The BCL2 inhibitor venetoclax induces high rates of durable remission in patients with previously treated chronic lymphocytic leukemia (CLL). However, despite continuous daily treatment, leukemia recurs in most patients. To investigate the mechanisms of secondary resistance, we analyzed paired pre-venetoclax and progression samples from 15 patients with CLL progression enrolled on venetoclax clinical trials. The novel Gly101Val mutation in BCL2 was identifi ed at progression in 7 patients, but not at study entry. It was fi rst detectable after 19 to 42 months of therapy, and its emergence anticipated clinical disease progression by many months. Gly101Val reduces the affi nity of BCL2 for venetoclax by ∼180-fold in surface plasmon resonance assays, thereby preventing the drug from displacing proapoptotic mediators from BCL2 in cells and conferring acquired resistance in cell lines and primary patient cells. This mutation provides new insights into the pathobiology of venetoclax resistance and provides a potential biomarker of impending clinical relapse. SIGNIFICANCE: Why CLL recurs in patients who achieve remission with the BCL2 inhibitor venetoclax has been unknown. We provide the fi rst description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is suffi cient to confer resistance.

Venetoclax induces high rates of response (~80%), including complete remissions (CR) in patients with heavily pre-treated chronic lymphocytic leukemia (CLL) through inhibition of BCL2. Despite achieving deep and durable responses, most patients will eventually experience disease progression on treatment. The molecular mechanisms that mediate clinical resistance to venetoclax in vivo are largely unknown. From a cohort of 67 relapsed CLL patients (Anderson et al, Blood 2017; 129:3362-3370) treated with venetoclax on three early phase clinical trials, we performed focussed genomic evaluation in those with CLL-type progressions (as opposed to large cell Richter's transformation). Targeted amplicon next generation sequencing of a panel of 33 genes recurrently mutated in lymphoid malignancy was performed where suitable pre- and post-progression samples were available. Twenty-one patients experienced CLL progression after a median of 36 months (range 6 - 73). Fifteen patients had paired samples for detailed analyses. A single heterozygous nucleotide variant was detected in BCL2 (NM_000633.2:c.302G>T, p.(Gly101Val)) in progression samples in 7 of 15 patients (Fig 1A). Further investigation using a highly sensitive (limit of detection 0.01%) and specific droplet digital PCR (ddPCR) assay indicated that the Gly101Val mutation was first detected at low variant allele fraction after 19-42 months on venetoclax, up to 25 months earlier than when standard disease progression criteria were met. The Gly101Val was not detected prior to venetoclax treatment in this cohort and was not detected in a series of samples from patients treated at our institution who had not received venetoclax (CLL [n=74], NHL [n=198], myeloma [n=103]) nor has it been described in cancer (COSMIC) or population (gnomAD) databases. To investigate whether Gly101Val directly causes resistance to venetoclax, we expressed it in two B-lineage cell lines (RS4;11 and KMS-PE-12). Gly101Val cells were ~30-fold less sensitive to venetoclax than cells expressing wild-type (WT) BCL2. The Gly101Val mutation conferred a selective advantage during continuous exposure to sublethal concentrations of venetoclax in 3-week cultures. The same phenomena was observed with primary patient Gly101Val mutant cells in both short-term survival assays and when cultured in a bone marrow stromal model (Thijssen et al, Haematologica 2015;100:302-6). On stroma, primary cells bearing the Gly101Val mutation demonstrated markedly increased resistance to venetoclax with concentrations higher than achievable clinically in vivo. In the absence of venetoclax, the Gly101Val mutant demonstrated preserved normal function by protecting cell lines from apoptosis induced by cytotoxics with similar effectiveness to WT BCL2. In binding assays, the capacity for venetoclax to compete in vitro with BIM for binding to the Gly101Val mutant was markedly reduced (~180-fold) compared to WT BCL2. This is most likely explained by the presence of a bulkier valine residue in a region juxtaposed to the venetoclax binding groove (Fig 1B). In cell-based assays, whilst venetoclax readily displaced BAX and BAK from WT BCL2 it was ineffective when these pro-apoptotic molecules were bound to the Gly101Val mutant. We observed that not all CLL cells at progression carried the Gly101Val mutation. One patient harbored distinct subclones with and without the BCL2 Gly101Val mutation at progression. The subclone with exclusively WT BCL2 was observed to have elevated BCL-xL by mass cytometry (CyTOF), while the Gly101Val clone had minimal BCL-xL expression. Together these data indicate that whilst the Gly101Val mutation is sufficient to enable clinical resistance to venetoclax, alternative mechanisms may also mediate resistance in the same patient. In conclusion, we have identified and functionally characterized a novel recurrent BCL2 mutation (Gly101Val) emerging in a cohort of patients with CLL-type progressions treated with venetoclax. The BCL2 Gly101Val impairs binding of venetoclax to BCL2, confers resistance to venetoclax in both patient leukemia cells and engineered cell lines, and provides a selective growth advantage over wild-type cells when maintained in the presence of the drug in vitro. This mutation provides new insights into the pathobiology of venetoclax resistance and provides a potential biomarker of impending clinical relapse. Figure 1 Figure 1. Disclosures Anderson: Walter and Eliza Hall: Employment, Patents & Royalties; AbbVie, Inc: Research Funding; Genentech: Research Funding. Gong:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Thijssen:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Birkinshaw:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Teh:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Xu:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Flensburg:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Lew:Walter and Eliza Hall: Employment, Patents & Royalties. Majewski:Abbvie: Patents & Royalties: I am an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Gray:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Tam:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding. Seymour:AbbVie: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Consultancy. Czabotar:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Huang:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Roberts:Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax; AbbVie: Research Funding; Genentech: Research Funding; Janssen: Research Funding.

