Acetylation of Mastermind-like 1 by p300 Drives the Recruitment of NACK to Initiate Notch-Dependent Transcription

Gene expression is a critical cellular process that is tightly controlled by coordination of many signals and transcriptional regulators. Transcription factors bind to specific DNA sequence and initiate the formation of higher-order complexes with cofactors and epigenetic enzymes, leading to chromosome structure remodeling and recruitment of general transcriptional machinery to initiate and maintain specific transcriptional profiles (1, 2). Accurate regulation of transcription factor activation is indispensable for all aspects of development and epistasis. Furthermore, dysregulation of transcription is an important driver of many diseases, including cancer.

Notch is an important transcription factor in both development and tumorigenesis. Upon activation through cell-to-cell interaction, the Notch intracellular domain is released from the cell membrane and translocates to the nucleus, where it initiates the formation of a ternary complex with Maml and CSL (3, 4). It is thought that this ternary complex serves as a scaffold to recruit additional transcriptional coactivators to drive a Notch-dependent transcriptional cascade (5–8). Therefore, deregulation of Notch leads to the aberrant enforcement of a transcriptional profile that drives a neoplastic program (3, 4, 9, 10).

Previously, we reported the identification of a novel Notch coactivator, termed NACK. We demonstrated that NACK binds to the Notch1 ternary complex in a Maml1-dependent manner and is required for both Notch target gene expression and Notch-driven tumorigenesis (11). Although there is a general acceptance that p300 plays a role in Notch-directed transcriptional activation, there is a significant lack of knowledge regarding the mechanistic details of p300 on initiation of Notch-directed transcription (12, 13). Evidence suggests that p300 is present on promoters prior to Notch localization, indicating that Notch does not recruit p300 to chromatin (14). Furthermore, p300 has been shown to acetylate Maml1 (15). However, the role that acetylation of Maml1 plays in activation of Notch-directed transcription remains unknown. Here, we report that Maml1 is acetylated by p300 on lysine residues 188 and 189, and that this acetylation of Maml1 drives the recruitment of NACK to the Notch ternary transcription complex and subsequent recruitment of RNA polymerase II, thereby initiating transcription.

293T cells were obtained from ATCC. OE33 and OE19 cell lines (human esophageal adenocarcinoma) were obtained from the European Collection of Cell Culture. 786-0 cell lines (human renal adenocarcinoma) was obtained from ATCC. All cell lines were tested for mycoplasma contamination and propagated in growth media as specified by the provider. All cell lines were used for experiment within 2 months of thawing. All cell lines were obtained between 2008 and 2016 and authenticated by ATCC (cell line authentication utilizing short tandem repeat profiling).

Transfections were performed using LipoJet transfection reagent (SL100468, SignaGen Laboratories) according to the manufacturer's recommended protocol.

DNA affinity purification (DAP) assay was performed in 6 cm dish. DNA (4 μg) was transfected (0.1 μg N1^ICD-pcDNA, 1 μg Maml1-pcDNA, 1.5 μg NACK-pcDNA, 1 μg p300-pcDNA, and the empty pcDNA vector was used where necessary to bring the total amount to 4 μg). Media were replaced 16 hours after transfection and cells were harvested 48 hours after transfection.

Chromatin immunoprecipitation (ChIP) assay was performed in 10 cm dish and the following DNAs were transfected: 0.1 μg N1^ICD-pcDNA, 1 μg Maml1-pcDNA, and 1 μg NACK-pcDNA. Media were replaced 16 hours after transfection and cells were harvested 48 hours after transfection.

p300, PCAF, and NACK siRNA smart pools were purchased from Santa Cruz Biotechnology and transfected using LipoJet transfection reagent (SL100468, SignaGen Laboratories) according to the manufacturer's recommended protocol.

DAP was performed as described previously (11).

Quantitative RT-PCR was performed as described previously (11). Gene expression was normalized to HPRT1.

OE33 and 786-0 cells were treated with 10 μmol/L C646 for 6 hours or transfected with p300 or PCAF siRNA smart pool for 24 hours. For ChIP assay in 293T, cells were transfected and harvested as described in “Plasmid Transfection” section.

