Resistance to second-generation androgen receptor antagonists in prostate cancer

Pharmacology Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,
2Department of Pharmacy & Pharmacology, Netherlands Cancer Institute, Amsterdam, Netherlands.
3Department of Clinical Pharmacy, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands.
4Genitourinary Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda,
✉e-mail: [email protected] s41585-021-00438-4

Suppression of androgen receptor (AR) signalling has been central to the treatment of prostate cancer since the discovery of the sensitivity of this disease to test­ osterone in 1941 (REF.1). Androgen deprivation therapy (ADT), defined as either surgical orchiectomy or medi­ cal castration using a luteinizing hormone­releasing hormone (LHRH) agonist or antagonist, remains the cornerstone of treatment in patients with advanced disease2,3. Complete androgen blockade is the addition of first­generation AR antagonists (FG­ARAs) (fluta­ mide, nilutamide and bicalutamide), which inhibit the AR, to treatment with ADT. This combination treatment significantly improved the overall survival of patients with treatment­naive prostate cancer in several clinical trials. However, a larger meta­analysis showed only a marginal improvement, which was not statistically significant, in overall survival with this treatment approach, emphasizing the limitations in the potency of FG­ARAs for AR inhibition4–7. Furthermore, in the setting of castration- resistant prostate cancer (CRPC), FG­ARAs did not improve survival, sug­ gesting androgen­independent mechanisms of cancerprogression. Docetaxel, a microtubule­stabilizing taxane chemotherapy, was approved by the Food and Drug Administration (FDA) for the treatment of meta­ static CRPC (mCRPC) in 2004 (REF.8). In 2011, the FDA also approved abiraterone acetate, a cytochrome P450 17A1 (CYP17) inhibitor that blocks the production of intra­tumoural androgens and is co­administered with prednisone for the treatment of mCRPC9.

Subsequent research in AR signalling resulted in the approval of enzalutamide by the FDA in 2012. This agent — the first of the second­generation ARAs (SG­ARAs) — improved overall survival in patients with mCRPC10,11. SG­ARAs are characterized by improved potency compared with FG­ARAs, resulting in higher affinity for the AR and the capability to translocate into the nucleus while bound to the AR to further disrupt mechanisms of AR­mediated transcription12. In 2018, the FDA expanded the licence of enzalutamide to include patients with non­metastatic CRPC (nmCRPC). Additional clinical trials led to the approval of two other SG­ARAs, apalutamide and darolutamide, for use in nmCRPC, approved in 2018 and 2019 respectively13.

Castration-resistant prostate cancer (CRPC). A diagnosis of prostate cancer with disease progression (defined by PSA progression in the non- metastatic setting (nmCRPC), and radiographic progression in the metastatic setting (mCRPC)) despite castration levels of testosterone, achieved through long-standing androgen deprivation therapy or via orchiectomy (surgical removal of the testes).Biomarkers Biological molecules isolated from patients with cancer (in blood, tumour, urine etc.) that are informative of molecular features of the disease and could be utilized to better understand response to treatment.

Enzalutamide and apalutamide were also approved for the treatment of patients with metastatic castration- sensitive prostate cancer (mCSPC) in 2019 (REF.14) (TABLE 1). Despite successes in drug development for the treatment of prostate cancer, SG-ARAs remain suscepti- ble to acquired resistance, which limits the durability of response and necessitates further research15,16, focusing on clinically relevant biomarkers of resistance (for exam- ple, circulating tumour cells (CTCs), AR splice variant 7 (AR-V7)) and treatment strategies to limit and/or over- come potential pathways of resistance16,17. The purpose of this Review is to discuss clinically relevant mecha- nisms of acquired resistance to SG-ARAs and outline strategies to combat acquired resistance and prolong overall survival.

Genomic alterations in SG-ARA resistance
Acquired resistance to SG- ARAs can result from intra-tumoural heterogeneity and/or the enrichment of tumour subclones that are inherently resistant to ther- apy. An analysis of data from the Stand Up To Cancer– Prostate Cancer Foundation (SU2C- PCF) cohort (n = 150) identified a framework of common genomic alterations in patients with mCRPC18. These findings high- lighted the AR, phosphoinositide 3-kinase (PI3K) and Wnt protein signalling pathways, as well as cell cycle and DNA repair pathways, as potential targets for preci- sion oncological therapies (TABLE 2). Another study that used the Memorial Sloan Kettering-integrated mutation profiling of actionable cancer targets (MSK-IMPACT) assay to sequence the genomes of 504 tumours, expanded upon this framework by summarizing changes in the fre- quency of clinically relevant genomic alterations from localized prostate cancer to mCSPC and mCRPC19 (TABLE 2). The MSK-IMPACT study showed increased prevalence in several genomic alterations following castration resistance, including ATM serine/threonine kinase (ATM), tumour protein p53 (TP53), retino- blastoma 1 (RB1), phosphatase and tensin homologue (PTEN) and, most significantly, the AR. Numerous sub- sequent studies have confirmed these findings20–30, which provide potential targets for current drug development
efforts. With the expansion of FDA approval for use of SG-ARAs earlier in the disease course (that is, mCSPC, nmCRPC), the presence of genomic aberrations poten- tially enables optimization of therapy and improved clin- ical outcomes. Mechanisms of resistance to SG-ARAs (and abiraterone) can involve reactivation or persistent activation of the AR, AR bypass pathways31 (FIG. 1) and androgen or AR-independent mechanisms18 (FIG. 2).

Androgen receptor pathway. AR genomic alterations are highly prevalent in patients with mCRPC in compar- ison with those with mCSPC, owing to the selectivity of cells able to adapt and proliferate in the low andro- gen environment established with continuous ADT18,19. A wide variety of AR gene alterations, which result in increased likelihood of activation of the AR path- way despite reduced presence of androgens, has been reported, including amplifications, point mutations and splice variants32 (FIG. 1); such alterations facilitate resist- ance to AR-targeted therapies. Additional genomic alter- ations that have been associated with the AR pathway in advanced prostate cancer but have not been as fully characterized in their role in promoting SG-ARA resis- tance include SPOP, FOXA1 and ZBTB16 mutations (missense and deletions) and TMPRSS2–ERG fusions18,33. At diagnosis of mCRPC, common AR alterations present in tumour biopsy samples are amplifications18. Analysis of cell-free DNA (cf-DNA) in plasma samples collected from patients following enzalutamide treat- ment shows a higher frequency of AR copy number gain (associated with AR amplification) than cf-DNA samples from patients treated with abiraterone28,29. Analysis of cf-DNA in 65 patients highlighted an increased risk of early disease progression in patients with AR amplifi- cation compared with patients without amplification (HR 2.92, 95% CI 1.59–5.37)30. The presence of these amplifications demonstrates a compensatory resistance mechanism via increasing AR expression in response to the potent AR inhibition of enzalutamide, which results in diminished efficacy of treatment over time34. AR copy number gain has considerable inter-patient variability in circulating tumour DNA (ctDNA), the calculated proportion of tumour-derived cf-DNA. In a study of patients with mCRPC treated with either abiraterone or enzalutamide, a median of 7.1 AR copies (maximum of 56) were reported in 65 of 115 patients24. AR gene structural rearrangements truncating the ligand-binding domain (LBD) identified in patients with AR copy gains were associated with primary resistance24.

