Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells

Abstract

Activation-induced cytidine deaminase (AID) is a B-cell-specific enzyme that targets immunoglobulin genes to initiate class switch recombination and somatic hypermutation1. In addition, through off-target activity, AID has a much broader effect on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in the development and progression of lymphoma2. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation3. The phosphatidylinositol 3-kinase δ (PI3Kδ) pathway regulates AID by suppressing its expression in B cells4. Drugs for leukaemia or lymphoma therapy such as idelalisib, duvelisib and ibrutinib block PI3Kδ activity directly or indirectly5,6,7,8, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both of these effects were completely abrogated in AID-deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumours in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IGH and AID off-target sites in human chronic lymphocytic leukaemia and mantle cell lymphoma cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased somatic hypermutation in AID off-targets. In summary, we show that PI3Kδ or Bruton’s tyrosine kinase inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism. This effect should be carefully considered, as such inhibitors can be administered to patients for years.

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Figure 1: PI3Kδ blockade increases AID expression and CSR in activated mouse B cells.
Figure 2: PI3Kδ blockade increases the frequency of genome-wide chromosomal translocations in activated mouse B cells.
Figure 3: Enhanced genomic instability in mouse B cells by PI3Kδ blockade.
Figure 4: PI3Kδ inhibitors increase AID expression and genomic instability in human B cells.

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Acknowledgements

We thank K. Okkenhaug and F. Garcon for providing wild-type and mutated PI3Kδ constructs, F. Zhang for providing CRISPR/Cas9 plasmids. We thank M. Fleming and M. M. Awad for critically reading the manuscript. This work was supported by NIH grants R01 CA196703-01 to R.C.; 1U10CA180861-01 to C.J.W., R01AI077595 to F.W.A; Associazione Italiana per la Ricerca sul Cancro grant IG-12023 to R.C. and MFAG 10708 to M.C.; Worldwide Cancer Research grant 12-0216 to R.C.; Compagnia di San Paolo-Comitato Gigi Ghirotti to M.C.; American Cancer Society Grant RSG-13-002-01-CCE to J.R.B.; T.C.C. is supported by a National Research Foundation of Korea(NRF) fellowship; L.S.Y. was a Cancer Research Institute postdoctoral fellow; F.M. was a Lymphoma Research Foundation postdoctoral fellow; J.S. is a recipient of a PhRMA Foundation Research Fellowship; C.J.W. is Scholar of the Leukemia and Lymphoma Society; F.W.A. is an investigator of the Howard Hughes Medical Institute. A. W. is supported by the intramural program of NHLBI, NIH.

Author information

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Authors

Contributions

M.C. and C.P. planned and performed experiments. Q.W. performed analysis of the data. T.-C.C., F.L., T.P., C.A., C.V., and M.G. performed experiments. E.K. and R.B contributed to CRISPR/Cas9 experiments. F.M. performed GRO-seq experiments. L.-S.Y. performed the mutational analysis on VB1-8 exon. A.W., S.N.K, J.R.B, J.S., and C.J.W. provided clinical samples. F.W.A contributed to the design and interpretation of the experiments, and contributed to writing the manuscript. R.C. conceived and designed all the experiments, analysed data, and wrote the manuscript with the help of M.C., Q.W., and C.P. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Roberto Chiarle.

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Competing interests

A. W. declares support for Pharmacyclics

Additional information

Reviewer Information Nature thanks D. Fruman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 PI3Kδ blockade increases AID expression and CSR in activated mouse B cells.

a, Schematic representation of the in vivo experiment in wild-type mice immunized with sheep red blood cells and treated with the indicated inhibitors (n = 6 biological replicates). b, Representative dot plots of IgG1 switched GL7+ B220+ germinal centre B cells from mice treated with vehicle or the PI3Kδ inhibitors. c, Mean IgG1 CSR analysed by flow cytometry in control and idelalisib- or duvelisib-treated mice. d, e, IgG1 CSR analysed by flow cytometry in activated B cells treated with 0.5 μM (d) or 0.1 μM (e) of the indicated drugs. f, Western blot for AID protein expression in activated B cells treated with the indicated inhibitors (0.1 μM) (n = 3 biological replicates). For gel source data, see Supplementary Fig. 1. g, Viable cells were counted at the indicated time points by Trypan Blue exclusion in activated B cells treated with the indicated inhibitors (1 μM). h, Representative western blot for AID and PI3K p110δ protein expression in mouse B cells transduced with retrovirus expressing PI3K p110δWT or the p110δ(E1021K) active mutant (n = 4 biological replicates). i, Aicda mRNA levels analysed by qRT–PCR in activated B cells transduced with PI3K p110δWT or the p110δ(E1021K) active mutant. P values calculated by two-tailed Student’s t-test by comparing PI3K p110δ(E1021K) versus PI3K p110δWT cells. j, IgG1 CSR analysed by flow cytometry in activated B cells expressing PI3K p110δWT or the p110δ(E1021K) active mutant. Data are expressed as mean ± s.d. (n = 3); *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-tailed Student’s t-test (ce, g, i, j). Source data