ChIP assays were performed as described previously (11). Lysates were immunoprecipitated with anti-Notch1 (Bethyl Laboratories), anti-Maml1 (Cell Signaling Technology), or anti-NACK (anti-pragmin, Bethyl Laboratories), anti-RNA polymerase II (EMD Millipore), anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5; Abcam), anti-Histone H3 (Abcam), anti-Histone H3 (acetyl K27; Cell Signaling Technology). The HES1 promoter was amplified by qPCR with primers (forward: 5'-CGTGTCTCCTCCTCCCATT-3'; reverse: 5'-GGGGGATTCCGCTGTTAT-3'). The HES5 promoter was amplified by qPCR with primers (forward: 5'-GGGAAAAGGCAGCATATTGA-3'; reverse: 5'-CACGCTAAATTGCCTGTGAA-3'). The negative site was amplified by qPCR with primers (forward: 5'-AATGCTGGGCTTCCAAGGA-3'; reverse: 5'-GACCTTGGTGACTGTTGAGGAAAC-3').

Western blot was performed as described previously (11). Primary antibodies were anti-Flag (1:5,000; Sigma), anti-NACK (1:1,000; against aa209-287 of NACK and affinity purified), anti-CSL (1:1,000; generated against full-length CSL and affinity purified), anti-p300 (1:1,000; Abcam), anti-p300 and CBP (1:1,000; Sigma-Aldrich), anti-HSP90 (1:5,000; Santa Cruz Biotechnology), anti-Ac-lysine (1:5,000; Cell Signaling Technology), anti-Maml1 (1:5,000; Cell Signaling Technology), anti-cleaved-Notch1 (1:1,000; Cell Signaling Technology).

OE33 and 786-0 cell lysates were incubated with anti-Maml1 antibody (Cell Signaling Technology) and rProtein G beads (15920-010, Invitrogen) for 4 hours. Beads were washed for 5 minutes three times. Protein bound to the beads was analyzed by Western blot analysis with anti-Maml1 (1:1,000; against aa1-305 of Maml1 and affinity purified); anti-Ac-K188-Maml1 (1:1,000; produced and affinity purified by Thermo Fisher Scientific) and anti-Ac-K189-Maml1 (1:1,000; produced and affinity purified by Thermo Fisher Scientific).

Cell viability assays were performed using the CellTiter-Glo Kit (G7572, Promega) according to the manufacturer's recommended protocol.

Animal experiments were approved by the University of Miami Institutional Animal Care and Use Committee. Patient-derived xenograft (PDX) cancer models were established in NSG mice (The Jackson Laboratory) as described (16). Drug treatment was initiated when the tumor size reached 200 mm³. Tumor volume was determined by the formula: volume = (S × S × L)/2.

Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay was performed using DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer's recommended protocol.

p300 protein was purified through the bac-to-bac system; Maml1-305 GST fusion protein was produced in bacteria. Proteins were incubated with ³H-labeled acetyl-CoA and radioactive activity was detected by film exposure. Protein level was determined by Western blot analysis and colloidal staining.

All statistical analyses were performed using Student t test. All data represent three independent experiments unless indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

For animal experiments, P value was calculated using χ² in contingency table. The sample size was chosen to be greater than the minimal sample size from power assessment as described previously (17). Data are presented as mean ± SEM and were analyzed by two-tailed Student t test. A P value of less than 0.05 was considered significant.

Previously, we identified NACK as a novel Notch coactivator and demonstrated that NACK is critical for Notch1-dependent transcriptional activity and tumorigenesis (11). NACK binds to the Notch1 ternary complex in a Maml1-dependent manner. However, the mechanism by which Maml1 mediates this interaction is unclear. Maml1 is acetylated by p300, which appears to act as a coactivator of Notch transcription, although it is unclear how p300 potentiates Notch-driven transcriptional activity (12, 13, 15). Taken together, we reasoned that the acetylation on Maml1 by p300 may be the critical event leading to the loading of NACK onto the Notch ternary complex and thereby initiates transcription.

