TW201910348A - TIFA antagonists and their use for treating diseases - Google Patents

Abstract

The present invention relates to use of TRAF-interacting protein with an FHA domain (TIFA) antagonists for treating diseases. Particularly, the present invention relates to an isolated peptide fragment from TIFA which acts as a dominant negative inhibitor of TIFA and is effective in treating cancer or an inflammatory disorder. The present invention also relates to a method for predicting cancer prognosis based on the TIFA expression level in a subject in need. The present invention further relates to a method for treating a disease via TIFA silencing.

Description

TIFA antagonists and their use in treating diseases

The present invention relates to the use of a TRAF interacting protein (TIFA) antagonist having a FHA domain for the treatment of a disease. In particular, the present invention relates to isolated peptide fragments from TIFAs that act as dominant negative inhibitors of TIFAs and are effective in treating cancer or inflammatory diseases. The invention also relates to methods of predicting cancer prognosis based on TIFA performance of an individual in need thereof. The invention further relates to methods of treating diseases via TIFA silencing.

Cancer is one of the leading causes of death worldwide. Chemotherapy and molecular target treatment resistance are the main problems facing current cancer research and treatment. For example, acute myeloid leukemia (AML) is a clonal hematologic malignancy with great variability in clinical features, pathogenesis, and treatment outcomes. This malignant disease is caused by abnormal differentiation of hematopoietic precursor cells, which causes them to lose their ability to respond to proliferative regulators. The standard treatment for patients with AML is initially induced by chemotherapy, followed by post-remission therapy, with additional chemotherapy cycles or allogeneic stem cell transplantation to prevent recurrence (1). Despite significant advances, current AML treatments offer only limited survival benefits, rather than providing a completely satisfactory response, presumably due to chemotherapy resistance and disease recurrence (1, 2). In order to reduce the recurrence rate and improve the efficacy of treatment, it is urgent to find novel targets (3, 4).

Nuclear factor-κB (NF-κB) controls the immune response of various aspects and preferentially regulates cell survival, proliferation, and differentiation (5). Recent evidence has attributed an increasing number of malignancies to abnormal activation of NF-κB, which interacts with other signaling molecules and pathways (6) and is therefore considered a risk factor for poor prognosis in several cancers including leukemia (7). It is agreed that sustained activation of NF-κB protects tumor cells from apoptotic stimuli and promotes their resistance to chemotherapy and ionizing radiation (8), presumably through transcriptional activation of the anti-apoptotic/promoting factor Bcl-2. With Bcl-X _L (9). According to this view, the role of NF-κB in the process of leukemia production is also applied to AML (10), and the inhibition of NF-κB in this hematopoietic malignant tumor again achieves chemical sensitivity, which may be due to the weakening of pro-survival response and promotion Due to activation of the death signal (11). These observations focused on the NF-κB signaling axis as a promising therapeutic target (12).

The Aurora family of serine/threonine kinases promotes tumor proliferation during mitosis by regulating chromosome alignment, isolation, and cytokinesis (13) and is considered an anti-cancer target (14). It has been reported that the Aurora A line of bone marrow (BM) monocytes in AML patients is upregulated (15) and is shown to be associated with adverse cytogenetic risks and higher white blood cell (WBC) counts (16). Furthermore, it has been shown that Aurora A promotes chemical resistance of cancer cells in vitro and in vivo by reducing chemotherapy-induced apoptosis by activating the NF-κB signaling pathway (17). With support, treatment with the Alorora A inhibitor VX680 was reported (18), which shows clinical utility for chronic myelogenous leukemia (CML) (19), increasing the ratio of Bax/Bcl-2, suggesting that cells are promoted Apoptosis.

TIFA is a TRAF-interacting protein that is a relatively novel participant in the NF-κB signaling pathway. Previous studies by the inventors have found that TIFA overexpression promotes NF-κB activity in a TNF-α-dependent manner, and TIFA silencing attenuates this TNF-α-stimulated NF-κB signaling (20). Mechanistically, this signal axis initiates TNF-α-dependent phosphorylation of threonine 9 (pThr9) on TIFA, which interacts with the forkhead-associated (FHA) domain of TIFA To promote oligomerization of TIFA dimers (20, 21). TIFA oligopolymerization then supports a high-ordered architecture of the TRAF family of components, such as TNF receptor-associated factor 2 (TRAF2) or TRAF6, and modulates the ubiquitination of TRAF6 to activate The IκB kinase (IκK) complex whereby IκB is subsequently phosphorylated and undergoes ubiquitin-dependent cleavage, allowing nuclear translocation of NF-κB to transactivate downstream factors (22). In addition to TNF-α stimulation, it has been shown that such TIFA phosphorylation-dependent oligopolymerization is also driven by Gram-negative bacteria-derived monosaccharide heptose-1,7-bisphosphate (HBP) to activate innate Immunization (23), and this TIFA mediates the innate immune response, which is based on the assembly of NLRP3 inflammasome at the endogenous sheer stress (24), indicating that TIFA regulates innate immune responses and inflammation. The important role of the reaction.

The FHA domain consists of about 80-120 amino acids, and it is known to specifically recognize phosphorylated threonine (pThr) to function as a signaling. The sequence homology in the different FHA-containing proteins is quite low, however, the overall structural architecture of the FHA domain is quite retained, with two b-strand b-sheaths forming a beta sandwich structure. The b-strands are connected by loops, and although the length varies widely, the function recognizes the specific pThr ligand. The FHA-pThr binding function can regulate a variety of biological functions ranging from DNA damage repair, cell cycle checkpoints, and even message transmission. The specificity, biological function, structure, and mechanism of the FHA domain are extracted from recent review literature (25).

In the present invention, it has been unexpectedly discovered that the TIFA peptide fragments provided herein can act as a dominant negative inhibitor of TIFA to block or attenuate the activation of interleukin-stimulated NF-κB. In particular, the TIFA peptide fragment contains a dimeric core segment that links the N-terminal Thr9 segment or the C-terminal TRAF6/TRAF 2 interaction segment to effectively treat cancer or inflammatory diseases via targeting TIFAs. Block or attenuate the activation of interleukin-stimulated NF-κB. In particular, it was found that the expression of TIFA peptide fragments can antagonize the growth of cancer cells stimulated by inflammatory mediator secretion and enhance chemical toxicity/reduction of chemical resistance to achieve therapeutic effects. The invention also provides a treatment of a disease/condition that is silenced via TIFA, and a method of predicting the prognosis of a cancer based on the amount of TIFA expression in an individual in need thereof.

