Fig 2: PI3Kδ inhibitors increase AID expression and genomic instability in human B cellsa,b AICDA mRNA relative expression in the human MEC1 or Mino B cell lines treated with the indicated inhibitors (1 μM). Data are mean ± s.d. (n=3 technical replicates, n = 3 biological replicates). ** P ≤ 0.01, *** P ≤ 0.001; two-tailed Student’s t-test. c, Western blot for AID protein expression in MEC1 and Mino B cell lines (n = 4 biological replicates). For gel source data, see Supplementary Figure 1. d, Translocation junction frequency in AID on-target (D2-21 and Sμ-J4 region in the IGH locus) and AID off-target sites in MEC1 B cell line treated with 1 μM DMSO, idelalisib or duvelisib. AID knock-out (KO) MEC1 cells were generated by CRISPR/Cas9 mediated deletion (Extended Data Fig. 8e). Data are from pooled HTGTS libraries from 3 independent experiments. ** FDR ≤ 0.01, *** FDR ≤ 0.001. Statistical analysis in Methods. e, Mutation frequency calculated on the regions downstream TSS for IRF4 and PIM1, and the PAX5 enhancer region in CLL patients treated with idelalisib or ibrutinib. Untreated CLL patients are used as control. Boxplots indicate cumulative frequencies of C-to-T or G-to-A transition mutation in DNA samples collected before (pre) and after (post) treatment in each patient (control n=8, idelalisib n=10, ibrutinib n=10, Supplementary Table 6). Whiskers extend to a maximum of 1.5X interquartile range (IQR) beyond the box. * P < 0.05, paired samples two-tailed Student’s t-test.

Fig 3: Ibrutinib increases AID expression and the frequency of translocations to AID on- and off-target sites in mouse activated B cellsa, AKT phosphorylation was detected by Western Blot in mouse activated B cells treated with DMSO, idelalisib, duvelisib or ibrutinib (1 μM) for the indicated time points (n = 2 biological replicates). For gel source data, see Supplementary Figure 1. b, MEC1 and Mino human lymphoma cells were treated with the indicated inhibitors (1 μM) and AKT phosphorylation was evaluated by Western Blot (n = 3 biological replicates). c, Viable cells were counted at the indicated time points by Trypan Blue exclusion in activated B cells treated with DMSO or ibrutinib (1 μM). Data are expressed as mean ± s.d. (n=3). P values calculated by two-tailed Student’s t-test. d, Western blot for AID protein expression in activated B cells treated with 1 μM ibrutinib. The DMSO panel from Fig. 1a is shown for comparison (n=3 biological replicates). e, Aicda mRNA levels analyzed by qRT-PCR in activated B cells treated with DMSO or ibrutinib (1 μM). Data are expressed as mean ± s.d. (n = 3 technical replicates, n = 3 biological replicates). * P < 0.05, two-tailed Student’s t-test. f, IgG1 CSR in activated B cells analyzed by flow cytometry. Data are expressed as mean ± s.d. (n = 3 biological replicates). * P < 0.05, *** P ≤ 0.001, two-tailed Student’s t-test. g, Histograms showing translocation junction frequency to AID on-target (Igh and Igk loci indicated with a #) and off-target sites in activated B cells treated with ibrutinib. Targets are divided based on the statistical significance of increased junctions frequency compared to DMSO treatment (FDR < 0.001 on the left; FDR < 0.01 on the right). Statistical analysis is indicated in the Method. For each treatment, data are from pooled HTGTS libraries of similar size from independent experiments as indicated in Supplementary Table 1 and 5. h, Venn diagrams showing the fraction of AID off-target sites shared in activated mouse B cells treated with ibrutinib, idelalisib or duvelisib.

Fig 4: Ibrutinib increases AID expression and the frequency of translocations to AID on- and off-target sites in human B cellsa, AICDA mRNA levels analyzed by qRT-PCR in MEC1, Mino, JeKo-1 and GM06990 cell lines after treatment with DMSO or ibrutinib. Data are expressed as mean ± s.d. (n = 3 technical replicates, n = 3 biological replicates). * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001, two-tailed Student’s t-test. b, Western blot for AID protein expression in MEC1, Mino, JeKo-1 and GM06990 B cell lines treated with the indicated inhibitors (1 μM) for 48 h. For comparison, Mino and GM06990 panels correspond to the panel shown in Fig. 4c and Extended Data Fig. 6d, respectively, with the addition of the ibrutinib lane (n = 3 biological replicates). For gel source data, see Supplementary Figure 1. c, Histograms showing translocation junctions frequency to AID on-target and off-target sites in MEC1 and JeKo-1 B cell lines treated with ibrutinib. For each treatment, data are from pooled HTGTS libraries of similar size from three independent experiments (Supplementary Table 1 and 4). Significance is calculated as FDR in ibrutinib over DMSO treated human B cells. Statistical analysis is indicated in the Method. *** FDR ≤ 0.001. d, Mutation frequency of control non AID off-targets: intragenic regions for PAX5, MTFR1, BCAT1, CLMN, UBXN7, ERICH6B, and random intergenic regions on Chr. 1, Chr. 8, and Chr. 18 were targeted re-sequenced in CLL patients untreated or treated with idelalisib and ibrutinib. Boxplots indicate cumulative frequencies of C-to-T or G-to-A transition mutation in DNA samples collected before (pre) and after (post) treatment in each patient (control n=8, idelalisib n=10, ibrutinib n=10, Supplementary Table 6). Whiskers extend to a maximum of 1.5X interquartile range (IQR) beyond the box. P values calculated by paired samples two-tailed Student’s t-test.

Fig 5: Phosphatidylinositol 3-Kinase (PI3K)δ blockade increases AID expression and CSR in activated mouse B cellsa, Western blot for AID protein from B cells treated with the indicated inhibitors (1 μM) (n = 3 biological replicates). For gel source data, see Supplementary Figure 1. b, Aicda mRNA levels were analyzed by qRT-PCR. Data are expressed as mean ± s.d. (n = 3 biological replicates).. ** P ≤ 0.01, *** P ≤ 0.001, two-tailed Student’s t-test from idelalisib or duvelisib vs DMSO-treated B cells. c, GRO-Seq profiles of Aicda gene in B cells at 48 h after activation (n = 2 biological replicates). Blue profiles: sense transcription, Red profiles: antisense transcription. d, Quantification of GRO-Seq sense and antisense reads per kilobase per million mapped reads (RPKM) in the Aicda gene, **P ≤ 0.01,***P ≤ 0.001, multiple test adjusted. e, IgG1 CSR in activated B cells. Data are expressed as mean ± s.d. (n = 3 biological replicates). ** P ≤ 0.01, *** P ≤ 0.001, two-tailed Student’s t-test from idelalisib or duvelisib vs DMSO treated B cells