To investigate the loading of NACK onto the Notch ternary complex, we isolated the Notch1 ternary complex by CSL DAP assay. The Notch1 ternary complex was assembled in 293T cells by cotransfecting Notch1 intracellular domain (N1^ICD), Maml1, and NACK. This complex was then isolated by DAP assay with CSL site containing oligonucleotides. In the presence of N1^ICD and Maml1, NACK was precipitated together with the complex. However, when NACK was transfected without N1^ICD and Maml1, NACK was not precipitated on DNA. These data indicate that the isolation of NACK by CSL DAP assays depends on the formation of the Notch1 ternary complex (Fig. 1A, lanes 6 and 7). To investigate whether p300 has an effect on NACK recruitment, we cotransfected p300, N1^ICD, Maml1, and NACK into 293T cells, and performed CSL DAP assays. When p300 was ectopically expressed, the levels of N1^ICD, Maml1, and CSL in the complex were unaffected, indicating that p300 does not directly affect ternary complex formation. However, the level of NACK in the complex was dramatically enhanced by inclusion of p300. Moreover, the total level of NACK was not changed, indicating that p300 regulates NACK recruitment instead of NACK protein level (Fig. 1A, lanes 7 and 8). To demonstrate whether the effect of p300 was a result of the acetyltransferase activity of p300, we performed the CSL DAP assay using an acetyltransferase-deficient p300 (termed p300.DY). p300.DY failed to enhance the recruitment of NACK (Fig. 1A, lanes 8 and 9), indicating that p300 enhances NACK recruitment to the Notch1 ternary complex through acetylation.

Consistent with previously published results (15), Maml1 in both input and isolated complex was acetylated by p300 (Fig. 1A, lanes 3 and 8). To identify the critical residues involved in acetylation-dependent NACK binding, we mutated five potential p300-acetylated lysine residues (K67, K69, K73, K188, and K189) in Maml1 to arginine [termed Maml1(5S)]. Maml1(5S) was expressed equally compared to wild-type Maml1, but the acetylation by p300 was largely blocked (Fig. 1A, lanes 3 and 5). CSL DAP assay was then performed with Maml1(5S), and NACK recruitment to the Notch1 ternary complex was largely blocked, although the Maml1 protein level in the complex remained equivalent to wild type Maml1 (Fig. 1A, lanes 8 and 10), indicating that acetylation on Maml1 plays a critical role in NACK recruitment.

As a member of the histone acetyltransferase (HAT) protein family, p300 shares similar properties with other family members. Therefore, we were interested in determining whether other HAT proteins can also regulate NACK recruitment to the Notch1 ternary complex. CREB-binding protein (CBP) and p300/(CREB-binding protein) associated factor (PCAF) were tested, because both of them had been reported to be involved in Notch signaling (13, 18). CBP and PCAF were cotransfected with N1^ICD, Maml1, and NACK into 293T cells and subjected to CSL DAP assays. p300 and CBP acetylated Maml1 (Fig. 1B, lanes 2–4) and increased NACK recruitment to the Notch1 ternary complex (Fig. 1B, lanes 7–9). In contrast, PCAF had no effect on Maml1 acetylation or NACK recruitment (Fig. 1B, lanes 5 and 10).

To further validate the regulatory function of p300 on the Notch1 complex, p300 siRNA smart pool and C646 (p300/CBP inhibitor) were used. The p300 siRNA smart pool was cotransfected and knocked down approximately 50% of endogenous p300 at the protein level (Fig. 1C, lanes 2 and 3). Although the NACK amount in the complex was largely reduced, N1^ICD, Maml1, and CSL protein levels remained the same (Fig. 1C, lanes 5 and 6). Consistent with this observation, C646 treatment of N1^ICD-, Maml1-, and NACK-transfected 293T cells inhibited NACK recruitment in a dose-dependent manner without affecting N1^ICD, Maml1, or CSL levels (Fig. 1D). These results further support the hypothesis that p300 regulates the interaction between NACK and the Notch1 ternary complex, but does not affect ternary complex formation.

