Core-binding Factor Subunit Research Articles

Core-binding factor subunit beta is not required for non-primate lentiviral Vif-mediated APOBEC3 degradation.

Viral infectivity factor (Vif) is required for lentivirus fitness and pathogenicity, except in equine infectious anemia virus (EIAV). Vif enhances viral infectivity by a Cullin5-Elongin B/C E3 complex to inactivate the host restriction factor APOBEC3. Core-binding factor subunit beta (CBF-β) is a cell factor that was recently shown to be important for the primate lentiviral Vif function. Non-primate lentiviral Vif also degrades APOBEC3 through the proteasome pathway. However, it is unclear whether CBF-β is required for the non-primate lentiviral Vif function. In this study, we demonstrated that the Vifs of non-primate lentiviruses, including feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), caprine arthritis encephalitis virus (CAEV), and maedi-visna virus (MVV), do not interact with CBF-β. In addition, CBF-β did not promote the stability of FIV, BIV, CAEV, and MVV Vifs. Furthermore, CBF-β silencing or overexpression did not affect non-primate lentiviral Vif-mediated APOBEC3 degradation. Our results suggest that non-primate lentiviral Vif induces APOBEC3 degradation through a different mechanism than primate lentiviral Vif. Importance: The APOBEC3 protein family members are host restriction factors that block retrovirus replication. Vif, an accessory protein of lentivirus, degrades APOBEC3 to rescue viral infectivity by forming Cullin5-Elongin B/C-based E3 complex. CBF-β was proved to be a novel regulator of primate lentiviral Vif function. In this study, we found that CBF-β knockdown or overexpression did not affect FIV Vif's function, which induced polyubiquitination and degradation of APOBEC3 by recruiting the E3 complex in a manner similar to that of HIV-1 Vif. We also showed that other non-primate lentiviral Vifs did not require CBF-β to degrade APOBEC3. CBF-β did not interact with non-primate lentiviral Vifs or promote their stability. These results suggest that a different mechanism exists for the Vif-APOBEC interaction and that non-primates are not suitable animal models for exploring pharmacological interventions that disrupt Vif-CBF-β interaction.

Acute myeloid leukemia with t(7;21)(p22;q22) and 5q deletion: a case report and literature review

The gene RUNX1 at chromosome 21q22 encodes the alpha subunit of Core binding factor (CBF), a heterodimeric transcription factor involved in the development of normal hematopoiesis. Translocations of RUNX1 are seen in several types of leukemia with at least 21 identified partner genes. The cryptic t(7;21)(p22;q22) rearrangement involving the USP42 gene appears to be a specific and recurrent cytogenetic abnormality. Eight of the 9 cases identified in the literature with this translocation were associated with acute myeloid leukemia (AML), with the remaining case showing refractory anemia with excess blasts, type 2. Herein, we present a patient with two preceding years of leukopenia and one year of anemia prior to the diagnosis of AML, NOS with monocytic differentiation (myelomonocytic leukemia) whose conventional cytogenetics showed an abnormal clone with 5q deletion. Interphase FISH using LSI RUNX1/RUNXT1 showed three signals for RUNX1. FISH studies on previously G-banded metaphases showed the extra RUNX1 signal on the short arm of chromosome 7. Further characterization using the subtelomeric 7p probe showed a cryptic 7;21 translocation. Our case and eight previously reported leukemic cases with the t(7;21)(p22;q22) appear to share similar features including monocytic differentiation, immunophenotypic aberrancies (often with CD56 and/or CD7), and a generally poor response to standard induction chemotherapy. About 80% of these cases had loss of 5q material as an additional abnormality at initial diagnosis or relapse. These findings suggest that t(7;21) may represent a distinct recurrent cytogenetic abnormality associated with AML. The association between the t(7;21) and 5q aberrancies appears to be non-random, however the pathogenetic connection remains unclear. Additional studies to evaluate for RUNX1 partner genes may be considered for AML patients with RUNX1 rearrangement and 5q abnormalities; however knowledge of the prognostic implications of this rearrangement is still limited.

Label-free quantitative proteomic analysis of the YAP/TAZ interactome

The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients. To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFβ, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFβ, and FLI-1. RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

Connective tissue growth factor induces osteogenic differentiation of vascular smooth muscle cells through ERK signaling

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of atherosclerosis by promoting vascular smooth muscle cell (VSMC) growth, migration, apoptosis, adhesion and the secretion of matrix components. The osteogenic differentiation of VSMCs is essential in the development of vascular calcification. However, the role of CTGF in the transdifferentiation and calcification of VSMCs is unclear. In the present study, we examined whether CTGF stimulates VSMC transdifferentiation. Primary VSMCs were obtained from mouse thoracic aortas by enzymatic digestion and identified by immunostaining for smooth muscle specific α-actin antibody (α-SMA). VSMC calcification was induced by the addition of CTGF to the osteogenic mediaum containing 5-10% FBS in the presence of 0.25 mM ascorbic acid and 10 mM β-glycerophosphate for 14 days. Calcified cells were determined by Alizarin Red S staining. Our results revealed that CTGF induced the expression of several bone markers, including alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and core-binding factor subunit α1 (Cbfα1)/runt-related transcription factor 2 (Runx2), as well as calcification. However, the inhibition of extracellular signal-regulated kinase (ERK) activity using the ERK-specific inhibitor, PD98059, blocked the induction of these proteins and VSMC calcification. Based on these data, we conclude that CTGF stimulates the transdifferentiation of VSMCs into osteoblasts and that the ERK signaling pathway appears to play a critical role in this process.

