Targeting BCL2 pathway to enhance immunogenicity in ALK + NSCLC

Background : Translocations of the Anaplastic Lymphoma Kinase (ALK) gene occur in 3-5% of Non-Small Cell Lung Cancer (NSCLC) patients and are treated with ALK tyrosine kinase inhibitors (TKIs). However, resistance to ALK TKIs develop through on-target mutations and bypass signaling. Treatment response to ALK TKIs also relies on apoptosis, a complex mechanism regulated by BCL2 family proteins. Therefore, dysregulation of this pathway promotes tumor progression and therapy resistance. It has been reported that cancer cells depend on anti-apoptotic proteins of BCL-2 family (e.g. BCL-2, MCL-1, BCL-xL) for their survival. This has led to the development of BH3 mimetics, which are inhibitors of anti-apoptotic proteins. ALK+ NSCLC is considered an immune “cold” tumor with low immunogenicity, demonstrated by fewer neoantigens and lack of cytotoxic tumor-infiltrating lymphocytes (TILs), leading to limited efficacy of immune checkpoint-targeting therapies in these patients. Some TKIs have been reported to elicit durable antitumor immunity by inducing immunogenic cell death (ICD) through the release of damage-associated molecular patterns (DAMPs). However, this has not been studied in the context of ALK+ NSCLC.We hypothesized that ALK-directed therapy in combination with BH3 mimetics would initiate ICD through the release of DAMPs and increase sensitivity to ALK TKIs. Methods: We evaluated the efficacy of ALK TKIs (Lorlatinib, Alectinib) alone and in combination with the BH3 mimetics navitoclax (BCL-2/BCL-xL inhibitor), venetoclax (BCL-2 inhibitor) and s63845 (MCL-1 inhibitor) to induce damage-associated molecular patterns (DAMPs) in ALK+ NSCLC cell lines. Results: ALK+ cancer cell lines exhibited high expression of MCL-1 and BCL-xL proteins, indicating their dependency on BCL-2 family proteins for survival. The addition of BH3 mimetics enhanced apoptosis induction, as evidenced by increased PARP cleavage, and further reduced colony formation efficiency compared to ALK TKI single-agent treatment. Both ALK TKI single agent and combination treatment with BH3 mimetics induced key DAMPs essential for ICD, including: (1) increased type I interferon signaling via IFN-beta (3-7-fold) and CXCL10 (2-3-fold); (2) peak release of extracellular ATP at 7 hours of treatment; and (3) enhanced phosphorylation of eIF2alpha compared to the control. Conclusions: ALK-directed therapy has the potential to induce immunogenic cell death (ICD) and elicit antitumor immune responses. Combining BH3 mimetics with ALK TKIs can be a promising therapeutic strategy to enhance anti-tumor immunity and overcome ALK TKI resistance in ALK+ NSCLC