Multiple BCL2 mutations cooccurring with Gly101Val emerge in chronic lymphocytic leukemia progression on venetoclax

Abstract

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Figure 1.

BCL2 mutations in patients with progressive CLL on venetoclax. (A) BCL2 mutations in a cohort of patients with CLL progression on venetoclax. Patients are ordered in descending Gly101Val cancer cell fraction (CCF). CCF was determined as (VAF/disease burden determined by flow cytometry) × 2 (assuming heterozygosity). Area of blue circles is proportional to CCF mutated. The top row shows the total CCF harboring BCL2 mutations (the sum of individual CCF and assumes occurrence in mutually exclusive cells). Mutations detected were c.302G>T, p.(Gly101Val); c.302_303delinsTT, p.(Gly101Val); c.307G>T, p.(Asp103Tyr); c.308A>T, p.(Asp103Val); c.309C>A, p.(Asp103Glu); c.319_330dup, p.(Arg107_Arg110dup); c.338C>G, p.(Ala113Gly); c.386G>T, p.(Arg129Leu); c.467T>A, p.(Val156Asp); BCL2 NM_000633.2. Patient CLL6 had both a c.302G>T and a complex variant (c.302_303delinsTT) leading to a p.(Gly101Val) in different reads; the Gly101Val area is the sum of the 2 CCFs for this patient. (B) Structure of BCL2 protein with venetoclax bound (PDB ID 6O0K) illustrating the positions of the mutated residues Asp103, Val156, Arg107 to Arg110, Ala113, and Arg129.

Figure 2.

Venetoclax/BIM binding characteristics and in vitro sensitivity of BCL2 Asp103Glu mutation. (A) Impact of Asp103Glu on the ability of BCL2 to bind BH3 ligands. (Left) BIMBH3 binding. A total of 0 to 40 nmol/L mutant BCL2 was used as an analyte against the BIMBH3 peptide immobilized on a surface plasmon resonance (BIAcore) sensor chip. The raw response (RU) curves (colored curves) from a representative experiment were fitted to 1 site-specific kinetic model (black curves) to derive on and off rates (supplemental Table 2), and calculate K_D values for interactions with Asp103Glu. (Right) Steady-state competition of various venetoclax concentrations (0-50 nmol/L) prebound to Asp103Glu (0-250 nmol/L), competing against a BIMBH3 immobilized chip. Fitted data were used to derive the K_I for venetoclax binding to Asp103Glu. Data are representative of 3 independent experiments, reporting means ± 1 SD. (B) Expression of BCL2 Asp103Glu in RS4;11 (top) or KMS-12-PE (bottom) cell lines reduces sensitivity to venetoclax (left) but not to navitoclax (right). Each of these mutants or wild-type (WT) BCL2 were expressed and the in vitro sensitivities to venetoclax (0-10 μmol/L) or to navitoclax (NAV; 0-10 μmol/L) measured 24 hours later. Data represent means ± 1 SD of at least 3 independent experiments. K_D,equilibrium binding constant for BIMBH3 peptide, from direct binding experiments; K_I, fitted equilibrium binding constant for venetoclax, from steady-state competition experiments.