Incidence and Clinical Features of Core Binding Factor Acute Myeloid Leukemia: A Collaborative Study of the Japan Adult Leukemia Study Group and the Korean Society of Hematology.

AbstractAbstract 2584 Background:Core binding factor (CBF) acute myeloid leukemia (AML) is a specific subgroup of AML, which is commonly related with rearrangements of genes encoding subunits of CBF, AML1 /RUNX1 (AML1-ETO) in t(8;21)(q22;q22) or CBFB (CBFB-MYH11) in inv(16)(p13q22)/t(16;16)(p13;q22), and both clinical outcomes are usually superior in de novo AML. According to literatures, the frequency of patients with t(8;21)(q22;q22) among AML is higher in Japan and Korea than Western countries. However, the accurate incidence and clinical features of CBF-AML are unclear in a large cohort. Patients and methods:We have analyzed 1,536 de novo AML patients from 2 clinical trials of Japan Adult Leukemia Study Group (JALSG, AML97 and AML201 studies, registered from December 1997 to December 2005) and 876 from 3 institutes in Korea (diagnosed from January 2007 to December 2011). Patients from 15 to 64 years old were included in this study, but those with FAB-M3/t(15;17)(q22;q12–21) were excluded. Results:The incidence of CBF-AML was similar in both Japan and Korea (23.3% and 21.2%, P=0.2), however, that of t(8;21) and inv(16) each was a little different between both countries. t(8;21) and inv(16) were observed in 18.6% and 4.9% in Japan, and in 15.0% and 6.3% in Korea, respectively. Of these, the incidence of t(8;21) in Korea was lower than that in Japan (P=0.02), but higher than those reported from Western countries [4.9–8.6% in MRC, CALGB, and SWOG/ECOG except t(15;17), P<0.001].The prognoses of patients with t(8;21) and inv(16) were similar in complete remission (CR) rate between both countries, but different in 5-year overall survival (OS) and disease-free survival (DFS) rates. CR rate was 91.3% (Japan) and 93.9% (Korea) in t(8;21) (p=0.3), and 98.7% and 100% in inv(16) (P=0.6), respectively. In t(8;21), OS was relatively better in Japan than in Korea (64.6% versus 54.9%, P=0.055), however, DFS tended to be worse in Japan than in Korea (47.6% versus 56.1%, P=0.47). In inv(16), almost the same results with t(8;21) were observed (72.6% versus 65.7% in OS, P=0.2, and 47.7% versus 62.2% in DFS, P=0.06).The white blood cell (WBC) counts and WBC index were prognostic factors in patients with t(8;21), and significantly related to clinical outcome in Japanese patients, however, no significant differences were observed in Korean patients. Patients with t(8;21) having high WBC count (>=10×109/L) were found to have a poor outcome in Japan (44.8% versus 74.5% in OS and 32.4% versus 57.5% in DFS; P<0.001 each), but not in Korea (53.0% versus 57.1% in OS, P=0.8, and 54.0% versus 58.2% in DFS; P=0.6).Additional chromosome abnormalities found in t(8;21) and inv(16) were similar in both countries. In t(8;21), a sole t(8;21) was observed in 25.5% and 25.2%, a loss of a sex chromosome (-X/-Y) in 60.8% and 60.3%, del(9q) in 8.4% and 6.1%, and other abnormalities including 7q- in 23.8% and 29.0% in Japan and Korea, respectively. However, correlations of additional chromosome abnormalities and clinical outcome were different in each country. In Japan, patients with -X/-Y had relatively favorable OS than those with sole t(8;21) [69.1% versus 57.5%, P=0.08]. However, opposite correlation was observed in Korea [48.1% in -X/-Y versus 59.6% in sole t(8;21), P=0.03], although sole t(8;21) had identical OS in both counties [57.5% versus 59.6%, P=0.4] Conclusions:High incidence of t(8;21) in AML was observed in both countries, however, prognostic significance of various factors, such as WBC counts, WBC index, and additional chromosome abnormalities, were different in each country. One of the reasons for these differences may be dependent on the different treatment protocols in each country, and further analysis of factors associated with prognosis in each country is needed. Disclosures:No relevant conflicts of interest to declare.

HIV Type 1 Viral Infectivity Factor and the RUNX Transcription Factors Interact with Core Binding Factor β on Genetically Distinct Surfaces

Human immunodeficiency virus type 1 (HIV-1) requires the cellular transcription factor core binding factor subunit β (CBFβ) to stabilize its viral infectivity factor (Vif) protein and neutralize the APOBEC3 restriction factors. CBFβ normally heterodimerizes with the RUNX family of transcription factors, enhancing their stability and DNA-binding affinity. To test the hypothesis that Vif may act as a RUNX mimic to bind CBFβ, we generated a series of CBFβ mutants at the RUNX/CBFβ interface and tested their ability to stabilize Vif and impact transcription at a RUNX-dependent promoter. While several CBFβ amino acid substitutions disrupted promoter activity, none of these impacted the ability of CBFβ to stabilize Vif or enhance degradation of APOBEC3G. A mutagenesis screen of CBFβ surface residues identified a single amino acid change, F68D, that disrupted Vif binding and its ability to degrade APOBEC3G. This mutant still bound RUNX and stimulated RUNX-dependent transcription. These separation-of-function mutants demonstrate that HIV-1 Vif and the RUNX transcription factors interact with cellular CBFβ on genetically distinct surfaces.

