Journal articles: 'CREBBP'

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Yu, Chuanjiang, Mara Holloman, Andrew Kim, et al. "Differential Role of Crebbp Missense and Truncating Mutations in the Malignant Transformation of Germinal Center B Cells." Blood 144, Supplement 1 (2024): 47. https://doi.org/10.1182/blood-2024-208876.

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Somatic mutations of the CREBBP acetyltransferase are highly recurrent in germinal center (GC)-derived lymphomas, including follicular lymphoma (FL, 60% of cases) and diffuse large B cell lymphoma (DLBCL; 40% of cases in the EZB/C3 genetic subtype). Mutations include prototypical inactivating events that abrogate the C-terminal acetyltransferase (AT) domain, as well as amino-acid changes clustering within this domain, which impair the protein enzymatic activity by decreasing its affinity for acetyl-coenzyme A (Pasqualucci et al., Nature 2011). These events are acquired early during lymphomagenesis, in a putative common precursor cell (CPC) that subsequently undergoes divergent evolution to FL or tFL through the acquisition of additional genetic lesions. While extensive work has focused on understanding the lymphoma-driving role of CREBBP protein loss, mimicking the outcome of truncating mutations, the functional significance of missense mutations remains largely unexplored. This is a critical gap because, different from truncated proteins, CREBBP missense mutants are expressed, suggesting they may contribute to malignant transformation through distinct mechanisms. In support of this view, missense and truncating mutations are differentially distributed in FL and DLBCL, with the former being preferentially enriched in FL (p&lt;0.01). The aim of this study was to investigate the in vivo role of CREBBP missense mutations during the initial phases of lymphomagenesis, and contrast it with the phenotype resulting from CREBBP loss. To address these questions, we took advantage of a novel, conditional knock-in mouse model where the most common R1446H mutant protein (R1447 in mice, hereafter CrebbpRH) is expressed specifically in GC B cells under the control of its endogenous promoter, following T cell-dependent immunization and Cγ1Cre-driven recombination. RT-PCR and sequencing confirmed efficient, GC-restricted activation of the knock-in allele, and Crebbp protein levels were comparable in homozygous CrebbpRH vs WT littermates, documenting a stable protein. While modest differences were noted in the GC B cell fraction of CrebbpKO mice, consistent with our previous work, expression of the CrebbpRH allele led to pronounced, dose-dependent expansion of the GC (p&lt;0.001), the structure from which FL and DLBCL arise. These GCs displayed an abnormal dark zone to light zone polarity manifesting as an increased proportion of centrocytes (p&lt;0.001), and persisted abnormally for several weeks after immunization, when the GC reaction is normally extinguished, indicating an altered dynamics. To investigate the mechanisms underlying this phenotype, we contrasted the transcriptomic and chromatin profiles of CrebbpRH and CrebbpKO GC B cells. We found shared but also unique transcriptional changes separating the two cohorts by unsupervised hierarchical clustering. Consistently, a significant number of differentially expressed genes were unique to CrebbpRH vs CrebbpKO GC B cells (q&lt;0.05, FC≥1.2). In particular, CrebbpRH GCs were positively enriched in light-zone-associated and pre-memory B cell differentiation programs (Ccr6, Gpr183, Klf2); in contrast, these signatures were attenuated upon Crebbp deletion, indicating missense mutations may favor distinct cell fates upon GC exit. While global H3K27Ac was reduced in both CrebbpRH and CrebbpKO cells, consistent with impaired AT activity, Cut&Run analysis revealed a significant CrebbpRH-mediated redistribution of the H3K27Ac mark at super-enhancers, including those linked to genes involved in GC exit and B:T-cell interactions. Accordingly, expression of CrebbpRH alleles led to a two-fold increase in memory B cell responses to the T cell-specific antigen NP-KLH (p&lt;0.01), which was not detected in CrebbpKO mice, as measured by immunophenotyping. Moreover, CrebbpRH but not CrebbpKO mice showed compositional changes in the T cell microenvironment, with expansion of T follicular helper cells (8.3%, vs 3.8% in WT mice; p&lt;0.05). Thus, Crebbp missense mutations may skew GC B cell fate decisions toward memory B cell differentiation and favor the persistence of enlarged GCs to facilitate the malignant transformation process, in part by shaping the GC microenvironment. These data have implications for the understanding of FL/DLBCL ontogeny and for the therapeutic targeting of tumors carrying missense vs truncating mutations.

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Meyer, Stefanie, Sofija Vlasevska, Laura Garcia Ibanez, Claudio Scuoppo, Riccardo Dalla-Favera, and Laura Pasqualucci. "Targeting Histone Acetyltransferase Gene Inactivation in Diffuse Large B Cell Lymphoma." Blood 132, Supplement 1 (2018): 671. http://dx.doi.org/10.1182/blood-2018-99-117542.

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Abstract Diffuse Large B-cell Lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma, accounting for ~30% of de-novo diagnoses and also arising as a frequent clinical evolution of indolent lymphomas. Although curable in a substantial fraction of cases, one third of patients do not achieve durable remissions, highlighting the need for novel, targeted therapies. Over the past decade, we and others have identified the CREBBP acetyltransferase and, less frequently, its paralogue EP300 as highly recurrent targets of inactivating somatic mutations/deletions in DLBCL and follicular lymphoma (FL) (30% and 60% of patients, respectively), indicating a prominent role in the pathogenesis of these tumors (Pasqualucci et al., Nature 2011). In most cases, mutations are heterozygous and the residual wildtype (WT) allele is expressed, suggesting a haploinsufficient tumor suppressor role. Indeed, germinal center (GC)-specific loss of Crebbp perturbs the expression of genes that are relevant to the normal biology of this structure, i.e. the lymphoma cell of origin, and cooperates with BCL2 deregulation to increase the incidence of tumors recapitulating the features of the human disease (Zhang et al., Cancer Discovery 2017). Intringuingly, while CREBBP binds to virtually all GC-specific superenhancers, no detrimental effects were observed upon its deletion in mice, suggesting the existence of compensatory mechanisms. Consistent with this hypothesis, inactivation of CREBBP and EP300 rarely coexist in human DLBCL and FL, suggesting that cells require a certain amount of acetyltransferase activity. To investigate whether EP300 compensates for CREBBP loss in the GC, we analyzed the GC responses in compound mouse models engineered to specifically delete these two genes (alone and in combination) upon SRBC immunization and induction of a Cγ1-driven Cre-recombinase. While CrebbpKOmice showed a mild increase in GC formation, as reported, loss of Ep300 led to ~40% reduction in the percentage of GC cells (mean: 1.8% vs 3.1% in WT littermates; p<0.05), documenting that these two enzymes play non-entirely overlapping roles in this population. Importantly, GC formation was completely abrogated in CrebbpKOEp300KO mice and dramatically impaired in CrebbpHETEp300KO mice, as compared to both WT and single EP300KO mice. These data suggest that GC B cells require a minimum amount of acetyltransferase activity, and reveal a potential therapeutically exploitable dependency of Crebbp-mutated GC B cells on Ep300. In order to probe if a similar dependency exists in neoplastic GC B cells, we used an inducible CRISPR/Cas9 system to delete EP300 (or a control non-genic region) in 4 DLBCL cell lines representative of the various CREBBP genotypes found in DLBCL, and monitored cell proliferation and survival in competition assays over 12 days. Compared to CREBBPWT, CREBBP heterozygous and homozygous mutant cells were significantly counter-selected from the total population following doxycycline induced EP300 deletion (~30% at day 7). Moreover, no EP300-edited clones were recovered from the CREBBP mutant lines in single cell plating assays, compared to CREBBP WT (p<0.01). Thus, DLBCL cells remain addicted to the residual EP300 aceyltransferase activity, supporting the existence of a therapeutic window for EP300 inhibitors. To explore this concept further, we generated isogenic DLBCL clones carrying WT or defective CREBBP alleles (n=4 each), and performed drug-sensitivity assays with 2 novel small molecule inhibitors that specifically target the CREBBP/EP300 HAT or BRD domain. While, at higher doses, both inhibitors interfered with cell growth in all clones, CREBBPKO cells were significantly more sensitive than their isogenic WT pairsat low nanomolar ranges (IC50: 60nM vs 300nM). Importantly, we were able to design an in vitro protocol that was toxic to CREBBPKO cells but tolerated by CREBBPWT cells, providing a proof of concept for therapeutically targeting these molecules. In conclusion, we show that CREBBP and EP300 have differential roles in normal GC B cell development and that CREBBP mutated cells are addicted to the residual EP300 activity. This dependency is maintained in DLBCL cells, providing the basis for the potential application of acetyl transferase inhibition into the clinical settings. Disclosures No relevant conflicts of interest to declare.

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Ying, Hsia-Yuan, Yanwen Jiang, Ana Ortega-Molina, et al. "Crebbp Mutations Disrupt Dynamic Enhancer Acetylation in B-Cells, Enabling HDAC3 to Drive Lymphomagenesis." Blood 128, no. 22 (2016): 735. http://dx.doi.org/10.1182/blood.v128.22.735.735.

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Abstract Somatic mutations in CREBBP occur frequently in germinal center derived lymphomas such as DLBCL and FL. However whether or how these mutations might contribute to lymphomagenesis is still largely unknown. Most CREBBP mutations are predicted to result in loss of function since they target the histone acetyltransferase (HAT) domain or give rise to premature stop codon prior to the HAT domain. Here, we show that Crebbp shRNA knockdown (KD) accelerated lymphomagenesis in VavP-Bcl2 transgenic mice, a model that recapitulates human GC-derived lymphomas. The median time to lymphoma onset in VavP-Bcl2/CrebbpKD mice was 114 days, significantly shorter than control VavP-Bcl2/GFP mice (193 days, p=0.04). Histopathology revealed that VavP-Bcl2/CrebbpKD lymphomas were more aggressive and widely disseminated than VavP-Bcl2/GFP lymphomas. CREBBP can regulate gene enhancer function through H3K27 acetylation. ChIP-seq in VavP-Bcl2/CrebbpKD lymphoma cells revealed significant reduction of H3K27ac peaks compared to control lymphoma cells (N = 1717, Kolmogorov-Smirnov test, p<2.2E-16). Loss of H3K27ac was markedly skewed towards enhancers. We observed similar loss of enhancer H3K27ac in human DLBCL cells after CREBBP shRNA KD. Enhancer H3K27ac loss was significantly associated with repression of nearby genes in both murine (FDR q=0.044) and human lymphoma cells (FDR q=0). RNA-seq performed in three independent FL or DLBCL patient cohorts revealed a characteristic CREBBP mutant gene expression signature featuring prominent transcriptional repression (p=1.32E-14, p=0.001, and p=0.0002 respectively). Notably, the human patient CREBBP mutant signature was highly enriched in murine and human cell line CREBBP shRNA profiles (FDR=0, GSEA), indicating that CREBBP KD signature was highly similar to CREBBP mutant signature in humans. Functional analysis of the CREBBP mutant/KD signature showed significant enrichment of GC exit pathways including genes induced by CD40, IRF4 and plasma cell differentiation; as well as immune response processes including antigen processing and presentation, such as MHC class II genes (BH-adjusted p<0.05). To better understand mechanism we performed an integrative analysis of CREBBP signatures against databases of B-cell transcription factor and epigenome profiles. This analysis yielded significant enrichment (BH-adjusted p<0.05) for i) enhancers bound by the BCL6 transcriptional repressor and its SMRT/HDAC3 corepressor complex, ii) enhancers that are normally deacetylated in GC B-cells, and iii) genes induced by BCL6 siRNA. This is notable because in normal GCs BCL6 represses enhancers by recruiting SMRT/HDAC3 complexes to deacetylate H3K27. Hence our data suggest that CREBBP is a counteracting HAT to BCL6/SMRT/HDAC3. Indeed, conditional knockout of Hdac3 in GC B-cells in mice resulted in impaired GC formation and a transcriptional signature featuring upregulation of the same genes that are repressed by CREBBP KD (GSEA FDR=0). Moreover, CREBBP KD in DLBCL cells resulted in H3K27ac loss at BCL6/SMRT/HDAC3 regulated enhancers, including those nearby CDKN1A, NFATC1, FOXP1, and MHC II genes, such as HLA-DQA1 and HLA-DRB5. CREBBP KD also resulted in silencing of these genes. Since we show HDAC3 is the opposing HDAC to CREBBP then we reasoned that CREBBP mutant DLBCLs might be especially dependent on HDAC3. Indeed we observed that HDAC3 shRNA resulted in profound suppression of CREBBP mutant DLBCL cells in vitro and in vivo (DLBCL xenografts in mice, p=0.005), whereas CREBBP WT cell lines were barely affected by HDAC3 KD. The opposing effects of BCL6/SMRT/HDAC3 and CREBBP on MHC class II could have implications for immune surveillance. Accordingly CREBBP KD induced significant loss of cell surface HLA-DR molecules (p<0.05), and these cells exhibited up to 90% less capability to stimulate T-cell response in allogeneic mixed lymphocyte reaction experiments. The loss of MHC class II molecules and T-cell response was rescued when CREBBP loss of function cells were exposed to a specific HDAC3 inhibitor. In summary, CREBBP mutations drive lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, resulting in a repressive transcriptional programming that disrupts GC exit and evades immune surveillance. HDAC3 targeted therapy may rescue these effects and serve as a precision approach for CREBBP mutant lymphomas. Disclosures Scott: Celgene: Consultancy; Roche: Honoraria; Janssen: Consultancy; BC Cancer Agency: Patents & Royalties: Inventor on a patent licensed to NanoString Technologies. Tam:Millennium Pharmaceuticals, Inc.: Consultancy. Melnick:Janssen: Research Funding.

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LeBlanc, Francis, Josh Bennett, Kwangmin Choi, and Daniel T. Starczynowski. "Targeting CREB-Binding Protein (CREBBP) Overcomes Resistance to Azacitidine and Venetoclax Therapy in Acute Myeloid Leukemia (AML)." Blood 142, Supplement 1 (2023): 5765. http://dx.doi.org/10.1182/blood-2023-187063.

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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.

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Lamble, Adam J., Robert B. Gerbing, Jenny L. Smith, et al. "Crebbp Alterations Are Associated with a Poor Prognosis in De Novo AML." Blood 138, Supplement 1 (2021): 3451. http://dx.doi.org/10.1182/blood-2021-154052.