Thus, in one aspect, the invention provides a TIFA inhibitor comprising an isolated TIFA peptide fragment comprising a dimeric core peptide segment, the N-terminus binding to a Thr9 peptide segment or the C-terminus binding to a TRAF6/TRAF2 interaction a peptide segment, wherein (i) the Thr9 peptide segment comprises an N-terminal phosphorylation/oligopolymerization motif MX ₁ X ₂ FEDX ₃ DTX ₄ EX ₅ X ₆ T, the sequence of which is set forth in SEQ ID NO: 13 Where X ₁ is serine (S) or threonine (T), X ₂ is serine (S), threonine (T), aspartic acid (N), X ₃ is propylamine Acid (A) or proline (V), X ₄ is glutamic acid (E) or glutamine proline (Q), X ₅ is threonine (T) or methionine (M), and X ₆ is valine (V) or leucine (L); (ii) the dimerized core peptide segment comprises six b chains comprising (a) a b3 chain having the b3 motif VKFG; b) a b4 chain having a b4 motif YX ₁ F, wherein X ₁ is threonine (T) or isoleucine (I); (c) a b5 chain having a b5 motif QFX ₁ LX ₂ X ₃ F, wherein X ₁ is serine (S), valine (V), or alanine (A), X ₂ is glutamine proline (Q) or histidine (H), and X ₃ is leucine (L), proline (P), or guanamine Acid (V); (d) a b6 chain having a b6 motif SFEIKN; (e) a b7 chain having a b7 motif LIV; and (f) a b8 chain having a b8 motif X ₁ X ₂ L, wherein X ₁ is arginine (R), glutamine proline (Q), or lysine (K), and X ₂ is glutamic acid (E) or threonine (T); wherein b3 Each of the b8 strands is sequentially linked from the N-terminus to the C-terminus to the next using a plurality of internal loop sequences; and (iii) the TRAF6/TRAF2 interaction peptide segment comprises four b-chains, including a) a b9 chain having the b9 motif LX ₁ KX ₂ D, wherein X ₁ is aspartic acid (N) or histidine (H), and X ₂ is methionine (M), white Amine acid (L), valine acid (V), or isoleucine (I); (b) a b10 chain having b10 motif X ₁ CX ₂ X ₃ RF, wherein X ₁ is arginine ( R) or from the amine acid (K), X ₂ is methionine (M) or leucine (L), and X ₃ is valine (V), leucine (L), or isoleamine Acid (I); (c) a b11 chain having b11 motif YQX ₁ LX ₂ X ₃ X ₄ E wherein X ₁ is phenylalanine (F) or isoleucine (I), and X ₂ is methyl sulfide leucine (M), leucine (L), valine (V), or isoleucine (I), X ₃ is glutamic acid (E) or bran Acid amide (Q), and X ₄ is lysine (K) or arginine (R &lt); and (d) a chain b12, b12 die body having _{X 1 FX 2 X 3 X 4} FX 5 X 6 Wherein X ₁ is phenylalanine (F) or serine (S), X ₂ is glutamic acid (E) or glutamine proline (Q), X ₃ is threonine (T) or isoleamine The acid (I), X ₄ is glutamine proline (Q), histidine (H), or glutamic acid (E), and X ₅ is isoleucine (I), valine (V), Serine (S), or phenylalanine (F), and X ₇ is leucine (L), methionine (M), or phenylalanine (F); wherein each of the b9 to b12 chains A plurality of internal loop sequences are sequentially linked from the N-terminus to the C-terminus to the next; and a C-terminal loop sequence is ligated to the B-terminus of the b12 chain; wherein the isolated TIFA peptide fragment does not contain the Thr9 peptide segment and the TRAF6 /TRAF2 interacts with both peptide segments.

In a specific embodiment, the N-terminal Thr9-phosphorylation motif system is located at positions 1-14 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b3 modular system is located at position 47-50 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b4 modular system is located at positions 58-60 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b5 modular system is located at positions 69-75 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b6 modular system is located at positions 84-89 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b7 modular system is located at positions 96-98 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b8 modular system is located at positions 101-103 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b9 modular system is located at positions 106-110 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b10 modular system is located at positions 114-119 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b11 modular system is located at positions 122-129 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b12 modular system is located at positions 136-143 corresponding to SEQ ID NO: 1.

In some embodiments, the Thr9 peptide segment further comprises two b-chains comprising (a) a b1 chain having a b1 motif X ₁ LX ₂ X ₃ TX ₄ Y, wherein X ₁ is a cysteamine Acid (C) or serine (S), X ₂ is glutamine proline (Q) or histidine (H), X ₃ is methionine (M), isoleucine (I), Leucine (L) or valine (V), and X ₄ is valine (V), isoleucine (I) or leucine (L); and (b) a b2 chain having The b2 motif is located at position X ₁ X ₂ X ₃ P, wherein X ₁ is glutamic acid (E) or aspartic acid (D), and X ₂ is lysine (K) or threonine (T), and X ₃ is leucine (L) or phenylalanine (F), wherein the b1 chain is sequentially linked from the N-terminus to the C-terminus to the b2 chain using an internal loop sequence.

In a specific embodiment, the b1 modular system is located at position 15-21 corresponding to SEQ ID NO: 1.

In a specific embodiment, the b2 modular system is located at positions 39-42 corresponding to SEQ ID NO: 1.

In some embodiments, the C-terminal loop sequence of the TRAF6/TRAF2 interaction peptide segment is at position 144-184 corresponding to SEQ ID NO: 1.

In some embodiments, the Thr9 peptide segment comprises an amino acid sequence corresponding to positions 1-45 of SEQ ID NO: 1, or an amino acid sequence, which is at least 70% identical to the sequence degree. In some examples, the Thr9 peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-34.

In some embodiments, the dimeric core peptide segment comprises an amino acid sequence corresponding to positions 46-103 of SEQ ID NO: 1, or an amino acid sequence having at least 70 % is equal. In some examples, the dimeric core peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-46.

In some embodiments, the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence corresponding to position 104-184 of SEQ ID NO: 1, or an amino acid sequence, and is associated with the sequence At least 70% identical. In some examples, the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-58.

In some embodiments, the C-terminal loop sequence of the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence corresponding to position 144-184 of SEQ ID NO: 1, or an amino acid sequence, It is at least 70% identical to the sequence. In some examples, the C-terminal loop sequence of the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-70.

In a specific embodiment, the TIFA peptide fragment of the present invention is an F1-F2 peptide fragment comprising a Thr9 peptide segment and a dimeric core peptide segment, wherein the Thr9 peptide segment C-terminus is linked to a dimerization The core peptide segment is N-terminal and lacks the TRAF 6 interaction peptide segment. In some examples, the F1-F2 peptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-82.

In a specific embodiment, the TIFA peptide fragment of the present invention is an F2-F3 peptide fragment comprising a dimerized core peptide segment and a TRAF 6 interaction peptide segment, wherein the second polymer core peptide segment C-terminal The line is linked to the N-terminus of the TRAF 6 interaction peptide segment and lacks the Thr9 peptide segment. In some examples, the F2-F3 peptide fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-94.

