In this webinar, Dr. Victor Arrieta highlights the link between p-ERK activation and improved survival in rGBM patients using anti-PD-1 immunotherapy. Recurrent glioblastoma (rGBM) has displayed a varied response to anti-PD-1 immunotherapy, necessitating the identification of predictive biomarkers. Through extensive analyses and 3 clinical studies, we have identified that activation of the MAPK/ERK signaling pathway, particularly ERK1/2 phosphorylation (p-ERK), is associated with longer overall survival (OS) in rGBM patients receiving PD-1 blockade. Initially, enrichment of BRAF/PTPN11 mutations was reported in 30% of responsive rGBM patients, prompting the investigation of p-ERK as a potential marker beyond these mutations. Our research has unraveled an association between p-ERK abundance and better clinical outcomes following PD-1 blockade, with p-ERK mainly localized in tumor cells. Notably, high p-ERK GBMs contained unique microglia and macrophage phenotypes with elevated MHC class II expression, suggesting a novel interplay between MAPK activation and the tumor immune microenvironment. While these insights establish a pivotal role for p-ERK in predicting PD-1 blockade response in rGBM, the implementation in clinical settings calls for further validation and accuracy. Nonetheless, these findings pave the way for more personalized and effective immunotherapy strategies, emphasizing the significance of the tumor microenvironment and its interaction with therapeutic interventions in GBM. Key Topics Include: - The activation of the MAPK signaling pathway, specifically ERK1/2 phosphorylation (p-ERK), is identified as a predictive biomarker for longer overall survival in recurrent glioblastoma (eGBM) patients undergoing PD-1 blockade - High p-ERK tumors in rGBM present a distinct myeloid cell phenotype with elevated MHC class II expression, signifying a connection between MAPK pathway activation and the immune microenvironment - The implementation of p-ERK as a predictive biomarker in clinical settings requires further validation and exploration of variables impacting its evaluation

2. Víctor Andrés Arrieta González
MD,PhD
Exploring Predictive
Biomarkers and ERK1/2
Phosphorylation: A New
Horizon in Forecasting Survival
and Response to Immune
Checkpoint Blockade in
Glioblastoma Treatment

4. Clinical GBM responder and non-responder to PD-1 blockade
49.4 0.11
16.7
33.8
0 10
3
10
4
CD4 APC-Cy7
0
10
3
10
4
CD8
PE-Cy7-A
74.2 0.54
18.4
6.91
0 10
3
10
4
Comp-APC-Cy7-A
0
10
3
10
4
Comp-PE-Cy7-A
38.4 0.12
56.8
4.72
0 10
3
10
4
CD4 APC-Cy7
0
10
3
10
4
CD8
PE-Cy7
Start of
anti-PD-1
therapy
Start of
anti-PD-1
therapy
Last seen
alive 21
months after
PD-1 blockade
2 weeks 10 months 11 months 13 months 15 months
Resection
Resection
Resection
2 months Death 6
months
after PD-1
blockade
Recurrence
Recurrence
Responder
Non-responder
CD3 Blood Resected tissue
CSF
200 µm
Arrieta VA, Chen AX, et al, Nat Cancer 2021

5. Enrichment of BRAF/PTPN11 mutations in responder patients
Zhao J et al, Nat Med 2019
MAPK pathway
Pratilas CA et al, Clin Cancer Res 2010
PTPN11

6. p-ERK is associated with longer survival in recurrent GBM patients treated with
anti-PD-1 therapy
250 µm 250 µm
High p-ERK Low p-ERK
Arrieta VA, Chen AX, et al, Nat Cancer 2021

7. Patients with longer overall survival exhibited high levels of p-ERK regardless
of the genetic status of the BRAF/PTPN11
Arrieta VA, Chen AX, et al, Nat Cancer 2021

8. Cloughesy TF et al, Nat Med 2019
p-ERK is associated with overall survival in an independent cohort of
recurrent GBM patients treated with adjuvant PD-1 blockade
Arrieta VA, Chen AX, et al, Nat Cancer 2021

