In the late 1980s, scientists developed a novel approach to treating acute myeloid leukemia (AML), a type of blood cancer. Called differentiation therapy, it amounted to a bona fide cure for many patients. The treatment works by triggering cells “stuck” with a cancerous identity to keep developing and maturing, giving rise to different, non-disease- causing types.
Unfortunately, this treatment only works for a small subset of patients who have a particular subtype of the disease, called acute promyelocytic leukemia (APL). “For a long time, it was seen as kind of a one-off,” says M. Andres Blanco, PhD, an assistant professor at the University of Pennsylvania School of Veterinary Medicine.
Now, Blanco and colleagues have identified a new way to trigger differentiation in AML—one with potential to treat a much wider array of AML patients, according to the team which published its research (“KAT6A and ENL form an epigenetic transcriptional control module to drive critical leukemogenic gene expression programs”) in Cancer Discovery.
Their study identifies an enzyme that regulates the process by which AML cells differentiate. In both cell lines and an animal model, the researchers found that inhibiting this enzyme, particularly in combination with other anti-cancer therapies, prompted AML cells to lose aspects of their identity associated with aggressive growth. The cells also began to exit the cell cycle, on the path toward maturing into a new cell type.
“Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells,” write the investigators. “
This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene expression programs. We show that KAT6A is the initiator of a newly-described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyllysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation.
“Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small molecule inhibitors could be of high therapeutic interest for mono or combinatorial differentiation-based treatment of AML.”
“AML typically has a poor prognosis, with five-year survival below 50%,” says Blanco, senior and a co-corresponding author on the study. “If an approach like this, in combination with other therapies, could make the cancer less aggressive, that is notable and could help a lot of patients.”
All cells derive from stem cells, following different pathways of differentiation to arrive at their eventual fate. Blanco’s lab is particularly interested in the epigenetic regulation of cell identity; in other words, how factors aside from an organism’s DNA sequence can influence how cells mature.
“I am just really taken by the idea that, from an epigenetic standpoint, every cell in the genome has the same DNA, save for any rare mutations, but can take on completely different functions,” Blanco continues. “It’s amazing how that can happen.”
Knowing of the success of differentiation therapy in patients with APL, Blanco and colleagues aimed to understand what molecular players are involved in the epigenetic regulation of cell differentiation in AML more generally. To select candidates, they ran a screen using the CRISPR-Cas9 system, which can direct pieces of RNA associated with particular proteins in the cell, cause them to be deleted, which then affects cell function in a way that can be assessed using various assays. In this case, they were looking for proteins that, when deleted, affected cellular differentiation.
One of the screened proteins that affected cell differentiation was KAT6A, an enzyme known as a histone acetyltransferase, which makes modifications to DNA that help activate gene expression epigenetically. Though KAT6A had not been studied in the context of AML previously, when the researchers looked in databases that cataloged gene expression data from cancer patients, they found AML patients had higher KAT6A levels than patients with any other type of cancer or those without cancer.
Learning more about its activity, the researchers eliminated KAT6A from a human AML cell line and found cells grew more slowly. When they manipulated cells to have lower levels of KAT6A, a marker of differentiation increased, signaling that the enzyme was somehow setting up an obstacle in the way of cellular differentiation.
To see how KAT6A might be acting in an animal, researchers blocked KAT6A in an AML cell line, which they then transferred to immunodeficient mice. They found that mice that received cells with KAT6A knocked out had a slower growing disease and lived longer than mice that received AML cells containing the protein.
Identifying the mechanism
Now confident that KAT6A supported AML growth by blocking differentiation, Blanco and his team aimed to identify the steps it took, and the other molecules it interacted with, to accomplish that task. As a histone acetyltransferase, KAT6A can add one of three different modifications to histones, proteins around which DNA winds. When the researchers looked closely at genes associated with leukemia, they found that one of those three, H3K9ac, was often associated with the genes turning on or off.
After searching a database that includes a vast amount of information about which genes are functionally linked and associated with proliferation and survival in cancer cells, they found a protein called ENL, which binds H3K9ac after it has received an acetyl modification—the same modification that KAT6A catalyzes. “There was definitely an ‘aha!’ moment when we searched this database,” notes Blanco.
The finding helped the researchers understand that KAT6A is what’s known as a “writer,” it “writes” the modification of H3K9ac, and ENL is a “reader,” taking in that modification and acting upon it. “KAT6A prepares the groundwork for ENL to do its thing,” according to Blanco.
With this new understanding of AML differentiation regulation in hand, the research team hopes to continue experimenting with inhibiting KAT6A to see if they can create a new class of differentiation therapies—ones that can treat many more types of AML patients.