Menin-MLL inhibitors block oncogenic transformation by MLL-fusion proteins in a fusion partner-independent manner

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.

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FGFR2 fusion proteins drive oncogenic transformation of mouse liver organoids towards cholangiocarcinoma

Journal of Hepatology ◽

10.1016/j.jhep.2021.02.032 ◽

2021 ◽

Author(s):

Giulia Cristinziano ◽

Manuela Porru ◽

Dante Lamberti ◽

Simonetta Buglioni ◽

Francesca Rollo ◽

...

Keyword(s):

Fusion Proteins ◽

Mouse Liver ◽

Oncogenic Transformation ◽

Liver Organoids

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Effective production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

Microbial Cell Factories ◽

10.1186/s12934-020-01502-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Young Su Kim ◽

Hye-Jeong Lee ◽

Man-ho Han ◽

Nam-kyung Yoon ◽

Yeu-chun Kim ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Factors ◽

Growth Factor ◽

Fusion Proteins ◽

Human Growth ◽

Soluble Form ◽

Fusion Partner ◽

Small Protein ◽

E Coli ◽

Tev Protease

Abstract Background Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. Results We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. Conclusions We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

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In Vivo Cytotoxic T Lymphocyte Elicitation by Mycobacterial Heat Shock Protein 70 Fusion Proteins Maps to a Discrete Domain and Is Cd4+ T Cell Independent

Journal of Experimental Medicine ◽

10.1084/jem.191.2.403 ◽

2000 ◽

Vol 191 (2) ◽

pp. 403-408 ◽

Cited By ~ 95

Author(s):

Qian Huang ◽

Joan F.L. Richmond ◽

Kimiko Suzue ◽

Herman N. Eisen ◽

Richard A. Young

Keyword(s):

T Cell ◽

Heat Shock ◽

T Lymphocytes ◽

Heat Shock Protein ◽

Fusion Proteins ◽

Cytotoxic T Lymphocyte ◽

Protein Domain ◽

Cd4 T Cell ◽

Fusion Partner

To gain insights into the mechanisms by which soluble heat shock protein (hsp) fusions can elicit CD8+ cytotoxic T lymphocytes (CTLs) against the fusion partner, mycobacterial (Mycobacterium tuberculosis) hsp70 was dissected to ascertain whether a particular hsp domain is necessary, and knockout mice were used to determine whether the fusion protein's immunogenicity is dependent on CD4+ T lymphocytes. We found that the ability to elicit CD8+ CTLs depends on a discrete 200–amino acid protein domain, indicating that the fusion protein's immunogenicity for CD8+ T cells does not require coupled chaperone function or peptide binding. Further, we found that ovalbumin (OVA).hsp70 fusion protein elicited anti-OVA CD8+ CTLs about equally well in CD4 knockout and wild-type C57BL/6 mice, and also when the hsp70 was of murine (self) origin. The ability of hsp70 fusion proteins to elicit CD4-independent CTL responses suggests that hsp70 fusion proteins may be useful for immunological prophylaxis and therapy against disease in CD4+ T cell–deficient individuals.

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Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Blood ◽

10.1182/blood-2020-138622 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 30-31

Author(s):

Hsiangyu Hu ◽

Nirmalya Saha ◽

Yuting Yang ◽

Sierrah Marie Grigsby ◽

Rolf Marschalek ◽

...

Keyword(s):

Cell Growth ◽

Acute Leukemia ◽

Transcriptional Activation ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Leukemic Cells ◽

Fusion Partner ◽

Wild Type

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

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Rabaptin-5 is a novel fusion partner to platelet-derived growth factor β receptor in chronic myelomonocytic leukemia

Blood ◽

10.1182/blood.v98.8.2518 ◽

2001 ◽

Vol 98 (8) ◽

pp. 2518-2525 ◽

Cited By ~ 97

Author(s):

Magnus K. Magnusson ◽

Kristin E. Meade ◽

Kevin E. Brown ◽

Diane C. Arthur ◽

Lisa A. Krueger ◽

...