Repairing cartilage in osteoarthritis

The cartilage damage that is characteristic of osteoarthritis might be repaired using compounds that promote the differentiation of multipotent mesenchymal stem cells into chondrocytes. Using a high-throughput screen, Johnson et al. identified kartogenin as such a molecule. In models of osteoarthritis, the compound (given at early stages of the disease) promoted regeneration of cartilage and alleviated pain. Mechanistic studies showed that kartogenin binds to the actin binding protein filamin A, disrupts its interaction with the transcription factor CBFβ (core binding factor subunit β) and induces chondrogenesis by regulating the CBFβ–RUNX1 (runt-related transcription factor 1) transcriptional programme.

Poor Outcome of RUNX1-Mutated (RUNX1-mut) Patients (Pts) with Primary, Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) and Associated Gene- and MicroRNA (miR) Expression Signatures,

Abstract 3454RUNX1 encodes the α subunit of core binding factor, a heterodimeric transcription factor required for normal hematopoiesis. Acquired RUNX1 mutations (muts) have been associated with poor clinical outcome in AML; however, prior studies analyzed pts heterogeneous for cytogenetics, age, AML type (primary or secondary), and treatment received [including allogeneic stem cell transplant (alloSCT) in 1st complete remission (CR1)] and contained limited data regarding the potential molecular drivers of the worse outcome. We report a relatively large study testing the prognostic impact of RUNX1 muts in primary CN-AML pts (n=392) treated similarly with intensive cytarabine/anthracycline-based 1st-line therapy and without alloSCT in CR1. This cohort comprised both younger [<60 years (y); n=173] and older (≥60 y; n=219) pts. Pretreatment marrow (n=303) and blood (n=89) were analyzed centrally for RUNX1 muts by PCR and direct sequencing, and for FLT3-ITD, FLT3-TKD, MLL-PTD and NPM1, CEBPA, WT1, IDH1, IDH2 and TET2 muts. Gene and miR expression profiles were derived using microarrays. RUNX1 muts were found in 12.5% of pts (8% younger, 16% older), and were associated with lower hemoglobin (P=.01), white blood cells (WBC; P=.04), and blood blasts (P=.006). RUNX1-mut pts harbored NPM1 (P<.001) and CEBPA muts (P=.06) less frequently than RUNX1-wild-type (RUNX1-wt) pts. RUNX1-mut pts had lower CR rates (P=.005 in younger; P=.006 in older), and shorter disease-free (DFS; P=.058 in younger; P<.001 in older), overall (OS; P=.003 in younger; P<.001 in older) and event-free (EFS; P<.001 for younger and older; Figures 1 and 2) survival than RUNX1-wt pts. In multivariable models, RUNX1 muts remained associated with lower CR rate (P<.001) and shorter DFS (P<.001), OS (P<.001), and EFS (P<.001; Table) after adjustment for clinical and molecular variables. [Display omitted] [Display omitted] Table 1Multivariable analysis for EFS according to RUNX1-mut status in all CN-AML ptsHREFSPRUNX1, mut v wt2.271.65–3.12<.001FLT3-ITD, ITD v no ITD1.571.27–1.95<.001WT1, mut v wt1.441.02–2.01.04WBC, continuous 50 unit increase1.131.04–1.23.006Age group, ≥60y v <60y1.801.46–2.22<.001Note: A hazard ratio (HR) >1 corresponds to a higher risk for higher values of continuous variables and the 1st level listed of a dichotomous variable.To gain biological insight, RUNX1 mut-associated gene and miR expression signatures were derived in CN-AML for the first time. Older, NPM1-wt pts were analyzed since RUNX1 muts are more common in this age group and are nearly exclusive from NPM1 muts, which have their own characteristic gene-expression signature. This yielded 484 probe sets representing 278 named genes differentially expressed between RUNX1-mut (n=31) and RUNX1-wt (n=45) pts (P<.001). Genes normally expressed in hematopoietic stem (HSC) and early progenitor cells, including DNTT, BAALC, MN-1, CD109, P2RY14, FOXO1 and FLT-3 were upregulated in RUNX1-mut pts, as were components of the Wnt-signaling pathway, LRP6 and TCF4, that promote self-renewal and proliferation of HSCs. Genes upregulated (SETBP1, RBPMS, and SLC37A3) and downregulated (CCNA1 and RNASE3) in AML stem cells relative to AML progenitors were similarly deregulated in the RUNX1-mut signature. B cell lineage genes BLNK, IGHM, IRF8 and several class II MHC molecules were upregulated in RUNX1-mut pts while CEBPA, a key promoter of granulopoiesis, was downregulated. Genes implicated in chemoresistance, GAS6, PRKCE, and PTK2, were upregulated and MYCN, a promoter of both proliferation and apoptosis of myeloid cells, was downregulated in RUNX1-mut pts. Seven miRs were differentially expressed between RUNX1-mut and RUNX1-wt pts. Two members of the let-7 tumor suppressor family, which represses self-renewal and promotes differentiation of stem cells, were downregulated, as was miR-223, a positive regulator of granulopoiesis. MiRs -99a and -100 were also downregulated and miRs -211 and -595 upregulated in association with RUNX1 muts.In summary, RUNX1 muts are twice as common in older CN-AML pts than younger. They negatively impact on outcome in both younger and older pts not receiving alloSCT in CR1. RUNX1-mut blasts have molecular features of normal/malignant stem cells and B cells, which may explain their chemoresistance and guide novel therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.

