Predicting Venetoclax Sensitivity in Multiple Myeloma Using Biologic Activity Assays

Multiple myeloma (MM) is a heterogeneous cancer of long-lived antibody-secreting plasma cells in the bone marrow. Exciting discoveries in recent years have led to FDA-approval of 3 highly effective classes of drugs for MM, proteasome inhibitors, immunomodulatory agents, and antibodies, revolutionizing the treatment of this disease. However, as is the case with previous therapies, response in each individual patient is not guaranteed, and relapse in those who do respond is almost a certainty. While this cycle of response and relapse has fueled the quest for better therapeutics, it has also led to an individualized approach to treatment.

Venetoclax (ABT-199, Ven) is a specific inhibitor of the anti-apoptotic protein Bcl-2, which along with other anti- and pro-apoptotic members of the Bcl-2 family, regulates the initiation of cellular apoptosis. Dependence on the expression of Bcl-2 for survival has been demonstrated in the myeloma subtype harboring the t(11;14) translocation. However, data collected during a phase I clinical trial of Venetoclax in relapsed/refractory MM demonstrated only 40% of the t(11;14) patients treated achieved an objective response, suggesting the presence of this translocation should not be the sole determinant for treatment with Ven.

We have developed an in vitro assay to gauge patient plasma cell sensitivity to a variety of therapeutics. Here we report on the data generated with Ven. Ficoll-isolated buffy coat cells from 56 samples collected from 50 MM patients (50% t(11;14)) were treated with increasing concentrations of Ven for 24 h in order to determine the IC₅₀. The percentage of apoptotic CD45-/CD38+ plasma cells was determined via flow cytometry, staining with Annexin V-FITC. We found the 56 samples naturally separated into 3 categories: 1 - high sensitivity (IC₅₀ range 8 nM - 100 nM), 2 - intermediate sensitivity (200 nM - 450 nM), and 3 - resistant (IC₅₀ ≥ 1 μM). Samples positive for the t(11;14) translocation were found in all 3 categories, with the percentage in each proportional to sensitivity (54% in category 1, 35% in 2, and 11% in 3).

In order to verify the clinical significance of this assay, we compared our calculated IC₅₀s to the clinical response of 9 patients who received Ven treatment (6 Ven monotherapy, 2 Ven + Dexamethasone, 1 Ven + Daratumamab) after sample collection, as well as 2 patients who had progressed on Ven monotherapy prior to collection. For 5 of the 9 samples tested prior to treatment, the IC₅₀s fell in category 1 and corresponded to clinical responses of CR (1, IC₅₀ = 60 nM) and VGPR (4, IC₅₀s = 8 nM, 16 nM, 20 nM). Two of the remaining 4 samples had IC₅₀s in category 2, one patient recently began treatment and currently has stable disease (IC₅₀ = 320 nM), and the other failed to respond (IC₅₀ = 330 nM). The IC₅₀s of the last two samples were in category 3 and both patients failed to respond to Ven (IC₅₀s = 1.98 μM and 7 μM). We also tested samples from two patients after clinical progression on Ven monotherapy. One patient achieved only SD before progression at which point the sample had an IC₅₀ in category 2 (200 nM). The second patient achieved a sCR, but at the time of progression the sample fell into category 3 (12 μM). Pretreatment samples were not available for either patient. Furthermore, in 4 of the 5 patients where multiple samples were obtained and tested, sensitivity to Ven was altered by intervening therapies, suggesting testing at each relapse may be beneficial.

While our sample size is small, our data suggest patients with plasma cell sensitivity falling into category 1 may be likely to receive clinical benefit from treatment with Venetoclax, whereas the probability of those in categories 2 and 3 responding is much lower. In addition to testing apoptosis at 24 h, we have modified the iBH3 profiling technique to test drug sensitivity. This abbreviated assay determines sensitivity to Ven by measuring cytochrome C release after 3 hours of drug treatment, minimizing the loss of viability of primary cells associated with ex vivo culture. In 6 myeloma cell lines, 3 sensitive and 3 resistant to Ven, this modified approach yielded similar results as the apoptosis-based assay. Taken together these data highlight the potential benefit of these assays to both patients and clinicians in making the most informed choices on treatment plans within 24 hours of testing.