Keyword(s):

Growth Factor ◽

Fusion Protein ◽

Tyrosine Kinase ◽

Fusion Proteins ◽

Growth Regulation ◽

Chronic Myelomonocytic Leukemia ◽

Platelet Derived Growth Factor ◽

Fusion Partner ◽

Β Receptor ◽

Myelomonocytic Leukemia

Abstract Chromosomal translocations involving the platelet-derived growth factor β receptor (PDGFβR) gene have been reported in some patients with chronic myelomonocytic leukemia (CMML). The resultant fusion proteins have constitutive PDGFβR tyrosine kinase activity, but the partner genes previously reported(tel, Huntingtin interacting protein 1[HIP-1], H4/D10S170) have poorly understood roles in the oncogenic activity of the fusion proteins. A novel PDGFβR fusion protein has been characterized in a patient with CMML and an acquired t(5;17)(q33;p13). Southern blot analysis on patient leukemia cells demonstrated involvement of the PDGFβR gene. Using 5′ rapid amplification of complementary DNA ends–polymerase chain reaction (RACE-PCR) on patient RNA, rabaptin-5 was identified as a novel partner fused in-frame to thePDGFβR gene. The new fusion protein includes more than 85% of the native Rabaptin-5 fused to the transmembrane and intracellular tyrosine kinase domains of the PDGFβR. Transduction with a retroviral vector expressing rabaptin-5/PDGFβRtransformed the hematopoietic cell line Ba/F3 to growth factor independence and caused a fatal myeloproliferative disease in mice. Rabaptin-5 is a well-studied protein shown to be an essential and rate-limiting component of early endosomal fusion through interaction with the Ras family GTPases Rab5 and Rab4. The fusion protein includes 3 of 4 coiled-coil domains (involved in homodimerization of native rabaptin-5), 2 caspase-3 cleavage sites, and a binding site for the tumor suppressor gene tuberin (tuberous sclerosis complex-2). Early endosomal transport is critical in regulation of various growth factor receptors, through ligand-induced clathrin-mediated endocytosis, and thus this new fusion protein links together 2 important pathways of growth regulation.

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Efficient production of human growth factors in Escherichia coli by fusing with small protein 6HFh8

10.21203/rs.3.rs-72264/v2 ◽

2021 ◽

Author(s):

Young Su Kim ◽

Hye-Jeong Lee ◽

Man-ho Han ◽

Nam-kyung Yoon ◽

Yeu-chun Kim ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Factors ◽

Growth Factor ◽

Fusion Proteins ◽

Human Growth ◽

Soluble Form ◽

Fusion Partner ◽

Small Protein ◽

E Coli ◽

Tev Protease

Abstract Background: Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms.Results: We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli. The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (>99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents.Conclusions: We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli, with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

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Targeting LEDGF Interactions in MLL Leukemia

Blood ◽

10.1182/blood.v118.21.2500.2500 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2500-2500

Author(s):

Tomasz Cierpicki ◽

Shihan He ◽

Trupta Purohit ◽

Marcelo Murai ◽

Thomas Hartley ◽

...

Keyword(s):

