IL-6 contributes to the suppression of T and NK cell anti-tumor activity in EGFR-mutant NSCLC Sonia A. Patel 1 , Monique B. Nilsson 1 , Yan Yang 1 , XiaoxingYu 1 , Fahao Zhang 1 , Alissa Poteete 1 , Xiaoyang Ren 1 , Xiuning Le 1 , Li Shen 2 , Jing Wang 2 , John V. Heymach 1 1 Department of Thoracic and Head and Neck Medical Oncology, 2 Department of Cancer Biology, 3 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA •
Introduction
IL-6 suppresses the activation of NK cells in the EGFR-mutant microenvironment A HCC4006 OR2 OR4 OR7 HCC4006 OR2 OR4 OR7
Depletion of IL-6 increases overall survival and number of infiltrating lymphocytes in EGFR mutant GEMMs
• Patients with NSCLC with activating mutations in epidermal growth factor receptor (EGFR) receive clinical benefit from treatment with EGFR tyrosine kinase inhibitors (TKIs) • The majority of these patients will acquire resistance which can be mediated by various mechanisms including secondary EGFR mutations such as T790M, MET amplification, or EMT. • Anti-PD-1/PD-L1 immune checkpoint-blockade demonstrated clinical benefit in NSCLC patients. However, among patients with EGFR-mutant NSCLC, response rates to immunotherapy are minimal. • Previous studies show that IL-6 is a critical mediator of EGFR-TKI resistance. Thus, we sought to investigate the impact of IL-6 on anti-tumor immunity in EGFR-mutant NSCLC.
B
A
B
0 20 40 60 80 100
100
Vehicle anti-IL-6
L858R L858R-IL6 tKO Wildtype
p=0.0385
50
p=0.0212
0
0
5
10
15
20
0
20
40
60
Weeks Weeks
Days of Treatment
0
0
Days of Treatment
-
+
-
+
-
+
-
+
Anti-IL-6
-
+ -
+
-
+
-
+ Anti-IL-6
T cells
C
NK cells
C
HCC4006
T follicular helper cell ****
Activated NK cells
OR2
OR7
NK resting cell
NK cell
T cell CD4+ *
CD8 T cell
T-regulatory cells
100
0.04
anti-IL-6 Vehicle
100
100
0.15
0.025
Vehicle anti-IL-6
0.05
0.020
0.020
p=0.201
anti-IL-6 Vehicle
p=0.0546
**
****
0.04
p=0.1004
*
0.020
80
0.04
80
80
0.03
0.015
0.015
0.03
*
0.10
*
Hypothesis
0.015
0.03
60
60
60
0.02
0.010
0.010
0.02
0.010
0.02
0.05
40
40
40
Given the immunosuppressive role of IL-6, we hypothesized that IL-6 in part mediates the immunosuppressive phenotype responsible for EGFR-TKI resistant NSCLC’s marginal response to anti-PD-1/PD-L1 therapy through altering the tumor infiltrating immune cell populations and modulating their cytotoxic potential.
0.01
0.005
0.005
0.01
0.005
0.01
20
20
20
0.00
0.00
0.000
0.000
0.00
0.000
0.00
IL-6 KO
- +
-
+
- +
-
+
-
+
-
+
-
+
0
0
0
10 5 2.5 1.25 Effector: Target Ratio
10 5 2.5 1.25 Effector: Target Ratio
10 5 2.5 1.25 Effector: Target Ratio
Figure 2. Survival analysis of EGFR L858R GEMMs and EGFR L858R GEMMs crossed with an IL-6 knockout mouse showed that knockout of IL-6 significantly increased overall survival (A). EGFR- mutant NSCLC tumors treated with mouse anti-IL-6 blocking antibody significantly increased overall survival of mice (B). Knockout (KO) of IL-6 resulted in increased NK cell populations and the CD8 T cell populations but a decreased T-regulatory and T follicular helper cell populations (C). EGFR-independent TKI resistant tumors display mesenchymal, immunologically cold phenotype and secrete IL-6 A B EGFR-dependent EGFR-independent
Blockade of IL-6 induces T cell activity in EGFR- mutant NSCLC microenvironment Figure 4. Blockade of IL-6 increased NKG2D (A) and Granzyme B (B) expression in the NK cells co- cultured with EGFR-mutant NSCLC tumor cell lines. Blockade of IL-6 in the EGFR-TKI refractory cells co-cultured with NK cells increased NK cell-mediated killing (C).
