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

Abstract

The activated B cell (ABC-like) subtype of diffuse large B cell lymphoma (DLBCL) is characterized by chronic activation of signaling initiated by immunoglobulin μ (IgM). By analyzing the DNA copy number profiles of 1000 DLBCL tumors, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data, we found that the TCF4 (E2-2) transcription factor gene was the target of these alterations. Overexpression of TCF4 in ABC-like DLBCL cell lines led to its occupancy on immunoglobulin (IGHM) and MYC gene enhancers and increased expression of these genes at the transcript and protein levels. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting these gains as an attractive potential therapeutic target. Furthermore, the TCF4 gene was one of the top BRD4-regulated genes in DLBCL cell lines. BET proteolysis-targeting chimera (PROTAC) ARV771 extinguished TCF4, MYC, and IgM expression and killed ABC-like DLBCL cells in vitro. In DLBCL xenograft models, ARV771 treatment reduced tumor growth and prolonged survival. This work highlights a genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

Figure 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 1,000 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-value<0.1). DNA copy gains of 18q21.2 were the most frequent alteration in ABC-like DLBCL cases. (C-D) A Kaplan-Meier plot of overall survival for patients treated with CHOP combination chemotherapy (C) or CHOP plus Rituximab (D) shows that the presence of 18q21.2 gain is associated with poor outcome. (E) The frequency of 18q21 gains is shown relative to other somatic mutations that are significantly associated with the ABC-like DLBCL subtype. This shows that gains of 18q21 are the most frequent genetic alteration in ABC-like DLBCL. (F) REVEALER analysis was performed to identify the set functionally-complementary genetic features that likely contribute to the ABC-like DLBCL molecular phenotype. 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).

Figure 2:. The TCF4 gene is the most significant 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 peaks are highlighted with arrows. The most statistically significant peak harbors the TCF4 gene, while the less significant peak harbors the BCL2 gene. However, it can be seen that in many cases the DNA copy number gains span both the TCF4 and BCL2 regions. (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) are shown for all tumors (left) and for the ABC-like only (right). This shows that the majority of 18q DNA copy gains include both TCF4 and BCL2, but TCF4 is more frequently gained independently of BCL2 than vice versa. (C) The gene expression level from microarrays are shown for GCB-like DLBCL with diploid 18q (green), GCB-like DLBCL with 18q copy gain (beige), ABC-like DLBCL tumors with diploid for 18q (orange), and ABC-like DLBCL tumors with 18q DNA copy number gains (red). The expression of TCF4 is significantly higher in ABC-like DLBCL compared to GCB-like DLBCL in diploid cases and further significantly increased by DNA copy number gain. P-values are from students T-test, p>0.05 is denoted as ns, ***p<0.001. (D) The protein level of TCF4 and BCL2 are shown in ABC-like DLBCL cell lines, ordered according to increasing DNA copy number of the TCF4 locus. There is a visible relationship between TCF4 protein abundance and DNA copy number that is less clear for BCL2. (E) The protein level of TCF4 and BCL2 are shown in GCB-like DLBCL cell lines, with the ABC-like cell lines with the highest (U2932) and lowest (SUDHL2) TCF4 expression shown as reference. All of the GCB-like DLBCL cell lines show lower expression of TCF4, and there is no clear relationship with 18q DNA copy number. (F) The frequency of TCF4 DNA copy gains, TCF3 mutation and ID3 mutation are shown for a cohort of 108 Burkitt lymphoma tumors. Gains of the TCF4 locus are present at the same frequency of TCF3 mutations and are significantly mutually exclusive from TCF3 and ID3 mutations.

Figure 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 identified a large set of genes with increased expression associated with TCF4 gain. This included the direct targets of 18q DNA copy number gains, TCF4 and BCL2, and multiple genes with an important role in the pathophysiology of DLBCL, such as IRF4, MYC and the immunoglobulin heavy chain μ (IGHM), that are upregulated as a secondary effect of TCF4 gain. ChIP-seq of TCF4 from SUDHL2 and TMD8 cell lines showed that the majority of these genes were marked with TCF4 binding in intragenic or distant enhancer elements, suggesting that their up-regulation may be driven by transcriptional activation by TCF4. (B) The significant TCF4 ChIP-seq peaks from SUDHL2 and TMD8 are shown, ordered from strongest (top) to weakest (bottom) signal ratio compared to the input control. Significant peaks were detected for important genes such as MYC and IRF4, but multiple IGHM peaks were amongst those with the highest TCF4 binding. (C) A violin plot shows that primary ABC-like DLBCL tumors with 18q21 gain express significantly higher transcript levels of IGHM compared to ABC-like DLBCL tumors with 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 (grey). Yellow shading indicates the statistically significant peak. For reference, ENCODE data for H3K27 acetylation (H3K27Ac) ChIP-seq in CD20+ B-cells is shown, which support the TCF4 bound regions as bona fide enhancer elements in B-cells. (E) The binding of TCF4 to the two immunoglobulin heavy chain loci and the MYC enhancer locus was confirmed 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, HBL1). The signal was significantly above that of the isotype control IgG antibody for all cell lines and loci (P<0.001). Each bar represents the mean +/− S.E.M. of 3 independent experiments.

