extrapolated to infinity; Cmax, highest observed plasma concentration; tmax, time at which Cmax occurred, QD; quaque die, every day. c, Growth of primary 4T1 tumours, inoculated in the breast fat pad, measured by calipers and expressed as tumour volume. Mice were dosed per os with vehicle or PI-3065 (75 mg/kg, daily) for 36 days. 10⁵ tumour cells were inoculated 12 h post first dosing. d, Percentage of tumour-free mice upon continuous per os treatment of mice with vehicle or PI-3065 (25 mg/kg, twice daily) for 37 days, with tumour cells inoculated on day 7 of PI-3065 dosing. 15 mice were used for each genotype. e, Class I PI3K isoform expression in 4T1 cells. f, Proliferation of 4T1 cells following a 4 h treatment with the indicated p110δ inhibitors, washing and MTS staining after 48 h culture. g, Percentage body weight change (from day 0) of 4T1 tumour-bearing mice upon daily per os administration of PI-3065 (75 mg/kg) or vehicle for 36 consecutive days. Statistically significant differences are indicated by * (P < 0.05) or ** (P < 0.01), as determined by the non-parametric Mann-Whitney t test. Between brackets: number of mice used per experiment.

Figure 1. Impact of genetic inactivation of p110δ on tumour growth and metastasis

a, percentage of mice with visible B16 ear tumours (left) or lymph nodes metastasis (right). Photographs show B16 metastases in cervical lymph nodes and representative excised lymph nodes. b-d, primary tumour burden of the indicated tumour lines. e, 4T1 metastasis as detected by luciferase activity (left and middle panel) or histology (right panel), expressed as a percentage of the total number of tumour-bearing animals/group. f, Growth of primary 4T1 tumours. g, Survival of 4T1 tumour-bearing mice. a-f, Statistically significant differences are indicated by * (P < 0.05) or ** (P < 0.01), as determined by the non-parametric Mann-Whitney t test. Between brackets: number of mice used per experiment. Each dot represents an individual mouse.

Figure 2. Inactivation of p110δ in Treg is sufficient to confer cancer resistance

a, Relative and total numbers of Treg in the draining lymph nodes of naive and 4T1 tumour-bearing mice. b, Impact of adoptive transfer of Treg into δ^D910A mice on EL4 tumour wet weight and tumour-infiltrating CD8⁺ T cells. c, Number of mice with visible B16 tumours and B16 tumour weight in mice of the indicated genotype. d, Survival of EL4 tumour-bearing mice of the indicated genotype. a-c, Statistically significant differences are indicated by * (P<0.05) or ** (P<0.01), as determined by the non-parametric Mann-Whitney t test or Anova. Between brackets: number of mice used per experiment. Each dot represents an individual mouse.

Figure 3. Impact of p110δ inactivation on T cell-mediated anti-tumour immunity

a, Growth of 4T1 in δ^D910A mice injected with antibodies to CD4 or CD8. Arrow indicates the time of antibody injection. b, Metastasis in CD4 or CD8 T cell-depleted 4T1 tumour-bearing δ^D910A mice. c, In vitro cytotoxic activity of splenocytes, isolated from 4T1 tumour-bearing WT and δ^D910A mice 21 days after inoculation and cultured for 4 days with mitomycin-treated 4T1 cells. E/T, effector to target (4T1 or LLC) ratio. d, Frequency of CD44^high CD4⁺ and CD8⁺ T cells in splenocytes from 4T1 tumour-bearing mice cultured for 5 days with mitomycin-treated 4T1 cells. e, Frequency of IFNγ⁺ T cells after 16h PMA+ionomycin stimulation of splenocytes from WT and δ^D910A 4T1 tumour-bearing mice. f, Relative levels of tumour-infiltrating CD3⁺ lymphocytes and OVA-specific CD8⁺ T cells in LLC-OVA tumours in WT or δ^D910A mice. g, In vitro cell killing of a 1:1 EL4-OVA:EL4 mix following 24h incubation with CTL from WT or a δ^D910A OT-I mice (at an E/T ratio of 10:1), incubated with or without the p110δ inhibitor IC87114 during the 8 day expansion phase, the 24h killing phase, or both. Cell killing efficiency is expressed as the ratio of EL4-OVA cells over EL4 cells remaining after incubation with effector cells. h, Effect of adoptive transfer in WT mice of OT-I CD8⁺ or OT-II CD4⁺ cells on growth of subsequently inoculated EL4-OVA. i, Survival of post-surgical 4T1 tumour-bearing mice. j, Survival of δ^D910A mice which had remained tumour-free >200 days after surgery, and of naïve WT mice, following injection of 10,000 4T1 cells. Statistics are as described in the legend to Figure 1.

Figure 4. Impact of p110δ inactivation on myeloid cells in 4T1 tumour-bearing mice

a, 4T1 primary tumour growth and lung metastasis in WT, δ^D910A, Rag^−/− and Rag^−/− xδ^D910A mice. b, 4T1 tumour growth and total numbers of splenic CD11b⁺Gr1^high myeloid cells in WT and δ^D910A mice. c, Gating strategy used to identify myeloid cell subsets and frequency of splenic PMN-MDSCs and neutrophils of naïve and 4T1 tumour-bearing WT and δ^D910A mice. d, Spearman correlation between accumulation of splenic PMN-MDSCs and Treg in WT or δ^D910A mice. e, Impact of depleting CD8⁺ T cells in δ^D910A mice on 4T1 tumour burden and presence of splenic myeloid cell populations. f, Impact of purified splenic myeloid cells on proliferation of anti-CD3-stimulated WT T cells. g, Cytokine production by splenocytes from 4T1 tumour-bearing (30 days after inoculation) from WT or δ^D910A mice, individually cultured for 4 days. Statistics are as described in the legend to Figure 1.

Figure 5. Impact of pharmacological inactivation of p110δ on tumour growth and T cell responses

a, Mice, dosed with vehicle or PI-3065 (75 mg/kg, daily) for 36 days and inoculated with 10⁵ 4T1 cells 12h post first dosing, were assessed for tumour growth by luciferase imaging (first panel), tumour weight (second panel) or luciferase activity in tumours excised 35 days after inoculation (third panel). Incidence of 4T1 metastasis (fourth panel), as detected by H&E staining and histology, expressed as percentage of the total number of tumour-bearing animals per group. b, Impact of PI-3065 (75 mg/kg) on KPC mouse survival (left) and macrometastases (as detected by H&E staining) and cancer-associated pathology (right). c, Proportion of Treg (% of CD4⁺) in the draining lymph nodes of KPC mice administered vehicle or PI-3065. d, Proportion of CD44^high T cells (% of CD8⁺) in the draining lymph nodes of KPC mice administered vehicle or PI-3065. e, Relative numbers of CD8⁺ T cells (% of CD45⁺) in normal pancreas and PDAC lesions of KPC mice treated or not with PI-3065. Statistics are as described in the legend to Figure 1.