Efficacy of IRAK 4 Kinase Inhibitor CA-4948 in Mantle Cell

CA-4948 Blocks the TLR/IL-1R Induced Canonical NF-κB signaling Pathway

Correspondence

CARD11 activating mutations

(8%)

CA-4948 is a potent, oral IRAK4 Ser/Thr kinase inhibitor with >500-fold against IRAK1 CA-4948 treatment in culture did not result in antiproliferative/cytotoxicity activity in the

6 MCL cell lines tested (>10 µM at 3 days) CA-4948 inhibited TLR-induced signaling and cytokine production in MCL cell lines

with an intact BCR-driven canonical NF-κB pathway CA-4948 resulted in potent in vivo anti-tumor activity in MCL models with intact

canonical NF-κB signaling, which was enhanced in combination with ibrutinib treatment

CA-4948 exhibited weakest in vivo activity in models with alternative NF-κB signaling These results underscore the therapeutic potential of targeted IRAK4 kinase inhibition

by CA-4948 in combination with BTK inhibitors for the treatment of MCL

Efficacy of IRAK4 Kinase Inhibitor CA-4948 in Mantle Cell Lymphoma Robert N. Booher, Ruzanna Atoyan, Maria Elena S. Samson, Holly Modafferi, Mylissa A Borek, Steven Dellarocca, and David P. Tuck

Curis, Inc., Lexington, MA

#C31 ACCR 2018 Advances in Malignant Lymphoma

Curis, Inc. 4 Maguire Road Lexington, MA 024221

Abstract Background: NF-κB signaling plays a critical role in MCL as evidenced by the high response rate observed in refractory/relapsed MCL patients treated with BTK inhibitor ibrutinib. However, the majority of these patients relapse with a dismal prognosis. Thus, identifying additional targeted agents that affect the eventual activation of NF-κB mediated through the B-cell receptor (BCR) or other signaling pathways is needed to address primary or relapsed ibrutinib-resistance. As an essential component in the IL1R/toll-like receptor (TLR) mediated NF-κB signaling pathway, IRAK4 is one such target. For these studies, we tested the novel oral IRAK4 kinase inhibitor CA-4948, which is currently in a Phase I trial for R/R non-Hodgkin lymphoma (clinicaltrials.gov NCT03328078). Experimental procedures: Six MCL cell lines (JeKo-1, MAVER-1, Mino, GRANTA-519, REC-1, and Z-138) were exposed to escalating doses of CA-4948 either alone or in combination with other targeted agents, and changes in viability were evaluated after 24-96 hr. These same six cell lines were also treated with CA-4948 to assess its effect on TLR- agonist-induced NF-κB signaling by evaluating cytokine production and western blot analysis of intracellular signaling pathway components. Finally, the in vivo efficacy of CA-4948 was evaluated in mouse xenograft subcutaneous tumor models of these six cell lines. Results: Similar to previously observed results in CA-4948 treated DLBCL cell lines, blocking IRAK4 kinase function was neither cytostatic nor cytotoxic in these six MCL lines under standard in vitro growth conditions (EC50 > 10 µM). In contrast, CA-4948 blocked TLR- agonist-induced pro-inflammatory cytokine production and TLR pathway activation markers (e.g. p-IKK) in MCL lines, including Mino and REC-1. Interestingly, GRANTA-519 and Z-138 cells exhibited constitutive production of a subset of cytokines in the absence of TLR stimulation, consistent with reports that these lines have deregulated alternative NF-κB signaling. In vivo, CA-4948 exhibited anti-tumor activity in the Mino and REC-1 xenograft models. Consistent with constitutive NF-κB activation independent of BCR and TLR signaling, CA-4948 demonstrated no activity against the GRANTA-591 and Z-138 xenograft tumor models. Conclusion: Our findings reveal a requirement for IRAK4 kinase function in TLR- agonist-induced NF-κB signaling and cytokine production in MCL cell lines. Oral administration of CA-4948 demonstrated an essential in vivo role for IRAK4 function in certain MCL cells grown as xenograft tumors. These results provide the rationale for continued testing of CA-4948 in combination with canonical and alternative NF-κB pathway-targeted agents.

