More than 60 years ago, British physician Denis Parsons Burkitt and his associates achieved one of the signal successes in cancer medicine when they cured children in sub-Saharan Africa with a form of lymphoma by treating them with high doses of the chemotherapy drug cyclophosphamide. Now, Dana-Farber Cancer Institute researchers have shown that the traditional understanding of the drug's mode of action is incomplete.
In the journal Cancer Discovery, the researchers demonstrated that large doses of cyclophosphamide not only kill cancer cells directly, as has been known, but also spur an immune system attack on the cells (2019; doi:10.1158/2159-8290.CD-18-1393). The discovery resolves longstanding questions about how cyclophosphamide and other alkylating agents—among the oldest and most widely used types of chemotherapy—work, and it suggests a novel way of sparking an immune system strike on certain cancers.
“Our results show that, at high doses, cyclophosphamide and other alkylating agents blur the line between chemotherapy and immunotherapy,” said Dana-Farber's David Weinstock, MD, the senior author of the study. “These findings offer insights into how to switch on key immune system cells to augment existing therapies.”
History of Chemo
Cyclophosphamide was just the eighth anti-cancer drug to enter standard therapy when it was approved by the FDA in 1954. It became a mainstay of cancer treatment after Burkitt and others used high doses to cure children with what's now known as Burkitt lymphoma—which had a 100 percent mortality rate at the time—sometimes with only one dose. Cyclophosphamide and other alkylating agents are now used at lower doses to treat many types of cancer, including breast, ovarian, and pediatric cancers.
Alkylating agents work by attaching chemical components called alkyl groups to cancer cells' DNA, leading to breaks in the DNA molecule. The damage undermines the cells' ability to duplicate their DNA and, ultimately, to divide.
Over the years, clues emerged that there's more to the drugs' effectiveness than damaging DNA. Researchers discovered, for example, that while high doses are much more effective against certain cancers than low doses, they inflict about the same amount of DNA damage, suggesting that something else comes into play at high doses. Sporadic data pointed to the immune system. Another clue came from pathology studies of Burkitt lymphoma tissue.
“Burkitt lymphoma and other high-grade lymphomas with rearrangements in the MYC gene have a ‘starry sky’ appearance under the microscope, with large numbers of macrophages dispersed among the lymphoma cells,” Weinstock remarked.
In the new study, investigators focused on the effect of high doses of cyclophosphamide on macrophages. In mouse models implanted with human lymphoma tissue, the researchers showed that high doses of the drug, but not normal doses, damaged tumor cells in a way that severely stressed the lymphoma cells. The stressed cells responded by secreting cytokines, substances that summon macrophages to eat the tumor cells.
The researchers analyzed thousands of these macrophages to determine which genes were active, or expressed, in each of them. They found that one subset, which expresses the proteins CD36 and FcgRIV, has a particularly voracious appetite for stressed lymphoma cells. Dubbed “super-macrophages,” they devour lymphoma cells, Weinstock noted.
Although high doses of cyclophosphamide and other alkylating agents may be too toxic for patients with diseases other than Burkitt lymphoma, researchers are investigating agents that mimic their ability to stress cancer cells, but with milder side effects.
The findings may be especially relevant for the treatment of “double-hit” lymphomas, which are marked by their aggressiveness and for a rearrangement in the MYC gene, Weinstock observed. Targeted therapies are currently lacking for this disease, which accounts for 6-10 percent of diffuse large B-cell lymphomas and generally has poor outcomes for patients.
Making Cancer Stem Cells Visible to Immune System
Leukemia stem cells protect themselves against the immune defense by suppressing a target molecule for killer cells. This protective mechanism can be tricked with drugs. In the journal Nature, scientists describe the new therapeutic approaches that can possibly be derived from these results (2019; doi: 10.1038/s41586-019-1410-1).
Patients with acute myeloid leukemia (AML) often relapse after apparently successful treatment. Leukemia stem cells that survive the therapy are responsible for the return of the disease. Scientists can partially explain this phenomenon: The stem cells have protective mechanisms that make them resistant to chemotherapy. But how do they manage to escape the immune defense?
A team of scientists from the Universities of Basel and Tübingen, the German Cancer Research Center, the Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM), and the German Cancer Consortium (DKTK) have now investigated this phenomenon and discovered a surprising mechanism.
The researchers analyzed the leukemia cells of 175 AML patients and found that the cancer stem cells suppress the NKG2D-L proteins on their surface. These proteins enable the natural killer cells (NK cells) to recognize damaged and infected cells, as well as cancer cells, and kill them if necessary. In this way, the leukemia stem cells escape destruction by the immune system. Leukemia cells without stem cell properties, on the other hand, present these target molecules on their surface and are therefore kept in check by the NK cells.
In mice to which AML cells from patients had been transferred, the researchers showed that while the normal AML cells (without stem cell properties) were controlled by the NK cells, the NKG2D-L-negative leukemia stem cells escaped the killer squad.
“Such a connection between stem cell properties and the ability to escape the immune system was unknown until now,” said Claudia Lengerke, MD, from the University Hospital of Basel and the University of Basel.
“An essential mechanism of this immune resistance in leukemia stem cells is apparently the suppression of danger signals such as NKG2D-L on the cell surface,” added Helmut Salih, MD, from the University Hospital of Tübingen and the German Cancer Consortium DKTK.
What is behind this extraordinary protective mechanism? The scientists noticed that leukemia stem cells produce a particularly high amount of PARP1, an enzyme that apparently blocks NKG2D-L production. Preclinical experiments with mice to which human leukemia cells had been transferred showed that PARP1 actually plays an important role in immune escape. If these animals were treated with drugs that inhibit PARP1, the leukemia stem cells again expressed NKG2D-L on their surface—and were then recognized and eliminated by NK cells.
Cancer therapies involving the immune system have been successfully applied for many years in the form of allogeneic stem cell transplantation in AML patients in certain disease situations. In recent years, further novel immunotherapeutic approaches have been developed which are currently being clinically tested.
“Our results show how cancer stem cells cleverly trick the immune system. The elucidation of the underlying mechanism now makes it possible to counterattack,” stated Andreas Trumpp, PhD, German Cancer Research Center and HI-STEM.
The results provide the basis for the possibility of combating malignant leukemia stem cells by combining PARP inhibitors with active NK cells. The scientists involved are now planning to evaluate this approach in a clinical study.