9. p-ERK is predominantly expressed by GBM cells
p-ERK
IHC
H&E
p-ERK
IHC
H&E
p-ERK
IHC
H&E
BRAFV600E
mutant GBM
High p-ERK
Wild-type BRAF
GBM
High p-ERK
Wild-type
BRAF GBM
Low p-ERK
200 µm 200 µm
200 µm 200 µm
200 µm 200 µm
p-ERK
SOX2 DAPI CD163 DAPI
SOX2 TMEM119
p-ERK
SOX2 DAPI CD163 DAPI
SOX2 TMEM119
p-ERK
SOX2 DAPI CD163 DAPI
SOX2 TMEM119
Arrieta VA, Chen AX, et al, Nat Cancer 2021

10. High-p-ERK GBMs contain abundant TMEM119+ microglia
p-ERK Iba1
250 µm 250 µm
Arrieta VA, Chen AX, et al, Nat Cancer 2021

11. TMEM119+ microglia are closer to SOX2+ p-ERK+ cells
TMEM119+ cells
CD163+ cells
SOX2+ p-ERK+
cells
Mean distance to the
nearest neighbor
From
To
SOX2+ p-ERK-
cells
TMEM119+ SOX2+ p-ERK+ TMEM119+ SOX2+ p-ERK+
High p-ERK Low p-ERK
200 µm 200 µm
Arrieta VA, Chen AX, et al, Nat Cancer 2021

12. CD163+ cells
GFAP+ p-ERK-
cells
Mean distance to the
nearest neighbor
From
To
GFAP+ p-ERK+
cells
Arrieta VA, Chen AX, et al, Nat Cancer 2021
CD163+ GFAP+ pERK+
CD163+ GFAP+ pERK+
High p-ERK Low p-ERK
200 µm 200 µm
CD163+ myeloid cells are closer to GFAP+ p-ERK+ cells

13. Analysis of GBM-infiltrating microglia and macrophages by single-cell RNA-seq
Arrieta VA, Chen AX, et al, Nat Cancer 2021

14. Myeloid cells from high p-ERK GBMs
have increased MHC II expression
Arrieta VA, Chen AX, et al, Nat Cancer 2021
GO
MHC
Class
II
protein
Complex
Binding
1.75
1.50
1.25
1.00
0.00
0.75
0.50
0.25
GO MHC Class II protein Complex Binding
0.8
0.6
0.4
0.2
0.0
1.0
0.5
0.0
-0.5
0 5000 10000 15000 20000 25000
High p-ERK
Low p-ERK

15. Myeloid cells from high p-ERK GBMs
have increased MHC II expression
Arrieta VA, Chen AX, et al, Nat Cancer 2021

16. • ERK1/2 phosphorylation shows that is a predictive biomarker of immune checkpoint
blockade including anti-PD-1 therapy in three independent cohorts of recurrent GBM
patients.
• GBMs that exhibit high numbers of p-ERK+ cells contain abundant TMEM119+ microglia.
• Myeloid cells derived from high p-ERK GBMs exhibit increased MHC class II expression
compared to myeloid cells derived from low p-ERK GBMs.
Conclusions

17. • There are several aspects to consider before implementing p-ERK as a biomarker for
response to anti-PD-1 therapy:
• Further validation in additional clinical cohorts
• Cut point value optimization
• Alternative techniques for MAPK pathway activation
• Standardized protocols for p-ERK assessment
Considerations

18. Acknowledgments
Columbia University
• Junfei Zhao
• David Cieremans
• Pavan
Upadhyayula
• Jinzhou Yuan
• Wenting Zhao
• Brice Laffleur
• Uttiya Basu
• Gerson Rothschild
• Peter Sims
• Peter Canoll
• Jeffrey N. Bruce
Northwestern University
• Seong Jae Kang
• Kirsten B. Burdett
• Crismita Dmello
• Andrew Gould
• Matthew McCord
• Li Chen
• Daniel Zhang
• Dinesh Jaishankar
• Joseph Shilati
• Christina Amidei
• Lee D Cooper
• Rimas V. Lukas
• Hui Zhang
• Jonathan T.
Yamaguchi
• Daniel J. Brat
• Bin Zhang
• Roger Stupp
MD Anderson Cancer Center
• Cynthia Kassab
• Xiaoyang Ling
• Jared K. Burks
UCLA
• Robert Prins
• Tim F.
Cloughesy
Andrew X. Chen J. Robert Kane
Adam Sonabend Fabio M. Iwamoto
Raul Rabadan
Amy Heimberger Craig Horbinski Catalina Lee Chang