AR point mutations are also prevalent in cf-DNA samples collected from patients with mCRPC. Common point mutations found in these patients include F877L, H875Y, T878A and L702H, which are all located in the AR LBD, the target of both FG- ARAs and SG-ARAs24,29,30. H875Y, T878A and L702H mutations
have been observed in patients with mCRPC pre- and post-enzalutamide treatment, with evidence of the ini- tial emergence of such mutations at the time of disease progression following enzalutamide treatment in 6 of 65 patients (9%)30. H875Y and T878A mutations are associated with AR promiscuity, enabling oestrogens and progesterone to activate the AR pathway35. H875YFG-ARA, first-generation androgen receptor antagonist; mCRPC, metastatic castration-resistant prostate cancer; mCSPC, metastatic castration-sensitive prostate cancer; MFS, metastasis-free survival; NA, not available; nmCRPC, non-metastatic castration-resistant prostate cancer; NR, not reported or evaluated; OS, overall survival; PFS, progression-free survival; SG-ARA, second-generation androgen receptor antagonist; TPP, time to PSA progression. aPrimary end point of the study. bNumber of deaths per group at the time of analysis was reported. cData reported in percentages indicate that the median was not reached at time of publication; thus, it is reported as a percentage of patients with the indicated end point and L702H AR mutations can enable glucocorticoid binding to the AR; L702H is frequently associated with abiraterone therapy and AR activation via prednisone31,36. Resistance to enzalutamide treatment has been well-characterized in patients, and research into resist- ance to other contemporary SG- ARAs is ongoing. Both enzalutamide and apalutamide have agonistic activity when binding to the ARF877L mutation in vitro, thereby activating the AR pathway37; enzalutamide was also shown to be a stronger agonist in the presence of both T878A and F877L mutations versus F877L alone in vitro38. Conversely, darolutamide has shown strong antagonistic activity to the F877L mutation in addition to other LBD-containing AR variants in vitro12,39. Despite established cell lines with this mutation in vitro, clini- cal detection of the F877L mutation in cf-DNA samples following SG-ARA treatment has been limited in com- parison with other mechanisms of resistance29,40. Two clinical studies showed that small populations of patients treated with apalutamide had a low prevalence of F877L mutations (3 of 29 patients40 and 3 of 82 patients41 in each study, respectively), which is similar to findings in patients treated with enzalutamide29.

Despite very similar resistance patterns in patients treated with abiraterone and enzalutamide, key differ- ences include an increased role of the ARL702H and ART878A mutations in abiraterone resistance, and an increased role of the F877L mutation and AR bypass facilitated via glucocorticoid receptor (GR)-mediated signalling in enzalutamide resistance31.Resistance to enzalutamide and abiraterone has also been associated with expression of AR-V7, a con- stitutively active AR variant that lacks the LBD that is critical for SG-ARA activity42,43. AR-V7 mRNA expres- sion in CTCs, assessed via the Adna test platform, was found in 39% of 31 patients with mCRPC treated with enzalutamide. Compared with AR-V7– patients, AR-V7+ patients treated with enzalutamide had shorter progression-free survival (PFS) (median 1.4 months ver- sus 6.0 months, P < 0.001) and overall survival (median
5.5 months versus ‘not reached’, P = 0.002)44. In addition to the mRNA-based Adna test platform, AR-V7 can also be detected using the EPIC sciences CTC-based plat- form, an immunofluorescence-based detection assay that measures AR-V7 protein localized in the nucleus45. A multicentre, prospective-blinded study of 118 men with mCRPC found that both platforms were sufficient to detect AR-V7 and show decreased PFS and overall survival in those who were positive for AR-V7 following treatment with either enzalutamide or abiratarone46.Preclinical studies have shown that apalutamide and darolutamide treatment confer similar resistance pat- terns to enzalutamide by increasing Aldo-ketoreductase shown to confer resistance to both apalutamide and enzalutamide50. Further characterization of apalutamide and darolutamide resistance is ongoing.

Lineage plasticity. In comparison to mCSPC, mCRPC is enriched for alterations in the key tumour suppres- sors RB1 and TP53 (REFS18,19,23). In patients treated with abiraterone or enzalutamide, alterations in AR, RB1 and TP53 were significantly associated with shorter time to progression (univariate analyses with P < 0.05), with RB1 alterations having a strong association with poor sur- vival (relative risk of death 3.31, 95% CI 1.64–6.67)21. Loss of both TP53 and RB1 enables tumours to become AR independent, upregulates genes associated with cellular plasticity and pluripotency (such as SOX2 and BRN2) and activates neuroendocrine differentiation51–53. Epigenetic reprogramming can alter DNA methylation and upregulate the histone methyltransferase epige- netic modifier, enhancer of zeste homologue 2 (EZH2). Inhibition of EZH2 has been shown to restore sensitivity of prostate cancer cells to SG-ARAs54. Overexpression of EZH2 and the MYCN proto-oncogene promotes ATM upregulation, facilitating enzalutamide resistance via neuroendocrine differentiation54,55. The resulting stem-cell-like phenotype is frequently associated with neuroendocrine or small-cell lineages of prostate can- cer, enabling adaptation to selective pressure to confer enzalutamide resistance56. In a study of 160 patients with progressive mCRPC and prior histological evidence of adenocarcinoma, 27 patients had small-cell histology upon biopsy of a metastasis; 4 of these patients had previously received enzalutamide and 6 had previously received both abiraterone and enzalutamide. The prev- alence of this treatment-emergent small-cell cancer in this population was higher than in previous reports of de novo small-cell cancer of the prostate (<1%), suggest- ing the role of an alternative transcriptional programme arising from the plasticity of cells in response to per- sistent AR targeting and potential utility of targeting epigenetic modifiers such as EZH2. Overall survival of patients with treatment-emergent small-cell cancer was significantly reduced in comparison with patients with- out such tumour features (44.5 versus 36.6 months, HR 2.02, 95% CI 1.07–3.82, P = 0.027)57.

RB1 loss alone alters cell cycle function by preventing the inhibition of transcription factor E2F, which upregu- lates cyclins and cyclin-dependent kinases (CDKs) to drive progression to S phase, resulting in uncontrolled cell proliferation58. Loss of chromatin remodeller chro- modomain helicase DNA-binding protein 1 (CHD1) was also found to be associated with chromatin dysreg- ulation and lineage plasticity, resulting in heterogene- ous mechanisms of enzalutamide resistance, including tumours driven by the GR, and the transcription factors Epigenetic reprogramming Changes in transcription and modifications to chromatin, resulting in loss of characteristics of the original cell, and acquisition of a new molecular signature, irrespective of genomic 1 member C3 (AKR1C3) expression via activa- tion of the steroid biosynthesis pathway. Targeting of AKR1C3 leads to AR-V7 inhibition and re-sensitization of prostate cancer cells to apalutamide and daroluta- mide, confirming a role of AR-V7 in conferring resist- ance to SG-ARAs47,48. Loss of TLE family member 3, transcriptional corepressor (TLE3) leads to upregula- tion of the GR49 and activation of this pathway has been T-box transcription factor (TBX2), POU domain, class 3, transcription factor 2 (BRN2) and nuclear receptor subfamily 2 group F member 1 (NR2F1)59.

In addition to neuroendocrine differentiation, enzalutamide treatment has been shown to induce epithelial–mesenchymal transition (EMT), a process that enables epithelial cells to acquire a more mesenchymal phenotype with increased metastatic potential. Snail,a transcription factor responsible for EMT-mediated plas- ticity, was shown to induce enzalutamide resistance via promotion of AR activity in the absence of full-length AR and AR-V7 (REFS60,61). The transforming growth factor-β (TGFβ) pathway can also activate EMT in response to enzalutamide treatment via interaction of TGFβ with mothers against decapentaplegic homologue 2 (SMAD2) via downregulation of forkhead box A1 (FOXA1)62. AR-mediated acquired resistance mechanisms to SG-ARAs and potential treatments under investigation. Testosterone synthesis (1) — inhibition of cytochrome P450 17A1 (CYP17) downregulates production of testosterone in prostate cancer cells. Abiraterone inhibits both CYP17 lyase and CYP17 hydroxylase9, whereas seviteronel and galeterone target CYP17 lyase specifically98,99,105,106. Androgen receptor (AR) (2) — AR amplification increases AR expression to outcompete inhibition28,29. AR-V7 is constitutively active and does not have a ligand-binding domain (LBD); thus, it is unable to bind to second-generation androgen receptor antagonists (SG-ARAs)42,43. Niclosamide degrades AR-V7 (REFS103,104). AR point mutations in the LBD can alter binding. Enzalutamide, apalutamide and darolutamide target wild-type (WT) AR and T878A, H875Y and L702H mutated AR, whereas darolutamide can also target the F877L mutation12. EPI-7386 targets the N- terminal domain in AR-V7 and ARs with LBD point mutations102.

