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.

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.

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.

Cancer-Associated Exosomal CBFB Facilitates the Aggressive Phenotype, Evasion of Oxidative Stress, and Preferential Predisposition to Bone Prometastatic Factor of Breast Cancer Progression.

Background: Despite therapeutic advancements, metastasis remains a major cause in breast cancer-specific mortality. Breast cancer cells are susceptible to oxidative damage and exhibit high levels of oxidative stress, including protein damage, DNA damage, and lipid peroxidation. Some breast cancer risk factors may change the level of endogenous oxidative stress. Circulating exosomes play critical roles in tumorigenesis, distant metastasis, and poor prognosis in patients with breast cancer. Methods: We used an online database to analyze the expression and prognostic value of core binding factor subunit ß (CBFB) and oxidative stress-related targets in patients with breast cancer. Serum from healthy controls and patients with primary breast cancer or bone metastatic breast cancer in the bone was collected. Exosomes were isolated from the sera or cell culture media. We used an MDA-MB-436-innoculated tumor xenograft mouse model for silencing CBFB. Results: Circulating exosomes from patients with breast cancer metastasis to the bone were rich in CBFB. The human mammary fibroblast cells HMF3A and fibroblasts derived from patient samples cocultured with exosomes had increased α-SMA and vimentin expression and IL-6 and OPN secretion. Similarly, nonmetastatic breast cancer cells cocultured with exosomes exhibited increased levels of certain markers, including vimentin, snail1, CXCR4, and Runx2, and the exosomes had high CBFB expression. Silencing CBFB in metastatic MDA-MB-436 and MDA-MB-157 cells resulted in suppressed migration and invasion and downregulation of vimentin, CXCR4, snail1, Runx2, CD44, and OPN. Conversely, CBFB overexpression resulted in upregulation of Runx2, vimentin, snail1, CD44, and OPN in nonmetastatic T47D and MCF12A cells. The CBFB-rich exosomes derived from MDA-MB-436 cells induced enhanced metastatic phenotypes in the low-metastatic T47D and MCF12A cell lines. Conclusion: Our results revealed that CBFB may promote bone metastasis in patients with breast cancer. Of therapeutic relevance, targeting CBFB resulted in decreased tumor burden and bone metastasis, downregulation of bone metastasis markers, and impaired regulation of oxidative stress-related proteins NAE1 and NOS1.

Comprehensive Mutation Profile in Acute Myeloid Leukemia Patients with RUNX1-RUNX1T1 or CBFB-MYH11 Fusions

Objective: This study was undertaken with the aim of better understanding the genomic landscape of core-binding factor (CBF) acute myeloid leukemia (AML). Materials and Methods: We retrospectively analyzed 112 genes that were detected using next-generation sequencing in 134 patients with de novo CBF-AML. FLT3-ITD, NPM1, and CEBPA mutations were detected by DNA-PCR and Sanger sequencing. Results: In the whole cohort, the most commonly mutated genes were c-KIT (33.6%) and NRAS (33.6%), followed by FLT3 (18.7%), KRAS (13.4%), RELN (8.2%), and NOTCH1 (8.2%). The frequencies of mutated genes associated with epigenetic modification, such as IDH1, IDH2, DNMT3A, and TET2, were low, being present in 1.5%, 0.7%, 2.2%, and 7.5% of the total number of patients, respectively. Inv(16)/t(16;16) AML patients exhibited more mutations of NRAS and KRAS (p=0.001 and 0.0001, respectively) than t(8;21) AML patients. Functionally mutated genes involved in signaling pathways were observed more frequently in the inv(16)/t(16;16) AML group (p=0.016), while the mutations involved in cohesin were found more frequently in the t(8;21) AML group (p=0.011). Significantly higher white blood cell counts were found in inv(16)/t(16;16) AML patients with c-KIT (c-KITmut) or NRAS (NRASmut) mutations compared to the corresponding t(8;21) AML/c-KITmut and t(8;21) AML/NRASmut groups (p=0.001 and 0.009, respectively). Conclusion: The mutation profiles of t(8;21) AML patients showed evident differences from those of patients with inv(16)/t(16;16) AML. We have provided a comprehensive overview of the mutational landscape of CBF-AML.

AM22, a novel synthetic microRNA, inhibits the proliferation of colorectal cancer cells by targeting core binding factor subunit ß (CBFB).

