Translational control of immune checkpoint proteins in lung cancer
Introduction
Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide. Despite recent advances, high rates of recurrence highlight the need for novel treatment options. Groundbreaking discoveries in identifying and exploiting the PD-1/PD-L1 (Programmed Death 1/ Programmed Death Ligand 1) immune checkpoint have resulted in the approval of monoclonal antibodies that disrupt this interaction as a first-line therapy for lung cancer patients. However, only ~20% of NSCLC patients benefit from checkpoint blockade. A critical question remains as to what mechanisms facilitate resistance to PD-1/PD-L1 therapy and whether other immune checkpoints or pathways can be pursued clinically in combination with this therapy. The integrated stress response (ISR) pathway represents an emerging therapeutic vulnerability. We previously demonstrated that ISR activation potently induces PD-L1 in NSCLC and suppression of anti-tumor immunity (Suresh et al, Nature Cancer, 2020). We discovered that ISR pathway activation enhances PD-L1 translation through the bypass of inhibitory upstream open reading frames (uORFs) in the PD-L1 5' UTR and a mechanistic link to the translation initiation factor eIF5B. Our latest studies demonstrate that the immune checkpoint protein, CD155 (Cluster of Differentiation 155), is induced by ISR activation.
Methods
Multiple KRAS and EGFR mutant human lung adenocarcinoma cell lines were subjected to ISR activation conditions and assessed by western blot and flow cytometry. RT-qPCR analysis and polysome profiling were performed to assess transcription and translation of PD-L1 and CD155. The CD155 5' UTR was analyzed for the presence of functional uORFs using dual luciferase reporter assays. IHC staining was performed on primary human lung adenocarcinomas with PD-L1, CD155, and p-eIF2α (ISR activation marker) antibodies. C57BL/6J mice were transplanted with syngeneic KRAS mutant CMT167 cells, and tumor burden was assessed in the presence and absence of ISR activation.
Results
Both PD-L1 and CD155 are induced by multiple arms of the ISR pathway, and CD155 harbors inhibitory uORFs in its 5' UTR. Importantly, we observed a significant correlation between PD-L1 and CD155 expression in a panel of primary human lung adenocarcinomas. We also demonstrated that ISR activation inhibits T cell function in co-culture assays and promotes tumor growth in the CMT167 syngeneic mouse model. Analysis of immune cell infiltration in this in vivo model is ongoing. Currently, we are determining the extent to which ISR inhibition suppresses tumorigenesis by promoting anti-tumor immunity and whether this may synergize with existing immune checkpoint therapies.
Conclusion
We have identified new regulatory circuits of immune checkpoint protein activation. Overall, these studies will set the stage for determining whether inhibition of the ISR pathway alone or in combination with immune checkpoint blockade will benefit lung cancer patients. Our studies may also lead to new therapeutic approaches to trigger anti-cancer immune responses and improve current strategies.