Prostate-specific membrane antigen (PSMA) (3) — PSMA is expressed on the extracellular surface of prostate cancer cells and is upregulated in response to enzalutamide116,117. 177Lu-PSMA-617 is a PSMA-targeted radiopharma- ceutical119,120. Bipolar androgen therapy (4) — administration of supra- physiological testosterone downregulates the overexpression of the AR, a result of enzalutamide resistance121–124. Glucocorticoid receptor (GR) signalling (5) — upregulation of the GR in response to persistent AR targeting leads to a pathway to bypass AR inhibition125. CORT125281 is a GR antagonist126. Transcriptional targeting (6) — the use of an antisense oligonucleotide such as ARRx could target AR at the mRNA level, eliminating problems associated with targeting the LBD107,108. Bromodomain and extraterminal (BET) inhibition (7) — combining a BET inhibitor such as ZEN003694 with an SG-ARA could downregulate AR-V7- mediated signalling and bromodomain-containing protein 4 (BRD4) expression111,112,115.

Non-AR-mediated acquired resistance to SG-ARAs and potential treatments under investigation. Phosphoinositide 3-kinase–protein kinase B–mammalian target of rapamycin (PI3K–AKT–mTOR) pathway (1) — genomic alterations such as phosphatase and tensin homologue (PTEN) loss result in dysregulated activation18,19,21,22. Therapeutic agents inhibiting this pathway include ipatasertib and capivasertib (target AKT)148–151, MLN0128 and everolimus (target mTOR)152,156, GSK2636771 (targets PI3K)155 and LY3023414 (targets both PI3K and mTOR)154. Wnt–β-catenin pathway (2) — mutations altering this pathway include β-catenin (CTNNB1) activating mutations and APC regulator of Wnt signalling pathway (APC) inactivating mutations19,26,86. Mismatch repair defects (3) — mutations of DNA mismatch repair (dMMR) proteins and/or the presence of increased microsatellite instability or tumour mutational burden can upregulatePDL1 expression in tumour cells73. Pembrolizumab is a PD1 inhibitor, and durvalumab and atezolizumab are PDL1 inhibitors74–76,145,146,163,164. Hypoxia and angiogenesis (4) — in response to hypoxic tumour environments, alternative cell signalling promotes angiogenesis94. Cabozantinib is a dual hepatocyte growth factor receptor (MET) and vascular endothelial growth factor receptor 2 (VEGFR2) inhibitor160–164. Hypoxia- inducible factor 1α (HIF1α), a transcription factor involved in cross-talk with the androgen receptor (AR) and β-catenin, can be modulated with indirect targeting via camptothecin94,165,166. CCS1477 is an inhibitor of CREB-binding protein-E1A binding protein (CBP-p300)167,168. Epithelial–mesenchymal transition (EMT) (5) — EMT increases the metastatic potential of cells. Snail induces EMT and promotes AR activation in the absence of AR proteins60,61.

Metformin inhibits enzalutamide-induced activation of EMT via inhibition of the transforming growth factor-β–mothers against decapentaplegic homologue 2–matrix metalloproteinase 9 (TGFβ–SMAD2–MMP9) and TGFβ–signal transducer and activator of transcription 3 (STAT3) axes131,132. Epigenetics and lineage plasticity (6) — loss of retinoblastoma 1 (RB1) leads to AR independence and upregulation of SRY-box transcription factor 2 (SOX2), which activates alternative signalling pathways51–53. RB1 loss prevents inhibition of E2 transcription factor (E2F)58 and leads to overexpression of enhancer of zeste homologue 2 (EZH2), which, together with overexpression of MYCN proto-oncogene, results in neuroendocrine differentiation54,55. Tazemetostat inhibits EZH2 (REF.130) and alisertib is an aurora kinase A (AURKA) inhibitor, which disrupts MYCN signalling127. DNA damage and homologous recombination (7) — mutations in DNA damage repair (DDR) genes affect DNA repair signalling17. In the presence of such mutations, poly(ADP-ribose) polymerase (PARP) inhibitors such as olaparib and rucaparib generate synthetic lethality17,68. Platinum agents and camptothecins are DNA-damaging agents69–71.

DNA repair. ADT-induced sensitivity to radiation and altered DNA repair gene expression following ADT provide evidence of cross-talk between androgen sig- nalling and DNA damage repair (DDR) pathways63. Although data suggest a role of DNA repair signalling in disease progression in mCRPC and in the setting of enzalutamide resistance, much is still to be learned63,64.Although a substantial number of patients have germline DDR mutations that are not a direct result of treatment resistance, evidence of somatic mutation accu- mulation is present, most notably with ATM in mCRPC compared with mCSPC27,63 (TABLE 2). Patients with meta- static prostate cancer and/or a familial history of cancer, notably ovarian and breast cancer, are encouraged to undergo HRR germline testing, as recommended by the National Comprehensive Cancer Network67. Defects of HRR pathways have been shown to result in sensitivity to PARP inhibition via synthetic lethality17,68. Tumours harbouring HRR mutations are also sensitive to platinum agents69–71.
Although prostate cancer is deemed an immunologically cold tumour, DNA repair defects suggest a potential role of PD1- or PDL1-targeted immunotherapy, especially in patients who have received numerous lines of previous therapy, including SG-ARAs72. Germline and somatic mutations in DNA mismatch repair (dMMR) genes (for example, MSH2, MLH1) can enable hypermutation and microsatellite instability (MSI) in tumours20. Patients with dMMR mutations and/or MSI- high tumours have increased T cell infiltration and upregulation of tumour neo-antigens, including PDL1 (REF.73). In 2017, these findings led to the approval of pembrolizumab, an anti-PD1 targeted agent, for use in patients with dMMR or MSI-high tumours of any histology once all standard-of-care treatments have been exhausted74. In 2020, the FDA approved pembrolizumab for patients with solid tumours and high tumour mutational bur- den (TMB; ≥10 mutations per megabase) based on data from the KEYNOTE-158 study75. This study showed a significant increase in objective response rate following treatment with pembrolizumab in patients with high TMB (29% (30 of 102 patients), 95% CI 21–39%) in comparison with patients with low TMB (6% (43 of 688 patients), 95% CI 5–8%)76.

A case series of 1,033 patients with prostate cancer who underwent clinical genomic profiling reported that 32 patients had MSI-high or dMMR-deficient tumours and 7 of these patients had a pathogenic germline MMRCross-talk Cellular processes in a tumour cell that can be activated by two or more cell-signalling pathways, which can result in unattenuated signalling via inhibition of only one of these pathways and/or amplification of the transcriptional pattern.Homologous recombination repair(HRR). DNA metabolic process involved in template- dependent repair or tolerance of complex DNA damages (such as DNA gaps, DNA double-strand breaks).Genomic alterations to numerous proteins involved in this process, including BRCA1/2, have been noted to be predictive markers for poly(ADP-ribose) polymerase inhibitor therapy.