Core-Binding Factor Acute Myeloid Leukemia

Core-binding factor acute myeloid leukemia (AML) is cytogenetically defined by the presence of t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22), commonly abbreviated as t(8;21) and inv(16), respectively. In both subtypes, the cytogenetic rearrangements disrupt genes that encode subunits of core-binding factor, a transcription factor that functions as an essential regulator of normal hematopoiesis. The rearrangements t(8;21) and inv(16) involve the RUNX1/RUNX1T1 ( AML1-ETO ) and CBFB/MYH11 genes, respectively. These 2 subtypes are categorized as AML with recurrent genetic abnormalities, and hence the cytogenetic fusion transcripts are considered diagnostic of acute leukemia even when the marrow blast count is less than 20%. The t(8;21) and inv(16) subtypes of AML have been usually grouped and reported together in clinical studies; however, recent studies have demonstrated genetic, clinical, and prognostic differences, supporting the notion that they represent 2 distinct biologic and clinical entities. This review summarizes the spectrum of this subset of AMLs, with particular emphasis on molecular genetics and pathologic findings.

RUNX1 Mutations in Pediatric AML: A Report From the Children's Oncology Group.

Abstract 2614Poster Board II-590The RUNX1 gene (previously AML1) encodes the alpha subunit of core binding factor (CBFα), which is implicated in normal and malignant hematopoiesis. Translocations involving RUNX1 have been associated with favorable prognosis in acute leukemias (e.g., t(8;21) in AML and t(12;21) in ALL). Point mutations of RUNX1 have been identified in exon 3 and exon 8 of the RUNX1 gene in a subset of AML patients and are most closely associated with MDS associated AML and AML subtype M0. We evaluated the prevalence and prognostic significance of RUNX1 mutations in pediatric patients with de novo AML treated on pediatric AML trials CCG-2941 and CCG-2961. Initial evaluation of exon 3 and exon 8 of the RUNX1 gene was conducted on a cohort of 100 randomly selected patient specimens. In this initial analysis, we identified 4 missense mutations of exon 3 that caused a change in codon 56. No exon 8 mutations were identified. Subsequent molecular genotyping of the remaining 484 patient specimens were limited to exon 3. Of the 584 diagnostic specimens tested, a missense mutation of exon 3 was detected in 19 patients (3.3%). All detected mutations occurred at nucleotide 167 (T to C) causing a leucine to serine substitution at codon 56 (L56S). Additionally, two patients had a synonymous mutation (G to A at nucleotide 183, P61P) and were considered wild type. Demographics, laboratory characteristics and clinical outcome were compared between those with and without RUNX1 mutations. There was no significant difference with regard to age, gender or race between these groups. Those with RUNX1 mutations had a significantly lower prevalence of organomegaly (16% vs. 42%, p=0.02) and significantly higher rate of extramedullary disease (chloroma 26% vs. 10%, p=0.04). There was no significant difference in association with other known cytogenetic abnormalities or risk groupings (standard, low, or high risk grouping on these studies). t(8;21) translocations were detected in 22% of those with RUNX1 mutations compared to 16% in those without mutation (p=0.6), and there was no RUNX1 mutation detected in patients with inv(16). Of the 85 patients with FLT3/ITD, NPM1 or CEBPA mutations, only 3 patients had a concomitant RUNX1 mutation (2 with FLT3/ITD and 1 with NPM1 mutation). Remission induction rate was compared between patients with and without RUNX1 mutations. Those with RUNX1 mutations had a similar CR rate to those without mutations (89% vs. 79%, p=0.4). Overall survival from remission for patients with and without RUNX1 mutation was 39 ± 25% and 60 ± 5% respectively (p=0.1) with a corresponding disease-free survival of 34 ± 24% vs. 49 ± 5% (p=0.25). RUNX1 mutations may represent a biologically distinct group that presents in 3.3% of pediatric AML patients across different morphologic and cytogenetic populations. Alterations of RUNX1 affect signal transduction pathways and may be exploited in defining a population for directed and risk based therapy. Disclosures:No relevant conflicts of interest to declare.

Survival Is Poorer in Patients with Secondary Core Binding Factor Acute Myelogenous Leukemia Compared with De Novo Core Binding Factor Leukemia.

