Small molecule inhibitors of JAK1/2 improve physiological and functional measures of cancer-associated cachexia

Jordan S. Fridman, Eian Caulder, Xiaoming Wen, James Rodgers, Andrew Combs, Timothy Burn, and Kris Vaddi Incyte Corporation, Wilmington, Delaware, USA

ABSTRACT # 2848
Cancer-associated cachexia (CAC) is a significant contributing factor to the morbidity and mortality of malignancy. Though poorly defined, CAC is broadly described as a significant unintended and undesirable weight loss often accompanied by fatigue, altered metabolism, and systemic inflammation. Here, we attempt to understand the role of inflammation in CAC and demonstrate that IL-6, amongst other cytokines, contributes to multiple aspects of cachexia. Furthermore, we show that pharmacological antagonism of inflammatory cytokine signaling - via inhibition of JAK1/2 - reduces aberrant JAK/STAT activity and dramatically improves body mass and physical performance in mouse models of CAC. We characterized CAC using 3 tumor models (Colon26, PC-3M, and A375.S2). Effects of tumors on body weight ranged from total inhibition of weight gain to 30% weight loss; muscle and fat were depleted with a maximum loss of ~90% of parametrial fat stores and ~50% of gastrocnemius muscle weight. These changes had a dramatic effect on physical performance as demonstrated by decrements in grip strength and locomotor activity approaching 50% and 90%, respectively. Splenomegaly was observed in the majority of models suggesting systemic inflammation may contribute to the cachectic state. We therefore performed an unbiased analysis of plasma samples from cachectic mice and identified a number of markedly elevated inflammatory cytokines and chemokines, many of which signal through the non-receptor tyrosine kinases JAK1/2 (e.g. IL-6, IL12, and IL17) or which are indirectly stimulated by such cytokines (e.g. IL-6 induced MCP1). Moreover, administration of physiologically relevant levels of IL-6 was sufficient to activate JAK/STAT in muscle and result in reduced lean body mass and grip strength. Because of the reported and observed inflammatory components of CAC, we hypothesized that pharmacological inhibition of JAK1/2 may prevent or improve the cachectic phenotype. Using selective inhibitors (INCB16562 and INCB19408), we demonstrated that systemic inhibition of JAK1/2 reduces aberrant JAK/STAT signaling in muscle in the aforementioned models, minimizes splenomegaly and has a marked tissue salvaging effect on both fat and muscle resulting in striking functional improvements (~200%) in strength and activity. These effects were not associated with changes in food consumption or tumor growth. In summary, the data suggest that multiple JAK-activating cytokines are elevated in CAC and contribute to functional deficits associated with the condition. Moreover, we show that systemic inhibition of JAK1/2 can reduce inflammation, improve total and lean body mass, and enhance functional performance in multiple cachexia models. Clinical exploration of selective JAK inhibition is therefore warranted for the prevention or alleviation of CAC.

1.

Biochemical and cellular characterization of Incyte JAK inhibitors Kinase Potency* (nM)

2.
A

Tumor growth (A) is associated with loss of total body weight (B), fat and muscle (C)
A375.S2 Human Melanoma PC-3M Human Prostate Cancer

3.
A

Cachectic mice have elevated plasma levels of inflammatory proteins (A) but no changes in food consumption (B)

Cl N

Tumor Volume (mm3)

Compound INCB16562 INCB19408

JAK1 9 10

JAK2 2 21

JAK3 1895 1945

Tyk2 32 94
*performed at 1mM ATP

Cl H N N

2500

2000

Colon 26 Murine Adenocarcinoma

B
Food Consumption

1500

Grams chow / interval

Cytokine levels relative to normal mice

Biochemical selectivity was confirmed at concentrations ~100x the IC50s of JAK1/2

120

INCB16562

N H

1000

220 Nave BALB/c nu/nu A375.S2 Human Melanoma 180 PC-3M Human Prostate Cancer Colon 26 Murine Adenocarcinoma 140 Nave BALB/c