Extended Data Figure 2 Frequency of AID-mediated translocation junctions in activated mouse B cells is increased by PI3Kδ blockade.

a, Frequency of chromosomal translocations between the c-myc and the Igh and Igk loci, represented as reads per million (RPM) in wild-type and Aicda−/− activated B cells treated with the indicated inhibitors. Significance is calculated as FDR by comparing AS-604850, idelalisib or duvelisib to DMSO-treated B cells as indicated in the Methods. **FDR ≤ 0.01, ***FDR ≤ 0.001, ****FDR ≤ 1 × 10−10. b, Detailed view of the distribution of translocation junctions in the Igh locus from wild-type and Aicda−/− mouse B cells treated with the indicated inhibitors. Numbers of translocation junctions in focal clusters are indicated in bold. c, Histograms showing the AID off-targets with significantly increased frequency of HTGTS junctions (FDR < 0.001) induced by idelalisib treatment as compared to DMSO (54 out of 59 off-target sites, 91.5%) in wild-type and Aicda−/− activated B cells. Significance is calculated as indicated in the Methods. Circos plots show the overview of genome-wide translocation distribution. Individual translocations from c-myc to AID target sites are represented as arcs originating from c-myc DSB breaks on chromosome 15. Thickness and red colour intensity of the arcs represent the fold increase of translocation frequency in idelalisib versus DMSO-treated cells. d, Histograms showing the AID off-targets with significantly increased frequency of translocation junctions (FDR < 0.001) induced by duvelisib treatment as compared to DMSO (55 out of 63 off-target sites, 87.3%) in wild-type and Aicda−/− activated B cells. Significance is calculated as indicated in the Methods. Circos plots show the overview of genome-wide translocation distribution. e, Translocation junction distribution (top), GRO-seq (middle) and ChIP–seq (bottom) profiles in three representative AID off-target genes (Pim1, Cd83, Bcl6) in wild-type and Aicda−/− activated B cells treated with idelalisib, duvelisib or ibrutinib. GRO-seq sense and antisense transcription is displayed in blue and red profiles. Numbers of translocation junctions in focal clusters are indicated in bold.

Extended Data Figure 3 Frequency of AID on-target and off-target translocation junctions is reduced in activated mouse B cells transduced with the PI3K p110δ(E1021K) active mutant.

a, Histograms showing junction frequency in activated mouse B cells transduced with retrovirus expressing PI3K p110δWT or the p110δ(E1021K) active mutant. Significance is calculated as FDR by comparing PI3K p110δWT to p110δ(E1021K)-transduced B cells as indicated in the Methods. **FDR ≤ 0.01, ***FDR ≤ 0.001. b Detailed view of the distribution of translocation junctions in one representative I-Scel off-target (Fermt2). c, Detailed view of the distribution of translocation junctions in the Igh locus. Numbers of translocation junctions in focal clusters are indicated in bold. d, Histograms showing frequency of translocation junctions as RPM for I-Scel off-targets in wild-type and Aicda−/− activated B cells treated with the indicated inhibitors. e, Detailed view of the distribution of translocation junctions in two representative I-Scel off-target sites (Aco2 and Fermt2). Numbers of translocation junctions in focal clusters are indicated in bold.

