1. AR-V7 Splice Variant in Prostate Cancer : Taking Centre Stage
Department of Medical Oncology
AR-V7 Splice Variant in Prostate Cancer : Taking Centre Stage
ARV7 in CRPC…focus of discussion
Presented by:
Mohsin Maqbool
Ph.D Student, MO, IRCH

2. Outline Introduction Splicing- AR Splice variants , ARV7
Clinical role of ARV7 in CRPC
Discussion and Conclusion

3. Prostate cancer : present therapies and Resistance
Androgen-deprivation therapy (ADT) remains the principal treatment, most patients eventually develop castrate resistance - Castration resistant prostate cancer(CRPC)
Enzalutamide and Abiraterone represent breakthroughs in CRPC treatment
20 to 40% of patients have no response (i.e., they have primary resistance)
C. B Huggins
Nobel Prize -1961
Androgen receptor (AR) signaling is a critical survival pathway for prostate cancer cells, and androgen-deprivation therapy (ADT) remains the principal treatment
Castration resistant prostate cancer(CPRC) : Majority of patients initially respond to ADT, most eventually develop castrate
Resistance
ADT is known to provide remission of the disease, best
evidenced by a decline of prostate-specific antigen (PSA) in about
90% of patients.2 After a mean time of 2–3 years, however, the
disease progresses despite continuous hormonal manipulation.
This type of cancer is known as castrate-resistant prostate cancer
(CRPC).2 Metastatic castration-resistant prostate cancer (mCRPC) is
associated with a poor prognosis and mean survival time of only
16–18 months.3
Docetaxel and cabazitaxel are the only United States Food and
Drug Administration (FDA)-approved chemotherapies for the
treatment of mCRPC (Table 1). These tubulin-binding taxanes
have been proven to decrease PSA levels and palliate symptoms
but survival benefits are modest
Disease progression despite serum testosterone levels of <50 ng/dl
Rising PSA from nadir(baseline) on ADT
Radiographic progression
Clinical Progression
Enzalutamide exerts its antitumor activity through its interaction with the ligand-binding domain of the androgen receptor and is ligand-independent and Abiraterone inhibits P-450 CYP17A1 which blocks androgen synthesis
Patients who initially have a response to enzalutamide or abiraterone, virtually all eventually acquire secondary resistance
Resistance to chemotherapy and molecularly targeted therapies is a major
problem facing current cancer research
One explanation for the resistance to both agents may involve the presence of androgen receptor splice variants
C V. Hodges
Ryan CJ et al , JCO 2011

4. Splicing and splice variants
mRNA
(Coding for final gene and protein)
Splice variant 1
Splice variant 2
Splice variant 3

5. Splice variants in drug resistance
Alternative splicing (Splice variants)- key molecular mechanism for protein diversity, but has implications in drug resistance too
Splicing profile of several cancer associated genes is altered- role in tumor progression
Cancer-associated alternative splicing variants- as new biomarkers in cancers ( Breast cancer, Pancreatic cancer)
Normal
Variants
Moreover, the characterization of splicing deregulation in cancer will lead to a better comprehension of malignant
transformation. Cancer-associated alternative splicing variants may be new tools for the diagnosis and classification of
cancers and could be the targets for innovative therapeutical interventions based on highly selective splicing correction
approaches.
Sharmistha Pal et al Pharm & therp 2012
C Holohan et al Nat Rev, 2013

6. AR Splice variants in Androgen receptor and ARV7

7. Splice variants : Role of ARV7 in resistance
ARV7(AR3) is most studied AR, one of the major splice variants expressed in human prostate tissues, is constitutively active, and its transcriptional activity is not regulated by androgens or antiandrogens
ARV7 is significantly up-regulated during PCA progression and ARV7 expression level is correlated with the risk of tumor recurrence
The androgen receptor (AR) plays a key role in progression to
incurable androgen ablation–resistant prostate cancer (PCA).
We have identified three novel AR splice variants lacking the
ligand-binding domain (designated as AR3, AR4, and AR5) in
hormone-insensitive PCA cells. AR3, one of the major splice
variants expressed in human prostate tissues, is constitutively
active, and its transcriptional activity is not regulated by
androgens or antiandrogens. Immunohistochemistry analysis
on tissue microarrays containing 429 human prostate tissue
samples shows that AR3 is significantly up-regulated during
PCA progression and AR3 expression level is correlated with
the risk of tumor recurrence after radical prostatectomy.
Overexpression of AR3 confers ablation-independent growth
of PCA cells, whereas specific knockdown of AR3 expression
(without altering AR level) in hormone-resistant PCA cells
attenuates their growth under androgen-depleted conditions
in both cell culture and xenograft models, suggesting an
indispensable role of AR3 in ablation-independent growth of
PCA cells. Furthermore, AR3 may play a distinct, yet essential,
role in ablation-independent growth through the regulation
of a unique set of genes, including AKT1, which are not regulated
by the prototype AR. Our data suggest that aberrant
expression of AR splice variants may be a novel mechanism
underlying ablation independence during PCA progression,
and AR3 may serve as a prognostic marker to predict patient
outcome in response to hormonal therapy. Given that these
novel AR splice variants are not inhibited by currently available
antiandrogen drugs, development of new drugs targeting these
AR isoforms may potentially be effective for treatment of
ablation-resistant PCA. [Cancer R
Zhiyong Guo, Feng Sun, et al Can Res 2009