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Abstract Introduction: The translocation, t(8;16)(p11;p13), results in the fusion between KAT6A and CREBBP and has been associated with a poor prognosis in both pediatric and adult acute myeloid leukemia (AML). This lesion has therefore been re-classified as high risk on the active Phase 3 Children's Oncology Group (COG) trial for de novo AML, AAML1831 (NCT04293562). Less is known about the prognostic significance of CREBBP sequence variants. Methods: CREBBP variant status was determined in patients with AML enrolled on 4 successive COG trials for de novo pediatric AML (NCT00003790, NCT00070174, NCT01407757, NCT01371981). Fusions involving CREBBP were prospectively obtained via conventional cytogenetics and retrospectively confirmed via RNAseq. Insertions and deletions (indels) leading to frameshift mutations and single nucleotide variants (SNVs) were retrospectively interrogated via next generation sequencing. Results: Of 2216 patients (age: 0-29.8 years), 55 (2.5%) patients had an alteration involving CREBBP. Sixteen (29%) of these were a fusion involving CREBBP (CREBBP/fus), with KAT6A being the most common translocation partner (n=15) and the remaining translocation involving ANK1. The remaining 39 patients (71%) had a CREBBP mutation (CREBBP/mut), including 19 with an indel (CREBBP/indel) leading to a frameshift mutation and 20 with a SNV (CREBBP/SNV). We compared clinical and biologic characteristics between the three cohorts. CREBBP/fus patients were significantly younger than CREBBP/indel and CREBBP/SNV patients (median ages of 2.6 vs. 7.8 vs. 11.9 years; p=0.027). There was a higher prevalence of t(8;21)/RUNX1-RUNX1T1 in CREBBP/indel patients compared to CREBBP/SNV patients (42.1% vs. 5%; p=0.008). In contrast, CREBBP/SNV patients were more likely to be associated with a normal karyotype (40% vs. 5.3%; p=0.02). There was a similar prevalence of co-occurring high-risk lesions in CREBBP/indel (n=5; CBFA2T3-GLIS2, KMT2A-AFF1, KMT2A-MLLT4, MLLT10-PICALM, NUP98-HOXA9) and CREBBP/SNV (n=7; DEK-NUP214, ETV6-FOXO1, FUS-ERG, NUP98-NSD1, ETV6-MNX1, FLT3-ITDx2) patients. There was otherwise no difference between presenting WBC count, FLT3-ITD, NPM1, CEBPA, remission rates or MRD status after Induction 1 therapy. Patients with any CREBBP alteration had a significantly worse 5-year event free survival (EFS) compared to patients without (25.9% vs. 45.2%; p=0.002) and this inferior EFS overlaps with contemporarily defined high-risk patients (Figure 1a). Evaluation of outcomes based on type of alteration demonstrated a similar 5-year EFS of 33.3% and 23.1% between CREBBP/fus and CREBBP/mut patients, respectively (Figure 1b; p=0.832). This poor EFS was maintained in the CREBBP/indel patients with a co-occurring t(8;21) (n=8, 5-year EFS 12.5%). When patients with co-occurring high-risk lesions were excluded from analysis, the remaining CREBBP/mut (n=27) patients maintained their poor EFS (29.6%). Despite their poor EFS, CREBBP/mut patients had an analogous overall survival (OS) to non-CREBBP patients (57.4% vs. 62.3%; p=0.499, Figure 1c), demonstrating that these patients could be successfully salvaged following relapse. In contrast, all patients with CREBBP/fus that relapsed subsequently died from their disease (OS 33.3%). Conclusions: In a large study of CREBBP alterations in pediatric patients with de novo AML, we show that these patients have a dismal EFS, regardless of alteration type. Further, despite enrichment of t(8;21), the favorable prognosis typically conferred by this alteration was abrogated by the co-occurrence of CREBBP/indel. Similarly, by excluding patients with co-occurring high-risk lesions from analysis, we show that these poor outcomes persist in a cohort of patients that would otherwise be considered low risk. Translocations between CREBBP and KAT6A in patients over 90 days of age are considered high risk on the active COG phase 3 trial. Given the inferior EFS and high salvage rates associated with other CREBBP alterations, intensification of upfront treatment, including hematopoietic stem cell transplant, should be considered in this population. The authors would like to acknowledge Astellas Pharma Global Development, Inc. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Zhu, Yu, Zi Wang, Yanan Li, et al. "The Role of CREBBP/EP300 and Its Therapeutic Implications in Hematological Malignancies." Cancers 15, no. 4 (2023): 1219. http://dx.doi.org/10.3390/cancers15041219.

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Disordered histone acetylation has emerged as a key mechanism in promoting hematological malignancies. CREB-binding protein (CREBBP) and E1A-binding protein P300 (EP300) are two key acetyltransferases and transcriptional cofactors that regulate gene expression by regulating the acetylation levels of histone proteins and non-histone proteins. CREBBP/EP300 dysregulation and CREBBP/EP300-containing complexes are critical for the initiation, progression, and chemoresistance of hematological malignancies. CREBBP/EP300 also participate in tumor immune responses by regulating the differentiation and function of multiple immune cells. Currently, CREBBP/EP300 are attractive targets for drug development and are increasingly used as favorable tools in preclinical studies of hematological malignancies. In this review, we summarize the role of CREBBP/EP300 in normal hematopoiesis and highlight the pathogenic mechanisms of CREBBP/EP300 in hematological malignancies. Moreover, the research basis and potential future therapeutic implications of related inhibitors were also discussed from several aspects. This review represents an in-depth insight into the physiological and pathological significance of CREBBP/EP300 in hematology.

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Hashwah, Hind, Corina A. Schmid, Sabrina Kasser, et al. "Inactivation of CREBBP expands the germinal center B cell compartment, down-regulates MHCII expression and promotes DLBCL growth." Proceedings of the National Academy of Sciences 114, no. 36 (2017): 9701–6. http://dx.doi.org/10.1073/pnas.1619555114.

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The genes encoding the histone acetyl-transferases (HATs) CREB binding protein (CREBBP) and EP300 are recurrently mutated in the activated B cell-like and germinal center (GC) B cell-like subtypes of diffuse large B cell lymphoma (DLBCL). Here, we introduced a patient mutation into a human DLBCL cell line using CRISPR and deleted Crebbp and Ep300 in the GC B cell compartment of mice. CREBBP-mutant DLBCL clones exhibited reduced histone H3 acetylation, expressed significantly less MHCII, and grew faster than wild-type clones in s.c. and orthotopic xenograft models. Mice lacking Crebbp in GC B cells exhibited hyperproliferation of their GC compartment upon immunization, had reduced MHCII surface expression on GC cells, and developed accelerated MYC-driven lymphomas. Ep300 inactivation reproduced some, but not all, consequences of Crebbp inactivation. MHCII deficiency phenocopied the effects of CREBBP loss in spontaneous and serial transplantation models of MYC-driven lymphomagenesis, supporting the idea that the mutational inactivation of CREBBP promotes immune evasion. Indeed, the depletion of CD4+ T cells greatly facilitated the engraftment of lymphoma cells in serial transplantation models. In summary, we provide evidence that both HATs are bona fide tumor suppressors that control MHCII expression and promote tumor immune control; mutational inactivation of CREBBP, but not of EP300, has additional cell-intrinsic engraftment and growth-promoting effects.

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Zimmer, Stephanie N., Qing Zhou, Ting Zhou, et al. "Crebbp haploinsufficiency in mice alters the bone marrow microenvironment, leading to loss of stem cells and excessive myelopoiesis." Blood 118, no. 1 (2011): 69–79. http://dx.doi.org/10.1182/blood-2010-09-307942.

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Abstract CREB-binding protein (CREBBP) is important for the cell-autonomous regulation of hematopoiesis, including the stem cell compartment. In the present study, we show that CREBBP plays an equally pivotal role in microenvironment-mediated regulation of hematopoiesis. We found that the BM microenvironment of Crebbp+/− mice was unable to properly maintain the immature stem cell and progenitor cell pools. Instead, it stimulates myeloid differentiation, which progresses into a myeloproliferation phenotype. Alterations in the BM microenvironment resulting from haploinsufficiency of Crebbp included a marked decrease in trabecular bone that was predominantly caused by increased osteoclastogenesis. Although CFU-fibroblast (CFU-F) and total osteoblast numbers were decreased, the bone formation rate was similar to that found in wild-type mice. At the molecular level, we found that the known hematopoietic modulators matrix metallopeptidase-9 (MMP9) and kit ligand (KITL) were decreased with heterozygous levels of Crebbp. Lastly, potentially important regulatory proteins, endothelial cell adhesion molecule 1 (ESAM1) and cadherin 5 (CDH5), were increased on Crebbp+/− endothelial cells. Our findings reveal that a full dose of Crebbp is essential in the BM microenvironment to maintain proper hematopoiesis and to prevent excessive myeloproliferation.

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Huntly, Brian J. P., Sarah Jayne Horton, George Giotopoulos, et al. "Early Loss of CREBBP Confers Malignant Stem Cell Properties on Lymphoid Progenitors." Blood 128, no. 22 (2016): 460. http://dx.doi.org/10.1182/blood.v128.22.460.460.

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Abstract Loss-of-function mutations of the cyclic-AMP response element binding protein, binding protein (CREBBP) gene have recently been described at high frequencies across a spectrum of lymphoid malignancies, particularly follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). The multiple effects of this epigenetic regulator on developmental and homeostatic processes have been extensively studied, however, exactly how CREBBP functions as a tumor suppressor and the reasons for its particular predilection for suppression of lymphoid tumors remains unclear. In addition, for many mature lymphoid malignancies, the existence of cancer stem cells is unproven and their provenance and the initial target cell for transformation a source of ongoing debate. Here we use multiple mouse strains to model loss of Crebbp in different lymphoid compartments to address these questions. We demonstrate that early loss of Crebbp in hematopoietic stem and progenitor cells (HSPC), through disruption by the Mx1-Cre recombinase, leads to increased development of hematological malignancies, particularly of the B-lymphoid lineage that mimic features of human lymphomas. Theses B-cell malignancies are of long latency and are preceded by significant alterations in the proliferation, self-renewal and differentiation of lymphoid progenitors, allowing hyperproliferative lymphoid progenitors whose differentiation is blocked to accumulate. Using an aberrant surface phenotype that resembles the eventual tumor, we identify a pre-malignant population in the peripheral blood of animals that is often evident many months before any disease characteristics. We demonstrate pre-malignant stem cell characteristics for this population in functional experiments, where it generates high level reconstitution of peripheral blood in transplant recipients, but only gives rise to disease in these animals after a long latency. We also utilize this unique cellular population in longitudinal genome scale analyses (clonality, RNA-Seq, ChIP-Seq and exome sequencing) to document the mechanisms of malignant evolution. Linking the increased rate of mutation we describe to Crebbp loss, we also demonstrate increased DNA damage and an altered DNA-damage response in premalignant lymphoid progenitors. Importantly, using a Cd19-Cre recombinase that excises only within committed lymphoid cells, we are able to demonstrate that when Crebbp is lost at a later stage of lymphoid development, the marked cellular abnormalities described above are completely lost and the development of tumors is no different from normal (Figure, below). Taken together, these findings define the developmental stage-specific tumor suppressor functions of Crebbp and shed light on the cellular origins and subsequent evolution of lymphoid malignancies. In addition, the altered response to DNA damage that we demonstrate upon loss of Crebbp, allied to the increased exposure to physiological DNA-damage during lymphoid ontogeny offers an explanation for the high incidence of CREBBP mutations in mature lymphoid malignancies. Figure Left panel, Kaplan Meier graph for Mx1-Cre Crebbp mice with loss of Crebbp in the HSPC compartment demonstrates significantly shorter survival vs WT littermates with intact expression of Crebbp. In contrast, when Crebbp is excised in a later lymphoid compartment through Cd19-Cre mediated recombination, right panel, no difference in survival is noted from WT littermate controls. Figure. Left panel, Kaplan Meier graph for Mx1-Cre Crebbp mice with loss of Crebbp in the HSPC compartment demonstrates significantly shorter survival vs WT littermates with intact expression of Crebbp. In contrast, when Crebbp is excised in a later lymphoid compartment through Cd19-Cre mediated recombination, right panel, no difference in survival is noted from WT littermate controls. Disclosures Huntly: Novartis: Speakers Bureau; BMS: Speakers Bureau; Ariad: Speakers Bureau; Pfizer: Speakers Bureau.

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Peck, Barrie, Philip Bland, Ionnana Mavrommati, et al. "3D functional genomics screens identify CREBBP as a targetable driver in aggressive triple-negative breast cancer." Cancer Res 81, no. 4 (2021): 847–59. https://doi.org/10.1158/0008-5472.CAN-20-1822.

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Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo–like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. Significance: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

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Jamileh, Malbin. "Deletion of SHANK3 and CREBBP gene in the patients with intellectual disability and mix phenotype." International Journal Of Genetic Medicine And Gene Therapy 1, no. 1 (2019): 15–20. https://doi.org/10.36811/ijgmgt.2019.110004.

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Rubinstein-Taybi Syndrome (RSTS) as a group of congenital anomalies mainly include, short broad thumbs and toes, short stature and intellectual disability are caused by either a micro-deletion in the CREBBP (CBP) or EP300 genes. Generally most RSTS patients have a deletion in the CREBBP gene but some patients have shown deletion in the EP300 gene. Here we introduce an affected case without some typical characteristics of RSTS with deletions in the CREBBP and SHANK3 genes. The patient was a 24 years old man with a history of infantile hypotonia and childhood developmental delay, heavy eyebrows, ptosis, speech difficulty without large thumb and toes. The conventional cytogenetic finding was normal male. Further investigation was performed using Multiplex Ligation Probe Amplification (MLPA) technique to screen micro-deletion syndromes and subtelomeric rearrangements and Micro-deletion was detected in CREBBP and SHANK3 gene and a detected in DECR2 gene. Deletion in the CREBBP or EP300 genes or both in the patients with broad thumb and toes (RSTS) has been detected but there are other patients with deletion in CREBBP gene without this sign of RSTS. However, we report SHANK3 gene deletion in the patient with deletion in CREBBP gene and without broad thumbs and toes. Keywords: Rubinstein-Taybi Syndrome (RSTS); CREBBP; SHANK3; Broad thumb and toes

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Idoia, García-Ramírez, Shashank Shrishrimal, Ines Gonzalez-Herrero, et al. "CREBBP Loss Cooperates with BCL2 Over-Expression to Promote Lymphoma in Mice." Blood 128, no. 22 (2016): 458. http://dx.doi.org/10.1182/blood.v128.22.458.458.

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Abstract Follicular lymphoma (FL) is genetically characterized by translocations of the BCL2 oncogene that are found in ~90% of patients, and mutations of chromatin modifying genes that are found in up to 96% of patients. The latter include inactivating mutations of KMT2D and CREBBP, and activating mutations of EZH2, among others. However, CREBBP has yet to be investigated using this approach. We recently defined the evolutionary hierarchy of somatic mutations in FL and found that CREBBP mutations were most frequently acquired as early events during disease evolution and were maintained throughout disease progression and transformation. Recent studies, using transgenic mouse models, have shown that inactivation of KMT2D and introduction of the activating EZH2 mutation results in perturbed B-cell development and lymphomagenesis. Here, we extended upon these observations by performing targeted next generation sequencing of an additional cohort of tumors allowing the identification of the spectrum of CREBBP mutations across 200 FLs. This identified CREBBP mutations in 55% of tumors, and found that 31% of these mutations reside within the lysine acetyltransferase domain. Furthermore, 30% of mutations altered a single amino acid, arginine 1408, to either a cysteine or histidine residue. We performed a sensitive in vitro acetyltransferase assay for these point mutants and show that they result in >90% loss of catalytic activity. As our results show that CREBBP mutations result in a loss of function, we modeled these events in mice by floxing one or both alleles of Crebbp and crossing with the Mb1-cre strain. This yielded mice that deleted Crebbp specifically in B-cells. We additionally crossed these mice with the EµBcl2 strain that over-expresses Bcl2 in B-cells. Inactivation of Crebbp in B-cells was associated with deficits in B-cell development, with significantly reduced numbers of total B-cells that were contributed to by reductions in multiple B-cell subsets. These deficits were partially rescued by the EµBcl2 transgene. After 14-21 months, some mice became ill and necropsy revealed lymphadenopathy and splenomegaly as a result of B-cell lymphoma. We noted increased penetrance and decreased latency of lymphoma with one vs two alleles of Crebbp deleted, and with absence vs presence of the EµBcl2 transgene (Figure 1). We investigated the molecular etiology of these tumors by isolating splenic B-cells from these mice and performing transcriptome profiling and epigenetic profiling for the histone H3 lysine 18 acetylation (H3K18Ac) mark that is catalyzed by Crebbp. Transcriptional profiling identified a signature of 335 genes with increased expression and 370 genes with decreased expression, including an incremental increase in Myc expression when one or both alleles of Crebbp were deleted, respectively. Surprisingly, changes in transcript abundance were not associated with changes in H3K18Ac in the proximal regulatory regions of those genes. Regions of significantly altered H3K18Ac were instead localized primarily to intragenic regions. Analysis of the DNA sequences in these regions identified a significant enrichment of motifs that contained Myc consensus sequences, and these were present in >60% of regions with altered H3K18Ac. In addition, ChIP-seq data from the ENCODE database showed a strong level of Myc binding to the center of these regions with altered H3K18Ac. Together, our results demonstrate that inactivating mutations of Crebbp may have a role in altering B-cell development. The significant induction of Myc expression that was associated with Crebbp deletion, and epigenetic changes in regions that are bound by Myc, suggest that Crebbp inactivation may have a role in the induction of Myc expression and activity. This may be important with respect to transformation of FL, which may proceed via induction of MYC. However, our results also demonstrate some important discrepancies between the role of CREBBP mutations in human FL, and the role of Crebbp deletion in murine models. Disclosures Lunning: Celgene: Consultancy; Spectrum: Consultancy; TG Therapeutics: Consultancy; Gilead: Consultancy; Genentech: Consultancy; Juno: Consultancy; Bristol-Myer-Squibb: Consultancy; AbbVie: Consultancy; Pharmacyclics: Consultancy.

