CREB-binding Protein Research Articles

Abstract A047: Discovery of potent and paralog selective PROTAC degraders of CBP or p300 proteins for the treatment of various cancers

Abstract E1A binding protein (p300) and its paralog CREB binding protein (CBP or CREBBP) are ubiquitously expressed histone acetyl transferases (HAT). They act by scaffolding or as co-activator and enhancer of different transcription factors like HIF1a, BRCA-1, p53, c-Myc, PD-L1, Estrogen receptor (ER) and Androgen receptor (AR). CBP and p300 are multidomain proteins that harbour different functional units imperative for chromatin remodelling and transcription like Bromodomain (BD), Histone acetyl transferase (HAT) domain, KIX domain etc. These two closely related epigenetic modulators are known to play oncogenic role in a variety of cancers. Conventional CBP/p300 inhibitors targeting either BD, HAT or KIX domain do not discriminate between CBP and p300 proteins due to high homology. Because of the indiscriminate inhibition of both proteins, HAT domain inhibitors showed tolerability issues preventing their clinical advancement. In contrast, bromodomain inhibitors are better tolerated because they only block coactivation function at the transcription level without impacting the scaffolding or global histone acetylation function. They show less than desirable efficacy in various cancer models within very well tolerated doses. The implication of selective inhibition of either of these proteins in various cancer has been reported in the literature due to differential dependency on either of the paralogs. A synthetic lethal relationship between these paralogs in cancer set up has also been well established recently. However, none of the reported inhibitors of the various domains described above could selectively target CBP or p300 alone due to high sequence homology. Paralog selectivity in recent time has been proved to be possible with degrader approach due to differentiated ternary complex formation. Structure-guided modeling and iterative medicinal chemistry approaches were applied to identify paralog selective and potent first in class degraders with diverse linker length and linker composition. Identified compounds showed pronounced paralog selective degradation of target proteins and cellular effects in a panel of cell lines with loss of function mutation for either of the paralog proteins but not in wild type cells. The paralog selective lead degraders are currently being further optimized to identify development candidates. Selective degraders of CBP and p300 are expected to enhance efficacy of SOC drugs or immune checkpoint blockers by governing the acetylation and transcription of oncogenic factors and co-stimulatory molecules involved in tumor progression. Citation Format: Chandrasekhar Abbineni, Saravanan Thiyagarajan, Krishna Chaitanya T, Achala Apte, Naveen Kumar R, Avinash Kumar, Dabbeeru Madhu Babu, Shwetha S, Aishwarya B Kamath, Monalisha Mandal, Rituparna Panigrahi, Sivapriya Marappan, Subhendu Mukherjee, Kavitha Nellore, Shekar Chelur, Murali Ramachandra, Susanta Samajdar. Discovery of potent and paralog selective PROTAC degraders of CBP or p300 proteins for the treatment of various cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A047.

Abstract C085: OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC

Abstract Metastatic castration-resistant prostate cancer (mCRPC) remains an urgent unmet medical need despite existing therapies that target androgen receptor (AR) signaling. Disease progression is commonly a result of acquired resistance to existing anti-androgen therapies. Biological models have revealed potential resistance mechanisms of CRPC through the reactivation of the AR pathway. Transcriptional coactivators CREB-binding protein (CBP) and E1A binding protein (EP300) are high value targets in mCRPC as they mediate androgen-dependent and -independent activity of the AR pathway. EP300 and CBP proteins harbor both histone acetyltransferase (HAT)- and bromo-domains, and the HAT activity of EP300 regulates AR function. EP300 bromodomain inhibition partially blocks its HAT activity, reducing AR-mediated gene expression and tumor growth. OPN-6602 and OPN-6742 are potent, selective, and orally active small molecule dual EP300/CBP bromodomain inhibitors which abrogate persistent AR signaling in CRPC by modulating H3K27 acetylation and the recruitment of EP300, AR full length (AR-FL), and AR variant 7 (AR-v7) to AR response elements. OPN-6602 and OPN-6742 obstruct the EP300 bromodomain leading to downregulation of the AR pathway and consequent anti-tumor effects in AR-dependent CRPC. OPN-6602 and OPN-6742 are highly selective (>100-fold) against other bromodomains based on a screening panel of 40 bromodomains. Biochemical screens using AlphaScreen technology show these compounds to be equipotent against EP300 and CBP. OPN-6602 is particularly potent by Surface Plasmon Resonance analysis, displaying a KD of 0.87 nM, with a fast association rate (1.5 E+06/Ms) and a slow dissociation rate (1.3 E-03/s). Consequently, OPN-6602 displays a noteworthy residence time of 13 minutes. Both compounds show an AR-selective mode of action with anti-proliferative activity in AR-dependent prostate cancer cell lines (e.g. VCaP, LNCaP, and 22Rv-1) but are inactive against AR-negative prostate cancer cell lines (e.g. DU145). VCaP and mCRPC patient-derived xenograft (PDX) RNA profiling provides evidence of a marked, dose-dependent, pharmacodynamic response indicative of EP300/CBP inhibition. In the VCaP xenograft model, OPN-6602 and OPN-6742 show dose-dependent tumor growth inhibition and corresponding pharmacodynamic responses with prostate-specific antigen level decreases and decreased H3K27 acetylation. In the QR (AR-v7+) PDX model of CRPC (post-androgen deprivation therapy), a synergistic effect was observed between OPN-6602 and enzalutamide. This combination further modulates the expression of AR-driven transcripts such as MYC compared to single agent treatments. The pharmacokinetic profiles of OPN-6602 and OPN-6742 are favorable for oral single daily dose administration due to a high Cmax and short half-life. Due to its higher potency, OPN-6602 has been chosen as clinical candidate and a first-in-human study will start in 2024. Citation Format: Bernice Matusow, Wayne Spevak, Chao Zhang, Yan Ma, Rafe Shellooe, Peipei Li, James Tsai, Pan- Yu Chen, Parmveer Singh, Jackie Walling, Christine Nichols, Jason Halladay, Kerry Inokuchi, Gaston Habets, Gideon Bollag. OPN-6602, a dual EP300/CBP bromodomain inhibitor modulates androgen-driven transcription, including MYC, in mCRPC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C085.

