Suppression of tumor cell lactate-generating signaling pathways eradicates murine PTEN/p53-deficient aggressive-variant prostate cancer via macrophage phagocytosis

Abstract

Purpose: PTEN loss-of-function/PI3K pathway hyperactivation occurs in ∼50% of metastatic, castrate-resistant prostate cancer patients, resulting in poor therapeutic outcomes and resistance to immune checkpoint inhibitors across multiple malignancies. Our prior studies in prostate-specific PTEN/p53-deleted genetically engineered mice (Pb-Cre;PTEN ^fl/fl Trp53 ^fl/fl GEM) with aggressive-variant prostate cancer (AVPC) demonstrated feedback Wnt/β-catenin signaling activation in 40% mice resistant to androgen deprivation therapy (ADT)/PI3K inhibitor (PI3Ki)/PD-1 antibody (aPD-1) combination, resulting in restoration of lactate cross-talk between tumor-cells and tumor-associated macrophages (TAM), histone lactylation (H3K18lac) and phagocytic suppression within TAM. Here, we targeted immunometabolic mechanism(s) of resistance to ADT/PI3Ki/aPD-1 combination, with the goal of durable tumor control in PTEN/p53-deficient PC.

Experimental design: Pb-Cre;PTEN ^fl/fl Trp53 ^fl/fl GEM were treated with either ADT (degarelix), PI3Ki (copanlisib), aPD-1, MEK inhibitor (trametinib) or Porcupine inhibitor (LGK 974) as single agents or their combinations. MRI was used to monitor tumor kinetics and immune/proteomic profiling/ ex vivo co-culture mechanistic studies were performed on prostate tumors or established GEM-derived cell lines.

Results: We tested whether Wnt/β-catenin pathway inhibition with LGK 974 addition to degarelix/copanlisib/aPD-1 therapy enhances tumor control in GEM, and observed de novo resistance due to feedback activation of MEK signaling. Based on our observation that degarelix/aPD-1 treatment resulted in partial inhibition of MEK signaling, we substituted trametinib for degarelix/aPD-1 treatment, and observed a durable tumor growth control of PI3Ki/MEKi/PORCNi in 100% mice via H3K18lac suppression and complete TAM activation within TME.

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.

Statement of translational relevance: PTEN loss-of-function occurs in ∼50% of mCRPC patients, and associated with poor prognosis, and immune checkpoint inhibitor resistance across multiple malignancies. Our prior studies have demonstrated that ADT/PI3Ki/PD-1 triplet combination therapy controls PTEN/p53-deficient PC in 60% of mice via enhancement of TAM phagocytosis. Here, we discovered that resistance to ADT/PI3K/PD-1 therapy occurred via restoration of lactate production via feedback Wnt/MEK signaling following treatment with PI3Ki, resulting in inhibition of TAM phagocytosis. Critically, co-targeting of PI3K/MEK/Wnt signaling pathways using an intermittent dosing schedule of corresponding targeted agents resulted in complete tumor control and significantly prolonged survival without significant long-term toxicity. Collectively, our findings provide "proof-of-concept" that targeting lactate as a macrophage phagocytic checkpoint controls growth of murine PTEN/p53-deficient PC and warrant further investigation in AVPC clinical trials.

Figure 1.. The addition of PORCNi is unable to control tumor growth in Pb-Cre;PTEN^fl/fl Trp53^fl/fl mice that are resistant to ADT/PI3Ki/aPD-1 combination therapy, due to feedback upregulation of MEK signaling.

(A) Pb-Cre;PTEN^fl/fl Trp53^fl/fl mice with established solid prostate tumors were treated with copanlisib (14 mg/kg, iv, every alternate day, 42 days) + degarelix (0.625 mg, every month, 42 days, ADT) + PD-1 antibody (100 _μg/dose, every alternate day, 42 days, aPD-1). On day 28, non-responder mice received LGK`974 (porcupine inhibitor, 3 mg/kg, po, daily, 14 days). The change in solid tumor volume is qualitatively represented by MRI for ADT + copanlisib+ aPD-1 + LGK`974 treated group. Total tumor area and cystic parts of tumors are outlined by yellow and red colors on MRI, respectively. (B) At the end of 42 days of treatment, tumor extracts obtained from mice treated with indicated drug(s) were profiled by western blot analysis for PI3K, MEK and Wnt signaling. (C) Flow cytometry was performed to assess infiltration frequency (upper panel) and activation status (MHC-II^hi, lower panel) of tumor-associated macrophages (TAM, CD11b+Ly6g-Ly6c-F4/80+), relative to control mice that responded to ADT + copanlisib + aPD-1 treatment. Bar graph represents. n=2 mice per group. Significances/p-values were calculated by one-way ANOVA and indicated as follows, *p<0.05 and **p<0.01.

Figure 2.. 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 PD-1 antibody (aPD-1, 100 _μg/mouse, ip, every alternate day) alone or in combination with ADT (degarelix, 0.625mg, single dose, castration) for 28 days. Western blot analyses were performed for indicated protein markers on prostate tumor extracts. (B) 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. (C) 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). (D) 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. For in vivo studies, n=5–6 mice per group and for ex vivo, n=2 independent experiments. Significances/p-values were calculated by Chi-square test (panel B), one-way ANOVA (panel C-D) and indicated as follows, *p<0.05, **p<0.01 and ***p<0.001; ns = not statistically significant.

Figure 3.. 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 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 4.. 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) 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. Western blot analyses were performed for indicated protein markers on PTEN/p53-deficient prostate tumor extracts. (B) 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. (C) 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. 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) (D) or analyzed for the indicated proteins by Western blotting (E). n = 5–6 mice per group for in vivo studies; for in vitro experiments, n = 3 independent experiments. Significances/p-values were calculated by one-way ANOVA (panel B and D), Chi-square test (panel C) and indicated as follows, *p<0.05, **p<0.01, ***p<0.001 and ^#p<0.05 for partial response (panel A); ns = not statistically significant.

Figure 5.. 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/<sub>μ</sub>L) 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 experiments. Significances/p-values were calculated by one-way ANOVA and indicated as follows, ***p<0.001.

Figure 6.. 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=3 mice per group. Significances/p-values were calculated by Log-rank test and indicated as follows, *p<0.05.