Background There has been renewed interest in immunotherapy for the treatment of advanced prostate cancer (PC), partly based on the anti-tumor immune activation that occurs with ADT, and partly based on the clinical responses to immune checkpoint inhibitors (ICI) targeting CTLA-4 and PD-1/PD-L1 in other cancers.^1-3 However, only 10-25% of metastatic castrate-resistant prostate cancer (mCRPC) patients respond to ICI, with a lack of durable benefit in the majority of patients.^4-5 PTEN LOF alterations, which occur in approximately 50-75% of mCRPC patients, are associated with poor prognosis, development of metastases,^6-8 and immunosuppressive tumor microenvironment.^9-11 Given the aggressive natural history and poor therapeutic outcomes of PTEN-mutant advanced PC to standard-of-care hormonal therapies,⁶ chemotherapies¹² and ICI,¹⁰ a deeper understanding of immune evasion mechanisms is critical for the discovery of new therapeutic strategies to effectively treat this molecular subset of AVPC.

Methods Prostate-specific PTEN/p53-deficient genetically engineered mice (GEM) (40) were screened for autochthonous prostate tumor development and monitored for response to therapy by ultrasound and MRI, respectively. Following the development of 150-200 mm³ solid tumors, the mice were treated with either androgen deprivation therapy (degarelix), PI3K inhibitor (copanlisib), or PD-1 antibody, as single agents or their combinations. Harvested tumors following in vivo treatment underwent flow cytometry, or utilized for ex vivo studies on single cell suspensions or sorted TAM. Single cell RNAseq on human metastatic bone and lymph node samples were performed using established methods.¹³

Results We performed co-clinical trials in prostate-specific PTEN/p53-deficient genetically engineered mice, and discovered that recruitment of PD-1-expressing tumor-associated macrophages (TAM) thwarts androgen deprivation therapy (ADT)/PI3K inhibitor (PI3Ki) combination-induced tumor control. Strikingly, we observed TAM-dependent ~3-fold increased anti-cancer response with ADT/PI3Ki/PD-1 antibody (aPD-1) combination. Mechanistically, decreased lactate production from PI3Ki-treated tumor cells suppressed histone lactylation within TAM, resulting in their phagocytic activation, which was augmented by ADT/aPD-1 treatment and attenuated by feedback activation of Wnt/β-catenin-pathway. Furthermore, single-cell RNA-sequencing analysis in mCRPC patient biopsy samples revealed a direct correlation between high glycolytic activity and TAM phagocytosis suppression.

Conclusions Our findings demonstrate that immunometabolic strategies to reverse lactate and PD-1-mediated TAM immunosuppression by PI3Ki and aPD-1, respectively, in combination with ADT, controls tumor growth and warrants further clinical investigation in PTEN/p53-deficient mCRPC.

References

1. Wolchok JD, Chiarion-Sileni V, Gonzalez R, Rutkowski P, Grob JJ, Cowey CL, et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N Engl J Med 2017;377(14):1345–56. doi: 10.1056/NEJMoa1709684.

2. Sharma P, Allison JP. Dissecting the mechanisms of immune checkpoint therapy. Nat Rev Immunol 2020;20(2):75–6. Doi: 10.1038/s41577-020-0275-8.

3. Gamat M, McNeel DG. Androgen deprivation and immunotherapy for the treatment of prostate cancer. Endocr Relat Cancer 2017;24(12):T297–T310. Doi: 10.1530/ERC-17-0145.

4. Antonarakis ES, Piulats JM, Gross-Goupil M, Goh J, Ojamaa K, Hoimes CJ, et al. Pembrolizumab for treatment-refractory metastatic castration-resistant prostate cancer: multicohort, open-label phase II KEYNOTE-199 Study. J Clin Oncol 2020;38(5):395–405. doi: 10.1200/JCO.19.01638.

5. Sharma P, Pachynski RK, Narayan V, Flechon A, Gravis G, Galsky MD, et al. Initial results from a phase II study of nivolumab (NIVO) plus ipilimumab (IPI) for the treatment of metastatic castration-resistant prostate cancer (mCRPC; CheckMate 650). Journal of Clinical Oncology 2019;37:142.

6. Jamaspishvili T, Berman DM, Ross AE, Scher HI, De Marzo AM, Squire JA, et al. Clinical implications of PTEN loss in prostate cancer. Nat Rev Urol 2018;15(4):222–34. Doi: 10.1038/nrurol.2018.9.

7. Wang S, Gao J, Lei Q, Rozengurt N, Pritchard C, Jiao J, et al. Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 2003;4(3):209–21. Doi: 10.1016/s1535-6108(03)00215-0.

8. Robinson D, Van Allen EM, Wu YM, Schultz N, Lonigro RJ, Mosquera JM, et al. Integrative Clinical Genomics of Advanced Prostate Cancer. Cell 2015;162(2):454. Doi: .1016/j.cell.2015.06.053.

9. Di Mitri D, Mirenda M, Vasilevska J, Calcinotto A, Delaleu N, Revandkar A, et al. Re-education of Tumor-Associated Macrophages by CXCR2 Blockade Drives Senescence and Tumor Inhibition in Advanced Prostate Cancer. Cell Rep 2019;28(8):2156–68. e5. Doi: 10.1016/j.celrep.2019.07.068. gene leads to metastatic prostate cancer. Cancer Cell 2003;4(3):209–21 doi 10.1016/s1535-6108(03)00215-0.

10. 10. Cetintas VB, Batada NN. Is there a causal link between PTEN deficient tumors and immunosuppressive tumor microenvironment? J Transl Med 2020;18(1):45. Doi: 10.1186/s12967-020-02219-w.

11. 11. Peng W, Chen JQ, Liu C, Malu S, Creasy C, Tetzlaff MT, et al. Loss of PTEN Promotes Resistance to T Cell-Mediated Immunotherapy. Cancer Discov 2016;6(2):202–16. Doi: 10.1158/2159-8290.CD-15-0283.

12. 12. Toso A, Revandkar A, Di Mitri D, Guccini I, Proietti M, Sarti M, et al. Enhancing chemotherapy efficacy in Pten-deficient prostate tumors by activating the senescence-associated antitumor immunity. Cell Rep 2014;9(1):75–89. doi: 10.1016/j.celrep.2014.08.044.

13. 13. Kfoury Y, Baryawno N, Severe N, Mei S, Gustafsson K, Hirz T, et al. Human prostate cancer bone metastases have an actionable immunosuppressive microenvironment. Cancer Cell 2021;39(11):1464–78 e8. doi: 10.1016/j.ccell.2021.09.005.

Ethics Approval Murine experiments were performed in accordance with NIH guidelines and protocol approved by the Institutional Animal Care and Use Committee (IACUC) at University of Chicago (ACUP 72483-12). Bone metastatic PC samples were collected and handled in accordance to the protocol approved by the Institutional Review Board (IRB, Dana Farber/Harvard Cancer Center protocol 13-416 and Partners protocol 2017P000635/PHS). For metastatic lymph nodes of PC patients, baseline biopsies were collected and processed as mentioned in the investigator-initiated, IRB-approved clinical trial (IRB-18-0154 of Chicago, NCT03572478) of rucaparib in combination with nivolumab, co-sponsored by Clovis Oncology and Bristol Myers Squibb.