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Dixon, Zach, Julie A. E. Irving, and Lindsay Nicholson. "Crebbp K nockdown Does Not Impact on Glucocorticoid Induced Apoptosis in Childhood Acute Lymphoblastic Leukemia." Blood 126, no. 23 (2015): 1429. http://dx.doi.org/10.1182/blood.v126.23.1429.1429.

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Abstract Childhood acute lymphoblastic leukemia (ALL) is the most common childhood cancer and, despite a cure rate approaching 90%, relapse is a significant cause of death in young people. Recently it has been shown that inactivating mutations in the histone acetyltransferase, CREB binding protein (CREBBP or CBP) are frequently seen at relapse in childhood ALL, with enrichment in high hyperdiploid and hypodiploid cases. Mutations are usually heterozygous, suggesting haploinsufficiency, and are often acquired at relapse, implying a role in drug resistance. Since glucocorticoid (GC) response genes are known targets of CREBBP and, given the pivotal role of GCs in ALL therapy, it has been postulated that CREBBP mutations confer GC chemoresistance. CREBBP is a multifunctional protein, playing a role in cAMP dependent signalling, acetylation mediated activation of p53 and inactivation of BCL6 and a range of DNA damage repair pathways including base excision repair (BER) and direct DNA damage repair. To assess the role of CREBBP haploinsufficiency in ALL, RNAi techniques were used to create isogenic CREBBP knockdown models of ALL. CREBBP knockdown was carried out using small hairpin RNA (shRNA) transduction (termed shCBP cells) or small interfering RNA (siRNA) transfection (termed siCBP cells) in the PreB 697 B-cell precursor cell line (t(1;19)) and the hypodiploid MHH-CALL-2 cell line, as well as high hyperdiploid primagraft ALL cells. Knockdown of at least 50% of control was confirmed at both mRNA and protein level. The functional impact of CREBBP knockdown in cells was determined by analysis of known CREBBP target residues; acetyl H3K18 and H3K27, and transcription of cAMP dependent genes (CXCR4, MKNK2, DUSP5, DUSP10 and RGS16). To assess the impact of CREBBP knockdown on response to GCs, cells were treated with dexamethasone and expression of the classic glucocorticoid receptor (GR) targets; GILZ and FKBP51, was assessed by quantitative reverse transcriptase PCR (QRT-PCR). Alamar blue cell viability assays were used to determine the sensitivity of each CREBBPknockdown model to dexamethasone compared to isogenic controls. Three out of four cell models displayed a reduction in H3K18 or H3K27 acetylation compared to isogenic control, indicating a relevant functional impact of CREBBP knockdown. Cell lines showed a trend towards reduced induction of some of the selected cAMP dependent targets but statistical significance was not achieved (p values >0.2). Gene expression profiling and Ingenuity Pathway Analysis of PreB 697 shCBP cells compared to isogenic control predicted that upstream transcription of NR3C1, the gene encoding the GR, would be affected in CREBBP knockdown cells. However, while induction of GILZ and FKBP51 in PreB 697 shCBP cells in response to GC was significantly impaired in knockdown compared to control cells (GILZ p=0.009, FKBP51 p=0.03), they were no more resistant to dexamethasone (p=0.9). This was mirrored in siCBP cell lines and primagraft cells, where a significant impairment in basal expression of GILZ and/or FKBP51 was seen in some lines (GILZ reduction; p=0.03 PreB 697 shCBP, p=0.02 PreB 697 siCBP, FKBP51 reduction; p=0.01 primagraft siCBP cells) but no significant impairment in the transcriptional induction of these genes in response to GC compared to isogenic control was observed (p values >0.5). Importantly, no decreased sensitivity to dexamethasone was seen in any model after CREBBP knockdown (p values >0.1). CREBBP knockdown in ALL cells had no significant effect on the induction of cAMP dependent genes, had a variable effect on GR target expression, but consistently showed no impact on GC sensitivity, regardless of cytogenetic context. These data show that the acquisition of CREBBP mutations at relapse in childhood ALL is not mediated through GC resistance and suggest that other CREBBP associated mechanisms, such as DNA damage repair, may influence drug response. Understanding the role of CREBBP in carcinogenesis and drug resistance is crucial as it is implicated as a tumour suppressor in a growing number of cancers, making it a potential multi-tumour target for novel therapies. Disclosures No relevant conflicts of interest to declare.

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Xu, Abai, and Tingting Chen. "Abstract 5102: Correlation analysis of CREBBP mutation with tumor mutation burden and effect of immune checkpoint therapy in bladder cancer." Cancer Research 82, no. 12_Supplement (2022): 5102. http://dx.doi.org/10.1158/1538-7445.am2022-5102.

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Abstract Background: CREB-binding protein (CREBBP, hereafter CBP) encoded the protein having intrinsic histone acetyltransferase activity, which stabilized the additional protein interactions with the transcription complex. The mutation of CREBBP has been reported that caused Rubinstein-Taybi syndrome (RTS) and associated with acute myeloid leukemia, but not been studied in bladder cancer (BLCA). Immune checkpoint therapy (ICI) has significantly became one of the primary treatment of BLCA, and Tumor mutation burden (TMB) has been regarded as the most prevalent biomarker to predict immunotherapy. The CREBBP mutation status correlation with TMB and immune response remain unknown. Methods: Whole exome sequencing (WES) data and clinical data of 406 BLCA patients was obtained from the Cancer Genome Altas (TCGA). The mutation data of 206 FFPE tumor samples from Chinese BLCA patients were sequenced by targeted next-generation sequencing (NGS, 3DMed panel). NGS data and clinical data of 192 advanced BLCA patients treated with ICI were obtained from the MSK-IMPACT Clinical Sequencing cohort (MSKCC). The association between CREBBP mutation and TMB level in BLCA were explored. Survival analysis was determined by Kaplan-Meier (KM) analysis. Results: In total, The CREBBP mutation frequency was 16.25% (66/406) of BLCA patients in TCGA, 14.56% (30/206) in Chinese cohort and 13.54 (26/192). In Chinese cohort, the alteration frequency of NOTCH signal pathways affected was 48.54% (100/206), in which the highest frequency genes was CREBBP. The TMB level in CREBBP mutant group was higher than wild-type group both in Chinese group (Wilcoxon test, p = 0.001) and MSKCC cohort (Wilcoxon test, p &lt; 0.001). The survival analysis were performed on patients from TCGA cohorts without treatment information and MSKCC BLCA patients treated with ICIs, separately. In TCGA cohort, there are not significantly difference between CREBBP mutation group (n=65) and wild-type group (n=340). While in MSKCC ICIs treatment cohort, the overall survival (OS) of CREBBP mutation group (n=26) were significantly longer than wild-type group (n=166) (median OS, mutation vs wild-type = NE vs 15 months; HR 0.468 [95% CI 0.26-0.84]; P = 0.0431). Conclusion: The CREBBP gene mutation was associated with higher TMB level. Clinical cohort analysis results suggested that CREBBP might be a predictive biomarker of immune checkpoint therapy but not a prognostic factor in BLCA. Citation Format: Abai Xu, Tingting Chen. Correlation analysis of CREBBP mutation with tumor mutation burden and effect of immune checkpoint therapy in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5102.

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Feng, Dapeng, Zhengwei Li, Liang Yang, et al. "BMSC-EV-derived lncRNA NORAD Facilitates Migration, Invasion, and Angiogenesis in Osteosarcoma Cells by Regulating CREBBP via Delivery of miR-877-3p." Oxidative Medicine and Cellular Longevity 2022 (March 1, 2022): 1–19. http://dx.doi.org/10.1155/2022/8825784.

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Bone marrow mesenchymal stem cells (BMSCs) can boost osteosarcoma (OS) cell proliferation and invasion, yet the function of extracellular vesicles (EVs) derived from BMSCs on OS is scarcely known. This study is aimed at examining the role of BMSC-EVs in OS cells. BMSCs and BMSC-EVs were isolated and identified. The effect of EVs and EVs-si-NORAD on OS cell proliferation, invasion, migration, and angiogenesis was determined. Expressions of NORAD, miR-877-3p, and CREBBP were detected. The binding relationship among NORAD, miR-877-3p, and CREBBP was verified. The miR-877-3p inhibitor or pc-CREBBP was delivered into OS cells treated with EVs-si-NORAD for in vitro analysis. The nude mouse model of the subcutaneous tumor xenograft was established for in vivo analysis. BMSC-EVs promoted OS cell proliferation, invasion, migration, and angiogenesis. BMSC-EVs carried NORAD into OS cells and upregulated CREBBP by sponging miR-877-3p. miR-877-3p downregulation or CREBBP overexpression partly inverted the inhibitory effect of EVs by silencing NORAD on OS cell proliferation, invasion, migration, and angiogenesis. In vivo experiments validated that BMSC-EV-derived NORAD facilitated tumor growth by upregulating CREBBP via miR-877-3p. To conclude, BMSC-EV-derived NORAD facilitated OS cell proliferation, invasion, migration, and angiogenesis by modulating CREBBP via miR-877-3p, which may offer new insights into OS treatment.

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Gimenez, Alicia Garcia, Jonathan Ditcham, Dhoyazan M. Azazi, et al. "Abstract 363: Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition." Cancer Research 84, no. 6_Supplement (2024): 363. http://dx.doi.org/10.1158/1538-7445.am2024-363.

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Abstract B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological malignancy of B lineage progenitors. It remains a leading cause of death in childhood, while outcomes in adults are dismal. There is therefore a need to better understand drivers of high-risk B-ALL and to develop novel therapeutic approaches targeting these challenging patient cohorts. Loss-of-function mutations affecting CREBBP are recurrent second-hit mutations across multiple genetic subtypes of B-ALL and are associated with adverse features, including high-risk genetic subtypes and persistent measurable residual disease. In addition, they have been mechanistically associated with chemoresistance and are more frequently found in relapse. We sought to identify novel treatment options for CREBBP-mutated high-risk B-ALL. CREBBP-mutated isogenic human B-ALL cell lines were genome-engineered to provide a platform for synthetic lethal drug screening. We subjected these cell lines to a targeted drug screen focused on clinically-actionable drugs in classes that have either been implicated or hypothesized to show differential sensitivity patterns in published models of B cell lymphoma and other CREBBP-mutated malignancies. Unexpectedly, CREBBP-mutated cells were not differentially sensitive to traditional cytotoxic chemotherapy, and paradoxically showed a degree of sensitization to the glucocorticoid Dexamethasone, used in current ALL induction regimens. As anticipated, and validating our screen design, inhibitors of the CREBBP paralogue EP300 (the CREBBP/EP300-specific bromodomain inhibitor Inobrodib and the CREBBP/EP300 acetylase inhibitor A485) exhibited synthetic lethality, consistent with previous reports in B-cell lymphoma. The most potent hit was the BCL2 inhibitor Venetoclax, which we show acts through a non-canonical, but BCL2-dependent mechanism resulting in ferroptotic programmed cell death. CREBBP-mutated cell lines were transcriptionally and functionally characterized, revealing underlying differences in cell-cycle, metabolism and response to oxidative stress. Acquisition of resistance to Venetoclax further dysregulated these pathways and resulted in a transcriptionally-convergent state. Lastly, we demonstrate that small-molecule inhibition of CREBBP sensitizes B-ALL cells, regardless of genotype, to Venetoclax-induced ferroptosis in vitro and in vivo, providing a potential novel drug combination for broader clinical translation in B-ALL. In summary, we have identified a number of actionable compounds that specifically target CREBBP-mutated high-risk B-ALL, demonstrate a novel mechanism-of-action for the BCL2 inhibitor Venetoclax in B-ALL and propose CREBBP-inhibitors and Venetoclax as a novel treatment combination for B-ALL across genotypes. Citation Format: Alicia Garcia Gimenez, Jonathan Ditcham, Dhoyazan M. Azazi, Eshwar Meduri, Ryan Asby, Nathalie Sakakini, Cecile K. Lopez, Nisha Narayan, Jaana Bagri, Tumas Beinortas, Shuchi Agrawal Singh, George Giotopoulos, Michael P. Murphy, Sarah J. Horton, Brian J. Huntly, Simon E. Richardson. Genetic or pharmacological inactivation of CREBBP sensitizes B-cell acute lymphoblastic leukemia to ferroptotic cell death upon BCL2 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 363.

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García-Ramírez, Idoia, Saber Tadros, Inés González-Herrero, et al. "Crebbp loss cooperates with Bcl2 overexpression to promote lymphoma in mice." Blood 129, no. 19 (2017): 2645–56. http://dx.doi.org/10.1182/blood-2016-08-733469.

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Key Points Crebbp inactivation perturbs B-cell development, but cooperates with Bcl2 overexpression to promote lymphoma. Transcriptional and epigenetic signatures of Crebbp loss implicate Myc in disease etiology.

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Tokunaga, Kenji, Shunichiro Yamaguchi, Eisaku Iwanaga, et al. "Crebbp HAT Domain Mutations Are Frequently Detected in Adult Acute Lymphoblastic Leukemia." Blood 120, no. 21 (2012): 1419. http://dx.doi.org/10.1182/blood.v120.21.1419.1419.