Targeting CREB-Binding Protein (CREBBP) Overcomes Resistance to Azacitidine and Venetoclax Therapy in Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is a clonal myeloid malignancy arising from hematopoietic stem or progenitor cells (HSPC) within the bone marrow (BM). Despite advancements in our understanding of AML pathogenesis, patients continue to face grim outcomes, with a five-year relative survival rate of approximately 20%. This highlights the pressing need to identify molecular features of AML that are amenable to therapeutic intervention. The ideal molecular targets would be those broadly dysregulated and integral to multiple signaling pathways implicated in AML pathogenesis. Recent therapeutic regimens have incorporated venetoclax, a small molecule inhibitor of BCL2, in combination with hypomethylating agents, showing moderate success. Venetoclax (VEN) acts as a BH3 mimetic, specifically inhibiting BCL2. While clinical outcomes of VEN combined with hypomethylating agents (decitabine or azacitidine - AZA) have been promising, a substantial subset of patients remains refractory or experiences relapse following treatment with these regimens. Due to the molecular and cellular heterogeneity of AML, identifying ideal mechanisms to circumvent resistance remains elusive. In our investigations, we have observed that patient-derived cells from AML exhibit a higher frequency of dysregulated immune and inflammatory pathways than previously appreciated, referred to as “oncogenic immune signaling states.” Furthermore, immune and inflammatory-related pathways are preferentially expressed in AML patients who are resistant/refractory to VEN/AZA compared to those who respond favorably. To identify critical immune and inflammatory-related pathways contributing to VEN/AZA responses, we developed an oncogenic immune signaling-targeted CRISPR-library, aimed at identifying potential therapeutic targets involved in VEN/AZA resistance in AML. Utilizing this targeted CRISPR-screen approach, we successfully identified CREB-binding protein (CREBBP) as the top candidate linked to resistance to VEN/AZA therapy. Notably, CREBBP alterations have been associated with a poor prognosis in de novo AML, making it a compelling target for further investigation. To validate CREBBP's role in VEN/AZA resistance, we treated a VEN/AZA-resistant AML cell line, THP1 (FAB subtype M5, KMT2A-r), with commercially available pharmacologic inhibitors of CREBBP, A485, and CCS1477, and assessed the response. Remarkably, inhibition of CREBBP with either A485 or CCS1477 significantly decreased cell proliferative ability and clonogenic potential. Importantly, co-treatment with either A485 or CCS1477 and VEN/AZA synergistically abrogated cell proliferation and clonogenic potential of THP1 cells, effectively overcoming resistance (Figure 1). Our ongoing studies include validating CREBBP as a potential therapeutic target for overcoming VEN/AZA resistance in AML. We have created CREBBP-knockdown cell lines through genetic manipulation (shRNA) and will utilize these to confirm the specificity of CREBBP in modifying resistance to VEN/AZA. In addition we will utilize patient-derived xenograft studies in immunocompromised (NRG) mouse models. By investigating CREBBP's role in mediating resistance, we aim to unlock new avenues for enhancing the efficacy of VEN/AZA therapy in AML patients, particularly those facing resistance to current therapies. In conclusion, our research highlights the critical involvement of CREBBP in VEN/AZA resistance in AML. Targeting this molecular pathway holds the promise of offering a potential therapeutic approach to improve treatment outcomes for AML patients, particularly those facing resistance to current therapies. Understanding and harnessing CREBBP's influence could pave the way for developing personalized and effective strategies to combat this challenging hematological malignancy.

Encouraging Outcome of AML with t(8;16) Following Allogeneic Stem Cell Transplantation in First Remission - a Retrospective Analysis on Behalf of the Acute Leukemia Working Party of the EBMT