Nevertheless, germline and somatic defects in DNA repair, notably homologous recombination repair (HRR), confer both prognostic and predictive implications in prostate cancer treatment. The most notable HRR genes include BRCA1 and BRCA2, with an indirect contribution of ATM, which encodes the serine-protein kinase ATM that senses DNA damage and activates HRR. Less established DDR genes of interest include CHEK2, BRIP1, RAD51D, PALB2 and FANCL17. The PROREPAIR B study found a significant decrease in cause-specific survival in patients with mCRPC carrying germline BRCA2 mutations in comparison with non- carriers (17.4 versus 33.2 months, P = 0.027), although researchers were unable to find a significant association of cause-specific survival with ATM, BRCA1 and PALB2 mutations (23.3 versus 33.2 months, P = 0.264)65. More recently, poly(ADP-ribose) polymerase (PARP) inhib- itors, including olaparib and rucaparib, were recently approved in patients with DDR mutations, offering a potential therapeutic option beyond SG-ARAs and abiraterone18,19,23,66.mutation. Among 11 patients with dMMR and/or MSI-high mCRPC who received PD1 or PDL1 ther- apy, 6 patients had significant PSA decline (>50%), with 4 showing a radiographic response to treatment20. Mutations that inactivate cyclin-dependent kinase 12 (CDK12), although initially proposed to provide sensi- tivity to PARP inhibition, have subsequently been found to lead to widespread focal tandem duplications and gene fusion-induced neoantigens via disrupted DNA replication-associated repair, promoting sensitivity to PD1- or PDL1-targeted therapy77,78. These mutations occur in 5–7% of patients with mCRPC and are asso- ciated with shorter time to progression of disease while on enzalutamide19,78,79.

PI3K–AKT–mTOR pathway. Loss of PTEN, a key tumour suppressor moderating the phosphoinos- itide 3-kinase–protein kinase B–mammalian target of rapamycin (PI3K–AKT–mTOR) pathway, is more prevalent in patients with mCRPC than in those with mCSPC and is associated with both enzalutamide and
abiraterone resistance18,19,21,22. Preclinical studies have shown that targeting both the PI3K and AR pathways significantly inhibits tumour growth in both PTEN– and enzalutamide-resistant preclinical models, the latter notably with GR-mediated resistance80,81. Assessment of CTCs in 16 patients with mCRPC identified frequent genomic alterations in AR, PI3K and hepatocyte growth as well as agents targeting alternative cell signalling pathways of established or potential clinical relevance (FIG. 2). Clinical trials examining alternative therapeu- tic approaches to current standard-of-care treatment are ongoing (Supplementary Table 1). Some of these approaches have been evaluated using novel methodol- ogies such as histology-agnostic biomarker-driven stud-Synthetic lethality A co-occurrence of multiple genetic events that results in cell death, whereas each event occurring independently would be tolerable for cell survival.

A frequently characterized ‘synthetic lethal’ combination in prostate cancer comprises a tumour cell with a DNA damage repair mutation
treated with a poly(ADP-ribose) polymerase inhibitor.Immunologically cold A term referencing tumours in which few to no immune cells are present and so they do not readily respond to immunotherapeutic treatment (e.g. PD1/PDL1 inhibition).Microsatellite instability (MSI). Observed differences of tumour cells in short tandem repeat DNA sequences (1–6 base pairs) in comparison with the inherited genome, frequently associated with defective mismatch repair.Tumours with high MSI have been proven to be more susceptible to immunotherapy, notably pembrolizumab.

WNT–β-catenin pathway A cell-signalling pathway that implicates important cellular functions, including stem cell regeneration and organogenesis. β-catenin, in addition to its activation via the canonical pathway, can specifically interact with the androgen receptor (AR) to enhance gene transcription.Basket trials Clinical trials that select a specific biomarker–treatment pairing and screen patients in a histologically agnostic approach (that is irrespective of tumour origin) in search of the specific biomarker to determine eligibility for one of the available biomarker– treatment arms.

Umbrella trials Clinical trials that prospectively screens patients with a pre-specified cancer to assign a molecular signature
to determine enrolment and/or placement into pre-specified treatment arms.factor receptor (MET) signalling25; MET has promiscu- ous functionality in cancer, with the potential to acti- vate the PI3K–AKT–mTOR, Ras–MAPK, JAK–signal transducer and activator of transcription (STAT) and Wnt–β-catenin pathways82.Enzalutamide treatment has also been demonstrated to activate PI3K–AKT–mTOR signalling via stabiliza- tion of AKT phosphatase. PTEN loss reduces feedback regulation of PI3K–AKT–mTOR signalling, further enabling AKT activation via cross-regulation of AR and PI3K pathways by reciprocal feedback83,84. The PI3K pathway is also enhanced by activating mutations (e.g. in PIK3CB or AKT1) or amplification (PIK3CA), although such alterations occur less frequently than PTEN loss18.

Wnt–β-catenin pathway. In metastatic prostate cancer, potentially clinically relevant mutations that stimulate the Wnt–β-catenin pathway signalling have been iden- tified in β-catenin (CTNNB1), adenomatous polypo- sis coli protein (APC) and E3 ubiquitin-protein ligase RNF43 (RNF43)85, with a similar prevalence in patients with mCSPC and mCRPC19. A retrospective study that included 137 patients with mCRPC who progressed on first-line abiraterone or enzalutamide reported that 11% of patients had either activating mutations in CTNNB1 or inactivating mutations in APC or RNF43. Patients with these Wnt-activating mutations had shorter time to PSA progression (6.5 versus 9.6 months, HR 2.34, P = 0.003) and shorter overall survival (23.6 versus 27.7 months, HR 2.28, P = 0.01) than those without Wnt-activating mutations26. Among 101 patients with mCRPC, CTNNB1 mutations were exclusive to those with enzalutamide resistance and were an independent predictor of worse overall survival (median 13.6 versus 41.7 months for those without CTNNB1 mutations, P = 0.025)86.β-Catenin signalling is also relevant in hypoxic tumours. Intratumoural hypoxia is a feature of aggres- sive prostate cancer that promotes the upregulation of hypoxia-inducible factor 1α (HIF1α), a transcription factor with roles in tumour angiogenesis, anaerobic metabolism, immunity, adaptation and metastatic potential87–91. A ternary complex comprising HIF1α, AR and β-catenin on androgen response elements was suggested to facilitate cross-talk between the HIF and AR pathways92,93. Evidence of such cross-talk has been demonstrated in vitro via dual inhibition of HIF1α and AR, which led to synergistic downregulation of AR- and HIF-mediated transcription, vascular endothelial growth factor (VEGF) expression and cell proliferation94.

Alternative therapeutic targets
Many investigational therapeutics and treatment reg- imens have been developed to address the multitude of resistance mechanisms in prostate cancer, including agents targeting AR-mediated acquired resistance (FIG. 1),ies (or basket trials) in which eligibility for enrolment is conferred by relevant genetic mutations regardless of cancer type and umbrella trials in which patients are pro- spectively screened for eligibility to take part in one of various sub-studies of targeted treatments selected based on the presence of biomarkers95,96. Basket and umbrella trials are meant to evaluate approaches that focus on potential predictive biomarkers in tumours resistant to the standard of care.

AR pathway inhibition. Efforts to develop more effec- tive AR- targeted therapies have focused on over- coming established SG-ARA resistance mechanisms, but have frequently resulted in negative trial results (Supplementary Table 1). The ARMOR3- SV trial investigated the efficacy of galeterone, an oral agent proposed to inhibit CYP17 lyase and degrade and antagonize the AR97, in 38 patients with chemotherapy- naive, AR-V7+ mCRPC. The investigators overlooked the limited prevalence of AR-V7 in newly diagnosed mCRPC and as it was deemed unlikely to enrol enough patients to meet the primary end point, the study was closed early98,99. EPI-506, an agent that targets the N-terminal domain of the AR to block AR and AR-V7 signalling, failed to improve PSA responses in patients (n = 28) with mCRPC. Additionally, EPI-506 exhibited low potency, extensive metabolism and a short half-life, resulting in a considerable pill burden100,101; EPI-7386, a more active, metabolically stable version of EPI-506, is currently in a phase I trial102. Similarly, niclosamide, an agent proposed to degrade AR-V7, was unable to pro- vide sufficient plasma concentrations needed for anti- tumour activity103,104. Seviteronel, a selective CYP17 lyase inhibitor with activity against the T878A and F877L AR mutations, showed limited anti-tumour activity and clinically significant central nervous system-related toxicities in 17 patients with mCRPC previously treated with enzalutamide105,106.

