Dr. Richard James’ team is gaining ground in its quest to understand drug resistance in patients with B-cell lymphoma.

In 2015, Dr. Richard James’ team discovered how a key enzyme – called Bruton’s tyrosine kinase (Btk) – influences the way certain immune cells respond to viruses and infections.

Richard JamesBtk turns some types of immune cells on and off, and drugs that block Btk can stop malignant cells from proliferating in patients with B-cell lymphoma. But no one understands the full breadth of Btk activity, how the drugs impact non-malignant cells, or how malignant cells develop drug resistance.

James and his colleagues are solving these mysteries by mapping out Btk’s role in immune cells. Their 2015 study pinpointed Btk’s effect on macrophage cells in mouse models. These cells serve as the first line of defense against infections and viruses.

The researchers were the first to illuminate exactly how Btk influences the toll-like receptor (TLR) pathway, which activates an immune response to molecules inside pathogens. The study showed that Btk stimulates TLR signaling in resident peritoneal macrophages, which live around the abdomen. But Btk inhibits TLR signaling – and therefore suppresses immune response – in bone marrow macrophages and thioglycollate-elicited macrophages.

As James’ team worked to explain this divergence, they identified a pathway – called Trem2/Dap12 – that Btk uses to inhibit immune response. Their research suggests that Btk plays a much more influential role in TLR signaling and immune response than was previously thought.

“If you think of cell signaling like a symphony, we thought Btk was just one of the players, but it’s more like the conductor – it’s telling the TLR pathway what to do,” James says.

Practical insights from proteomics

James’ lab uses proteomics to map out how proteins drive cell behavior, while other researchers typically use DNA sequencing to look for gene mutations that cause cells to function or malfunction.

“When DNA sequencing finds a gene variant, it can be hard to see why it’s significant because you often don’t know what the gene does in the first place,” James says.

Btk and other kinases trigger a biological domino effect, activating a series of proteins that work together to tell cells to perform a particular function.

“Proteomics lets us identify each step in this process,” James says. “That leads to opportunities to use drugs to improve cell function.”

How Btk affects macrophages

For their 2015 study, James and his colleagues blocked Btk and two related kinases, Tec and Bmx, in several types of mouse macrophages. Then they stimulated the TLR pathway in a way that normally provokes an immune response.

Resident peritoneal macrophages reacted by secreting fewer proinflammatory cytokines, which signal the immune system to attack bacteria or viruses. But bone marrow macrophages and thioglycollate-elicited macrophages became hyperactive, secreting more proinflammatory cytokines than normal cells. The study was published in the Journal of Immunology in July.

“We thought Btk was going to either positively or negatively regulate this process – we didn’t expect it to do both,” James says.

Identifying a key mechanism

James and his colleagues, including Seattle Children’s Drs. David Rawlings and Giacomo Tampella, used proteomics and pathway analysis to explain this divergence.

David Rawlings They found that Btk positively regulates the Trem2/Dap12 pathway, which inhibits TLR signaling in bone marrow macrophages and thioglycollate-elicited macrophages. This means Btk stimulates the pathway to suppress TLR signaling and, consequently, immune response.

“Removing Btk is like taking the brakes off,” James says. “The bacterial response is heightened because the proteins aren’t phosphorylated.”

In resident peritoneal macrophages, the researchers found that Btk promotes TLR signaling at points downstream of the TLR receptor, enabling the cells to mount an immune response.

Unraveling drug resistance

The research is part of James’ larger quest to understand how drugs that block Btk affect patients with lymphoma, and to find ways to reverse the drugs’ unwanted effects – such as leaving patients susceptible to infections and viruses that macrophages normally fend off.

“Now that we’re understanding Btk’s role in macrophages, it could lead to ways to reactivate them and keep patients as healthy as possible,” James says.

A larger problem is that B cells eventually become drug resistant, and lymphoma comes back and is fatal.

“We’re using what we learned to start piecing together how B cells stay alive when they lose Btk for a long time,” James says. “That could help us find ways to block those mechanisms and stop lymphoma long-term.”

If you think of cell signaling like a symphony, we thought BTK was just one of the players, but it’s more like the conductor.
– Dr. Richard James