Abstract
In this issue, Blombery and colleagues show that the chronic lymphocytic leukemia (CLL) cells bearing Gly101Val mutation confer resistance to venetoclax by reducing the affinity of BCL2 for venetoclax by 180-fold in cell lines and in patient cells. Detection of this mutation provides a potential biomarker for impending disease progression and an opportunity for targeted and combinational therapy to treat CLL.
See related article by Blombery et al., p. 342.
Venetoclax is a highly selective BCL2 inhibitor that disrupts the interaction of this protein with BH3 domain proteins thereby permitting apoptosis. Venetoclax as monotherapy or in combination with rituximab has been approved for marketing in patients with chronic lymphocytic leukemia (CLL) with or without 17p deletion and in patients who have received at least one prior therapy (1, 2). The overall response rate of venetoclax in CLL as monotherapy was 79% with 20% of these being complete responses. However, in the setting of relapsed disease, although remissions are durable, relapses virtually always are eventually observed, suggesting an acquired mechanism of resistance. Venetoclax has also been tested to treat other malignancies including acute myeloid leukemia, multiple myeloma, non-Hodgkin's lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, marginal zone lymphoma, and Waldenström macroglobulinemia (3). Indeed, venetoclax demonstrates some activity in all these diseases either as monotherapy or combination with other treatments, making any finding relevant to resistant mechanisms quite broad to many hematologic malignancies. Therefore, it is of high importance to further explore pathways and mechanisms involved in venetoclax resistance, for which CLL serves as an outstanding patient in vivo model to acquire serial samples over time and analytically examine serial tumor cells for relevant mechanisms of resistance. Blombery and colleagues report that Gly101Val point mutation in BCL2 reduces venetoclax binding to the hydrophobic groove and is sufficient to confer resistance (3) to venetoclax.
Studies have reported acquired mutations in BCL2 that lead to resistance in CLL (4, 5). However, using 67 patients from three clinical trials with venetoclax, Blombery and colleagues performed a genomic evaluation and discovered a recurrent novel BCL2 mutation (Gly101Val) in patients with relapsed CLL treated with this agent (6). This mutation was not detected at treatment initiation, but was found at disease progression. In this study, 21 patients treated with venetoclax had CLL-type progression and 18 developed Richter transformation. Fifteen of these 21 patients with CLL had paired samples (pre- and post-progression), and targeted amplicon next-generation sequencing was performed on these samples. Assessing the entire coding region of BCL2, Blombery and colleagues found a single variant in 4 patients and they studied the emergence of this mutation in serial samples of these patients. The authors also did not find BCL2 Gly101Val in general population or in patients with other B-cell malignancies who had not received treatment with venetoclax.
This study shows that CLL cells harboring Gly101Val are less sensitive to both the BCL2 inhibitors venetoclax and ABT-737 killing in vitro. ABT-737 targets BCL2/BCL-XL and induces apoptosis via BAK/BAX. Additionally, to confirm that Gly101Val mutation alone is sufficient to cause resistance, the authors overexpressed this mutation in human B-cell lines RS4;11 and KMS-12PE. Mutated cell lines were 30-fold less sensitive to venetoclax than the wild-type (WT). These data suggest that the effect is highly specific for BCL2 inhibition by venetoclax. To address how venetoclax competes with proapoptotic proteins such as BIM, BAX, and BAK for BCL2 binding, the authors used competition surface plasmon resonance experiments. From these experiments, the authors were able to see that BIM binding to BCL2 was unaffected by the addition of venetoclax in both WT and Gly101Val-mutant cell lines. Interestingly, venetoclax could not displace the other proapoptotic proteins BAX and BAK when they were bound to Gly101Val or Phe104Leu mutants. To complement these data, the authors exposed the WT and mutant cells to venetoclax continuously in long-term culture and found the outgrowth of mutant-bearing clones. Taken together, these data suggest that Gly101Val mutation is sufficient to confer venetoclax resistance in patients with CLL (Fig. 1).
