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.

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.

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.

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.

Molecular dissection of a hyper-aggressive CBFB-MYH11/FLT3-ITD-positive acute myeloid leukemia.

Acute Myeloid Leukaemia (AML) is a haematological malignancy showing a hypervariable landscape of clinical outcomes and phenotypic differences, explainable by heterogeneity at the cellular and molecular level. Among the most common genomic alterations, CBFB-MYH11 rearrangement and FLT3-ITD gene mutations, have opposite clinical significance and are unfrequently associated. We present here a Molecular Case Report in which these two events co-exist an ultra-aggressive phenotype resulting in death in 4 days from hospital admittance. Somatic and germline Whole Exome Sequencing analysis was performed to uncover other putative driver mutations, de-novo genomic structural events or germline clusters increasing cancer insurgence. Only three mutations in LTK, BCAS2 and LGAS9 were found, unlikely causative of the exhibited phenotype, prompting to additional investigation of the rare CBFB-MYH11/ FLT3-ITD scenario.

Jumping translocation involving chromosome 13q in a patient with Crohn's Disease and inv(16)(p13.1q22)/CBFB-MYH11 acute myeloid leukemia.

Jumping translocations (JT) are rare chromosomal rearrangements caused by the translocation of one donor chromosome segment to two or more recipient chromosomes. In the setting of myeloid neoplasms, JT are typically associated with disease transformation to acute myeloid leukemia (AML), and studies to date have found JT to be associated with poor prognosis and short overall survival. However, JT have been only very rarely reported in AML associated with a favorable AML prognostic cytogenetic marker. Additionally, JT have infrequently been described in hematological malignancies associated with autoimmune diseases (AID) such as Crohn's Disease (CD). Here we describe a case of a 40-year-old female with a 24-year history of CD diagnosed with AML harbouring the inv(16)(p13.1q22)/CBFB-MYH11 rearrangement in conjunction with sideline clones containing trisomy 13, tetrasomy 13, and a JT of chromosome 13q12 jumping to 7q32 and 18p11.2. The patient attained molecular remission one month post diagnosis after induction 7 + 3 chemotherapy. Morphologic relapse of disease occurred 27 months post diagnosis. A second molecular remission was attained 3 months later after re-induction chemotherapy. The patient received a sibling bone marrow transplant 32 months post diagnosis and is currently in remission 7 months post allogeneic transplant. To the best of our knowledge, this case represents the first report of JT occurring in inv(16)(p13.1q22)/CBFB-MYH11 AML and the second of JT occurring in an AML patient with prior clinical history of CD. This case provides further insight into the rare occurrence of JT in AML, particularly AML with a favorable cytogenetic marker in conjunction with AID.

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.

3'CBFB deletion in CBFB-rearranged acute myeloid leukemia retains morphological features associated with inv(16), but patients have higher risk of relapse and may require stem cell transplant.

Fluorescence in situ hybridization analysis (FISH) using a CBFB breakapart probe is widely used to detect CBFB rearrangement (CBFBr) in cases of acute myeloid leukemia (AML). However, detection of 3'CBFB deletion (3'CBFBdel) often poses a challenge for interpretation, and the clinical importance of 3'CBFBdel associated CBFBr remains largely unknown. We identified 16 AML patients with 3'CBFBdel, 11 (69%) of which were confirmed to have CBFB::MYH11 fusion. These 11 patients presented with de novo AML; 10 showed myelomonocytic differentiation, 8 had a prominent eosinophilic component, and 7 showed characteristic eosinophils with basophilic granules. Next generation sequencing showed mutations in 7/8 patients, 5 with KRAS/NRAS, 3 with FLT3-TKD, but none with KIT mutations. Except for one patient who died 5 days after diagnosis of AML, all 10 patients received chemotherapy and achieved remission initially. However, within 3 years, 5 (50%) patients had relapsed, of whom, 1 died and 4 received hematopoietic stem cell transplant. After a median follow-up of 76 months, 3 patients died and 8 were alive in complete remission. Our study shows that detection of 3'CBFBdel is not equivalent to unbalanced CBFB rearrangement, and therefore, an alternative confirmatory test is warranted. AML with 3'CBFBdel/CBFBr often shows similar pathological features to AML with inv(16), but appears to have different mutation profiles and a higher risk of relapse requiring hematopoietic stem cell transplant.

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.

Non-age-related neoplastic loss of sex chromosome correlated with prolonged survival in real-world CBF-AML patients.

In this real-world clinical study, in which we determined eligibility for allogenic hematopoietic stem cell transplantation by prognostic factors and minimal residual disease status, we retrospectively evaluated cytogenetic, genetic, and clinical features in 96 patients with core-binding factor acute myeloid leukemia (CBF-AML) including 62 patients with RUNX1/RUNX1T1 and 34 patients with CBFß/MYH11. Multivariate analyses for 5-year overall survival (OS) in CBF-AML patients revealed that age of 50 years or older (HR: 3.46, 95% CI 1.47-8.11, P = 0.004) and receiving 2 or more induction cycles (HR: 3.55, 95% CI 1.57-8.05, P = 0.002) were independently associated with worse OS and that loss of sex chromosome (LOS) was independently associated with better OS (HR: 0.09, 95% CI 0.01-0.71, P = 0.022). At the time of complete remission, all 21 karyotyped patients with LOS had a normal karyotype. Furthermore, in all 9 patients with LOS who had a mosaic of metaphase cells with and without t(8;21) or inv(16), the metaphase cells without t(8;21)/inv(16) showed a normal karyotype. These results proved that LOS was not age-related and physiological, but rather a neoplastic chromosomal abnormality.

All-trans retinoic acid induces differentiation in primary acute myeloid leukemia blasts carrying an inversion of chromosome 16.

All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.