Venetoclax dose-dependent increase in BCL-2 protein detected in CLL cells. (A) Mean proportions of different CLL clusters from all patients in the cohort at the indicated timepoints during treatment. (B) Graph of the results of the LDA of data from the CLL clusters from patient CLL19 across increasing doses of venetoclax. The magnitude of the projection of each marker direction onto the plane of the first 2 LDA components is represented by blue dots. This has a maximum value of 1 if the marker direction lies in the planes of the first 2 LDA components. To reveal the markers that drive the changes, the markers are ordered on the x-axis by the magnitude of this projection. The green shaded area represents the distribution of ranking curves of randomly oriented planes (see “Methods”). (C) Summary of markers ranked by contributions to the first 2 LDA components in all patients for venetoclax dose escalation (seen in panel B for patient CLL19). The higher the ranking of a marker, the more it is affected by venetoclax dose changes with 20 being the highest ranked marker and 0 the lowest. (D) Representative histograms showing BCL-2 protein expression of curated pairs of patients with CLL at screening (blue) and after 200 mg VEN treatment (red) in patient CLL13, CLL19, and CLL22 as measured by flow cytometry. The distribution of BCL-2 levels in each sample is represented by the box and whisker plots in the lower panel. The box represents the 25th, 50th, and 75th percentiles of the population with x marking the mean and the whiskers representing the minimum and maximum values (excluding outliers). (E) Fold change in BCL-2, MCL-1, and BCL-XL protein expression in samples after 200 mg treatment normalized to screening in individual patients. P values were calculated using raw data and Student 2-tailed paired t test. (F) Representative results of venetoclax sensitivity in an in vitro assay of CLL cells from patient CLL19. (G) Summary of the LC50 values of CLL cells at screening and after 200 mg venetoclax treatment. (H) Representative results of venetoclax sensitivity in an in vitro assay in nontransformed CD4⁺ T cells from patient CLL19. (I) Summary of the LC50 values of CD4⁺ T cells at screening and after 200 mg venetoclax treatment. (J) Schematic representation of the hypothesis. CLL cells express a range of BCL-2 amounts, from BCL-2⁺ to BCL-2⁺⁺⁺. CLL cells with relatively lower levels of BCL-2 (BCL-2⁺) are more sensitive to venetoclax treatment, thereby enriching for CLL cells with higher BCL-2 levels (BCL-2⁺⁺⁺) after venetoclax monotherapy. (K) Distribution of BCL-2 in CLL cells before (Scr) and 1 week after 200 mg dose of venetoclax with a dashed line indicating the threshold distinguishing the top 20% of BCL-2 expressors (BCL-2^top20) and 80% lower BCL-2 expressors (BCL-2^lower80) at screening. This threshold was then applied to each paired profile after 200 mg treatment. (L) Estimated concentration of BCL-2^top20 and BCL-2^lower80 in CLL cells in the blood at screening and after 200 mg venetoclax treatment based on the BCL-2^top20/BCL-2^lower80 threshold assigned in the screening sample. P values calculated using ratio paired t test. For box and whisker plots in panel D, outliers were omitted from the plot with an outlier factor of 1.5. Outliers represented less than 2.5% of the total population in all samples. For panels G and I, Student 2-tailed paired t test was used (patients with missing values were excluded from analysis). For panels E and L, the mean ± standard error of the mean (SEM) is shown. ∗P < .05; ∗∗∗P < .005; ∗∗∗∗P < .001. BIM, BCL-2 interacting mediator of cell death; cMyc, myelocytomatosis oncogene protein; Ikba, NK-κ-B inhibitor α; LC50, lethal concentration 50; ns, not significant; pERK, phosphorylated extracellular signal-regulated kinase; pPLCg, phosphorylated phospholipase C γ; pRb, retinoblastoma protein.