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Predicting response to therapy with BH3 profiling

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Amy Carpenter Aquino

June 2020—Precision medicine in oncology, which today is nearly universally about genetics, needs to move beyond omics and static approaches, Anthony Letai, MD, PhD, professor of medicine at Dana-Farber Cancer Institute and Harvard Medical School, said at last year’s meeting of the Association for Molecular Pathology.

Dr. Letai reported how his laboratory uses dynamic BH3 profiling, a novel assay that detects BCL2 protein dependence in cancer cells and measures changes in their apoptotic priming, to predict clinical response to therapy. He and others call it functional precision medicine.

“The fundamental part of what we call functional precision medicine is taking the drugs you’re interested in and putting them in contact with the tumors you’re interested in and measuring something fast and something relevant. We choose to use BH3 profiling.” Others doing the same work use different methods, he said.

“But the fundamental change is that we realize the incredible value of exposing actual patient samples to drugs, and the technology now exists to do this in a rigorous way.”

“There is an enormous amount of actionable information that can be obtained from taking the actual cancer cell you’re interested in and subjecting it to a relevant perturbation, that is, exposing it to the actual drugs,” he said. “We nearly completely overlook this in today’s precision medicine approaches, and I think there is enormous unrealized potential in this general approach.”

Dr. Letai

Uniting these efforts worldwide is what led he and colleagues to form the Society for Functional Precision Medicine, of which he is president, and they need the pathology community, he said. “In order for these functional approaches to penetrate more, we have to think twice before we immediately kill the samples [with formalin]. As soon as we kill these samples, the ability to gain functional information is lost,” along with the ability to choose the drugs that will work in cancer patients.

The phenotype of increased apoptotic priming is likely to be the main reason chemotherapy ever works, he said. “It can be very useful in assigning this exciting new class of drugs called BH3 mimetics, of which venetoclax is the first member to gain FDA approval” for chronic lymphocytic leukemia and acute myeloid leukemia. The work of his laboratory sped those approvals.

“If you were waiting for genetics to tell you that you should use venetoclax in CLL or AML—a very effective drug in these two diseases—you would still be waiting. There are no mutations, no copy number variations, in either of these two diseases in BCL2 or in related genes. It is not related to genetics; it is related to a functional phenotype to which genetics is completely blind,” he said.

“Help us. Join us,” Dr. Letai urged the molecular pathologists at the meeting. “The pathologists are the ones who know how to do these tests, who know how to convert an interesting laboratory assay into something that can be used in the clinic, that is rigorous enough analytically that it can be reproducible” and used to make patient treatment decisions. He credits pathologists Annette Kim, MD, PhD, and Fabienne Lucas, MD, PhD, of Brigham and Women’s Hospital, in “making us think a little more like clinical pathologists in understanding what it takes to take an interesting laboratory finding and turn it into a rigorous laboratory test.”

His laboratory has focused on the interaction of BH3 peptides with the BCL2 (B-cell-lymphoma-2) family of proteins, of which there are pro- and antiapoptotic members, and their roles in apoptosis. “The point of commitment to programmed cell death is that event of permeabilization of the mitochondrial outer membrane, which is regulated by the BCL2 family of proteins,” he said.

Cell damage signals can prompt BH3 activator peptides Bid and Bim to activate the proapoptotic members of the BCL2 family, Bax and Bak, causing them to homo-oligomerize at the mitochondrial outer membrane, resulting in permeabilization and cell death. The antiapoptotic BCL2 molecules can bind to and sequester the proapoptotic molecules to thwart interaction and prevent permeabilization.

“How can we use this knowledge to figure out how close a cell is to the threshold of apoptosis?” Dr. Letai said. His original plan to measure all the proteins and total them up in “some weird equation” was too complicated.

“Instead we took a more simple and functional approach, which is we synthesized these BH3 peptides,” he said. Drawing on his postdoctoral work in the laboratory of the late Stanley J. Korsmeyer, MD, Dr. Letai showed that the synthetic BH3 peptides “are essentially equivalents of the proapoptotic proteins” and can be easily measured.

This led to the next question: “How much BH3 peptide do we need to add until the mitochondrion permeabilizes?” A cell with a mitochondrion that required a large amount of BH3 peptide for permeabilization was considered far from the threshold and unprimed for apoptosis. “We simply take mitochondria, expose them to BH3 peptides, and then measure the mitochondrial outer membrane permeabilization,” he said.

