A protein too big to be ignored as a potential drug target for reversing blood diseases

It all started with a simple question: “How do cells communicate with the outside world to sense their environment?”

The pursuit of the answer turned out to reveal far more than what Amittha Wickrema, PhD, professor of medicine at the University of Chicago, had dreamed. Focusing on how the production of red blood cells is triggered, Wickrema and his long-time collaborators Amit Verma, MD, from Albert Einstein College of Medicine and Chuan He, PhD, the John T. Wilson Distinguished Service Professor of Chemistry at UChicago, identified a key protein that could serve as a drug target to cure a group of blood diseases.

Like all other types of cells, blood cells are produced by stem cells. Upon receiving special molecular signals known as cytokines, these impressionable stem cells start to differentiate into various types of blood cells. The process seems straightforward enough, until you consider the large diversity and immense number of blood cells in our bodies. In the case of red blood cells, the production can exceed a rate of one billion cells per minute. Any flaws in the regulation of this process can have dire consequences. If the generation of these cells spirals out of control, the result is blood cancer; a dramatic decline in the production leads to anemia.

Pre-cancerous blood diseases associated with the overproduction of blood cells are often treated by targeting the mutated Janus kinase 2 (JAK2) protein, which is essential for cell growth and division. JAK2 resides in the cell and is activated when exterior cytokines bind to the cell surface.

“There is a whole group of blood diseases where JAK2 is mutated, leading to the overproduction of red blood cells,” Wickrema said. “Unfortunately, JAK2 drugs do not permanently eliminate the cells carrying mutant JAK2 and hence aren’t a cure.”

Until recently, the interconnectedness between JAK2, cytokines, and the DNA in the nucleus for blood cell production remained an enigma. In a new study published in the June 2019 issue of Cancer Discovery, Wickrema and his coauthors were the first to complete this connection by identifying a crucial second protein in this communication pathway, the TET methylcytosine dioxygenase 2 (TET2).

TET2 directly communicates with the DNA in the nucleus, and it can be indirectly activated by cytokines through the JAK2 protein. Where the JAK2 drug falls short in regulating blood cell production, now drugs tailored towards TET2 activation may compensate—with perhaps greater effectiveness. Given TET2’s direct interaction with the DNA in the nucleus, “TET2 drugs may give a more direct access to gene activation, and TET2’s more downstream role can perhaps provide us with greater control over the production of blood cells,” Wickrema said.

From cytokine, to JAK2, to TET2, to nucleus

Not every gene in our body is active all at once. TET2 is able to activate silenced genes by carrying out a simple but specific chemical reaction at a DNA strand. Just like a light switch turning on, the gene will come to life and is free to participate in gene expression once again. Gene expression enables the synthesis of proteins needed for various cellular functions.

Wickrema and his team focused on blood-specific cytokine molecules that can activate TET2 to initiate this chemical reaction. Subsequently, they discovered what causes the TET2 to be active: Attached to the protein molecule is a wayward phosphoryl group consisting of one phosphorus and three oxygen atoms.

This research revealed that a phosphoryl group is transferred onto very specific locations on the TET2 molecule by the JAK2 protein in a process called phosphorylation. The TET2 protein is massive, more than twelve thousand times heavier than a water molecule. Yet the research successfully pinpointed the phosphorylation sites to two amino acids among TET2’s two thousand constituents. This finding is crucial for drug design, where specificity is key. Specificity enables drugs to carry out highly precise functions without interfering in other processes which may bring about adverse side effects.

Without this phosphorylation process, TET2 cannot communicate with the DNA and activate the necessary cohort of genes that commands the transformation of stem cells to red blood cells. As further proof, the researchers supplied natural TET2 and TET2 without the phosphorylation sites to cultured stem cells. Only when provided with TET2 whose phosphorylation sites remained intact, the blob of stem cells took on a distinct brownish hue synonymous with hemoglobin after several days, indicating its conversion into red blood cells.

“This experiment truly identified the connection between mechanism and function, which has practical applications in blood disease treatment,” Wickrema said.

TET2 holds promise not just for drug design

For the first time, this study also revealed that one could potentially target TET2 to treat patients with JAK2 mutations, regardless of whether these patients also carry certain TET2 mutations. Indeed, the development of drugs that can modulate TET2 activity is one of Wickrema and his collaborators next big goal.

“The bottom line is our discovery has opened up new ways to treat blood diseases,” Wickrema said. “Currently, there are no drugs that directly target TET2.”

It would be one big missed opportunity not to study the TET2 further for drugs development. But remember, TET2 is a gigantic molecule, almost twice the size of JAK2. For this study, the researchers only scrutinized the one section of the protein, its catalytic domain. This is where most of the functions executed by the protein take place as it interacts with JAK2 and DNA. Only two amino acid sites out of two thousand are needed as the stars of phosphorylation, but Wickrema believes that Mother Nature does not do things without reason. TET2 is big—too big to be ignored. The cell has invested a lot of energy in making such a large protein; the question is, why? Surely there are more functions to TET2 than meet the eye, and the researchers also intend to discover them.

In addition to Jong Jeong, Ph.D., the first author and the lead scientist in the Wickrema group, Amit Verma, M.D., head of hemato/oncology and his group at Albert Einstein College of Medicine, and Chuan He, Ph.D., distinguished professor of chemistry, and his graduate student Ji Nie were pivotal in making these discoveries possible. A host of other collaborators, both at the University of Chicago and several other institutions also contributed to this work.