1. The Androgen Receptor: Mechanisms of Resistance
Ignacio Duran, MD
UGC Oncología Integral
Hospital Univ. Virgen del Rocío
Sevilla

2. Outline Introduction The Androgen Receptor Mechanisms of Resistance
Strategies to overcome resistance
Conclusions

3. Basic Principles: Transcription Factor
DNA
A transcription factor: It is a protein that binds to specific DNA sequences, thereby regulating the transcription of genetic information from DNA to mRNA. Transcriptions factors perform this function alone or creating a complex with other proteins by promoting the recruitment of RNApol (enzyme that activate the transcription of genetic information from DNA to RNA

4. Genes/RNA/Splicing
DNA
mRNA

5. Molecular Biology

6. The Androgen Receptor (AR)
≈180 kb
DNA
Xq11-12
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Dominio
N-Terminal
(NTD)
Dominio
de unión a
DNA (DBD)
Región
Bisagra
Dominio
de unión a
ligando (LBD)
Transcripcion y
“splicing” o unión del exoma
mRNA codificante
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
1116 ATG
3876 TGA
Traducción
Proteína
Aminoácidos 1
559
672
919
Schaufele F, et al. The structural basis of androgen receptor activation: intramolecular and intermolecular amino-carboxy interactions. Proc Natl Acad Sci USA 2005; 102: 9802–9807.
Sack JS, et al. Crystallographic structures of the ligand-binding domains of the androgen receptor and its
T877A mutant complexed with the natural agonist dihydrotestosterone. Proc Natl Acad Sci USA 2001; 98: 4904–4909.
Slide Courtesy of Dr. G. Billalabeitia

7. How the AR works TRAP220 SRC DHT CARM1 LSD1 FoxA1 NCoR/SMRT CBP/ p300
Chromatin is packed in the nucleosome and needs to be untangle and expose in order to expose the target genes
AR Target genes have some kind of “locators” denominated Androgen Response Elements (AREs)
To accomplish this complicated process the AR needs the collaboration of other proteins (Co-Activators)
pCAF
TRAP220
DHT
FoxA1
LSD1
SRC
CARM1
CBP/
p300
NCoR/SMRT

8. How the AR works
The transcription induced by the AR is a complex process with multiple players & actions:
Some gene’s expression is induced
Proteins in Sem. Liq (PSA)
Proteins involved in metab
Some gene’s expression is repressed
Testosterone synthesis enzymes ((AKR1C3, HSD17B6)
Androgen Receptor Genes (Self repression; negative feedback)
Cell Cycle Progression (In physiological conditions induce differentiation)
The AR transcription profile can vary depending on prostate cell status (normal Vs tumoral) and undifferentiation
TMPRSS2 is one of the target genes of the AR so in the eventual presence of a TMPRSS2-ERG fusion gene, AR would also control ERG
Tomlins SA, et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 2005; 310: 644–648.
Wang Q, et al. Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer. Cell 2009; 138: 245–256.

9. AR & CRPC
“It is now accepted that CRPC continues to rely on androgen signaling…”
“Owing to this new understanding, several drugs have recently emerged for the treatment of CRPC
Some of these agents either suppress the synthesis of extragonadal androgens (adrenal and/or intratumor) or target the androgen receptor directly 8 4
Pmol/g
Testosterone Levels in prostate tissue
Normal prostatic tissue
CRPC tissue
Overexpression of the AR by IHC
Antonarakis ES et al. N Engl J Med ;11

10. The good side
The requirement for intratumoral androgen synthesis in driving CRPC progression is most clearly demonstrated by the survival benefit conferred by ABIRATERONE ACETATE, (17α -hydroxylase/17,20-lyase inhibitor), and ENZALUTAMIDE, (AR antagonist ) in both the pre-chemo and post-chemo setting in patients with CRPC
De Bono J et al. NEJM 2011Scher H et al. NEJM 2012 Ryan C et al. NEJM 2013 Beer T. NEJM 2014

11. The bad side
Despite the dramatic improvement in CRPC therapeutics, the benefit is still “insufficient” and eventually all patients develop resistance to the currently available treatments
Hence, there is a need to better understand the biology of the disease and identify these mechanisms of resistance and develop appropriate drugs to overcome those

