Several US Food and Drug Administration (FDA)-approved anti-malarial and anti-fungal drugs might be able to overcome a previously unexploited mechanism that allows some leukemia cells to escape programmed cell death, according to preclinical experiments conducted at the University of New Mexico in Albuquerque.[1,2]
In some healthy tissues, intracellular accumulation of cyclic adenosine monophosphate (cAMP) can trigger apoptosis. In leukemias, cAMP can play a role in cancer cell maturation and can be both pro- and antiapoptotic. In acute myeloid leukemia (AML), cAMP elevation appears to trigger apoptosis.
However, the team found that AML and B-cell acute lymphoblastic leukemia (B-ALL) cells are able to excrete cAMP, circumventing its pro-apoptotic signaling, explained senior study author Alexandre Chigaev, PhD, a research associate professor at the University of New Mexico’s Center for Molecular Discovery.
“The idea that malignant cells can evade apoptosis through cyclic AMP pumping, that’s new,” Chigaev said. “It’s never been reported, previously.”
Unfortunately, it is unclear exactly how leukemia cells manage to remove cytoplasmic cAMP. Different leukemia cell lines differentially express multidrug resistance protein 4 (MRP4/ABCC4), a cAMP transporter—an observation that led the researchers to predict that the presence of ABCC4 protein on leukemia cells would correlate with cAMP efflux. But when they loaded B-ALL cell lines with fluorescently tagged cAMP (F-cAMP), that was not the case; leukemia cell ABCC4 expression did not predict F-cAMP efflux.
ABC transporters are upregulated in stem-like cells, which are associated with resistance to chemotherapeutic agents, “which may suggest that these cells require active removal of cAMP or other structurally related compounds in order to remain in a pluripotent state,” the study coauthors noted.
Chigaev and colleagues reason that another transporter protein—or perhaps another mechanism altogether—must be involved in leukemia cells’ ability to regulate cAMP levels to circumvent apoptosis.
Rather than attempting to develop a new drug to target leukemia cells’ apoptotic evasion, Chigaev’s team used high-volume screening of several libraries of FDA-approved drugs to see if existing small-molecule medications might already be able to do so.
Screening existing drugs to identify compounds that might be repurposed in this way is vastly less time-consuming and expensive than developing new drugs, Chigaev explained.
Using auto-sampling high-throughput flow cytometry and a novel assay of leukemia cell cAMP efflux, the research team initially identified 51 candidate cAMP efflux inhibitors. Of these, subsequent dose-response validation work narrowed that list to six compounds: two structurally related anti-malarial agents (artesunate and dihydroartemisinin); the anti-fungal drug clioquinol; and the other biologically active compounds cryptotanshinone, patulin, and parthenolide.
cAMP efflux inhibition is a mechanism of action that has not previously been reported for these drugs, Chigaev said.
These compounds (and MK-571, first developed as a leukotriene receptor antagonist and later described as a cAMP efflux inhibitor that was used as a control) all decreased cAMP efflux and cell viability in AML and B-ALL cell lines, and in primary B-ALL samples from patients, but not in normal blood cells. Six patients’ B-ALL cells were tested in vitro and exhibited dose-dependent declines in cell viability following exposure to the compounds.
“They showed very good, very promising effects,” Chigaev said. “All of these drugs showed efficacy in our tests.”
Clioquinol, a treatment for “traveler’s diarrhea” in the 1960s, is “very safe” and “surprisingly, worked alone” against leukemia cells in the team’s study, Chigaev said.
The slopes of dose-response curves for the different drugs were more B-ALL cell line– and patient-specific rather than drug-specific, the authors reported. That might mean that small or transient increases in intracellular cAMP can be antiapoptotic and promote cell survival in some leukemia cells, the authors suggested. Future studies with larger numbers of patients’ samples might help identify gene and phenotype profiles that correlate with stronger dose-response relationships with candidate cAMP efflux inhibitors, the authors noted.
The cAMP efflux inhibitors also modulated Very Late Antigen-4 (VLA-4, an integrin and adhesion molecule involved in hematopoietic progenitor cell retention in bone marrow), to varying degrees. VLA-4 anchors healthy but immature immune and blood cells in bone marrow during their maturation. cAMP-associated loss of VLA-4 cell adhesion might explain leukemic cells’ escape from bone marrow.
“Our data suggest that targeting cAMP efflux with small molecules could be an efficient way to raise cAMP in certain types of cancer, and this could potentially result in the development of a new class of pathway-specific therapeutics,” the authors concluded. “Because increased cAMP efflux is not a typical trait of healthy cells, the identified ICE [inhibitors of cAMP efflux] exhibited specificity toward leukemic cells.”
The efficacy of targeting cAMP efflux by cancer cells would hinge on those cells’ ability to remove cAMP, however, they cautioned—which might vary importantly between cell lineages and patients.
The researchers are pursuing additional testing with mouse models but hope to conduct human clinical trials in the future.
1. Perez DR, Smagley Y, Garcia M, et al. Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia. Oncotarget. 2016;7:33960-82.
2. University of New Mexico Cancer Center. Press release: UNM Cancer Center scientists use already-approved drugs to force cancer cell death. July 19, 2016.
3. Chigaev A, Waller A, Amit O, Sklar LA. Galphas-coupled receptor signaling actively down-regulates alpha4beta1-integrin affinity: a possible mechanism for cell de-adhesion. BMC Immunol. 2008;9:26.