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Abstract Abstract 1419 Aims: Molecular pathogenesis of acute lymphoblastic leukemia (ALL) has largely been verified in pediatric patients and the identification of genetic alterations have contributed to stratifying therapeutic applications. In adult patients with ALL, cytogenetic and genetic abnormalities have not sufficiently been elucidated and therapeutic improvement has been hindered. CREB binding protein (CREBBP) is a transcriptional coactivator that interacts with a diverse range of transcription factors and regulates transcription by histone acetylation in hematopoiesis. Mutations of the CREBBP gene are recently found in approximately 2–4% of pediatric patients with ALL. Especially in relapsed cases, the mutations prevail (18–63%) and are possible markers for prediction of relapse in pediatric ALL. In adult patients with ALL, the clinical significance of CREBBP mutations remains to be determined. Here we examined adult ALL patients in an attempt to determine the incidence, clinical characteristics and prognostic impact of the CREBBP mutations. Methods: We investigated 71 adult patients with newly diagnosed ALL treated with JALSG protocols between 1986 and 2010. Age ranged from 15 to 86 years, with a median of 54 years. CREBBP mutations are dominantly identified in histone acetyltransferase (HAT) domain. HAT domain in the CREBBP gene was amplified with RT-PCR using RNA isolated from the peripheral blood or bone marrow mononuclear cells at diagnosis and was subjected to direct sequencing. We compared clinical profiles between patients with and without CREBBPHAT domain mutations. This study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results: CREBBP HAT domain mutations were detected in 8 of 71 (11.3%) patients: one nonsense mutation, five insertion mutations with frameshifts, and five missense mutations. Two patients harbored biallelic mutations. The mutations at diagnosis in adult patients were seen more frequently than those in pediatric patients ever reported. Such mutations were not completely identical to those detected in pediatric ALL, but were seen in the region within the HAT domain, indicating that such mutations are loss-of-function mutations. The mutations were found in both B-cell (6/53: 11.3%) and T-cell (1/9: 11.1%) ALL, and distributed in patients harboring IKZF1 alterations (3/31: 9.7%) or the BCR-ABL fusion gene (2/19: 10.5%). There were no statistical difference in age, sex, leukocyte, platelet counts and complete remission rate between patients with and without the CREBBP HAT domain mutations. Patients with the mutations had a trend with worse cumulative incidence of relapse (P=0.4637), relapse-free survival (P=0.4195) and OS (P=0.2349) compared to patients lacking the mutations, but statistical significance was not detected in this small cohort. Conclusions: CREBBP HAT domain mutations at diagnosis in adult ALL are found more frequently than in pediatric ALL. This may be one of the mechanisms that adult ALL has been associated with poor OS compared with pediatric ALL. In this study, CREBBP HAT domain mutations were observed in various subtypes of ALL: both B-cell and T-cell ALL, and both Philadelphia chromosome positive and negative ALL. In pediatric ALL, CREBBP mutations were frequently seen in relapsed patients but not in previously untreated patients. These observations suggest that CREBBP mutations play an important role in an additional late event(s) leading to the development and progression of ALL. Our study implies the possibility that mutations of the CREBBP gene are associated with the pathogenesis and prognostic marker of adult ALL and represent specific epigenetic modifiers in adult ALL, serving as potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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Camos, Mireia, Jordi Esteve, Dolors Colomer, et al. "Gene Expression Signature of Acute Myeloid Leukemia (AML) with T(8;16)(P11;P13) and MYST3-CREBBP Rearrangement: A Microarray Study Validated by Multiple Real-Time PCR." Blood 106, no. 11 (2005): 3009. http://dx.doi.org/10.1182/blood.v106.11.3009.3009.

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Abstract AML with t(8;16)(p11;p13) is an infrequent leukemia subtype with characteristic clinical-biological features. The t(8;16)(p11;p13) translocation leads to the fusion of MYST3 and CREBBP genes, probably resulting in a disturbed transcriptional program of a myelo-monocytic precursor. In this study, the genetic signature of MYST3-CREBBP AML was compared with other well-defined AML subtypes. Genotypic analyses using oligonucleotide U133A arrays (Affymetrix) were performed on RNA of 23 AML patients, including three MYST3-CREBBP cases, PML-RARa (n=3), RUNX1-CBF2T1 (n=3), CBFβ-MYH11 (n=3), t(9;11)/AF9-MLL (n=1), monocytic AML (FAB M4/M5), n=8, and two cases of AML with multilineage dysplasia. Forty-six genes differentially expressed in MYST3-CREBBP cases were analyzed by multiple real-time RT-PCR using low-density arrays in an additional series of 40 patients, which included 7 MYST3-CREBBP cases, 18 AML samples with well characterized rearrangements (PML-RARa, n=3; RUNX1-CBF2T1, n=3; CBFβ-MYH11, n=3, MLL-rearranged AML, n=9), and 15 patients with normal karyotype AML. After unsupervised analysis, MYST3-CREBBP cases clustered together, displaying a distinctive expression signature. The analysis by RT-PCR confirmed the gene expression pattern found in the high-density array study. Thus, overexpressed genes included oncogene RET, several homeobox (HOXA9, HOXA10), genes involved in apoptosis (DAP) and prolactin gene. In contrast, cyclinD2, STAT5A, STAT5B and WT1 were underexpressed. Interestingly, MYST3-CREBBP cases showed up-regulation of a subgroup of genes (HOXA9, MEIS1, AKR7A2, CHD3, FLT3 and APBA2) that were also found overexpressed in MLL-rearranged leukemias. In summary, this study showed the distinctive genetic signature of MYST3-CREBBP AML, which harbours some similarities with MLL-rearranged AML. In addition, the low-density array methodology validated the results of the microarray analysis and allowed to study a larger series of patients, including samples not suitable for microarray analysis.

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Chafai Elalaoui, Siham, Wiam Smaili, Julien Van-Gils, et al. "Clinical description and mutational profile of a Moroccan series of patients with Rubinstein Taybi syndrome." African Health Sciences 21, no. 2 (2021): 960–67. http://dx.doi.org/10.4314/ahs.v21i2.58.

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Background: Rubinstein-Taybi syndrome (RSTS; OMIM 180849) is a rare autosomal dominant developmental disorder with an estimated prevalence of one case per 125,000 live births. RSTS is characterized by typical face, broad thumbs and halluces, short stature, and intellectual disability. Facial dysmorphy is characteristic with microcephaly, low frontal hairline, arched eyebrows, long eyelashes, convex profile of nose, narrow palate, and micrognathia. RSTS is mainly due to mutations or microdeletions of the CREBBP gene (about 60%) and more rarely of the EP300 gene (8%). Objective: Clinical description and identification of mutations of patients with Rubinstein Taybi syndrome. Methods: PCR and direct sequencing of CREBBP gene. Results: We report here, the clinical and molecular data of a series of six Moroccan patients with a phenotype of RSTS. The molecular study of the major gene CREBBP (by Sanger Sequencing followed by CGH array, if sequence normal) revealed point mutations in five patients. For the sixth patient, CGH array revealed a microdeletion carrying the CREBBP gene. Through this work, we emphasize the importance of clinical expertise in the diagnosis, management and genetic counseling in Rubinstein Taybi syndrome. Keywords: Rubinstein Taybi syndrome; CREBBP gene; mutation; Moroccan.

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Al-Qattan, Mohammad M., Zuhair A. Rahbeeni, Zuhair N. Al-Hassnan, et al. "Chromosome 16p13.3 Contiguous Gene Deletion Syndrome including the SLX4, DNASE1, TRAP1, and CREBBP Genes Presenting as a Relatively Mild Rubinstein–Taybi Syndrome Phenotype: A Case Report of a Saudi Boy." Case Reports in Genetics 2020 (January 9, 2020): 1–5. http://dx.doi.org/10.1155/2020/6143050.

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The classic Rubinstein–Taybi syndrome Type 1 (RSTS1, OMIM 180849) is caused by heterozygous mutations or deletions of the CREBBP gene. Herein, we describe the case of a Saudi boy with chromosome 16p13.3 contiguous gene deletion syndrome (OMIM 610543) including the SLX4, DNASE1, TRAP1, and CREBBP genes, but presenting with a relatively mild RSTS1 syndrome phenotype. Compared with previously reported cases with severe phenotypes associated with 16p13.3 contiguous gene deletions, our patient had partial deletion of the CREBBP gene (with a preserved 5′ region), which might explain his relatively mild phenotype.

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Meyerhöfer, Markus, Viral Shah, Aarón Gallego-Crespo, et al. "The Transcriptional Activators p300/Crebbp Are Context-Dependent Functional Repressors of Interferon Genes, Offering Therapeutic Promise in AML." Blood 144, Supplement 1 (2024): 152. https://doi.org/10.1182/blood-2024-194781.

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The lysine acetyltransferase (KAT) enzymes p300 and CREBBP, primarily recognized as histone acetyltransferases, are pivotal co-activators in acute myeloid leukemia (AML) maintenance. However, the traditional view of histone acetylation as a basic mechanism for transcriptional activation is increasingly being challenged, while the role of p300/CREBBP in acetylating non-histone proteins remains largely unexplored. This gap confounds the rationale for advancing novel p300/CREBBP KAT inhibitors to clinical trials, as a comprehensive understanding of p300/CREBBP functions is crucial for their effective clinical evaluation. Here, we set out to dissect the roles of p300/CREBBP in AML beyond histone acetylation, assess the implications of inhibition of their KAT domain and explore potential effective therapeutic combinations to facilitate their clinical application in treating AML. Initially, we methodically analyzed the impact of p300/CREBBP inhibition across various AML subtypes using RNASeq and qPCR as preliminary screens, followed by confirmatory single-cell RNASeq and mass spectrometry-based total proteomics. We assessed the effects of KAT domain inhibition (hereafter KATi - via A-485 or CPI-1612) in comparison to a p300/CREBBP bromodomain inhibitor and a PROTAC degrader. All these compounds suppressed key AML maintenance genes, but uniquely, KATi also induced a massive expression of interferon-stimulated genes (ISG). This was particularly pronounced in monocytic AML and contributed to cell cycle arrest, differentiation and apoptosis. In SLAMSeq and qPCR, we observed that ISG induction began shortly after KATi and intensified progressively within the first 24 hrs, illustrating a dynamic ‘feed-forward’ transcriptional activation. We hypothesized that this unexpected induction of ISG upon KATi might reveal a previously unrecognized suppressive role exerted by the p300/CREBBP KAT activity. To investigate this, we employed a multidimensional approach, comparing cells treated with KATi to DMSO controls. We began with acetylproteomics and RIME-based chromatin pull-down of p300 to pinpoint the interactors of p300/CREBBP in monocytic AML and complemented these with ChIPSeq of identified acetylation-interactors, PROSeq to track real-time transcription at promoters and enhancers, and a PerturbSeq screen paired with scRNASeq to assess the functional importance of the interactors. p300 and CREBBP emerged as their own most significant interactors and targets for (auto)acetylation. They were followed by the cBAF remodeling complex members ARID1A and SMARCA4. Other significant partners included transcription(al) (co)factors like RUNX1, IRF8, PU.1, MEF2D or IRF2BP2. PROSeq revealed a pronounced and almost exclusive increase in transcription at ISG promoters. At these sites, ChIPSeq revealed KATi-induced ‘trapping’ of p300 and CREBBP, increased remodeling activity by ARID1A/SMARCA4 and exclusion of IRF8 from the chromatin. In PerturbSeq, the ISG induction triggered by KATi was particularly reduced when disrupting ARID1A/SMARCA4, highlighting the cBAF complex's critical role in mediating ISG responses to KATi. This was further validated by employing PROTAC-mediated degradation of the cBAF subunit SMARCA4, which mitigated the ISG release and alleviated AML cell death associated with KATi. Finally, RIME-based chromatin pull-down and mass spectrometry for the cBAF subunit ARID1A revealed new interactions with STAT1 and other positive ISG regulators upon KATi, while ChIPSeq showed exclusive new binding of STAT1 to critical ISG promoters, confirming a shift to a transcriptionally active state. Testing the therapeutic potential of this ISG release beyond single compound treatment, we combined KATi with Interferon alpha (IFNa). This demonstrated an exponential increase of the ISG response and a significant, highly synergistic induction of apoptosis in human and murine monocytic AML cell lines and primary samples, suggesting a promising clinical combination. In vivo treatment combinations are currently underway. In conclusion, active p300/CREBBP maintain a repressive environment for ISGs in conjunction with the cBAF complex in monocytic AML. p300/CREBBP KAT domain inhibition shifts this to a permissive state, releasing ISGs, which can be further amplified by IFNa. In our view, this combination holds significant translational potential for AML treatment.

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Bommi-Reddy, Archana, Sungmi Park-Chouinard, David N. Mayhew, et al. "CREBBP/EP300 acetyltransferase inhibition disrupts FOXA1-bound enhancers to inhibit the proliferation of ER+ breast cancer cells." PLOS ONE 17, no. 3 (2022): e0262378. http://dx.doi.org/10.1371/journal.pone.0262378.

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Therapeutic targeting of the estrogen receptor (ER) is a clinically validated approach for estrogen receptor positive breast cancer (ER+ BC), but sustained response is limited by acquired resistance. Targeting the transcriptional coactivators required for estrogen receptor activity represents an alternative approach that is not subject to the same limitations as targeting estrogen receptor itself. In this report we demonstrate that the acetyltransferase activity of coactivator paralogs CREBBP/EP300 represents a promising therapeutic target in ER+ BC. Using the potent and selective inhibitor CPI-1612, we show that CREBBP/EP300 acetyltransferase inhibition potently suppresses in vitro and in vivo growth of breast cancer cell line models and acts in a manner orthogonal to directly targeting ER. CREBBP/EP300 acetyltransferase inhibition suppresses ER-dependent transcription by targeting lineage-specific enhancers defined by the pioneer transcription factor FOXA1. These results validate CREBBP/EP300 acetyltransferase activity as a viable target for clinical development in ER+ breast cancer.

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Bertulfo, Kalay, Hannah Miller, Ryan Najac, et al. "Crebbp Inhibition Potentiates JAK2 Inhibition in Post-MPN Acute Myeloid Leukemia." Blood 144, Supplement 1 (2024): 823. https://doi.org/10.1182/blood-2024-201988.