Our lab previously demonstrated that N^Δ2105 (N1^ICD containing internal deletion of aa 2105-2114) is unable to initiate transcription or recruit NACK to the Notch1 ternary complex (11, 19). Furthermore, it was shown that an overlapping region of Notch1 (aa 2109-2123) is critical for the interaction between Notch1 and p300 (12). Therefore, we reasoned that aa 2105-2114 of Notch1 may be important for the acetylation of Maml1 by p300, leading to the regulation of NACK recruitment and Notch transcriptional activity. To test this hypothesis, we cotransfected 293T cells with p300, N1^ICD, N^Δ2105, Maml1, and NACK, and performed CSL DAP assays. In the absence of ectopically expressed p300, N^Δ2105 was able to form the ternary complex, but unable to recruit NACK to the complex (Fig. 1E, lanes 5 and 7). Moreover, in the presence of ectopically expressed p300, acetylation of Maml1 is dramatically blocked when N^Δ2105 is cotransfected (Fig. 1E, lanes 2 and 4), and the recruitment of NACK to the ternary complex containing N^Δ2105 is attenuated (Fig. 1E, lanes 6 and 8). These data indicate that Notch1 plays a role in regulation of Maml1 acetylation, leading to the subsequent recruitment of NACK.

p300 regulates NACK recruitment to the isolated Notch1 ternary complex, therefore we reasoned that p300 should also play a role in regulating NACK occupancy on Notch target gene promoters. In contrast to the ectopically expressed proteins in 293T cells, a Notch-dependent esophageal cancer cell line, OE33, was used to monitor the relationship between p300 and endogenous Notch-dependent transcriptional activity. ChIP assays determined the occupancy of NACK, Notch1, and Maml1 on the HES1 promoter (Supplementary Fig. S1A). The p300 siRNA smart pool was transfected into OE33 cells to knockdown p300 and greater than 80% knockdown was achieved (Supplementary Fig. S1B). When the p300 level was significantly reduced, the occupancy of NACK on the HES1 promoter was dramatically reduced. However, the occupancy of Notch1 and Maml1 on the HES1 locus was not significantly changed, indicating that in these cells, p300 regulates NACK recruitment to the Notch ternary complex, as was observed in the 293T reconstitution experiments (Fig. 2A). Moreover, RT-qPCR assay demonstrated that the Notch target genes HES1 and HEY1 were downregulated, whereas TBP, which is not a Notch target gene, remained unchanged (Fig. 2B). Since C646 is a potent inhibitor of p300 and CBP, we reasoned that treatment of cells with C646 should duplicate the p300 siRNA smart pool results. Cells treated for 6 hours with 10μmol/L C646 were subjected to ChIP assay and the occupancy of NACK on the HES1 locus was determined. Treatment of cells with C646 dramatically inhibited localization of NACK to the HES1 locus without affecting the occupancy of Notch1 and Maml1. Similarly, C646 reduced transcription of the HES1 and HEY1 loci without affecting the TBP control (Fig. 2A and C). To extend these results to additional cell lines, the same experiments were performed in another Notch-dependent cell line, 786-0. Both p300 siRNA smart pool and C646 blocked NACK presence on the HES1 promoter and inhibited Notch target gene expression (Supplementary Fig. S1C, S1D, S1E, and S1F). These results indicate that p300 does not affect Notch1–Maml1–CSL ternary complex assembly on the HES1 promoter, but regulates NACK recruitment to the complex. Considering Notch1 and Maml1 occupancy remained the same and NACK functions as a coactivator of Notch signaling (11), these data indicate that p300 critically regulates Notch transcriptional activity through control of NACK occupancy.

Because PCAF had no effect on the recruitment of NACK in CSL DAP assay (Fig. 1B), we were interested in determining whether PCAF regulates the occupancy of Notch1, Maml1, and NACK on the HES1 promoter. We transfected PCAF siRNA smart pool into OE33. When PCAF was knocked down, the acetylation on Histone 3 lysine 9, one of the substrates of PCAF (20), was attenuated (Fig. 2D). However, the mRNA level of Notch target genes, HES1 and HEY1, remained the same (Fig. 2E and F). Moreover, the occupancy of Notch1, Maml1, and NACK on the HES1 promoter remained unchanged. These results demonstrate the specificity of p300 in the regulation of Notch transcriptional complex formation.

