REAKT Manuscript Supplementary Material

Table of Contents:1. Inclusion Criteria2. Dose escalation/de-escalation schedule 3. Definition of Dose Limiting Toxicity4. Research Sample Collection Schedule5. Research Sample Methods6. Supplementary Tables7. Supplementary Figure Legends8. Supplementary References

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1. Inclusion Criteria:1. Written informed consent.

2. Histological diagnosis of adenocarcinoma of the prostate and with tumour tissue accessible

for research analyses for this trial (e.g. PTEN testing). Patients who have no histological

diagnosis must be willing to undergo a biopsy to prove prostate adenocarcinoma.

3. Metastatic Castration‐Resistant Prostate Cancer (mCRPC).

4. Progressed after 1 or 2 lines of taxane based chemotherapy.

5. Progressed after abiraterone or enzalutamide (pre or post chemotherapy). Patients must

have received at least 12 weeks of treatment with either abiraterone or enzalutamide.

6. Age ≥18 years.

7. Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0 – 2.

8. PSA ≥ 10ng/ml.

9. Documented willingness to use an effective means of contraception while participating in the

study and for 12 months post last dose of treatment (see section 4.5).

10. Documented ongoing castrate serum testosterone <50 ng/dL (<2.0 nM).

11. Received prior castration by orchiectomy and/or ongoing Luteinizing Hormone‐Releasing

Hormone (LH‐RH) agonist treatment.

12. Progression of disease by PSA utilizing PCWG2 criteria and at least another of the following

criteria:

a. Bone scan: disease progression as defined by at least 2 new lesions on bone scan.

b. Soft tissue disease progression defined by modified RECIST 1.1.

c. Clinical progression with worsening pain and the need for palliative radiotherapy for

bone metastases.

13. Willing to have a biopsy to obtain tumor tissue for biomarker analyses prior to and after

treatment.

Exclusion Criteria

1. Prior treatment with PI3K, AKT, TOR kinase or mTOR inhibitors (see Appendix C).

2. Surgery, chemotherapy, or other anti‐cancer therapy within 4 weeks prior to trial entry /

randomization into the study (6 weeks for bicalutamide). Any other therapies for prostate

cancer, other than GnRH analogue therapy, such as progesterone, medroxyprogesterone,

progestins (megesterol), or 5‐alpha reductase inhibitors (e.g., finasteride or dutasteride),

must be discontinued at least 2 weeks before the first dose of study drug.

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3. Participation in another clinical trial and any concurrent treatment with any investigational

drug within 4 weeks prior to trial entry / randomization.

4. Prior limited field radiotherapy within 2 weeks or wide field radiotherapy within 4 weeks of

trial entry / randomization.

5. History of seizure or any condition that may predispose to seizure including, but not limited

to underlying brain injury, stroke, primary brain tumours, brain metastases, or alcoholism.

6. History of loss of consciousness or transient ischemic attack within the previous 12 months

of trial entry / randomization.

7. Known brain or leptomeningeal involvement.

8. Use of potent inhibitors or inducers of CYP3A4, CYP2C9 and CYP2C19 (see Appendix B)

within 2 weeks before trial entry / randomization (3 weeks for St John’s Wort) must be

avoided.

9. Clinically significant abnormalities of glucose metabolism as defined by any of the following:

a. Diagnosis of diabetes mellitus type I or II (irrespective of management).

b. Glycosylated hemoglobin (HbA1C) ≥8.0% at screening (64 mmol/mol) (conversion

equation for HbA1C [IFCC‐HbA1C (mmol/mol) = [DCCT‐HbA1C (%) – 2.15] x

10.929).

c. Fasting Plasma Glucose ≥ 8.9mmol/L at screening. Fasting is defined as no caloric

intake for at least 8 hours.

10. Inadequate organ and bone marrow function as evidenced by:

a. Hemoglobin <8.5 g/dL

b. Absolute neutrophil count <1.0 x 109/L

c. Platelet count < 75 x 109/L

d. Albumin ≤25 g/dL.

e. AST / SGOT and/or ALT / SGPT ≥ 2.5 x ULN (≥ 5 x ULN if liver metastases present)

f. Total bilirubin ≥ 1.5 x ULN (except for patient with documented Gilbert’s disease)

g. Serum Creatinine > 1.5 x ULN

11. Inability or unwillingness to swallow oral medication.

12. Malabsorption syndrome or other condition that would interfere with enteral absorption.

13. Any of the following cardiac criteria;

a. Mean resting corrected QT interval (QTcF) >470msec obtained from 3 consecutive

ECGs taken within 5 minutes.

b. Any clinically important abnormalities in rhythm, conduction, or morphology of a

resting ECG (e.g., complete left bundle branch block, third degree heart block).