8. Clinical relevance of ARV-7 in CRPC
Clinical relevance of androgen- receptor variants in castration- resistant prostate cancer receiving enzalutamide or abiraterone not known
Prospectively evaluated androgen receptor splice variant-7 messenger RNA (AR-V7) in circulating tumor cells from patients receiving enzalutamide or abiraterone
Even though AR-V7 results in continuous activation of the androgen receptor in animal models and human prostate cancer cell lines, it was not previously clear whether AR-V7 would confer resistance to enzalutamide.
Enzalutamide was given at a dose of 160 mg daily; abiraterone was given at a dose of 1000 mg daily,
with prednisone at a dose of 5 mg twice daily
Hypothesized that detection of AR-V7 in circulating tumor cells may be associated with resistance to enzalutamide and abiraterone In CRPC
Prospectively enrolled men with metastatic CRPC treatment with enzalutamide or abiraterone
Progressive disease despite “castration levels” of serum testosterone (<50 ng per deciliter), with continued ADT, and documented metastases, as confirmed on computed tomography (CT)
Patients were excluded if they planned to receive additional concurrent anticancer therapies
Analysis of circulating tumor cells (Kit Method- AlereTM CTC AdnaTest)
Detection and quantification of AR-V7 by quantitative real-time PCR using custom primers specific for AR-V7 and also in tissue based samples

9. Methods
Prospectively enrolled men with metastatic CRPC treatment with enzalutamide or abiraterone
Progressive disease despite “castration levels” of serum testosterone (<50 ng per deciliter), with continued ADT, and documented metastases, as confirmed on computed tomography (CT)
Patients were excluded if they planned to receive additional concurrent anticancer therapies
Analysis of circulating tumor cells (Kit Method- AlereTM CTC AdnaTest)
Detection and quantification of AR-V7 by quantitative real-time PCR using custom primers specific for AR-V7 and also in tissue based samples
Analysis of circulating tumor cells (Kit Method- AlereTM CTC AdnaTest)
Prostate Cancer Detect (Product No. T-1-521) kit was used to make cDNA for detection of prostate cancer-associated RNA transcripts using polymerase chain reaction (PCR)
Detection and quantification of AR-V7 by quantitative real-time PCR using custom primers specific for AR-V7
Detection and quantification of AR-V7 by quantitative real-time PCR using custom primers specific for AR-V7 and also in tissue based samples

10. Patient Characteristics
62 patients with detectable circulating tumor cells, of whom 31 received enzalutamide and 31 received abiraterone
Median follow-up time was 5.4 months (range, 1.4 to 9.9) among enzalutamide- treated patients and 4.6 months (range, 0.9 to 8.2) among abiraterone-treated patients
Emmanuel S. Antonarakis et al, NEJM 2014

11. Patient Characteristics
62 patients with detectable circulating tumor cells, of whom 31 received enzalutamide and 31 received abiraterone
Median follow-up time was 5.4 months (range, 1.4 to 9.9) among enzalutamide- treated patients and 4.6 months (range, 0.9 to 8.2) among abiraterone-treated patients

12. Overall proportion of patients who had a PSA response while receiving enzalutamide was 32%
In enzalutamide cohort, PSA response rate among AR-V7–positive patients was 0% and the rate among AR-V7–negative patients was 53% (P = 0.004)
Panel A shows the 31 enzalutamide-treated patients, and Panel B the 31
abiraterone-treated patients. The dotted line shows the threshold for defining a PSA response (=50% reduction in PSA level from baseline). Asterisk indicate an increase of more than 100% in best PSA response. Daggers indicate patients in the enzalutamide cohort who had previously received
abir aterone and patients in the abiraterone cohort who had previously received
enzalutamide.
Overall proportion of patients who had a PSA response while receiving abiraterone was 55%
In the abiraterone cohort, the PSA response rate among AR-V7–positive patients was 0%, and the rate among AR-V7–negative
patients was 68%((P = 0.004)
AR-V7 status remained predictive of PSA response after adjustment for the expression of full-length androgen receptor mRNA
Emmanuel S. Antonarakis et al, NEJM 2014

