Cannabidiol mitigates radiation-induced intestine ferroptosis via facilitating the heterodimerization of RUNX3 with CBFß thereby promoting transactivation of GPX4.

Radiation enteritis remains a major challenge for radiotherapy against abdominal and pelvic malignancies. Nevertheless, there is no approved effective therapy to alleviate irradiation (IR)-induced gastrointestinal (GI) toxicity. In the current study, Cannabidiol (CBD) was found to mitigate intestinal injury by GPX4-mediated ferroptosis resistance upon IR exposure. RNA-sequencing was employed to investigate the underlying mechanism involved in the radio-protective effect of CBD, wherein runt-related transcription factor 3 (RUNX3) and its target genes were changed significantly. Further experiment showed that the transactivation of GPX4 triggered by the direct binding of RUNX3 to its promoter region, or by stimulating the transcriptional activity of NF-κB via RUNX3-mediated LILRB3 upregulation was critical for the anti-ferroptotic effect of CBD upon IR injury. Specially, CBD was demonstrated to be a molecular glue skeleton facilitating the heterodimerization of RUNX3 with its transcriptional chaperone core-biding factor ß (CBFß) thereby promoting their nuclear localization and the subsequent transactivation of GPX4 and LILRB3. In short, our study provides an alternative strategy to counteract IR-induced enteritis during the radiotherapy on abdominal/pelvic neoplasms.

RUNX1::ETO and CBFß::MYH11 converge on aberrant activation of BCAT1 to confer a therapeutic vulnerability in core-binding factor-acute myeloid leukaemia.

Effectively targeting transcription factors in therapeutic interventions remains challenging, especially in core-binding factor-acute myeloid leukaemia (CBF-AML) characterized by RUNX1::ETO and CBFß::MYH11 fusions. However, recent studies have drawn attention towards aberrant amino acid metabolisms as actionable therapeutic targets. Here, by integrating the expression profile and genetic makeup in AML cohort, we found higher BCAT1 expression in CBF-AML patients compared with other subtypes. Metabolic profiling revealed that high BCAT1 expression led to reprogrammed branch amino acid metabolism in CBF-AML and was associated with sphingolipid pathway relating to the fitness of leukaemia cells, supported by transcriptomic profiling. Mechanistically, we demonstrated in cell lines and primary patient samples that BCAT1 was directly activated by RUNX1::ETO and CBFß::MYH11 fusion proteins similarly in a RUNX1-dependent manner through rewiring chromatin conformation at the BCAT1 gene locus. Furthermore, BCAT1 inhibition resulted in blunted cell cycle, enhanced apoptosis and myeloid differentiation of CBF-AML cells in vitro, and alleviated leukaemia burden and prolonged survival in vivo. Importantly, pharmacological inhibition of BCAT1 using the specific inhibitor Gabapentin demonstrated therapeutic effects, as evidenced by delayed leukaemia progression and improved survival in vivo. In conclusion, our study uncovers BCAT1 as a genetic vulnerability and a promising targeted therapeutic opportunity for CBF-AML.

Design of Vif-Derived Peptide Inhibitors with Anti-HIV-1 Activity by Interrupting Vif-CBFß Interaction.

One of the major functions of the accessory protein Vif of human immunodeficiency virus type 1 (HIV-1) is to induce the degradation of APOBEC3 (A3) family proteins by recruiting a Cullin5-ElonginB/C-CBFß E3 ubiquitin ligase complex to facilitate viral replication. Therefore, the interactions between Vif and the E3 complex proteins are promising targets for the development of novel anti-HIV-1 drugs. Here, peptides are designed for the Vif-CBFß interaction based on the sequences of Vif mutants with higher affinity for CBFß screened by a yeast surface display platform. We identified two peptides, VMP-63 and VMP-108, that could reduce the infectivity of HIV-1 produced from A3G-positive cells with IC50 values of 49.4 µM and 55.1 µM, respectively. They protected intracellular A3G from Vif-mediated degradation in HEK293T cells, consequently increasing A3G encapsulation into the progeny virions. The peptides could rapidly enter cells after addition to HEK293T cells and competitively inhibit the binding of Vif to CBFß. Homology modeling analysis demonstrated the binding advantages of VMP-63 and VMP-108 with CBFß over their corresponding wild-type peptides. However, only VMP-108 effectively restricted long-term HIV-1 replication and protected A3 functions in non-permissive T lymphocytes. Our findings suggest that competitive Vif-derived peptides targeting the Vif-CBFß interaction are promising for the development of novel therapeutic strategies for acquired immune deficiency syndrome.