The BCL2 inhibitor venetoclax has complete response rates of up to 50% in chronic lymphocytic leukemia patients, but secondary resistance reflecting acquired mutations in BCL2 can lead to treatment failure. Blombery et al report that an unexpectedly large number of patients carry multiple BCL2 mutations with subclonal variation in their occurrence.

Highly active BTK inhibitors (BTKis) and the BCL2 inhibitor venetoclax have transformed the therapeutic landscape for chronic lymphocytic leukemia (CLL). Results of prospective clinical trials demonstrate the efficacy of venetoclax to salvage patients with disease progression on BTKis, but data on BTKi therapy after disease progression on venetoclax are limited, especially regarding durability of benefit. We retrospectively evaluated the records of 23 consecutive patients with relapsed/refractory CLL who received a BTKi (ibrutinib, n = 21; zanubrutinib, n = 2) after stopping venetoclax because of progressive disease. Median progression-free survival (PFS) and median overall survival after BTKi initiation were 34 months (range, <1 to 49) and 42 months (range, 2-49), respectively. Prior remission duration ≥24 months and attainment of complete remission or undetectable measurable residual disease on venetoclax were associated with longer PFS after BTKi salvage (P = .044 and P = .029, respectively). BTKi therapy achieved durable benefit for patients with the BCL2 Gly101Val venetoclax resistance mutation (estimated 24-month PFS, 69%). At a median survivor follow-up of 33 months (range, 2-53), 11 patients remained on BTKi and 12 had stopped therapy because of disease progression (n = 8) or toxicity (n = 4). Our findings indicate that BTKi therapy can provide durable CLL control after disease progression on venetoclax.