Figure 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 a significant increase in IGHM transcript compared to control cells. (B) Tetracycline-induced expression of TCF4 led to a marked increase in IgM protein in ABC-like DLBCL cell lines with low TCF4 copy number. An increase in MYC was also observed in SUDHL2 and HBL1 cell line, but was not significant in TMD8. No change was observed for BCL2. The quantification of triplicate experiments is shown in Figure S5. (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. TCF4-induced up-regulation of IgM alone did not significantly increase BTK or BLNK phosphorylation, but it significantly enhanced phosphorylation following BCR stimulation. (D) The quantification of western blots from triplicate experiments combining TCF4 induction and BCR stimulation, as shown in C, demonstrate the significant increase in BCR-induced signaling through BTK and BLNK when TCF4 is over-expressed in each of 3 ABC-like DLBCL cell lines. Each bar represents the mean +/− S.E.M. of 3 independent experiments. Groups were compared by Student’s T-test. P-value>0.05 is denoted at ns. *p<0.05, **p<0.01.

Figure 5:. Functional dependency upon TCF4 in ABC-like DLBCL.

(A) A schematic of the two TCF4 dominant-negative (TCF4dn) constructs. The TCF4^ΔBR construct has an in-frame deletion of the basic region prior to the helix-loop-helix domain. The TCF4^R582P construct has a single amino acid change within the helix-loop-helix domain. The TCF4dn constructs (orange) are predicted to form heterodimers with wild-type TCF4 (purple) and inhibit DNA binding. (B) A cell competition assay was performed by mixing equal fractions of GFP+ cells possessing the either the TCF4^ΔBR or TCF4^R582P tetracycline-inducible dominant-negative construct, with the parental cell line. Cells were exposed to doxycycline and the GFP+ fraction measured every 2–3 days for 10 days. The ABC-like DLBCL cell lines with high-level (maroon) or low-level (red) TCF4 DNA copy number gain declined over time with expression of either TCF4dn. This was not observed for ABC-like DLBCL cell lines with diploid TCF4 (orange) or GCB-like DLBCL cell lines selected based upon variable copy number and TCF4 expression. Each point represents the mean +/− S.E.M. of 3 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. Both constructs significantly inhibited TCF4 binding at these loci in the U2932 and RIVA cell lines with high TCF4 DNA copy number gain and protein expression. Each bar represented the mean +/− S.E.M. of 3 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 TCF4 target genes, IgM and MYC. Tetracycline-inducible expression of either TCF4dn constructs reduced IgM and MYC expression relative to uninduced cells and doxycycline-treated EV control cells. (E) Quantification of triplicate experiments from D shows that the attenuation of IgM and MYC expression is statistically significant for both of the unique TCF4dn constructs and in both the U2932 and RIVA ABC-like DLBCL cell lines.

Figure 6:. BET proteolysis-targeting chimeras (PROTAC) effectively inhibit TCF4.

(A) The treatment of ABC-like DLBCL cell lines with high TCF4 DNA copy number using small molecule BET inhibitors, JQ1 and OTX015, leads to an accumulation of BRD4 but can reduce the BRD4-targets TCF4 and MYC. The BET PROTAC, ARV771, effectively degrades BRD4 and leads to a more potent reduction of TCF4 and MYC at 10-fold lower doses than the small molecule inhibitors. (B) The treatment of U2932 and RIVA cell lines with 50nM of ARV771 for 24h led to broad changes in transcript levels. This included the down-regulation of known BRD4 target genes, BCL6 and PAX5, as well as the down-regulation of TCF4 and its target gene, IGHM. (C) Gene set enrichment analyses are overlaid 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 Figure 3A. Treatment with ARV771 led to a significant and coordinate down-regulation of this TCF4-associated signature in both the U2932 and RIVA cell lines. (D) The TCF4 target genes IgM and MYC are reduced by ARV771 treatment, but can be partially rescued by enforced expression of DDK-tagged tetratcyline-inducible TCF4 (t.o.TCF4). As expected, the expression of BRD4 was not rescued. (E-F) Quantification of triplicate experiments for U2932 (E) and RIVA (F) shows that IgM and MYC expression are significantly rescued by enforced expression of TCF4. Each bar represents the mean +/− S.E.M. of 3 independent experiments, compared with a Student’s T-test. *p<0.05, **p<0.01. (G) The rescue of TCF4 expression ameliorated, but did not eliminate, 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 represented the mean +/− S.E.M., compared using a Student’s T-test. *p<0.05.

Figure 7:. In vivo activity of ARV771 in ABC-like DLBCL.

(A-E) Murine xenografts of the U2932 cell line were allowed to become established and then treated with 30mg/kg of ARV771 daily x 5 days per week for 3 weeks. At the end of treatment the luminescence was significantly lower in ARV771-treated mice compared to vehicle control (A, B) and a representative tumor shows on-target reduction of BRD4, TCF4, IgM and MYC expression (C). Treatment significantly inhibited tumor growth (D) and led to significantly prolonged survival in ARV771-treated mice (E) despite the short duration of treatment (E, shaded blue). (F-K) Murine xenografts of the RIVA cell line were allowed to become established and then treated with 30mg/kg of ARV771 daily x 5 days per week for 2 weeks. At the end of treatment the luminescence was significantly lower in ARV771-treated mice compared to vehicle control (F, G) and a representative tumor shows on-target reduction of BRD4, TCF4, IgM and MYC expression (H). Treatment significantly inhibited tumor growth (I) and led to significantly prolonged survival in ARV771-treated mice (K) despite the short duration of treatment (K, shaded blue).