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Post-MyeloProliferative Neoplasm Acute Myeloid Leukemia (Post-MPN AML) is an aggressive and lethal hematologic malignancy arising from myeloproliferative neoplasms. Currently, there is no standard of care treatment for post-MPN AML patients, with allogeneic stem cell transplantation being the only curative option. However, the advanced age and comorbidities of post-MPN AML patients render them unfit for allo-SCT, emphasizing the urgent unmet need to find novel treatment for this disease. Hyperactivation of JAK/STAT signaling is highly prevalent in post-MPN AML pathogenesis, with as much as 50-90% of MPN cases harboring driver mutations in JAK2, CALR or MPL. The JAK2 inhibitor ruxolitinib has been tested in clinical trials for post-MPN AML and was reported to improve the patient quality of life, however, it was not effective in modifying the course of the disease. Given the limitations of targeting the JAK/STAT pathway, we aimed to identify genes and pathways synergistic with JAK2 inhibitors in post-MPN AML. Here, we demonstrate through genome-wide CRISPR screens in post-MPN AML line HEL treated with four different JAK2 inhibitors (i.e. ruxolitinib, momelotinib, pacritinib and fedratinib) that depletion of CREBBP sensitizes cells to JAK2 inhibition. CREBBP is a histone acetyltransferase which also acts as a coactivator of essential transcription factors in diverse hematopoietic lineages. CREBBP is overexpressed in AML and is ubiquitously expressed in blood cancer cell lines, underscoring its potential as a therapeutic target. Genetic depletion of CREBBP via CRISPR/Cas9 editing showed that CREBBP, but not its paralog EP300, sensitizes HEL to JAK2 inhibition. In addition, pharmacological approaches showed that JAK2 inhibitors synergize with CREBBP/EP300 inhibitors to effectively kill post-MPN AML lines. In both human and murine models of post-MPN AML, the combination treatment of ruxolitinib plus CREBBP/EP300 inhibitor SGC-CBP30 or CCS1477 substantially induced apoptosis and cell cycle arrest at G1. Moreover, bioluminescence imaging of HEL luciferase mouse xenografts treated with vehicle, ruxolitinib (80 mg/kg), CCS1477 (20mg/kg) and combination showed significant decrease in leukemia burden in the combination-treated animals after 3 weeks of treatment. Integration of the CRISPR viability screen and transcriptome profiles of HEL cells treated with JAK and CREBBP inhibitors identified 118 genes that are both essential for the proliferation and are downregulated with the combination treatments. GSEA showed enrichment of MYC targets in the said genes with E2F4, MYC and HSF1 being the top transcription factors associated with them. Notably, 21 of the 118 genes identified are part of the epichaperome network which is regulated by MYC. Accordingly, western blot analysis showed decreased MYC, E2Fs, STAT3 and STAT5 protein levels in HEL cells treated with the combinations. In addition, combination treatments also reversed the accumulation of JAK2 protein levels induced by ruxolitinib, which is a well-studied mechanism driving disease persistence. We hypothesize that this is due to the downregulation of MYC-regulated epichaperome genes which are known to regulate JAK/STAT signaling by physically interacting and stabilizing JAK2. Further investigation on the epigenetic changes induced by the ruxolitinib/CREBBP inhibitors is underway to identify their modulatory role of MYC and STAT3/5 regulatory regions. Overall, our results demonstrate that CREBBP/EP300 inhibition potentiates JAK2 inhibition in post-MPN AML by further attenuating MYC expression and activity, and repressing JAK/STAT and other pathways associated with JAK2 inhibitor persistence. Therefore, we propose CREBBP inhibition as a potential therapeutic strategy to potentiate JAK2 inhibition in post-MPN AML.

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Mullighan, Charles, Jinghui Zhang, Lawryn H. Kasper, et al. "CREBBP Mutations In Relapsed Acute Lymphoblastic Leukemia." Blood 116, no. 21 (2010): 413. http://dx.doi.org/10.1182/blood.v116.21.413.413.

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Abstract Abstract 413 Relapsed acute lymphoblastic leukemia (ALL) is a leading cause of death due to disease in young people, but the biologic determinants of treatment failure remain poorly understood. To identify novel sequence mutations contributing to relapsed in ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. The cohort included B-progenitor ALL with high hyperdiploidy (N=3), TCF3-PBX1 (N=1), ETV6-RUNX1 (N=3), rearrangement of MLL (N=3), BCR-ABL1 (N=3), and low hyperdiploid, pseudodiploid, or miscellaneous karyotypes (N=10). This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including mutations in the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 63 diagnosis-relapse cases and 200 acute leukaemia cases that did not relapse found that 19% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP). The mutations were either present at diagnosis, acquired at relapse, or duplicated to homozygosity at the time of relapse. Moreover, several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations confer a selective advantage and promote resistance to therapy. The mutations either resulted in truncated alleles or deleterious substitutions in highly conserved residues of the HAT domain. To examine the functional consequences of the mutations, we introduced wild type or mutant Crebbp alleles into Cbp/Ep300flox/flox murine embryonic fibroblasts, (dKO MEFs), and examined histone acetylation, expression of CREBBP target genes, and cellular proliferation. The HAT domain mutations resulted in impaired acetylation of the key Crebbp substrate, H3K18, and resulted in impaired transcriptional regulation of multiple CREBBP targets and pathways, including cAMP, dsRNA and dexamethasone responsive genes. The latter observation suggests that CREBBP mutations may directly result in resistance to corticosteroid therapy, which is a hallmark of high risk ALL. Together, these data these results extend the landscape of genetic alterations in leukemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL. Disclosures: Pui: EUSA Pharma: Honoraria; Enzon: Honoraria; Sanofi-Aventis: Honoraria.

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Schroers-Martin, Joseph G., Joanne Soo, Gabriel Brisou, et al. "Recurrent Crebbp Mutations in Follicular Lymphoma Appear Localized to the Committed B-Cell Lineage." Blood 136, Supplement 1 (2020): 30–31. http://dx.doi.org/10.1182/blood-2020-142761.

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Background: Follicular lymphoma (FL) is genetically characterized by translocations involving the BCL2 locus on chromosome 18q21. However, up to 70% of healthy individuals also carry detectable t(14;18)-positive cells, suggesting BCL2 translocation is critical but not sufficient for FL development. Chromatin modifying genes (CMGs) including KMT2D, CREBBP, EZH2, and EP300 are almost ubiquitously mutated in FL. We previously reported the direct characterization by ultra-deep sequencing of pre-diagnostic blood and tissue specimens from 19 subjects who ultimately developed FL. CREBBP lysine acetyltransferase (KAT) domain mutations were the most commonly observed precursor lesions, detected in blood a median of 7.5 years before diagnosis in patients developing FL (8/19, 42%) but not in healthy adults with or without detected BCL2 translocations (0/13, p=0.01 and 0/20, p&lt;0.001, respectively) (Schroers-Martin et al, ASH Annual Meeting 2017). While the BCL2 translocation is thought to occur in pre-B-cell precursors in the bone marrow, the high prevalence of CMG mutations raises the possibility that, analogous to myeloid age-related clonal hematopoiesis, such early lesions could occur in hematopoietic stem cells (HSCs) rather than in the committed B-cell lineage. Methods: To address this question of mutational hierarchy, we studied blood, bone marrow aspirate, and lymph node samples from 6 patients with CREBBP or KMT2D mutations in their FL tumor. We analyzed flow-sorted purified hematopoietic cell populations by deep sequencing, including isotype-specific mature B-cell, mature T-cell, and precursor populations encompassing HSCs and common lymphoid progenitors (CLPs). Patients additionally underwent routine clinical sequencing of tumor biopsy and marrow specimens. Results: Bulk sequencing of a diagnostic bone marrow aspirate from patient FL002 revealed a CREBBP mutation concordant with FL tumor biopsy. To ascertain the population bearing this mutation, we sorted to high purity viable marrow aspirate cells (Fig. A). The CREBBP mutation was confirmed in the mature B-cell compartment at an AF of 40.3% but was not detected in other cell populations. To validate this finding, a similar sorting strategy was employed on viable bone marrow aspirate or peripheral blood samples from another 4 FL patients bearing CREBBP mutations (Fig B). In each case, CREBBP was absent from the CD34+ precursor population. In patient CIML004 a characteristic KMT2D stop mutation was likewise absent in precursors. An atypical case sheds additional light on the localization of early FL mutations. Patient LYM267 was diagnosed with Grade 1-2 FL bearing CREBBP and NRAS mutations. Eight years into a prolonged remission after chemoimmunotherapy, he developed cutaneous and gingival myeloid sarcoma without radiographic or histopathological evidence of FL recurrence. While the CREBBP mutation was not detected in myeloid sarcoma or bone marrow biopsies, concordant NRAS mutations and clonal VDJ rearrangements were seen in all 3 compartments (Fig. C). This unusual clonal lineage favors the occurrence of the CREBBP mutation later than the branch point between morphologically distinct lymphoid and myeloid tumors, likely in the committed B-cell lineage after pre-BCR rearrangements (Fig D). Conclusions: HSCs are believed to be the cell of origin in several lymphoid leukemias, and mouse models have demonstrated lymphoma development with induced CREBBP lesions in HSCs (Horton et al Nat Cell Bio 2017). However, in sorted hematopoietic cell populations from marrow and peripheral blood, we observed CREBBP mutations in B-cell lineages but never in CD34+/CD20- precursor populations or paired lymphoid/myeloid disease. Our data therefore are not in support of HSCs as a precursor reservoir in FL. Given that cells harboring the t(14;18) translocation in healthy individuals appear derived from the germinal center, recurrent mutations in CREBBP are likely to occur after the pre-B-cell stage. Figure Disclosures Kurtz: Genentech: Consultancy; Foresight Diagnostics: Other: Ownership; Roche: Consultancy. Khodadoust:Kyowa Kirin: Consultancy; Seattle Genetics: Consultancy. Nadel:Innate Pharma: Research Funding; Institut Roche: Research Funding. Diehn:RefleXion: Consultancy; Varian Medical Systems: Research Funding; Illumina: Research Funding; Roche: Consultancy; BioNTech: Consultancy; AstraZeneca: Consultancy. Roulland:Celgene/BMS: Research Funding; Roche: Honoraria. Alizadeh:Pharmacyclics: Consultancy; Genentech: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Gilead: Consultancy; Pfizer: Research Funding; Roche: Consultancy.

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Lazaro‐Navarro, Juan, Clara Alcon, Mathurin Dorel, et al. "Inhibiting H3K27 Demethylases Downregulates CREB‐CREBBP, Overcoming Resistance in Relapsed Acute Lymphoblastic Leukemia." Cancer Medicine 14, no. 1 (2025): 1–7. https://doi.org/10.1002/cam4.70596.

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ABSTRACTBackgroundCREB binding protein (CREBBP) is a key epigenetic regulator, altered in a fifth of relapsed cases of acute lymphoblastic leukemia (ALL). Selectively targeting epigenetic signaling may be an effective novel therapeutic approach to overcome drug resistance. Anti‐tumor effects have previously been demonstrated for GSK‐J4, a selective H3K27 histone demethylase inhibitor, in several animal models of cancers.MethodsTo characterize the effect of GSK‐J4, drug response profiling, CRISPR‐Dropout Screening, BH3 profiling and immunoblotting were carried out in ALL cell lines or patient derived samples.ResultsHere we provide evidence that GSK‐J4 downregulates cyclic AMP‐responsive element‐binding protein (CREB) and CREBBP in B‐cell precursor‐ALL cell lines and patient samples. High CREBBP expression in BCP‐ALL cell lines correlated with high GSK‐J4 sensitivity and low dexamethasone sensitivity. GSK‐J4 treatment also induced Bcl‐2 and Bcl‐XL dependency and apoptosis.ConclusionsThis study proposes H3K27 demethylase inhibition as a potential treatment strategy for patients with treatment‐resistant ALL, using CREBBP as a biomarker for drug response and combining GSK‐J4 with venetoclax and navitoclax as synergistic partners.

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Haase, Jacob, Talia A. Gebhard, Qi Liu, et al. "Abstract 3068: CREBBP/EP300 disruption promotes tumor progression and confers synthetic lethality in anaplastic thyroid cancers." Cancer Research 83, no. 7_Supplement (2023): 3068. http://dx.doi.org/10.1158/1538-7445.am2023-3068.

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Abstract Anaplastic thyroid cancers (ATC) are fast-growing, undifferentiated tumors and almost invariably fatal, primarily due to the lack of effective therapeutic options. The recent approval of dabrafenib plus trametinib for the treatment of BRAFV600E-mutant ATCs improved the prognosis of a subset of patients, but ineligibility and acquired resistance still limit their use. Overall, ATC patients remain in great need of tailored therapeutic options. We previously showed that loss-of-function alterations targeting histone acetyltransferase (HAT) genes, namely CREBBP and EP300, occur in 15-20% of ATCs, but only in &lt;1% of their well-differentiated counterparts. What remains unknown are the specific mechanisms by which HAT disruptions perturb chromatin architecture, impact gene homeostasis, and unleash cellular processes in these aggressive tumors. We are assessing the HAT-mediated thyroid cancer progression. CREBBP/EP300 knockouts enhanced thyroid cancer cell proliferation in vitro and induced thyroid gland growth in a thyroid-specific mouse model of HAT loss. We are leveraging these animals to characterize the in vivo effects of Crebbp/Ep300 knockout, alone or in combination with BrafV600E, in thyroid cancer phenotypes and epigenetic reconfiguration. In addition, we are exploiting the molecular consequences of HAT loss to explore tailored treatments. CRISPR/Cas9 screens identified a mutual CREBBP/EP300-dependency of HAT-mutant human cancer cell models. Our experiments in thyroid cancer cells employing CREBBP/EP300-targeting proteolysis-targeting chimera (PROTAC) compounds specifically degraded these proteins and decreased histone acetylation. Our findings prove the oncogenicity of HAT loss in thyroid cancer progression and support exploring synthetic lethality dependencies in CREBBP/EP300-mutant ATCs. In summary, we provide pre-clinical basis to inform genomics-driven and mechanism-oriented decisions for the clinical management of patients with HAT-altered ATC. Citation Format: Jacob Haase, Talia A. Gebhard, Qi Liu, Sara G. Bernabé, Jingzhu Hao, Chisom Unegbu, Athanasios Bikas, Jun Qi, Iñigo Landa. CREBBP/EP300 disruption promotes tumor progression and confers synthetic lethality in anaplastic thyroid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3068.

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Sima, Aurora, Roxana Elena Smădeanu, Anca Angela Simionescu, Florina Nedelea, Andreea-Maria Vlad, and Cristina Becheanu. "Menke–Hennekam Syndrome: A Literature Review and a New Case Report." Children 9, no. 5 (2022): 759. http://dx.doi.org/10.3390/children9050759.

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Background: Menke–Hennekam syndrome (MHS) is a rare and recently described syndrome consecutive to the variants in exon 30 or 31 in CREBBP (CREB-binding protein gene). The CREB-binding protein (CREBBP) and EP300 genes are two commonly expressed genes whose products possess acetyltransferase activity for histones and various other proteins. Mutations that affect these two genes are known to cause Rubinstein–Taybi syndrome (RTS); however, with the application of whole exome sequencing (WES) there were reports of variants that affect specific regions of exon 30 or 31 of these two genes but without the specific phenotype of RTS. Material and Methods: A review of the available literature was conducted, aimed at underscoring the difficulties in diagnosing MHS based on phenotype particularities. Results: Five applicable studies were identified by searching PubMed, Web of Science, and Scopus databases for publications up to November 2021 using the key terms “Menke–Hennekam syndrome” and “CREBBP”. Conclusions: In this paper, we present a new case and highlight the importance of exome sequencing to identify different mutations of exons 30 and 31 of the CREBBP gene involved in MHS, and we make formal recommendations based on our literature review.

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Melanie, Schoof, Gefion Dorothea Epplen, Carolin Walter, et al. "MODL-11. The tumor suppressor CREBBP and the oncogene MYCN cooperate to induce malignant brain tumors in mice." Neuro-Oncology 24, Supplement_1 (2022): i170—i171. http://dx.doi.org/10.1093/neuonc/noac079.634.

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Abstract CREBBP (cAMP response element-binding protein binding protein) and MYCN (v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog), two essential proteins in central nervous system development that are described to be genetically altered in the same brain tumor entities, such as high grade glioma or medulloblastoma. Therefore, we hypothesized that alterations in both genes cooperate to induce brain tumor formation. In order to investigate molecular mechanisms and potential tumor formation, we established a novel mouse model combining a deletion of Crebbp with an overexpression of MYCN in neural stem cells (hGFAP-cre::CrebbpFl/Fl::lsl-MYCN). The animals developed highly aggressive forebrain tumors with 75 % penetrance already detectable in seven-day old mice, leading to tumor-related death within eight months. Although tumors mostly developed in the olfactory bulb region, we hypothesized that these tumors develop from subventricular stem cells that migrate into the olfactory bulb, where they settle and establish large solid tumors. In order to test this hypothesis, we analyzed neural stem cells (NSCs) derived from our mouse model. Compared to control cells, NSCs from transgenic mice have a significantly increased cell viability and proliferation in vitro and did not undergo full glial differentiation. In order to gain further insights into the cellular origin and biology of the tumors, we finally employed single cell RNA sequencing. Apart from a high intratumoral heterogeneity within the tumor cell population, data comparison with non-neoplastic CNS cell populations revealed a high similarity with transit-amplifying NSCs of the subventricular zone. Together, we were able to show the oncogenic potential of a combined loss of Crebbp and aberrant expression of MYCN in neural stem cells. This novel mouse model will be an important tool for understanding tumor-driving mechanisms and a valuable model for preclinical testing and will thereby help to improve patient care.