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c. Any factors that increase the risk of QTc prolongation or risk of arrhythmic events

such as heart failure, hypokalemia, congenital long QT syndrome, family history of

long QT.

d. syndrome or unexplained sudden death under 40 years‐of‐age, or any concomitant.

e. medication known to prolong the QT interval or with a potential for Torsades de

pointes.

f. Experience of any of the following procedures or conditions in the preceding six

months: coronary artery bypass graft, angioplasty, vascular stent, myocardial

infarction, angina pectoris, congestive heart failure NYHA ≥ Grade2.

g. Uncontrolled hypotension defined as – systolic blood pressure (BP) <90mmHg

and/or diastolic BP <50mmHg.

14. Clinically significant history of liver disease consistent with Child‐Pugh Class B or C,

including viral or other hepatitis, current alcohol abuse, or cirrhosis.

15. Any other finding giving reasonable suspicion of a disease or condition that contraindicates

the use of an investigational drug or that may affect the interpretation of the results or

renders the patients at high risk from treatment complications.

16. Need for chronic corticosteroid therapy of >10 mg of prednisolone or >0.5mg of

dexamethasone per day or an equivalent dose of other anti‐inflammatory corticosteroid, for

the use of concomitant steroids on this trial please refer to section 12.1. Patients in which

corticosteroids cannot be stopped prior to entering the trial are allowed a maximum of 10mg

of prednisolone per day or equivalent. In the case of corticosteroid discontinuation, a 2‐week

(14 days) washout is required with a mandatory PSA check prior to starting the trial. If the

PSA has declined compared to the value obtained prior to stopping corticosteroids, patients

will not be eligible for study. Patients can only enter the study with a confirmed PSA

increase.

17. Malignancies other than prostate cancer within 5 years prior to trial entry / randomization,

except for adequately treated basal or squamous cell skin cancer.

18. Unresolved clinically significant toxicity from prior therapy except for alopecia and Grade

peripheral neuropathy.

19. Inability to comply with study and follow‐up procedures.

20. Patients with predominately small cell or neuroendocrine differentiated prostate cancer are

not eligible.

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2. Dose escalation/de-escalation schedule:

Dose Level AZD5363 - Commence Cycle 0 Day 1 MDV3100 - Commence Cycle 1 Day 1

-2 160mg bid 4 days on 3 days off 160mg daily

-1 240mg bid 4 days on 3 days off 160mg daily

1 320mg bid 4 days on 3 days off 160mg daily

1A (optional) 400mg bid 4 days on 3 days off 160mg daily

2 480mg bid 4 days on 3 days off 160mg daily

2A (optional) 560mg bid 4 days on 3 days off 160mg daily

3 640mg bid 4 days on 3 days off 160mg daily

3. Definition of Dose Limiting Toxicity (DLT): A DLT is defined as one of the following toxicities occurring mainly but not exclusively during the

DLT assessment window of Cycle 0 Day 1 to Cycle 1 Day 28 (35 day assessment window) and

is highly probably or probably related to either AZD5363 or enzalutamide (see causal attribution

guidance in Table 8):

1. Grade ≥3 non‐hematologic, non–hepatic major organ adverse event, excluding the following:

a. Grade 3 nausea, vomiting, or diarrhea that resolves to Grade ≤1 within 7 days with

appropriate supportive care.

b. Grade 3 rash that resolves rapidly upon discontinuation of drug with appropriate

supportive measures.

c. Grade 3 laboratory abnormality that is asymptomatic and deemed by the investigator

not to be clinically significant.

2. Grade ≥3 febrile neutropenia.

3. Grade ≥4 neutropenia (absolute neutrophil count <500/uL) lasting >7 days.

4. Grade ≥3 thrombocytopenia associated with acute hemorrhage.

5. Grade ≥4 thrombocytopenia.

6. Grade ≥4 anemia.

7. One episode of fasting Grade ≥4 hyperglycemia (>27.8mmol/l) or two episodes of

symptomatic fasting.