In another aspect, the invention provides a recombinant nucleic acid comprising a nucleotide sequence encoding any of the peptides described herein. Such a nucleic acid can be a vector comprising the coding sequences described herein. In some examples, the vector is a viral vector.

Any of the peptides or nucleic acids can be tuned to form a composition, for example, a pharmaceutical composition, which further comprises a physiologically acceptable carrier.

Also provided herein is a method for treating a disease or condition associated with TIFA activation comprising administering to an individual an agent of an effective amount of a peptide or encoding any of the nucleic acids as described herein, or a combination thereof Things. The disease or condition associated with TIFA activation can be a cancer or inflammatory disease associated with TIFA activation, or an interleukin-stimulated NF-κB activation associated with cancer or an inflammatory disease. In some embodiments, the individual has cancer and the peptide fragment, the encoding nucleic acid, and the composition comprising the peptide or encoding nucleic acid are administered in combination with an anti-cancer therapy. Specifically, the amount of the peptide fragment, the encoding nucleic acid, and the composition administered is effective to enhance the cytotoxicity of the anticancer treatment. In some embodiments, the anti-cancer treatment involves administration of an anti-cancer agent or radiation exposure. Anticancer agents include, but are not limited to, chemotherapeutic agents and molecular targeted drugs. Examples of anticancer agents are etoposide, idarubicin, cytarabine, sorafenib, regorafenib, bleomycin. ), bortezomib, bubuan sulfate, and metosulfam.

The present inventors have also discovered that TIFA protein expression is associated with prognosis of leukemia. In particular, the present invention provides a method for predicting the prognosis of leukemia comprising collecting a blood sample from a leukemia patient, measuring the amount of TRAF-interacting protein in the blood sample by the FHA domain (TIFA), and determining the amount of TIFA in the blood sample. Determine the prognosis of leukemia in patients, in which the increase in TIFA in blood samples represents a poor prognosis.

Further provided is a method for inhibiting the activation of interleukin-stimulated NF-κB in a subject comprising administering to the individual an effective amount of a TIFA antagonist. Examples of TIFA antagonists include interfering nucleic acids that target TIFAs or TIFA dominant negative inhibitors, such as the TIFA peptide fragments described herein. In some instances, the individual to be treated can be a human patient with a disease or condition associated with interleukin-stimulated NF-κB activation. Such diseases or conditions may be cancers or inflammatory diseases due to interleukin-stimulated NF-κB activation.

In particular, the cancers to be treated according to the invention are associated with chronic inflammatory or inflammatory diseases. Specific examples of the present invention for treating cancer include, but are not limited to, hematological cancers (such as leukemia or lymphoma), liver cancer, gastrointestinal cancer (such as gastric cancer or rectal cancer), lung cancer, prostate cancer, pancreatic cancer, ovarian cancer, and breast cancer. . In some embodiments, the TIFA antagonists described herein can be administered in combination with an anticancer agent that is treated alone or in combination with an anti-inflammatory agent, such as enanercept (antanercept), anti-survival agent, as compared to an anticancer agent. Target drugs, such as ABT-263, and NF-kB antibody agents, such as bortezomib, provide a synergistic effect in the treatment of cancer.

In some embodiments, the inflammatory disease is hepatitis, atherosclerosis, pulmonary hypertension, cardiomyopathy, rheumatoid arthritis, inflammatory bowel disease, and Fabry disease.

Also disclosed within the scope of the invention is a pharmaceutical composition for treating a disorder associated with the activation of interleukin-stimulated NF-κB, comprising any of the peptides described herein or a nucleic acid encoding the same, and pharmaceutically acceptable Carrier. The invention further describes the use of a peptide or a nucleic acid encoding the same as described herein to produce an agent for use in a method described herein, such as a disease associated with the activation of interleukin-stimulated NF-κB.

The following description sets forth the details of one or more embodiments of the invention. Other features and advantages of the present invention will be apparent from the following detailed description of the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

The singular forms "a", "the", and "the" Thus, for example, reference to "a component" shall include a plurality of such components and their equivalents as are known to those skilled in the art.

The words "comprising" or "comprising" are used to include or encompass the meaning of the meaning of one or more features, components, or components. Words such as "including" or "including" include the words "composition" or "consisting of".

As used herein, "polypeptide" refers to a polymer composed of amino acid residues linked by a peptide bond. The word "peptide" means a short multi-peptide consisting of a linked amino acid, for example, 200 amino acids or less, 175 amino acids or less, and 150 amino acids or less, such as 140 or less. 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, or 40 or less amino acid lengths.

The words "about" or "approximately" as used herein mean the degree of acceptable deviation understood by one of ordinary skill in the art, which may vary to some extent depending on the use herein. In general, "about" or "approximate" may refer to values in the range of ±10% near the value used.

As used herein, "corresponding to" means the position of a residue in a protein or peptide, or a similar, homologous, or equivalent position in a protein or peptide.

As used herein, the term "substantially identical" means that the second sequence has more than 70%, preferably 75%, more preferably 80%, even more preferably 85%, even more preferably 90%, and preferably It is 95% or 100% homologous.

TIFA protein

As used herein, "TRAF-interacting fork-binding protein A (FHA)", "TRAF-interacting protein with FHA domain", "TIFA", "TIFA protein", and "TIFA multi-peptide" are used. Can be used interchangeably. The TIFA proteins described herein may include amino acid sequences as shown in SQE ID NO: 1 (human TIFA), and may also include amino acid sequences (i) that are substantially identical to those described herein. An amino acid sequence of any TIFA protein; and (ii) encoded by a nucleic acid sequence capable of hybridizing to at least moderately stringent conditions to any nucleic acid sequence encoding any TIFA protein shown herein, or capable of at least Any nucleic acid sequence encoding any TIFA protein shown herein is hybridized under moderate stringency conditions, but using a synonymous codon (eg, the codon does not have the same nucleotide sequence but encodes the same amino acid). The TIFA proteins described herein include human TIFAs and their homologues derived from vertebrates, and in particular, homologs derived from mammals. In particular, the TIFA proteins described herein include a human-derived TIFA amino acid sequence (SEQ ID NO: 1) derived from other mammalian TIFA amino acid sequences (SEQ ID NOs: 2 to 12), Having at least 70%, at least 75%, or at least 80% SEQ ID NO: 1 identity (see Figure 7). The TIFA proteins described herein further comprise any recombinantly (genetically engineered) TIFA multipeptide encoded by a cDNA copy encoding a native polynucleotide sequence of TIFA.

Structurally, the TIFA protein used herein, from N-terminus to C-terminus, includes (i) an F1 domain, ie, a Thr9 peptide segment, comprising an N-terminal phosphorylation/oligomerization motif And optionally a b1-b2 chain; (ii) an F2 domain, that is, a di-polymeric core peptide segment comprising six b-strands comprising a b3-b8 chain; and (iii) an F3 domain That is, a TRAF6/TRAF2 interaction peptide fragment comprising four b-strands including a b9-b12 chain and a C-terminal loop sequence. Table 1 describes the F1, F2, and F3 domains and their structural features.