In the 2022 European Leukemia Net (ELN) risk classification, acute myeloid leukemia (AML) with translocation t(8;16) was newly classified as AML defined by specific cytogenetic abnormalities, associated with unfavorable prognosis. Translocation (8;16)(p11;p13) is a rare abnormality in AML with female preponderance and occurs more frequently in secondary AML (sAML), especially therapy-related, than in de novo disease. Clinically, AML with t(8;16) is associated with high risk of bleeding, occurrence of extramedullary manifestations, myelomonocytic differentiation, and signs of hemophagocytosis in the bone marrow. The translocation leads to the fusion of acetyltransferase KAT6A (also known as MOZ or MYST3) and CREB binding protein ( CREBBP) genes, both of them involved in hematopoiesis. ELN risk stratification of AML with t(8;16) was based on studies that included relatively low numbers of patients receiving allogeneic stem cell transplantation (alloSCT). In the largest report by Kayser et al. ( Br J Haematol 2021) 21 patients (15 transplanted in first complete remission [CR1], median follow-up 5.5 years) were analyzed. A 5-year overall survival (OS) of 38% was achieved after alloSCT compared to 11% in patients who received chemotherapy only. The aim of our study was to evaluate the outcome of patients with AML harboring t(8;16) after alloSCT in a larger cohort from the EBMT registry. All types of donor, conditioning regimen and graft were included. Sixty patients were identified, of whom 44 had been transplanted in CR1. Median follow-up from alloSCT was 72.3 months, median age was 46.3 years, and 73.3% were female. Karnofsky performance status was >80% in 59 patients, one had missing data. At 2 years after alloSCT, OS and leukemia free survival (LFS) of the entire cohort were 42.7% and 42.2%, respectively, (28.6% and 14.3% in patients transplanted in advanced disease). Cumulative incidence of relapse (RI) and non-relapse mortality (NRM) at two years was 36.9% and 15.3%, respectively, in patients transplanted in CR1, and 78.6% and 7.1%, respectively, in patients transplanted in advanced disease. The 44 patients transplanted in CR1 were analyzed in detail. Basic characteristics were comparable to the entire cohort as described above. Twenty-two of the patients had de novo, 22 had sAML. According to the ELN 2017 classification (not yet including t(8;16) as a factor for poor prognosis), 54.5% had an intermediate and 45.5% an adverse cytogenetic risk. Furthermore, 38.8% had a complex karyotype. Donors were matched related in 34%, matched unrelated in 54%, haploidentical in 5% and cord blood in 7%. Conditioning was myeloablative in 52.3%, whereas 47.7% had received reduced intensity conditioning. Among patients transplanted in CR1, 2- and 5-year OS from alloSCT was 48% and 39.9%, the respective LFS was 46.8% and 35.5%. On multivariate analysis (MVA), harboring t(8;16) within a complex karyotype was the major risk factor for outcome, being associated with higher RI (hazard ratio [HR] 3.38, p=0.01), lower LFS (HR 4.17, p=0.016), and lower OS (HR 3.08, p=0.017). In contrast, patients with t(8;16) outside of a complex karyotype achieved excellent results (Figure 1). Besides a complex karyotype, MVA revealed sAML as an additional, independent risk factor with a higher RI (HR 3.73, p=0.026). Age was the major factor for NRM, reaching 5% vs. 26.5% in patients below or above the median (p=0.02). In conclusion, according to the largest series analyzed in this setting so far, overall results after alloSCT in AML with t(8;16) transplanted in CR1 were favorable, especially in patients without multiple additional cytogenetic abnormalities, achieving a low risk of relapse and a 5-year OS >60%. Hence, alloSCT in CR1 appears to abrogate the unfavorable prognostic value of this translocation. Whether or not t(8;16) does have any additional influence on the outcome of patients with a complex karyotype remains to be evaluated. Figure Legend Figure 1: Outcome of AML with t(8;16) with and without complex karyotype

Inhibiting CBP Decreases AR Expression and Inhibits Proliferation in Benign Prostate Epithelial Cells.

(1) Background: CREB-binding protein (CBP) is a key transcriptional coactivator of androgen receptors (AR). We conducted this study to investigate the effects of CBP on AR expression and proliferation in benign prostatic hyperplasia (BPH) prostate epithelial cells. (2) Methods: By analyzing a published data set, we found that CBP was closely related to the gene expression of AR in prostate cells. We enrolled 20 BPH patients who underwent transurethral resection of the prostate (TURP) in Peking University First Hospital in 2022, and analyzed the expressions of CBP and AR in BPH prostate tissues. Then, we used ICG-001 and shRNA to inhibit CBP in prostate epithelial cells (BPH-1 cells and RWPE-1 cells), and conducted immunofluorescence, cell viability assay, flow cytometry analysis, and Western blot to analyze the effects of CBP on AR expression and proliferation in prostate epithelial cells. We also studied the interaction between CBP and AR through a co-immunoprecipitation assay. (3) Results: CBP is consistent with AR in expression intensity in prostate tissues. Inhibiting CBP decreases AR expression, and induces proliferation inhibition, apoptosis, and cell cycle arrest in BPH prostate epithelial cells. The co-immunoprecipitation assay showed that CBP binds with AR to form transcription complexes in prostate epithelial cells. (4) Conclusions: Inhibiting CBP decreases AR expression and inhibits proliferation in benign prostate epithelial cells. CBP may be a potential target to affect AR expression and the proliferation of prostate epithelial cells in BPH.

Ibrutinib and Rituximab Versus Fludarabine, Cyclophosphamide, and Rituximab for Patients in NCRI FLAIR Study with Previously Untreated CLL: Assessment of Mutational Landscape of Patients at Baseline and Prognostic Impact