Clinical implications of additional chromosomal abnormalities in adult acute myeloid leukemia with inv (16)/t(16;16)/CBFB::MYH11.

OBJECTIVES: This study assesses the clinical significance of additional cytogenetic abnormalities (ACAs) and/or the deletion of 3'CBFB (3'CBFBdel) resulting in unbalanced CBFB::MYH11 fusion in acute myeloid leukemia (AML) with inv (16)/t(16;16)/CBFB::MYH11. METHODS: We retrospectively evaluated the clinicopathologic features of 47 adult de novo AML with inv (16)/t(16;16)/CBFB::MYH11 fusion. There were 44 balanced and 3 unbalanced CBFB::MYH11 fusions. Given the low frequency of unbalanced cases, the latter group was combined with 19 published cases (N = 22) for statistic and meta-analysis. RESULTS: Both balanced and unbalanced cases were characterized by frequent ACAs (56.5% and 72.7%, respectively), with +8, +22, and del(7q) as the most frequent abnormalities. The unbalanced group tends to be younger individuals (p = .04) and is associated with a lower remission rate (p = .02), although the median overall survival (OS) was not statistically different (p = .2868). In the balanced group, "ACA" subgroup had higher mortality (p = .013) and shorter OS (p = .011), and patients with relapsed disease had a significantly shorter OS (p = .0011). Cox multivariate regression analysis confirmed that ACAs and history of disease relapse are independent risk factors, irrespective of disease relapse status. In the combined cohort, cases with ACAs had shorter OS than those with "Sole" abnormality (p = .0109). CONCLUSIONS: ACAs are independent high-risk factors in adult AML with inv (16)/t(16;16)/CBFB::MYH11 fusion and should be integrated for risk stratification in this disease. Larger studies are needed to assess the clinical significance of the unbalanced CBFB::MYH11 fusion resulting from the 3'CBFBdel.

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID-interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

Therapy-related acute myeloid leukemia with CBFB/MYH11 in a patient with ovarian cancer after exposure to chemotherapy.

In patients with acute myeloid leukemia (AML), about 25%-35% of patients have a history of other hematological diseases, 10% of patients have a history of malignant tumors in other systems and have received cytotoxic treatment including chemotherapy and/or radiation, and the disease is categorized as therapy-related acute myeloid leukemia (t-AML) according to the World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues. Two subsets of t-AML are generally recognized based on the nature of prior treatments and the characteristics of the disease. The most common type occurs after exposure to alkylating agents and/or radiation, with a latent period of 5 to 10 years. The less common type occurs after treatment with agents targeting topoisomerase II and has a shorter latent period of 1 to 5 years. The majority of these cases are associated with balanced recurrent chromosomal translocations frequently involving MLL at 11q23, RUNX1 at 21q22, or CBFB at 16q22 and morphologically resemble the features of de novo AML associated with these translocations. Here, we describe a rare case of a 48-year-old female with ovarian cancer who developed AML with CBFB/MYH11 fusion, less than two years after exposure to paclitaxel and carboplatin chemotherapy.

Cbfß Is a Novel Modulator against Osteoarthritis by Maintaining Articular Cartilage Homeostasis through TGF-ß Signaling.

TGF-ß signaling is a vital regulator for maintaining articular cartilage homeostasis. Runx transcription factors, downstream targets of TGF-ß signaling, have been studied in the context of osteoarthritis (OA). Although Runx partner core binding factor ß (Cbfß) is known to play a pivotal role in chondrocyte and osteoblast differentiation, the role of Cbfß in maintaining articular cartilage integrity remains obscure. This study investigated Cbfß as a novel anabolic modulator of TGF-ß signaling and determined its role in articular cartilage homeostasis. Cbfß significantly decreased in aged mouse articular cartilage and human OA cartilage. Articular chondrocyte-specific Cbfb-deficient mice (Cbfb△ac/△ac) exhibited early cartilage degeneration at 20 weeks of age and developed OA at 12 months. Cbfb△ac/△ac mice showed enhanced OA progression under the surgically induced OA model in mice. Mechanistically, forced expression of Cbfß rescued Type II collagen (Col2α1) and Runx1 expression in Cbfß-deficient chondrocytes. TGF-ß1-mediated Col2α1 expression failed despite the p-Smad3 activation under TGF-ß1 treatment in Cbfß-deficient chondrocytes. Cbfß protected Runx1 from proteasomal degradation through Cbfß/Runx1 complex formation. These results indicate that Cbfß is a novel anabolic regulator for cartilage homeostasis, suggesting that Cbfß could protect OA development by maintaining the integrity of the TGF-ß signaling pathway in articular cartilage.