We identified five potential p300 acetylation lysine sites on Maml1 (K67, K69, K73, K188, and K189) and mutations at all these sites [termed Maml1(5S)] were able to block the majority of Maml1 acetylation (Supplementary Fig. S2A). In addition, NACK recruitment on the Notch1 ternary complex was largely blocked when these sites were mutated (Fig. 1A, lanes 8 and 10), indicating that one or more of these 5 residues regulate the interaction between NACK and the Notch1 ternary complex.

To determine which residues are important for the interaction between NACK and Maml1, five lysine residues were mutated to arginine in two groups [Maml1(3S) for K67R/K69R/K73R and Maml1(2S) for K188R/K189R]. The mutations in both groups reduced acetylation on Maml1 by p300 (Supplementary Fig. S2A), indicating at least one lysine residue in each group is acetylated by p300. Wild-type Maml1, Maml1(5S), Maml1(3S), and Maml1(2S) were individually cotransfected with N1^ICD and NACK into 293T cells. CSL DAP assays of cell lysates revealed that although all mutants maintained the ability to form the ternary complex with N1^ICD and CSL at similar levels of N1^ICD, Maml1, and CSL, Maml1(2S) failed to recruit NACK, but Maml1(3S) was not affected (Supplementary Fig. S2B, lanes 5–8), indicating that one or both of K188 and K189 are critical for NACK recruitment. Single mutations (K188R and K189R) were generated and tested in a similar manner. The result demonstrated that both K188R and K189R dramatically reduced the level of NACK without reducing N1^ICD and Maml1 (Fig. 3A, lanes 7, 9, and 10), indicating that both K188 and K189 are important for the interaction between NACK and the Notch1 ternary complex.

To explore whether endogenous Maml1 is acetylated at the K188 and K189 sites, antibodies directed against Ac-K188 Maml1 and Ac-K189 Maml1 were generated and validated (Supplementary Fig. S2C). Endogenous Maml1 from OE33 and 786-0 cell lines was immunoprecipitated by anti-Maml1 antibody and immunoblotted with Ac-K188- and Ac-K189-specific antibodies. This analysis revealed that endogenous Maml1 is acetylated on both K188 and K189 and that acetylation on both of these residues can be blocked by C646 in a dose-dependent manner (Fig. 3B), indicating that endogenous Maml1 is acetylated by p300/CBP on both K188 and K189 residues.

The Maml family is composed of three paralogs in mammals, termed Maml1, Maml2, and Maml3. To determine whether Maml2 and Maml3 also form complexes with NACK, CSL DAP assays were performed with Maml2- and Maml3-transfected 293T cells. Both Maml2 and Maml3 formed a complex with N1^ICD and CSL; however, Maml3, but not Maml2, was able to recruit NACK (Fig. 3C, lanes 6–8), indicating that Maml3 has the ability to recruit NACK, but Maml2 does not.

Sequence alignment of Maml1 and Maml3 reveals that amino acid residue K245 on Maml3 is in a similar context to K188/K189 in Maml1 (Fig. 3D), indicating that K245 in Maml3 may play an important role in NACK recruitment. Therefore, we replaced residue K245 by arginine (termed Maml3^K245R) to test this hypothesis. Maml3^K245R was cotransfected with N1^ICD and NACK into 293T cells and CSL DAP assays were performed. The K245R mutation completely blocked recruitment of NACK to the Notch1^ICD–Maml3–CSL complex without affecting ternary complex formation (Fig. 3E, lanes 5 and 6), indicating that residue K245 regulates the interaction between NACK and the Notch1–Maml3–CSL ternary complex.

Sequence alignment between Maml1 and Maml2 shows less homology than the alignment between Maml1 and Maml3. Furthermore, Maml2 aa sequence does not have lysine residues with similar contexts to K188/K189 in Maml1. However, all of Maml paralogs have a Notch-CSL binding region at the N-terminus (Fig. 3D). This likely explains why Maml2 is able to form the Notch1^ICD ternary complex, but cannot recruit NACK.

Although NACK acts as a Notch coactivator, the mechanism is unclear. Because NACK seems to function downstream of the Notch ternary complex, we reasoned that NACK could be responsible for recruitment of the general transcriptional machinery, such as RNA polymerase II. To investigate this hypothesis, we performed ChIP assays on the promoter of Notch target genes. Following the transfection of NACK siRNA smart pool into OE33 cells, ChIP assays revealed that the occupancy of both RNA polymerase II and serine-5-phosphorylated RNA polymerase II on the HES1 promoter was reduced by similar percentage (Fig. 4A), indicating that NACK is responsible for the recruitment, but not the activation of RNA polymerase II. mRNA levels of HES1 and HEY1 were significantly reduced (Fig. 4B), indicating that the transcriptional activity of Notch signaling was attenuated. These data indicate that NACK plays a role in the initiation of transcription through the recruitment of RNA polymerase II.

NACK is predicted to have a kinase-like domain at the C-terminus (11). Therefore, we were interested whether ATP regulates the recruitment of NACK to the Notch1 ternary complex. To address this question, we performed CSL DAP assays with N1^ICD-, Maml1-, and NACK-transfected 293T cells. ATP was added to the cell lysates being incubated with CSL-binding DNA-conjugated beads. The results revealed that ATP enhances the recruitment of NACK to the Notch1 ternary complex in a dose-dependent manner, without affecting the level of N1^ICD, Maml1, and CSL (Fig. 4C). To further validate this result, the key residue of NACK for ATP binding, K1002, was mutated into alanine [termed NACK(K)] and a CSL DAP assay was performed. The result revealed that NACK(K) does not affect the level of N1^ICD, Maml1, and CSL in the Notch1 ternary complex; however, NACK(K) cannot bind to the ternary complex (Fig. 4D).

To further determine the role of NACK in RNA polymerase II recruitment, 293T cell line was transfected with N1^ICD, Maml1, and NACK. HES5 was dramatically induced (Fig. 4E). ChIP assay was performed on the promoter of HES5. After the cotransfection of N1^ICD, Maml1, and NACK, the mRNA level of HES5 was dramatically induced, as well as the occupancy of RNA polymerase II. Then, wild-type Maml1 was replaced with Maml1(2S) or wild-type NACK was replaced with NACK(K). Although protein expression levels were similar (Fig. 4F), there was no increase in either the mRNA level of HES5 or the occupancy of RNA polymerase II (Fig. 4G and H). Because Maml1(2S) and NACK(K) only affect recruitment of NACK to the Notch1 ternary complex, but not the stability of the complex (Fig. 3A and 4D), these data indicate that NACK is critical for RNA polymerase II recruitment and subsequent Notch transcriptional activity.

Given that C646 inhibits NACK recruitment and NACK is required for Notch-directed transcription, we reasoned that C646 and DAPT (a gamma-secretase inhibitor) should have a synergistic effect on inhibiting Notch-dependent tumor cell growth.

Both Notch and NACK are critical for the survival of EAC tumors (11, 21). Oncomine analysis revealed that CBP has a higher expression in EAC tumors, compared to the normal esophageal tissue (Supplementary Fig. S3A). Therefore, we used EAC cell lines, OE33 and OE19, to test our hypothesis through CellTiter-Glo viability assays. OE33 cells are sensitive to DAPT with an EC₅₀ of 400 nmol/L, whereas for OE19 the EC₅₀ with DAPT is more than 10 μmol/L in this assay. C646 treatment of OE33 cells revealed an EC₅₀ of approximately 3 μmol/L, whereas OE19 cells were largely refractory (Supplementary Fig. S3B and S3C). However, when DAPT was combined with C646 we observed a reduction of DAPT EC₅₀ in both OE19 and OE33 cell lines (EC₅₀ of OE19 decreased from more than 10 μmol/L to 30 nmol/L; EC₅₀ of OE33 decreased from 300 nmol/L to 30 nmol/L; Fig. 5A and B).