Novel approaches to differentially target the AR pathway to overcome acquired resistance are under investigation (Supplementary Table 1). ARRx is an antisense oligonucleotide designed to hybridize AR mRNA to inhibit full-length AR and AR-V7 expression107. This agent is currently being evaluated in combination with enzalutamide in patients with treatment-naive mCRPC108. ARV-110 utilizes a technology called PROteolysis TArgeting Chimera (PROTAC) to promote ubiquitination and degradation of the AR. Following successful evaluation in enzalutamide-resistant preclin- ical models, ARV-110 is currently being investigated in a phase I clinical trial in patients with mCRPC following progression on at least two lines of therapy, including enzalutamide and/or abiraterone109,110.

In preclinical models, use of bromodomain and extraterminal (BET) chromatin reader inhibitors in
N-terminal domain of the AR The non-ligand-binding domain of the androgen receptor (AR), as opposed to the C-terminal domain containing the
ligand-binding domain.The N-terminal domain is conserved in full-length AR, splice variant AR, and AR with ligand-binding domain mutations, and provides a potential advantage owing to non-competitive inhibition.

Antisense oligonucleotide A small single-stranded nucleic acid designed to bind to a specific RNA sequence in tumour cells to bring about gene silencing and result in tumour cell growth inhibition.PROteolysis TArgeting Chimera (PROTAC). A heterobifunctional molecule designed with two ligands, one to bind a target protein and the other to bind E3 ubiquitin ligase, connected by a linker. The function is to facilitate the degradation of target protein via thubiquitin–proteasome system.Bromodomain and extraterminal (BET) chromatin reader A family of proteins that serve as epigenetic readers of acetylated histones and can regulate gene transcription.combination with either enzalutamide or apalutamide were shown to overcome enzalutamide resistance via downregulation of AR-V7 expression and AR signalling, providing a rationale for their clinical development111,112. A first-in-human dose escalation study of mivebresib, a pan-BET inhibitor, suggested modest clinical acti- vity in patients with advanced prostate cancer, with 6 of 10 patients included in an expansion cohort having stable disease following treatment113,114. The efficacy of another BET inhibitor, ZEN003694, in combination with enzalutamide in patients with mCRPC, is currently being investigated in a phase Ib/IIa safety and tolerability study115.
Prostate-specific membrane antigen (PSMA) is a type II transmembrane protein serving as a receptor and enzyme with high expression in normal prostate cells in comparison with other organs (for example, brain, kidney, intestine) and is expressed 8–12 times more in prostate cancer cells than in normal prostate cells116. PSMA expression has previously been shown to be suppressed following androgen treatment in an AR-dependent manner, and conversely is upregulated in response to enzalutamide treatment in vivo117. Thus, PSMA represents a specific target for enhanced PET–CT diagnostic imaging in patients with prostate cancer and suspected metastases118, with recent FDA approval for the imaging agent 68Ga-PSMA-11. In addition to aiding diagnosis, PSMA-targeted radioligands could be used for treating PSMA-expressing prostate cancer lesions, notably through the use of radiation delivered via a radio- pharmaceutical, such as 177Lu-PSMA-617. In patients with mCRPC previously treated with enzalutamide or abiraterone, 177Lu-PSMA-617 treatment resulted in a PSA decline of greater than 50% in 34 of 104 patients (33%) after the first cycle119; further clinical evaluation of this treatment in mCRPC is currently ongoing120.

Alternative hormonal approaches. Numerous clinical trials are investigating the use of alternative approaches to target the AR and/or androgen biosynthesis to over- come acquired resistance. Bipolar androgen therapy (BAT) via the re-introduction of supraphysiological concentrations of testosterone has been proposed to target the adaptive overexpression of the AR resulting from continuous enzalutamide therapy in mCRPC cells by forcing downregulation of the overexpressed AR. However, treatment with BAT is controversial as it goes against the common clinical standard of care empha- sizing the importance of continuous testosterone sup- pression, especially following castration resistance121. An open-label, phase II, multicohort study in patients with asymptomatic mCRPC who had progressed fol- lowing enzalutamide treatment demonstrated that the addition of BAT to ADT was associated with a PSA decline of >50% in 9 of 30 patients (30%, 95% CI 15–49, P < 0.0001). Moreover, 15 of 21 patients who finished the 28-day course of BAT and were then re-challenged with enzalutamide achieved a >50% PSA decline121,122. The TRANSFORMER phase II, randomized, open-label study randomly assigned 94 patients with mCRPC to BAT and 101 patients to enzalutamide, with cross-over permitted at radiographic progression. Median overall
survival among patients who received sequential BAT and enzalutamide was 37.3 months (n = 34), compared with 28.6 months (n = 55) among those who received enzalutamide alone (HR 0.50, P = 0.03)123,124. These studies suggest the potential for therapeutic benefit with the re-introduction of testosterone following prolonged suppression with ADT and SG-ARA therapy.In addition to paradoxical stimulation of the AR, treatments have been developed to target the GR, which can bypass AR inhibition to induce tumour proliferation125. An open-label, phase I/IIa dose esca- lation study is currently evaluating a GR antagonist, CORT125281, in combination with enzalutamide in patients with SG- ARA or abiraterone- resistant mCRPC126.

Epigenetics and lineage plasticity. Investigational treat- ments have frequently sought to target epigenetic dys- regulation to remedy non-AR-dependent signalling in tumours with neuroendocrine features. Aurora kinase A serves to stabilize and prevent degradation of MYCN, which is capable of suppressing AR signalling to induce lineage plasticity, promote tumour aggressiveness, and facilitate AR-independent tumour growth, thus render- ing AR-targeted therapies ineffective127. An aurora kinase A inhibitor, alisertib, was proposed to disrupt MYCN sig- nalling and restore AR signalling to target neuroendo- crine tumours that are resistant to SG-ARAs. In a study of 60 patients with mCRPC and evidence of neuroendocrine pathology who received alisertib, only 8 patients (13.4%) achieved a meaningful clinical response of radiographic progression-free survival (rPFS) at 6 months127,128.
Like MYCN, targeting EZH2 also represents a way of restoring AR signalling in neuroendocrine-differentiated tumour cells129. Use of the EZH2 inhibitor, tazemetostat, in combination with either abiraterone or enzalutamide is currently being evaluated for safety in patients with mCRPC130.
In preclinical animal models, treatment with met- formin was shown to downregulate EMT induction fol- lowing enzalutamide treatment via repression of STAT3 activation and TGFβ1 expression following enzaluta- mide treatment, counteracting tumour cell invasion and metastasis. Enzalutamide also promotes EMT via the TGFβ1–SMAD3–matrix metalloproteinase 9 axis, with evidence of upregulation of N-cadherin, Vimentin, and Twist, and downregulation of E-cadherin; metformin treatment was also shown to counteract these factors responsible for EMT131. Findings in these preclinical models provide a rationale for combination treatment with metformin and AR antagonists in patients with prostate cancer. A clinical trial is currently investigating the efficacy of metformin in combination with bicalut- amide in patients with biochemically recurrent prostate cancer on ADT132.

DNA repair. The success of biomarker-driven PARP inhibitor treatment in patients with mCRPC following treatment with abiraterone or enzalutamide has changed the standard of care, providing an additional line of therapy for patients with relevant predictive biomark- ers. The randomized, open-label, phase II TOPARP-Astudy in 50 patients with mCRPC who had previously received taxane and AR-targeted therapies established the potential role of mutations in DDR genes as predic- tors of responses to the PARP inhibitor, olaparib. In this study, patients with alterations in DDR genes, such as BRCA2 and ATM, who were treated with olaparib had significantly longer rPFS (median 9.8 versus 2.7 months, P < 0.001) and overall survival (median 13.8 months ver- sus 7.5 months, P = 0.05) than those who did not have mutations in these genes133. Another phase II study, TOPARP-B, confirmed that olaparib treatment led to significant reductions in PSA and an increase in PFS in patients with mCRPC and BRCA1 or BRCA2 mutations who had previously been treated with taxane chemother- apy and/or enzalutamide or abiraterone. Additionally, PSA declines of 50% or greater were observed in patients with ATM, PALB2, FANCA or CHEK2 aberrations68.