One of the most common secondary resistance mechanisms to ibrutinib (IBR, a first-generation, irreversible Bruton's tyrosine kinase [BTK] inhibitor) in CLL is the development of mutations in BTK involving Cys481, leading to impaired drug binding (Woyach et al, NEJM 2014; Furman et al, NEJM 2014). The mechanisms of resistance to second generation BTK inhibitors are currently unknown. We aimed to assess the spectrum of acquired BTK mutations in patients with CLL progression on zanubrutinib (ZANU), a second-generation, irreversible inhibitor of BTK. We identified 38 CLL patients, treated with ZANU on clinical trials (NCT02343120, NCT02569476, NCT03336333, NCT02795182) at three centres, for whom serial samples were available. Four of 38 patients (10.5%) had CLL progression on ZANU (time to progression 5, 26, 29 and 48 months) and underwent amplicon next generation sequencing (NGS) of BTK (exon 11, 15, 16) and PLCG2 (exon 16, 19-20, 24, 27-28). Remarkably, we detected a BTK kinase domain mutation, BTK Leu528Trp (NM_000061.2:c.1583T>G), in all four patients progressing on ZANU. In addition, all four patients had detectable Cys481 mutations at lower variant allele frequency (VAF) than the BTK Leu528Trp (median BTK Leu528Trp 34.9% vs BTK Cys481 9.1%). Analysis of sequence reads from amplicon NGS and RNA-sequencing data demonstrated that BTK Leu528Trp and BTK Cys481 mutations were present on different alleles. Assessment of the BTK Leu528Trp and BTK Cys481 mutations with high sensitivity droplet digital PCR (ddPCR) confirmed the absence of both mutations prior to ZANU exposure in all patients (sensitivity 0.1% VAF). Longitudinal analysis of the four patients with the BTK Leu528Trp mutation demonstrated the appearance of the Leu528Trp coincident with rising measurable disease and subsequent clinical CLL progression. We then went on to test patients on ZANU without disease progression but with persistent measurable disease (n=34) by ddPCR and detected three further patients harbouring low level BTK Leu528Trp mutations (VAF <1%). These mutations were first detected after a median of 40 months on ZANU therapy. The BTK Leu528Trp mutation has been described only once previously in a patient in the context of IBR resistance (who transformed with Richter's syndrome) where it co-occurred with Cys481 mutations (Maddocks et al, JAMA Oncol 2015). As the prevalence of BTK Leu528Trp among progressive disease samples in our cohort exceeds all prior reports in IBR-treated patients, we sought to further understand the specificity of BTK Leu528Trp for ZANU progression. Targeted sequencing in a cohort of 49 patients progressing on IBR from the European Research Initiative on CLL (ERIC) did not detect the BTK Leu528Trp in any patients (sensitivity 1% VAF). We went on to perform biochemical and cellular studies on the BTK Leu528Trp mutation. Assessment of enzymatic activity of BTKLeu528Trp demonstrated a significant loss of activity compared to both BTKWT and BTKCys481Ser. This was further confirmed by assessing BTK autophosphorylation in HEK293 cells. Autophosphorylation at BTK Tyr223 was markedly reduced in HEK293 cells stably expressing BTKLeu528Trp compared to both BTKCys481Ser and BTKWT. In addition, a crystal structure of apo-BTKLeu528Trp was solved to understand effects of BTKLeu528Trp on ZANU binding to BTK. The alignment of the crystal structure of apo-BTKLeu528Trp with that of BTKWT-ZANU or the modeled structure of BTK-ATP suggested potential steric clashes between BTKLeu528Trp and ZANU (Figure 1A), as well as BTKLeu528Trp and ATP (Figure 1B). In conclusion, we have described the novel enrichment of BTK Leu528Trp mutations occurring in patients with CLL progressing on ZANU and both structural and experimental data consistent with this mutation resulting in interference with both ATP and ZANU binding to BTK. These findings emphasize the potential for agent-specific resistance mutations with second generation BTK inhibitors and the need to include these mutations in diagnostic screening for BTK resistance in the clinic. SH/CPST co-first authors, CT/PB co-senior authors Disclosures Handunnetti: Abbvie: Other: Travel Grant; Gilead: Honoraria. Zhou:Beigene: Employment. Sun:Beigene: Employment. Xing:Beigene: Employment. Zhang:Beigene: Employment. Guo:Beigene: Employment. Sutton:Abbvie: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Ghia:Dynamo: Consultancy, Honoraria; ArQule: Consultancy, Honoraria; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Acerta/AstraZeneca: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy; Novartis: Research Funding; Juno/Celgene: Consultancy, Honoraria. Scarfo:AstraZeneca: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Seymour:Takeda: Consultancy; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; Janssen: Research Funding; BeiGene: Research Funding. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Liu:Beigene: Employment. Cheah:Roche, Janssen, MSD, Gilead, Loxo Oncology, Acerta, BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Abbvie: Research Funding; Roche: Other: Travel expenses. Tam:Janssen: Honoraria, Research Funding; BeiGene: Honoraria; Pharmacyclics LLC, an AbbVie company: Honoraria; Roche: Honoraria; Novartis: Honoraria; AbbVie: Honoraria, Research Funding. Blombery:Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy.

The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment for CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (³ 1 of neutropenia, thrombocytopenia, anemia) lasting ³4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (tMNs). tMNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with tMNs. Single cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.

No abstract

The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to BTK inhibitors and BCL2 inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of eight patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.