To investigate whether DAPT and C646 have a synergistic effect on Notch transcriptional activity, OE33 cells were treated with DAPT and C646 individually and in combination, and the mRNA level of the Notch target gene, HES1, was determined by q-PCR assay. Treatment with both DAPT (0.3 μmol/L) and C646 (0.3 μmol/L) caused a greater inhibition than DAPT (0.3 μmol/L) alone (Fig. 5C, lanes 2 and 6), whereas the protein levels of activated Notch1 (Notch1 cleaved at Val1744) were equivalent (Fig. 5D, lanes 2 and 6), indicating C646 does not change activated Notch1 protein levels, but attenuates the activity of Notch1. Moreover, the synergy analysis using Calcusyn software for the combination of DAPT and C646 at ratio of 1:10 showed that the combination displays synergism (CI < 1; Fig. 5E). To determine whether the synergistic effect also works on mice, the EAC47 PDX model was treated with DAPT (5 mg/kg) and C646 (0.5 mg/kg) individually or in combination. The combination treatment led to a significant reduction in tumor weight and size, whereas the single treatments displayed no efficacy, similar to DMSO treatment (Fig. 5F and G). TUNEL assay on the tumor samples revealed that the combination treatment led to an induction of apoptosis, whereas the single treatments did not (Fig. 5H). These results indicate that the combination of subefficacious doses of C646 and DAPT cooperate, and provide proof of concept for combination therapy of a p300/CBP and a Notch inhibitor as a treatment for cancer.

Assembly of the Notch transcriptional activation complex proceeds in a stepwise manner that can be described as having two stages: the formation of Notch–Maml–CSL core complex (ternary complex) and the subsequent recruitment of additional cofactors that mediate activity. This idea is based on experimental evidence demonstrating that specific mutations in Notch and Maml that are competent in ternary complex formation fail to initiate Notch-dependent transcription (19, 22). We recently described NACK as an important Notch transcription complex cofactor (11). However, the mechanistic details of how NACK functions are not understood. Herein, we demonstrate that p300-and CBP-mediated acetylation of Maml1 regulates the interaction between NACK and Notch ternary complex. In addition, Maml paralogs have differential abilities to recruit NACK to the ternary complex. Furthermore, NACK functions in the initiation of transcription through recruitment of RNA polymerase II to the promoter. Moreover, we demonstrate that simultaneous inhibition of p300/CBP and Notch displays a synergistic effect and, therefore, provide a rationale for a combinatorial therapeutic strategy for Notch-dependent cancers.

It has long been appreciated that p300 acts as an important Notch coactivator. p300 has been demonstrated to interact with Notch1 and Maml1 and potentiate Notch-mediated transcriptional activation (12, 13). However, the mechanistic details describing the role of p300 in Notch-mediated transcription are not clear. Although several reports have shown that Maml1 is acetylated by p300 on multiple lysine residues, no activity has yet been attributed to acetylation of Maml, and it is not clear in which step of Notch-mediated transcription process p300 is involved (15, 23). Fryer and colleagues demonstrated that p300 was localized on the HES1 promoter prior to activation of Notch, indicating that p300 may play a role in assembly and/or modulation of the Notch transcriptional complex (14). Herein, we demonstrate the functional role of p300 in Notch-mediated transcriptional activation. We propose that p300 acetylates Maml1 on lysines 188 and 189, creating a high-affinity template for NACK recruitment. NACK recruits RNA polymerase II, leading to transcription initiation. Although we do not fully understand how NACK recruits RNA polymerase II, we now can provide a sequence of events in Notch-mediated transcription activation and a direct role of p300 in the process. What does this imply for Notch signaling? As proposed, this model indicates that the activity of Notch transcriptional complexes can be regulated locally and that Notch target genes could be activated differentially, depending on the presence of p300. For example, on promoters with preloaded p300, the Notch transcriptional complex would form and Maml would subsequently be acetylated, resulting in the recruitment of NACK, thereby initiating transcription. Conversely, on promoters without preloaded p300, Notch transcriptional complexes may form, however, they would be blocked at the acetylation step, resulting in no recruitment of NACK and failure to initiate or sustain a Notch-dependent transcriptional response. Therefore, epigenetic modulation of promoters would influence the Notch transcriptional response. This model can explain, at least in part, why Notch target gene profiles are cell-context dependent.