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8. Grade 3 hyperglycemia (>13.9 – 27.8mmol/l) on separate days within 7 days, as determined

by laboratory blood glucose evaluation.

9. Any Grade >3 elevation of hepatic transaminases (ALT or AST) OR total bilirubin lasting >48

hours will be considered a DLT with the following exceptions:

a. For patients with elevated hepatic transaminase at baseline due to documented liver

metastasis (i.e. <5 x ULN), hepatic transaminase >8x ULN for >48 hours will be

considered a DLT.

b. For patients with elevated total bilirubin at baseline due to documented liver

metastasis or Gilbert's disease (i.e. ³1.5 x ULN), total bilirubin of >2.5x ULN will be

considered a DLT.

10. Any case involving an increase in hepatic transaminase >3 x ULN and an increase in total

bilirubin >2 x ULN, without any findings of cholestasis AND in the absence of other

contributory factors (e.g. worsening of metastatic disease or concomitant exposure to known

hepatotoxic agent) is suggestive of potential drug‐induced liver injury according to Hy’s Law

and will be considered a DLT.

11. Any other toxicity, which in the view of the investigators is considered to be a DLT, at any

time during the study. These cases will be discussed at the SRC meetings.

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4. Research Sample Collection Schedule:

5. Research Sample Methods:Pharmacokinetic analysis:

Plasma concentration was measured by a fully validated liquid chromatography-tandem mass

spectrometry (LCMSMS) method and pharmacokinetic (PK) parameters maximum plasma

concentration (Cmax), time to reach Cmax (Tmax), the area under the plasma concentration

time curve (AUC) were derived from non-compartmental analysis (Phoenix version 6.4).

Geometric means of AUC and Cmax were derived at each dose level and dose normalized for

comparison with or without enzalutamide with a 90% confidence interval

Pharmacodynamic analysis:

Measurement of phosphorylated Ser9 and total GSK3β were undertaken on PRP using an

assay validated to Good Clinical Practice standards on the MesoScale Discovery (MSD®)

technology platforms1,2. In addition, phosphorylated Thr246 and total PRAS40 were

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immunofluorescent stained, imaged by confocal microscopy and quantified using IN Cell

translator and developer software (GE Healthcare Life Sciences) in fixed hair follicles3,4.

Targeted Next Generation Sequencing (NGS):

Targeted sequencing was performed as previously described5. Libraries were constructed from

40ng of DNA using GeneRead Mix-n-Match V2 (Qiagen®; Hilden, Germany) customized 113

genes panel and pooled libraries were sequenced on the MiSeq (Illumina®; San Diego, CA,

USA). FASTQ files were generated using the Illumina MiSeq Reporter v2.5.1.3. Sequence

alignments were performed using BWA tools and the GATK variant annotator by the Qiagen

GeneRead Targeted Exon Enrichment Panel Data Analysis web Portal.

PTEN Immunohistochemistry:

PTEN protein expression was determined by IHC on 4-mm-thick FFPE sections as previously

described6. Nuclear and cytoplasmic staining intensity were semi-quantitatively assessed by a

pathologist blinded to clinical outcome data using the H-score formula: formula: [(% of weak

staining cells) x 1] + [(% of moderate staining cells) x 2] + [(% of strong staining cells) x 3]. IHC

quantification is presented as H-scores (HS), ranging from 0 (minimum) to 300 (maximum)7. A

binary classification scheme as previously published8 was used with cases considered PTEN

normal if H-score >10 and PTEN loss if H-score≤10.

AdnaTestTM mRNA extraction, cDNA conversion and AdnaTestTM CTC Call

Isolation and enrichment of CTCs from CRPC patient peripheral blood (PB) draws was carried

out as previously described9. CTC calls for each sample are presented as CTC call positive

(actin positive and either PSMA and/or PSA and/or EGFR positive; CTC+), CTC call negative

(actin positive all other markers negative; CTC-) or failed cDNA conversion (actin negative;

failed).

mRNA quantification of AR-FL and AR-V7

CTC+ samples were used for the measurement and quantification of AR-V7 transcripts as

previously described9. AR-V7 status is presented as continuous (copies/mL) and binary (present

≥ 1 copy/mL and absent < 1 copy/mL) outcomes.