Each of the b strands can be ligated to the next in sequence from the N-terminus to the C-terminus, which can vary in length and have a relatively low degree of sequence homology. Specifically, each of the aforementioned TIFA protein motifs is located substantially at the position where the N-terminal Thr9-phosphorylation motif system is located at positions 1-14 corresponding to SEQ ID NO: 1; the b1 motif system is located corresponding to SEQ ID NO: 1 position 15-21; b2 modular system is located corresponding to position 39-42 of SEQ ID NO: 1; b3 modular system is located corresponding to position 47-50 of SEQ ID NO: 1; The b4 motif system is located corresponding to positions 58-60 of SEQ ID NO: 1; the b5 motif system is located at position 69-75 corresponding to SEQ ID NO: 1; the b6 motif system is located corresponding to SEQ ID NO: 1 Positions 84-89; b7 mode system is located at position 96-98 corresponding to SEQ ID NO: 1; b8 mode system is located at position 101-103 corresponding to SEQ ID NO: 1; b9 mode system is located at phase Corresponding to position 106-110 of SEQ ID NO: 1; b10 is located at position 114-119 corresponding to SEQ ID NO: 1; b11 is located at position 122-129 corresponding to SEQ ID NO: 1. And/or the b12 modular system is located at a position corresponding to positions 136-143 of SEQ ID NO: 1. In some embodiments, the C-terminal loop sequence of the TRAF6/TRAF2 interaction peptide segment is located corresponding to positions 144-184 of SEQ ID NO: 1. Furthermore, each of the F1, F2, and F3 domains can be located substantially at the position: the F1 domain corresponds to position 1-45 of SEQ ID NO: 1; the F2 domain corresponds to position 46 of SEQ ID NO: 1 103; and/or the F3 domain corresponds to positions 104-184 of SEQ ID NO: 1.

Functionally, TIFA proteins can be activated by phosphorylation after stimulation with interleukins, bacterial-derived sugars (23, 26), or inflammatory conditions (24) (eg, TNF-α), thereby driving TIFA oligomerization, which results in TRAF6 or TRAF2 interactions, including NF-κB activation. In particular, it is disclosed herein that a dimeric core segment (F2 domain) can form an intrinsic dimer between two TIFA monomers. In addition, the Thr9 peptide segment (F1 domain) can be phosphorylated at the Thr9 residue of the N-terminal phosphorylation/oligomerization motif (SEQ ID NO: 13) using a kinase such as aolola A kinase. And, once phosphorylation occurs, the phosphorylated Thr9 residue of the N-terminal phosphorylation/oligopolymerization motif of the F1 domain may be derived from the pThr-recognition motif located in the F2 domain (eg, at position 51 of SEQ ID NO: 1 -89, see Figure 7-8) Identification, thus driving the oligomerization of TIFAs (eg, a TIFA dimer via the F1 domain N-terminal phosphorylation/oligopolymerization motif and F2 domain pThr recognition motif) Intermolecular interactions between the bodies, combined with additional one or more TIFA dimers). TIFA oligomers formed from at least two TIFA dimers can subsequently interact with tumor necrosis factor receptor-associated factor 6 (TRAF6) or TRAF2 via TRAF6/ of each TIFA monomer of TIFA oligomers The TRAF2 interaction peptide fragment (F3 domain) is carried out to promote TRAF2/TRAF6 oligomerization. The functional characteristics of each of the F1, F2, and F3 domains can be determined by methods known in the art. For example, Thr9 phosphorylation can be assayed using the in vitro kinase assay (20), which can be determined by non-denaturing PAGE (native PAGE) (20, 23, 24) and TIFA via the dimerization of the F2 domain. The interaction of oligomers with TRAF6 or TRAF2 via the F3 domain can be confirmed by immunoprecipitation.

In some embodiments, the Thr9 peptide segment (F1 domain) comprises an amino acid sequence corresponding to positions 1-45 of SEQ ID NO: 1 (ie, SEQ ID NO: 23), or The amino acid sequence is at least 70%, 80%, or 90% identical to the sequence. In some examples, the Thr9 peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-34.

In some embodiments, the polymeric core peptide segment comprises an amino acid sequence corresponding to positions 46-103 of SEQ ID NO: 1 (ie, SEQ ID NO: 35), or an amino acid sequence possesses The sequence is at least 70%, 80%, or 90% identical. In some examples, the dimeric core peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-46.

In some embodiments, the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence corresponding to positions 104-184 of SEQ ID NO: 1 (ie, SEQ ID NO: 47), or an amino acid. The sequence is at least 70%, 80%, or 90% identical to the sequence. In some examples, the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-58.

In some embodiments, the C-terminal loop sequence of the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence corresponding to position 144-184 of SEQ ID NO: 1, or an amino acid sequence, It is at least 70% identical to the sequence. In some examples, the C-terminal loop sequence of the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59-70.

2. TIFA Peptide fragment and composition containing the same

A TIFA peptide fragment (or interchangeably termed a TIFA fragment) refers to a non-naturally occurring truncated fragment derived from TIFA, or a functional variant thereof. Such a peptide differs from a naturally occurring full-length TIFA protein, at least such that the peptide fragment can serve as a dominant negative peptide of the full-length activated TIFA protein, which competes with the activated TIFA protein, thereby reducing the effect of the activated TIFA protein. . Specifically, TIFA peptide fragments have an inhibitory effect on interleukin-induced NF-κB activation. Thus, TIFA peptide fragments can be considered as TIFA antagonists for inhibiting TIFA-mediated signaling pathways, thereby facilitating the treatment of diseases and conditions associated with abnormal activation of TIFAs.

According to the present invention, the TIFA peptide fragment as a dominant negative peptide of the full-length activated TIFA protein must include a dimerized core peptide segment (F2 domain) which can be linked to the Thr9 peptide segment at the N-terminus (F1 structure) Domain), or the TRAF6/TRAF2 interaction peptide segment (F3 domain) is ligated at the C-terminus. In a specific embodiment, the TIFA peptide fragment of the invention comprises a Thr9 peptide segment (F1 domain) and a dimeric core peptide segment (F2 domain), wherein the C-terminus of the Thr9 peptide segment is linked to two The N-terminus of the core peptide segment is polymerized and the TRAF6/TRAF2 interaction peptide segment (F3 domain) is absent. In some examples, a TIFA peptide fragment of the invention is selected from the group consisting of SEQ ID NOs: 71-82.