Autophagy Inhibition as a Potential Therapeutic Strategy for Breast Cancer with Mitochondrial Translation Defect Caused by CBFB-Deficiency.

ABBREVIATIONS: AMPK, AMP-activated protein kinase; BioID, biotinylation identification; CBFB, core-binding factor subunit beta; HCQ, hydroxychloroquine; HNRNPK, heterogeneous nuclear ribonucleoprotein K; PDX, patient-derived xenograft; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; TUFM, Tu translation elongation factor, mitochondrial; ETC, electron transport chain.

The RUNX/CBFß Complex in Breast Cancer: A Conundrum of Context.

Dissecting and identifying the major actors and pathways in the genesis, progression and aggressive advancement of breast cancer is challenging, in part because neoplasms arising in this tissue represent distinct diseases and in part because the tumors themselves evolve. This review attempts to illustrate the complexity of this mutational landscape as it pertains to the RUNX genes and their transcription co-factor CBFß. Large-scale genomic studies that characterize genetic alterations across a disease subtype are a useful starting point and as such have identified recurring alterations in CBFB and in the RUNX genes (particularly RUNX1). Intriguingly, the functional output of these mutations is often context dependent with regards to the estrogen receptor (ER) status of the breast cancer. Therefore, such studies need to be integrated with an in-depth understanding of both the normal and corrupted function in mammary cells to begin to tease out how loss or gain of function can alter the cell phenotype and contribute to disease progression. We review how alterations to RUNX/CBFß function contextually ascribe to breast cancer subtypes and discuss how the in vitro analyses and mouse model systems have contributed to our current understanding of these proteins in the pathogenesis of this complex set of diseases.

Dysregulation of Mitochondrial Translation Caused by CBFB Deficiency Cooperates with Mutant PIK3CA and Is a Vulnerability in Breast Cancer.

Understanding functional interactions between cancer mutations is an attractive strategy for discovering unappreciated cancer pathways and developing new combination therapies to improve personalized treatment. However, distinguishing driver gene pairs from passenger pairs remains challenging. Here, we designed an integrated omics approach to identify driver gene pairs by leveraging genetic interaction analyses of top mutated breast cancer genes and the proteomics interactome data of their encoded proteins. This approach identified that PIK3CA oncogenic gain-of-function (GOF) and CBFB loss-of-function (LOF) mutations cooperate to promote breast tumor progression in both mice and humans. The transcription factor CBFB localized to mitochondria and moonlighted in translating the mitochondrial genome. Mechanistically, CBFB enhanced the binding of mitochondrial mRNAs to TUFM, a mitochondrial translation elongation factor. Independent of mutant PI3K, mitochondrial translation defects caused by CBFB LOF led to multiple metabolic reprogramming events, including defective oxidative phosphorylation, the Warburg effect, and autophagy/mitophagy addiction. Furthermore, autophagy and PI3K inhibitors synergistically killed breast cancer cells and impaired the growth of breast tumors, including patient-derived xenografts carrying CBFB LOF and PIK3CA GOF mutations. Thus, our study offers mechanistic insights into the functional interaction between mutant PI3K and mitochondrial translation dysregulation in breast cancer progression and provides a strong preclinical rationale for combining autophagy and PI3K inhibitors in precision medicine for breast cancer. SIGNIFICANCE: CBFB-regulated mitochondrial translation is a regulatory step in breast cancer metabolism and synergizes with mutant PI3K in breast cancer progression.

Heterozygous pathogenic variants involving CBFB cause a new skeletal disorder resembling cleidocranial dysplasia.