12. Molecular Mechanisms Two major mechanisms of resistance:
1.- Intratumoral synthesis of androgens?
2.- AR aberrant signalling
AR amplification/ Splice variants/ AR mutants
AR post transcriptional modifications
AR activation NO ligand dependent
The characterisation of potential driving genomic alterations in CRPC may provide the opportunity for a better understanding of mechanisms of resistance to targeted therapies and stimulate a molecularly driven clinical research

13. Mechanism 1: Intratumoral synthesis of DHT

14. Mechanism 1: Intratumoral synthesis of DHT
Intratumoral synthesis of DHT from precursors that are secreted from the adrenal gland occurs through a pathway that circumvents T
Chang et al., 2011; Knudsen and Penning, 2010
This DHT synthesis requires three enzymes:
3β -hydroxysteroid dehydrogenase (3β HSD)
Steroid-5α -reductase (SRD5A)
17β -hydroxysteroid dehydrogenase (17βHSD)

15. HSD3281 (3BHSD1)
HSD3B1 encodes for the peripherally expressed isoenzyme (3βHSD1) and has a germline single nucleotide polymorphism (SNP) at position 1245 of HSD3B1, converting A → C, which exchanges an asparagine (N) for a threonine (T) at 3βHSD1 amino acid position 367.
Chang KH, Li R, Kuri B et al. A gain-of-function mutation in DHT synthesis in
castration-resistant prostate cancer. Cell 2013; 154: 1074–1084.

16. HSD T
CRPC sometimes expresses the 367T form of 3β HSD1 (3β HSD1(367T)), which increases metabolic flux from dehydroepiandrosterone (DHEA) via the 5α -androstanedione (5α -dione) pathway to DHT
This happens by protein resistance to ubiquitination and degradation rather than increased catalytic activity.
Chang KH, Li R, Kuri B et al. A gain-of-function mutation in DHT synthesis in
castration-resistant prostate cancer. Cell 2013; 154: 1074–1084.

17. Mechanisms 2. AR aberrant signalling
The role of AR gene amplification
Somatic mutations of AR as mechanisms of resistance
AR gene rearrangement (Splice Variants)
AR signalling ligand-independent
Dillard PR,. Androgen-independent prostate cancer cells acquire the complete steroidogenic potential of synthesizing testosterone from cholesterol.Mol Cell Endocrinol 2008; 295: 115–120
Edwards J, et al. Androgen receptor gene amplification and protein expression in hormone refractory prostate cancer. Br J Cancer 2003; 89:552–556..
Taplin ME, et al. Androgen receptor mutations in androgen-independent prostate cancer: cancer and Leukemia Group B Study J Clin Oncol 2003; 21: 2673–2678.
Steinkamp MP, et al. Treatment-dependent androgenreceptor mutations in prostate cancer exploit multiple mechanisms to evade therapy. Cancer Res 2009; 69: 4434–4442.

18. 1.-AR gene amplification
West Coast Prostate Cancer Dream Team
Androgen receptor (AR) amplification in patients with metastatic castration-resistant prostate cancer (mCRPC) refractory to therapy with abiraterone acetate or enzalutamide: Preliminary results from the SU2C/PCF/AACR West Coast Prostate Cancer Dream Team (WCDT). J Clin Oncol 32:5s, 2014 (suppl; abstr 5020) Author(s): Eric Jay Small et al.
Background: ...As part of the WCDT project which aims to identify genetic pathways underlying primary and acquired resistance to Abi and Enz, AR+ was assessed in mCRPC biopsies.
Methods: Following central radiologic review, eligible mCRPC pts underwent biopsy at one of 5 WCDT clinical sites, using a uniform biopsy protocol. Tissue was both frozen, and formalin fixed/paraffin embedded (FFPE). FFPE tissue underwent a CLIA-certified assessment of a mutational panel, IHC for PTEN, and fluorescence in situ hybridization (FISH) for AR+.

