BCL6 confers KRAS-mutant non-small-cell lung cancer resistance to BET inhibitors

Abstract

The bromodomain and extra-terminal domain (BET) proteins are promising therapeutic targets to treat refractory solid tumors; however, inherent resistance remains a major challenge in the clinic. Recently, the emerging role of the oncoprotein B cell lymphoma 6 (BCL6) in tumorigenesis and stress response has been unveiled. Here, we demonstrate that BCL6 was upregulated upon BET inhibition in KRAS-mutant cancers, including non-small-cell lung cancer (NSCLC). We further found that BRD3, not BRD2 or BRD4, directly interacted with BCL6 and maintained the negative autoregulatory circuit of BCL6. Disrupting this negative autoregulation by BET inhibitors (BETi) resulted in a striking increase in BCL6 transcription, which further activated the mTOR signaling pathway through repression of the tumor suppressor death-associated protein kinase 2. Importantly, pharmacological inhibition of either BCL6 or mTOR improved the tumor response and enhanced the sensitivity of KRAS-mutant NSCLC to BETi in both in vitro and in vivo settings. Overall, our findings identify a mechanism of BRD3-mediated BCL6 autoregulation and further develop an effective combinatorial strategy to circumvent BETi resistance in KRAS-driven NSCLC.

Keywords: Cancer; Drug therapy; Oncology.

Figure 1. Clinical BETi promote BCL6 expression.

(A) BCL6 expression in KRAS-mutant NSCLC cells in response to clinical drug treatments. Cells were treated with the indicated drugs at a concentration of their one-half IC_50s for 48 hours. Graph shows the relative BCL6 protein levels, normalized to GAPDH. **P < 0.01, by unpaired, 2-tailed Student’s t test, comparing the relative BCL6 protein level of the OTX015-treated group with that of the vehicle-treated group. (B and C) OTX015 upregulated BCL6 expression at the (B) protein and (C) transcription levels in a time-dependent manner. A549, H292, and H441 cells were treated with OTX015 for the indicated durations. BCL6 expression was detected by Western blotting and PCR assays. (D) BCL6 protein levels in cells upon treatment with BETi (OTX015, JQ1, I-BET762, and AZD5153) or a BRD9i (BI7273). (E) BETi upregulated BCL6 expression in a set of KRAS-mutant NSCLC cells. Cells were treated with OTX015 (300 nM), JQ1 (300 nM), or DMSO for 6 hours. Cell lysates were probed with antibodies against BCL6 and GAPDH. (F) OTX015 upregulated BCL6 levels in 2 primary NSCLC cell lines harboring a KRAS^G12C mutation. Left: DNA-Seq of exon 2 in the KRAS gene. Right: BCL6 levels in LC087A05 and LC308B01 cells exposed to OTX015 (300 nM) or DMSO for 6 hours. (G) BCL6 protein levels in orthotopic xenografts. During the 1-week treatment with OTX015, tumor tissue was isolated on day 2 and day 7, followed by immunoblot analysis for BCL6 expression. Three biologically independent samples per group are shown.

Figure 2. BCL6 is required for the therapeutic efficacy of BETi.

(A) Efficacy of BCL6 overexpression in A549 cells by immunoblot analysis. (B and C) BCL6 overexpression (BCL6^OE) impaired the inhibitory effects of OTX015 on A549 cells, as indicated by (B) growth curves and (C) relative cell viability of cultured colonies. (D) Efficacy of BCL6 overexpression in H23 cells by immunoblot analysis. (E and F) BCL6 overexpression impaired the inhibitory effects of OTX015 on H23 cells, as indicated by the (E) growth curves and (F) relative viability of the cultured colonies. (G) BCL6 silencing potentiated OTX015 efficacy in H441 cells. Left: Representative images of colony formation assays. Right: Relative viability of the cultured colonies. siNC, siRNA negative control. (H) BI3802 enhanced the efficacy of OTX015 in A549 cells. A549 cells were treated with concentration gradients of OTX015 with or without BI3802 for 48 hours. Shown are cell viability curves and immunoblot analysis of BCL6 expression. Results in C, F and G are representative of 3 independent experiments. Data represent the mean ± SEM of biological triplicates. ***P < 0.001, by unpaired, 2-tailed Student’s t test.

Figure 3. BET inhibition disrupts the BCL6 autoregulatory circuit.