IHC staining for AR-V7 and pERK protein expression

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IHC for AR-V7 (Clone RM7, GTX33604, GeneTex) and pERK (Clone D13.14.4E, 4370, Cell

Signalling Technology) was performed on CRPC patient biopsies as previously described10. All

tissue blocks from CRPC biopsies were sectioned and only considered for IHC analyses if

adequate tumour material was present (≥ 50 tumour cells by author DNR). For AR-V7 IHC

(Clone RM7, GTX33604, GeneTex), following antigen retrieval (Tris/EDTA buffer, pH8.1) in

microwave for 18 minutes at 800W, the antibody was diluted (1:500) in Dako REAL diluent

(Dako, Agilent Technologies) and tissue was incubated for 1 hour. For pERK IHC (Clone

D13.14.4E, 4370, Cell Signalling Technology), following antigen retrieval (Citrate buffer, pH6.0)

in microwave for 18 minutes at 800W, the antibody was diluted (1:400) in Dako REAL diluent

(Dako, Agilent Technologies) and tissue was incubated for 1 hour. After washes, bound

antibody was visualized using the Dako EnVision Detection System (Dako, Agilent

Technologies). Sections were counterstained with hematoxylin. Mouse xenografts from 22Rv1

(AR-V7 positive) and PC3 (AR-V7 negative), and cell pellets from VCaP (AR-V7 positive) and

DU145 (AR-V7 negative), were used as controls for AR-V7 IHC. Cell pellets from 22Rv1 treated

with vehicle (pERK positive) or trametinib (pERK negative) were used as controls for pERK IHC.

Rabbit IgGs were used as negative controls. Nuclear AR-V7 expression was determined for

each case by an experienced pathologist (DNR) in a blinded fashion using a modified H-score

method and pERK expression was reported as percentage positive cells11.

CellSearchTM CTC enumeration

CTC counts were determined from mCRPC patient PB draw using the CellSearchTM CTC Kit

(Menarini, Silicon Biosystems) according to the FDA-cleared manufacturer’s method as

previously described9,12,13. Identification and enumeration of CTC was done in a blinded fashion

and reported as CTC count per 7.5mLs of peripheral blood.

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6. Supplementary Table Legends:

Supplementary Table S1: Dose escalation of capivasertib with number of patients treated and corresponding number of DLTs. *All DLT’s encountered were grade 3 maculopapular rash.

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Supplementary Table S2: Summary of Treatment Related Adverse Events.

Supplementary Table S3: Pharmacokinetic data from individual patients at cycles 0 and 2. AUC8h and Cmax derived from

non-compartmental analysis.

Supplemental Table S4: Cmax and AUC calculated at different dose levels. Cycle 2 data have been normalized to Cycle 0 dose. Mean difference of log transformed data shows a statistical difference based on 90% CI.

Supplementary Table S5: Response data with PTEN status and targeted next generation

sequencing results on an individual patient level. *Indicates this patient had stable disease on

treatment for 6 months. **Indicates this patient had a >30% PSA decline at 4 weeks but

withdrew consent from trial. ***Indicates this patient discontinued trial due to grade 3

hyperglycemia.

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Supplementary Table S6: Response Assessment. *Includes 2 non-confirmed CTC count

conversions.

Supplementary Table S7: Tissue samples obtained for correlative studies and their outcomes.

(+) indicates sample was obtained and analysis was successfully performed. (NS) indicates

that no sample was available for testing, or in the case of fresh tissue biopsy there was either no

biopsy performed or there was not adequate tumor sample in the biopsy for testing. (FQC)

indicates that a sample was available but testing failed quality control. (NP) indicates that this

test was not performed on this sample; note that this only applies to PTEN IHC, as testing was

only performed on either the archival sample or fresh tissue biopsy at screening.

Supplementary Table S8: Next generation sequencing data.

7. Supplementary Figure Legends:

Supplementary Figure S1: Treatment schedule. Each vertical line represents a day, with (x) representing days where

AZD5363 and/or enzalutamide were administered, respectively.

Supplementary Figure S2: Percentage change from baseline of pThr246 PRAS40 4h post dose of AZD5363 at cycle 0 day

1 and cycle 2 day 1 in hair follicles. Dotted line represents baseline set at pre-dose cycle 0 day

1. Horizontal line, median.; +, mean; box, quartiles 1 to 3; whiskers extend from the quartiles to

the most extreme observation within 1.5x interquartile range. To take into account the pThr246

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PRAS40 signals represented a sub-population of the PRAS40 levels in the hair follicles, the

pThr246 PRAS40 levels were normalized to the total PRAS40 levels in the same sample prior to

calculation of percentage change from baseline.