19. Results: 60 of 300 planned mCRPC pts have undergone a metastasis biopsy. To date, 33 pts have had biopsies successfully evaluated by AR FISH, including 12 from bone, 11 from lymph nodes, 8 from liver and 2 from other soft tissues. Of 16 Abi and Enz naive pts, 13 (81%) were AR+. By contrast, only 1/11 Abi resistant pts (9%) was AR+. 4/6 Enz resistant pts (67%) were AR+. Of 6 AR+ patients that went on to receive Abi or Enz, 3 (50%) had a response (PCWG2 criteria), whereas of 5 pts without AR+ who went on to receive Abi/Enz, just 1 (20%) had a response.
Conclusions: AR amplification can be evaluated by FISH in small biopsies of mCRPC, including bone, and it was observed in 18/33 samples (55%). AR+ was common in mCRPC patients prior to therapy with Abi or Enz (81%). However, once resistance developed, Abi resistant pts had a far lower likelihood of AR+ (9%) than Enz resistant pts (67%).
These data support the hypothesis that treatment with Abiraterone selects for non-AR amplified cells, whereas Enzalutamide does not. In this relatively small cohort, mCRPC pts with AR+ are more likely to respond to subsequent AR targeted therapeutics than pts without AR+.

20. 2.-AR Mutant
A novel AR mutant (AR F876L) was recently identified in enzalutamide-sensitive cell lines, as well as xenograft models with prolonged exposure to enzalutamide. Acquisition of AR F876L mutation converts enzalutamide and ARN-509 into AR agonist, and induces treatment resistance in vitro as well as in vivo.
Balbas MDet al. Overcoming mutation-based resistance to antiandrogens with rational drug design. Elife 2013; 2: e00499.
Joseph JD,et al. A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509. Cancer Discov 2013; 3: 1020–1029.
Korpal M, et al. An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 \Cancer Discov 2013; 3: 1030–1043.

21. AR F876L
AR F876L allows the repositioning of the helix in AR (helix 12) to an agonist-like conformation that permits coactivation recruitment when tumor cells are treated with enzalutamide.
Interestingly, bicalutamide remained an antagonist in these AR-mutant cells.

22. 3.- Splice Variants
One emerging mechanism is AR gene rearrangement, resulting in constitutively active truncated AR splice variants (AR-Vs W with lack of LBD
The expression of some exons is lost and some others that were cryptic in between the introns become expressed
The most frequent splicing variants are AR-V7, V9, V5, V1 and V3
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
E2b AR
E3b
E3c
E3d
E3e
RA3
AR-V7
AR-V5
AR-V9
AR-V1
RA4
AR-V3
RAEx1/2/2b
AR ΔHR- DBD
AR Δ Ex3-HR- DBD
CRYPTIC EXONS
Simon Haile and Marianne D. Sadar. Androgen receptor and its splice variants in prostate cancer. Cell Mol Life Sci, 2011 ;68(24): 3971–3981.
Slide Courtesy of Dr. G. Billalabeitia

23. Splice Variants
AR-Vs determined in CTCs have been recently associated with resistance to both enzalutamide and abiraterone acetate
N Engl J Med 371;11 September 11, 2014

24. 4. Ligand Independent AR signaling
Through different mechanisms ligand independent aberrant AR signalling might occur
These include some of the post-translational modifications of the AR [i.e. Phosphorilation of AR through HER expression]
as well as the loss of expression of some key tumour suppressor genes such as pTEN [loss of pTEN is a common event in CRPC]
Gioeli D and Paschal BM. Post-translational modification of the androgen receptor. Molecular and Cellular Endocrinology 2012; 352: 70–78.
van der Steen T, Tindall DJ and Huang H. Posttranslational Modification of the Androgen Receptor in Prostate Cancer. Int. J. Mol. Sci. 2013, 14,
Carver B., et al. Reciprocal Feedback Regulation of PI3K and Androgen Receptor Signaling in PTEN-Deficient Prostate Cancer. Cancer Cell 2011, 19, 575–586

25. Another mechanisms of resistance
“Although targeting the androgen axis has a clear terapeutic benefit, its effectiveness is temporary as prostate tumor cells adapt to survive and grow. Androgen deprivation might activate EMT and neuroendocrine transdifferentiation
Nouri M et al. Androgen-targeted therapy-induced epithelial mesenchymal plasticity and neuroendocrine transdifferentiation in prostate cancer.
Front Oncol Dec 23;4:370.

