Mechanisms of Resistance to Prostate-Specific Membrane Antigen-Targeted Radioligand Therapy in a Mouse Model of Prostate Cancer

Abstract

Prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) is effective against prostate cancer (PCa), but all patients relapse eventually. Poor understanding of the underlying resistance mechanisms represents a key barrier to development of more effective RLT. We investigate the proteome and phosphoproteome in a mouse model of PCa to identify signaling adaptations triggered by PSMA RLT. Methods: Therapeutic efficacy of PSMA RLT was assessed by tumor volume measurements, time to progression, and survival in C4-2 or C4-2 TP53^-/- tumor-bearing nonobese diabetic scid γ-mice. Two days after RLT, the proteome and phosphoproteome were analyzed by mass spectrometry. Results: PSMA RLT significantly improved disease control in a dose-dependent manner. Proteome and phosphoproteome datasets revealed activation of genotoxic stress response pathways, including deregulation of DNA damage/replication stress response, TP53, androgen receptor, phosphatidylinositol-3-kinase/AKT, and MYC signaling. C4-2 TP53^-/- tumors were less sensitive to PSMA RLT than were parental counterparts, supporting a role for TP53 in mediating RLT responsiveness. Conclusion: We identified signaling alterations that may mediate resistance to PSMA RLT in a PCa mouse model. Our data enable the development of rational synergistic RLT-combination therapies to improve outcomes for PCa patients.

Keywords: DNA damage response; [177Lu]Lu-PSMA; [225Ac]Ac-PSMA; prostate cancer; proteomics/phosphoproteomics.

Graphical abstract

FIGURE 1.

Optimizing treatment activities for [²²⁵Ac]Ac- and [¹⁷⁷Lu]Lu-PSMA RLT. (A) Individual tumor growth curves after [¹⁷⁷Lu]Lu-PSMA (12 tumors, 6 mice per group; NT vs. RLT, P ≤ 0.0018 [7 wk]; 30 vs. 120 MBq, P > 0.99 [7 wk] and P = 0.032 [16 wk]). (B, top) Survival: 4.8 wk (NT), 15 wk (30 MBq), not reached (120 MBq) (6 mice per group: all P ≤ 0.001). (B, bottom) TTP: 6.6 wk (NT), not reached (30 and 120 MBq) (6 mice per group; NT vs. 30 MBq, P = 0.153; all other P ≤ 0.014). (C) Individual tumor growth curves after [²²⁵Ac]Ac-PSMA RLT (8 mice per group; NT vs. RLT, P ≤ 0.027 [6 wk]; 20 vs. 40 or 100 kBq, P < 0.023 [15 wk], 40 vs. 100 kBq, P > 0.99). (D, top) Survival: 4.5 wk (NT), 16 wk (20 kBq), 30 wk (40 kBq), 19 wk (100 kBq) (mice per group; P ≤ 0.0137, except 40 vs. 100 kBq [P = 0.0783]; 20 vs. 100 kBq [P = 0.6203]). (D, bottom) TTP: 3.5 wk (NT), 15 wk (20 kBq), not reached (40 kBq, 100 kBq) (8 mice per group; P ≤ 0.0012, except 40 vs. 100 kBq [P = 0.679]).

FIGURE 2.

Proteomic analyses of PCa tumors reveal PSMA RLT–induced alterations. (A) Experimental workflow (also applies to Fig. 3). (B) Volcano plots highlighting changes in protein levels relative to untreated groups (188 significant proteins for [¹⁷⁷Lu]Lu-PSMA RLT and 241 for [²²⁵Ac]Ac-PSMA RLT). (C) Gene ontology analysis of 2 datasets revealing commonly activated pathways. (D) Transcription factor enrichment analysis on differentially expressed proteins. Identified transcription factors have at least 4 associated targets. Graphs represent data from 3 tumors per group for [¹⁷⁷Lu]Lu-PSMA RLT and 5 tumors per group for [²²⁵Ac]Ac-PSMA RLT. FDR = false-discovery rate; LC = liquid chromatography.

FIGURE 3.

Phosphoproteomic analyses of PCa tumors reveal PSMA RLT–induced alterations. (A) Volcano plots of identified phosphopeptides (512 significant phosphopeptides for [¹⁷⁷Lu]Lu-PSMA RLT and 405 for [²²⁵Ac]Ac-PSMA RLT). (B) Kinase-substrate enrichment analysis identifying kinases with at least 5 substrates. Graphs represent data from 3 tumors per group for [¹⁷⁷Lu]Lu-PSMA RLT and 5 tumors per group for [²²⁵Ac]Ac-PSMA RLT. FDR = false-discovery rate.

FIGURE 4.

Nucleotide levels are significantly altered by [²²⁵Ac]Ac-PSMA RLT compared with untreated controls (P ≤ 0.0499). Mean NT fold-change ± SD is shown (5 tumors per group). *Significant difference. dADP = deoxyadenosine diphosphate; dAMP = deoxyadenosine monophosphate; dATP = deoxyadenosine triphosphate; dCMP = deoxycytidine monophosphate; dGTP = deoxyguanosine triphosphate.

FIGURE 5.

TP53 loss renders PCa resistant to PSMA RLT. (A) Tumor growth of C4-2 (left) and C4-2 TP53^−/− (right) tumors treated with 15 MBq of [¹⁷⁷Lu]Lu-PSMA RLT. Geometric mean and 95% CI (n = 8–10 mice per group) are shown. (B) TTP: C4-2 NT, 27 d; C4-2 RLT, undefined (P = 0.0016); C4-2 TP53^−/− NT, 14 d; C4-2 TP53^−/− RLT, 30.5 d (P = 0.3250). (C) Survival: C4-2 NT, 38 d; C4-2 RLT, undefined (P = 0.0044); C4-2 TP53^−/− NT, 21 d; C4-2 TP53^−/− RLT, 42 d (P = 0.33939). *Significant difference. C4-2p53^−/− NT = C4-2 TP53^−/− NT.