BACKGROUND: Cleidocranial dysplasia (CCD) is a rare skeletal dysplasia with significant clinical variability. Patients with CCD typically present with delayed closure of fontanels and cranial sutures, dental anomalies, clavicular hypoplasia or aplasia and short stature. Runt-related transcription factor 2 (RUNX2) is currently the only known disease-causing gene for CCD, but several studies have suggested locus heterogeneity. METHODS: The cohort consists of eight subjects from five unrelated families partially identified through GeneMatcher. Exome or genome sequencing was applied and in two subjects the effect of the variant was investigated at RNA level. RESULTS: In each subject a heterozygous pathogenic variant in CBFB was detected, whereas no genomic alteration involving RUNX2 was found. Three CBFB variants (one splice site alteration, one nonsense variant, one 2 bp duplication) were shown to result in a premature stop codon. A large intragenic deletion was found to delete exon 4, without affecting CBFB expression. The effect of a second splice site variant could not be determined but most likely results in a shortened or absent protein. Affected individuals showed similarities with RUNX2-related CCD, including dental and clavicular abnormalities. Normal stature and neurocognitive problems were however distinguishing features. CBFB encodes the core-binding factor ß subunit, which can interact with all RUNX proteins (RUNX1, RUNX2, RUNX3) to form heterodimeric transcription factors. This may explain the phenotypic differences between CBFB-related and RUNX2-related CCD. CONCLUSION: We confirm the previously suggested locus heterogeneity for CCD by identifying five pathogenic variants in CBFB in a cohort of eight individuals with clinical and radiographic features reminiscent of CCD.

[Acute myeloid leukemia with type I CBFB::MYH11 fusion gene not detected by screening test for leukemia-related chimeric genes].

A 27-year-old woman with pancytopenia was admitted to our hospital. Bone marrow aspiration revealed 52.2% myeloperoxidase-positive myeloblasts, leading to a diagnosis of acute myeloid leukemia. While a screening test for chimeric genes related to leukemia initially yielded negative results, including for the CBFB::MYH11 fusion gene, G-banded karyotyping uncovered the presence of inv (16)(p13.1q22). Further investigation by fluorescence in situ hybridization (FISH) confirmed the split signals for CBFB. A second screening test for leukemia-related chimeric genes with different PCR primers revealed the elusive CBFB::MYH11 fusion gene. Subsequently, the type I CBFB::MYH11 fusion gene was identified through exhaustive exploration using RNA sequencing for fusion gene discovery. This exceptional case highlights the existence of a distinctive subtype of CBFB::MYH11 that may yield false-negative results in conventional chimeric fusion screening, thus emphasizing the indispensable utility of PCR primer modification, FISH, and RNA sequencing in the investigative process.

PPP1R7 Is a Novel Translocation Partner of CBFB via t(2;16)(q37;q22) in Acute Myeloid Leukemia.

In a subset of acute myeloid leukemia (AML) cases, the core binding factor beta subunit gene (CBFB) was rearranged via inv(16)(p13.1q22) or t(16;16)(p13.1;q22), in which the smooth muscle myosin heavy chain 11 gene (MYH11) was the partner (CBFB::MYH11). Rare variants of CBFB rearrangement occurring via non-classic chromosomal aberrations have been reported, such as t(1;16), t(2;16), t(3;16), t(5;16), and t(16;19), but the partners of CBFB have not been characterized. We report a case of AML with a complex karyotype, including t(2;16)(q37;q22), in which the protein phosphatase 1 regulatory subunit 7 gene (PPP1R7) at chromosome 2q37 was rearranged with CBFB (CBFB::PPP1R7). This abnormality was inconspicuous by conventional karyotype and interphase fluorescence in situ hybridization (FISH), thus leading to an initial interpretation of inv(16)(p13.1q22); however, metaphase FISH showed that the CBFB rearrangement involved chromosome 2. Using whole genome and Sanger sequencing, the breakpoints were identified as being located in intron 5 of CBFB and intron 7 of PPP1R7. A microhomology of CAG was found in the break and reconnection sites of CBFB and PPP1R7, thus supporting the formation of CBFB::PPP1R7 by microhomology-mediated end joining.

Dominant Negative Mutants of Human Immunodeficiency Virus Type 1 Viral Infectivity Factor (Vif) Disrupt Core-Binding Factor Beta-Vif Interaction.

Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3s) are host restriction factors that inhibit viral replication. Viral infectivity factor (Vif), a human immunodeficiency virus type 1 (HIV-1) accessory protein, mediates the degradation of APOBEC3s by forming the Vif-E3 complex, in which core-binding factor beta (CBFß) is an essential molecular chaperone. Here, we screened nonfunctional Vif mutants with high affinity for CBFß to inhibit HIV-1 in a dominant negative manner. We applied the yeast surface display technology to express Vif random mutant libraries, and mutants showing high CBFß affinity were screened using flow cytometry. Most of the screened Vif mutants containing random mutations of different frequencies were able to rescue APOBEC3G (A3G). In the subsequent screening, three of the mutants restricted HIV-1, recovered G-to-A hypermutation, and rescued APOBEC3s. Among them, Vif-6M showed a cross-protection effect toward APOBEC3C, APOBEC3F, and African green monkey A3G. Stable expression of Vif-6M in T lymphocytes inhibited the viral replication in newly HIV-1-infected cells and the chronically infected cell line H9/HXB2. Furthermore, the expression of Vif-6M provided a survival advantage to T lymphocytes infected with HIV-1. These results suggest that dominant negative Vif mutants acting on the Vif-CBFß target potently restrict HIV-1. IMPORTANCE Antiviral therapy cannot eliminate HIV and exhibits disadvantages such as drug resistance and toxicity. Therefore, novel strategies for inhibiting viral replication in patients with HIV are urgently needed. APOBEC3s in host cells are able to inhibit viral replication but are antagonized by HIV-1 Vif-mediated degradation. Therefore, we screened nonfunctional Vif mutants with high affinity for CBFß to compete with the wild-type Vif (wtVif) as a potential strategy to assist with HIV-1 treatment. Most screened mutants rescued the expression of A3G in the presence of wtVif, especially Vif-6M, which could protect various APOBEC3s and improve the incorporation of A3G into HIV-1 particles. Transduction of Vif-6M into T lymphocytes inhibited the replication of the newly infected virus and the chronically infected virus. These data suggest that Vif mutants targeting the Vif-CBFß interaction may be promising in the development of a new AIDS therapeutic strategy.

Circ-CBFB exacerbates hypoxia/reoxygenation-triggered cardiomyocyte injury via regulating miR-495-3p in a VDAC1-dependent manner.

A large body of literature has identified that circular RNAs play critical roles in regulating the occurrence and development of cardiovascular disease. In the present study, we intended to provide new ideas and perspectives on the functional role of circ-CBFB in hypoxia/reoxygenation (H/R)-injured cardiomyocytes. We observed that circ-CBFB expression was enhanced which was accompanied by a miR-495-3p reduction in response to H/R exposure. Functionally, deletion of circ-CBFB obviously potentiated cell viability and restrained cell apoptosis, which was accompanied by a remarkable elevation of antiapoptotic Bcl-2 but the repression of proapoptotic Bax and cleaved caspase-3 in response to H/R. Additionally, the absence of circ-CBFB dramatically prohibited H/R-evoked cardiomyocyte oxidative stress, as revealed by a decrease in reactive oxygen species overproduction, diminution in MAD content, and enhancement in SOD, CAT, and GSH-Px activities. Moreover, elimination of circ-CBFB resulted in improvement of mitochondrial dysfunction, as assessed by mitochondrial membrane potential, adenosine triphosphate production, and the release of cyto-c. Interestingly, circ-CBFB inversely regulated miR-495-3p expression via acting as a competing endogenous RNA. VDAC1 was identified to be a functional target of miR-495-3p and positively modulated by circ-CBFB. Mechanically, dissipation of miR-495-3p or augmentation of VDAC1 manifestly counteracted the beneficial effects of circ-CBFB knockdown on H/R-elicited cardiomyocyte insult. Collectively, these observations demonstrated that absence of circ-CBFB offered cardio-protection against H/R-triggered cardiomyocyte injury by relieving apoptosis, oxidative stress, and mitochondria dysfunction through miR-495-3p/VDAC1 axis. This work unveiled an innovative axis of circ-CBFB/miR-495-3p/VDAC1 in H/R-challenged cardiomyocyte damage, exerting its potential in providing new thoughts in acute myocardial infarction management.