Our previous studies have revealed the important roles of the nonseed regions of microRNAs (miRNAs) in gene regulation, which provided novel insight into the development of miRNA analogs for cancer therapy. Here, we altered each nucleotide in the nonseed region of miR-34a and obtained novel synthetic miRNA analogs. Among them, AM22, with a base alteration from G to C at the 17th nucleotide of miR-34a, showed extensive antiproliferative activity against several colorectal tumor cell lines and achieved effective inhibition of core binding factor subunit ß (CBFB) expression. Subsequent investigations demonstrated that AM22 directly targeted CBFB by binding to its 3'-untranslated region (3'-UTR). Inhibition of CBFB showed obvious antiproliferative activity on HCT-116 and SW620 cells. Furthermore, the antiproliferative effects of AM22 on these cells were also measured in xenograft mouse models. In conclusion, this study identified AM22 as a potential antitumor miRNA by targeting CBFB and provided a new design approach for miRNA-based cancer treatment by changing the nonseed region of miRNA.

Circular RNA_0062582 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via regulation of microRNA-145/CBFB axis.

Osteoporosis poses a threat to human health worldwide. To date, there have been few studies regarding targeted treatment of osteoporosis. We aimed to identify the possible molecular mechanism of circular RNA (circ)_0062582 in osteogenic differentiation, and the interactions among circ_0062582, microRNA-145 (miR-145) and core-binding factor subunit ß (CBFB). The proliferation of human bone marrow mesenchymal stem cells (hBMSCs) was tested with a cell counting kit-8 assay. Circ_0062582, miR-145 and CBFB were overexpressed by transient transfection. Dual-luciferase reporter assay system was used to analyze the combination among circ_0062582, miR-145 and CBFB. Additionally, the levels of circ_0062582, miR-145, CBFB, osterix (OSX), osteocalcin (OCN) and collagen type 1 (COL1) were detected by means of RT-qPCR or western blot analysis. Alkaline phosphatase and Alizarin red stainings were performed to analyze the degree of osteogenic differentiation under the control of circ_0062582, miR-145 and CBFB. The results demonstrated that circ_0062582 level was notably elvated during osteogenic differentiation of hBMSCs. Circ_0062582 overexpression significantly promoted osteogenic differentiation and upregulated the levels of osteogenic differentiation-related proteins, including OSX, OCN and COL1. In addition, miR-145, which was identified as the target gene of circ_0062582, could specifically target CBFB 3'-UTR regions. Next, these changes caused by the overexpression of circ_0062582 were reversed following the addition of miR-145 mimic. Following overexpression of CBFB, osteogenic differentiation was increased. In summary, these results demonstrated that the role of circ_0062582 in osteoporosis is mediated through regulating the expression level of CBFB via miR-145.

CBFß promotes colorectal cancer progression through transcriptionally activating OPN, FAM129A, and UPP1 in a RUNX2-dependent manner.

Colorectal cancer (CRC) is commonly associated with aberrant transcription regulation, but characteristics of the dysregulated transcription factors in CRC pathogenesis remain to be elucidated. In the present study, core-binding factor ß (CBFß) is found to be significantly upregulated in human CRC tissues and correlates with poor survival rate of CRC patients. Mechanistically, CBFß is found to promote CRC cell proliferation, migration, invasion, and inhibit cell apoptosis in a RUNX2-dependent way. Transcriptome studies reveal that CBFß and RUNX2 form a transcriptional complex that activates gene expression of OPN, FAM129A, and UPP1. Furthermore, CBFß significantly promotes CRC tumor growth and live metastasis in a mouse xenograft model and a mouse liver metastasis model. In addition, tumor-suppressive miR-143/145 are found to inhibit CBFß expression by specifically targeting its 3'-UTR region. Consistently, an inverse correlation between miR-143/miR-145 and CBFß expression levels is present in CRC patients. Taken together, this study uncovers a novel regulatory role of CBFß-RUNX2 complex in the transcriptional activation of OPN, FAM129A, and UPP1 during CRC development, and may provide important insights into CRC pathogenesis.

CBFB cooperates with p53 to maintain TAp73 expression and suppress breast cancer.

The CBFB gene is frequently mutated in several types of solid tumors. Emerging evidence suggests that CBFB is a tumor suppressor in breast cancer. However, our understanding of the tumor suppressive function of CBFB remains incomplete. Here, we analyze genetic interactions between mutations of CBFB and other highly mutated genes in human breast cancer datasets and find that CBFB and TP53 mutations are mutually exclusive, suggesting a functional association between CBFB and p53. Integrated genomic studies reveal that TAp73 is a common transcriptional target of CBFB and p53. CBFB cooperates with p53 to maintain TAp73 expression, as either CBFB or p53 loss leads to TAp73 depletion. TAp73 re-expression abrogates the tumorigenic effect of CBFB deletion. Although TAp73 loss alone is insufficient for tumorigenesis, it enhances the tumorigenic effect of NOTCH3 overexpression, a downstream event of CBFB loss. Immunohistochemistry shows that p73 loss is coupled with higher proliferation in xenografts. Moreover, TAp73 loss-of-expression is a frequent event in human breast cancer tumors and cell lines. Together, our results significantly advance our understanding of the tumor suppressive functions of CBFB and reveal a mechanism underlying the communication between the two tumor suppressors CBFB and p53.

