Downregulation of BAX and PUMA and upregulation of MCL-1 in vitro and in vivo. (A) Top: IC₅₀ values for VEN in 10 sensitive (blue) vs VEN-resistant (red) B-cell lymphoma cell lines: WSU-NHL, OCI-LY-19, DOHH-2, DB, KARPAS-422, HBL-1, 697, P30-OH-KUBO, Nalm6, and OSU. Middle: densitometric analyses of immunoblots of BCL2 proteins (supplemental Figure 1A-B). Mean ± SD of at least 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, compared with parental (VEN naïve) cells, Students t test. Lower: results from WES. Genomic alterations are annotated according to the color panel below the image. (B) Immunohistochemistry of BAX in 6 primary CLL samples. Scale bar, 100 μm. Pictures 1 through 5: lymph node sections posttherapy; picture 6: bone marrow post–VEN therapy. Relative MCL1 (left panel) and BBC3 (PUMA) (right panel) and BAX mRNA expression level for CLL patients 1 and 2 pre- (blue) and post (red)-VEN therapy determined by bulk 3′RNA-seq. (C) Results for Bax, Bbc3, and Mcl1 guide RNAs from CRISPR/Cas9-Screen in murine lymphoma cell line after 28 days with/without VEN (10 nM). (D) Immunoblot for MCL1, BCL-xL, BCL2, PUMA, and BAX in 2 T-PLL patients before and after VEN resistance. (E) Immunoblot for MCL1, BAK1, PUMA, and BAX in 3 sensitive and S63845-resistant B-cell lymphoma cell lines, respectively. (F) Densitometric analyses of immunoblots against MCL1, BAK1, PUMA, and BAX normalized to β-actin. Data illustrated as mean ± SD of at least 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .001, compared with parental (blue) cells, Student t test. 3′RNA-seq, 3’RNA-sequencing; IC₅₀, median inhibition concentration.