Suppression of Tumor Cell Lactate-generating Signaling Pathways Eradicates Murine PTEN/p53-deficient Aggressive-variant Prostate Cancer via Macrophage Phagocytosis

Abstract

Purpose: Phosphatase and tensin homolog (PTEN) loss-of-function/PI3K pathway hyperactivation is associated with poor therapeutic outcomes and immune checkpoint inhibitor resistance across multiple malignancies. Our prior studies in Pb-Cre;PTENfl/flTrp53fl/fl genetically engineered mice (GEM) with aggressive-variant prostate cancer (AVPC) demonstrated tumor growth control in 60% mice following androgen deprivation therapy/PI3K inhibitor (PI3Ki)/programmed cell death protein 1 (PD-1) antibody combination, via abrogating lactate cross-talk between cancer cells and tumor-associated macrophages (TAM), and suppression of histone lactylation (H3K18lac)/phagocytic activation within TAM. Here, we targeted immunometabolic mechanism(s) of PI3Ki resistance, with the goal of durable tumor control in AVPC.

Experimental design: Pb-Cre;PTENfl/flTrp53fl/fl GEM were treated with PI3Ki (copanlisib), MEK inhibitor (trametinib) or Porcupine inhibitor (LGK'974) singly or their combinations. MRI was used to monitor tumor kinetics and immune/proteomic profiling/ex vivo coculture mechanistic studies were performed on GEM tumors or corresponding tumor-derived cell lines.

Results: Given our proteomic profiling showing persistent MEK signaling within tumors of PI3Ki-resistant GEM, we tested whether addition of trametinib to copanlisib enhances tumor control in GEM, and we observed 80% overall response rate via additive suppression of lactate within TME and H3K18lac within TAM, relative to copanlisib (37.5%) monotherapy. The 20% resistant mice demonstrated feedback Wnt/β-catenin activation, resulting in restoration of lactate secretion by tumor cells and H3K18lac within TAM. Cotargeting Wnt/β-catenin signaling with LGK'974 in combination with PI3Ki/MEKi, demonstrated durable tumor control in 100% mice via H3K18lac suppression and complete TAM activation.

Conclusions: Abrogation of lactate-mediated cross-talk between cancer cells and TAM results in durable ADT-independent tumor control in PTEN/p53-deficient AVPC, and warrants further investigation in clinical trials.

Figure 1.. PI3Ki/MEKi combination therapy induces activated TAM-mediated tumor growth control in 80% of Pb-Cre;PTEN^fl/flTrp53^fl/fl mice.

(A) Pb-Cre;PTEN^fl/flTrp53^fl/fl mice with established prostate tumors were treated with trametinib (3 mg/kg, po, every day), singly and in combination with copanlisib (14 mg/kg, iv, every alternate day) for 28 days. Tumor growth was monitored using MRI and response rates (Partial response plus Stable disease) were quantified as described in Methods. (B) At the end of treatment, prostate tumors were profiled using flow cytometry and analyzed for % frequency of total and MHC-II expressing TAM (CD45⁺CD11b⁺F4/80⁺ cells). (C) Single cell suspensions of PTEN/p53-deficient prostate GEM tumors were treated with copanlisib (C, 100 nM), trametinib (T, 5 nM) or their combination for 24 hours as described in Methods. Ex vivo CM were collected from these groups and analyzed for lactate content using colorimetry kits. (D) Prostate tumor extracts harvested from PI3Ki, MEKI and combinatorial treatment groups underwent western blot analysis for the indicated proteins. For in vivo studies, n=5-6 mice per group (experimental male mice with the correct genotype were obtained following screening of 79 littermates derived from 11 breeding pairs and randomized until the indicated number of mice achieved in each group). For ex vivo studies, n=2 independent biological experiments. Significances/p-values were calculated by Chi-square test (panel A), one-way ANOVA (panel B-C) and indicated as follows, *p<0.05, **p<0.01 and ***p<0.001; ns = not statistically significant.

Figure 2.. PI3Ki/MEKi combination therapy potently suppresses histone lactylation and enhances phagocytosis within activated TAM, relative to single agent controls.

(A) Single cell suspensions of PTEN/p53-deficient prostate GEM tumors were treated with copanlisib (C, 100 nM), trametinib (T, 5 nM) or their combination for 24 hours, and conditioned media (CM) was collected at the end of treatment. FACS-sorted TAM were incubated ex vivo in CM for 24 hours, followed by co-culture with CTV dye stained-AC1/SC1 cells for 2 hours. Bar graphs demonstrate histone lactylation status (B) and phagocytic activity (C) of MHC-II^hi/PD-1^hi/lo expressing TAM, relative to untreated group. FC = fold change. n=2 independent biological experiments. Significances/p-values were calculated by one-way ANOVA and indicated as follows, *p<0.05, **p<0.01 and ***p<0.001.