26. Some Strategies Ongoing
New compounds are being developed/tested targetting some of these mechanisms responsible of resistance:
DR103 is a compound that might restore the position of helix 12 into the antagonist form in both wild-type and F876L-mutant AR.
EPI-001 covalently binds to the NTD, inhibits protein–protein interactions necessary for transcriptional activity of the AR and its splice variants.
BGB324 is a small molecule inhibitor of the AXL receptor
Trametinib (MEK Inh) is being tested in the neoadjuvant setting
GI-6301 is a Brachyury vaccine in Phase I
ARN-509 is an oral novel AR antagonist that binds to AR, and blocks nuclear translocation and binding of AR to ARE, and recruitment of co-activators.
Galeterone, a triple mechanism of action functioning as CYP17 lyase inhibitor, as well as an antiandrogen and as an AR-degrading agent.

27. Summary
The AR is a tightly regulated key transcription factor in both the hormone-sensitive and castration-resistant stages of advanced prostate cancer
The receptor and its major ligands are critical targets and some drugs have been successfully developed in this setting
Mechanisms of resistance to the current treatments have been identified and strategies to overcome them represent an intense area of research

28. Gracias por su atención !!

29. But things may not be so easy….
and still there is a lot to do.

30. Another mechanisms of resistance
“Although targeting the androgen axis has a clear terapeutic benefit, its effectiveness is temporary as prostate tumor cells adapt to survive and grow. Androgen deprivation might activate EMT and neuroendocrine transdifferentiation
Nouri M et al. Androgen-targeted therapy-induced epithelial mesenchymal plasticity and neuroendocrine transdifferentiation in prostate cancer.
Front Oncol Dec 23;4:370.

31. ….Tenemos tiempo aun?

32. BACK-UP

33. Search of new compounds
New Androgen Biosynthesis Inhibitors

34. ORTERONEL: TAK
Orteronel (TAK-700; Millennium Pharmaceuticals, The Takeda Oncology Company, Cambridge, MA) is an oral, relatively selective, nonsteroidal CYP17 17,20-lyase inhibitor
The selectivity of orteronel to inhibit 17,20-lyase compared with 17-hydroxylase could afford it a safer toxicity profile than agents that inhibit both steps in the testosterone synthesis pathway
Ferraldeschi, R Cancer J 2013;19: Yamaoka M, et al . J Steroid Biochem Mol Biol. 2012;129:.

35. TAK Development: The End
Based on positive preclinical data and solid background
Phase I and II studies were progressively conducted achieving good results
However when taken to Phase III (pre & post chemo)...

36. VT-464 (VIAMET)
VT-464 (Viamet), is a novel oral CYP17 inhibitor with a higher selectivity for 17, 20-lyase over 17-hydroxylase.
In animal models, VT-464 induces androgen suppression without a concomitant increase of upstream steroids or cortisol suppression, thus providing the opportunity to be potentially used without concomitant steroids
Eisner JR, Abbott DH, Bird IM et al. VT-464: a novel, selective inhibitor of P450c17 (CYP17)-17, 20 lyase for castration-refractory prostate cancer (CRPC).J Clin Oncol 2012; 30: abstr 198.

37. NEW ANTIANDROGENS

38. ARN-509
ARN-509. Like enzalutamide, ARN-509 is an oral novel AR antagonist that binds to AR, and blocks nuclear translocation of AR, binding of AR to androgen response elements, and recruitment of co-activators by the AR.

39. ARN-509
In a murine xenograft model of mCRPC, ARN-509 showed greater antitumor activity than enzalutamide, for a given dose and plasma concentration. Furthermore, ARN-509 achieved significantly lower steady-state brain levels than those observed with enzalutamide, suggesting its lower seizurogenic potential
Clegg NJ, Wongvipat J, Joseph JD et al. ARN-509: a novel antiandrogen for
prostate cancer treatment. Cancer Res 2012; 72: 1494–1503.