Dr. Letai described an intracellular-based assay his laboratory developed to measure the effect of BH3 profiling on a single cell suspension of cells, such as leukemia cells. Instead of extracting the mitochondria, “we gently permeabilize the plasma membrane of the cells, so that the peptides that we add can gain access directly to the mitochondria,” he said. After 60 to 90 minutes of exposure to Bim BH3 peptides, the cells would be stained with a cytochrome c antibody to measure cytochrome c release from the mitochondria (Ryan J, et al. Methods. 2013;​61[2]:​156–164).

“Note that the cytochrome c is an intermembrane space protein, so when the outer membrane is ruptured, it leaves the mitochondria,” Dr. Letai said. The cell membrane permeabilization results in a cytochrome c-negative cell.

“Why do we care?” he said.

His laboratory first used BH3 profiling to understand BCL2-inhibitor drugs and which diseases to target with venetoclax (ABT-199), the latest version of the BCL2-inhibitor drugs. “It is a great [BCL2] antagonist, very selective, very potent.” And the question is, for what diseases can it be used?

Dr. Letai’s team used fluorescence polarization binding assays to determine the sensitivity of BH3 peptides to the BCL2 antiapoptotic proteins. “Certain peptides are promiscuous but some are very selective. For instance, the HRK BH3 peptide selectively binds and inhibits BCL-XL,” he said. A cell that readily permeabilizes its mitochondria in response to the HRK peptide, for example, is a BCL-XL-dependent cell, while the Noxa BH3 peptide can be used to identify MCL1-dependent cells. The Bad BH3 peptide is selective for BCL2-dependent cells (Certo M, et al. Cancer Cell. 2006;​9[5]:351–365).

They tested the theory in model systems of known BCL2 and MCL1 dependents, and then turned their attention to human cancers, “to diseases found in the wild,” Dr. Letai said. They looked first at CLL, for which there were discrepancies in the literature about the apoptotic protein dependence of CLL. “And this pretty much puts it to rest. The mitochondria of CLL patients are uniformly sensitive to the Bad peptide but not to the HRK peptide,” which is diagnostic of BCL2 dependence, he said. “When we exposed CLL cells to ABT-737, a primordial BCL2 inhibitor, it was universally sensitive to the inhibitor” (Del Gaizo Moore V, et al. J Clin Invest. 2007;117[1]:112–121).

Dr. Letai’s team further showed that introducing a BCL2 inhibitor, such as ABT-737, to CLL cells quickly caused the displacement of proapoptotic proteins and the activation of the proapoptotic protein Bax to induce oligomerization and cell permeabilization. “Within hours you could see the normalization of the white blood count in the periphery,” he said. “That’s how rapidly this effect happens.”

Similar work with AML myeloblasts showed BCL2 dependence in AML, though “we ran into enormous skepticism,” Dr. Letai said. “People just didn’t believe that BCL2 could be involved in a myeloid malignancy.”

His team’s work showed that most AML myeloblast samples were more sensitive to the BH3 Bad peptide, and thus more BCL2 dependent, than normal hematopoietic stem cells. “This suggests that there was already built in for us a therapeutic index for BCL2 inhibition in AML,” Dr. Letai said.

At this point, Dr. Letai connected and shared data with Marina Konopleva, MD, PhD, a physician-scientist and professor in the departments of leukemia and stem cell transplantation at MD Anderson Cancer Center, and they presented the data to Abbvie “and convinced them we should start a clinical program in AML with BCL2 inhibition, because the preclinical work was very strong,” he said.

The first human trial of venetoclax in AML patients showed “definite evidence of clinical activity of BCL2 antagonism in the case of AML,” Dr. Letai said. He and Dr. Konopleva reported an objective response rate of 19 percent in the trial patients, the majority of whom were elderly and had received at least one prior therapy (Konopleva M, et al. Cancer Discov. 2016;6[10]:1106–1117).

In subsequent trials of venetoclax with hypomethylating agents Vidaza (azacitidine) or decitabine, Dr. Letai said, “responses were remarkable. In treatment-naïve elderly patients, the CR/CRi [complete remission/complete remission with incomplete marrow recovery] is upward of 70 to 75 percent. Just a remarkable achievement with an induction regimen that is essentially taken at home” (DiNardo CD, et al. Blood. 2019;​133​[1]:7–17).

When BCL2 inhibitors made it into the clinic for CLL, he said, “it was a remarkable result—79 percent response rate with complete remission in 20 percent of patients” (Roberts AW, et al. N Engl J Med. 2016;​374​[4]:311–322).

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