Research ArticleCancer

Targetable genetic alterations of TCF4 (E2-2) drive immunoglobulin expression in diffuse large B cell lymphoma

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Science Translational Medicine  19 Jun 2019:
Vol. 11, Issue 497, eaav5599
DOI: 10.1126/scitranslmed.aav5599
  • Fig. 1 DNA copy number gains of 18q21.2 are the most frequent genetic alteration in ABC-like DLBCL.

    (A) GISTIC analysis of DNA copy number profiles form 1000 DLBCL tumors identified 21 peaks of DNA copy loss (blue, left) and 20 peaks of DNA copy gain (red, right). The green line indicates the significance threshold of Q value = 0.1. (B) The GISTIC peaks from (A) are shown with reference to their frequency in ABC-like (orange) compared to GCB-like (green) cell of origin subtypes (*Q < 0.1). DNA copy gains of 18q21.2 were the most frequent alteration in ABC-like DLBCL cases. (C and D) A Kaplan-Meier plot of overall survival for patients treated with (C) CHOP combination chemotherapy or (D) R-CHOP shows that the presence of 18q21.2 gain is associated with poor outcome. (E) The frequency of 18q21 gains (+18q21) is shown relative to other somatic mutations that are associated with the ABC-like DLBCL subtype. (F) REVEALER analysis was performed to identify the set functionally complementary genetic features that likely contribute to the ABC-like DLBCL molecular phenotype. COO, cell of origin. Mutations of MYD88 were used as the seed feature. Mutations of IRF4, PIM1, and CD79B, and DNA copy gains of 18q21 were selected as additional features that likely also contribute to the phenotype §Seed feature. IC, information coefficient; CIC, conditional information coefficient.

  • Fig. 2 The TCF4 gene is a key target of 18q DNA copy number gains.

    (A) A schematic of 18q DNA copy number gains is shown, with each line representing a single tumor and deeper shades of red indicating higher DNA copy number. The GISTIC Q value is shown at the top of the diagram and the two significant (Q value of <0.1) peaks are highlighted with arrows. (B) The frequency of tumors with DNA copy number gains that include both the TCF4 and BCL2 genes (purple), the TCF4 gene and not the BCL2 gene (pink), or the BCL2 gene and not the TCF4 gene (yellow) is shown for all tumors (left) and for the ABC-like only (right). (C) TCF4 expression from microarrays is shown for GCB-like DLBCL with diploid 18q (green, n = 74), GCB-like DLBCL with 18q copy gain (beige, n = 22), ABC-like DLBCL tumors with diploid for 18q (orange, n = 59), and ABC-like DLBCL tumors with 18q DNA copy number gains (red, n = 52). P values are from Mann-Whitney test. P > 0.05 is denoted as ns, **P < 0.01, and ***P < 0.001. (D) Protein expression of TCF4 and BCL2 in ABC-like DLBCL cell lines, ordered according to increasing DNA copy number of the TCF4 locus. The GCB-like cell line, OCI-Ly1, is shown for reference. (E) Protein expression of TCF4 and BCL2 in GCB-like DLBCL cell lines. The ABC-like cell lines, U2932 and SUDHL2, are shown for reference. (F) The frequency of TCF4 DNA copy gains, TCF3 mutation, and ID3 mutation in a cohort of 108 BL tumors. A Fisher’s exact test was used to compare the frequency of TCF3 and ID3 mutations (black bars) in tumors with TCF4 gain (red bars) compared to those without TCF4 gain, P = 0.0191.

  • Fig. 3 TCF4 regulates IgM expression in ABC-like DLBCL.

