Reversal of Lactate and PD-1-mediated Macrophage Immunosuppression Controls Growth of PTEN/p53-deficient Prostate Cancer

Abstract

Purpose: Phosphatase and tensin homolog (PTEN) loss of function occurs in approximately 50% of patients with metastatic castrate-resistant prostate cancer (mCRPC), and is associated with poor prognosis and responsiveness to standard-of-care therapies and immune checkpoint inhibitors. While PTEN loss of function hyperactivates PI3K signaling, combinatorial PI3K/AKT pathway and androgen deprivation therapy (ADT) has demonstrated limited anticancer efficacy in clinical trials. Here, we aimed to elucidate mechanism(s) of resistance to ADT/PI3K-AKT axis blockade, and to develop rational combinatorial strategies to effectively treat this molecular subset of mCRPC.

Experimental design: Prostate-specific PTEN/p53-deficient genetically engineered mice (GEM) with established 150-200 mm3 tumors, as assessed by ultrasound, were treated with either ADT (degarelix), PI3K inhibitor (copanlisib), or anti-PD-1 antibody (aPD-1), as single agents or their combinations, and tumors were monitored by MRI and harvested for immune, transcriptomic, and proteomic profiling, or ex vivo co-culture studies. Single-cell RNA sequencing on human mCRPC samples was performed using 10X Genomics platform.

Results: Coclinical trials in PTEN/p53-deficient GEM revealed that recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts ADT/PI3Ki combination-induced tumor control. The addition of aPD-1 to ADT/PI3Ki combination led to TAM-dependent approximately 3-fold increase in anticancer responses. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their anticancer phagocytic activation, which was augmented by ADT/aPD-1 treatment and abrogated by feedback activation of Wnt/β-catenin pathway. Single-cell RNA-sequencing analysis in mCRPC patient biopsy samples revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression.

Conclusions: Immunometabolic strategies that reverse lactate and PD-1-mediated TAM immunosuppression, in combination with ADT, warrant further investigation in patients with PTEN-deficient mCRPC.

Figure 1.. The combination of ADT and PI3Ki demonstrates partial anti-tumor response in PTEN/p53-deficient murine PC via increased infiltration/activation of tumor-associated macrophages (TAM) within the TME.

(A) Pb-Cre; PTEN^fl/fl Trp53^fl/fl mice were followed by serial ultrasound until they developed solid prostate tumors (100–150 mm³) by 16–20 weeks of age. Mice were randomized to untreated, degarelix (0.625 mg, single dose, ADT) treated, copanlisib (14 mg/kg, iv, every alternate day, PI3Ki, C) treated, and their combination treated groups. Tumor growth was monitored using MRI, and ORR (partial response + stable disease) were calculated, as described in Methods. (B-F) Following acute treatment for 7 days, prostate tumors were harvested and analyzed by flow cytometry for the following cell populations: proliferating tumor cells (Ki67⁺CD45^-, (B)), total TAM (CD45⁺CD11b⁺F4/80⁺ cells, (C)), PD-1 (D) and MHC-II (E) expressing TAM. (F) Representative flow plots demonstrate % frequency of MHC-II^hi/lo/PD-1^hi/lo expressing TAM (CD45⁺CD11b⁺F4/80⁺ cells). (G) Correlation plot highlights association of MHC-II^hi/PD-1^lo, MHC-II^hi/PD-1^hi, MHC-II^lo/PD-1^lo and MHC-II^lo/PD-1^hi TAM subsets with % ORR observed following 28-days treatment of single agents or their combination and in untreated group (U). n=2–8 mice per group. Significances/p-values were calculated by Chi-square test (panel A), one-way ANOVA (panel B-E), Pearson test (panel G) and indicates as follows, *p<0.05 and **p<0.01; ns = not statistically significant.

Figure 2.. ADT/PI3Ki combination therapy significantly enhances phagocytosis of PTEN/p53-deficient murine cancer cells by activated TAM.

(A) Experimental schema for phagocytosis experiment, in which sorted TAM from PTEN/p53-deficient prostate GEMM tumors were co-cultured with CTV dye stained-AC1/SC1 cells under normal and AD conditions, and treated with either DMSO (negative control) or copanlisib (C, 100 nM) for 24 hours. Bar graphs demonstrate fold change (FC) in phagocytosis of AC1 (B) and SC1 (C) cells by MHC-II^hi/PD-1^lo, MHC-II^hi/PD-1^hi, MHC-II^lo/PD-1^lo and MHC-II^lo/PD-1^hi TAM subsets, relative to untreated group (U). n=2 independent experiments. Significances/p-values were calculated by one-way ANOVA and indicates as follows, *p<0.05, **p<0.01 and ***p<0.001; ns = not statistically significant.

Figure 3.. The addition of PD-1 blockade to androgen depletion/PI3Ki enhances phagocytic capacity of suppressive PD-1^hi macrophages.

(A) Schema illustrating co-culture of FACS-sorted TAM subsets (MHC-II^hi/lo/ PD-1^hi) from PTEN/p53-deficient prostate tumors with CTV dye stained-AC1/SC1 cells under normal and AD conditions. Co-cultures were treated with copanlisib (C, 100 nM), PD-1 antibody (P, 10 μg/mL) or their combination for 24 hours, followed by phagocytosis assay measuring CTV dye uptake within TAM. Bar graphs demonstrate fold change (FC) in phagocytic activity of MHC-II^hi/lo/PD-1^hi expressing TAM, relative to untreated group (U) in AC1 (B) and SC1 (C) cells. n=2 independent experiments. Significances/p-values were calculated by one-way ANOVA and indicates as follows, *p<0.05, **p<0.01 and ***p<0.001; ns = not statistically significant.