Figure 3.. Addition of PORCNi overcomes resistance to PI3Ki/MEKi combination therapy and induces activated TAM-mediated 100% response rate in Pb-Cre;PTEN^fl/flTrp53^fl/fl mice.

(A) PTEN/p53-deficient GEM tumor-derived AC1/SC1 cells were treated with copanlisib (C, 100 nM), trametinib (T, 5 nM) or their combination for 24 and 72 hours. In vitro CM were collected following treatment and analyzed for lactate content using colorimetry kits. (B) Pb-Cre;PTEN^fl/flTrp53^fl/fl mice with established prostate tumors were treated with indicated combinations of trametinib (T, 3 mg/kg, po, every day), copanlisib (C, 14 mg/kg, iv, every alternate day), LGK`974 (L, 3 mg/kg, po, every day), PD-1 antibody (aPD-1, 100μg/mouse, ip, every alternate day) and ADT (degarelix, 0.625 mg, single dose) for 28 days. Tumors were monitored with MRI and response rates/partial response/stable disease were determined as described in Methods. (C-D) Tumors from untreated or copanlisib+trametinib+LGK`974-treated mice were analyzed by flow cytometry for % frequency of total and MHC-II expressing TAM (CD45⁺CD11b⁺F4/80⁺ cells) (C) or analyzed for the indicated proteins by Western blotting (D). n = 5-6 mice per group for in vivo studies (experimental male mice with the correct genotype were obtained following screening of 92 littermates derived from 15 breeding pairs and were randomized until the indicated number of mice achieved in each group). For in vitro experiments, n = 3 independent biological experiments. Significances/p-values were calculated by one-way ANOVA (panel A), Chi-square test (panel B), student t-test (panel C) and indicated as follows, *p<0.05, **p<0.01, ***p<0.001 and ^#p<0.05 for partial response (panel B); ns = not statistically significant.

Figure 4.. PI3Ki/MEKi/PORCNi combination therapy suppresses lactate production from PTEN/p53-deficient GEM tumor-derived PC cells and secondary histone lactylation within activated TAM, resulting in enhanced TAM phagocytosis.

(A) AC1/SC1 cells were treated with copanlisib (C, 100 nM), trametinib (T, 5 nM), LGK`974 (L, 50 nM) or their combination for 72 hours. For mechanistic dissection, lactate (lac, 100 nmol/μL) and LGK`974 (eL, 50nM) were added to the CM collected after C+T+L and C+T treatments of AC1/SC1 cells, respectively. FACS-sorted TAM were incubated with the indicated CM for 24 hours followed by co-culture with CTV dye stained-AC1/SC1 cells for 2 hours. (B) Bar graphs demonstrate fold change (FC) in histone lactylation and phagocytic activity of MHC-II^hi/PD-1^hi/lo expressing TAM, relative to untreated group. n = 2 independent biological experiments. Significances/p-values were calculated by one-way ANOVA and indicated as follows, ***p<0.001.

Figure 5.. Intermittent long-term dosing of PI3Ki + MEKi + PORCNi results in tumor clearance of established tumors and significant prolongation of survival in Pb-Cre;PTEN^fl/fl Trp53^fl/fl mice.

(A) Schema illustrating Pb-Cre;PTEN^fl/fl Trp53^fl/fl mice dosed intermittently with copanlisib (C, 14 mg/kg, iv, every alternate day) + trametinib (T, 3 mg/kg, po, every day) + LGK`974 (L, 3 mg/kg, po, every day). (B) Kaplan-Meier survival curves were plotted for C+T+L treatment, relative to untreated control. (C) Tumors were harvested following short-term and long-term C+T+L treatment, and representative histopathologic H&E staining of formalin-fixed paraffin-embedded tissue is depicted. (D) Model illustrating the decrease in lactate production from PTEN/p53-deficient PC cells in response to PI3Ki+MEKi+PORCNi treatment suppresses histone lactylation and enhances phagocytosis of PC cells within activated TAM. n=5 mice per group (experimental male mice with the correct genotype were obtained following screening of 33 littermates derived from 4 breeding pairs and randomized until the indicated number of mice achieved in each group). Significances/p-values were calculated by Log-rank test and indicated as follows, *p<0.05.