Translocation (8;21)(q22;q22) and inversion of chromosome 16 [inv(16) (p13q22)] are considered good-risk cytogenetic abnormalities in acute myeloid leukemia (AML) accounting for 15% of primary AML cases (Blood. 2002 Dec 15;100(13):4325–36) and are characterized at the molecular level by disruption of genes encoding subunits of core binding factor (CBF). Increasing age, increasing peripheral blast percentage and complex cytogenetics are associated with poor overall survival in these patients (Br J Haematol. 2006 Oct;135(2):165–73). Here we assess differences in patient characteristics and outcomes between the primary and secondary core binding factor AML. One hundred eighty nine CBF AML patients treated at our institution were studied; 18 (9.5%) had secondary AML. Patients with secondary CBF AML were older (p= 0.02, median ages 57 vs. 44 years) and had lower WBC counts (p=0.03) (Table1). Overall survival (OS) was worse in the secondary AML patients (94 weeks versus 621 weeks, p=0.0016) (Figure 1). Age, hemoglobin, platelet count, and bilirubin were significantly associated with OS in the univariate analysis. In the multivariate analysis, after adjusting for age, hemoglobin, WBC, and bilirubin, secondary CBF AML was marginally significantly associated with worse OS (hazard ratio 1.884, CI95% 0.979–3.625, p=0.058) but not worse PFS (p= 0.15) (Table 2). These data suggest that the secondary CBF AML has much poorer prognosis than the primary CBF AML, further indicating that “CBF AML” is not a homogeneous entity with a uniformly good prognosis.Table 1:Summary statistics of patients' continuous characteristics by abnormality statusPatient characteristicsTotal patientsPrimary AML (n=171, male=96)Secondary AML (n=18, male=8)p-value (primary vs secondary AML)Median (range)Median (range)Median (range)Age (years)44 (16–88)44 (16–88)57 (31–75)0.02WBC (103/mm3)14.7 (0.6–387)16.4 (0.6–387)6.6 (0.8–103.8)0.03Hemoglobin (g/dl)8.1 (2.5–14.3)8.2 (2.5–14.3)7.8 (4.8–10.8)0.33Platelet count (103/mm3)38 (5–350)38 (5–350)39 (9–139)0.88Bilirubin (mg/dl)0.6 (0.0–15.3)0.6 (0.0–5.0)0.6 (0.1–15.3)0.94Creatinine (mg/dl)0.9 (0.5–2.9)0.9 (0.5–2.9)0.9 (0.5–1.8)0.66Table 2:Multivariate Cox proportional hazards model in estimating the association between covariates and patients' survivalVariableHazard ratio (95% CI)p-valueAbnormality (secondary vs primary)1.884 (0.979–3.625)0.058Age (1 year increase)1.028 (1.012–1.044)0.0004Hemoglobin (1gm/dl increase)0.864 (0.769–0.970)0.014WBC (103/mm3 increase)1.003 (1.000–1.007)0.061Bilirubin (1 mg/dl increase)1.179 (1.044–1.332)0.008 [Display omitted]

Development of Allosteric Inhibitors of the Interaction of AML1 and CBFβ for the Treatment of Leukemia.

The protein-protein interaction between the subunits of the nuclear transcription factor core binding factor, RUNX1 (CBFα) and CBFβ, plays a critical role in hematopoiesis. Chromosomal rearrangements that target CBF genes are among the most common mutations in leukemia, including t(8;21) found in approximately 12% of cases of AML and resulting in the protein AML1-ETO. We describe the development of small molecule inhibitors of the interaction between RUNX1 and CBFβ. We used virtual screening to identify lead compounds. These leads were used to generate a library of compounds to explore the structure-activity relationships and optimize activity, resulting in the identification of low micromolar IC50 inhibitors of this protein-protein interaction. We confirmed by FACS FRET that inhibition also occurs in mammalian cells. HEK-293 cells were transfected with Cerulean-Runt domain and Venus-CBFβ. FRET emission from cells was monitored on a flow cytometer to assess binding of CBFβ and the Runt domain. Results with drug were compared to FRET between Runt and wild-type CBFβ and FRET between Runt and a mutant CBFβ with 2 point mutations abrogating binding to Runt. The small molecule inhibitor KG-3-275 inhibited CBFβ - Runt domain binding in a dose dependent manner (Table 1). Treatment of the t(8;21) leukemia cell lines Kasumi-1 and SKNO-1 results in inhibition of proliferation. SKNO-1 cells are inhibited in a dose-dependent manner by KG-3-275 but not by the weak inhibitor KG-1-253 (Fig 1). KG-3-275 does not inhibit the growth of renal tubular and hepatocellular cell lines (Fig 1). Kasumi-1 and SKNO-1 undergo apoptosis in a dose-dependent manner upon treatment with KG-3-275 (Table 2). Finally, KG-3-275 shows synergy with ATRA in increasing differentiation of Kasumi-1 cells as measured by CD11b expression, consistent with recent published results establishing a link between AML1-ETO and repression of RAR signaling (Fig 2). These data indicate drugs inhibiting CBF interactions hold promise as targeted agents in the treatment of CBF leukemias.Fluorescence Resonance Energy Transfer Geometric Mean in HEK-293 CellsCerulean-Runt domain + Venus-CBFb4.07 +/− 0.1Cerulean-RD + Venus-CBFb61/1041.85 +/− .09C-RD + V-CBFb + KG-3-275 200 mcm3.05 +/− .15C-RD + V-CBFb + KG-3-275 100 mcm3.25 +/− 0.1C-RD + V-CBFb + KG-3-275 50 mcm3.45 +/− .25C-RD + V-CBFb + KG-3-275 25 mcm3.6 +/− 0.2% Cells Annexin V/PI Negative at 72 HrsKasumi-10.25% DMSO91.2 +/− 0.9KG-3-275 25 mcm91.2 +/− 1.2KG-3-275 50 mcm85.9 +/− 2.4KG-3-275 100 mcm66.7 +/− 6.7SKNO-10.25% DMSO77.6 +/− 3.4KG-3-275 25 mcm77.6 +/− 2.3KG-3-275 50 mcm63.7 +/− 3.6KG-3-275 100 mcm13.7 +/− 6.8 [Display omitted] [Display omitted]

Development of Small Molecule Inhibitors of the AML1-ETO and CBFβ-SMMHC Oncoproteins.

