



Checkpoint inhibitors, drugs designed to “release the brakes” on cancer-fighting immune cells, have shown promise in treating cancer patients, albeit a minority of cancer patients. To help checkpoint inhibitors help the immune system fight cancer, scientists are investigating combination treatments, such as pairings of the most potent checkpoint inhibitors and new drugs that could effectively shut down molecular pathways that serve as programmed cell death protein 1 (PD-1) escape mechanisms.

The trick, however, is to identify these molecular pathways. A new means of doing so has been demonstrated by scientists based at Dana-Farber/Boston Children's Cancer and Blood Disorders Center. These scientists report that they have used CRISPR/Cas9 genome-editing technology to test the function of thousands of tumor genes in mice. Essentially, the scientists systematically screened for drug targets that could potentially enhance the effectiveness of PD-1 checkpoint inhibitors.

The scientists, led by pediatric oncologist W. Nick Haining, B.M., B.Ch., tested 2368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. This work allowed the scientists to recover the known immune-evasion molecules programmed death-ligand 1 (PD-L1) and CD47, and confirm that defects in interferon-γ signaling caused resistance to immunotherapy.

Additional details of the work appeared July 19 in the journal Nature, in an article entitled “In Vivo CRISPR Screening Identifies Ptpn2 as a Cancer Immunotherapy Target.” Encouragingly, the article uncovered previously unidentified cancer immunotherapy targets.

“Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation, and the unfolded protein response,” the article’s authors wrote. “In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression.”

These findings not only indicate that in vivo genetic screens in tumor models can identify new immunotherapy targets in unanticipated pathways, they also raise a more specific possibility: Deleting the Ptpn2 gene in tumor cells could made these cells more susceptible to PD-1 checkpoint inhibitors.

“PD-1 checkpoint inhibitors have transformed the treatment of many cancers,” noted Dr. Haining, senior author on the new paper, who is also associate professor of pediatrics at Harvard Medical School and associate member of the Broad Institute of MIT and Harvard. “Yet despite the clinical success of this new class of cancer immunotherapy, the majority of patients don't reap a clinical benefit from PD-1 blockade.”

That, Dr. Haining added, has triggered a rush of additional trials to investigate whether other drugs, when used in combination with PD-1 inhibitors, can increase the number of patients whose cancer responds to the treatment.

“The challenge so far has been finding the most effective immunotherapy targets and prioritizing those that work best when combined with PD-1 inhibitors,” Dr. Haining explained. “So, we set out to develop a better system for identifying new drug targets that might aid the body's own immune system in its attack against cancer.

“Our work suggests that there's a wide array of biological pathways that could be targeted to make immunotherapy more successful. Many of these are surprising pathways that we couldn't have predicted. For instance, without this screening approach, it wouldn't have been obvious that Ptpn2 is a good drug target for the immunotherapy of cancer.”

To cast a wide net, the paper's first author Robert Manguso, a graduate student in Dr. Haining's lab, started by engineering the melanoma skin cancer cells so that they all contained Cas9, the “cutting” enzyme that is part of the CRISPR editing system. Then, using a virus as a delivery vehicle, he programmed each cell with a different single-guide RNA (sgRNA) sequence of genetic code. In combination with the Cas9 enzyme, the sgRNA codes—about 20 amino acids in length—enabled 2368 different genes to be eliminated.

By injecting the tumor cells into mice and treating them with PD-1 checkpoint inhibitors, Manguso was then able to tally up which modified tumor cells survived. Those that perished had been sensitized to PD-1 blockade as a result of their missing gene.

“Ptpn2 usually puts the brakes on the immune signaling pathways that would otherwise smother cancer cells,” Dr. Haining pointed out. “Deleting Ptpn2 ramps up those immune signaling pathways, making tumor cells grow slower and die more easily under immune attack.”

With the new screening approach in hand, Dr. Haining's team is quickly scaling up their efforts to search for additional novel drug targets that could boost immunotherapy.

Dr. Haining says the team is expanding their approach to move from screening thousands of genes at a time to eventually being able to screen the whole genome, and to move beyond melanoma to colon, lung, and renal carcinomas, and more. He's assembled a large team of scientists spanning Dana-Farber/Boston Children's and the Broad Institute to tackle the technical challenges that accompany screening efforts on such a large scale.

In the meantime, while additional potential drug targets are likely around the corner, Dr. Haining's team is taking action based on their findings about Ptpn2. “We're thinking hard about what a Ptpn2 inhibitor would look like,” said Dr. Haining. “It's easy to imagine making a small-molecule drug that turns off Ptpn2.”



