Figure 4.. Pre-treatment of TAM ex vivo with androgen depletion alone and in combination with aPD-1, increases phagocytosis of PTEN/p53-deficient GEMM tumor cells by MHC-II^hi/PD-1^lo and MHC-II^hi/PD-1^hi TAM subsets, respectively.

(A) Schema illustrating pre-treatment of FACS-sorted TAM subsets from PTEN/p53-deficient prostate tumors with copanlisib (C, 100 nM), PD-1 antibody (P, 10 μg/mL) or their combination under normal and AD condition for 24 hours. After PBS wash, treated TAM were co-cultured with CTV dye stained-AC1/SC1 cells for 2 hours. Bar graphs demonstrate fold change (FC) in phagocytosis of AC1 (B) and SC1 (C) cells by MHC-II^hi/PD-1^hi/lo expressing TAM, relative to untreated group (U). n=2 independent experiments. Significances/p-values were calculated by one-way ANOVA and indicates as follows, **p<0.01 and ***p<0.001; ns = not statistically significant.

Figure 5.. PI3Ki suppresses lactate production from treated PTEN/p53-deficient PC cells and secondary histone lactylation within activated TAM, resulting in enhanced TAM phagocytosis.

(A) Schema illustrating lactate add-back phagocytosis experiment on ex vivo CM. Single cell suspensions of PTEN/p53-deficient prostate GEMM tumors were treated with copanlisib (C, 100 nM) and CM was collected 24 hours following treatment. FACS-sorted TAM from PTEN/p53-deficient prostate tumors were cultured ex vivo with CM for 24 hours in presence or absence of lactate (100 nmol/μL). After PBS wash, TAM were co-cultured with CTV dye stained-AC1/SC1 cells for 2 hours. Bar graphs demonstrate fold change (FC) in phagocytic activity (B) and histone lactylation status (C) of activated MHC-II^hi/PD-1^lo and MHC-II^hi/PD-1^hi TAM, relative to untreated group (U). (D-E) Single cell suspensions were prepared from human bone/lymph node metastatic PC patient samples (BMET-1/−2 and LMET-1/−2/−3) and underwent single-cell RNA using Chromium controller 10x Genomics platform. Annotated tumor cells, TAM and myeloid cells were characterized for aerobic glycolysis (D, E), phagocytosis suppression (D) and M1-polarization status (E), respectively, using a normalized gene score, as described in the methods section. For ex vivo studies, n=2 independent experiments and for bioinformatics analysis, n = 355 tumor cells and 389 TAM in bone metastatic PC patients; n = 367 tumor cells and 101 myeloid cells in lymph node metastatic PC patients. Significances/p-values were calculated by one-way ANOVA (panel B-C), Wilcoxon rank-sum text (panel D-E) and indicates as follows, *p<0.05, **p<0.01 and ***p<0.001; ns = not statistically significant.

Figure 6.. ADT + PI3Ki + aPD-1 antibody leads to tumor control in PTEN/p53-deficient GEMM via activated TAM, with feedback Wnt/β-catenin activation mediated restoration of lactate and histone lactylation observed in non-responders.

(A) Pb-Cre; PTEN^fl/fl Trp53^fl/fl mice with established prostate tumors were treated with PD-1 antibody (aPD-1, 100 μg/mouse, ip, every alternate day, P) alone or in combination with ADT (degarelix, 0.625mg, single dose, castration) or copanlisib, (14 mg/kg, iv, every alternate day, C) or ADT + copanlisib. For in vivo macrophage depletion, clodronate (200 μg/mouse, ip, every week, Cl) was administered concurrently with ADT + copanlisib + PD-1 antibody. Tumor volume was monitored non-invasively using MRI and ORR (partial response + stable disease) was calculated, as described in Methods. (B) Pb-Cre; PTEN^fl/fl Trp53^fl/fl mice were treated with the indicated drug(s) for 7 days, and analyzed for % frequency of total and MHC-II expressing TAM (CD45⁺CD11b⁺F4/80⁺ cells). (C) Tumor RNA was isolated following 28 days treatment with indicated agents and sequenced to perform pathway enrichment analysis. Reactome plot shows pathway analysis for differentially expressed genes in non-responder tumors, relative to responder and untreated. (D) Western blot analyses were performed for indicated protein markers on PTEN/p53-deficient prostate GEMM tumor extracts following treatment with ADT, copanlisib, PD-1 antibody or their combinations for 28 days. (E-F) AC1 cells were treated with copanlisib (C, 100 nM), PD-1 antibody (P, 10 μg/mL) or their combinations under normal or AD conditions for 24 and 72 hours. Western blot analyses (E) were performed for indicated protein markers on AC1 cell lysates, and active β-catenin level was quantified by Image J software, and lactate levels in the supernatant were analyzed by fluorimetry. (F). (G) Model illustrating the decrease in lactate production from PTEN/p53-deficient PC cells in response to PI3Ki inhibitor treatment overcomes histone lactylation mediated TAM suppression and enhances phagocytosis of PC cells within TAM. Concurrent ADT and aPD-1 treatment directly activates TAM within TME, thereby accentuating the effects of single-agent PI3Ki on TAM functionality/polarization. Wnt/β-catenin activation drives resistance to ADT/PI3Ki/aPD-1 combination therapy. For in vivo studies, n=6–10 mice per group (panel A), n=2 mice per group (panel B), and for in vitro, n=3 independent experiments. Significances/p-values were calculated by Chi-square test (panel A), one-way ANOVA (panel B and F), Reactome analysis (panel C) and indicated as follows, *p<0.05, **p<0.01 and ***p<0.001; ns = not statistically significant.