In another specific embodiment, the TIFA peptide fragment of the invention comprises a dimeric core peptide segment (F2 domain) and a TRAF6/TRAF2 interaction peptide segment (F3 domain), wherein the dimeric core peptide region The C-terminus of the segment is ligated to the N-terminus of the TRAF6/TRAF2 interaction peptide segment, which lacks the Thr9 peptide segment (F1 domain). In some examples, a TIFA peptide fragment of the invention is selected from the group consisting of SEQ ID NOs: 83-94.

In additional specific embodiments, the TIFA fragments described herein can be variants of TIFA fragments that have one or more mutations. It will be appreciated that the multi-peptide may have a limited number of alterations or modifications which may be made within a particular portion of the peptide and are not related to its activity or function, and still produce an equivalent or similar biological activity or function to an acceptable degree. Variant. In particular, the activity exhibited by the TIFA fragments of the invention reduces TIFA activation and subsequent NF-κB activation. Thus, the amino acid position necessary or non-essential for the activity of the TIFA fragment of the invention can be discerned, which reduces TIFA activation/NF-κB activation. For TIFA activation assays, for example, in vitro kinase assays, non-denaturing PAGE, immunoprecipitation, or luciferase reporter gene assays can be utilized. Stimulation of cells with inflammatory mediators or pathological stress results in activation of NF-κB, which can be determined via luciferase assay using, for example, the NF-κB reporter gene system. If the degree of TIFA activation/NF-κB activation declines when overexpressing a test TIFA fragment, the test TIFA fragment can be considered to be effective in reducing TIFA activation/NF- compared to the non-overexpression test TIFA fragment. κB activation. In some instances, the amino acid residue is mutated to a retentive amino acid substitution, meaning that the amino acid residue chemical structure is similar to another amino acid residue, and for multi-peptide function, activity, or other organism Effects such as effects have little or no effect. Variants can be prepared by methods known to those of ordinary skill in the art for altering multi-peptide sequences, such as references using such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. For example, a retaining substitution of an amino acid includes an amino acid substitution performed in the following groups: (i) A, G; (ii) S, T; (iii) Q, N; (iv) E, D; (v) M, I, L, V; (vi) F, Y, W; and (vii) K, R, H.

The TIFA fragments of the present invention can be produced by chemical synthesis using conventional techniques of protein chemistry, such as solid phase synthesis or synthesis in homogeneous solutions.

Alternatively, the TIFA fragments of the invention can be prepared by recombinant techniques. In this aspect, a recombinant nucleic acid comprising a nucleotide sequence encoding a TIFA fragment of the invention and a host cell comprising the recombinant nucleic acid are provided. Host cells can be cultured under suitable conditions to express the multi-peptide of interest. The performance of the multi-peptide may be in the form of a composition such that it is continuously produced or inducible, which requires stimulation to initiate performance. In the case where the performance can be induced, an inducing substance such as isopropyl β-D-1-thiogalactoside (IPTG) or methanol can be added to the medium, for example, to initiate protein production. Polypeptides can be recovered and purified from host cells using a variety of techniques well known in the art, such as chromatography, such as FPLC or affinity columns.

The terms "polynucleotide" or "nucleic acid" may refer to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs, including those having non-naturally occurring nucleotides. The synthesis of polynucleotides can be utilized, for example, as an automated DNA synthesizer. The "nucleic acid" B dictionary type means a large polynucleotide. It should be understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), it also includes an RNA sequence (ie, A, U, G, C), wherein "U" is substituted. "T". The word "cDNA" means that a DNA is complementary or identical to an mRNA, whether in single or double stranded form.

The term "complementary" means that the topological compatibility or surface interaction of the dinucleotide is matched. Thus, two molecules can be described as complementary, and in addition, the contact surface features are complementary to each other. If the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, the first polynucleotide is complementary to the second polynucleotide. Thus, a polynucleotide of sequence 5'-TATAC-3' is complementary to a polynucleotide of sequence 5'-GTATA-3'.

The word "encoding" means the intrinsic property of a particular nucleotide sequence in a polynucleotide (eg, a gene, cDNA, or mRNA) that can be used as a template to synthesize other polymers and macromolecules of a biological process. A defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, if a gene produces mRNA transcription and translation to produce a protein in a cell or other biological system, the gene is said to encode a protein. Those skilled in the art will appreciate that many different polynucleotides and nucleic acids can encode the same multi-peptide due to the degeneracy of the genetic code. It will also be appreciated that the skilled artisan can employ conventional techniques for nucleotide substitutions that do not affect the multi-peptide sequence encoded by the polynucleotide to reflect the codon usage of any particular host organism in which the peptide is to be expressed. (codon usage). Thus, unless otherwise indicated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate in form and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA can include introns.

By "recombinant nucleic acid" is meant a polynucleotide or nucleic acid having sequences that are not naturally joined together. The recombinant nucleic acid can exist in the form of a vector. A "vector" can contain a given nucleotide sequence of interest and regulatory sequences. Vectors can be used to represent a given nucleotide sequence (expression vector) or to maintain a given nucleotide sequence for replication, manipulation, or transfer between different positions (eg, between different organisms). For the above purposes, the vector can be introduced into a suitable host cell. "Recombinant cell" means the introduction of a recombinant nucleic acid into a host cell. "Transformation" means a cytogenetic change (i.e., extracellular DNA) after incorporation of new DNA. "Transfection" means the process of exogenous DNA transfer from cells. "Transduction" particularly means the process by which exogenous DNA is introduced into a cell via a viral vector. "Transformed cells" means cells that are introduced into a DNA molecule encoding a protein of interest using recombinant DNA techniques.

The vector may be of various types including plastids, vesicles, F-type fosmids, episomes, artificial chromosomes, phage, viral vectors and the like. Typically, in a vector, a given nucleotide sequence is operably linked to a regulatory sequence such that when the vector is introduced into a host cell, the given nucleotide sequence can be expressed in the host cell under the control of regulatory sequences. Regulatory sequences may include, for example, without limitation, promoter sequences (eg, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) early promoter, T7 promoter, and alcohol oxidase gene (AOX1) promoter ), initiation codon, origin of replication, enhancer, operator sequence, secretion signal sequence (eg, alpha-mating factor signal), and other control sequences (eg, Shiine-Dalgarno sequence and Termination sequence). Preferably, the vector may further comprise a marker sequence (eg, an antibiotic resistance marker sequence) for subsequent screening procedures. To produce a protein, in a vector, a given nucleotide sequence of interest can be ligated to another nucleotide sequence other than the above regulatory sequences, such that a fusion polypeptide is produced and facilitates subsequent purification procedures. The fused multi-peptide comprises a tag for purification which binds to the end of the multi-peptide and is preferably small in size which does not affect the desired activity of the multi-peptide. In particular, the tag is below about 30 amino acid residues, specifically about 20 amino acid residues, more specifically about 10 amino acid residues or less; or has a molecular weight of about 10 Below kDa, specifically below about 5 kDa, more specifically below about 2.5 kDa. Examples of such tags include, but are not limited to, six (6) to fourteen (14) His-tags or one (1) to two (2) Myc-tags. The tag can be linked to the N- or C-terminus of the peptide. In some embodiments, the tag is cleavable in vitro or in vivo. In vitro or in vivo cleavage can be carried out by proteases.

