Manipulating a master regulator prevents tumor growth in mice

Manipulating a master regulator prevents tumor growth in mice

A protein identified nearly 40 years ago for its ability to stimulate red blood cell production plays a surprising and critical role in dampening the immune system's response to cancer.

Blocking the protein's activity turns previously "cold" or immune-resistant liver tumors in mice into "hot" tumors teeming with cancer-fighting immune cells. Combined with immunotherapy, which further activates these immune cells against the cancer, the treatment led to complete regression of existing liver tumors in most mice. Treated animals lived for the duration of the experiment. In contrast, control animals only survived a few weeks.

This is a fundamental breakthrough in our understanding of how the immune system is turned off in cancer. I couldn't be more excited about this discovery, and I hope treatments targeting the mechanism we uncovered will quickly advance to human trials. “

Edgar Engleman, MD, PhD, professor of pathology and medicine

Engleman is the lead author of the research, which will be published online April 24 inScience. Basic Life research scientist David Kung-Chun Chiu, PhD, is the lead author of the study.

Probably applicable to many types of cancer

Although the work was completed in mice, there is strong evidence that the protein, erythropoietin, or EPO, plays a similar role in many types of cancers.

“Research from more than a decade ago showed that giving EPO to cancer patients with anemia to stimulate red blood cell production accelerates tumor growth,” Engleman said.

The compound was so notable that in 2007 the Food and Drug Administration required a black box warning label for the drug, warning against its use in people with cancers. The researchers also saw a clear correlation between patients' prognosis and levels of naturally occurring EPO and its receptor in the tumor.

“These old reports clearly showed that the more EPO or Epor there was in tumors, the worse the patients were,” Engleman said. "But the link between EPO and cancer immunity has never been established before. In fact, because EPO is so well established as a growth factor for red blood cells, it took a long time and many experiments to convince us that EPO plays a fundamental role in blocking the immune response to cancer."

Chiu developed and studied genome editing techniques to create multiple mouse models of liver cancer to study how liver tumors develop and respond to treatment. Each model recapitulates specific mutations, histology, and response to approved therapies found in subtypes of human liver cancers. Tumor formation was induced either by injecting a combination of DNA-encoded proteins associated with liver cancer into the animals' tail vein or by implanting liver cancer cells into the animals' livers.

The researchers were interested in the effect on cancer growth of a common immunotherapy that targeted a molecule called PD-1 on immune cells called T cells. Binding to PD-1 blocks the ability of cancer cells to dampen T cell activity. Anti-PD-1 therapies, including one marketed commercially as Keytruda, are routinely used to treat many types of human cancers, including melanoma, Hodgkin's lymphoma and some types of lung cancer. In some cases, they have changed patient outcomes. However, a large majority of tumors, including most liver, pancreatic, colon, breast and prostate cancers, are resistant to treatment.

The researchers found that some combinations of mutations, similar to human liver cancer, led to the development of liver tumors that were largely ignored by the immune system, becoming immune privileged or cold. These tumors did not shrink when the animals were treated with anti-PD-1 because there were few T cells present in the tumor.

In contrast to the cold tumors, other mutations resulted in hot or “inflamed” tumors that were weakened with T cells. These tumors were highly sensitive to anti-PD1 treatment, which directed the T cells to attack the cancer.

Unexpectedly, the cold tumors showed increased EPO levels compared to hot tumors. This increase is likely caused by the oxygen-depleted microenvironment - a condition called hypoxia - prevalent in cold tumors. Hypoxia induces the production of proteins in cancer cells, which in turn increase the production of EPO to create more red blood cells to combat the low oxygen levels.

“Hypoxia in tumors has been studied for decades,” Engleman said. "It is beyond anyone's knowledge, including me, that EPO in this context could be doing anything other than serving as a growth factor for red blood cells."

Intrigued, the researchers turned to existing databases to confirm that elevated EPO levels correlate with poorer survival in people with cancer of the liver, kidney, breast, colon and skin. They then tinkered with the tumor cells' ability to make EPO and were surprised at what happened in the animals' liver tumors.

They found that mutations that had led to the development of cold tumors instead caused hot tumors when the tumors were modified so that they were unable to make EPO. Conversely, hot tumors that had previously been successfully eradicated by the immune system thrived when engineered to achieve elevated levels of EPO.

Further exhaustive research showed that in cold tumors, tumor cells produce and secrete EPO, which binds to receptors on the surface of immune cells called macrophages. The macrophages then switch to an immunosuppressive role, scaring away cancer-mutating T cells and taming their activity.

The importance of this EPO-moderated crosstalk between tumor cells and macrophages was clearly demonstrated when the researchers examined the combinatorial effect of blocking the EPO signaling pathway and the anti-PD-1 pathway simultaneously.

In these experiments, no mice with cold liver tumors treated with control or anti-PD-1 lived more than eight weeks after tumor induction. In contrast, 40% of mice with macrophages were unable to survive the EPO receptor after termination of the experiment 18 weeks after tumor induction. When mice lacking the EPO receptor were given anti-PD-1 treatment, all animals lived for the duration of the experiment.

“It’s easy,” Engleman said. “If you remove that EPO signaling, either by lowering the hormone levels or by blocking the receptors on the macrophages, you not only get a reduction in tumor growth, but you get tumor regression along with sensitivity to anti-PD-1 delivery.”

Engleman and his colleagues are now designing treatments that target EPO signaling in human cancers. Nonspecifically targeting the EPO protein can cause anemia, which Engleman speculates may be an acceptable compromise for effective cancer therapy. An alternative approach is to selectively block the EPO receptors on the surfaces of macrophages in cancer.

“I continue to be amazed by this finding,” Engleman said. “Not every tumor will respond the same way, but I am very optimistic that this discovery will lead to powerful new cancer therapies.”

Researchers from the New York Blood Center and the pharmaceutical company Immunedge Inc. contributed to the research.

The study was funded by the National Institutes of Health (grants R01CA262361, P01CA244114, U54CA2745115, and P01HL149626).

Chiu is a co-founder of Immunedge Inc. Engleman is a founder, shareholder and board member of Immunedge Inc. Chiu and Engleman are Stanford-affiliated inventors of PCT/US2023/063997 entitled “EPO receptor agonists and antagonists.”

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