The PROfound randomized, open-label phase III study compared olaparib with the physician’s choice of hormonal agent in patients with mCRPC and an alter- ation in a DDR gene who had previously been treated with a hormonal agent (62% of patients had previously received enzalutamide)17. Patients were divided into two cohorts: cohort A (n = 245) had at least one alteration in BRCA1, BRCA2 or ATM; cohort B (n = 142) had altera- tions in any of 12 other DDR genes. In cohort A, patients who received olaparib had significantly longer rPFS than those who received hormonal treatment (7.4 versus 3.6 months, HR 0.34, 95% CI 0.25–0.47, P < 0.001). Median
overall survival was also longer in the olaparib group of cohort A (19.1 versus 14.7 months, HR 0.69, 95% CI 0.50–0.97, P = 0.02), and 81% of patients in the control group who experienced disease progression crossed over to receive olaparib. Conversely, overall survival in cohort B showed no statistically significant difference between olaparib and control therapy (14.1 versus 11.5 months, HR 0.96, 95% CI 0.63–1.49). Analysis of the overall study population (cohorts A and B) also showed an improve- ment in rPFS with olaparib treatment (5.8 months versus 3.5 months, HR 0.49, 95% CI 0.38–0.63, P < 0.001)17,134.

Another PARP inhibitor, rucaparib, was granted accelerated approval in 2020 by the FDA based on the results of the TRITON2 phase II study in patients with mCRPC and BRCA mutations who were previously treated with a taxane and a hormonal agent. In patients with BRCA mutations, this study showed a clinically sig- nificant objective radiological response rate (complete or partial response on Response Evaluation Criteria in tumours exhibiting TMB-high whose disease has pro- gressed, despite standard-of-care therapy, provides a new treatment option for mCRPC. Evidence of pembroli- zumab efficacy is limited in patients with prostate can- cer, and the approved indications (MSI-high; TMB-high) for patient enrolment in clinical trials appear critical for treatment efficacy20. The IMbassador250 phase III, ran- domized clinical trial evaluated the PDL1 inhibitor ate- zolizumab plus enzalutamide versus enzalutamide alone in 759 patients with mCRPC who had previously been treated with either abiraterone or docetaxel. Regardless of MSI status, the combination treatment was associated with considerable toxicity and did not improve overall survival138,139. Ongoing phase III studies are evaluating the addition of pembrolizumab to enzalutamide irre- spective of biomarkers in mCSPC140 and treatment-naive mCRPC141. Further evaluation of biomarkers associated with response to PD1 or PDL1 inhibitors is needed in patients with SG-ARA-resistant prostate cancer.

Additional trials are investigating the use of com- bination approaches to exploit DNA repair pathways. The National Cancer Institute (NCI) is conducting two phase II clinical trials of such approaches in patients with mCRPC following enzalutamide and/or abiraterone therapy. One study is investigating the use of NLG207, a nanoparticle–drug conjugate of the potent topoisomer- ase I inhibitor camptothecin142,143, as a DNA-damaging agent in combination with olaparib irrespective of DDR mutation status144. A phase I/II study that evaluated the efficacy of the PDL1 inhibitor durvalumab in combina- tion with olaparib in 17 patients with mCRPC reported a substantial rPFS in men with DDR alterations (median of 16.1 months, 95% CI 7.8–18.1 months); additionally, the probability of 12-month PFS in patients with DDR alterations increased by 46.9% (P = 0.031) in comparison with patients without DDR mutations145,146. Outside of the NCI, the KEYLYNK-010 phase III study is currently investigating the efficacy of pembrolizumab and olapa- rib versus either enzalutamide or abiraterone in patients with mCRPC who experienced disease progression dur- ing treatment with docetaxel and either enzalutamide or abiraterone147.

PI3K–AKT–mTOR pathway inhibition. Studies that focus on PTEN loss and targeting of the PI3K–AKT–mTOR pathway are also ongoing. A phase II randomized study in 250 patients with mCRPC who had previously been treated with taxane and AR-targeted PI3K–AKT–mTOR pathway An intracellular cell signalling pathway that promotes metabolism, growth, proliferation, survival and angiogenesis following activation via an extracellular signal under normal conditions. In prostate cancer, this pathway can become dysregulated, mos frequently via the absence of phosphatase and tensin homologue (PTEN), to promote tumorigenesis, and is thus a viable target for drug development.Solid Tumours and Prostate Cancer Working Group Criteria) of 47.5% and time to PSA progression of 6.5 months (95% CI 5.7–7.5) compared with historical controls (study did not include a control arm)135,136. TRITON3, a confirmatory phase III study evaluating rucaparib, is currently ongoing137. Of note, rucaparib has only been approved for use in patients with BRCA muta- tions who have previously been treated with a taxane and either abiraterone or enzalutamide, whereas olaparib is approved for patients with any DDR mutation who have received either abiraterone or enzalutamide, but prior chemotherapy is not necessary for eligability17,135.

The histology-agnostic approval of the PD1 inhibitor pembrolizumab for patients with MSI-high tumours or therapy evaluated the efficacy of the AKT inhibitor ipatasertib in combination with abiraterone versus pla- cebo plus abiraterone148. When stratified for PTEN loss, adding low-dose ipatasertib (200 mg) to abiraterone improved rPFS by 6.5 months compared with placebo (11.1 versus 4.6 months, HR 0.46, 95% CI 0.25–0.83);
such benefit in rPFS was non-existent in patients with- out PTEN loss, with no significant difference between patients treated with and those without ipatasertib (7.5 months versus 5.6 months, HR 0.84, 95% CI 0.51–1.37)149. Use of another AKT inhibitor, capivasertib, in combination with enzalutamide resulted in a treatment response (defined as ≥50% decline in PSA, CTC con- version (that is a decrease in the number of CTCs in the blood from baseline), and/or radiological response) in 3 of 15 patients with progressive mCRPC who had pre- viously received abiraterone or enzalutamide; the three responders had either PTEN loss or an AKT-activating mutation (for example, E17K)150,151. Treatment with a dual mTOR inhibitor, MLN0128, had limited success in 9 patients with mCRPC previously treated with abi- raterone or enzalutamide152; the median time on treat- ment was only 11 weeks (range 3 to 30) and 4 patients had immediate PSA decline following MLN0128 discontinuation153. Other PI3K–AKT–mTOR pathway inhibitors are being evaluated in combination with enza- lutamide in patients with mCRPC, including LY3023414 (REF.154), GSK2636771 (REF.155) and everolimus156.

Angiogenesis. To date, no biomarkers have been vali- dated for assessment of angiogenic activity in prostate cancer. However, the utility of anti-angiogenic treatment has been investigated in patients with CRPC without SG-ARA resistance, though with disappointing results that have not resulted in FDA approvals157–159. Cabozantinib, a dual MET and VEGF receptor 2 (VEGFR2) inhib- itor, was evaluated as a monotherapy in patients with mCRPC who had previously been treated with docetaxel plus enzalutamide or abiraterone in two separate phase III trials with either prednisone or mitoxantrone as the com- parator (COMET-1 and COMET-2, respectively)160,161. Although neither study reported a significant over- all survival benefit with cabozantinib, a retrospective analysis of combined data from the two study cohorts suggested that patients with high-risk disease and/or prognostic factors (for example, age, albumin, PSA, site of metastases) might benefit from this therapy162. Combination treatment with cabozantinib and the PDL1 inhibitor atezolizumab was shown to be tolerable and to have clinically meaningful activity in a phase Ib study that enrolled 44 patients with mCRPC who had previ- ously been treated with enzalutamide or abiraterone163. The objective response rate was 32% with two complete responses and 12 partial responses, and 21 patients (48%) had stable disease following treatment164.