40. ARN-509
A phase I trial accrued 30 men with mCRPC and reported promising activity of ARN-509 At 12 weeks, 42% ≥50% PSA declines & FDHT-PET imaging demonstrated AR blockade at 4 weeks ARN-509 was safe and exhibited linear pharmacokinetics. A phase II showed at 12 weeks, PSA response of 91% in therapy naïve and 60% in post-AA mCRPC patients and 89.5% in non metastatic CRPC patients
A Phase III trial in M0 is ongoing and results are pending
Rathkopf DE, Antonarakis ES, Shore ND et al. ARN-509 in men with metastatic castration-resistant prostate cancer. In ESMOSeptember Abstract 964 TIP. Smith MR, Antonarakis ES, Ryan CJ et al. ARN-509 in men with high-risk nonmetastatic castration reistant prostate cancer. In ESMO, September Abstract 920P

41. ODM-201
ODM-201
This is a novel pure oral AR antagonist that does not penetrate the blood–brain barrier in preclinical models.
In a phase I/II study, treatment with ODM-201 was well tolerated and was associated with a high activity in mCRPC, including those with prior treatment with docetaxel and a CYP17 inhibitor
Fizazi K, Bono P, Jones RH et al. An open-label, phase I/II safety, pharmacokinetic,and proof-of concept study of ODM-201 in patients with progressive metastatic castration-resistant prostate cancer (CRPC). In European Cancer Congress 2013,

42. AZD 3514
AZD3514.
This is an oral drug which inhibits AR signaling through two distinct mechanisms, an inhibition of ligand-driven nuclear translocation of AR, and downregulation of androgen receptor levels.
Preclinically, AZD3514 has been shown to have antitumor activity in both androgen-sensitive and castration resistant prostate tumors

43. AZD 3514
In a first-in-man phase I trial, 49 CRPC pts were treated with escalating dose levels of AZD3514 Encouraging responses were seen, even in those with prior disease progression on AA, including: PSA decline of ≥30% in 11 of 49 (23%), PSA decline of ≥50% in 7 of 49 (14%),and objective soft tissue responses in 2 of 26 (8%) men with measurable disease. The most common toxicities were nausea and vomiting, and were manageable with oral antiemetics. None met the definition of dose limiting toxicity.
Loddick SA, Ross SJ, Thomason AG et al. Mol Cancer Ther 2013; 12: 1715–1727.
Omlin A, Jones RJ, van der Noll R et al. J Clin Oncol 2013; 31(suppl): abstr 4511

44. Galeterone: TOK-001
Galeterone (VN/124-1, TOK-001; Tokai Pharmaceuticals, Cambridge, MA)
It is an oral, semisynthetic, steroidal agent with a triple mechanism of action functioning as CYP17 lyase inhibitor,
as well as an antiandrogen and as an AR-degrading agent.
ARMOR (Androgen Receptor Modulation Optimized for Response) clinical development program for the evaluation of galeterone in patients with CRPC, has been completed

45. TOK-001
In a phase I study of chemo-naïve men with CRPC, TOK-001 was well tolerated and demonstrated clinical activity. ARMOR-1 Of 49 patients,22% demonstrated a >50% PSA decline and an additional 26% had PSA declines of 30%–50% [34]. Based on these preliminary results Galeterone received fast-track designation from FDA

46. ARMOR-2
The most mature dataset is in metastatic treatment naïve CRPC where interim data in 21 patients show 90% achieved a PSA30 and 81% achieved a PSA50.
In addition, initial data in abiraterone refractory patients shows both biochemical activity and stable disease.

47. EPI-001
EPI-001 covalently binds to the NTD, inhibits protein–protein interactions necessary for transcriptional activity of the AR and its splice variants, and reduces the growth of CRPC in xenografts. EPI-001 has the potential to be efficacious in CRPC progressing on treatment with enzalutamide,where AR-Vs are believed to play an increasing role as driver of tumor progression .
Myung JK, Banuelos CA, Fernandez JG et al. An androgen receptor N-terminal domain antagonist for treating prostate cancer. J Clin Invest 2013; 123:2948–2960.

57. Molecular Facts behind clinical observations
Prostate cancer is very heterogeneous disease
Prostate cancer progression is grouped into three categories:
Endocrine-driven
Microenvironment-dependent
Tumor cell–autonomous
Cancer Discov; 3 (8):
M. Fraser et al. Urologic Oncology: Seminars and Original Investigations ] (2013) 1–10