Extended Data Figure 4 PI3Kδ blockade increases SHM frequency in Igh V region in activated mouse B cells and transcription of genes.

a, Map of mutations on the VB1-8 productive allele sequence in activated B cells treated with the indicated inhibitors. The y axis indicates the mutation frequency at each nucleotide in sequences that have 0 to 2 mutations per sequence at day 4 of stimulation after subtraction of mutation frequency at day 0 of stimulation. Green shading indicates top s.e.m. from three independent mice. Orange and yellow bars mark the positions of AGCT and other RGYW motif that is not AGCT, respectively. One experiment is represented out of three biological replicates with similar results. b, Mutation frequency of nucleotide 454 (top) and 455 (bottom) of the VB1-8 productive allele at day 4 after subtraction of mutation frequency at day 0. Data represent mean of three independent stimulations of B cells from three independent mice. Error bars indicate s.d. Fold changes between each mean are indicated. c, Stacking bar chart shows percentage of sequences that have the indicated number of mutations per sequence. Data are displayed as the mean from three independent stimulations of B cells derived from three mice. Error bars indicate s.e.m. d, The number of reads from individual mice for the data are shown in a. e, MA plots of log fold changes generated by DESeq2 in R package against mean expression (normalized counts) from GRO-seq data for idelalisib or duvelisib over DMSO. Red dots indicate significantly upregulated genes (n = 39 idelalisib, n = 47 duvelisib) or downregulated genes (n = 32 idelalisib, n = 33 duvelisib) with multiple test adjusted P value < 0.1 (Supplementary Table 3).

Extended Data Figure 5 PI3Kδ inhibitors and ibrutinib increase c-myc DSB formation and the incidence of plasma cell tumour in mice.

a, Detailed view of the distribution of rearrangements (deletions or inversions) in the c-myc locus in mice treated as above. Numbers of translocation junctions in focal clusters are indicated in bold. b, RPM frequency of rearrangements (deletions or inversions) in the c-myc locus in germinal centre B cell from mice treated in vivo with the idelalisib or duvelisib. Junctions within ± 300 bp of primer region were excluded. Significance is calculated as FDR by comparing idelalisib or duvelisib to DMSO-treated mouse as indicated in the Methods. **FDR ≤ 0.01. c, Schematic representation of the experimental outline of pristane-induced plasma cell tumours in mice treated with PI3Kδ inhibitors and ibrutinib. The mice were treated in two independent biological experimental replicates, each consisting of 6 mice per group. d, Direct PCR assay for Ighc-myc translocation in mice with plasma cell tumours. Translocations from c-myc to the IgHα locus are shown. Translocations for the only mouse in the vehicle group and from three example mice from treated groups are shown. Bands were purified from gels and were sequenced to confirm the Ighc-myc translocation junction. For gel source data, see Supplementary Fig. 1. e, Development of plasma cell tumour in mice induced with pristane and treated with idelalisib, duvelisib or ibrutinib is plotted over time. The presence of plasma cell tumours was confirmed by histology (n = 12 for each treatment in 2 independent cohorts of 6 mice). P values calculated by log-rank (Mantel–Cox) test. f, Example histology of plasma cell tumours in mice induced with pristane and treated with the indicated drugs. Magnification, 40×; scale bar, 50 μm; insets: high magnification image of clusters of atypical plasma cells.

Extended Data Figure 6 PI3Kδ blockade increases AID expression in human B-cell lines.

a, Viable cells were counted at the indicated time points by Trypan Blue exclusion in MEC1, Mino, JeKo-1 and GM06990 cell lines treated with the indicated inhibitors (1 μM). b, Histograms showing the AICDA mRNA relative expression in JeKo-1 and GM06990 cell lines treated with 1 μM inhibitors. c, Western blot for AID protein expression in MEC1 and Mino B-cell lines treated with 0.1 μM inhibitors (n = 2 biological replicates). d, Western blot for AID protein expression in JeKo-1 and GM06990 B-cell lines treated with 1 μM inhibitors (n = 4 biological replicates). For gel source data, see Supplementary Fig. 1. e, Histograms showing the AID protein relative expression at the indicated time points in MEC1, Mino, JeKo-1 and GM06990 cell lines treated with 1 μM inhibitors. AID abundance was measured by ImageJ software and normalized for the β-actin intensity of the corresponding lane. Data are expressed as mean ± s.d. (n = 3 biological replicates). *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, two-tailed Student’s t-test (a, b, e). Source data

Extended Data Figure 7 Strategy to generate chromosomal translocations in human B cells from DSBs introduced in the c-MYC locus by CRISPR/Cas9 technology.