Novel approaches to targeting angiogenesis are cur- rently in development. One such approach involves tar- geting HIF1α to downregulate cross-talk between the AR, HIF and β-catenin pathways94,165. A clinical trial is currently investigating the efficacy of NLG207-mediated inhibition of HIF1α accumulation in combination with enzalutamide to overcome AR and hypoxia-mediated resistance in patients with mCRPC previously treated with enzalutamide166. Another open-label phase I/IIa study is evaluating the safety and efficacy of CCS1477, which targets CREB-binding protein-E1A binding protein (CBP-p300), a co-activator of HIF1α and AR167,168.

Limiting acquired resistance to SG-ARAs
As the available options for treating patients with advanced prostate cancer have increased, investiga- tors have sought to optimize treatment selection and maximize the effectiveness of each line of therapy169. Approaches to limiting the impact of acquired resistance on clinical outcomes of SG-ARA treatment focus on treatment sequence, earlier introduction of SG-ARAs,use of combination regimens, and implementation of precision medicine approaches (FIG. 3). Treatment optimi- zation requires appropriate treatment goals and clinical trial end points, defined by the Prostate Cancer Working Group170. Several completed and ongoing phase III clinical trials have focused on optimizing the use of approved therapies for prostate cancer to limit acquired resistance to SG-ARAs (Supplementary Table 2).Optimizing treatment sequence. As abiraterone, enzal- utamide and docetaxel are equally efficacious first-line treatment options for mCRPC, the development of opti- mal strategies for sequential use of these agents to avoid acquired resistance and optimize efficacy is of clinical importance171. However, approaches to limiting acquired resistance and maximizing overall survival are unclear. A retrospective cohort study of 260 patients with mCRPC who had previously received first-line docetaxel therapy evaluated treatment sequence permutations of up to three subsequent therapies: enzalutamide, abiraterone or cabazitaxel (a microtubule-stabilizing taxane chemother- apy)172. The researchers reported similar PSA response, PFS, and overall survival for the second- and third-line treatment options, regardless of the agent used172.
The CARD study used a randomized, open-label design to compare cabazitaxel with abiraterone or enzalutamide in 255 patients with mCRPC who had previously received docetaxel and either abiraterone or enzalutamide (patients received the hormonal therapy to which they were naive). Patients who received cabazitaxel showed a small but significant benefit with respect to PFS (4.4 versus 2.7 months, HR 0.52, 95% CI 0.40–0.68,
P < 0.001) and overall survival (13.6 versus 11.0 months, HR 0.64, 95% CI 0.46–0.89, P = 0.008) compared with those who received the other therapies173,174. Another randomized, open-label phase II study showed that treat- ment with abiraterone followed by enzalutamide once PSA had progressed lengthened time to subsequent PSA progression in comparison with treatment with enzal- utamide followed by abiraterone (median 19.3 months, 95% CI 16.0–30.5 versus 15.2 months, 95% CI 11.9–19.8, HR 0.66, 95% CI 0.45–0.97, P = 0.036), but had no effect on overall survival175,176. An ongoing phase III study is evaluating second-line docetaxel or hormonal therapy in patients with asymptomatic or oligometastatic mCRPC following abiraterone or enzalutamide treatment177.

The available data from these studies suggest that accumulation of acquired resistance with successive lines of therapies diminishes clinical improvement over time. However, alternating between anti-hormonal therapy and chemotherapy is generally accepted as the standard-of-care approach in mCRPC178. Further research is needed to identify the optimal sequence for incorporating treatment with other licensed therapies, such as the autologous cellular immunological agent sipuleucel-T or the radiopharmaceutical radium-223.Evaluating new indications and combinations. Ongoing phase III studies are exploring indications for SG-ARAs in localized prostate cancer and characterizing potential differences in acquired resistance patterns in this set- ting compared with those seen in mCSPC and mCRPC.

a Default monotherapeutic sequencing in patients with mCRPC

b Earlier introduction of therapy in high-risk localized prostate cancer

c Potential combination therapeutic approaches in mCRPC

Several studies are evaluating the efficacy of SG-ARA therapy in localized prostate cancer, including combi- nation treatment with radiation, neoadjuvant treatment prior to radical prostatectomy, and salvage therapy post- radical prostatectomy (Supplementary Table 2). The efficacy of apalutamide and abiraterone in combination

with ADT in patients with non-metastatic, high-risk or N1 prostate cancer who have detectable PSA follow- ing radiation and ADT for 6–8 months is also being investigated179. A trial of stereotactic body radiotherapy in combination with enzalutamide and ADT to treat oligometastases is also underway180.

Examples of treatment implementation strategies for advanced prostate cancer. a | Default monotherapeutic sequencing in patients with metastatic castration-resistant prostate cancer (mCRPC): in addition to continuing androgen deprivation therapy (ADT), the current standard-of-care approach to treating mCRPC involves alternating between chemotherapy and anti-hormonal treatment171–173,175,178. b | Early introduction of therapy in high-risk localized prostate cancer: early intensive therapeutic interventions such as neoadjuvant hormonal therapy followed by radical prostatectomy or radiotherapy are currently being investigated in patients with high-risk localized prostate cancer216–218. If the patient’s disease progresses following radical treatment, the patient could be treated with ADT with or without a second-generation androgen receptor antagonist (SG-ARA) until their disease progresses (darolutamide is in clinical trials for this indication)14,219. Once the disease has transitioned from metastatic castration-sensitive prostate cancer (mCSPC) to mCRPC, monotherapeutic sequencing (as per part a) would commence. c | Potential combination therapeutic approaches in mCRPC: various combination therapies for mCRPC are being investigated in clinical trials, including radium-223 in combination with either enzalutamide or darolutamide192,193, and PDL1 inhibition in combination with poly(ADP-ribose) polymerase (PARP) inhibition144,145. Different combination therapies could potentially be used sequentially in these patients. d | Precision medicine approaches in mCRPC: evaluation of biomarkers such as circulating tumour cells or cell-free DNA at diagnosis of mCRPC and following each instance of disease progression could potentially enable the identification of the most appropriate treatment intervention for individual patients199,200.

In addition, combination approaches are being investigated to potentially maximize clinical benefit and limit potential routes of resistance. The Alliance A031201 randomized controlled study, which evalu- ated enzalutamide plus abiraterone versus enzalutamide alone in 1,311 patients with treatment-naive mCRPC, demonstrated no significant overall survival benefit and increased toxicity with the combination therapy181,182. A similar study is currently investigating apalutamide plus abiraterone in chemotherapy-naive mCRPC183; data from a phase Ib study suggest that this combination has anti-tumour activity and no drug–drug interactions184. Two phase III studies are evaluating abiraterone in com- bination with SG-ARA therapy: STAMPEDE is investi- gating the addition of enzalutamide in mCSPC185 and the Androgen Annihilation study is investigating the addi- tion of apalutamide in biochemically recurrent prostate cancer following radical prostatectomy186. A phase III placebo-controlled study of the addition of darolutamide to ADT plus docetaxel in patients with mCSPC is due to complete in 2022 (REF.187).

SG-ARA trials have also investigated the implemen- tation of radium-223 in combination treatments. The ERA-223 study in patients with mCRPC was terminated early, as abiraterone plus ADT and radium-223 treat- ment was found to be associated with a high-risk of frac- ture (112 of 392 patients, 29%) compared with treatment with abiraterone plus ADT alone (45 of 394 patients, 11%)188. However, the addition of bone-protecting agents (e.g. denosumab) and removal of prednisone from treatment regimens could potentially enable safe administration of SG-ARAs in combination with radium-223 (REFS189–191). Phase III studies currently eval- uating radium-223 in combination with SG-ARAs and a bone-protecting agent in the absence of prednisone include PEACE-III192 and ESCALATE193.

