By Deborah Borfitz

March 7, 2024 | Researchers in California have shown that breast cancer patients with RNA expression levels of ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) in the bottom 50th percentile can confidently be treated with Keytruda (pembrolizumab) as well as surgery. Their cancer would have a zero percent chance of metastasizing in seven years, according to Lingyin Li, Ph.D., associate professor of biochemistry at Stanford University and an investigator at the Arc Institute. 

The finding that ENPP1 levels can serve as a biomarker for patient stratification and should be targeted for immunotherapy emerged from an analysis of data from the groundbreaking I-SPY2 trial of neoadjuvant treatment for locally advanced breast cancer. Breast cancer patients with low ENPP1 expression have “hot” tumors characterized by high infiltration of T cells, part of a relatively small group of solid cancers that also include melanoma and a subset of lung cancers targetable by immunotherapy. 

As revealed in a study that was published recently in Proceedings of the National Academy of Sciences (DOI: 10.1073/pnas.2313693120), ENPP1 is an innate immune checkpoint of the anticancer cGAMP–STING pathway in breast cancer. STING is the acronym for stimulator of interferon genes, and cGAMP for extracellular 2′3′-cyclic-GMP-AMP. 

Keytruda is a PD-1 (programmed death receptor-1)-blocking antibody. It is the subject of literally thousands of completed and ongoing trials across a wide variety of cancers and treatment settings, says Li, but to date the immunotherapy drug has only been approved in a limited number of cancers where clinical efficacy was observed—a list that includes triple-negative breast cancer.  

Based on results of the latest study, Keytruda “shouldn’t be experimental anymore” for patients with breast cancer. Given that ENPP1 is highly upregulated in many cancer types, and correlates with poor prognosis, it’s likely that the same “on-off switch” for controlling immunotherapy resistance and metastasis exists more universally. 

Li previously discovered that cGAMP is exported outside the cells but is often prevented from triggering a response because ENPP1 chews up the molecular danger signals (Nature Center, DOI: 10.1038/s43018-020-0028-4). In effect, ENP1 helps keep cold tumors cold, she proposed. 

Breast cancer patients with high ENPP1 expression are not without hope, Li points out. They might be treated with ENPP1 inhibitors that are already entering clinical trials to pharmacologically inhibit ENPP1 activity so they can respond to Keytruda. 

For diagnostic purposes in the clinic, ENPP1 levels can be easily determined by bulk sequencing of RNA or even histological staining, says Li. But it was single-cell RNA sequencing used in the study that revealed ENPP1 can be found in both healthy and cancerous cells.  

This was a surprise, given that ENPP1 levels are normally associated with cancer rather than the tumor microenvironment, Li says. On the other hand, she adds, it makes complete sense since ENPP1 functions as a checkpoint of PDL-1, the ligand of PD-1, that is expressed by cancer and immune cells alike.  

Springboarding Off I-SPY2 

The ongoing I-SPY2 neoadjuvant platform trial started in 2010 and nearly 1,000 of its participants had their tumor biopsy samples sent for RNA sequencing at the time of diagnosis, says Li, in addition to having ER/PR (estrogen receptor/progesterone receptor) and HER2 (human epidermal growth factor receptor-2) status testing done.

Patients with ER/PR-positive tumors were funneled to the arm for tamoxifen treatment, while the HER2-positives were directed to Herceptin or another monoclonal antibody, and those who were triple-negative for ER, PR, and HER2 but had a BRCA mutation were assigned to the PARP (poly-ADP ribose polymerase) inhibitor treatment arm. “Later, triple-negative patients or just HER2-negative patients were also tested in a Keytruda arm,” Li continues. 

The women subsequently underwent surgery when their surgeon and pathologist would decide if they had or hadn’t responded to the treatments completely. “Right away we could see that patients who had lower ENPP1 levels were more likely to have a pathological complete response to two therapies—Keytruda and PARP inhibitor,” says Li. 

“So, when taken together, low ENPP1 patients have enhanced cGAMP stability,” she continues. STING signaling is thereby strengthened, inducing an antitumor innate immune response, which synergized with Keytruda. cGAMP-STING signaling is further amplified in patients who cannot repair their DNA due to a lack of both BRCA and PARP activity, explaining the synergistic effects seen in the PARP inhibitor arm.  

The main objective of the I-SPY2 trial was to identify indicators of response to neoadjuvant chemotherapy that predict survival in women with high-risk breast cancer. Only two decades ago, Li notes, every cancer patient was almost guaranteed to get chemotherapy. 

The I-SPY2 study ultimately succeeded in transforming how breast cancer patients get treated, says Li, by providing a framework for identifying a response-predictive subtyping schema for prioritizing therapies (Cancer Cell, DOI: 10.1016/j.ccell.2022.05.005). “We took advantage of this extremely well-run clinical trial,” the likes of which don’t exist for glioblastoma, lung, and pancreatic cancers. 

Clinical Trials Begin

In 2018, Li co-founded a biotech company known as Angarus Therapeutics that is focused on developing ENPP1 inhibitors. “Angarus” is Latin for messenger, a fitting term since cGAMP is an anticancer messenger, she says. 

Angarus Therapeutics is hardly alone in its quest, since the role of ENPP1 in immune modulation and tumor promotion is now well recognized. Companies in the space are like “mushrooms after the rain,” says Li, who welcomes the competition. Several such agents are already in clinical trials and recruiting patients.  

Angarus has received authorization from the U.S. Food and Drug Administration to begin phase 1 clinical trials of its ENPP1 inhibitor candidate and is now actively organizing centers to run it, with the first patients getting dosed this year, says Li. Between 15 and 30 metastatic patients will ultimately be enrolled across indications (e.g., breast, lung, pancreatic, and glioblastoma) with different readout timelines. 

Given that ENPP1 has been shown to be upregulated and correlated with poor prognosis in cancer, Li says she encourages patients falling in the high expression bucket to seek out a ENPP1 inhibitor trial and, if they fall in the low expression bucket, think about getting Keytruda. Li also asks that investors “keep an open mind” about immunotherapy despite some disappointments over the last decade, including the failure of vadimezan in phase 3 trials. (Chemical Biology & Drug Design, DOI: 10.1111/cbdd.13166). 

Keep in mind the STING pathway was only discovered 14 years ago and cGAMP a decade ago, and it takes that long to understand the biology to effectively alter it, she says. “We are finally at a time when we can make an impact. Now is not the time to lose hope.” 

Clinical oncologists should be aware, as noted in the PNAS paper, that radiation treatment activates the cGAMP pathway, she says. “We expect radiation therapy to combine well with Keytruda, which relies on the cGAMP-STING pathway to be activated.” Radiation oncologists might therefore consider trials taking a combination approach with the ENPP1 inhibitors.