Core binding factor (CBF) is a heterodimeric transcription factor composed of RUNX1 (CBFα) and CBFβ subunits which are essential for normal blood cell development. CBFβ functions to increase the DNA-binding of the RUNX1 subunit 20–40 fold and to protect the RUNX1 subunit against ubiqitination and proteasome degradation, making this protein-protein interaction critical for CBF function. Two of the most common translocations involving the subunits of CBF are the inv(16) and the t(8;21) which produce the chimeric proteins CBFβ-SMMHC and AML1-ETO, respectively, which are associated with the development of Acute Myeloid Leukemia (AML). The AML1-ETO fusion protein is a dominant inhibitor of wildtype RUNX1-CBFβ activity in vivo and causes a blockage in normal hematopoiesis, predisposing for the development of leukemia. The interaction between CBFβ and AML1-ETO is critical for its function, therefore treatments targeting AML1-ETO and blocking its interaction with CBFβ are highly likely to be therapeutically beneficial. The CBFβ-SMMHC fusion protein causes dysregulation of CBF function by means of anomalously tight binding to RUNX1. Since binding to RUNX1 is required for the dysfunction associated with CBFβ-SMMHC, this interaction represents an excellent target for inhibition as a potential therapeutic strategy. We have initiated efforts to develop small molecule inhibitors of the RUNX1-CBFβ interaction as possible therapeutics for the treatment of the associated leukemias. Both virtual screening searches, focused on the X-ray structures of RUNX1 Runt domain and CBFβ, and high-throughput screening of NCI (National Cancer Institute) and Maybridge fragment libraries were used to identify initial lead compounds interacting with these proteins and blocking heterodimerization of CBF. Compounds were tested experimentally by FRET (Fluorescence Resonance Energy Transfer) and ELISA for their inhibition of RUNX1-CBFβ interaction. This resulted in a number of initial lead compounds targeting either the Runt domain or CBFβ and inhibiting this protein-protein interaction. Based on the docking mode selected lead compounds were further optimized using medicinal chemistry approaches to increase their affinity and determine the structure-activity relationships (SAR). This resulted in several compounds with low micromolar affinity (IC50 < 10 μM) which effectively block the heterodimerization of CBF in vitro and in a cell-based assay. Interestingly, compounds targeting CBFβ bind to a site displaced from the binding interface for RUNX1 as shown by the NMR-based docking, i.e. these compounds function as allosteric inhibitors of this protein-protein interaction. The most potent compounds were tested either in the Kasumi-1 leukemia cell line harboring t(8;21) translocation or in the ME-1 cell line with inv(16), resulting in a blockage of proliferation, induction of apoptosis and differentiation of these cells. These compounds represent the first small molecule inhibitors targeting CBF and inhibiting this interaction. They represent good starting points for the development of therapeutically useful inhibitors. Several approaches are being explored to modify these compounds to achieve selectivity towards AML1-ETO or CBFβ-SMMHC oncoproteins versus wild type proteins.

Allosteric Inhibition of the Protein-Protein Interaction between the Leukemia-Associated Proteins Runx1 and CBFβ

The two subunits of core binding factor (Runx1 and CBFbeta) play critical roles in hematopoiesis and are frequent targets of chromosomal translocations found in leukemia. The binding of the CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein to Runx1 is essential for leukemogenesis, making this a viable target for treatment. We have developed inhibitors with low micromolar affinity which effectively block binding of Runx1 to CBFbeta. NMR-based docking shows that these compounds bind to CBFbeta at a site displaced from the binding interface for Runx1, that is, these compounds function as allosteric inhibitors of this protein-protein interaction, a potentially generalizable approach. Treatment of the human leukemia cell line ME-1 with these compounds shows decreased proliferation, indicating these are good candidates for further development.

The Ubiquitin Specific Peptidase 42 Gene (USP42) Is Recurrently Involved as a Fusion Gene with RUNX1 in Acute Myeloid Leukemia with the t(7;21)(p22;q22) Cryptic Translocation.

The RUNX1 gene encodes the DNA-binding subunit of the heterodimeric transcription factor core binding factor (CBF) and is involved in the regulation of several genes important for hematopoiesis. RUNX1 is frequently rearranged by chromosomal translocations in a spectrum of hematologic malignancies, generating a RUNX1 fusion gene. Some of these RUNX1 fusion genes have been shown to be important in leukemogenesis, such as the ETV6/RUNX1 fusion in B-lineage acute lymphoblastic leukemia and the RUNX1/RUNX1T1 fusion in M2 acute myeloid leukemia (AML). We here describe the first case of adult AML with the t(7;21)(p22;q22) cryptic translocation. Morphologic analysis showed an AML without maturation (AML M1). The karyotype was 46, XY, del(5)(q22q33),?del(21)(q22) in twenty metaphases and analysis by spectral karyotyping (SKY) revealed a t(7;21). The translocation breakpoints at bands 21q22 and 7p22 were studied using bacterial artificial chromosomes (BACs) and showed RUNX1 and USP42 rearrangements. An in-frame fusion between RUNX1 exon 7 and USP42 exon 3 was confirmed by RT-PCR and sequencing. The RUNX1/USP42 fusion includes both the DNA binding RUNT domain of RUNX1 and the catalytic region of USP42. The RUNX1/USP42 fusion gene has recently been reported in one case of pediatric AML-M0 with normal karyotype at diagnosis (Paulsson et al., Leukemia 2006; 20:224). A complex karyotype including a deletion of the long arm of chromosome 5 was found at relapse in this case. Interestingly, blast cells in our t(7;21) positive leukemia share similar features with the previously published case. By flow cytometry, leukemic cells were positive for HLA-DR, CD34, CD33, CD13 markers and aberrantly expressed CD7 and CD56 without B-lineage markers. We next analyzed samples of CD7 and CD56 positive AMLs with different karyotypes (normal and complex) by FISH using probes to detect the RUNX1/USP42 fusion gene. The USP42 gene encodes a cysteine protease involved in the ubiquitin pathway as a deubiquitinating enzyme. Ubiquitin-specific proteases (USPs) are a large group of enzymes with highly conserved amino acid sequences that form the catalytic region. Some USPs have been shown to be involved in the stabilization of proteins, such as p53, and in the regulation of gene silencing. USP42 is part of a novel class of genes involved in the pathogenesis of leukemia and its role in hematologic cancers remains to be studied. To elucidate the possible implication of USP42 in hematologic malignancies, the expression of USP42 was also investigated by real-time PCR in our case with t(7;21) and in a series of acute leukemias.