Summary CA-4948 ± Ibrutinib Inhibition of NF-κB p65 Signaling

and Cytokine Production

ITAM

ITAM

SFK

SYK

CD79B /A ITAM

mutations (23%)

P

P

BTK

TLR

IRAK4 IRAK1

MYD88 activating mutations

(29%)

BCR Signaling TLR Signaling

PI3K

BLNK PLCγ

P

P P

P

P

Nucleus

Cytosol

MYD88

P

TRAF6

TAK1 TAB1/2

MKKs

p38 JNK ERK

P

P

P P P

AP1

Pro-inflammatory cytokines

TRAF6 MALT1 BCL10

CARD11 P

PKCb

P P

CD79

B

CD79

A

p50 p65 NF-κB

p50 p65

IκBα

p50 p65

Pro-inflammatory cytokines

IKKγ

A20

Ub

A20 loss

silencing (23%)

Ub

IKKβ IKKα P

Ub Ub

A20

AP1 AP1

Illustration modified from L. Staudt & Karin, 2010 CSH Perspect Biol. Mutation frequencies from Ngo et al. 2011 Nature 470:115

Ub Ub

Ibrutinib Idelalisib

P

CA-4948

Antiapoptosis (Bcl2)

BCL2 overexpression/ translocation

Venetoclax

Copies of this poster obtained using the QR code are for personal use only and may not be reproduced without written permission of the authors.

Poster PDF Copy

In vitro CA-4948 Viability Assays in MCL Cell Lines

Contact Information: Robert Booher, Ph.D. [email protected]

CA-4948 Exhibits Anti-Tumor Efficacy in Xenograft MCL Models with Canonical NF-κB Activation

In vivo efficacy studies of xenograft subcutaneous MCL tumor models. MCL cell lines with chronic activation of (A) BCR-driven classical and (B) alternative NF-κB pathways (Rahal R. et al., (2014) Nature Medicine 20(1) 87-94)

Drug Dosage (mg/kg)

TGI % (Day 14)

P value (T-test)

# mice (Day 14)

Vehicle (Ibrutinib) - na na 10/10 CA-4948 100 50 < 0.0001 9/10 Ibrutinib 12.5 15 ns 9/10

CA-4948 + Ibrutinib 100 + 12.5 81 < 0.0001 9/10

R E C -1 T u m o r G ro w th

0 5 1 0 1 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

Tu

mo

r V

olu

me

(m

m3

)+

SE

M

C A -4 9 4 8 , 1 0 0 m g /k g

v e h ic le ( Ib ru tin ib )

Ib ru t in ib , 1 2 .5 m g /k g

C A -4 9 4 8 , 1 0 0 m g /k g+ Ib ru t in ib , 1 2 .5 m g /k g

M IN O T u m o r G ro w th

0 5 1 0 1 5 2 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

C A -4 9 4 8 , 1 0 0 m g /k g

v e h ic le 1 + 2



Drug Dosage (mg/kg)

TGI % (Day 20)

P value (T-test)

# mice (Day 20)

Vehicle 1 + 2 - na na 10/10 CA-4948 100 na na 10/10 Ibrutinib 12.5 na na 10/10

CA-4948 + Ibrutinib 100 + 12.5 na na 10/10

Drug Dosage (mg/kg)

TGI % (Day 18)

P value (t-test)

# mice (Day 18)

Vehicle 1 + 2 - na na 11/12 CA-4948 100 81 < 0.001 12/12 Ibrutinib 12.5 62 0.002 12/12

CA-4948 + Ibrutinib 100 + 12.5 85 < 0.001 12/12

J e K o -1 T u m o r G ro w th

0 5 1 0 1 5 2 0 2 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

C A -4 9 4 8 , 1 0 0 m g /k g

v e h ic le 1 + 2



G R A N T A -5 1 9 T u m o r G ro w th

0 5 1 0 1 5 2 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

Tu

mo

r V

olu

me

(m

m3

)+

SE

M

C A -4 9 4 8 , 1 0 0 m g /k g

v e h ic le 1 + 2



Drug Dosage (mg/kg)

