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Functional genomics yields drivers, mutations, and pathways in leukemia

The AML cell line, U937, differentiates with a SYK-directed shRNA.

The study of genes, mutations, and collective pathways contributing to leukemia is a cornerstone of the DF/HCC Leukemia Program, led by Thomas Look, MD (DFCI) and co-leaders David Scadden, MD (MGH), Pier Paolo Pandolfi, MD, PhD (BIDMC), and Scott Armstrong, MD, PhD (CHB). The mutations that cause leukemia are being discovered at a rapid pace and DF/HCC researchers are using these genes to identify effective ways to inhibit the pathways specific to leukemia.

“The exciting challenge at the moment is functional genomics,” says Look. “Which genes and mutations contribute to driving leukemia development and growth and which do not? For those that do contribute to leukemia— how are they linked in common pathways so researchers can better understand how they are functioning?”  Many mutations now seem to affect relatively few numbers of patients overall, but DF/HCC researchers are searching for links grouping some of these disparate mutations into common pathways that can be targeted with new therapies.  “We need more unifying concepts,” says Look.  “It is clear that many mutations are found relatively infrequently so it is an exciting time of pulling everything together with the idea of inhibiting these pathways.”

To better study the mutations and pathways driving leukemia development, DF/HCC leukemia researchers have focused on several tools, including the use of human cell lines and several animal models including immunodeprived and syngeneic mice and zebrafish to validate the many new emerging mutations and candidate mutations. Look’s own laboratory studies use zebrafish to better understand leukemia functional genomics. For example, his group determined that Notch mutations in T-cell ALL (T-ALL) may be amenable to treatments with gamma secretase inhibitors or other small molecule inhibitors. “Notch, a transmembrane receptor, is mutated in more than half of T-cell leukemias and is one of our more exciting connections,” he says.

Driving AML differentiation

Along the lines of exploring leukemia pathways, Kimberly Stegmaier, MD (DFCI) has been pursuing an unexpected response to EGF receptors (EGFR) in acute myeloid leukemia (AML). A focus of her laboratory has been to apply genomic approaches to the discovery of new small molecule modulators of leukemia pathways such as AML differentiation. “We wanted to see if we could mature AML cells into more normal white blood cells,” she says. AML arises from a primitive white blood cell blocked in its ability to mature. By triggering the AML differentiation process, the cancer cells cease to divide and ultimately undergo cell death. Differentiation-based therapy has already been used to treat patients with acute promyelocytic leukemia (APL), an AML subtype.

Her group is now interested in applying this differentiation concept to other forms of AML. However, there is not one druggable target responsible for making a normal hematopoietic stem cell become a white blood cell. Nor is there one critical enzyme known that if altered would force the differentiation of an AML cell into a normal white blood cell. To tackle this dilemma, she used gene expression profiles (the genes whose expression are turned on or off in a biological state) and screened chemical libraries for those molecules that switched the gene fingerprint from that of an AML cell to that of a mature white blood cell. From that screen, the Stegmaier lab determined that a group of EGFR inhibitors switched on the differentiation process. But, EGFR is not expressed in AML cells. “This led us to believe that the EGFR inhibitors were working via an off-target, non-EGFR protein,” she says.

What Stegmaier next determined using integrated genetic and proteomic approaches is that EGFR inhibitors also inhibit a protein called spleen tyrosine kinase (SYK). Her lab confirmed in vitro and in animal studies that inhibiting SYK—either with RNA interference or via SYK chemical inhibitors—has anti-AML activity. “What is so exciting about this is that a chemical genomics screen led us to EGFR inhibitors, to a new target, SYK, and now to a new SYK inhibitor (R788),” she says. Stegmaier is now working with Daniel D’Angelo, MD, PhD (DFCI) to initiate a clinical trial of a SYK inhibitor in patients with AML.

RNA interference finds functionally relevant genes

Benjamin Ebert, MD, PhD (BWH) works to improve the RNA interference technology currently applied by researchers investigating leukemia and other myeloid malignancies.

“RNA interference (RNAi) is a functional approach to screen and look for novel genes that may be playing an important role in myeloid malignancies,” explains Ebert. “The idea is to target and knock down the expression of individual genes and test the genes one by one to see whether they may be playing a functional role in disease. RNAi knocks down the mRNA of a gene thereby blocking its ability to make protein.”

Ebert’s work involves the study of human hematopoietic progenitors that acquire mutations and lead to cancer. “We conduct RNA interference studies in those cells and find genes that are important for differentiation, self-renewal, or proliferation, which are the central abnormalities in hematologic malignancies,” he explains.

“One of the great things about RNA interference is that you can look at hundreds of candidate genes and test each individually to determine its role in disease,” explains Ebert. “We can conduct very detailed studies to find genes that are playing a functional role in human cells without having to grow them in mice or other model organisms.”  With this approach Ebert hopes to identify and shut down mechanisms involving specific genes playing a role in myeloid diseases such as leukemia.

Studying the leukemia microenvironment

DF/HCC researchers are also looking beyond the leukemia cell itself to identify factors potentially leading to its development or maintenance. David Scadden, MD (MGH), Leukemia Program co-leader, studies the leukemia microenvironment for contributions to disease. “For the most part researchers see cancer as the result of a cell gone bad,” says Scadden. “We think it is a tissue that goes bad.”  A focus of the Scadden laboratory is to better understand how the microenvironment contributes to or may be manipulated to inhibit the growth of leukemia. “In short, we are interested in how the microenvironment can be modified,” he says.

One of the aims of this work is to identify novel therapeutic targets important in altering the support of malignant cells to see if they preferentially inhibit malignant leukemia while not destroying normal stem cells.  “We are interested in looking at whether or not modification of the environment stops leukemia initiation,” he says. Scadden’s lab is working on modifying a bone cell—a mesenchymal cell—that contributes to the microenvironment and secondarily changes blood development, leading to leukemia in some animal studies.

Scadden believes this represents one crucial step in the multistep process leading to leukemia. “We are suggesting that there may be interactions between the microenvironment and the leukemic cell that are important both in the initiation of leukemia and its maintenance that may offer novel types of approaches,” says Scadden. “These are potential therapeutic targets with great promise because they interact at the cell-to-cell level.”

As DF/HCC researchers continue to make strides in functional genomics and microenvironment contributions to leukemia, the potential for highly-specific treatments is expansive. Says Look, “It is the age of targeted therapy and a tremendously exciting time to be doing research in leukemia.”

—Alice McCarthy
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1Cancer Cell, Volume 16, Issue 4, 281-294, 6 October 2009