Introduction: Recent studies using next generation sequencing have revealed recurrent mutated genes in CLL are associated with clinical outcome. We assessed the baseline mutational profile of 771 patients from FLAIR treated with either fludarabine, cyclophosphamide and rituximab (FCR) or ibrutinib and rituximab (IR). The prognostic impact of individual gene mutations on disease progression was investigated. Method: FLAIR is a phase III, multicentre, randomised, controlled, open, parallel group trial for previously untreated CLL requiring therapy according to IWCLL criteria. Patients with >20% chromosome 17p deletion were excluded. Patients were randomised to FCR or IR. Somatic hypermutation (SHM) status was determined by PCR amplification of IGHV-IGHD-IGHJ gene rearrangements using IGHV leader/FR1 primers. Bidirectional Sanger sequencing was analysed using IMGT V-Quest and the ARResT/AssignSubsets tool. Extracted DNA was sequenced using an Illumina MiSeq and analysed using an in-house pipeline. Amplicon based targeted sequencing of 33 recurrently mutated genes in lymphoid malignancies were performed in parallel. Detected variants were reported down to minimum variant allele fractions of 3-5% and coverage of 100X. Low level variants were confirmed by repeat sequencing. Results: 771 patients were randomly assigned to receive FCR (385) or IR (386). IGHV gene SHM status was available for 728/771 (94.4%): 388 (53.3%) IGHV unmutated (>98% of nucleotide identity to germline), 294 (40.4%) IGHV mutated and 46 (6.3%) stereotyped subset #2 (n=28 IGHV mutated, n=18 IGHV unmutated). Gene mutations were assessed in 767/771 (99.5%) patients at baseline and mutations (>1) were detected in 480/767 (62.6%). Mutation frequencies ranged from 0.1-18.8% with mutations in SF3B1 (18.8%), ATM (14.5%), NOTCH1 (10.0%), MYD88 (6.1%), POT1 (5.7%), TP53 (5.0%), BRAF (4.4%) and RPS15 (4.0%) being the most frequent. No detectable mutations at baseline were found in 287/767 (37.4%) and there was no significant difference in PFS or OS compared to patients with >1 mutation. A significantly shorter PFS and OS was observed for TP53 mutated patients compared to wildtype (HR 2.23 [95% CI 1.31-3.79]; p=0.003 and HR 2.43 [95% CI 1.04-5.66]; p=0.039 respectively). When further subdivided by treatment, a trend for shorter PFS was observed in TP53 mutated patients in both arms compared to wildtype, but significance was only achieved for FCR (HR 2.48 [95% CI 1.25-4.91]; p=0.009) and not IR (HR 2.27 [95% CI 0.97-5.30]; p=0.059). A significantly shorter PFS was observed for FCR compared to IR in patients with ATM mutations at baseline (HR 0.35 [95% CI 0.16-0.80]; p=0.012) and RSP15 (HR 0.19 [95% CI 0.04-0.96]; p=0.044). A trend for shorter PFS was also observed in NOTCH1 mutated patients treated with FCR however, this was not found to be significant when compared to wildtype or NOTCH1 mutated IR patients. CREBBP mutation at baseline (n=19; 2.5%) was associated with shorter PFS (HR 2.63 [95% CI 1.29-5.35]; p=0.008) and OS (HR 4.37 [95% CI 1.75-10.94]; p=0.002) compared to wild type (Fig). When further subdivided by treatment arm, shorter PFS and OS was also observed for CREBBP mutated patients treated with FCR compared to wildtype (n=11; HR 4.37 [95% CI 1.75-10.94]; p=0.002 and HR 10.14 [95% CI 3.83-26.83]; p<0.001 respectively). A similar trend was not observed with IR (n=8; PFS HR 0.79 [CI 0.11-5.74]; p=0.819 and OS HR NE [95% CI NE-NE]; p=0.988). CREBBP mutated patients treated with FCR had a shorter PFS compared to IR (HR 0.1 [95% CI 0.01-0.86]; p=0.036). The PFS and OS for IGHV subset #2 was similar to IGHV mutated patients (Fig). In FCR treated patients there was no difference between mutated (n=13) and unmutated subset #2 (n=7) but with IR there was a trend that IGHV mutated subset #2 patients (n=15) had better PFS and OS than their IGHV unmutated counterparts. Conclusions: Our study confirms that TP53 mutations result in shorter PFS and OS in CLL. We demonstrate improved PFS when patients with ATM or RPS15 mutations are treated with IR compared to FCR. Mutations in the CREBBP gene have recently been described as a novel candidate driver in CLL. We report a significant improvement in disease progression in CREBBP mutated patients treated with IR when compared to FCR. In contrast to previous studies in frontline CLL IGHV subset #2 patients had a similar outcome to good risk IGHV mutated patients.

Crebbp Mutations Are Associated with Slow Minimal Residual Response to Upfront Induction Chemotherapy in Pediatric High-Hyperdiploid B-Cell Precursor Acute Lymphoblastic Leukemia Treated in the AIEOP-BFM ALL 2009 Protocol

Introduction High-hyperdiploidy (HHD) represents the most common cytogenetic abnormality found in B-cell precursor acute lymphoblastic leukemia (B-ALL). Despite the overall good outcome of this entity, up to 15% of patients suffer from disease relapse. Genome-wide ALL studies with next-generation sequencing (NGS) approaches identified a distinct pattern of somatic mutations in HHD ALL, enriched in mutations occurring in genes activating the RAS pathway and in CREBBP gene, especially in relapsed patients. Although several studies have attempted to explore the prognostic significance of these mutations that are potentially susceptible of targeted therapies, their impact in uniformly treated patients with newly diagnosed B-ALL is not well characterized, and there is no consensus on their clinical significance. In this study, we aimed to investigate the frequency of recurrent mutations and to define their prognostic role in the setting of newly diagnosed HHD B-ALL, by using end of induction minimal residual disease (MRD) and event-free survival (EFS). Methods We included 101 consecutive patients, aged between 1-17 years, with newly diagnosed HHD B-ALL and enrolled in the AIEOP-BFM ALL 2009 protocol (NCT01117441). HHD was defined by the presence of a DNA index ≥1.16 and lack of ETV6::RUNX1, BCR::ABL1, KMT2A::AFF1 translocations. Diagnostic leukemia samples of 89 patients were sequenced using a validated clinical NGS panel profiling 39 genes involved in leukemia pathogenesis. Mutations affecting RAS pathway ( NRAS, KRAS, PTPN11, NF1, BRAF) and mutations affecting CREBBP gene were included. In addition, digital multiplex ligation-dependent probe amplification (digital MLPA) analysis for the identification of IKZF1 deletion and IKZF1plus phenotype was evaluable for 67 patients. A Fisher exact test and Wilcoxon rank sum test were used for categorical and continuous variables. MRD was assessed by an IG/TR PCR assay at day +33 of induction (timepoint 1, TP1) and at day +78 (timepoint 2, TP2). EFS was estimated by using Kaplan and Meier method and differences between groups were tested using the log-rank test. Resistance to treatment, relapse, death or second malignant neoplasm were considered as events, whichever occurred first. Results Patients' characteristics according to the presence of studied mutations are shown in Table 1. Mutations affecting RAS pathway and CREBBP occurred in 57.3% and 12.3% of HHD B-ALL patients, respectively. Clonal RAS pathway mutations, defined by variant allele frequency (VAF) ≥25%, were found in 16.8% of cases. Among the 11 patients with mutations of CREBBP, 7 had also a mutation affecting KRAS and the association was significant (p=0.03). Seven patients (10.4%) had deletion of IKZF1, of whom 5 (7.4%) were IKZF1plus. There was no association between any mutations or IKZF1 status and patient presenting characteristics, as including sex, age and WBC counts. MRD was evaluable in 84 patients at the end of induction (TP1). MRD level ≥5x10 -4 was found in 36.4% of patients with CREBBP mutations and in 9.6% of those without, and this association was statistically significant (p=0.02). Additionally, MRD negativity was found in 9.1% and 38.4%, respectively. At TP2, MRD was still detectable in 45.5% of patients with CREBBP mutations compared to 20.6% of those with CREBBP wildtype (p=0.08). Similarly, we observed an association between RAS pathway mutations and MRD at TP1 (p=0.01) with a marked difference in MRD negative patients (21.3% vs 51.4% in patients with and without mutations). No association was observed between MRD level and clonal RAS pathway mutations or IKZF1 status. Despite the association between CREBBP mutations and high MRD at TP1, no significant difference in final high-risk group was found (27.3% vs 11.5%, p=0.17). A total of 8 events were observed, of which 6 were relapses. No impact of any mutations or IKZF1 status in term of 5-years EFS was found in our cohort. Conclusion Our study shows that CREBBP mutations in HHD B-ALL are associated with inferior early response to treatment in terms of MRD level at the end of induction (TP1) but not with worse EFS with our whole current chemotherapy backbone. Therefore, the inclusion of CREBBP mutated patients in very low risk protocols should be carefully evaluated. The validation of these findings is ongoing through the analysis of patients treated by other international study groups adopting different chemotherapy backbones.