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Derheimer, Frederick A., Ninvita Givarkes, Natalie Miller, et al. "Abstract LB028: Discovery and characterization of selective heterobifunctional degraders of EP300 in hematopoietic malignancies." Cancer Research 84, no. 7_Supplement (2024): LB028. http://dx.doi.org/10.1158/1538-7445.am2024-lb028.

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Abstract The paralogous histone acetyltransferases EP300 and CREBBP are two of the master regulators of transcription by controlling both enhancer and promoter activity through histone and non-histone acetylation. EP300 and CREBBP activity is primarily driven by their acetylation of K27 and K18 on histone H3 leading to open and active chromatin state. Targeting the EP300/CREBBP axis of chromatin regulation may be a way to disrupt the signaling pathways downstream of key oncogenic signaling networks like MYC/IRF4 in hematopoietic cancers. Successful development of dual EP300/CREBBP BRDi and HATi has led to promising pre-clinical efficacy, but due to observed tolerability issues may potentially limit their utility in the clinic. We hypothesized, based upon published mouse KO phenotypes, that a selective EP300 compound may improve tolerability. Due to the high sequence homology between EP300 and CREBBP in both the BRD and HAT domains, developing selective inhibitors has proven to be challenging. To combat this, we have utilized heterobifunctional protein degrader technology (ProDegs, also known as PROTACS) to develop EP300 selective ProDegs with greater than 100-fold selectivity over CREBBP. We observe rapid targeted degradation of EP300 with maximal degradation occurring within the first 2-4 hours after treatment. Concomitant with the loss of EP300, we see modulation of the associated H3K27ac and H3K18ac biomarkers as well as alterations in key oncogenic transcription networks. In both in vitro and in vivo assays looking at impact to platelet maturation, we observed that selective loss of EP300 largely abrogated observed thrombocytopenia compared to dual EP300/CREBBP inhibitors. Upon treatment, MM and NHL models show strong downregulation of MYC/IRF4 oncogenic signaling pathways, both of which culminate in a loss of cellular viability and cell death. Additionally, we observe in vivo dose responsive tumor growth inhibition in OPM2 and Pfeiffer xenograft models. Together, the data demonstrate the potential utility of EP300 selective ProDegs for the treatment of hematopoietic malignancies. Citation Format: Frederick A. Derheimer, Ninvita Givarkes, Natalie Miller, Rita Grantner, Casey Quinlan, Sonja Brun, Thomas Paul, Astrid Ruefli-Brasse, Stefan Steyn, Derek Bartlett, Matthew Hayward, Matthew Brown, Daniel Uccello, Benjamin J. Burke, Kris Borzilleri, Adam Gilbert, Michelle Hemkens. Discovery and characterization of selective heterobifunctional degraders of EP300 in hematopoietic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB028.

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Wijetunga, N. Ari, Emily Lebow, Alexander Chan, et al. "Genetic Alterations of Follicular Lymphoma Can Predict Response to Very Low Dose Radiotherapy." Blood 142, Supplement 1 (2023): 6099. http://dx.doi.org/10.1182/blood-2023-185338.

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Introduction Follicular lymphoma (FL) is typically indolent, but relapses and transformation to higher grade disease are common. FL is often radiosensitive and can show complete response (CR) even to very low dose radiotherapy (VLDRT, 4Gy). There are no known genetic markers of FL radiosensitivity. We sought to identify molecular signatures of FL radiosensitivity to aid in patient selection for VLDRT. Methods We analyzed our institutional database and identified 110 FL patients with 113 tumors treated with radiotherapy (RT) and preexisting MSK-IMPACT, a targeted exon sequencing panel. For each patient, we collected key clinicodemographic information including pre- and post-RT treatment history, prior large cell lymphoma, and Ann Arbor stage at RT. For treated tumors, we obtained tumor site, grade, diameter, maximum SUV, BCL2 translocation (trans) status through fluorescence in situ hybridization, RT dose and fractionation, and the treatment strategy ( i.e. treating all sites of disease vs a subsite of disease). Tumor site was dichotomized by pelvic tumors vs all others, as this was the most frequently altered site. The presence of mutant (mut) genes compared to wild type (WT) were associated with RT response, measured by PET scan within 6 months of RT, using logistic regression. Log-rank testing and Cox proportional hazards models were used to analyze local progression free survival (LPFS), censored at start of unplanned therapy, with 2-year (2y) survival reported. Multivariate modeling was used to adjust gene associations with outcomes, controlling for the clinicodemographic and tumor characteristics mentioned above. Results The patient and tumor characteristics are summarized in table 1. We found CREBBP to be the most frequently altered gene (66% of tumors). We identified 5 signatures of altered genes, most of which showed altered CREBBP and relatively long LPFS (e.g. one signature showed CREBBP mut, TNFRSF14 mut and IRF8/STAT6 mut with a 2y LPFS 78%); however, one signature with concurrent KMT2D mut and altered BCL2 including trans and mut had significantly shorter LPFS than all other signatures (2y LPFS 47%, p &lt; 0.01) and lower odds of CR relative to the other signatures (p= 0.03). CREBBP mut was the only alteration associated with increased odds of CR (Odds ratio: 2.39 (95% CI: 1.06-5.37, p = 0.04), and this effect remained significant after adjusting for pelvic disease site (p=0.04). Mutations of BCL2, IRF8, and KMT2D were associated with LPFS using log-rank testing (p&lt;0.01, p=0.02, and p=0.04, respectively). BCL2 mut or concurrent BCL2 mut and BCL2 trans (mut/trans) had shorter LPFS for the overall cohort (p&lt;0.01 for both), while for VLDRT, only BCL2 mut/trans was associated with shorter LPFS (p=0.02). In the VLDRT cohort, we found that CREBBP histone acetyltransferase (HAT) mut was associated with improved LPFS compared to WT (n=39, 2y LPFS 74% vs 52%, HR:0.41 (95% CI: 0.18-0.93, p=0.03)), but this was not observed for tumors receiving &gt;4Gy (n=44, HR: 1.27 (95% CI: 0.32-5.08), p=0.74)). We observed that either BCL2 mut, BCL2 trans, or BCL2 mut/trans and CREBBP HAT WT had a 2y LPFS of 13% whereas BCL2 mut, BCL2 trans or BCL2 mut/trans and CREBBP HAT mut had a 2y LPFS of 83% (p&lt;0.01), and this relationship persisted on multivariate analysis (Figure 1). Conclusions Incorporating genetic signatures associated with radiosensitivity, and specifically alterations involving BCL2 and CREBBP, may independently improve patient selection for RT. CREBBP HAT domain mutations are potentially targetable and may have important implications for augmenting radiosensitivity to VLDRT.

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Dougherty, Bonnie V., Beatrice C. Thomas, Alice M. Walsh, et al. "Abstract 6544: Targeting resistance mechanisms to AR-targeted therapy in prostate cancer through inhibition of CREBBP/EP300." Cancer Research 84, no. 6_Supplement (2024): 6544. http://dx.doi.org/10.1158/1538-7445.am2024-6544.

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Abstract Standard initial treatment for men with metastatic prostate cancer involves targeting the androgen receptor (AR) with androgen deprivation therapy or androgen-receptor signaling inhibitors. Resistance eventually develops, such as through AR-activating alterations, leaving few targeted options for patients. Here, we utilize data from 4,509 prostate cancer patients with longitudinal clinical and DNA/RNA sequencing data to elucidate mechanisms of resistance and identify potential therapeutic vulnerabilities in resistant disease. By constructing comprehensive regulatory networks, termed hybrid networks, built across different disease stages, combining features from WGCNA, causal mediation analysis, probabilistic causal network inference, and public knowledge graphs, we uncover subnetworks strongly associated with disease progression and resistance. These subnetworks reflect biological processes for cell cycle, E2F, DNA repair, MYC, and AR, comprehensively identifying broad transcriptional changes that drive disease progression and resistance. Selected subnetwork scores, derived from RNA expression, were significantly associated with outcomes in both the hormone-sensitive and castration-resistant settings, demonstrating that the subnetworks capture early and late resistance. The hybrid networks identified the transcriptional co-activators CREBBP and EP300 as key modulators of these subnetworks, suggesting that targeting CREBBP/EP300 may be effective in treating both early and late-stage disease. Prostate cancer cell lines were treated with FT-6876, a small molecule inhibitor of CREBBP/EP300, and growth inhibition was measured in both enzalutamide sensitive and resistant cell lines, covering a wide range of resistance mechanisms. The selected subnetworks were enriched for genes down-regulated following treatment with FT-6876, suggesting that targeting CREBBP/P300 addresses multiple resistance-related pathways. Differential changes in protein abundance and protein acetylation between enzalutamide sensitive and resistant cell lines identified changes in fatty acid metabolism as a common, downstream event in response to treatment with FT-6876, suggesting an additional mechanism for response to FT-6876. Together, our integrated theoretical and experimental analyses support the hypothesis that targeting CREBBP/EP300 may provide therapeutic benefits to prostate cancer patients. Further, the subnetworks identified may serve as mechanistically-informed biomarkers for identifying patients who may benefit from inhibition of CREBBP/EP300. Citation Format: Bonnie V. Dougherty, Beatrice C. Thomas, Alice M. Walsh, Veronica Calvo, Kathryne E. Ware, Andrew J. Armstrong, Jens Renstrup, Jason A. Somarelli, Eric E. Schadt. Targeting resistance mechanisms to AR-targeted therapy in prostate cancer through inhibition of CREBBP/EP300 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6544.

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Camos, Mireia, Jordi Esteve, Pedro Jares, et al. "Gene Expression Profile of Acute Myeloid Leukemia (AML) with t(8;16)(p11;p13) and MYST3/CREBBP Rearrangement." Blood 104, no. 11 (2004): 2054. http://dx.doi.org/10.1182/blood.v104.11.2054.2054.

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Abstract Translocation t(8;16)(p11;p13) is an infrequent chromosomal abnormality in de novo and secondary AML cases, leading to the fusion of MYST3 (MOZ) and CREBBP (CBP) genes, both of them harboring histone lysine acetyl-transferase activity. This AML variety displays specific clinical and biological features, although its gene expression profile is currently unknown. In this study, the genetic signature of AML cases with MYST3/CREBBP rearrangement was compared with the genetic profile of other well-defined AML subtypes. Genotypic analyses using oligonucleotide U133A arrays (Affymetrix) were performed on RNA of 19 AML samples, including t(8;16)-AML (n=3), t(15;17) (n=3), t(8;21) (n=2), inv(16)/t(16;16) (n=3), t(9;11) with AF9/MLL rearrangement (n=2), 3 cases with normal karyotype and flt-3 internal tandem duplication (flt-3 ITD), the three remaining samples corresponding to monocytic cases (M4/M5) without MLL rearrangement nor flt-3 ITD. After unsupervised analysis, cases of AML with t(8;16) clustered together, displaying a differential expression profile. Supervised analysis allowed the identification of the top 53 up-regulated and 28 down-regulated genes. Among the set of genes overexpressed, genes involved in chromatin remodelling and transcription (HOXA9, HOXA10, MEIS1, CHD3, SATB1) and protooncogenes (RET, flt-3, LMO2) were identified. In contrast, CREBBP gene and several members of the JAK-STAT pathway (STAT3, STAT5B, JAK2) were underexpressed. Interestingly, overexpression of multiple homeobox genes was detected in flt-3 ITD cases, some of them as a distinctive finding (HOXA2, HOXA3, HOXB6), and others (HOXA9, HOXA10, MEIS1) were found to be highly expressed in MYST3/CREBBP and MLL-rearranged samples. In conclusion, AML with t(8;16) and MYST3/CREBBP rearrangement shows a distinctive gene expression profile, with some similarities with MLL rearranged leukemias and flt-3 ITD AML cases, thus suggesting a partially common leukemogenic pathway.

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Bruno Marcelo Rocha Freire. "Identificação da fusão gênica MYST3-CREBBP em crianças com LMA e hemofagocitose." Revista Científica Hospital Santa Izabel 1, no. 4 (2020): 42–44. http://dx.doi.org/10.35753/rchsi.v1i4.167.

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LMA apresenta uma translocação (8;16) (p11; p13) que gera a fusão gênica MYST3-CREBBP, que é mais comumente vista em adultos. Essa translocação (8;16) (p11; p13) está associada à idade precoce, diferenciação monocítica e hemofagocitose e sendo indicativo de pior prognóstico. Método: série de 266 casos de LMA em pacientes pediátricos no período de 2003 a 2012. Foram incluídas crianças com menos de 2 anos de idade, com LMA e presença de hemofagocitose. Foram incluídos 48 casos de LMA sem hemofagocitose. O método de hibridização fluorescente in situ (FISH) para os rearranjos MLL foi realizado ao diagnóstico, assim como o FISH para rearranjo MYST3-CREBBP. Resultados: dos casos analisados, 5 estavam associados à fusão MYST3-CREBBP e tinham associação com: hepatoesplenomegalia, lesões de pele ou cloroma localizados. Discussão: a alta frequência de LMA MYST3-CREBBP em bebês (idade < 24 meses) e casos de LMA congênita favorecem a hipótese de que a leucemia ocorre durante a vida intrauterina e pode ser utilizada para explorar melhor o entendimento da leucemogênese da LMA.  Publicado na Rev. Bras. Hematol hemoter. 2016;38(4):291–297, com mesmo título. Autores: Francianne Gomes Andrade, Elda Pereira Noronha, Rosania Maria Baseggio, Teresa Cristina Cardoso Fonseca, Bruno Marcelo Rocha Freire, Isis M. Quezado Magalhães, Ilana R. Zalcberg, Maria S. Pombo-de-Oliveira.

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Gotti, Giacomo, Daniela Silvestri, Grazia Fazio, et al. "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." Blood 142, Supplement 1 (2023): 4344. http://dx.doi.org/10.1182/blood-2023-187833.

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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.

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Green, Michael R., Shingo Kihira, Chih Long Liu, et al. "Mutations in early follicular lymphoma progenitors are associated with suppressed antigen presentation." Proceedings of the National Academy of Sciences 112, no. 10 (2015): E1116—E1125. http://dx.doi.org/10.1073/pnas.1501199112.

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Follicular lymphoma (FL) is incurable with conventional therapies and has a clinical course typified by multiple relapses after therapy. These tumors are genetically characterized by B-cell leukemia/lymphoma 2 (BCL2) translocation and mutation of genes involved in chromatin modification. By analyzing purified tumor cells, we identified additional novel recurrently mutated genes and confirmed mutations of one or more chromatin modifier genes within 96% of FL tumors and two or more in 76% of tumors. We defined the hierarchy of somatic mutations arising during tumor evolution by analyzing the phylogenetic relationship of somatic mutations across the coding genomes of 59 sequentially acquired biopsies from 22 patients. Among all somatically mutated genes, CREBBP mutations were most significantly enriched within the earliest inferable progenitor. These mutations were associated with a signature of decreased antigen presentation characterized by reduced transcript and protein abundance of MHC class II on tumor B cells, in line with the role of CREBBP in promoting class II transactivator (CIITA)-dependent transcriptional activation of these genes. CREBBP mutant B cells stimulated less proliferation of T cells in vitro compared with wild-type B cells from the same tumor. Transcriptional signatures of tumor-infiltrating T cells were indicative of reduced proliferation, and this corresponded to decreased frequencies of tumor-infiltrating CD4 helper T cells and CD8 memory cytotoxic T cells. These observations therefore implicate CREBBP mutation as an early event in FL evolution that contributes to immune evasion via decreased antigen presentation.