    (A) Differential gene expression analysis of 110 primary ABC-like DLBCL tumors with or without TCF4 DNA copy number gain. ChIP-seq signal for TCF4 from SUDHL2 and TMD8 cell lines shows TCF4 binding at intragenic or distant enhancer elements. (B) Significant TCF4 ChIP-seq peaks from SUDHL2 and TMD8 cells (P < 0.01) are shown, ordered from strongest (top) to weakest (bottom) signal ratio compared to the input control. (C) A violin plot shows expression of IGHM in ABC-like DLBCL tumors with either a 18q21 gain or diploid 18q21. (D) Two of the TCF4 peaks at the immunoglobulin heavy-chain locus are shown for TCF4 ChIP (blue) compared to the equivalent input control (gray). Yellow shading indicates the statistically significant peak (P < 0.01). For reference, Encyclopedia of DNA elements (ENCODE) data for H3K27 acetylation (H3K27Ac) ChIP-seq in CD20+ B cells are shown. (E) The binding of TCF4 to the two immunoglobulin heavy-chain loci and the MYC enhancer locus by ChIP-qPCR in two cell lines with high TCF4 DNA copy number and protein expression (U2932 and RIVA) and three cell lines with tetracycline-inducible TCF4 expression (SUDHL2, TMD8, and HBL1). IP, immunoprecipitation. The signal was significantly above that of the isotype control IgG antibody for all cell lines and loci. Student’s t test (P < 0.001). Each bar represents the mean ± SEM of three independent experiments.

  • Fig. 4 Induced expression of TCF4 in ABC-like DLBCL cell lines drives MYC and IgM expression and potentiates BCR signaling.

    (A) Tetracycline-induced expression of TCF4 in ABC-like DLBCL cell lines with low TCF4 copy number resulted in an increase in IGHM transcripts compared to control cells. (B) The effect of tetracycline-induced expression of TCF4 on IgM protein in ABC-like DLBCL cell lines with low TCF4 copy number. The quantification of triplicate experiments is shown in fig. S8. (C) A representative Western blot shows the phosphorylation of downstream kinases from IgM, BTK, and BLNK, with or without tetracycline-induced TCF4 and/or BCR stimulation with an αIgM cross-linking antibody. (D) The quantification of Western blots from triplicate experiments combining TCF4 induction and BCR stimulation, as shown in (C). Each bar represents the mean ± SEM of three independent experiments. Groups were compared by Student’s t test. P > 0.05 is denoted as ns, *P < 0.05, and **P < 0.01.

  • Fig. 5 Functional dependency upon TCF4 in ABC-like DLBCL.

    (A) A schematic of the two TCF4 dominant-negative (TCF4dn) constructs. The TCF4ΔBR construct has an in-frame deletion of the basic region before the helix-loop-helix domain. The TCF4R582P construct has a single amino acid change within the helix-loop-helix domain. (B) A cell competition assay was performed by mixing equal fractions of green fluorescent protein–positive (GFP+) cells having either the TCF4ΔBR or TCF4R582P tetracycline-inducible dominant-negative construct with the parental cell line. Cells were exposed to doxycycline and the GFP+ fraction measured every 2 to 3 days for 10 days. One-way analysis of variance (ANOVA) comparing ABC-like cell lines with TCF4 DNA copy gain either to ABC-like cell lines with diploid TCF4 (**P < 0.01 and ***P < 0.001) or to GCB-like cell lines (###P < 0.001). Each point represents the mean ± SEM of three independent experiments. (C) TCF4 ChIP-qPCR was performed for the two immunoglobulin enhancers and the MYC enhancer in the presence or absence of dominant-negative constructs in the U2932 and RIVA cell lines with high TCF4 DNA copy number gain and protein expression. Each bar represents the mean ± SEM of three independent experiments, with statistical significance assessed by Student’s t test compared to empty vector (EV) control. **P < 0.01. (D) A representative Western blot shows the expression of the DDK-tagged TCF4dn and the expression of TCF4 target genes IgM and MYC. (E) Quantification of triplicate experiments from (D) shows the attenuation of IgM and MYC expression for both of the unique TCF4dn constructs and in both the U2932 and RIVA ABC-like DLBCL cell lines. Groups were compared by Student’s t test. *P < 0.05 and **P < 0.01.

  • Fig. 6 BET proteolysis-targeting chimeras inhibit TCF4 expression.