Sevoflurane suppresses ALG13 transcription in a CREBBP-dependent manner to induce hippocampal damage and cognitive impairment

BackgroundSevoflurane (Sev) is a common clinical anesthetic but may lead to cognitive impairment. This study aims to deconstruct the underpinning molecular mechanism involved in Sev-induced neurological damage. MethodsBioinformatics analyses was conducted to investigate candidate cognitive impairment-related physiological targets of Sev. C57BL/6 mice and SH-SY5Y cells were exposed to Sev to generate animal and cellular models, respectively. Neurological impairment in mice was evaluated by Morris water maze test, modified Longa scoring, and pathological changes and cell apoptosis in the hippocampal tissues. In vitro, viability, apoptosis, and inflammatory cytokine concentration in SH-SY5Y cells were measured. Gain- or loss-of-function studies of CREB binding protein (CREBBP) and its predicted target asparagine-linked glycosylation 13 (ALG13) were performed in mice and in SH-SY5Y cells to investigate their roles in neural damage. ResultsSev treatment induced neurological deficit in mice and damage on SH-SY5Y cells, and it reduced protein level of CREBBP in both models. CREBBP overexpression restored learning and memory ability of mice, reduced neurological deficit score, and reduced cell apoptosis while enhancing neuronal viability in the hippocampus. In vitro, the CREBBP overexpression increased viability while suppressing apoptosis and inflammation in SH-SY5Y cells. CREBBP bound to the ALG13 promoter to increase its transcription. Further knockdown of ALG13 negated the neuro-protective functions of CREBBP in vivo and in vitro. ConclusionThis study demonstrates that Sev targets CREBBP to inhibit ALG13 transcription to induce hippocampal damage and cognitive impairment.

The histone acetyl transferases CBP and p300 regulate stress response pathways in synovial fibroblasts at transcriptional and functional levels

The activation of stress response pathways in synovial fibroblasts (SF) is a hallmark of rheumatoid arthritis (RA). CBP and p300 are two highly homologous histone acetyl transferases and writers of activating histone 3 lysine 27 acetylation (H3K27ac) marks. Furthermore, they serve as co-factors for transcription factors and acetylate many non-histone proteins. Here we showed that p300 but not CBP protein expression was down regulated by TNF and 4-hydroxynonenal, two factors that mimic inflammation and oxidative stress in the synovial microenvironment. We used existing RNA-sequencing data sets as a basis for a further in-depth investigation of individual functions of CBP and p300 in regulating different stress response pathways in SF. Pathway enrichment analysis pointed to a profound role of CBP and/ or p300 in regulating stress response-related gene expression, with an enrichment of pathways associated with oxidative stress, hypoxia, autophagy and proteasome function. We silenced CBP or p300, and performed confirmatory experiments on transcriptome, protein and functional levels. We have identified some overlap of CBP and p300 target genes in the oxidative stress response pathway, however, with several genes being regulated in opposite directions. The majority of stress response genes was regulated by p300, with a specific function of p300 in regulating hypoxia response genes and genes encoding proteasome subunits. Silencing of p300 suppressed proteasome enzymatic activities. CBP and p300 regulated autophagy on transcriptome and functional levels. Whereas CBP was indispensable for autophagy synthesis, silencing of p300 affected late-stage autophagy. In line with impaired autophagy and proteasome function, poly-ubiquitinated proteins accumulated after silencing of p300.

A Deeply Quiescent Subset of CML LSC depend on FAO yet Avoid Deleterious ROS by Suppressing Mitochondrial Complex I.

