For the first time, researchers have followed the path of acute lymphoblastic leukemia (ALL) cells crossing the blood-brain barrier and invading the central nervous system (CNS). This discovery opens the door to new approaches for preventing CNS involvement in patients with ALL.

Hisayuki Yao, MD, PhD, of the Division of Hematologic Malignancies and Cellular Therapy at Duke, presented findings on behalf of the research team led by Dorothy A. Sipkins, MD, PhD, at the American Society of Hematology's 59th Annual Meeting & Exposition, December 9 to 12, 2017, in Atlanta, GA.

Burden of CNS involvement
Up to 70% of adults with ALL develop CNS involvement, at which point the median overall survival is approximately 6 months. Few treatment options are available to reduce the risk of CNS involvement, in part because the underlying disease mechanisms are not well understood.

In the lab directed by Sipkins, researchers previously showed that inhibiting phosphoinositide 3-kinase delta (PI3Kd) selectively prevented CNS involvement in animal models of ALL. In the current set of experiments, the team identified the integrin alpha-6 (ITGA6) gene as a key mediator of ALL cell migration. Next, the research group sought to follow ITGA6 and ALL cells across the blood-brain barrier.

How to cross the blood brain barrier?
The ITGA6 cell surface receptor is highly specific for the laminin family of extracellular matrix (ECM) proteins. Laminin is localized to 2 regions within the CNS: the basement membranes of vessels within the brain parenchyma and the basement membranes of the meningeal vessels and meninges. Sipkins and her team hypothesized that ITGA6-positive ALL cells enter the CNS through their interactions with laminin within these vessels.

To test this hypothesis, the team first examined the interaction between ALL cells and laminin-positive parenchymal vessels. In the mouse model, the movements of injected fluorescent ALL cells were tracked across multiple time points following engraftment. Although ALL cells were detected within the lumen of vessels outside the CNS, these cells were not able to migrate across the blood-brain barrier.

Answers in an anatomy book
With the parenchymal route ruled out, researchers next examined the meninges. But how could ALL cells travel from the bone marrow to the meninges?

"To answer that question, we had to turn to our anatomy textbook," Yao says. Pouring over images of the vascular anatomy of the meninges and the adjacent vertebral bone marrow, they found it: a spot where meningeal vessels directly connect to the bone marrow vasculature in the skull and spine.

In particular, the team focused on emissary vessels in the spine that pass directly between the vertebral bone marrow and the subarachnoid space. When imaging the emissary vessels in the spines of mice engrafted with ALL, the path of ALL cell migration was clear. "We found that these cells came streaming through the bony channels into the CNS," Yao says.

Further testing confirmed that emissary blood vessels, meningeal vessels, and adjacent nerve roots within the CNS are positive for laminin expression. The team also showed that ALL cells favor higher concentrations of laminin and can be induced to migrate toward increasing laminin concentrations in a 3-dimensional ECM assay. These findings support the proposed mechanism whereby ALL cells migrate along laminin-rich emissary blood vessels that pass directly through the vertebral or calvarial bone marrow and into the CNS.

PI3Kd inhibition and CNS migration
After identifying how ALL cells migrate to the CNS, the research team then showed that they could suppress this process by blocking the PI3Kd and ITGA6 pathways. In vitro experiments showed that treatment with GS-649443 or an anti-ITGA6 antibody inhibited the migration of ALL cells in a laminin-rich matrix.

In vivo experiments showed similar results. In the mouse model, compared with placebo (vehicle) treatment, GS-649443 significantly decreased the number of bony channels filled with ALL cells. Similarly, treatment with an anti-ITGA6 antibody was associated with a significant decrease in the number of ALL cells present in the cerebrospinal fluid and a decrease in the rate of limb paralysis. These findings confirm the role of ITGA6 activity for ALL invasion into the CNS.

Implications for ALL treatment
In summary, researchers identified a novel mechanism whereby ALL cells travel along laminin-rich emissary vessels from the bone marrow, through the subarachnoid space, and into the CNS. Treatment with a PI3Kd inhibitor disrupts key signaling pathways along this route, crippling ALL cell motility and blocking CNS invasion. Based on these findings, PI3kd inhibition may play a promising role in preventing and treating CNS recurrence in patients with ALL.

Source: Yao H, Warner M, Price T, et al. Acute lymphoblastic leukemia cells bypass the blood brain barrier to enter the CNS via a neuronal developmental pathway. Presented at: American Society of Hematology 59th Annual Meeting; December 9-12, 2017; Atlanta, GA. Abstract 144.