References 1. Engelman, et al. Clin Cancer Res 14 , 2895-2899 (2008). 2. Nilsson , et al. Sci Transl Med 9 (2017). 3. Lee et al., JAMA Oncology ( 2018) .
Results
OR2
OR7
A
HCC4006
100
100
100
anti-IL-6 Vehicle
anti-IL-6 Vehicle
anti-IL-6 Vehicle
*
Acquired EGFR-TKI resistance is associated with increased levels of IL-6
*
80
80
80
60
60
60
****
40
40
40
B
C
A
3000
3000
1500
****
20
20
20
***
***
***
2000
0
0
0
2000
1000
**
**
10 5 2.5 1.25 Effector: Target Ratio
10 5 2.5 1.25 Effector: Target Ratio
10 5 2.5 1.25 Effector: Target Ratio
**
**
**
D
C
NK cell
B
*
T cell
Gene signature analysis (EGFR-dependent vs EGFR- independent)
T follicular helper cells
Activated NK cells
NK resting cells
1000
NK cells
1000
CD8 T cell *
500
CD4 T cells
T-regulatory cells
*
Figure 5. Blockade of IL-6 in the EGFR- TKI refractory cells transduced with OKT3 co-cultured with T cells moderately increased T cell-mediated killing (A). Anti-PD-1 therapy did not significantly improve overall survival in the EGFR L858R GEMM model. However, combination treatment of anti-IL-6 with anti-PD-1 treatment significantly increased overall survival (B).
Vehicle anti-IL-6 anti-PD-1 anti-IL-6/ anti-PD-1
100
0.00 0.01 0.02 0.03 0.04 0.05
0.10
0.05
0.4
0.008
0.135
0.020
*
***
****
*
****
80
**
0.08
0.04
0.130
0.3
0.006
0.015
0
0
0
60
0.06
0.03
0.125
0.2
0.004
0.010
40
0.04
0.02
0.120
0.1
0.002
0.005
20
0.02
0.01
0.115
D
0.00
0
0.000
0.0
0.00
Figure 1. Osimertinib-resistant (OR) variants secreted significantly greater levels of IL-6 compared to EGFR- mutant parental cells HCC4006 (A) and H1975 (B). Patient-derived models (MDA-L-004K, MDA-L0011, MDA-L-0024, MDA-L-0046, and MDA-L-0065) of EGFR-TKI resistance also secreted higher amounts of IL-6 as compared to EGFR-mutant TKI-naïve cells (C). From TCGA analysis, patients with EGFR-mutant NSCLC, altered levels of IL-6 were associated with a worse disease-free survival (D).
0.110
0.000
0
20
40
60
EGFR dependence (dox)
- +
-+
- +
- +
- +
- +
- +
Days of Treatment
E
Figure 3. Schematic of the development EGFR- independent EGFR-mutant model using doxycycline- inducible EGFR L858R GEMMs (A). CT imaging after 2- week treatment with osimertinib indicates strong sensitivity to EGFR-TKI in the EGFR-dependent but not EGFR-independent model (B). EGFR-independent tumors displayed an enrichment in an EMT gene expression signature (C). EGFR-independent tumors showed evidence of a colder immune microenvironment (decreased CD8 T cells, CD4 T cells, total NK cells, and decreased activated NK cells) (D). IL-6 was differentially overexpressed in EGFR- independent tumors as assessed by multiplex cytokine analysis of serum (E).
Conclusion
8
*
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IL-6 is upregulated in EGFR-mutant NSCLC tumors with acquired resistance to EGFR-TKIs and impairs anti-tumor immunity through suppression of T and NK cell function.
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2
Acknowledgements
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Emerson Collective, Lung SPORE grant 5 P50 CA070907, 1R01 CA190628, 1R01 CA234183- 01A1, Stading Fund for EGFR inhibitor resistance, CPRIT Training Award (RP210028).
Disease Free (Months)
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