Disease relapse and therapy resistance remain serious impediments to treating cancer. Leukemia stem cells (LSC) are therapy resistant and the cause of relapse. A state of deep quiescence appears to enable cancer stem cells (CSC) to acquire new somatic mutations essential for disease progression and therapy resistance. Both normal hematopoietic stem cells (HSC) and LSC share many common features, thereby complicating the safe elimination of LSC. A recent study demonstrated that long lived normal oocytes exist without mitochondrial complex I (MC-1), expressing it in a developmentally regulated fashion, thereby mitigating their vulnerability to ROS. Quiescent CSC rely on mitochondrial FAO, without complex I expression, thereby avoiding the generation of damaging ROS, similar to long lived normal human stem cells. A deeper understanding of the biology of therapy resistance is important for the development of optimal strategies to attain complete leukemia cures. Here, using scRNA-sequencing and ATAC-seq on primary chronic myelogenous leukemia (CML) patient samples, combined with bioinformatics analyses, we further examine the heterogeneity of a previously characterized in vitro imatinib-selected CD34-CD38- CML LSC population. We utilized a series of functional analyses, including single-cell metabolomic and Seahorse analyses, to validate the existence of the deepest quiescent leukemia initiators (LI) subset. Current study revealed heterogeneity of therapy resistant LSC in CML patients and their existence of two functionally distinct states. The most deeply quiescent LI suppress the expression of MC-1, yet are highly dependent on fatty acid oxidation (FAO) for their metabolic requirements and ATAC-seq demonstrated increased chromatin accessibility in this population, all consistent with an extremely primitive, quiescent stemness transcriptional signature. Importantly, the specific CREB binding protein (CBP)/β-catenin antagonist ICG-001 initiates the differentiation of LSC, including LI, decreases chromatin accessibility with differentiation and increasing expression of MC-1, CD34, CD38 and BCR-ABL1, thereby resensitizing them to imatinib. We investigated the biological aspects related to LSC heterogeneity in CML patients and demonstrated the ability of specific small molecule CBP/β-catenin antagonists to safely eliminate deeply quiescent therapy resistant CSC. These observations may represent an attractive generalizable therapeutic strategy that could help develop better protocols to eradicate the quiescent LSC population.

The relationship between neurodevelopmental transcriptional programs and insomnia: From Rubinstein-Taybi syndrome into energy metabolism

Neurodevelopmental disorders (NDD) are characterized by cognitive, emotional, and/or motor skills impairment since childhood, and sleep disturbances are a common comorbidity. Rubinstein-Taybi syndrome (RSTS), a rare genetic syndrome associated with NDD, is caused by CREBBP haploinsufficiency. This gene encodes an acetyltransferase with crucial role on the establishment of transcriptional programs during neurodevelopment. Although insomnia has been reported in RSTS patients, the convergent mechanisms between this sleep disturbance and CREBBP loss-of-function are not fully understood. We tested weather the genetic architecture underlying CREBBP regulatory targets and insomnia-associated genes is significantly shared. We then identified the biological pathways enriched among these shared genes. The intersection between CREBBP regulatory targets and genes associated with insomnia included 7 overlapping genes, indicating significantly more overlap than expected by chance. An over-representation analysis on these intersect genes identified pathways related to mitochondrial activity. This finding indicates that the transcriptional programs established by CREBBP might impact insomnia-related biological pathways through the modulation of energy metabolism. The overlapping gene set and biological pathways highlighted by this study may serve as a primer for new functional investigations of shared molecular mechanisms between insomnia and CREBBP regulatory targets.

The C-terminal transactivation domain of MITF interacts promiscuously with co-activator CBP/p300

The microphthalmia-associated transcription factor (MITF) is one of four closely related members of the MiT/TFE family (TFEB, TFE3, TFEC) that regulate a wide range of cellular processes. MITF is a key regulator of melanocyte-associated genes, and essential to proper development of the melanocyte cell lineage. Abnormal MITF activity can contribute to the onset of several diseases including melanoma, where MITF is an amplified oncogene. To enhance transcription, MITF recruits the co-activator CREB-binding protein (CBP) and its homolog p300 to gene promoters, however the molecular determinants of their interaction are not yet fully understood. Here, we characterize the interactions between the C-terminal MITF transactivation domain and CBP/p300. Using NMR spectroscopy, protein pulldown assays, and isothermal titration calorimetry we determine the C-terminal region of MITF is intrinsically disordered and binds with high-affinity to both TAZ1 and TAZ2 of CBP/p300. Mutagenesis studies revealed two conserved motifs within MITF that are necessary for TAZ2 binding and critical for MITF-dependent transcription of a reporter gene. Finally, we observe the transactivation potential of the MITF C-terminal region is reliant on the N-terminal transactivation domain for function. Taken together, our study helps elucidate the molecular details of how MITF interacts with CBP/p300 through multiple redundant interactions that lend insight into MITF function in melanocytes and melanoma.

Molecular insight into CREBBP and TANGO2 variants causing intellectual disability.

Intellectual disability (ID) can be associated with different syndromes such as Rubinstein-Taybi syndrome (RSTS) and can also be related to conditions such as metabolic encephalomyopathic crises, recurrent,with rhabdomyolysis, cardiac arrhythmias and neurodegeneration. Rare congenital RSTS1 (OMIM 180849) is characterized by mental and growth retardation, significant and duplicated distal phalanges of thumbs and halluces, facial dysmorphisms, and an elevated risk of malignancies. Microdeletions and point mutations in the CREB-binding protein (CREBBP) gene, located at 16p13.3, have been reported to cause RSTS. By contrast, TANGO2-related metabolic encephalopathy and arrhythmia (TRMEA) is a rare metabolic condition that causes repeated metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias and encephalopathy with cognitive decline. Clinicians need more clinical and genetic evidence to detect and comprehend the phenotypic spectrum of this disorder. Exome sequencing was used to identify the disease-causing variants in two affected families A and B from District Kohat and District Karak, Khyber Pakhtunkhwa. Affected individuals from both families presented symptoms of ID, developmental delay and behavioral abnormalities. The validation and co-segregation analysis of the filtered variant was carried out using Sanger sequencing. In the present study, two families (A and B) exhibiting various forms of IDs were enrolled. In Family A, exome sequencing revealed a novel missense variant (NM 004380.3: c.4571A>G; NP_004371.2: p.Lys1524Arg) in the CREBBP gene, whereas, in Family B, a splice site variant (NM 152906.7: c.605 + 1G>A) in the TANGO2 gene was identified. Sanger sequencing of both variants confirmed their segregation with ID in both families. The in silico tools verified the aberrant changes in the CREBBP protein structure. Wild-type and mutant CREBBP protein structures were superimposed and conformational changes were observed likely altering the protein function. RSTS and TRMEA are exceedingly rare disorders for which specific clinical characteristics have been clearly established, but more investigations are underway and required. Multicenter studies are needed to increase our understanding of the clinical phenotypes, mainly showing the genotype-phenotype associations.