a, Schematic representation of the experimental strategy employed to generate chromosomal translocation from the c-MYC locus by introducing DSBs in c-MYC intron 1 by two different sgRNAs (c-MYC-#1 or c-MYC-#2). Arrowheads (black) indicate the cleavage sites introduced by Cas9 and PAM sequence is in red. b, Surveyor Assay to measure the cutting efficiency of the c-MYC gene targeted in JeKo-1 cell line. Locus modification efficiencies are analysed 5 days after transduction of c-MYC-#1 or c-MYC-#2 Cas9 lentivirus. Estimated indel formation is indicated below each target. Black arrowheads indicate the size of the observed bands. N.D.:, not detectable. One representative experiment out of three performed with comparable results is shown. For gel source data, see Supplementary Fig. 1. c, Histograms showing frequency of translocation junctions in Cas9 off-target sites for c-MYC-#2 sgRNAs in MEC1 cell line treated with idelalisib or duvelisib. AID-knockout MEC1 cells were generated by CRISPR/Cas9-mediated deletion (Extended Data Fig. 8e). For each treatment, data are from pooled HTGTS libraries of similar size from independent experiments as indicated in Supplementary Table 1. d, Detailed view of the distribution of translocation junctions in representative Cas9 off-target site for c-MYC-#2. Numbers of translocation junctions in focal clusters are indicated in bold. e, f, Histograms showing translocation junctions frequency in Cas9 off-targets sites for c-MYC-#1 (e) or c-MYC-#2 (f) sgRNAs in JeKo-1 cell line treated with idelalisib or duvelisib. For each treatment, data are from pooled HTGTS libraries of similar size as indicated in Supplementary Table 1. g, h, Detailed view of the distribution of translocation junctions in representative Cas9 off-target sites for c-MYC-#1 (g) or c-MYC-#2 (h). Numbers of translocation junctions in focal clusters are indicated in bold.

Extended Data Figure 8 Translocations to AID off-targets are increased by idelalisib and duvelisib treatment in MEC1 and JeKo-1 cell line.

a, b, Distribution of translocation junctions in the IGH locus (a) and in the IRF4 AID off-target gene (b) in MEC1 cells. AID-knockout MEC1 cells were generated by CRISPR/Cas9-mediated deletion. Numbers of translocation junctions in focal clusters are indicated in bold. c, Translocation junction frequency in AID on-target and AID off-target sites in JeKo-1 B-cell line treated with DMSO, idelalisib or duvelisib (1 μM). Data are from pooled HTGTS libraries of similar size (Supplementary Tables 1, 4) from 3 independent experiments. Statistical analysis in Methods. *FDR ≤ 0.1, **FDR ≤ 0.01. d, Distribution of translocation junctions in the PIM1 AID off-target gene in JeKo-1 B-cell line. Numbers of translocation junctions in focal clusters are indicated in bold. e, AID-knockout MEC1 cells were generated by CRISPR/Cas9-mediated deletion, cloned and validated by indel sequencing of the Cas9 target site and by AID protein expression. Western blot for AID showing the parental cells (wild type), two AID-knockout clones (4 and 10) and one AID wild-type clone (6). AID-knockout clone 4 was treated with 1 μM DMSO, idelalisib or duvelisib (right panel) (n = 2 biological replicates). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 9 Ibrutinib increases AID expression and the frequency of translocations to AID on- and off-target sites in mouse activated B cells.

a, 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 Fig. 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 analysed by qRT–PCR in activated B cells treated with DMSO or ibrutinib (1 μM). f, IgG1 CSR in activated B cells analysed 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 (e, f). g, Histograms showing translocation junction frequency to AID on-target (#, Igh and Igk loci) and off-target sites in activated B cells treated with ibrutinib. Targets are divided on the basis of 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 Tables 1, 5. h, Venn diagrams showing the fraction of AID off-target sites shared in activated mouse B cells treated with ibrutinib, idelalisib or duvelisib. Source data

Extended Data Figure 10 Ibrutinib increases AID expression and the frequency of translocations to AID on- and off-target sites in human B cells.

a, AICDA mRNA levels analysed by RT–qPCR 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 Fig. 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 Tables 1, 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 chromosome 1, chromosome 8, and chromosome 18 were targeted re-sequenced in patients with CLL untreated or treated with idelalisib and ibrutinib. Box plots 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 1.5× interquartile range beyond the box. P values calculated by paired samples two-tailed Student’s t-test. Source data

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Compagno, M., Wang, Q., Pighi, C. et al. Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells. Nature 542, 489–493 (2017). https://doi.org/10.1038/nature21406

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