    (A) Protein expression after the treatment of ABC-like DLBCL cell lines with high TCF4 DNA copy number using small-molecule BET inhibitors JQ1 and OTX015. (B) Changes in gene expression induced by the treatment of U2932 and RIVA cell lines with 50 nM of ARV771 for 24 hours. (C) Gene set enrichment analyses are shown for U2932 (green) and RIVA (blue) for the set of genes that were more highly expressed in primary ABC-like tumors with TCF4 DNA copy number gain compared to those tumors without DNA copy number gain, as shown in Fig. 3A. (D) The TCF4 target genes IgM and MYC are reduced by ARV771 treatment but can be partially rescued by enforced expression of DDK-tagged tetracycline-inducible TCF4 (t.o.TCF4). (E and F) Quantification of triplicate experiments for (E) U2932 and (F) RIVA. Each bar represents the mean ± SEM of three independent experiments compared using Student’s t test. *P < 0.05 and **P < 0.01. (G) The effect of rescuing TCF4 expression on the apoptosis induced by ARV771 treatment, as shown by the percentage of annexin-V+ TOPRO-3+ cells with or without TCF4 rescue by doxycycline-induced expression. Bars represent means ± SEM. Student’s t test, *P < 0.05.

  • Fig. 7 In vivo activity of ARV771 in ABC-like DLBCL.

    (A to E) Murine xenografts of the U2932 cell line were allowed to become established and then treated with ARV771 (30 mg/kg) daily, 5 days/week for 3 weeks. (A and B) At the end of treatment, the luminescence was quantified for ARV771-treated and vehicle control–treated mice. (C) A representative tumor shows on-target reduction of BRD4, TCF4, IgM, and MYC expression. (D) Treatment significantly inhibited tumor growth (Mann-Whitney test, ***P < 0.001, **P < 0.01, and *P < 0.05) and (E) led to significantly prolonged survival in ARV771-treated mice despite the short duration of treatment (E, shaded blue). Mantel-Cox log-rank test, P = 0.033. (F to J) Murine xenografts of the RIVA cell line were allowed to become established and then treated with ARV771 (30 mg/kg) daily, 5 days/week for 2 weeks. (F and G) At the end of treatment, the luminescence was quantified in ARV771-treated and vehicle control–treated mice. (H) A representative tumor shows on-target reduction of BRD4, TCF4, IgM, and MYC expression. (I) Treatment significantly inhibited tumor growth (Mann-Whitney test, *P < 0.05) and (J) led to significantly prolonged survival in ARV771-treated mice despite the short duration of treatment (K, shaded blue). Mantel-Cox log-rank test, P = 0.028.

Additional Files

  • The PDF file includes:

    • Fig. S1. Validation of cell of origin subtyping.
    • Fig. S2. Comparison of outcome in CHOP-treated patients with DLBCL stratified by cell of origin subtype and 18q21 gain.
    • Fig. S3. Recurrently mutated genes enriched in the ABC-like DLBCL subtype.
    • Fig. S4. Expression patterns of TCF3 and TCF4 in normal and malignant B cells.
    • Fig. S5. Tetracycline induction of TCF4 expression.
    • Fig. S6. ChIP-seq peaks for TCF4.
    • Fig. S7. Cellular localization of TCF4-induced IgM.
    • Fig. S8. Quantification of TCF4-induced MYC and BCL2 protein expression.
    • Fig. S9. shRNA-mediated knockdown of TCF4.
    • Fig. S10. BRD4 regulates TCF4 in DLBCL cell lines.
    • Fig. S11. Western blot quantification of triplicate BET inhibitor experiments.
    • Fig. S12. ARV771 induces apoptosis in RIVA and U2932 cell lines.
    • Fig. S13. Effect of ARV-771 on IgM expression in GCB-like DLBCL cell lines.
    • References (5763)

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 contains the following supplementary tables:
    • Table S1 (Microsoft Excel format). Genomic and clinical data from DLBCL tumors included in this study.
    • Table S2 (Microsoft Excel format). NGS statistics.
    • Table S3 (Microsoft Excel format). Genes in GISTIC peaks.
    • Table S4 (Microsoft Excel format). Cell of origin association of GISTIC peaks.
    • Table S5 (Microsoft Excel format). Cell of origin association of recurrently mutated genes.
    • Table S6 (Microsoft Excel format). Integrative analysis of DNA CNAs.
    • Table S7 (Microsoft Excel format). Differential gene expression analysis of ABC-like tumors with or without TCF4 copy gain.
    • Table S8 (Microsoft Excel format). ChIP-seq peaks for TCF4 signature genes.
    • Table S9 (Microsoft Excel format). Differentially expressed genes following ARV-771 treatment.
    • Table S10 (Microsoft Excel format). Primer sequences.

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