(A) OTX015 treatment shifted BCL6 from binding its promoter regions (region 1 and region 2) to its coding region (region 3). A549 cells were treated with DMSO or OTX015 (300 nM) for 6 hours. Chromatin was sheared and subsequently precipitated using a specific antibody against BCL6. Cellular DNA for the ChIP assay was isolated and analyzed on an Illumina NextSeq 500 instrument. Reads were mapped to the human reference genome (GRCh37/hg19) using Bowtie2 (version 2.2.9). UCSC’s Genome Browser tracks showed BCL6 ChIP-Seq signals in the BCL6 gene locus. Blue shading marks the peaks located in the promoter region. (B) OTX015 treatment promoted BCL6 to bind to its coding region. qPCR was performed with primers specifically targeting region 1, region 2, and region 3 of the BCL6 gene. The data are plotted relative to the values obtained with the IgG control antibody. Data represent the mean ± SEM of biological triplicates. P values were analyzed by unpaired, 2-tailed Student’s t test, comparing the OTX015 treatment group with the control group. (C) RNA Pol II–binding level at the 3 indicated regions examined by ChIP-qPCR. Data represent the mean ± SEM of biological triplicates. P values were determined by unpaired, 2-tailed Student’s t test, comparing the OTX015 treatment group with the control group. (D) Response of different BCL6 promoter (P) regions to OTX015 treatment. Luciferase reporter vectors with promoters containing the indicated BCL6 promoter regions (region 1 and region 2) or the control sequence were equivalently transfected into A549 cells. Transfected cells were then exposed to 300 nM OTX015. Cells were harvested to detect luciferase expression 48 hours after transfection. Data represent the mean ± SD of biological triplicates. P values were determined by unpaired, 2-tailed Student’s t test, comparing the OTX015 treatment group with the control group. (E) Response of different BCL6 promoter regions to BCL6 silencing. Luciferase reporter vectors with promoters containing the indicated BCL6 promoter regions (mentioned in D) or the control sequence were equivalently transfected into BCL6-knockdown A549 cells. Transfected cells were harvested to detect luciferase expression 48 hours after transfection. Data represent the mean ± SD of biological triplicates. P values were determined by unpaired, 2-tailed Student’s t test, comparing the siBCL6 groups with the control group.

Figure 4. BRD3 acts as a cofactor to maintain BCL6 autoregulation.

(A) Knockdown of BRD2 or BRD3 upregulated BCL6 expression. A549 cells transfected with 20 nM siRNAs targeting BRDs were collected 48 hours after transfection. BRD2, BRD3, BRD4, and BCL6 expression were detected by immunoblot analysis. (B) BRD4-specific inhibitors did not affect BCL6 expression. A549 and H292 cells were treated with BETi (OTX015 and JQ1), BRD4-specific inhibitors (DC-1 and DC-2), or a negative control compound (DC-control) for 6 hours. (C and D) Endogenous interaction of BCL6 and BRD3. A549 cell lysates were subjected to immunoprecipitation experiments using an anti-BCL6 or anti-BRD3 antibody. Immunoprecipitates were analyzed with antibodies against BRD2, BRD3, BRD4, and BCL6. Ten percent of total lysates was used as the input control. The anti-IgG antibody was used as a negative control. (E) Knockdown of BRD3 increased BCL6 mRNA levels. Data represent the mean ± SEM of biological triplicates. P values were analyzed by comparing siBRD3 groups with the control group. *P < 0.05 and ***P < 0.001, by unpaired, 2-tailed Student’s t test. (F) ChIP and re-ChIP were performed to test the cobinding of the BCL6 and BRD3 complex at the loci on the BCL6 promoter regions mentioned in Figure 3A. The first ChIP was performed using anti-BCL6 and anti-IgG antibodies. The second ChIP was performed using anti-BRD3 and anti-IgG antibodies to analyze the first ChIP (anti-BCL6) components. Data represent the mean ± SEM of biological triplicates. P values were analyzed by Student’s t test analysis for the first and second ChIPs. (G) BRD3 binding at the 3 indicated regions analyzed by ChIP-qPCR. Data represent the mean ± SEM of 3 independent experiments. P values were determined by unpaired, 2-tailed Student’s t test. (H) BRD3 maintained the BCL6 autoregulatory circuit. A549 cells were cotransfected with P1-Fluc, together with an equivalent amount of pcDNA3.1-BRD3, pcDNA3.1-BCL6, and/or pcDNA3.1-Ctrl. Cells were harvested for a luciferase assay 48 hours after transfection. Western blotting was subsequently conducted using specific antibodies against BCL6, BRD3, and GAPDH. Data represent the mean ± SD of biological triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA. Immunoblots in A, C, and D were contemporaneous and run in parallel from the same biological replicate.

Figure 5. Increased BCL6 activates the mTOR pathway through suppression of DAPK2.