Small Molecules ◽

Fusion Proteins ◽

High Throughput Screening ◽

Hox Genes ◽

Conflicts Of Interest ◽

Leukemia Cells ◽

Screening Methods ◽

Fusion Partner ◽

Acute Leukemias ◽

N Terminus

Abstract Abstract 2500 Chromosomal translocations of MLL (Mixed Lineage Leukemia) gene result in aggressive acute leukemias, affecting both children and adults. Fusion of MLL to one of more than 60 partner genes results in MLL fusion oncoproteins which upregulate expression of Hox genes required for normal blood cell development, ultimately leading to development of acute leukemia. Regardless of the fusion partner, the presence of MLL translocations is associated with early relapse and poor prognosis. Survival rates are particularly low for infants and there is a pressing need for the development of targeted therapies against leukemias with MLL translocations. The oncogenic activity of MLL fusion proteins is dependent on association with LEDGF (lens epithelium-derived growth factor) and menin, both of which interact with the N-terminus of MLL retained in all MLL fusion proteins. LEDGF is a chromatin-associated protein, which interacts conjointly with MLL and menin on the chromatin of the cancer associated genes, and both interactions are required for the MLL-mediated leukemogenesis and misregulation of HOXA9 expression. Therefore, LEDGF functions as an essential oncogenic cofactor in MLL related leukemias, and may represent a valuable molecular target for therapeutic intervention with small molecules. We have performed rigorous biophysical and biochemical studies and revealed that LEDGF is involved in simultaneous interaction with menin and with the N-terminus of MLL. Interestingly, the association of LEDGF with the menin-MLL complex has relatively low affinity which limits the application of conventional screening methods for lead identification. To develop small molecule inhibitors targeting LEDGF interactions we have employed two strategies: Fragment Based Drug Discovery (FBDD) approach and High Throughput Screening (HTS). We have identified several compounds that bind directly to LEDGF. By applying NMR spectroscopy we discovered that these compounds interact with the menin binding site on LEDGF. Then we have assessed the activity of these compounds using a broad range of cell based assays. We found that compounds targeting LEDGF specifically inhibit proliferation of the MLL leukemia cells without affecting the non-MLL leukemia cells. They also induce apoptosis and differentiation of MLL leukemia cells as assessed by increased expression of CD11b differentiation marker and substantial change in morphology of these cells. Furthermore, these compounds reduce transforming properties of MLL fusion proteins and downregulate expression of Hoxa9 and Meis1 genes confirming a highly specific mode of action. Overall, our results demonstrate that targeting of LEDGF by small molecules is feasible and may results in development of potent inhibitors of LEDGF interaction with menin and MLL fusion proteins in leukemias with MLL rearrangements. Such compounds might provide a new therapeutic approach for the treatment of MLL-rearranged leukemias. Disclosures: No relevant conflicts of interest to declare.

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Novel SWI/SNF Chromatin-Remodeling Complexes Contain a Mixed-Lineage Leukemia Chromosomal Translocation Partner

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2942-2952.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2942-2952 ◽

Cited By ~ 60

Author(s):

Zuqin Nie ◽

Zhijiang Yan ◽

Everett H. Chen ◽

Salvatore Sechi ◽

Chen Ling ◽

...

Keyword(s):

Chromatin Remodeling ◽

Fusion Proteins ◽

Chromosomal Translocation ◽

Chromosomal Translocations ◽

Fusion Partner ◽

Regulate Gene Expression ◽

Downstream Target ◽

Chromatin Remodeling Complexes ◽

Gain Access ◽

Common Target

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

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The Leucine Zipper Motif of the Drosophila AF10 Homologue Can Inhibit PRE-Mediated Repression: Implications for Leukemogenic Activity of Human MLL-AF10 Fusions

Molecular and Cellular Biology ◽

10.1128/mcb.23.1.119-130.2003 ◽

2003 ◽

Vol 23 (1) ◽

pp. 119-130 ◽

Cited By ~ 18

Author(s):

Laurent Perrin ◽

Sébastien Bloyer ◽

Conchita Ferraz ◽

Namita Agrawal ◽

Pradip Sinha ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Transcriptional Repression ◽

Fusion Proteins ◽

Leucine Zipper ◽

Full Length ◽

Fusion Partner ◽

Leucine Zipper Motif ◽

Dominant Mutation ◽

Leucine Zipper Domain ◽

Phd Domain

ABSTRACT In a screen for Drosophila genes that interfere with transcriptional repression mediated by the Polycomb group of genes, we identified a dominant mutation affecting the Alhambra (Alh) gene, the fly homologue of the human AF10 gene. AF10 has been identified as a fusion partner of both MLL and CALM in infant leukemias. Both fusion proteins retain the leucine zipper domain of AF10 but not its PHD domain. We show here that, while the full-length ALH protein has no activity on Polycomb group-responsive elements (PREs), overexpression of the isolated ALH leucine zipper domain activates several PREs. Within the ALH full-length protein, the PHD domain inhibits the PRE deregulation mediated by the leucine zipper domain. This deregulation is conserved in the human AF10 leucine zipper domain, which confers the same activity on an oncogenic MLL-AF10 fusion protein expressed in Drosophila melanogaster. These data reveal new properties for the leucine zipper domain and thus might provide new clues to understanding the mechanisms by which AF10 fusion proteins in which the PHD domain is lost might trigger leukemias in humans.

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