Predicting response to therapy with BH3 profiling

“Now there are FDA approvals in CLL and AML, in a variety of settings, for venetoclax in the United States and around the world,” Dr. Letai said.

What about general chemosensitivity to conventional chemotherapy agents?

“It’s a bit of a mystery why these should selectively kill cancer cells,” Dr. Letai said. “So we tested the hypothesis that perhaps these types of drugs perform damage and cause apoptotic signaling in every cell in our body, but there are some cells we’ll call primed cells, that are right on the edge.” These primed cells respond to apoptotic signaling by going over the threshold and committing to apoptosis, “whereas less primed cells, like chemoresistant cancer cells or most normal cells, when subjected to these death insults, move toward the cliff but not over it.”

Cue the promiscuous BH3 peptides. Bim, Bid, and Puma interact with all of the antiapoptotic proteins and are good measures of net apoptotic reserve—“basically, how far you are from the edge of the cliff.”

“We asked, ‘Does sensitivity to these peptides at the mitochondrial level correlate with sensitivity at the clinical level to these conventional chemotherapeutic agents?’” he said. “And it turns out it did.”

For example, results of studies of patients with either multiple myeloma, ALL, or ovarian cancer found that patients who responded well to standard clinical therapies were more primed at the mitochondrial level than patients who responded poorly (Ni Chonghaile T, et al. Science. 2011;334​[6059]:​1129–1133).

“We have since found this in many different diseases,” Dr. Letai said. Examinations of normal chemotherapy-resistant tissue, such as the heart, lung, and brain, find these tissue cells tend to be significantly less primed for apoptosis. The unprimed tissue in the adult body “is the main reason why there is a therapeutic index for chemotherapy, and why we can kill blood cancers but we can’t, in general, cure solid tumors with chemotherapy. Blood cancers and normal blood cells are the most primed cells in our body.”

The theory held true during his team’s study of myeloblasts with a comparison of AML patient responses to conventional chemotherapy—stable complete remission, complete remission that relapsed, or poor response. “It correlated very well with how primed their mitochondria were,” as measured by response to the Bim peptide, he said. AML patients with highly primed mitochondria achieved better outcomes; those patients with myeloblasts that were more primed than normal hematopoietic stem cells were found to stand a better chance of cure with chemotherapy alone.

 

 

What can be done for unprimed patients? “Maybe we could make unprimed cancer cells more primed,” Dr. Letai said. “Are there ways we could expose tumors to chemotherapies and see whether they evoke apoptotic signaling ex vivo, in the lab, using primary patient tumor cells?”

Dr. Letai and his team found that their dynamic BH3 profiling technique could be used ex vivo to predict the cytotoxic response of cancer cells to various treatments. “Turns out our tool could do this because the induction, the initiation of apoptotic signaling, happens within hours. And that means we do not demand long-term ex vivo culture for our primary cancer cells,” he said. “This has long been the hang-up of ex vivo approaches to measuring chemotherapy sensitivity in cancer” (Montero J, et al. Cell. 2015;160[5]:977–989).

“Now we have a way.” His laboratory takes liquid or solid cancer cells, turns them into a single-cell suspension, distributes them to 384-well plates, then exposes the cells to treatments. Next they measure: After six to 24 hours of exposure to the drug, what is the apoptotic priming of the cancer cells compared with a well containing untreated cancer cells? “That’s what we call our delta priming,” Dr. Letai said, “and we have subsequently found that the bigger the delta priming, the better the in vivo response.”

Does it work? In one trial, 21 different B-cell acute lymphoblastic leukemia patient-derived xenografts (PDX) in mice were exposed to the HDM2 (MDM2) inhibitor CGM097. The results: “We can, with perfection in this relatively small number of samples, completely discriminate the responders from the nonresponders based on this ex vivo assay,” Dr. Letai said (Townsend EC, et al. Cancer Cell. 2016;29[4]:574–586).

A human study using dynamic BH3 profiling to predict imatinib response in CML patients also had encouraging results. “We were able to discriminate, not with the same perfection but still quite well, between those who responded well and those who responded poorly to imatinib in the clinic.”

The next step, Dr. Letai said, would be to identify one therapy among several choices that would work best in a particular patient. Using AML PDX models, his laboratory performed dynamic BH3 profiling with a set of molecules and then measured the in vivo response to four different types of single-agent therapies: birinapant (second mitochondria-derived activator of caspase mimetic), quizartinib (FLT3 inhibitor), JQ1 (BDR4 inhibitor), and venetoclax.

“In each of these cases, our assay predicts very well the magnitude of the in vivo response, suggesting that across many different drugs we can continually predict the in vivo response,” Dr. Letai said of the unpublished study results.

“We can do this in the clinic,” he said. In a clinical trial with cohorts at Stanford Medicine and Massachusetts General Hospital, Dr. Letai’s team used dynamic BH3 profiling to evaluate AML patient response to treatment with lenalidomide plus chemotherapy with mitoxantrone, etoposide, and cytarabine. By studying the patient myeloblasts prior to therapy, exposing them ex vivo to lenalidomide and MEC, and measuring the delta priming, Dr. Letai’s laboratory was able to predict with good precision the patient response (Garcia JS, et al. Am J Hematol. 2020;95[3]:​245–250).

“This works also in solid tumors,” Dr. Letai said. A high throughput automated microscopic assay developed in his laboratory enables testing for thousands of drugs using the same sample, provided there are enough cells to distribute to the different wells. “We can do primary discovery directly on patient primary tissues in addition to smaller, clinical diagnostic studies” (Bhola P, et al. Sci Signal. In press.).

This assay uses immunofluorescence and automated analysis to measure the release of cytochrome c. Comparing the amount of cytochrome c positive cells in drug-treated versus untreated wells informs the evaluation of whether the drugs affect the sensitivity of the mitochondria to the promiscuous BH3 Bim peptide. “We get different delta primings for each drug and rank the quality of the drugs on that basis.”

Confident in their ability to discriminate between active and inactive compounds, Dr. Letai and his colleagues took an in vivo tumor model, MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen), and tested it across a 1,600-member bioactive library.

“Our question was, ‘Can we pick the drugs that will cause remission to these breast tumors by purely functional means?’”

While most of the 1,600 drugs “did nothing, which is great,” Dr. Letai said, his laboratory identified and prioritized for testing the drugs that selectively caused apoptotic signaling in the tumor cells but not the normal cells. “We chose a handful of positive and negative controls to see if we could predict the ones that would work.”

They tested two drugs for mouse breast tumor regression—an HSP90 inhibitor and a BCR-ABL kinase inhibitor—as single agents and in combination therapy. After 14 days of treatment with the drug combination, “we essentially had a complete remission of these murine breast tumors.”

Although removal of the two agents resulted in tumor regrowth, “it’s still strong proof that we’re able to identify active drugs that can be combined into effective combination regimens,” Dr. Letai said. A more comprehensive view of testing of drugs they predicted would work and not work showed “good correlation between what we measured in terms of delta priming and what was observed in terms of treatment of the actual mouse.”

Dr. Letai said the BH3 profiling assay is not far from being able to be performed in a CLIA-certified laboratory, thanks to the expertise of pathologists Dr. Kim and Dr. Lucas. Once it is, he said, it should be transferable. “There’s no reason why it wouldn’t be transferable to another center with proper training. We don’t do any magic. These are recipes, cookbooks, protocols that could be performed by a cell biologist.”

Amy Carpenter Aquino is CAP TODAY senior editor.