Development of Small Molecule Inhibitors of the CBFβ-SMMHC Oncoprotein.

The two subunits of core binding factor (RUNX1 and CBFβ) play critical roles in hematopoiesis and are frequent targets of chromosomal translocations found in leukemia. CBFβ functions to increase the DNA-binding of the RUNX1 subunit 20–40 fold and to protect the Runx1 subunit against ubiqitination and subsequent proteasome degradation, making this protein-protein interaction critical for CBF function. Two of the most common translocations involving the subunits of CBF are the inv(16) and the t(8;21) which produce the chimeric proteins CBFβ-SMMHC and AML1-ETO, respectively. We are characterizing both the structures and biochemical properties of these translocation products to gain insights into their function and provide novel avenues for therapeutic development. The inv(16) results in the fusion of the N-terminal 165 amino acids of CBFβ to the coiled-coil region of smooth muscle myosin protein. The CBFβ-SMMHC fusion protein causes dysregulation of CBF function by means of anomalously tight binding to RUNX1. As there is still one copy of the normal CBFβ present in those leukemic cells harboring the inv(16), inhibition of CBFβ-SMMHC may enable the wildtype CBFβ to restore normal CBF function. Since binding to RUNX1 is required for the dysfunction associated with this protein, this binding represents an excellent target for inhibition as a potential therapeutic strategy. A small molecule inhibitor of this kind has the potential to be a useful and highly specific therapeutic agent. As an initial step in this direction, we have developed small molecules which bind to CBFβ and inhibit RUNX1 binding. This represents the first step toward our overall goal of developing compounds which can specifically inhibit CBFβ-SMMHC while minimally perturbing the activity of CBFβ itself. We previously solved the 3D structure of CBFβ using solution NMR methods and mapped the binding interface with RUNX1 by both chemical shift perturbation as well as Ala mutagenesis of the binding interface. Using this data, we employed virtual screening of CBFβ and experimental screening with NMR spectroscopy and FRET to identify initial lead compounds that could bind to CBFβ and inhibit its interaction with the RUNX1 Runt domain. Using a traditional medicinal chemistry approach, we have elaborated these compounds to identify structure-activity relationships (SAR). Based on the SAR results and NMR-based docking of the compounds to CBFβ, we have optimized the initial leads to generate compounds with low micromolar affinity which effectively inhibit the binding of RUNX1 to CBFβ. These compounds represent the first small molecule inhibitors of this protein-protein interaction. These compounds demonstrate activity in a protein localization assay in mammalian cells with no generalized toxicity, indicating they are good leads for further development. NMR-based docking of these molecules to CBFβ shows these compounds bind at a site displaced from the binding interface for RUNX1 on CBFβ, i.e. these compounds function as allosteric inhibitors of this protein-protein interaction, an approach which may be generalizable to other protein-protein interactions. We are pursuing several approaches to modify these compounds to achieve selective inhibition of CBFβ-SMMHC with minimal perturbation of native CBFβ activity.

A Genome-Modified Mouse Line with an Insertion Mutation at the AML1/Runx1 Gene Locus Detected from a Patient with Myelodysplastic Syndrome.