TGI % (Day 22)

P value (T-test)

# mice (Day 22)

Vehicle 1 + 2 - na na 8/10 CA-4948 100 20 0.26 10/10 Ibrutinib 12.5 3 0.81 9/10

CA-4948 + Ibrutinib 100 + 12.5 21 0.18 10/10

Z -1 3 8 T u m o r G ro w th

0 5 1 0 1 5 2 00

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

C A -4 9 4 8 , 1 0 0 m g /k g

v e h ic le 1 + 2



M A V E R -1 T u m o r G ro w th

0 5 1 0 1 5 2 0 2 50

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

C A -4 9 4 8 , 1 0 0 m g /k g

v e h ic le 1 + 2



Drug Dosage (mg/kg)

TGI % (Day 18)

P value (T-test)

# mice (Day 18)

Vehicle 1 + 2 - na na 10/10 CA-4948 100 na na 10/10 Ibrutinib 12.5 na na 10/10

CA-4948 + Ibrutinib 100 + 12.5 na na 10/10

Drug Dosage (mg/kg)

TGI % (Day 22)

P value (T-test)

# mice (Day 22)

Vehicle 1 + 2 - na na 10/10 CA-4948 100 58 <0.0001 9/10 Ibrutinib 12.5 17 ns 10/10

CA-4948 + Ibrutinib 100 + 12.5 38 0.0003 9/10

CA-4948 Combination*

Cell Line Palbociclib (CDK4/6i)

Ibrutinib (BTKi)

Venetoclax (BCL2i)

Bortezomib (Proteasome)

REC-1 no synergy mild synergy mild synergy mild synergy

Mino no synergy no synergy no synergy no synergy

JeKo-1 no synergy no synergy no synergy mild synergy

GRANTA-519 no synergy no synergy no synergy no synergy

Z-138 no synergy no synergy no synergy mild synergy

MAVER-1 no synergy no synergy no synergy no synergy

*: summary results of CellTiter Glo viability assays after 24, 48, 72 and 96 hr treatments

CA-4948 EC50 (µM)*

Cell Line 3 days 5 days 7 days

REC-1 >10 4.7 2.9

Mino >10 >10 >10

JeKo-1 >10 >10 >10

GRANTA-519 >10 >10 >10

Z-138 >10 >10 >10

MAVER-1 >10 >10 >10

*: average of 2 or more CellTiter Glo viability assays

Uns

timul

ated

D

MSO

10

1.

0 0.

1 µM

+ TLR agonist

IKK-β P-IKKα/β

NF-κB P-NF-κB

ERK P-ERK

CA-4948

TLR-induced cytokine release assays (IC50 = 150-220 nM, THP1)

TLR-induced NF-κB reporter assays (IC50 = 520 nM, THP1)

DiscoverX Kinase Kd (nM)

IRAK4 23 IRAK1 12,000

CA-4948 Binding Affinity Activity

CA-4948: Small molecule inhibitor ATP-competitive, reversible Oral bioavailable

In vitro THP1 monocytic cell assays

CA-4948 inhibition of NF-kB reporter, secreted cytokine levels, and phospho-signals in THP1 monocytic cells

Activation of NF-κB pathway and Cytokine Production in

Response to TLR Agonists in MCL Cell Lines

REC

-1

GR

AN

TA-5

19

30 min 1 hr

IKK-α P-IKKα/β

NF-κB P-NF-κB

Moc

k

TLR agonist

1/2 4 5 7/8 Moc

k

TLR agonist

1/2 4 5 7/8

α-Tubulin

Moc

k

TLR agonist

1/2 4 5 7/8 Moc

k

TLR agonist

1/2 4 5 7/8

30 min 1 hr

MIN

O

JeK

o-1

MAV

ER-1

TH

P-1

IKK-α P-IKKα/β

NF-κB P-NF-κB

α-Tubulin

IKK-α P-IKKα/β

NF-κB P-NF-κB

α-Tubulin

A. B.

0

2 0 0

4 0 0

6 0 0

IL -6(p g /m l)

