Despite the clinical success of T-cell centric cancer immunotherapy in the treatment of several types of cancer, significant challenges need to be overcome in order to make immunotherapy more effective. Not all patients respond to the current FDA-approved checkpoint inhibitors and many tumor types are found to be resistant to these therapies. However, the mechanisms underlying this acquired resistance are not well understood. Neoantigens on the surface of the tumor cells elicit a cascade for immune cell targeting of the tumor. Cytotoxic lymphocytes, such as CD8+ T cells and natural killer (NK) cells, promote antitumor effects through a combination of perforin-dependent tumor cell killing and increasing tumor immune sensitivity through release of inflammatory cytokines, such as interferon-ɣ (IFN-ɣ) and tumor necrosis factor (TNF). However, tumor cells can disrupt antigen presentation to evade immune cells, which is demonstrated by loss of function mutations in β2-microglobulin (B2M), IFN-ɣ, and Janus kinases (JAK1/2) in patients that fail to respond to immunotherapy. In order to improve effectiveness of our current cancer immunotherapies, the genes that are involved in sensitization or protection of tumors from cytotoxic lymphocytes need to be identified to better understand the mechanisms of tumor mediated immune evasion.
In a recent study published in Science Immunology, researchers conducted both genome-wide and customized CRISPR-based screens both in vitro and in vivo on key genes within the inflammatory cytokine and antigen presentation pathways to identify mechanisms of tumor mediated immune evasion. Expression of a whole-genome guide RNA (gRNA) library in both a colon adenocarcinoma and a melanoma cell line revealed deletion of genes from three key pathways, antigen presentation, IFN-ɣ signaling, and TNF signaling protected tumor cells from T cell killing, showing that tumors selectively disrupt pathways which are sensitive to T cell killing in order to evade immune cell attack. In addition, deletion of genes in the TNF pathway prevented NK-mediated tumor cell death, indicating TNF derived from CD8+ T cells and NK cells is an important factor in mediating immune mediated tumor cell killing. In order to confirm these findings, a customized sgRNA pool encoding the top scoring 2000 guides from the genome-wide screen was introduced into cells, which were then exposed to three rounds of TNF treatment. Similarly to the genome-wide screen, the customized sgRNA screen enriched for genes known to modulate TNF-induced cell death as well as uncharacterized genes in the TNF pathway.
To validate the in vitro findings, tumor cells were transduced with the customized 2000-sgRNA pool and then injected into tumor-bearing mice. There was an enrichment in sgRNAs targeting genes involved in IFN-ɣ signaling, antigen presentation, and TNF signaling. To further validate these pathways, immune evasion genes from each pathway were individually depleted in tumor cells and then injected into mice. Deletion of these genes did not affect the growth of these cells in vivo, however, it did reduce the efficacy of adoptive cellular therapy, indicating the importance of all three pathways in tumor mediated immune evasion. Interestingly, decreased expression of at least two genes from each of the three pathways was associated with poor prognosis in patients with colorectal cancer.
By utilizing customized CRISPR-based screening, these researchers revealed that TNF, IFN-ɣ, and antigen presentation pathways were the major players for, and used by, cytotoxic lymphocytes for tumor cell death both in vitro and in vivo. Suppression of these pathways by the tumor cells is a potential mechanism for tumor mediated immune evasion and shows the importance of inflammatory cytokines to antitumor immunity. Currently, agents that sensitize tumor cells to TNF-induced apoptosis are undergoing clinical trials in solid and hematological cancers and may play an important role in improving the efficacy of current immunotherapies when paired together.