Supplementary Figure S3: Consort flow diagram. *1 patient (Patient 4) withdrew prior to starting any study treatment. **1

patient (Patient 7) had a multiple treatment interruptions during the DLT window due to non-

treatment related AEs, and was therefore considered non-evaluable. ***1 patient (Patient 10)

withdrew consent prior to completing the DLT window without experiencing a DLT.

Supplementary Figure S4: Individual patient plasma concentration of AZD5363 over time at cycles 0 (A) and 2 (B). Each

patient is represented by a colored line.

Supplementary Figure S5: Percentage change from baseline of pSer9 GSK3β post dose of AZD5363 at cycles 0 and 2 in

PRP. A) Percentage change at 4h after different doses of AZD5363 with (C2D1) and without

(C0D1) enzalutamide. Bars represent individual patients. Data are ranked in order of

descending change for each dose in C0D1 and the same patient rank order is used for C2D1. X

indicates missing data or data excluded due to hemolyzed samples. Dotted lines indicate -30

and -50% change. B) Temporal change from baseline in pSer9 GSK3β in PRP at various time

points after 400mg dose of AZD5363 with (C2D1) or without (C0D1) enzalutamide. Horizontal

line, median.; +, mean; box, quartiles 1 to 3; whiskers extend from the quartiles to the most

extreme observation within 1.5x interquartile range. *p<0.05 One Way repeated measures

ANOVA with Dunnett’s multiple comparison test compared to C0D1 pre.

For both A and B, baseline is pre-dose at cycle 0 day 1.  To take into account the pSer9 GSK3β

signals represented a sub-population of the GSK3β levels in the PRP, the pSer9 GSK3β levels

were normalized to the total GSK3β levels in the same sample prior to calculation of percentage

change from baseline.

Supplementary Figure S6:Circulating tumor cell (CTC) counts by CellSearch for each patient at screening (SC), cycle 0

day 1 (C0D1), cycle 2 day 1 (C2D1), cycle 4 day 1 (C4D1), and end of treatment (EOT). Patient

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4 did not receive any study treatment and was therefore not included in the CTC analysis.

*indicates patients meeting response criteria.

Supplementary Figure S7:IHC for AR-V7 (A.) and pERK (B.) expression by H-score for each patient at pre-dose, post-

dose, and end of treatment (EOT). *Indicates patients meeting criteria for response.

Supplementary Figure S8:(A) AdnaTestTM CTC AR-V7 analysis was performed on peripheral blood draws within RE-AKT

at screening (Sc), Cycle 4 Day 1 (C4D1) and end of treatment (EOT) where samples were

available. Continuous (mean ± minimum and maximum value) AR-V7 mRNA expression

(copies/ml; red bars) from technical replicates is shown. Dichotomized CTC AR-V7 (red boxes

positive; white boxes negative) status for each technical replicate is shown. CTC call (red boxes

positive; white boxes negative) performed in singlet for each sample is shown. Patients without

samples for analysis at a specific time point are shown (grey boxes and *). Patients

demonstrating a response to treatment are shown (*). (B) Nuclear AR-V7 protein expression

was determined by immunohistochemistry performed on CRPC biopsies pre- and post-

treatment, and at EOT. AR-V7 protein expression (H-score; red bars) is shown from each CRPC

tissue biopsies. Patients with (red boxes) and without (grey boxes and *) samples for IHC

analysis at each time point are shown (grey boxes and *). Patients demonstrating a response to

treatment are shown (*). (C) Percentage pERK positive cells was determined by

immunohistochemistry performed on CRPC biopsies pre- and post-treatment, and at EOT.

pERK positivity (% cells positive; red bars) is shown from each CRPC tissue biopsies. Patients

with (red boxes) and without (grey boxes and *) samples for IHC analysis at each time point are

shown (grey boxes and *). Patients demonstrating a response to treatment are shown (*). (D)

CellSearchTM CTC enumeration was performed on peripheral blood draws within RE-AKT at

screening (Sc), Cycle 0 Day 1 (C0D1), Cycle 2 Day 1 (C2D1), Cycle 4 Day 1 (C4D1), Cycle 7

Day 1 (C7D1) and end of treatment (EOT) where samples were available. CTC count per

7.5mLs of peripheral blood is shown. Patients demonstrating a response to treatment are shown

(*).

8. Supplementary References:

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