( - ) T L R a g o n s t (+ ) T L R a g o n s t

0

5 0 0

1 ,0 0 0

IL -8(p g /m l)


0 .1

1

1 0

1 0 0

1 ,0 0 0

1 0 ,0 0 0

T N F -α(p g /m l)


Min

o

R E C -1

G R A N T A -51 9

J eK o -1

MA V E R -1

Z -13 8

Min

o

R E C -1

G R A N T A -51 9

J eK o -1

MA V E R -1

Z -13 8

1

1 0

1 0 0

1 ,0 0 0

1 0 ,0 0 0

IL -1 0(p g /m l)


Cytokine Levels in Media From ± TLR-Stimulated MCL

Cell Lines (24 hr)

A. MCL cell lines were stimulated with agonists for TLR1/2 (Pam3CSK4), TLR4 (LPS), TLR5 (FLA-ST), or TLR7/8 (R878). NF-κB p65 blots are shown

B. MCL cells were stimulated with TLR cocktail (Pam3CSK4, LPS, FLA-ST, and R848)

A.

B.

IKK-α

P-IKKα/β

NF-κB

P-NF-κB

α-Tubulin

Uns

timul

ated

TLR1/2 agonist CA-4948

(µM)

− + + + − + + +

Uns

timul

ated

TLR4 agonist CA-4948

(µM)

Uns

timul

ated


(µM)

− + + + − + + +

Uns

timul

ated


(µM)

Uns

timul

ated


(µM)

− + + + − + + +

Uns

timul

ated


(µM)

REC-1 MINO JeKo-1

A. CA-4948 inhibition of TLR signaling pathway components after 1 hr stimulation B. Combination effect of CA-4948 + ibrutinib on TLR-induced cytokine production

0 .0

0 .5

1 .0

M IN O

p-I

KK

α/β

/ I

KK

α

T L R 7 /8(R 8 4 8 )

1 .0

C A -4 9 4 8(µ M )

0 .51 .0

C A -4 9 4 8(µ M )

0 .5

T L R 1 /2(P a m 3 C S K 4 )

0 .0

0 .5

1 .0

R E C -1

p-I

KK

α/β

/ I

KK

α

T L R 4(L P S )

1 .0

C A -4 9 4 8(µ M )

0 .51 .0

C A -4 9 4 8(µ M )

0 .5

T L R 1 /2(P a m 3 C S K 4 )

0 .0

0 .5

1 .0

J e K o -1

p-I

KK

α/β

/ I

KK

α

T L R 7 /8(R 8 4 8 )

1 .0

C A -4 9 4 8(µ M )

0 .51 .0

C A -4 9 4 8(µ M )

0 .5

T L R 1 /2(P a m 3 C S K 4 )

0

5

1 0

1 5

IL -8(p g /m l)

Un s tim

u late

dD

MS O 1 0 3 .3

1 .1

0 .3

7 0 .1

2 10 .3

30 .1

10 .0

3 70 .0

1 2 10 .3

30 .1

10 .0

3 70 .0

1 2 10 .3

30 .1

10 .0

3 70 .0

1 2 10 .3

30 .1

10 .0

3 70 .0

1 2

0

5 0

1 0 0

1 5 0

T N F -α(p g /m l)

- + + + + + +

C A -4 9 4 8(µ M )

T L R 1 /2 a g o n is t:(P a m 3 C S K 4 )

+ + + + +

1 0 µ MC A -4 9 4 8

3 µ MC A -4 9 4 8

1 .1 µ MC A -4 9 4 8

Ibrutin ib (µ M )

Ib ru tin ib (µ M )

+ + + + ++ + + + ++ + + + +

A. B. IL-8 and TNF-α levels in tissue culture medium of REC-1 cells after 5 hr TLR1/2 agonist stimulation

Documents

Efficacy of IRAK 4 Kinase Inhibitor CA-4948 in Mantle Cell