AML1 (Acute Myeloid Leukemia 1; also known as Runt-related transcription factor 1: Runx1) encodes the DNA binding subunit of Core-Binding Factor (CBF) transcription factor complex which plays important roles in initiating definitive hematopoiesis, thymocyte development, and platelet production. AML1's DNA-binding is mediated through its Runt domain, and their further association with the non-DNA-binding subunit, CBFβ stabilizes the resultant ternary complex to be functional. AML1 is frequently involved in leukemia-associated chromosome translocations, through which AML1 fusion-proteins with strong dominant-negative effects to normal CBF are generated, thus contributing to the leukemic transformation. Recently, genomic mutations of the AML1 gene locus have been described to associate with cases of acute myeloblastic leukemia and myelodysplastic syndromes (MDS), and pedigrees of familial platelet disorder with propensity to develop acute myeloblastic leukemia (FPD/AML). Frequent sites of the point-mutations are confined to the residues within the Runt domain, such as, R139, R174, or R177, which are important for the DNA-contact of the molecule. Preliminary results from studies using knockin mice carrying mis-sense mutations for these residues so far revealed that the mutants are biologically inactive when homozygously inherited. However, the mechanisms how the mutations contribute to the onset of the leukemia remain largely to be elucidated. In order to further characterize the aberration of AML1, we generated another genome-modified mouse line which harbors an insertion mutation of this gene locus. As reported previously, we identified a case of refractory anemia with excess of blasts (RAEB) whose blast cells have a mutated allele where three base-pair, ATC, is inserted after codon 150 (I150ins) of the molecules. By this mutation, an additional isoleucine residue is inserted into the 10th β-strand of the Runt domain sequences which provides a facet to interact with CBFβ. We introduced the cDNA of I150ins into AML1/Runx1 gene locus by means of a gene-knockin approach in mouse ES cells, and transmitted the allele into mouse germline through conventional methods. Subsequent heterozygous mice were viable and fertile. In contrast, homozygous mice die in utero similarly to the mice with simple disruption of this gene. These results indicate that this insertion mutation leads to the complete loss of its biologic function as were observed for the point-mutant mice. Further careful examinations of the heterozygous mice for this mutant allele in comparison with those for point-mutation animals and simple haplo-insufficient ones will provide valuable insights into the molecular mechanisms of AML1-related leukemic transformation.

Somatic Point Mutations in RUNX1/CBFA2/AML1 Are Common in High-Risk Myelodysplastic Syndrome, but Not in Myelofibrosis with Myeloid Metaplasia.

Introduction: The core-binding factor subunit RUNX1/CBFA2/AML1 (Runt-related transcription factor 1, Core-binding factor alpha 2 subunit, Oncogene AML1) is critical for generation of hematopoietic stem cells during embryogenesis and for normal megakaryopoiesis in adults. RUNX1/CBFA2/AML1 has long been recognized as an oncogene, and is frequently involved in leukemia-associated translocations that create aberrant fusion proteins (e.g., AML1-ETO). Recently, acquired somatic point mutations in the RUNX1/CBFA2/AML1 gene have been described in a subset of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). These point mutations are associated with the loss of normal RUNX1 trans-activation potential and/or altered binding to the non-DNA-binding subunit of core-binding factor, CBFB. Given the importance of core-binding factor in megakaryocytic differentiation and platelet production and the central role of megakaryocytes in the pathophysiology of myelofibrosis with myeloid metaplasia (MMM), and in light of the myelofibrotic phenotype of GATA-1low mice (GATA-1 and RUNX1 are co-expressed and co-operate in megakaryocytic differentiation), we hypothesized that RUNX1 gene mutations might be common in MMM. In addition, we wanted to know whether patients with MDS-associated acquired alpha thalassemia (i.e., ATMDS), a special MDS subgroup with a very high incidence of point mutations in the X-linked ATRX gene, have an especially high incidence of RUNX1 mutations that might account for the more severe hematopoietic defect found in these patients compared with boys with inherited ATR-X syndrome (detailed in Steensma et al, Blood 2004; 103:2019).Methods and Results: We analyzed samples from 26 well-characterized patients with MMM and from 52 with MDS (11 RA, 3 RARS, 8 RCMD, 3 RAEB-1, 7 RAEB-2, 2 MDS in transition to AML-M6, 14 atypical/unclassifable MDS, 4 MDS subtype unknown - including 18 with ATMDS) for RUNX1 point mutations by denaturing high-performance liquid chromatography (DHPLC) followed by subcloning and sequencing of samples exhibiting heteroduplex peaks. We found 5 RUNX1 mutations in MDS patients (9.6%), all of whom had RAEB-2 (4 patients) or a history of treated AML (1 patient), but detected no mutations in MMM patients. All patients with RUNX1 mutations progressed to overt leukemia within 1 year. ATMDS patients did not have an increased risk of RUNX1 point mutations (2/18 patients with mutations, 11.1%) when compared with MDS without thalassemia (3/34 patients, 8.8%; p=0.58). Additionally, 1 MDS patient had a clonally restricted mutation in GATA-1 (c.1066 C>T) (also screened by DHPLC), but this is not predicted to change the GATA-1 amino acid sequence and is likely a random event reflecting the genomic instability characteristic of MDS.Conclusion:RUNX1 point mutations are common in high-risk MDS, but not in MMM, and that ATMDS patients do not have a risk of RUNX1 mutations in excess of that expected for MDS in general.

AML1-FOG2 Fusion Protein in Myelodysplasia.

Core binding factor (CBF) participates in specification of the hematopoietic stem cell and functions as a critical regulator of hematopoiesis. Translocation or point mutation of AML1, which encodes the DNA-binding subunit of CBF, plays a central role in the pathogenesis of acute myeloid leukemia and myelodysplasia. We characterized the translocation t(X;21)(p22.3;q22.1) in a patient with myelodysplasia that fuses AML1 to the novel partner gene Friend of GATA-2 (FOG2). The reciprocal gene fusions AML1-FOG2 and FOG2-AML1 are both expressed. AML1-FOG2, which fuses the DNA-binding domain of AML1 to most of FOG2, represses transcription from the promoter of a hematopoietic target of AML1. AML1-FOG2 retains a motif that recruits the corepressor C-terminal binding protein (CtBP) and both proteins associate in a protein complex. These results suggest a role for CtBP in AML1-FOG2 transcriptional repression and implicate coordinated disruption of the AML1 and GATA developmental programs in the pathogenesis of myelodysplasia.