RNF6 promotes gastric cancer progression by regulating CCNA1/CREBBP transcription

ABSTRACT Ring finger protein 6 (RNF6) is a member of the E3 ubiquitin ligase family. Previous studies have reported the involvement of RNF6 as a ubiquitin ligase in the progression of gastric cancer (GC). However, this study found that RNF6 has a clear localization in the nucleus of GC, indicating a role other than ubiquitin ligase. Further chromatin immunoprecipitation sequencing (ChIP-seq) analysis revealed that RNF6 has DNA binding and transcriptional regulatory effects and is involved in important pathways such as tumor cell cycle and apoptosis. Cyclin A1 (CCNA1) and CREB binding protein (CREBBP) are downstream targets for RNF6 transcription regulation in GC. RNF6 binds to the promoter region of CCNA1/CREBBP and is actively regulating their expression in GC cells. Silencing CCNA1/CREBBP partially reversed the promoting effect of RNF6 overexpression on the biological function of GC cells. Our study suggests that RNF6 promotes the progression of GC by regulating CCNA1/CREBBP transcription.

Cardiac Aging Is Promoted by Pseudohypoxia Increasing p300-Induced Glycolysis.

Heart failure is typical in the elderly. Metabolic remodeling of cardiomyocytes underlies inexorable deterioration of cardiac function with aging: glycolysis increases at the expense of oxidative phosphorylation, causing an energy deficit contributing to impaired contractility. Better understanding of the mechanisms of this metabolic switching could be critical for reversing the condition. To investigate the role of 3 histone modifications (H3K27ac, H3K27me3, and H3K4me1) in the metabolic remodeling occurring in the aging heart, we cross-compared epigenomic, transcriptomic, and metabolomic data from mice of different ages. In addition, the role of the transcriptional coactivator p300 (E1A-associated binding protein p300)/CBP (CREB binding protein) in cardiac aging was investigated using a specific inhibitor of this histone acetyltransferase enzyme. We report a set of species-conserved enhancers associated with transcriptional changes underlying age-related metabolic remodeling in cardiomyocytes. Activation of the enhancer region of Hk2-a key glycolysis pathway gene-was fostered in old age-onset mouse heart by pseudohypoxia, wherein hypoxia-related genes are expressed under normal O2 levels, via increased activity of P300/CBP. Pharmacological inhibition of this transcriptional coactivator before the onset of cardiac aging led to a more aerobic, less glycolytic, metabolic state, improved heart contractility, and overall blunting of cardiac decline. Taken together, our results suggest how epigenetic dysregulation of glycolysis pathway enhancers could potentially be targeted to treat heart failure in the elderly.

β-catenin inhibitor ICG-001 suppress cell cycle progression and induce autophagy in endometrial cancer cells.

The incidence of endometrial cancer has been rising in recent years. Gene mutation and high protein expression of β-catenin are commonly detected in endometrioid endometrial cancer. ICG-001 is a β-catenin inhibitor via blocking the complex formation of β-catenin and cAMP response element-binding protein (CREB)-binding protein (CBP). This study aims to investigate the effect of ICG-001 on endometrial cancer inhibition. First, endometrial carcinoma patient-derived xenograft (PDX)-derived organoids and primary cells were used to verify the inhibiting ability of ICG-001 on endometrial cancer. Furthermore, endometrial cancer cell lines were used to investigate the anticancer mechanism of ICG-001. Using MTT assay and tumor spheroid formation assay, ICG-001 significantly reduced the cell viability of HEC-59 and HEC-1A cells. ICG-001 enhanced cisplatin-mediated cytotoxicity. ICG-001 decreased cancer stem cell sphere formation. ICG-001 decreased the protein expressions of CD44, hexokinase 2 (HK2), and cyclin A. ICG-001 lowered the cell cycle progression by flow cytometer analysis. Autophagy, but no apoptosis, was activated by ICG-001 in endometrial cancer cells. Autophagy inhibition by ATG5 silencing enhanced ICG-001-mediated suppression of cell viability, tumor spheroid formation, and protein expression of cyclin A and CD44. This study clarified the mechanism and revealed the clinical potential of ICG-001 against endometrial cancer.

SADS-CoV nsp1 inhibits the IFN-β production by preventing TBK1 phosphorylation and inducing CBP degradation.

Swine acute diarrhea syndrome (SADS) is first reported in January 2017 in Southern China. It subsequently causes widespread outbreaks in multiple pig farms, leading to economic losses. Therefore, it is an urgent to understand the molecular mechanisms underlying the pathogenesis and immune evasion of Swine acute diarrhea syndrome coronavirus (SADS-CoV). Our research discovered that SADS-CoV inhibited the production of interferon-β (IFN-β) during viral infection. The nonstructural protein 1 (nsp1) prevented the phosphorylation of TBK1 by obstructing the interaction between TBK1 and Ub protein. Moreover, nsp1 induced the degradation of CREB-binding protein (CBP) through the proteasome-dependent pathway, thereby disrupting the IFN-β enhancer and inhibiting IFN transcription. Finally, we identified nsp1-Phe39 as the critical amino acid that downregulated IFN production. In conclusion, our findings described two mechanisms in nsp1 that inhibited IFN production and provided new insights into the evasion strategy adopted by SADS-CoV to evade host antiviral immunity.

