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This article is adapted from the original, which first appeared on The Bench, the Synthego blog.

The medical community has been striving for decades to develop effective treatments for treating cancer. As there are more than 100 different types of cancer, each with their niche characteristics, there are hundreds of drugs targeting pathways active in specific cancer types.

Needless to say, researchers are aiming to develop a universal therapy for all types of cancer — without harming normal cells. In this regard, immunotherapy has emerged as a promising option in cancer research in recent years.

Now, James Hodge and his team at the National Cancer Institute at National Institutes of Health report a new method that combines traditional cancer drugs and immunotherapy agents. Although their findings, published in the Journal for ImmunoTherapy of Cancer, are currently tested in cell lines, this approach could lead to potent new cancer therapies in the future. Read on to learn more about their research.

What was known: Genotoxic stress kills cells

Poly ADP ribose polymerase (PARP) is an enzyme responsible for DNA repair in cells. As cancer cells frequently bear mutations in their DNA, blocking PARP-mediated repair results in their apoptosis, or cell death. Therefore, PARP inhibitor drugs have been approved for certain cancer types.

Olaparib, for example, is one such PARP inhibitor that is approved for treating ovarian and breast carcinoma. Patients with these cancers often have mutations in the gene responsible for tumor suppression, BRCA, which mess up homologous recombination in cells. This, together with faulty DNA repair triggered by olaparib, results in genomic instability, ultimately leading to cell death.

Genotoxic stress is known to trigger the immune system by inducing expression of ligands that are recognized by receptors of natural killer (NK) cells. This means that these cells put a target on themselves and are easily recognized by immune cells, even in the absence of BRCA mutation or similar genetic mutations. Additionally, an ongoing clinical study showed that olaparib and supplementary agents reduced antigen count in prostate cancer patients, even in those who did not show mutations affecting DNA repair.

Hodge and his team were intrigued by these findings. They saw this information as valuable pieces of a puzzle, and came up with further questions to fill in the missing gaps for developing a new potent cancer treatment.

Could the presence of olaparib (or PARP inhibitors in general) augment NK-mediated cell death even in the absence of BRCA mutation? If so, what is the mechanism of its action? Could this treatment be extended to other cancer types?

Mixing up the old and new ways of treating cancer

Inspired by the clinical study data mentioned above, the researchers focused on prostate carcinoma for their investigation. They used two cell lines: one that has known BRCA gene mutations and another without BRCA mutations (status confirmed using NGS). NK cells were isolated from healthy donors.

Their experimental design and main findings are highlighted in the sections below.

Olaparib exposure enhances immune system-mediated cell lysis

The researchers pretreated both cell types with olaparib and then exposed them to NK cells. Cell lysis was monitored using a real-time cell analysis (RTCA) lysis assay. The researchers observed that pretreatment with olaparib significantly increased NK cells related cell lysis (1.6-fold difference after 36 h), irrespective of BRCA mutation status. Cell lysis was further increased by the addition of cetuximab or avelumab, which are drugs mediating antibody-dependent cellular cytotoxicity (ADCC).

Death receptor upregulation causes cell death

Next, the researchers wanted to understand the mechanism by which olaparib increased NK-mediated target cell lysis. They suspected that death receptors were being expressed on these cells, making them an easy target. But which one of the many death receptors should be tested?

To answer this question, the team isolated RNA from tumor cells exposed to olaparib for different time intervals (6h, 12h, 18h) and analyzed it using NanoString array, which screens several hundred genes simultaneously to identify the ones upregulated in cancer. The results showed that the expression of tumor necrosis factor superfamily (TNFSF) genes was modulated in both cell lines. One particular receptor, TNFRSF10B (TRAIL-R2), seemed to be in the center of pathways involved in apoptosis.

Based on the RNA analysis data, the team focused on the TRAIL-R2 receptor. They further noted upregulated levels of cell-surface TRAIL-R2 receptors on olaparib exposure, measured by flow cytometry experiments.

Synthego’s engineered cells validate the hypothesis

The final test for confirming the role of TRAIL R2 death receptor in cell lysis was to deactivate the corresponding gene. They turned to trusted experts at Synthego to generate gene knockouts in their desired prostate cancer cell line using CRISPR. Synthego also characterized gene deletion in the Knockout Cell Pools by sequencing and provided the researchers with ready-to-use Engineered Cells for their experiments.

The team then exposed these TRAIL R2 knockout cells and normal control cells exposed to olaparib and NK cells and noted significantly lower lysis in the former, as compared to the control cells.

These results validate that increased TRAIL-R2 death receptor levels on the cell surface on olaparib exposure is largely responsible for increased NK-mediated cell death.

Study extends scope of PARP inhibitors to other cancer types

Olaparib has so far been approved only for cancers with BRCA gene mutations. As their experiments showed increased NK-lysis irrespective of BRCA mutation, the researchers wondered if its use could be more versatile. They tested other cancer cell lines including prostate, breast, non-small cell lung carcinoma, and chordoma, and saw that olaparib treatment significantly enhanced NK killing in all these tumor cells in presence of ADCC-mediating agents.

The implication that PARP inhibitors could effectively treat other cancer types in conjunction with immunotherapy opens its applications for broader cancer treatment in the future.

Do you want to use CRISPR Engineered Cells in your research? If so, check out Synthego’s free Cell Engineering 101 eBook.