In some embodiments, the peptide of the present invention may be referred to as "isolated" or "purified" if it is substantially free of cellular material or chemical precursors or other chemicals involved in the peptide preparation process. It should be understood that the terms "isolated" or "purified" do not necessarily reflect the extent to which the peptide is "absolutely" isolated or purified, such as by removal of all other materials (eg, impurities or cellular components). In some cases, for example, the isolated or purified peptide comprises a peptide-containing preparation having less than 50%, 40%, 30%, 20%, or 10% by weight of other proteins (eg, cellular proteins) a medium having less than 50%, 40%, 30%, 20%, or 10% by volume, or having less than 50%, 40%, 30%, 20%, or 10% by weight of a chemical precursor Or other chemicals involved in the synthesis process. The terms "isolated" or "purified" are also applicable to the nucleic acids of the invention.

In accordance with the present invention, an effective amount of the active ingredient (TIFA fragment) can be formulated in a physiologically acceptable carrier in a suitable form for delivery and absorption. The compositions of the present invention specifically comprise from about 0.1% to about 100% by weight of active ingredient, wherein the weight percentages are based on the weight of the entire composition. In some embodiments, the compositions of the invention may be pharmaceutical compositions or medicaments for use in therapy.

As used herein, "physiologically acceptable" means that the carrier is compatible with the active ingredient of the composition, and preferably stabilizes the active ingredient, and is safe for the recipient. The carrier can be a diluent, carrier, excipient, or matrix of the active ingredient. Some examples of suitable excipients include lactose, sucrose, glucose, sorbose, mannose, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium citrate, microcrystalline cellulose, polyvinylpyrrolidone. , cellulose, sterile water, syrup, and methyl cellulose. The composition may additionally comprise a lubricant such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier and a suspending agent; a preservative such as methyl and propyl hydroxybenzoate; a sweetener; and a flavoring agent . The compositions of the present invention provide a rapid, sustained, or delayed release of the active ingredient after administration to a patient.

According to the present invention, the composition may be in the form of tablets, pills, powders, troches, packages, lozenges, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injections, And packaging powder.

The compositions of the invention may be delivered via any physiologically acceptable route, such as oral, parenteral (e.g., muscle, intravenous, subcutaneous, and peritoneal), transdermal, suppository, and intranasal methods. With regard to parenteral administration, it is preferably used in the form of a sterile aqueous solution which may contain other substances, such as salts or glucose, sufficient to cause the solution to be isotonic with blood. The aqueous solution can be suitably buffered as required (eg pH 3 to 9). The preparation of a parenteral composition under sterile conditions can be accomplished by standard pharmacological techniques well known to those skilled in the art.

In some embodiments, a viral vector carrying a nucleic acid encoding a peptide of the invention is administered to an individual in need thereof. Many viral vectors are known in the art and include, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia, poliovirus vectors. Preferably, the replication-deficient virus is used as a viral vector such that the transformation of the recombinant cell strain and the recombinant viral vector does not result in the production of a replication competent virus, for example, introduction into a viral vector by homologous recombination of the recombinant cell strain viral sequence. The DNA of the present invention can also be administered using a non-viral vector, for example, a DNA or RNA liposome complex formulation. Such complexes comprise a lipid mixture that binds to a nucleic acid (DNA or RNA), providing a hydrophobic outer layer that allows delivery of genetic material into the cell.

3. Take TIFA Peptide fragment as TIFA Antagonist for treatment TIFA Therapeutic use of associated diseases

TIFAs are involved in NF-κB activation using various stimuli such as inflammatory interleukins or lipopolysaccharide (LPS). Unexpectedly, the present inventors have found that the TIFA fragments described herein inhibit TIFA activation and the resulting NF-κB activation (TIFA activation/NF-κB activation). Thus, any of the TIFA fragments described herein have inhibitory activity against TIFA activation and NF-κB activation, and are useful for inhibiting TIFA activation and NF-κB production in individuals with such therapeutic needs. Activation to facilitate treatment of a disease or condition associated with abnormal TIFA activation and/or NF-κB activation.

According to the present invention, the TIFA peptide fragment must include a dimeric core peptide segment (F2 domain) which can be linked to the Thr9 peptide segment (F1 domain) at the N-terminus or to the TRAF6/TRAF2 interaction at the C-terminus. Peptide segment (F3 domain). In a specific embodiment, the TIFA peptide fragment of the invention comprises an F1/F2 fragment, wherein the Thr9 peptide segment (F1 domain) is linked to the dimeric core peptide segment (F2 domain), from N-terminus to C End, which lacks the TRAF6/TRAF2 interaction peptide segment (F3 domain). In another specific embodiment, the TIFA peptide fragment of the invention comprises an F2/F3 fragment, wherein the dimeric core peptide peptide segment (F2 domain) is linked to the TRAF6/TRAF2 interaction peptide segment (F3 domain), From the N-terminus to the C-terminus, it lacks the Thr9 peptide segment (F1 domain). The present invention first demonstrates that this TIFA peptide fragment can act as a dominant negative inhibitor of TIFA, which effectively interferes with the self-binding of endogenous TIFA, thereby attenuating TIFA activation and NF-κB activation, specifically Inducing NF-κB activation, thus treating diseases or conditions associated with abnormal TIFA activation and/or NF-κB activation.

To carry out the methods disclosed herein, an effective amount of a composition, such as a pharmaceutical composition described herein, comprising a TIFA fragment or a nucleic acid encoding the same, can be administered to a subject in need (e.g., a human) treated by a suitable route. As used herein, the term "effective amount" means the amount of active ingredient that imparts the desired biological effect in the individual or cell being treated. The effective amount may vary depending on various reasons, such as the route and frequency of administration, the weight and species of the individual receiving the drug, and the purpose of administration. Based on the disclosure, methods of establishment, and its own experience, one skilled in the art can determine the dosage in each case.

The individual to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment can be a human patient at risk or suspected of having the underlying disease/condition (eg, cancer or inflammatory disease). An individual suspected of having any of the diseases/conditions of this subject may exhibit one or more symptoms of the disease/condition. An individual at risk of having a disease/condition can be an individual having one or more risk factors for the disease/condition.