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Marchetti, Giulia Bruna, Donatella Milani, Livia Pisciotta, et al. "The Phenotype-Based Approach Can Solve Cold Cases: The Paradigm of Mosaic Mutations of the CREBBP Gene." Genes 15, no. 6 (2024): 654. http://dx.doi.org/10.3390/genes15060654.

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Rubinstein–Taybi syndrome (RTS) is a rare genetic disorder characterized by intellectual disability, facial dysmorphisms, and enlarged thumbs and halluces. Approximately 55% of RTS cases result from pathogenic variants in the CREBBP gene, with an additional 8% linked to the EP300 gene. Given the close relationship between these two genes and their involvement in epigenomic modulation, RTS is grouped into chromatinopathies. The extensive clinical heterogeneity observed in RTS, coupled with the growing number of disorders involving the epigenetic machinery, poses a challenge to a phenotype-based diagnostic approach for these conditions. Here, we describe the first case of a patient clinically diagnosed with RTS with a CREBBP truncating variant in mosaic form. We also review previously described cases of mosaicism in CREBBP and apply clinical diagnostic guidelines to these patients, confirming the good specificity of the consensus. Nonetheless, these reports raise questions about the potential underdiagnosis of milder cases of RTS. The application of a targeted phenotype-based approach, coupled with high-depth NGS, may enhance the diagnostic yield of whole-exome sequencing (WES) in mild and mosaic conditions.

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Zhou, Ting, Stephanie N. Zimmer, Ziming Cheng, and Vivienne I. Rebel. "Myelodysplastic/Myeloproliferative Neoplasm in Crebbp+/− Mice Is a Transplantable Disease in Which Multiple Hematopoietic Cell Populations Are Involved, As Well As the Microenvironment." Blood 120, no. 21 (2012): 1705. http://dx.doi.org/10.1182/blood.v120.21.1705.1705.

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Abstract Abstract 1705 Myelodysplastic/myeloproliferative neoplasms (MDS/MPNs) are myeloid malignancies that display features of both MDS and MPN, but cannot be properly assigned to either MDS or MPN. It is currently not known whether it originates from the hematopoietic stem cell (HSC) compartment (like MDS), from a more committed myeloid progenitor population, or a combination thereof. Fifteen to 40% of MDS/MPN patients develop acute myeloid leukemia (AML); whether the transformation occurs in a particular cell population is also unknown. We previously demonstrated that mice heterozygous for the CREB binding protein gene (Crebbp) develop MDS/MPN at 9–12 months of age and ∼40% of them progress to develop a hematologic malignancy. Thus, Crebbp+/− mice are an excellent model to address the before mentioned questions, which is important for the development of better strategies to treat MDS/MPN. For this purpose, we harvested and combined bone marrow from 1.5-year old Crebbp+/− mice (10 donors per experiment, thereby ensuring that the marrow of ∼4 donors harbored malignant hematopoietic cells) and transplanted it into lethally irradiated, wild-type recipients. Groups of mice either received unfractionated whole bone marrow (WBM) or populations purified by fluorescence-activated cell sorting. Naive Crebbp+/− mice had demonstrated functional and/or quantitative abnormalities in long-term and short-term HSCs, common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs) and we therefore focused on these populations. All transplant recipients also received unfractionated wild-type “helper cells” to increase survival. Mice were closely monitored and those suspected of having developed a hematopoietic disease were sacrificed and their hematopoietic system analyzed. Four independent experiments were performed and data were combined for analysis. Among the 18 recipients who received Crebbp+/− WBM, 8 recipients (44%) developed an early-onset AML with myelofibrosis, 2–7 months after the transplant, which was not preceded by MDS. The other 10 recipients (56%) developed MDS/MPN, 12–18 months after the transplant. These mice displayed ineffective hematopoiesis, evidenced by a normocellular bone marrow, significant leukopenia, and trilineage dysplasia. One of these 10 Crebbp+/− WBM recipients that developed MDS/MPN subsequently progressed to AML. In contrast, none of the 15 recipients of Crebbp+/− HSCs (defined as Lin−Sca-1+c-Kit++ (LSK) cells, including long-term and short-term progenitors, as well as lymphoid-restricted progenitors) developed early-onset AML. Instead, 1 developed MDS/MPN while the remainder developed MDS by 11–17 months after the transplant, with one of them progressing to a disease resembling human mature T-cell leukemia. Transplantation of Crebbp+/− CMPs and GMPs also failed to cause early-onset AML and, as expected, gave rise to extremely low long-term reconstitution. Thus, these mice were mostly reconstituted by the co-transplanted wild-type “helper cells”. However, unexpectedly, 9 out of 24 (38%) showed <10% dysplastic cells in 1 or more lineages, while 4 (17%) developed overt MDS, i.e. >10% dysplastic cells and 2 (8%) developed MPD or AML with myelofibrosis. Control mice, i.e., recipients of wild-type BM cells remained healthy for the duration of the experiments. The results of these transplantation experiments show that in this mouse model, MDS/MPN is transplantable. However, it requires transplantation of WBM, since the transplantation of LSK cells resulted in MDS, suggesting that the microenvironment may play a crucial role in the etiology of MDS/MPN. This notion is in concordance with our previous study, demonstrating that Crebbp+/− mice transplanted with wild-type cells developed MPD that originated from the transplanted wild-type cells. This notion is further supported by the outcome of the CMP and GMP transplantation experiments, suggesting that abnormal myeloid progenitors are also important factors for MDS/MPN disease development. Moreover, malignant transformation seems to occur in a non-LSK cell that is more differentiated than CMPs and GMPs. Alternatively, malignant transformation requires all hematopoietic and non-hematopoietic cells to be present, again suggesting that MDS/MPN is a complex disease where both the hematopoietic compartment and its bone marrow microenvironment are affected. Disclosures: No relevant conflicts of interest to declare.

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Sokol, Ethan, Smruthy Sivakumar, Brennan Decker, Jeffrey Ross, and Priti Hegde. "Abstract P5-13-02: Serially biopsied BRCA1/2 mutant breast tumors frequently acquire alterations in BRCA1, BRCA2, and CREBBP." Cancer Research 82, no. 4_Supplement (2022): P5–13–02—P5–13–02. http://dx.doi.org/10.1158/1538-7445.sabcs21-p5-13-02.

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Abstract Introduction Tumors with alterations in BRCA1 or BRCA2 (BRCAm) are sensitive to poly ADP ribose polymerase inhibitors (PARPi), with significant benefit in ovary, breast, prostate, and pancreatic cancers. Despite strong early responses on PARPi, many patients eventually exhibit relapse. Previous literature has identified BRCA1/2 reversion as a common class of acquired alteration in the resistance setting; however, additional resistance pathways have not been well characterized. Here, we examined 100 BRCAm patients who were profiled with serial biopsy during clinical care to identify possible therapy resistance alterations. Methods Comprehensive genomic profiling (CGP) was carried out in a Clinical Laboratory Improvement Amendments (CLIA)-certified, CAP (College of American Pathologists)-accredited laboratory (Foundation Medicine Inc., Cambridge, MA, USA). Tissue biopsy CGP was performed on FFPE blocks examining at least 324 genes for all classes of alterations (FoundationOne® and FoundationOne® CDx). Liquid biopsy CGP was performed examining at least 62 genes (FoundationACT®, FoundationOne®Liquid).One hundred breast cancer patients with baseline BRCAm were serially profiled with CGP testing during routine clinical care (n=50 tissue then liquid; n=50 tissue for both tests). As a comparator, we examined 1,294 patients without a baseline BRCAm (BRCAwt) profiled with serial biopsy (n = 585 tissue then liquid; n=709 tissue for both tests). Co-occurrence analyses (Fisher’s exact) were run on the full research dataset, including 12,198 breast-biopsied (local) and 16,586 metastatic-biopsied samples. All classes of genomic alterations were included in the analysis. Results Of the serially biopsied samples, 7.2% (100/1,394) had baseline BRCAm. In patients with baseline BRCAm, acquired alterations were frequently observed in BRCA1/2 (17%), ESR1 (16%), TP53 (15%), MYC (12%), CREBBP (10%), RB1 (9%), PIK3CA (9%), and NF1 (5%). To understand if any of these are specific to the BRCAm population, we compared the milieu of acquired alterations to a serially biopsied BRCAwt cohort (n=1,294). While many of the alterations were common and shared across both cohorts, likely due to endocrine therapy use, acquired alterations in BRCA1, BRCA2, and CREBBP were specifically enriched in the BRCAm population (all p&lt;0.005). Acquired BRCA1/2 mutations were predicted reversion mutations and spanned a range of mechanisms, including large deletion/rearrangement events that remove the exon of the truncal mutation, point mutations that change a nonsense BRCA1/2 to a missense event, upstream indels that revert the frame of the truncal BRCA1/2 event, and splice site mutations that lead to a skipping of the truncal BRCA1/2. Of note, a majority of cases with a reversion involved a rearrangement event (9/17; 53%). For patients with a baseline BRCA1/2 short variant mutation, there was a trend towards a higher rate of reversion in BRCA2 v BRCA1 (23.1% v 18.5%, p&gt;0.05). Acquired BRCAm were never observed in cases with baseline deletion of BRCA1/2. The high rate of acquired CREBBP alterations was specific to the BRCAm population (10% BRCAm v 1% BRCAwt; p = 0.0013), suggesting a possible role for CREBBP in platinum or PARPi resistance. Consistent with this, BRCAm and CREBBP significantly co-occur in the metastatic setting (Odds ratio, OR = 1.6; p = 0.016) but not in the local setting (OR = 1.1; p = 0.68). Conclusions Analysis of serially biopsied BRCAm breast cases revealed frequent acquisition of BRCA1/2 reversion mutations and CREBBP alterations that are not frequently observed in BRCAwt samples. Additional studies are warranted to investigate the possible role of CREBBP in PARPi therapy resistance. Citation Format: Ethan Sokol, Smruthy Sivakumar, Brennan Decker, Jeffrey Ross, Priti Hegde. Serially biopsied BRCA1/2 mutant breast tumors frequently acquire alterations in BRCA1, BRCA2, and CREBBP [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-02.

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Zhang, Hui, Maoxiang Qian, Shirley, Kow Yin Kham, et al. "Whole Transcriptome Sequencing Identified a Distinct Subtype of Acute Lymphoblastic Leukemia with Abnormalities of CREBBP and EP300." Blood 128, no. 22 (2016): 3912. http://dx.doi.org/10.1182/blood.v128.22.3912.3912.

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Abstract While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384 gene with different partners (i.e., histone acetyl-transferases EP300 and CREBBP, TAF15, and TCF3). In 9 of 11 ALL cases with ZNF384 rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384 coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1 and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1 and BTLA loci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1 promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384 fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLA promoter. In human ALL cells, CLCF1 and BTLA promoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitro upon ectopic expression of ZNF384 fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384 or CREBBP-ZNF384 significantly stimulated preB cell colony formation. However, neither EP300- nor CREBBP-ZNF384 fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384 led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384 or CREBBP-ZNF384 substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300 and CREBBP may cause global epigenetic deregulation in ALL with potentials for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.

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Pasqualucci, Laura, David Dominguez-Sola, Annalisa Chiarenza, et al. "Genome-Wide Analysis Reveals Frequent Inactivating Mutations of Acetyltransferase Genes In B-Cell Lymphoma." Blood 116, no. 21 (2010): 474. http://dx.doi.org/10.1182/blood.v116.21.474.474.

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Abstract Abstract 474 B cell non-Hodgkin lymphoma (B-NHL) comprises a variety of biologically and clinically distinct diseases whose pathogenesis is associated with largely unique genetic lesions affecting oncogenes and tumor suppressor genes. The identification of the complete set of genes and cellular pathways that are altered in the lymphoma cell is critical for a full understanding of the genesis and maintenance of these malignancies. Using genome-wide copy number analysis and high throughput whole exome sequencing, we have recently found that the two most common types of B-NHL, follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), are associated with frequent structural alterations inactivating the genes that encode CREBBP and, in a smaller fraction of cases, EP300, two highly related histone and non-histone acetyltranferases (HAT) that act as transcriptional coactivators in multiple signaling pathways. Overall, ~29% (n=39/134) of DLBCL and 32% (n=15/47) of FL samples display genomic deletions and/or somatic point mutations that remove or inactivate the HAT domain of CREBBP. In 19 additional cases (15 DLBCL and 4 FL), mutations and deletions were found in the EP300 gene, also predicting its functional loss. With few exceptions, structural alterations of CREBBP and EP300 were mutually exclusive, suggesting that inactivation of these loci is at least in part functionally equivalent. Thus, in total ~36% of all DLBCL (n=49/134) and at least 40% of FL cases (n=19/47) display genetic aberrations targeting acetyltransferase genes. Interestingly, while these lesions were observed in both DLBCL phenotypic subtypes, their frequency appears to be significantly higher in germinal center B cell type DLBCL, where they account for ~46% of the cases (n=30/65) as compared to 27% in activated B cell type DLBCL (n=11/69; p<0.01). CREBBP and EP300 structural alterations commonly affect a single allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. While the functional consequences of these lesions are likely to be broad on gene transcription, we demonstrate specific defects in the acetylation-mediated inactivation of the BCL6 onco-protein and activation of the p53 tumor suppressor, both of which contribute to transformation. These results identify CREBBP/EP300 alterations as a major pathogenetic mechanism shared by common forms of B-NHL, and have direct implications for the development of therapeutic regimens targeted to acetylation/deacetylation mechanisms. Disclosures: No relevant conflicts of interest to declare.

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Cortopassi, Wilian A., Kiran Kumar та Robert S. Paton. "Cation–π interactions in CREBBP bromodomain inhibition: an electrostatic model for small-molecule binding affinity and selectivity". Organic & Biomolecular Chemistry 14, № 46 (2016): 10926–38. http://dx.doi.org/10.1039/c6ob02234k.

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Tokunaga, Kenji, Shunichro Yamaguchi, Taizo Shimomura, et al. "Accumulation Of Gene Alterations Of TP53, Crebbp and IKZF1 Is a Prognostic Factor In Adult Acute Lymphoblastic Leukemia." Blood 122, no. 21 (2013): 1386. http://dx.doi.org/10.1182/blood.v122.21.1386.1386.