To understand whether Gly101Val mutation interacts with other factors in the microenvironment and how these factors could protect CLL cells from venetoclax, the authors used an established coculture model (7). CD40 stimulation in CLL cells inhibits cell apoptosis and induces resistance to venetoclax (7, 8). Therefore, the authors cocultured CLL cells from progression and from venetoclax-naïve patients on CD40L-expressing stromal cells. Within a week in culture, the venetoclax-naïve CLL cells became less sensitive to venetoclax. After 1 week in coculture, the cells became highly resistant to venetoclax. These data suggest that the microenvironmental factors play a major role in resistance mechanisms. To check additional resistance mechanisms that coexist with Gly101Val mutation, the authors performed cytotoxicity experiments in cell lines and mass spectroscopy in patient samples. First, they looked at the subclonality for the Gly101Val mutation in patient samples and observed significant subclonality in 2 patients (70% in CLL3 and 25% in CLL2). For example, even though the patient had 25% of resistant leukemic cells with Gly101Val mutation, the LC50 shift was significantly higher than the other samples and cell lines. Therefore, they investigated the BCL2 family expression by mass spectrometry for this sample and found the expression of another prosurvival protein, BCL-XL. Then, they sorted the cells based on BCL-XL expression levels and found that Gly101Val mutation was confined to the cell population with low BCL-XL expression levels. This indicates that two populations from same patient have distinct resistance mechanisms and that these BCL-XL–expressing cells were sensitive to the BCL-XL–specific inhibitor A1331852. Altogether, these data suggest that additional resistance mechanism coexist with Gly101Val mutation.
Venetoclax is a targeted therapy and has shown promising results in clinical trials for a variety of hematologic malignancies, but most dramatic to this point in CLL. It is of great interest that the pattern of resistance to venetoclax in CLL mimics one similar to irreversible inhibitors of BTK, where mutation in the C481-binding site of ibrutinib results in resistance to this treatment (9, 10). Notably as well, both the BCL2 mutation described herein and also the C481S mutation seen with ibrutinib are not typically observed in cases of Richter transformation, suggesting distinct mechanism of escape from targeted therapy for each. As with the C481S mutation identified with irreversible BTK inhibition by ibrutinib, the Gly101Val mutation with BCL2 also provides a method to identify early evidence of molecular relapse before actual clinical progression occurs. Hence, both of these resistant mutations provide an avenue to employ genomic studies in the blood to identify early relapse without the need of imaging and potentially direct early targeted therapy toward resistant clones before patients become overtly ill with disease. This approach as part of clinical trials is currently ongoing in C481S-mutant CLL following ibrutinib therapy (NCT03513562 and NCT03400176) and could potentially be relevant for venetoclax as well. In addition, given the emergence of two distinct targeted therapy escape mechanisms that converge on the development of a new mutation that impairs drug action, it will be key to identify the pathogenesis of how this develops in CLL and ultimately develop strategies to prevent this in at-risk patients. Moving forward, it will also be of interest to see if the BCL2 Gly101Val mutation identified by Blombery and colleagues exists in other hematologic malignancies and solid tumors which are responsive to venetoclax. The article highlights the importance of studies of resistance mechanisms of effective targeted therapies such as venetoclax, as their identification early in the development process has the potential to lead to strategies to overcome them. Blombery and colleagues are to be commended in this regard for their commitment to the development of venetoclax and acquired resistance to this agent in CLL.
Disclosure of Potential Conflicts of Interest
J.C. Byrd is a consultant/advisory board member for Pharmacyclics. No potential conflicts of interest were disclosed by the other author.
Acknowledgments
This work was supported by grants from the NIH (R35 CA197734), The Connie Brown CLL Foundation, the Kevin Sullivan Foundation, the D. Warren Brown Foundation, and the Four Winds Foundation.