What role do Maml proteins serve in directing a Notch transcriptional event? In Drosophila, there is a single Maml gene. However, in mammals, there are three Maml paralogs (24). One interpretation of this is that Notch transcriptional activation complexes can vary depending on the specific Maml protein that is utilized. Notch, Maml, and CSL form the core ternary complex, which is the template for further recruitment of cofactors to form a higher-order transcriptional complex. Therefore, by extrapolation, Maml proteins may initiate specific Notch-dependent transcriptional events. Many studies have demonstrated that all three Maml paralogs are able to form ternary complexes with Notch and CSL, and act as Notch coactivators (25–28). Thus, the different roles of Maml paralogs may depend on the cofactors that they in turn recruit. We present the first evidence to support the hypothesis that Notch transcription cofactors have selectivity to Maml paralogs. That is, NACK is readily recruited to ternary complexes composed of Maml1 and Maml3, but not Maml2. Why do mammalian cells need Notch transcriptional complexes with different cofactors? Although at this time we cannot adequately answer this question, one explanation may be that the intensity and/or duration of Notch signaling may be required to be differentially regulated based on specific contextual needs, which can be precisely controlled through the use and modification of Maml paralogs.

Precision medicine cancer therapy is based on the idea that targeting specific pathways critical to the neoplastic phenotype of specific neoplasms will have greater efficacy and limited toxicity due to a greater therapeutic index. This principle can be further extended to the idea that targeting a pathway at multiple nodes will be more effective by inhibiting that pathway in a synergistic manner. If this is indeed the case, then targeted combination therapies can be utilized at much lower doses to achieve a greater effect than doses of individual drugs approaching the maximum tolerated dose or those with observable adverse effects. Because p300 activity is required for Notch transcriptional output by directing the recruitment of NACK, we reasoned that simultaneous inhibition of p300 and Notch would be synergistic in esophageal adenocarcinoma. To this end, we utilized cell lines and PDX models of esophageal carcinoma to clearly demonstrate that at doses for C646 and DAPT that are less than EC₁₀ individually, there is a dramatic inhibition of viability when they are combined to treat EAC cell lines. Furthermore, when mice harboring PDX tumors were treated with doses of DAPT and C646 that individually had no effect on the growth of the tumor, the combination caused significant inhibition of tumor growth.

In summary, this study provides a deeper mechanistic understanding of the assembly of the Notch transcriptional complex and the Notch transcription activity. Furthermore, we provide mechanistic details for the role of p300 in the assembly of the Notch transcriptional complex, and present evidence for the specificity of Maml paralogs in the function of Notch transcriptional activation complexes. Therefore, this study provides the rationale and proof of concept for a combinatorial therapeutic attack on Notch-dependent cancers.

No potential conflicts of interest were disclosed.

Conception and design: K. Jin, S. Jeffries, D.J. Robbins, A.J. Capobianco

Development of methodology: K. Jin, X. Han, Z. Wang, B. Li, S. Jeffries

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Jin, W. Zhou, Z. Wang, B. Li, S. Jeffries, W. Tao

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Jin, W. Zhou, D.J. Robbins

Writing, review, and/or revision of the manuscript: K. Jin, W. Zhou, X. Han, W. Tao, A.J. Capobianco

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Jin

The authors thank members of the Capobianco and Robbins laboratory for support and technical assistance, and Shuhua Zheng (University of Miami) for assistance in synergy analysis.

This work was supported by the NCI (NCI R01CA083736-12A1, NCI R01CA125044-02 to A.J. Capobianco). This project was also generously supported by funding from the Dewitt Daughtry Family Department of Surgery and the Sylvester Comprehensive Cancer Center to A.J. Capobianco.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.