Maybe you can’t teach an old dog new tricks, but you can still find out new things about the old tricks that old dog has been up to.

Isolated in 1977 and first marketed in 1989 in a recombinant form for anemia, erythropoietin (EPO) definitely is one of biotech’s old dogs. EPO is the main hormone regulating red blood cell production, primarily by stimulating bone marrow progenitor cells to differentiate into red blood cells.

But for all of its commercial and clinical successes, it has been known only for a few years that EPO stimulates separate pathways under normal circumstances and in times of cellular stress, such as anemia. In the March 2006 issue of the Journal of Clinical Investigation, researchers from the Maine Medical Center Research Institute teased apart the molecular machinery of the erythropoietin stress pathway.

Recombinant EPO was Thousand Oaks, Calif.-based Amgen’s first approved product; Amgen sells the drug under the trade name Epogen for dialysis-associated anemia and has licensed it to Johnson & Johnson for nondialysis uses like chemotherapy-related anemia. J&J sells it under the name Procrit. (Amgen also has a patent dispute with F. Hoffmann-La Roche Ltd. over a chemically modified version of the drug.) (See BioWorld Today, Nov. 10, 2005.)

The JCI study builds on the 2001 discovery that though the EPO receptor normally has no fewer than eight intracellular sites that are phosphorylated upon EPO binding, when it has to do without all those phosphorylation sites, the effect is - well, not much. Mice with a receptor that didn’t contain the phosphorylation sites had only about 20 percent fewer red blood cells than normal, and near-normal levels of progenitor cells. Humans with a naturally occurring truncated form of the receptor actually have accelerated blood cell formation.

Don Wojchowski, senior author of the current JCI study and director of the Institute’s stem and progenitor cell biology program, and his colleagues used a combination of bone marrow transplants and studies on primary bone marrow red blood cell progenitors to investigate the role of one specific phosphorylation site, amino acid 343, and the protein it binds when phosphorylated, Stat5.

Neither the EPO receptor nor red blood cell formation is exclusive to bone marrow, but Wojchowski called bone marrow stress erythropoiesis "a very important and perhaps understudied problem." To determine the effects of the EPO receptor specifically in bone marrow, Wojchowski and his colleagues transplanted normal mice with one of three types of bone marrow cells. The cells had either normal EPO receptors, EPO receptors lacking all eight phosphorylation sites or receptors with only the first site, amino acid 343.

The EPO receptor containing only the first phosphorylation site was able to induce red blood cell production that was similar to that in mice with wild-type receptor. The receptor that was totally without phosphorylation sites, however, was not able to efficiently support red blood cell formation during chemically induced anemia.

The authors also investigated whether genes that might be regulated by EPO and Stat5 are affected differently by the three receptors.

One surprising finding was that Stat5 activation had little effect on the anti-apoptotic factor Bcl-x; for that matter, none of the three receptor forms the researchers studied had much of an effect on Bcl-x.

"One consensus has been that EPO stimulates many targets, but as long as Bcl-x is stimulated, erythropoiesis will proceed," Wojchowski said, because Bcl-x is "a potent survival factor."

Bcl-x is a member of the first family of apoptosis proteins, Bcl-2; that family is being targeted by biotechnology firms such as Berkeley Heights, N.J.-based Genta Inc. and Montreal’s Gemin X Biotechnologies Inc. for the development of cancer therapeutics. (See BioWorld Today, Dec. 13, 2005, and Dec. 30, 2005.)

But Wojchowski said that for red blood cell formation, "we do not see evidence that Bcl-x is an immediate or strong EPO target, at least in the progenitor cells we studied."

They did find that two other molecular targets of Stat5, Pim-1 kinase and Oncostatin-M, were up-regulated by both wild-type EPO receptor and the truncated receptor that still contained amino acid 343, but not the fully truncated version.

"The question is - and it’s currently a $12 billion a year question - are either of those factors candidates for anemia therapy?" Wojchowski said.

His answer: "Probably not."

Pim-1 was one of the first oncogenes to be discovered, and stimulating it for anemia therapy might cause leukemia at a significant rate. Oncostatin-M is very widely expressed, making it an excellent candidate for side effects.

But, Wojchowski said, his laboratory is working on further characterizing the consequences of Pim-1 and Oncostatin-M activation, and also is investigating the nature of new EPO-plus-Stat5 target genes.

Asked whether Stat5, the first link in the stress erythropoiesis molecular chain, might itself be an alternative target for stimulating red blood cell formation, Wojchowski said, "Well, it’s another oncogene, and a fairly potent one."

But he added that "the fact that one can now separate steady-state from stress erythropoiesis based on EPO receptor signaling does open new doors for the development of new anemia therapeutics."

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