(A) Venn diagram shows the overlap of BCL6-bound gene promoters between the DMSO and OTX015 treatment groups. These data were generated by the ChIP-Seq experiments described in Figure 3A. (B) Heatmap of differentially expressed genes (fold change >2, adjusted P < 0.05) in the OTX015-specific region described in A. RNA-Seq was performed using the same cell samples as the ChIP-Seq in Figure 3A. Blue represents gene downregulation, and red represents gene upregulation, with 0 as the median. (C) Genome Browser tracks showing BCL6 ChIP–Seq signals in the DAPK2 gene locus in A549 cells treated with DMSO or OTX015 (300 nM) for 6 hours. Blue shading marks the peaks located in the promoter region. (D) BCL6 binding level at the promoter region of DAPK2 examined by ChIP-qPCR assays. (E) RNA Pol II–binding level at the promoter region of DAPK2 examined by ChIP-qPCR assays. Data represent the mean ± SEM of biological triplicates. The P value was determined by comparing the OTX015 treatment group with the control group, using an unpaired, 2-tailed Student’s t test. (F) Relative DAPK2 expression at different time points. Cells were treated with 300 nM OTX015, and DAPK2 mRNA levels were detected by qPCR. Data represent the mean ± SEM of 3 independent experiments. P values were determined by comparing OTX015-treated groups with the untreated group, using an unpaired, 2-tailed Student’s t test. (G) Effects of OTX015 on the DAPK2/mTOR signaling pathway on A549 cells. A549 cells were treated with OTX015 (300 nM) for the indicated durations. Treated cells were collected and probed with antibodies against DAPK2, p-mTOR (Ser2448), mTOR, p-P70S6K (Thr389), P70S6K, p–4E-BP1 (Thr37/46), 4E-BP1, and GAPDH, respectively. (H) Effects of OTX015 on the DAPK2/mTOR signaling pathway in H292 and H441 cells. (I) BCL6 knockdown impaired OTX015-mediated DAPK2 suppression and mTOR activation. BCL6 silencing was conducted by RNA interference in A549 cells. Cells were treated with 300 nM OTX015 for 48 hours. Cells lysates were collected and probed with antibodies against BCL6, DAPK2, p-mTOR (Ser3448), mTOR, and GADPH. Immunoblots in G, H, and I were contemporaneous and run in parallel from the same biological replicate, respectively. The immunoblots are representative of at least 3 independent experiments.

Figure 6. BCL6 inhibitors synergize with BETi in vitro and in vivo.

(A) Synergistic interaction between BCL6 inhibitors (FX1 and Compound 7) and BETi (OTX015, JQ1, and I-BET762) in KRAS-mutant NSCLC cells. CI values at ED₅₀, ED₇₅, and ED₉₀ were calculated using CalcuSyn software. CI values of less than 1 represent synergism. (B) Representative images of 3D colony formation assays of A549 cells in response to the indicated treatments. Scale bars: 0.1 cm (dark) and 1 cm (white). (C) Clonogenic assay of primary KRAS-mutant NSCLC cells. (D) Cell-cycle profiles. Data represent the mean ± SD of biological triplicates. P values were determined by unpaired, 2-tailed Student’s t test, comparing the combination treatment group with the control group. (E) Tumor volume (fold change) of patient-derived NSCLC (LACPDX) xenografts in mice (n = 10 per group). The mean AUC of tumor volumes (AUC_TV) on day 21 is shown. Mice were treated according to the schedule in E. Data represent the mean ± SEM. *P < 0.05 and ***P < 0.001, by 1-way ANOVA. (F) Representative lung micro-CT images of LSL-Kras(G12D) mice. Yellow arrowheads indicate lung tumors. (G) Representative images of H&E-stained unilateral lung lobe tissues after treatment. (H) Kaplan-Meier survival analysis of LSL-Kras(G12D) mice (n = 6 per group). *P < 0.05 and **P < 0.01, by log-rank test. (I) Representative micro-CT images of the lungs of LSL-Kras^G12D/+ Trp53^fl/fl genetically engineered mice. Areas marked by yellow dotted lines indicate lung tumors. (J) Box plots showing the tumor volumes at the endpoint of the indicated treatments based on micro-CT (n = 6 per group). The horizontal lines represent the median, the bottom and top of the boxes represent the 25th and 75th percentiles, respectively, and the vertical bars represent the range of the data. The P value in J was determined by unpaired, 2-tailed Student’s t test. All data are shown as the mean ± SEM.

Figure 7. mTOR inhibition sensitizes KRAS-mutant NSCLC to BETi.

(A) DAPK2 overexpression suppressed mTOR and S6K phosphorylation. H441 cells infected with a pCDH-DAPK2 or pCDH control virus were subjected to immunoblot analysis. Immunoblots were contemporaneous and run in parallel from the same biological replicate, respectively. (B) Colony formation of H441 and A549 cells with DAPK2 overexpression. Results are representative of 3 independent experiments. Data represent the mean ± SEM of biological triplicates. ***P < 0.001, by unpaired, 2-tailed Student’s t test, comparing the DAPK2^OE group with the vector group in the presence of OTX015. (C) Synergistic interaction between BETi (OTX015 and JQ1) and mTORi (rapamycin and ridaforolimus) in KRAS-mutant NSCLC cells. A549 and H441 cells were treated for 48 hours with various concentrations of the indicated inhibitors. The concentrations of BETi or mTORi were used in a 2-fold dilution series (0.78, 1.56, 3.13, 6.25, 12.5, and 25 μM for individual BETi; 0.78, 1.56, 3.13, 6.25, 12.5, and 25 nM for individual mTORi). Relative cell viability was subsequently measured. Data represent the mean ± SD of biological triplicates. CI values at each concentration were calculated using CalcuSyn software. (D) Tumor growth curves of LACPDX (n = 8 per group). The mean AUC of tumor volumes on day 23 is shown. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA. (E) Tumor weights at the end of therapy. Each dot represents a tumor from an individual mouse. Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA.