ACK1 Tyrosine Kinases: A Critical Regulator of Prostate Cancer

Nupam Mahajan Moffitt Cancer Center

Learners Objectives

�   How Androgen Receptor (AR) signaling is accomplished in absence of androgen

�   What are the epigenetic events that takes place to achieve androgen-independent AR signaling

�   What are the new therapeutic options being developed to overcome enzalutamide-resistant prostate cancer

Prostate Cancer

�   Prostate cancer is second most common cancer in American men

�   Almost 225,000 men are diagnosed and ~25,000 die due to prostate cancer in US every year

�   Androgen receptor (AR) is a key regulator of both initiation and progression of prostate cancer to metastatic stage

Androgen Receptor Signaling

AR

TATA ARE

PSA/hK2/TMPRSS2

+DHT

Nuclear Translocation

AR Coactivator RNA Pol

AR D

AR D

Cell survival and growth

Transcription

Castration Resistant Prostate Cancer

AR + Androgen

Androgen dependent or Hormone-sensitive

Tumor Growth

18-24 M

AR + Androgen

Tumor Growth

Castration Resistant Prostate Cancer

CRPC

Second generation of anti-androgen

�   Enzalutamide (Xtandi), is a new type of anti-androgen. It prevents AR entry into the nucleus, lowers PSA levels, slow the growth of tumors

�   It is effective in a subset of CRPC patients (1/3rd ) and survival advantage is modest (4-6 months).

�   Even the most responding patients relapsed within 2 years, often with lethal consequences

Tran et al., Science, 2009

Mechanism of Enzalutamide-resistance �   AR-V7 splice variant expression has emerged to be the major cause

of Enzalutamide and Abiraterone resistance

�   AR-V7 lacks the C terminal ligand-binding domain (& Enzalutamide binding) and remains constitutively active as a transcription factor.

Antonarakis et al., NEJM, 2014

CRPC: AR dependence

�   Overall, the most common genetic aberration in CRPC is AR mRNA upregulation, also reflected in AR-V7 increase in Enzalutamide resistant

�   These data have opened a new paradigm:

�   To achieve complete remission of CRPC, ablation of AR is the key.

�   However, targeted inhibition of AR transcription with small molecule inhibitors has not yet been accomplished.

ACK1 or TNK2

�  Ubiquitously expressed non-receptor tyrosine kinase

�   Plays a central role in integrating signal from receptor tyrosine kinases e.g. EGFR, HER2 and IR

�   ACK1 binds to AR and ACK1/AR complex translocate to nucleus activating transcription of AR target genes

SAM Kinase SH3 C CL MHR UB

Novel ACK1 inhibitor, (R)-9bMS

�  We identified (R)-9bMS as a potent ACK1 inhibitor

�   In vitro IC50=48 nM �   Mesylate salt is highly

soluble in aqueous media

�   (R)-9bMS exhibit significant selectivity

�   Inhibited 11 kinase out of 369 kinase (90% inhibition)

�   Except ACK1 others not known to express in prostate

Lawrence. et al., J Med Chem 2015

AR

pACK1

DM

SO(R

)-9bM

S

DH

T

Enza

luta

mid

e

1.0 0.1 2.9 3.0

Actin

siRNA

Con

trol

ACK1

VCaP

1.0 0.3 2.0 0.37

DM

SO

(R)-9bM

S

MG

132

MG

132+

(R)-9bM

S

AR

Actin

AR

Actin

LAPC4

.ACK1

1.0 0.30

Con

trol

ACK1

C4-2B

1.0 0.34

AR

DM

SO

(R)-9bM

S

DH

TEn

zalu

tam

ide

1.0 0.13 0.65 1.9

Actin

C4-2B

pACK1

(R)-9b suppresses AR expression

(R)-9b inhibits AR transcription

0.00.20.40.60.81.01.21.41.6

(R)-9b

DM

SO

DH

TEn

zalu

t

(R)-9b

0.0

0.5

1.0

1.5

2.0

2.5(R

)-9b

DM

SO DH

TEn

zalu

t

(R)-9b

VCaPAR

/Act

in R

atio

0.0

0.5

1.0

1.5

2.0

* **D

MSO DH

TEn

zalu

t

(R)-9b

(R)-9b

0.0

0.4

0.8

1.2

1.6

DM

SO DH

TEn

zalu

t

(R)-9b

(R)-9b

* **

PSA

/Act

in R

atio

DM

SO DH

TEn

zalu

t

(R)-9b

(R)-9b

LNCaP

0

1

2

3

4

DM

SO DH

TEn

zalu

t

(R)-9b

(R)-9b

LAPC4

0.0

0.51.0

1.5

2.02.5

3.0

3.5

* *** **

* **

0

1

1.5

2

DM

SO(R

)-9b

C4-2B

*

0

1

1.5

2

DM

SO(R

)-9b

*2.5

2.5

*

*****

1.8VCaP LNCaP LAPC4 C4-2B

0.00.2

0.40.6

0.8

1.0

1.2D

MSO

(R)-9b

AR-V7/

Act

in R

atio

AR-V7

AR

Actin

VCaP

AR-V

DM

SO

(R)-9b

.

Actin

LAPC4

AR-V

DM

SO(R

)-9b

Enza

luta

mid

e

DH

T

*

(R)-9b suppresses AR-V expression

How does ACK1 regulates AR/AR-V7

transcription?

TATA

AR Coactivator

Transcription Androgen

AR

ACK1

•   ACK1 `piggybacks’ AR to nucleus •   DNA is not exposed, but bound to histones to form chromatin

•   Does ACK1 phosphorylate histone to activate AR transcription?

P

P

ACK1 translocates to nucleus

Histone H4 phosphorylation at Tyr88

Antibody generation Mass Spectrometry

pY88-H4

H4

pY88-H4 Ab

pY88-H4 Ab+phosphopeptide

Biotin

100 400 700 1000 1300 1600m/z0

50

100

Intensity

b1

558.8 559.5 560.2m/z

[M+3H]3+, 558.9605

MS1

y1

y2 y4

y5

b7

y6y7

KT V T A M D V V Y AL KRMS/MS

pY Immonium Ion

CRPC Sample #1

ACK1 phosphorylates H4 at Tyr88

Endogenous H4 Y88-phosphorylation

+ + + H4

pY88-H4

+ + ACK1 _ _ + (R)-9bMS

ACK1

H4

_

Inpu

t

+ + _ + _ _

LAPC4(PDGF)

pY88-H4

+ + Ligand

pACK1

H4

_ + (R)-9bMS _ _

LNCaP(IGF)

pY88-H4

H4

ACK1

Y88F

Y72F

Vec

+ ACK1

Myc

Inpu

t

Inpu

t

In vitro Kinase assay

+ + + H4

pY88-H4

+ + ACK1 _ _ + (R)-9bMS

ACK1

H4

_

Inpu

t

+ + _ + _ _

LAPC4(PDGF)

pY88-H4

+ + Ligand

pACK1

H4

_ + (R)-9bMS _ _

LNCaP(IGF)

pY88-H4

H4

ACK1

Y88F

Y72F

Vec

+ ACK1

Myc

Inpu

t

Inpu

t

+ IGF

DM

SO

DH

T

(R)-9b

MS

Enz

alut

C4-2B

pY88-H4

H4

pY-ACK1

Input

H4 Y88-phosphorylation is insensitive to Enzalutamide and DHT

pY88-H4 marks deposited upstream of AR

Exon 1 2 3 4 5 6 7 866,7

63,8

74

66,6

69,6

00

66,6

49,5

50

66,6

31,3

00

AREM1 AREM2 AREM3

ChIP:pY88ChIP:IgG

nt position

Peak regions

Primers 4 137

AREM 3 2 1

(R)-9b compromises pY88-H4 deposition

ChIP: pY88-H4 IgG

AREM1

Per

cent

ofIn

put

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

DMSO(R)-9b

AREM2

0.0

0.5

1.0

1.5

2.0

2.5AREM3

DMSO(R)-9b

ChIP: pY88-H4 IgG ChIP: pY88-H4 IgG

* *

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

DMSO(R)-9b

*** *

Control Primer

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

DMSO(R)-9b

ChIP: pY88-H4 IgG

How do pY88-H4 epigenetic marks regulate AR

transcription?

WDR5-MLL2 complex is an epigenetic reader of pY88-H4 marks

�   pY88-H4 peptide used as a bait to identify interacting proteins:

Ø   MLL2 (KMT2D)- H3K4 lysine methyltransferase, modifies histones to activate transcription

Ø   WDR5- WD repeat domain 5

WDR5 `reads’ pY88-H4 marks, MLL2 acts as a `scribe’ to regulate deposition of H3K4me3 activating marks

ChIP: H3K4me3ChIP: MLL2

Per

cent

ofIn

put

0.0

0.2

0.4

0.6

0.8

1.0

1.2

DMSO(R)-9b

0.0

0.2

0.4

0.6

0.8

1.0DMSO(R)-9b

AREM1 ControlAREM1 Control

ChIP: Pol II

0.0

0.2

0.4

0.6

0.8

1.0DMSO(R)-9b

AREM1 Control

** * *

0

1

2

3

4

DMSO(R)-9b

ChIP: WDR5

AREM1 Control

**

Per

cent

ofIn

put

Per

cent

ofIn

put

Per

cent

ofIn

put

(R)-9bMS overcomes Enzalutamide Resistance

LNCaP-C4-2B 0.40 uM VCaP 0.45 uM LAPC4 0.75 uM LNCaP 1.75 uM RWPE 10.0 uM

(R)-9bMS Inhibits CRPC Growth

n=9

n=9

Days post injection

Vehicle(R)-9bMS

0 10 20 30

Tum

orVo

lum

e(m

m3)

0

200

400

600

800

1000

1200

C4-2B XenograftTumorss0.0

0.2

0.4

0.6

0.8

1.0DMSO(R)-9b

n=6

n=3

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4ARDMSO(R)-9b

PSA

n=6

n=3

0.0

0.5

1.0

1.5

2.0

2.5

3.0TMPRSS2DMSO(R)-9b

n=3n=6

** ** **

ACK1 AR MLL2 WDR5

P P

AREMs 1-3 AR Transcription

ACK1 Inhibitor

Mahajan K. et al., Cancer Cell, 2017

M

Epigenetic dynamics at AR locus

9b

Conclusion

�   We uncovered a new signaling nexus - ACK1 mediated H4-Y88-phosphorylation that maintains AR transcript levels in absence of androgen

�   H4-Y88-phosphorylation can be suppressed by small molecule inhibitor (R)-9bMS, compromising AR and AR-V7 transcription

�   ACK1 inhibitor sensitizes Enzalutamide-resistant CRPC cells, opening new treatment option for patients that are:

Ø   (i) not-responsive to this second generation of antiandrogen Ø   (ii) those CRPC tumors that have developed resistance

�   Mahajan K, Malla P, Lawrence H, Chen Z, Sinha CK, Malik R, Shukla S, Kim J, Coppola D, Lawrence N and Mahajan NP*.

ACK1 regulates histone H4 Tyr88-phosphorylation and AR gene expression in castration resistant prostate cancer. Cancer Cell, 31:790-803, 2017.

v   Mahajan K and Mahajan NP*. ACK1/TNK2 tyrosine kinase: molecular signaling and evolving role in cancer. Oncogene, 34:4162-7, 2015

v   Lawrence HR, Mahajan K, Liu Y, Zhang D, Tindal N, Huseyin M,

Gevariya H, Kazi S, Ozcan S, Mahajan NP*, Lawrence NJ*. Development of novel ACK1 inhibitors using a fragment based approach. J. Med. Chem., 58:2746-63, 2015.

References

v   Kiran Mahajan v   Nick Lawrence v   Harshani Lawrence v   Domenico Coppola v   Pavani Malla v   Chandan Sinha, Michigan

Acknowledgements

�   NIH/NCI (R01 CA208258) �   Bankhead Coley Award (6BC08) �   Department of Defense Breakthrough Award

(W81XWH-15-1-0312) �   Celgene award (02-20000-01-01) �   Prostate Cancer Foundation, Challenge Award (17CHAL06)

Current Funding

Questions

Audience Response question

�   Question: What is the `third generation’ of AR inhibitor

�   Answer: (R)-9bMS