500

0 0 5 10 15 20 25 30 35 40 45 50 55 60

Proliferation

100 80 60 40 20 0 -20 1

Days after inoculation

B
20

100

TF-1
GM-CSF INCB16562 (nM) 0 + + + 0 300 1000 p-STAT5 STAT5

% Body Weight Change

C
100
Parametrial Fat Gastrocnemius Muscle

60

10

20 0 5 10 15 20 25 30 35 40 45 50 55 60

% of Controls

10

100

1000

10000

75 50 25 0 -67%

-52% -78%

-36% -53%

Days after inoculation Fold change (log2)

INCB16562 (nM)
IC50 (nM) TF-1 TF1-BcrAbl 102 36 > 4,000 N 3 3

-10

Nave BALB/c nu/nu A375.S2 Human Melanoma

-20

PC-3M Human Prostate Cancer Colon 26 Murine Adenocarcinoma Nave BALB/c

-90%

-30 0 5 10 15 20 25 30 35 40 45 50 55 60

Days after inoculation

A375.S2

PC-3M

Colon-26

4.
A
2800

JAK1/2 inhibition prevents loss of muscle and fat stores independently of effects on tumor growth resulting in functional improvement in the Colon26 tumor model
Tumor Growth
Vehicle INCB16562 INCB19408

5.
A
1000

In the PC-3M human prostate cancer model, selective JAK inhibition improves cachexia-associated functional endpoints
Tumor Growth
Vehicle INCB16562 Enbrel

6.
Nave

Infusion of IL-6 causes loss of muscle mass (A) and strength (B) through activation of JAK signaling (C) reversal by JAK1/2 inhibition

CONCLUSIONS
Tumor growth is associated with weight loss and decreased fat and muscle stores without altered food consumption Administration of selective JAK1/2 inhibitors improves body composition and physical performance in mouse models of CAC High levels of inflammatory proteins were observed in multiple CAC models Infusion of IL-6 activates JAK/STAT signaling in skeletal muscle resulting in decreased muscle mass and strength that is prevented by JAK1/2 inhibition The selective JAK1/2 inhibitor INCB018424 improves body mass and physical activity in patients suffering from myeloproliferative neoplastic diseases (Verstovsek, ASH 2008)

B
250

Body Composition
Parametrial Fat Gastrocnemius Muscle 161% 132% 121% 204%

B
5

Total Body Weight

Vehicle INCB16562 Enbrel 0

A
115

Fat and Muscle

Parametrial Fat Gastrocnemius Muscle

B
103%

Grip Strength
Control IL-6 / Vehicle IL-6 / INCB16562

% Change from Baseline

Tumor Volume (mm3)

Tumor Volume (mm3)

% of Vehicle Control

2400 2000 1600 1200 800 400 0 0 2 4

30

% of Controls

200 150 100 50 0

800

-1%

103%

400

-10

Grams

600

-5

100

-17%

20

85

10

200

-15

Days of Treatment

10

12

14

INCB16562

INCB19408

0 0 2 4 6 8 10 12

-20 0 2 4 6 8 10 12

70 IL-6 / Vehicle IL-6 / INCB16562

0 0 4

Day of Treatment
7 12 14

C
25
Nave Vehicle INCB16562 INCB19408

Grip Strength

D
100

Exercise Wheel Activity

250 236% 213%

C
16

Days of Treatment
Days Post-Treatment

Grip Strength
Nave Vehicle INCB16562 Enbrel

D
100

Days Post-Treatment Days of Treatment

Exercise Wheel Activity

400 300 200 100 0 414%

C
% of Controls
500 400 300 200 100 0 Control

Muscle pSTAT5
140

Muscle pSTAT3 % of Controls

Ave. Strength (g)

% of Vehicle Control

Ave. Strength (g)

% of Naive Control

75 50 25 0

12 8 4 0

% of Naive Control

200 150 100 50 0

15 10 5 0 -1

75 50 25 0

% of Vehicle Control

20

110

80 50 Control IL-6 / Vehicle IL-6 / INCB16562

-90%

-80%

IL-6 / Vehicle

IL-6 / INCB16562

Days of Treatment

12

Nave

Vehicle

INCB16562 INCB19408

-1

10

No significant total body weight loss or changes in food consumption during this experiment

Days of Treatment TNF inhibition not tested in wheel activity assay

Nave

Vehicle

Vehicle

INCB16562

No significant loss of body weight or change in food consumption noted

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