Impact Of Sequencing Of Androgen Receptor-Signaling Inhibition (ARSI) And Ionizing Radiotherapy (RT) In Prostate Cancer: Importance Of Homologous Recombination (HR) Disruption

Abstract

The benefit of combining ARSI with RT for the treatment of localized prostate cancer is well established. The mechanism of action has been attributed to disruption of non-homologous end-joining (NHEJ) repair via impaired DNA-dependent protein kinase (DNA-PK) function. However, clinical trials have demonstrated inconsistent benefits from combining ARSI and RT, which we hypothesized might be explained by the treatment sequence of ARSI and RT. We exposed four prostate cancer cell lines (LNCaP, LNCaP-AR, C42B, LAPC-4) to ARSI via charcoal stripped serum and/or enzalutamide, a next-generation anti-androgen. ARSI was combined with fractionated RT in three sequences: neoadjuvant, concurrent, or adjuvant. The effects of ARSI sequencing were measured with clonogenic assays, immunoblotting for signaling proteins, qPCR of AR target genes, cell cycle analysis, DNA damage and repair pathway assays, and xenograft animal studies. Clinical validation was performed by meta-analysis of individual patient data from two randomized trials comparing neoadjuvant versus adjuvant ARSI with RT. Across all prostate cancer cell lines, adjuvant sequencing of ARSI after RT was significantly more effective at killing colony forming cells (20 to 50-fold, p<0.01) and decreasing the transcription and translation of downstream AR target genes (5-fold, p<0.01). This effect was recapitulated in xenograft studies showing that adjuvant ARSI was the only regimen that slowed prostate cancer tumor growth more than RT alone. Adjuvant inhibition of the NHEJ pathway with a DNA-PK inhibitor (NU 7441) did not recapitulate the effect of adjuvant ARSI as confirmed with functional NHEJ assays. Rather, we found adjuvant ARSI treatment impaired HR by decreasing the phosphorylation of nuclear ATM, CHK2, and p53 in addition to decreasing nuclear Rad51 and increasing γH2AX staining. Adjuvant ARSI doubled (p<0.05) the proportion of cells in the G2/M phase which—in the setting of double-strand breaks (DSBs) unrepaired by HR—may explain the greater cell death seen with adjuvant treatment. Finally, we validated our preclinical findings in a meta-analysis of patient-level data, confirming that adjuvant ARSI was associated with superior progression-free survival compared to neoadjuvant/concurrent ARSI (HR: 1.25 (95%CI: 1.07-1.47), p = 0.01). We demonstrate that sequencing of ARSI and RT is of critical importance and clarify a possible mechanism. Adjuvant ARSI—rather than neoadjuvant or concurrent—is associated with the greatest tumor control with RT. The beneficial effect of adjuvant ARSI is not explained by impaired NHEJ. Rather it appears to be mediated through disruption of HR and cell cycle checkpoints in response to DNA damage, which allows prostate cancer cells to proceed to G2 and mitosis with unrepaired DSBs.