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Abstract Aims Mutations of the genes associating with cell differentiation or proliferation are recognized as factors of tumorigenesis or prognosis in hematological malignancies. In pediatric acute lymphoblastic leukemia (ALL), alterations of IKZF1 (a factor of lymphocyte differentiation), TP53 (a cell cycle regulator) and CREBBP (a histone modifier) are found as possible prognostic markers for stratification of treatments. On the other hand, in adult ALL, clinical significance of such alterations remains to be determined. In the present work, we examined whether the mutations in those genes affected the incidence and prognosis in adult ALL patients. Methods We investigated 87 adult patients with newly diagnosed ALL treated with JALSG protocols between 1986 and 2011. Age ranged from 15 to 86 years, with a median of 51 years. We obtained cDNA and genomic DNA from the peripheral blood or bone marrow mononuclear cells at diagnosis. CREBBP mutations are dominantly identified in the histone acetyltransferase (HAT) domain. HAT domain in the CREBBP gene was amplified by PCR using cDNA and was subjected to direct sequencing. Additionally other histone modifiers, EZH2, EED, and UTX, were sequenced as the same as in CREBBP. TP53 exons 5 – 8 and 10, in which mutations were commonly reported, were sequenced using genomic DNA. We amplified IKZF1 using RT-PCR for detecting aberrant dominant negative isoforms: Ik6 and Ik10. Genomic deletions of IKZF1 were assessed with RQ-PCR or genomic DNA PCR. We compared clinical profiles between patients with and without such gene mutations. The present study was approved by the Institutional Review Boards and informed consent was obtained from each patient according to guidelines based on the revised Declaration of Helsinki. Results In 87 adult patients with ALL, alterations of CREBBP, EED, TP53 and IKZF1 were detected in 7 (9.5%), 3 (4.8%), 6 (6.9%) and 42 (50%), respectively. None of EZH2 and UTX mutation was found. The alterations of CREBBP and IKZF1 at diagnosis in adult patients were more frequent than those in pediatric patients ever reported. Some gene mutations were not found frequently. Each gene mutation per se did not significantly affect prognosis. We tried to predict the prognosis by scoring gene mutations and chromosomal abnormalities. Philadelphia chromosome (Ph) has great impact to prognosis of patients with ALL. We scored the number of mutated genes and Ph for each patient. As the score was higher, adult patients with ALL had poorer relapse-free survival (P=0.0439) and OS (P=0.4819), but statistical significance was not detected in this small cohort. Conclusions and Discussion Single gene mutations, such as IKZF1, can predict the prognosis in pediatric ALL. In adult ALL, however, only few gene mutations are reported to be promising prognostic factors which have impacts to treatment outcomes. Scoring system may be a useful method for predicting prognosis and stratifying treatment in adult ALL. Our study implies the possibility that a variety and heterogeneity of genetic alterations in adult ALL are associated with the pathogenesis for treatment resistance and prognostic marker of adult ALL. Disclosures: No relevant conflicts of interest to declare.

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Mosquera Orgueira, Adrián, Roi Ferreiro Ferro, José Ángel Díaz Arias, et al. "Detection of new drivers of frequent B-cell lymphoid neoplasms using an integrated analysis of whole genomes." PLOS ONE 16, no. 5 (2021): e0248886. http://dx.doi.org/10.1371/journal.pone.0248886.

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B-cell lymphoproliferative disorders exhibit a diverse spectrum of diagnostic entities with heterogeneous behaviour. Multiple efforts have focused on the determination of the genomic drivers of B-cell lymphoma subtypes. In the meantime, the aggregation of diverse tumors in pan-cancer genomic studies has become a useful tool to detect new driver genes, while enabling the comparison of mutational patterns across tumors. Here we present an integrated analysis of 354 B-cell lymphoid disorders. 112 recurrently mutated genes were discovered, of which KMT2D, CREBBP, IGLL5 and BCL2 were the most frequent, and 31 genes were putative new drivers. Mutations in CREBBP, TNFRSF14 and KMT2D predominated in follicular lymphoma, whereas those in BTG2, HTA-A and PIM1 were more frequent in diffuse large B-cell lymphoma. Additionally, we discovered 31 significantly mutated protein networks, reinforcing the role of genes such as CREBBP, EEF1A1, STAT6, GNA13 and TP53, but also pointing towards a myriad of infrequent players in lymphomagenesis. Finally, we report aberrant expression of oncogenes and tumor suppressors associated with novel noncoding mutations (DTX1 and S1PR2), and new recurrent copy number aberrations affecting immune check-point regulators (CD83, PVR) and B-cell specific genes (TNFRSF13C). Our analysis expands the number of mutational drivers of B-cell lymphoid neoplasms, and identifies several differential somatic events between disease subtypes.

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Zhang, Lingling, Oscar Atkins, Miu Shing Hung, Hans Christian Reinhardt, and Dinis Pedro Parente Calado. "Abstract PR03: Immunosurveillance of precancerous germinal center B cells." Blood Cancer Discovery 5, no. 3_Supplement (2024): PR03. http://dx.doi.org/10.1158/2643-3249.lymphoma24-pr03.

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Abstract Most Non-Hodgkin Lymphomas (NHLs) originate from germinal center (GC) or post-GCB cells. While the immune system actively targets precancerous cells in epithelial-derived cancers, it is uncertain if the same applies to precancerous GCB cells. Knowledge on Follicular Lymphoma (FL), the most common low-grade NHL derived from GCB cells, prompted us to develop novel mouse models to mimic the precancerous state. In FL, BCL2 translocation and CREBBP loss-of-function (LoF) mutations are characteristic of a precancerous state. These mutations can be detected years before FL diagnosis, hinting at potential immune involvement in preventing FL. Our research affirms the effectiveness of the mouse models in replicating the FL precancerous state. Mice with BCL2 overexpression (OE) alone or together with CREBBP LoF showed increased GCB cell expansion, up to 6-fold compared to control. This expansion involved reduced GCB cell death rather than increased proliferation, consistent with FL slow-growing nature. Mice harboring mutant GCB cells also exhibited a significant increase in memory B cells, up to 20-fold when compared to control. As the GC reaction progressed, the impact of CREBBP LoF emerged, profoundly altering GCB cell phenotypes, and triggering a remarkable surge in cell expansion, escalating up to 80-fold compared to mice with BCL2 OE alone or control. However, this hyperplasia was swiftly followed by a decline, indicative of immunosurveillance targeting precancerous GCB cells. Prior to the reduction of GCB cells with BCL2 OE and CREBBP LoF, we observed a notable expansion of PD1+ CD8+ T cells expressing granzyme A and perforin. These activated CD8+ T cells also expressed CXCR5, facilitating their infiltrating into the B cell follicle to specifically eliminate GCB cells with BCL2 OE and CREBBP LoF. Preemptive depletion of CD8+ T cell effectively prevented the loss of precancerous GCB cells. The examination of the GC reaction over time provided new perspectives into the immunosurveillance of precancerous GCB cells and FL development. The data supports the concept that precancerous GCB cells must overcome robust anti-tumor immunity for Lymphoma to ensue. This knowledge offers potential explanation for the infrequent and extended duration of the transition from precancerous GCB cells to full-blown FL. These models provide a foundation to explore mechanisms underlying immune evasion by precancerous GCB cells. Citation Format: Lingling Zhang, Oscar Atkins, Miu Shing Hung, Hans Christian Reinhardt, Dinis Pedro Parente Calado. Immunosurveillance of precancerous germinal center B cells [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR03.

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dos Anjos, Laura G., Daniela Bizinelli, and Katia C. Carvalho. "Abstract 6021: Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas." Cancer Research 83, no. 7_Supplement (2023): 6021. http://dx.doi.org/10.1158/1538-7445.am2023-6021.

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Abstract Background: Uterine leiomyosarcoma (LMS) comprises 60% to 70% of the uterine sarcoma (US). These tumors present high rates of recurrence and metastasis, and the patients’ survival rates are very low (20% in five years). It is known that somatic mutations and modifications in the DNA methylation levels are associated with several types of neoplasms. Previously, our group identified missense and loss of function mutations in KMT2D, CREBBP, ATM, TSC2, and GNAS, using a genetic screening method in LMS samples. Based on these results, we decided to investigate whether a genetic and epigenetic crosstalk could help to understand the biological processes involved in the LMS origin, development, and pathogenesis. The present study aimed to evaluate the KMT2D, CREBBP, ATM, TSC2 and GNAS gene expression profile in LMS cell line, and their methylation levels in formalin-fixed and paraffin-embedded (FFPE) patients´ samples. Methods: We selected 15 LMS - FFPE samples obtained via surgical procedures performed between 2012 and 2019 at the Instituto do Cancer do Estado de São Paulo (ICESP). Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit and we evaluated methylation levels using the Illumina Infinium Methylation EPIC BeadChip system 850k, including samples treated with bisulfite-treated DNA. Leiomyoma (LM) (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were cultivated for gene expression analysis. Cells were growth for 24, 48, 72, and 96 hours, and the total RNA was extracted by TRIzol. High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis and for gene expression evaluation, real-time PCR reactions were performed using TaqMan Universal PCR Master Mix and inventoried TaqMan probes. Results: KMT2D and TSC2 showed increased expression in LMS compared to LM (cut-off ≤ -2 and ≥ 2 for down and upregulation, respectively), with higher expression in 24 hours of KMT2D [Fold Regulation (FR): 2.77] and TSC2 (FR: 2.58). In contrast, CREBBP was downregulated, with lowest expression in 72 hours (FR: -6.57). ATM was upregulated at 24, 48 with higher expression in 72 hours (FR: 2.32), and GNAS was downregulated, with lowest expression in 96 hours (FR: -4.31). Moreover, the methylation analyzes showed an expressive hypomethylation in LMS samples, compared to myometrium, for KMT2D (β value: 0.45; p= 0.004); CREBBP (β value: 0.51 p &lt; 0.0001); ATM (β value: 0.47 p &lt; 0.0001); TSC2 (β value: 0.53 p &lt; 0.0001) and GNAS (β value: 0.41 p &lt; 0.0001). In conclusion, our study showed that potentially pathogenic mutations may be associated with an aberrant hypomethylation profile, as well as increased expression of KMT2D, TSC2, ATM and loss expression of CREBBP and GNAS in LMS. The interconnection of these genetic and epigenetic events is essential for understanding the complex biology of these tumors, in addition to enabling identification of biomarkers. Citation Format: Laura G. dos Anjos, Daniela Bizinelli, Katia C. Carvalho. Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6021.

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Li, Jie, Christopher Chin, Hsia-Yuan Ying, et al. "Cooperative Super-Enhancer Inactivation through Loss of Crebbp and KMT2D Skews B Cell Fate Decisions and Yields T Cell-Depleted Lymphomas." Blood 142, Supplement 1 (2023): 2776. http://dx.doi.org/10.1182/blood-2023-174501.

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Mutations in chromatin modifiers are a hallmark of many tumors, especially lymphomas arising from germinal center (GC) B cells. Given that most of these lymphoma mutations induce aberrant gene repression, it is surprising that they often co-occur in individual patients. The most common pairing are mutations affecting CREBBP and KMT2D, even though regulating overlapping gene enhancers and pathways. Hence their co-occurrence is especially puzzling. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d (C+K) do indeed induce a more severe lymphoma phenotype (vs either allele alone) and unexpectedly conferred an immune evasive phenotype manifesting as reduced CD8+ T cell infiltration. This was linked to profound repression of immune synapse genes that provide costimulatory effects to T-cells and others. Repression of immune synapse genes was reflected in impaired response to T-cell directed cell fate decisions and weakening of costimulatory signals. From the epigenetic perspective we observed interaction and mutually dependent cooperative binding of C+K to chromatin. Notably loss of C+K cooperativity was selectively severe at superenhancers (vs enhancers), especially those driving expression of immune synapse signaling genes and pointing to a particular dependency for both co-activators at these specialized regulatory elements. Therefore, we show that critical features of the lymphoma microenvironment are shaped by specific combinations of chromatin modifier somatic mutations, induced by the dependency of immune synapse superenhancers on cooperative actions of CREBBP and KMT2D.

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Los-de Vries, G. Tjitske, Wendy B. C. Stevens, Erik van Dijk, et al. "Genomic and microenvironmental landscape of stage I follicular lymphoma, compared with stage III/IV." Blood Advances 6, no. 18 (2022): 5482–93. http://dx.doi.org/10.1182/bloodadvances.2022008355.

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Abstract Although the genomic and immune microenvironmental landscape of follicular lymphoma (FL) has been extensively investigated, little is known about the potential biological differences between stage I and stage III/IV disease. Using next-generation sequencing and immunohistochemistry, 82 FL nodal stage I cases were analyzed and compared with 139 FL stage III/IV nodal cases. Many similarities in mutations, chromosomal copy number aberrations, and microenvironmental cell populations were detected. However, there were also significant differences in microenvironmental and genomic features. CD8+ T cells (P = .02) and STAT6 mutations (false discovery rate [FDR] &lt;0.001) were more frequent in stage I FL. In contrast, programmed cell death protein 1–positive T cells, CD68+/CD163+ macrophages (P &lt; .001), BCL2 translocation (BCL2trl+) (P &lt; .0001), and KMT2D (FDR = 0.003) and CREBBP (FDR = 0.04) mutations were found more frequently in stage III/IV FL. Using clustering, we identified 3 clusters within stage I, and 2 clusters within stage III/IV. The BLC2trl+ stage I cluster was comparable to the BCL2trl+ cluster in stage III/IV. The two BCL2trl– stage I clusters were unique for stage I. One was enriched for CREBBP (95%) and STAT6 (64%) mutations, without BLC6 translocation (BCL6trl), whereas the BCL2trl– stage III/IV cluster contained BCL6trl (64%) with fewer CREBBP (45%) and STAT6 (9%) mutations. The other BCL2trl– stage I cluster was relatively heterogeneous with more copy number aberrations and linker histone mutations. This exploratory study shows that stage I FL is genetically heterogeneous with different underlying oncogenic pathways. Stage I FL BCL2trl– is likely STAT6 driven, whereas BCL2trl– stage III/IV appears to be more BCL6trl driven.

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Zhao, Haifeng, Yutian Kan, Xinyuan Wang, Leiyuan Chen, Peng Ge, and Zhengzi Qian. "Genetic polymorphism and transcriptional regulation of CREBBP gene in patient with diffuse large B-cell lymphoma." Bioscience Reports 39, no. 8 (2019). http://dx.doi.org/10.1042/bsr20191162.

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Abstract In the present study, we aim to examine the relationship between genetic polymorphism and transcriptional expression of cyclic AMP response element binding protein (CREBBP) and the risk of diffuse large B-cell lymphoma (DLBCL). Two hundred and fifty healthy individuals and 248 DLBCL patients participated in the present study. The CREBBP rs3025684 polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The mRNA expression of CREBBP was tested by the real-time quantitative PCR (RT-qPCR). The allele A frequency of CREBBP rs3025684 in DLBCL patients was obviously higher than that of controls (P=0.01). No significant difference was detected between CREBBP rs3025684 polymorphism and clinical characteristics of DLBCL patients when subgrouped according to different parameters. The results demonstrated that the allele A of CREBBP rs3025684 increased the susceptibility to DLBCL (P=0.004), with a worse overall survival (OS) rate (P=0.002), a worse progression-free survival (PFS) rate (P=0.033) and poor prognosis (P=0.003) in DLCBL patients. Furthermore, the expression of CREBBP mRNA was considerably decreased in DLBCL patients as compared with controls (P<0.001), and the expression in patients with GG genotype was up-regulated in comparison with patients with GA and AA genotype (P=0.016 and P=0.001, respectively). However, no statistical differences were found in OS (P=0.201) and PFS (P=0.353) between the lower CREBBP mRNA level subgroup and higher CREBBP mRNA level subgroup. These data suggested that the CREBBP gene may be an important prognostic factor in DLBCL patients and perform an essential function in the development of DLBCL.

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Journal articles on the topic 'CREBBP'

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