Hypoxia-inducible factor 2α is a novel inhibitor of chondrocyte maturation.

Hypoxic environment is essential for chondrocyte maturation and longitudinal bone growth. Although hypoxia-inducible factor 1 alpha (Hif-1α) has been known as a key player for chondrocyte survival and function, the function of Hif-2α in cartilage is mechanistically and clinically relevant but remains unknown. Here we demonstrated that Hif-2α was a novel inhibitor of chondrocyte maturation through downregulation of Runx2 stability. Mechanistically, Hif-2α binding to Runx2 inhibited chondrocyte maturation by Runx2 degradation through disrupting Runx2/Cbfß complex formation. The Hif-2α-mediated-Runx2 degradation could be rescued by Cbfß transfection due to the increase of Runx2/Cbfß complex formation. Consistently, mesenchymal cells derived from Hif-2α heterozygous mice were more rapidly differentiated into hypertrophic chondrocytes than those of wild-type mice in a micromass culture system. Collectively, these findings demonstrate that Hif-2α is a novel inhibitor for chondrocyte maturation by disrupting Runx2/Cbfß complex formation and consequential regulatory activity.

A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

RUNX2 regulates leukemic cell metabolism and chemotaxis in high-risk T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

Allosteric interference in oncogenic FLI1 and ERG transactions by mithramycins.

ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with function of other factors. Mithramycin (MTM) is an anti-cancer, DNA binding natural product that functions as a potent antagonist of ERG and FLI1 by an unknown mechanism. We present a series of crystal structures of the DNA binding domain (DBD) of ERG/FLI1 culminating in a structure of a high-order complex of the ERG/FLI1 DBD, transcription factor Runx2, core-binding factor beta (Cbfß), and MTM on a DNA enhancer site, along with supporting DNA binding studies using MTM and its analogues. Taken together, these data provide insight into allosteric mechanisms underlying ERG and FLI1 transactions and their disruption by MTM analogues.

A novel HIV-1 inhibitor that blocks viral replication and rescues APOBEC3s by interrupting vif/CBFß interaction.

HIV remains a health challenge worldwide, partly because of the continued development of resistance to drugs. Therefore, it is urgent to find new HIV inhibitors and targets. Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 family members (APOBEC3) are important host restriction factors that inhibit HIV-1 replication by their cytidine deaminase activity. HIV-1 viral infectivity factor (Vif) promotes proteasomal degradation of APOBEC3 proteins by recruiting the E3 ubiquitin ligase complex, in which core-binding factor ß (CBFß) is a necessary molecular chaperone. Interrupting the interaction between Vif and CBFß can release APOBEC3 proteins to inhibit HIV-1 replication and may be useful for developing new drug targets for HIV-1. In this study, we identified a potent small molecule inhibitor CBFß/Vif-3 (CV-3) of HIV-1 replication by employing structure-based virtual screening using the crystal structure of Vif and CBFß (PDB: 4N9F) and validated CV-3's antiviral activity. We found that CV-3 specifically inhibited HIV-1 replication (IC50 = 8.16 µm; 50% cytotoxic concentration >100 µm) in nonpermissive lymphocytes. Furthermore, CV-3 treatment rescued APOBEC3 family members (human APOBEC3G (hA3G), hA3C, and hA3F) in the presence of Vif and enabled hA3G packaging into HIV-1 virions, which resulted in Gly-to-Ala hypermutations in viral genomes. Finally, we used FRET to demonstrate that CV-3 inhibited the interaction between Vif and CBFß by simultaneously forming hydrogen bonds with residues Gln-67, Ile-102, and Arg-131 of CBFß. These findings demonstrate that CV-3 can effectively inhibit HIV-1 by blocking the interaction between Vif and CBFß and that this interaction can serve as a new target for developing HIV-1 inhibitors.

Runx-mediated regulation of CCL5 via antagonizing two enhancers influences immune cell function and anti-tumor immunity.

CCL5 is a unique chemokine with distinct stage and cell-type specificities for regulating inflammation, but how these specificities are achieved and how CCL5 modulates immune responses is not well understood. Here we identify two stage-specific enhancers: the proximal enhancer mediates the constitutive CCL5 expression during the steady state, while the distal enhancer located 1.35 Mb from the promoter induces CCL5 expression in activated cells. Both enhancers are antagonized by RUNX/CBFß complexes, and SATB1 further mediates the long-distance interaction of the distal enhancer with the promoter. Deletion of the proximal enhancer decreases CCL5 expression and augments the cytotoxic activity of tissue-resident T and NK cells, which coincides with reduced melanoma metastasis in mouse models. By contrast, increased CCL5 expression resulting from RUNX3 mutation is associated with more tumor metastasis in the lung. Collectively, our results suggest that RUNX3-mediated CCL5 repression is critical for modulating anti-tumor immunity.