Use of clinical biomarkers. Characterization of clinically relevant prostate cancer biomarkers could enable the identification of optimal therapies and identify potential barriers to SG-ARA efficacy. In addition to the use of mutations in DNA repair genes as predictive biomark- ers, CTC enumeration and AR analysis could potentially inform precision medicine approaches.Number of CTCs was retrospectively assessed as a biomarker of treatment response in 245 patients with mCRPC receiving abiraterone while enrolled on the COU-AA-301 study. The results showed that 162 of these patients (73.3%) had a CTC response, catego- rized as a 30% decrease in CTC number after 4 weeks of treatment. Patients with CTC response were shown to have increased overall survival in comparison with non-responders (14.7 versus 8.5 months, HR 0.43, 95% CI 0.31–0.6, P < 0.001)194. Other potential approaches to assessing treatment responses include the detection of AR-V7 in CTCs195 or ctDNA analysis of full-length AR and AR-V7 (REF.196). As a marker of resistance to SG-ARAs, monitoring for full-length AR and AR-V7 expression could prove to be useful tools for monitoring alterations to the AR pathway and predicting disease progression prior to radiographic confirmation as per the current stand- ard of care. Additionally, it has been proposed that AR-V7+ patients could be more optimally treated with a taxane over abiraterone or enzalutamide owing to the intrinsic resistance to hormonal therapies associated with AR-V7. Although several studies have suggested that knowledge of AR-V7 status could enhance clini- cal decision-making195,197, results from ongoing studies (such as VARIANT (ISRCTN10246848)) are needed to confirm the predictive utility198.

The phase III ProBio study is currently evaluating the use of genomic analysis of ctDNA and germline DNA to determine the optimal treatment sequence for individual patients with mCRPC199. This study is using a pre-enrolment biomarker signature analysis to assign one of six treatments that will be continued until the disease progresses200. The biomarker signature will then be reassessed and a subsequent treatment selected based on the updated signature.Adaptive therapy. As tumours develop acquired resist- ance to standard treatments owing to evolving drug- resistant tumour populations, a more responsive and individualized approach is required. Adaptive therapy incorporates mathematical models to predict the growth of drug-sensitive and drug-resistant sub-clones present in tumours in a patient-specific manner, with treatment paradigms focused on treatment to contain disease as opposed to treatment for cure201. The goal is to cycle drug selection using real-time data to prevent over- population of resistant tumour cell phenotypes, while also controlling and retaining the population of existing sensitive sub-clones that can compete against the resist- ant clones, without the treatment goal of tumour erad- ication. This evolutionary dynamics-based approach has been proposed to limit the development of highly aggressive disease by eliminating the length of exposure to one selective pressure (that is, the treatment course) in an effort to prolong time to progression. Patient-specific multi-drug adaptive therapy requires the understand- ing of several factors: the frequency-dependent cycles of tumour evolution, the appropriate timing and selection of treatment, and the velocity of tumour evolution201. Although trials of adaptive therapy to reduce SG-ARA resistance are currently lacking, the similar patterns of acquired resistance to abiraterone suggests the poten- tial to implement SG-ARAs in the adaptive therapy approach if current abiraterone clinical trials confirm improvement in clinical outcomes.

In a pilot study that investigated adaptive abiraterone monotherapy, that is, cycling the treatment on and off, 10 of 11 patients with mCRPC maintained stable oscilla- tions of tumour burden, with oscillations being defined as pre-set parameters of tumour size to prevent eradi- cation of sub-clones while setting a maximum tumour burden. The study reported a time to progression of at least 27 months and a 47% reduction in cumulative drug use compared with standard dosing202. A follow-up trial to assess cycling on and off both abiraterone and an LHRH agonist together in patients with mCSPC is ongoing203. The use of a ‘primary-secondary’ treatment strategy with abiraterone and docetaxel has also been proposed as a potential adaptive strategy. Although no patients have been enrolled to evaluate such a strategy, mathematical models incorporating data from adaptive abiraterone-only therapy have suggested the potential for significant increases in time to progression through abiraterone/docetaxel adaptive therapy, including a pro- posed improvement of 132 days for one patient204. More ‘real-world’ clinical data are needed to fully assess the viability and success of adaptive therapeutic approaches.

Conclusions and future directions
The past decade has seen exciting research and break- throughs for patients with high-risk and advanced prostate cancer. With continued clinical investigations, several important questions associated with SG-ARA therapy need to be addressed.Clinical decisions regarding SG-ARA selection are of particular interest, especially within the context of acquired resistance. The majority of data outlining acquired resistance to SG-ARA treatment have been obtained from studies of enzalutamide28; thus, possible differences in resistance patterns across the drug class remain to be determined. Unlike enzalutamide and apalutamide, darolutamide has activity against AR with the F877L mutation, suggesting that treatment with this SG-ARA might be able to more effectively target the AR to further improve responses12. Enhanced AR potency and activity, in addition to a more favourable adverse-effect profile12,205–207, could result in increased use of darolutamide over enzalutamide or apalutamide in patients with nmCRPC.Initiating SG- ARA therapy earlier in the treat- ment armamentarium with or without the inclusion of other agents could introduce an additional toxicity risk and could affect acquired resistance in later lines of therapy14. Current analyses have established patterns of biomarker enrichment from treatment-naive mCSPC to mCRPC, noting the critical differences between these stages of disease19. Whether patterns of resistance to SG-ARA treatment in the setting of early disease dif- fer in comparison with those in mCRPC remains to bedetermined. The selection of appropriate patients for more rigorous treatment with SG-ARAs (as opposed to standard ADT monotherapy) on the basis of poor prog- nostic features (such as visceral metastases, high number of bone lesions) is also necessary. A subgroup analysis of the open-label, randomized phase III ENZAMET study suggested that patients with mCSPC who were treated with docetaxel in combination with either enzaluta- mide or a FG-ARA had similar overall survival, indi- cating that use of enzalutamide might not be necessary in this population208. Limiting the number of patients who receive more aggressive treatment regimens could promote increased tolerability, primarily less fatigue owing to less complete suppression of androgens209, and reduce the development of more aggressive tumour phenotypes201. More research is needed to determine whether earlier introduction of SG-ARA therapy is ben- eficial for all patients, including the impact on overall survival and the efficacy of later lines of therapy.

Tumour heterogeneity and the identification of bio- markers have led to numerous potential drug targets, including the first of hopefully many approved thera- pies to implement precision medicine in prostate cancer (that is, the PARP inhibitors olaparib and rucaparib). As methods of characterizing tumours become more sophisticated (such as with genomic profiling), it is essential to identify mechanisms that underlie critical biomarker alterations. A preclinical study has uncovered substantial heterogeneity in the mechanisms governing AR re-arrangement and amplifications32, indicating complexity beyond current AR-focused biomarker sig- natures. Following the FDA approval of olaparib and rucaparib, patients should routinely undergo genomic testing after discontinuation of SG-ARA or abiraterone therapy to determine whether treatment with a PARP inhibitor would be appropriate. Expanded genomic and transcriptomic profiling to characterize alterations of the AR and other pathways could enable selection of the optimal therapies for individual patients as well as answer important research questions, such as what are the underlying mechanisms that limit taxane efficacy in some patients with AR-V7+ tumours197? Furthermore, improved tracking of tumour evolutionary dynamics following various lines of therapy might aid in clin- ical decision- making and promote new treatment approaches, such as adaptive therapy201. As the reper- toire of therapeutic options with predictive biomarkers continues to expand, so too will the role of precision medicine to manage acquired resistance to SG-ARA treatment.
Drug development efforts in prostate cancer continue to evolve to meet the demands of a diverse patient popu- lation. Although many lessons about acquired resistance to SG-ARA therapy can be learned from the plethora of completed and ongoing clinical trials, it is remarkable to remember that SG-ARA therapy did not have an indica- tion in advanced prostate cancer 10 years ago. Continued diligence and persistence is required to further optimize and refine clinical decision-making regarding SG-ARA therapy.


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