MCL1 copy number gain is a recurrent mark of venetoclax resistance in patients with chronic lymphocytic leukemia

Abstract

Abstract Introduction. The advent of the BCL2 inhibitor (BCL2i) venetoclax (VEN) has been transformative for patients with chronic lymphocytic leukemia (CLL). Beyond continuous monotherapy, time-limited combinations have become cornerstones of standard-of-care for both first-line and relapse settings. Resistance to continuous VEN therapy has been shown to result from mutations affecting the BCL2 protein binding site (Blombery Cancer Discovery 2019). We and others have previously revealed that resistance is also driven by the overexpression of the BCL2 family anti-apoptotic protein MCL1, which resulted from a copy number gain of the MCL1 gene (located at 1q21.2) in 4 cases of a limited cohort of 6 patients (Guièze Cancer Cell 2019). However, how screening these resistance marks improves patient management remains to be determined. Indeed, these have been described independently, in limited patient cohorts, and rarely across different treatment lines or regimens.Here, we report the incidence of both BCL2 mutations and MCL1 gain in a large cohort of CLL patients relapsing after VEN (RAVEN study, NCT05246345). Methods This retrospective and prospective, multicenter French study assembled a cohort of patients selected as follows: CLL/SLL, relapsing or progressing as either CLL/SLL or Richter transformation (RT), on or after VEN as the last line of therapy. Tumor samples were required to be available at the time of relapse/progression. Samples prior to VEN initiation were also collected when available.BCL2 mutations were investigated using NGS and the BCL2 G101V mutation was also screened using digital PCR. A NGS panel screening for 35 CLL drivers was also performed. MCL1 gain was evaluated using fluorescence in situ hybridization with a specific probe (MCL1/CCP1 Cytotest). Results. Between March 2022 and May 2025, 74 patients were included across 15 French centers. At inclusion, median age was 74 years (range, 37–91), and sex ratio (M/F) was 1.2. The median number of prior therapeutic lines was 3 (range, 1–6). All patients were treated with VEN as the last line of therapy; 45 (60.8%) were double-exposed to BTK and BCL2 inhibitors, and 59 (79.7%) had prior chemo-immunotherapy. IGHV status was unmutated in 58 of 62 patients. TP53 disruption was seen in 26/52 (50%) patients before VEN. VEN was given as frontline therapy in 2 (2.7%) patients and as a further therapeutic line in 72 (97.3%) patients. The VEN regimen was monotherapy for 27 (36.5%) patients and in combination with an anti-CD20 antibody for 44 (59.5%). The median duration of VEN therapy was 24 months (range, 1-67). Disease relapse/progression after VEN occurred as CLL/SLL for 58 (78.4%) patients and RT for 16 (21.6%) (including 7 with CLL samples, 5 with RT samples, and 4 with both). Relapse/progression was on-therapy for 37 patients and after the end of therapy for the remaining 37. BCL2 mutations were detected in 18/63 (28.9%) patients with a median VAF of 2.2% (range, 0.02- 30%). The most frequent mutations were G101V (12/18, 66.7%), A113G (9/17, 52.9%) and R107_R110dup (8/17, 47.1%). MCL1 gain was found in 25/69 (36.2%) patients with a median proportion of affected interphase and metaphase cells of 17% (range, 0-100%) and 9.5% (range, 2.5-100%), respectively. Neither BCL2 mutation nor MCL1 gain was observed in the 17 pre-VEN samples. Interestingly, BCL2 mutations and MCL1 gain tended to be mutually exclusive, with only 3 patients with both. Moreover, relapses associated with MCL1 gain appear to be significantly earlier than those with BCL2 mutations (median time from VEN initiation, 32 months (95%CI, 23.1-35.4) vs 42 months (95%CI, 35.1-50.6); p=0.006). Of the 2 patients treated with VEN in the frontline setting, 1 showed MCL1 gain after 41 months of VEN monotherapy. Among the patients relapsing after the end of VEN therapy, 20.6% had BCL2 mutation and 27% had MCL1 gain. Conversely, patients progressing on therapy had BCL2 mutation and MCL1 gain in 37.9 % and 46.8% of cases, respectively. Interestingly, analysis of RT tissues did not reveal the occurrence of BCL2 mutation or MCL1 gain, suggesting alternative mechanisms. Overall, median OS from time to relapse to VEN was 19.3 months (95%CI, 15.5-NA), and no impact of BCL2 mutation or MCL1 gain on survival was noted.Conclusions: Besides BCL2 mutations, our study reveals that CLL patients relapsing after VEN present with MCL1 copy number gain, which should be included in the screening for BCL2i resistance markers.