Abstract: 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 identified at progression in 7 patients, but not at study entry. It was first detectable after 19 to 42 months of therapy, and its emergence anticipated clinical disease progression by many months. Gly101Val reduces the affinity 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 first description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is sufficient to confer resistance. See related commentary by Thangavadivel and Byrd, p. 320. This article is highlighted in the In This Issue feature, p. 305

Abstract: Venetoclax is a first-in-class cancer therapy that interacts with the cellular apoptotic machinery promoting apoptosis. Treatment of patients suffering chronic lymphocytic leukaemia with this BCL-2 antagonist has revealed emergence of a drug-selected BCL-2 mutation (G101V) in some patients failing therapy. To understand the molecular basis of this acquired resistance we describe the crystal structures of venetoclax bound to both BCL-2 and the G101V mutant. The pose of venetoclax in its binding site on BCL-2 reveals small but unexpected differences as compared to published structures of complexes with venetoclax analogues. The G101V mutant complex structure and mutant binding assays reveal that resistance is acquired by a knock-on effect of V101 on an adjacent residue, E152, with venetoclax binding restored by a E152A mutation. This provides a framework for considering analogues of venetoclax that might be effective in combating this mutation.

Abstract: 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.

Abstract: 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.

Abstract: The BCL2 Gly101Val mutation may be acquired in patients with chronic lymphocytic leukaemia (CLL) treated with venetoclax (VEN), leading to reduced drug binding affinity and secondary resistance. In the majority of patients, the Gly101Val mutation is subclonal within the CLL compartment consistent with the presence of alternative resistance mechanisms in CLL cells not harboring the Gly101Val mutation. To date, two Gly101Val mutated patients have been identified with co-existing candidate resistance mechanisms in Gly101Val non-mutated cells; one with BCL-XL over-expression (Blombery et al, Cancer Discov., 2019) and another with a second subclonal candidate BCL2 resistance mutation - Asp103Tyr (Tausch et al, Haematologica 2019). Given the possibility of additional resistance mechanisms, we investigated patients with progressive CLL on VEN harboring the Gly101Val mutation for the presence of additional acquired resistance mutations in BCL2. Ten patients with progressive CLL on VEN with Gly101Val mutations were identified by sensitive allele-specific droplet digital PCR. To further assess for alternative BCL2 mutations in this cohort we performed ultra-deep amplicon-based next generation sequencing (NGS) (median depth ~50,000X) targeting BCL2. An amplicon variant caller (Canary) specifically designed for low level variant calling was used (Doig et al, BMC Bioinformatics, 2017). To achieve enhanced specificity we performed digital NGS with PCR error-correction using unique molecular indexes (UMI) (QiaSEQ Targeted DNA Panel). Given the high GC content of BCL2 we also used hybridization-based NGS using a custom targeted panel (Blombery et al, BJH 2017) combined with a sensitive unpaired variant caller (GATK4/Mutect2). In 7/10 (70%) patients, BCL2 mutations in addition to the Gly101Val were detected. Recurrent mutations (detected in more than one patient) were Asp103Tyr, Asp103Glu, Arg107_Arg110dup, and Val156Asp. All additional recurrent mutations were confirmed to be absent prior to commencing VEN (sensitivity 1% variant allele frequency[VAF]). Phase-analysis of NGS reads was consistent with the presence of the additional recurrent mutations on different alleles (and therefore cells, assuming heterozygosity) to both each other and to Gly101Val. Multiple addition recurrent mutations were observed in patients in the cohort with one patient harboring three recurrent mutations in addition to the Gly101Val (Asp103Tyr, Asp103Glu, Val156Asp). In multiple patients in the cohort, the VAF of non-Gly101Val mutations exceeded that of the Gly101Val mutation. Importantly, in all patients a significant (albeit variable) proportion of CLL cells were found to be BCL2 wild-type consistent with the presence of as yet unidentified resistance mechanisms unrelated to BCL2 mutations. In one patient, two additional non-recurrent mutations were observed (Ala113Gly and Arg129Leu) in addition to Gly101Val and Val156Asp. Again, all four mutations in this patient were observed to be in mutually exclusive NGS reads. Strikingly, all of the recurrent acquired BCL2 mutated residues identified in our cohort are situated in the BCL2 binding groove that binds VEN (Figure 1). The Asp103 codon in the P4 pocket is critical for VEN binding through hydrogen bonding between its sidechain and the azaindole moiety of VEN. The Asp103Glu mutation is noteworthy given that the equivalent residue to Asp103 in BCL-XL is a Glu, which reduces VEN binding to BCL-XL. The Val156 mutation situated at the base of the P2 pocket is close to the chlorophenyl moiety of VEN and a change to Asp in this position may disrupt VEN binding. Ongoing binding experiments and modeling in cellular systems will further elucidate the mechanism and contributions of these new recurrent mutations to VEN resistance. In summary, we have extended the landscape of acquired candidate resistance mutations occurring in patients treated with VEN to include four novel recurrent BCL2 mutations. Moreover, our data are consistent with the emerging observation of multiple acquired resistance mechanisms operating in different CLL cells in a single patient contributing to an "oligoclonal" pattern of clinical relapse on VEN therapy. Figure 1 - BCL2 protein structure surface bound to venetoclax (VEN) in orange. The Asp103Tyr, Asp103Glu and Val156Asp mutation sites are shown in red and Arg107_Arg110dup region in blue Disclosures Blombery: Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Seymour:Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. 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; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; BeiGene: Research Funding.