Characterization of molecular mechanisms driving Merkel cell polyomavirus oncogene transcription and tumorigenic potential.

Merkel cell polyomavirus (MCPyV) is associated with approximately 80% of cases of Merkel cell carcinoma (MCC), an aggressive type of skin cancer. The incidence of MCC has tripled over the past twenty years, but there are currently very few effective targeted treatments. A better understanding of the MCPyV life cycle and its oncogenic mechanisms is needed to unveil novel strategies for the prevention and treatment of MCC. MCPyV infection and oncogenesis are reliant on the expression of the early viral oncoproteins, which drive the viral life cycle and MCPyV+ MCC tumor cell growth. To date, the molecular mechanisms regulating the transcription of the MCPyV oncogenes remain largely uncharacterized. In this study, we investigated how MCPyV early transcription is regulated to support viral infection and MCC tumorigenesis. Our studies established the roles of multiple cellular factors in the control of MCPyV gene expression. Inhibitor screening experiments revealed that the histone acetyltransferases p300 and CBP positively regulate MCPyV transcription. Their regulation of viral gene expression occurs through coactivation of the transcription factor NF-κB, which binds to the viral genome to drive MCPyV oncogene expression in a manner that is tightly controlled through a negative feedback loop. Furthermore, we discovered that small molecule inhibitors specifically targeting p300/CBP histone acetyltransferase activity are effective at blocking MCPyV tumor antigen expression and MCPyV+ MCC cell proliferation. Together, our work establishes key cellular factors regulating MCPyV transcription, providing the basis for understanding the largely unknown mechanisms governing MCPyV transcription that defines its infectious host cell tropism, viral life cycle, and oncogenic potential. Our studies also identify a novel therapeutic strategy against MCPyV+ MCC through specific blockage of MCPyV oncogene expression and MCC tumor growth.

Primary mucosal melanomas of the urogenital tract: a clinical, pathological, and genetic nationwide survey of Danish patients 1990-2019.

To describe the epidemiologic, clinical, histopathological, and genetic features of primary mucosal melanoma of the urinary tract in a national Danish cohort with cases included from the year 1990 to 2019. Patients of the Danish cohort were found using national databases. Only primary tumours were included in the cohort. Appropriate formalin-fixed paraffin-embedded blocks underwent next-generation sequencing. Eight cases of primary urinary bladder melanomas and 18 cases of primary urethral melanomas were included. Bladder melanomas had an incidence of 0.05 cases/million/year. Mean age at diagnosis was 67 years. The most frequent primary treatment was cystectomy. Adjuvant treatment was given in three cases and consisted of chemotherapy or radiotherapy. Mutations were found in the NF1, KRAS, ATRX, TP53, RAC1, and BRAF genes. Urethral melanomas were found to have an incidence of 0.12 cases/million/year. Average age at diagnosis was 77 years. The most frequent treatment was excision of the tumour. Adjuvant treatment was given in nine cases and most frequently consisted of radiotherapy. Mutations were found in the NF1, TERT PROMOTOR, NRAS, ATRX, TP53, ATM, TSC2, and CREBBP genes. The 5-year survival of patients with bladder melanoma was 12.5% and 22.2% for patients with urethral melanoma. Our study highlights the rarity of urinary tract melanomas and their poor prognosis. The most widely used treatment for urogenital mucosal melanoma remains surgical while adjuvant therapy strategies are evolving. Next-generation sequencing showed mutational patterns with no location-specific patterns. The most frequent mutations were in the NF1, ATRX, NRAS, and TP53 genes.

CREB-binding protein and HIF-1α/β-catenin to upregulate miR-322 and alleviate myocardial ischemia-reperfusion injury.

Myocardial ischemia/reperfusion injury (MIRI) is a prevalent condition associated with numerous critical clinical conditions. miR-322 has been implicated in MIRI through poorly understood mechanisms. Our preliminary analysis indicated potential interaction of CREB-binding protein (CBP), a transcriptional coactivator and acetyltransferase, with HIF-1α/β-catenin, which might regulate miR-322 expression. We, therefore, hypothesized that CBP/HIF-1α/β-catenin/miR-322 axis might play a role in MIRI. Rat cardiomyocytes subjected to oxygen-glucose deprivation /reperfusion (OGD/R) and Langendorff perfused heart model were used to model MIRI in vitro and in vivo, respectively. We used various techniques such as CCK-8 assay, transferase dUTP nick end labeling staining, western blotting, RT-qPCR, chromatin immunoprecipitation (ChIP), dual-luciferase assay, co-immunoprecipitation (Co-IP), hematoxylin and eosin staining, and TTC staining to assess cell viability, apoptosis, and the levels of CBP, HIF-1α, β-catenin, miR-322, and acetylation. Our results indicate that OGD/R in cardiomyocytes decreased CBP/HIF-1α/β-catenin/miR-322 expression, increased cell apoptosis and cytokines, and reduced cell viability. However, overexpression of CBP or miR-322 suppressed OGD/R-induced cell injury, while knockdown of HIF-1α/β-catenin further exacerbated the damage. HIF-1α/β-catenin bound to miR-322 promoter to promote its expression, while CBP acetylated HIF-1α/β-catenin for stabilization. Overexpression of CBP attenuated MIRI in rats by acetylating HIF-1α/β-catenin to stabilize their expression, resulting in stronger binding of HIF-1α/β-catenin with the miR-322 promoter and subsequent increased miR-322 levels. Therefore, activating CBP/HIF-1α/β-catenin/miR-322 signaling may be a potential approach to treat MIRI.