13. Progression–free Survival
Kaplan –Meier Analysis of PSA Progression free survival
Kaplan –Meier Analysis of Clinical/ Radiographic Progression free survival
Among enzalutamide-treated patients, PSA progression–free survival was shorter among men with detectable AR-V7 at baseline than among those with undetectable AR-V7 (P<0.001)
Among abiraterone-treated patients, PSA progression– free survival was shorter among men with detectable AR-V7 at baseline than among those with undetectable AR-V7 (P<0.001
The median PSA progression–free survival in enzalutamide-treated patients (Panel A) was 1.4 months (95% CI, 0.9 to not reached) in
AR-V7–positive patients and 6.0 months (95% CI, 3.8 to not reached) in AR-V7–negative patients (hazard ratio for PSA progression with
AR-V7 positivity, 7.4; 95% CI, 2.7 to 20.6; P<0.001 by the log-rank test). The median PSA progression–free survival in abiraterone-treated
patients (Panel B) was 1.3 months (95% CI, 0.9 to not reached) in AR-V7–positive patients and more than 5.3 months (95% CI, 5.3 to
not reached) in AR-V7–negative patients (hazard ratio for PSA progression with AR-V7 positivity, 16.1; 95% CI, 3.9 to 66.0; P<0.001 by
the log-rank test). The median clinical or radiographic progression–free survival in enzalutamide-treated patients (Panel C) was 2.1
months (95% CI, 2.0 to not reached) in AR-V7–positive patients and 6.1 months (95% CI, 4.7 to not reached) in AR-V7–negative patients
(hazard ratio for clinical or radiographic progression with AR-V7 positivity, 8.5; 95% CI, 2.8 to 25.5; P<0.001 by the log-rank test).
The median clinical or radiographic progression–free survival in abiraterone-treated patients (Panel D) was 2.3 months (95% CI, 1.4 to
not reached) in AR-V7–positive patients and more than 6.3 months (95% CI, 6.3 to not reached) in AR-V7–negative patients (hazard ratio
for clinical or radiographic progression with AR-V7 positivity, 16.5; 95% CI, 3.3 to 82.9; P<0.001 by the log-rank test).

14. Overall Survival
Preliminary survival analysis was conducted at 32% maturity in the enzalutamide-treated cohort (i.e., after 32% of the patients [10 patients] had died) (median follow-up, 8.4 months) and at 16% maturity in the abiraterone-treated cohort (i.e.,after 16% of the patients [5 patients] had died)(median follow-up. 9.3 months)
Overall survival was shorter in men with detectable AR-V7 at baseline than among those with undetectableAR-V7 both in the enzalutamide and abiraterone cohort (median,5.5 months vs. not reached; hazard ratio fordeath, 6.9; 95% CI, 1.7 to 28.1; P = by the log-rank test)
Clinical outcomes (PSA responses, PSA-PFS and PFS) for the entire patient
population according to their AR-V7 ‘conversion’ rates.
PSA- PFS
R- PFS

15. No effect on PSA in AR-V& positive patients
Expression of PSA suppressed in AR-V7–negative patients during
treatment with enzalutamide or abiraterone
PSA expression did not decrease in post-treatment samples of circulating tumor cells from men with detectable AR-V7 at baseline

16. Discussion
Treatment options for patients with castration-resistant prostate cancer (CRPC) has changed with FDA(US) approved agents have shown survival benefit for patients with CRPC
Enzalutamide and abiraterone- two new therapies directed at the androgen receptor, represent important advances in the management of castration- resistant prostate cancer
20 to 40% of patients have no response to these agents with respect to prostate-specific antigen (PSA) levels and that detection of ARV7 in tumor cells appears to be associated with resistance to both enzalutamide and abiraterone
Recent study shows a strong association between the presence of AR-V7 and resistance to enzalutamide and abiraterone

17. Discussion
Efforts directed towards ablating androgen-receptor activity, particularly by interfering with the functions of the N-terminal domain of the androgen receptor, are likely to be fruitful

18. Conclusion
ARV7/AR3 is important most studied and constitutive splice variant in prostate cancer appears to be upregulated during prostate cancer progression during hormone therapy, and is associated with androgen-resistant growth
AR-V7 could be used as a biomarker to predict resistance to enzalutamide and abiraterone and to facilitate treatment selection-of more advanced disease or a higher disease burden
AR-V7/AR3 is important most studied and constitutive splice variant in prostate cancer and detected in circulating tumor cells from patients with castration-resistant prostate cancer CRPC
- Development of new drugs targeting these AR isoforms(ARV7) may potentially be effective for treatment of ablation-resistant CRPC

19. Thankyou ..!!

20. Cause of Resistance ??
Studies in castration-resistant prostate cancer have shown that androgen receptor variants are often expressed in metastases, with faster disease Progression and shorter cancer-specific survival
Protein isoforms of AR function may be dependent on the activity of full- length androgen receptor??
One explanation for the resistance to both agents (Enzalutamide and Abiraterone) may involve the presence of androgen receptor splice variants
Resistance to chemotherapy and molecularly targeted therapies is a major
problem facing current cancer research
Pharmacokinetic (PK) factors such as drug absorption, distribution,
metabolism and elimination (ADME) limit the amount of a systemically administered drug that reaches the tumour. In the
tumour, the effects of the drug on the cancer cell are collectively termed its pharmacodynamic (PD) properties. The
anticancer activity of a drug can be limited by poor drug influx or excessive efflux; drug inactivation or lack of activation;
alterations such as changes in expression levels of the drug target; activation of adaptive prosurvival responses; and a lack
of cell death induction due to dysfunctional apoptosis, which is a hallmark of cancer.
Attard G et al JCO,2008
Scher HI et al NEJM, 2012