Abnormal TIFA activation is associated with a variety of diseases and conditions. In some embodiments, the disease or condition associated with TIFA activation is an inflammatory disease due to TIFA activation, such as interleukin-stimulated NF-κB activation. Examples of such diseases or conditions include, but are not limited to, hepatitis, atherosclerosis, pulmonary hypertension, cardiomyopathy, rheumatoid arthritis, inflammatory bowel disease, and Fabry disease. In other specific embodiments, the disease or condition associated with TIFA activation is cancer, in particular cancer associated with chronic inflammatory or inflammatory conditions. An example of a cancer to be treated according to the invention is a hematological cancer selected from lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphoma, and lymphocytes and cutaneous lymphoma), leukemia (including children with leukemia, hairy cell leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, and mast cell leukemia), bone marrow tumors, and mast cell tumors . Other examples of cancers to be treated by the present invention are cancers associated with chronic inflammatory or inflammatory conditions, including rectal cancer, lung cancer, gastrointestinal cancer, prostate cancer, pancreatic cancer, lymphoma, ovarian cancer, and breast cancer.

In a particular embodiment, the individual or subject can be a mammal having a risk of leukemia.

As used herein, "treatment" refers to the application or administration of a composition comprising one or more active agents to a subject suffering from a condition (eg, diabetes, atherosclerosis, and other inflammatory diseases), a condition The condition or condition, or the progression or tendency of the condition, is intended to heal, heal, alleviate, alleviate, alter, remedy, improve, improve, or affect the condition, the condition or condition of the condition, the disability caused by the condition, or the progression of the condition or tendency. Thus, the term "treatment" can also include, depending on the condition of the individual to be treated, preventing the condition, including preventing the onset of the condition or any symptoms associated therewith, and reducing or reducing the severity of the disease or any symptoms prior to its onset.

The term "anti-cancer treatment" as used herein may refer to administration of an anticancer agent or radiation to an individual having a tumor or cancer, for example, to provide inhibition of tumor or cancer cell growth or proliferation, or to induce poisoning. The term "anticancer agent" as used herein may include a chemotherapeutic agent or a molecular target agent. "Chemoresistant" may mean that a tumor or cancer cell exhibits little or no significant detectable therapeutic response to a chemotherapeutic agent used in chemotherapy. Some chemotherapeutic agents induce chemical resistance, which can lead to the survival of tumor cell populations, followed by recurrence after treatment. As used herein, "standard dose" can refer to an effective dose of a therapeutic agent that is recommended by an authoritative source of the pharmaceutical industry, including the Food and Drug Administration, and is commonly used in routine practice. As used herein, "reduced dose" can mean that one of the same therapeutic agents is below the standard dose but still retains substantially the same therapeutic effect. In particular, according to the present invention, the reduced dose of the chemotherapeutic agent is about 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less of the standard therapeutic dose of the same pharmaceutical agent.

Some cancer patients may be responsive to anticancer agents and may require high doses to achieve the desired therapeutic effect. The present inventors have surprisingly found that inhibition of TIFA activation enhances the chemical toxicity/reduction of chemoresistance of anticancer agents, which leads to the promotion of therapeutic efficacy. In particular, when inhibiting TIFA activation of cancer cells (eg, using TIFA fragments as a dominant negative inhibitor of TIFA), the dose of anticancer agent required to inhibit proliferation of cancer cells is reduced compared to uninhibited TIFA activation; and anticancer The combination of a agent with a TIFA antagonist (such as the TIFA fragment used herein) provides a synergistic increase in anti-cancer effects, such as a reduction in tumor volume, compared to treatment with an anti-cancer drug alone.

As used herein, the term "TIFA antagonist" means a substance or agent that substantially reduces, inhibits, blocks, and/or mitigates TIFA activation, including, but not limited to, phosphorylation and oligomerization, and subsequently Activation of NF-κB, in particular by stimulation of inflammatory interleukins or pathological stress. In addition to the TIFA fragment as a dominant negative inhibitor of TIFA described above, TIFA antagonists may include antisense nucleic acid molecules directed against the TIFA gene or small interfering RNA (siRNA) directed against TIFA nucleic acids, which may be used in the present invention.

In some embodiments, the TIFA antagonist comprises at least one antisense nucleic acid molecule capable of blocking or reducing the expression of a functional TIFA. The nucleotide sequence of TIFA is well known and readily available in publicly available databases. Antisense oligonucleotide molecules can be prepared conventionally that will specifically bind to the target mRNA without cross-reacting with other polynucleotides. Exemplary sites for targeting include, but are not limited to, a start codon, a 5' regulatory region, a coding sequence, and a 3' non-translated region. In a specific embodiment, an exemplary site of targeting is located in the coding sequence of the TIFA. In some embodiments, the oligonucleotide is from about 10 to 100 nucleotides in length, from about 15 to 50 nucleotides in length, from about 18 to 25 nucleotides in length, and the like. Oligonucleotides may comprise backbone modifications, such as phosphorothioate linkages, and 2'-0 sugar modifications as are known in the art.

4. TIFA As AML Mark of poor prognosis

The present invention is also based on the discovery that TIFA can be used as a marker for poor prognosis of AML. As shown in the examples below, AML patients with higher amounts of TIFA protein appear to have higher white blood cell (WBC) and blast counts than AML patients with lower TIFA levels. In addition, higher TIFA manifestations in AML patients were associated with lower response rates, and after tracing, patients with higher TIFA performance had overall survival (OS) and no disease compared with those with lower TIFA performance. Survival (DFS) was significantly shortened.

Accordingly, the present invention provides a method for predicting the prognosis of leukemia based on the amount of TIFA protein. Specifically, the method of the present invention comprises determining the amount of TIFA expression taken from a leukemia patient sample, and determining the prognosis of the leukemia of the patient based on the amount of TIFA expression in the sample, wherein an increase in the amount of TIFA in the sample indicates a poor prognosis.

As used herein, an increase in amount refers to an increased amount compared to the amount of a subject having no cancer or inflammatory disease or a reference or control amount. For example, the amount increase can be greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% above the reference or control amount. A reference or control amount can refer to an amount measured in a normal individual or sample type (eg, tissue or cells that are not diseased).

The biological sample analyzed herein by any of the methods described herein can be any type of sample, preferably a blood sample, taken from the individual to be diagnosed. Typically, the blood sample can be whole blood or a fraction thereof, such as peripheral monocytes, or plasma, which is heparinized or EDTA treated to avoid blood coagulation (serum). Alternatively, the biological sample can be a tissue sample or a biopsy sample.

Specifically, the method of the present invention for predicting the prognosis of a leukemia patient comprises: (a) measuring the amount of TIFA taken from the first biological sample of the patient at the first time point; (b) taking the measurement at the second time point. The amount of TIFA in the second biological sample of the patient; and (c) determining the progression of leukemia in the patient based on the amount of the first and second biological samples, wherein the amount of TIFA in the second biological sample is increased compared to the first biological sample High represents the progress of leukemia.

The invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. In view of the present invention, it will be appreciated by those skilled in the art that the invention may be

Example

1.1 Cell culture, human peripheral blood mononuclear cells (PBMCs) Separation, and TNF-α Stimulating effect

Hela, 293T, and HL-60 cell lines were taken from Dr. Li-Jung Juan (Taipei, Taiwan, 2014), and the KG-1 cell line was taken from Professor Shih-Lan Hsu of Taichung Veterans General Hospital (Taiwan, Taiwan, 2015), and the remaining cell lines were taken from Dr. Shui-Tein Chen of the Central Research Institute (Taipei, Taiwan, 2015). All cell lines were amplified and stored in liquid nitrogen upon receipt. The vial was thawed for their original experiments, while using the Promega GenePrint ^â 10 System for STR DNA fingerprinting, and with the ABI PRISM 3730 GENETIC ANALYZER GeneMapper ^â analysis software. All resulting STR profiles coincided with the conventional ATCC fingerprint (ATCC.org). Each cell line was maintained according to the inventors' previous methods (20, 27, 28). 293T and HeLa cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Gibco). Primary PBMCs, HL-60, KG-1, THP-1, U937, Jurkat, and K562 cells were maintained in Roswell Park Memorial Institute medium (RPMI-1640, Invitrogen). All media supplemented with 10% fetal bovine serum (FBS, Gibco), 200 mM L-glutamic acid (Gibco), 100 U/mL penicillin (Gibco), and 100 mg/mL streptomycin (Gibco) ). All cells were cultured in a humidified environment at 37 ° C and 5% CO ₂ . To isolate human PBMC, freshly obtained whole blood was layered on Ficoll (GE healthcare) as previously described (28). The isolated primary cells (29) were maintained according to the methods previously described and assayed in two weeks. When necessary, the cells were starved for 8 hours under serum-free conditions by adding 10 ng/mL of TNF-α (R&D Systems) for 30 minutes or before the indicated time point.

1.2 Platinum and siRNA synthesis

The full-length TIFA wild type was carried in the pCDNA3.1 vector, followed by the synthetic HA-tag, and the full-length Alora A was constructed on the same vector except that the Flag-tag was used. For retroviral transduction, full-length (wild-type or T9A mutant with TIFA siRNA-resistant silencing mutation) and TIFA fragment were amplified by polymerase chain reaction (PCR) and carried by pLNCX vector (Clontech), followed by two Continuous Myc-tag, as previously described (27). The pNF-kB-Luc system was presented by Dr. L.-P. Ting (National Yangming University, Taiwan). All constructs were sequenced prior to experimental application. The double stranded siRNA oligomer corresponds to the sequence 5'-UCA GGA CAA ACA GGU UUC CCG AGU U-3' (SEQ ID NO: 95) to target TIFA (20), which is synthesized by GeneScript, Aurora A Target siRNA was purchased from Cell Signalling (#8883) and control oligomers were purchased from GeneScript.

1.3 Reverse transcription PCR (RT-qPCR)

The degree of transcription of a particular gene was examined by real-time quantitative PCR as described above with minor modifications (27). On day 3 after siRNA transfection, total RNA was extracted from the harvested cells with TRIzol reagent according to the manufacturer's instructions (Invitrogen) and was present in oligo-dT (Invitrogen) from total RNA treated with DNase I (Invitrogen). First-strand cDNAs were synthesized using the SuperScript® III kit (Invitrogen) according to the manufacturer's protocol. SYBR Green (Roche) was supplemented with gene-specific primers for RT-qPCR and analyzed by LightCycler® 480 (Roche).

1.4 Platinum and siRNA Transient transfection

For transient transfection of 293T cells, 2 μg of indicated plastid DNA was transfected into 6 x 10 ⁵ cells for 48 hours using Jet-PEI (Polyplus Transfection) (pCDNA3.1 vector, HA-tagged TIFA, and pNF-kB- Luc) or 24 hours (Flag-labeled Aurora A), according to the manufacturer's instructions. For U937 cells transiently transfected using Lipofectamine 2000 (Invitrogen), 3 μg of the pNF-kB-Luc plasmid DNA is transfected ¹⁰⁶ suspension cells for 48 hours, the system according to the manufacturer instructions. To silence specific genes, add 9 or 15 μL of Lipofectamine RNAiMAX (Invitrogen) in the presence of Opti-MEM (Invitrogen) with 30 pmol (293T cells) or 50 pmol (suspended cells), transfected 6 x 10 ⁵ 293T cells, or a ¹⁰⁶ cell suspension, which line according to the manufacturer instructions. Six hours after the first transfection, the cells were cultured in a conventional medium for 24 hours, and a second siRNA transfection was performed in the same manner. After 72 hours of culture, cells or conditioned medium were collected.

1.5 Preparation of retroviral stable strains

For retroviral transduction, a pseudotyped virus is packaged in the presence of VSV-G in a pantropic retroviral expression system, according to the manufacturer's instructions (Clontech), and is infected with the designated cells. , as described in (27) above. After several weeks of screening with 400 mg/mL G418, Western blot analysis was used to confirm ectopic protein expression in stable cells prior to experimental analysis or siRNA transfection for phenotypic rescue experiments.

1.6 Cell processing

All chemotherapy drugs used in this study were obtained as follows: Etoposide (VP-16) was purchased from Sigma; Idarubicin and Cytarabine (Ara-C) were purchased from Phizer; Cisplatin was purchased from The European Directorate for the Quality of Medicines & HealthCare (EDQM); Rafi was purchased from Santa Cruz Biotechnology; Enbrel (TNF-α inhibitor) was purchased from Wyeth; Bortezomib (Velcade, NF-κB inhibitor) was purchased. From Janssen Pharmaceutica; ABT-263 (Navitoclax, BCL-2 inhibitor) was purchased from AdooQ Bioscience. The cell treatment with X-ray radiation was performed with a Faxitron RX-650 irradiator (Faxitron X-ray Corporation) at a dose rate of 0.46 Gy/min. When necessary, the cells were starved for 8 hours under serum-free conditions, after which 10 ng/mL of TNF-α (R&D Systems) was added for 30 minutes or at the indicated time points.

Kinase inhibitors and conditions for the treatment of 293T cells are as follows: 100 nM casein kinase II inhibitor III TBCA (CK2i), 500 nM 4-amino-5-(bromomethyl)-2-methylpyrimidine dihydrobromide Acid salt (GRKi), and 25 nM sorafenib (Rafi) were purchased from Santa Cruz Biotechnology; 200 nM necrostatin-1 (RIPK1i) and 10 nM GÖ 6976 (PKCi) were purchased from Tocris Bioscience; 50 nM Akt inhibitor VI (Akti), 300 nM IRAK-1/4 inhibitor (IRAK1/4i), and 10 nM (5Z)-7-oxy violet alcohol (oxozeaenol) (TAK1i) were purchased from Merck Millipore; nM caffeic acid phenethyl ester (CAPE, NF-κBi) was purchased from Sigma; 10 nM VX680 (Aolora Ai) was purchased from Selleck Chemicals; 50 nM MK-5108 (Aurora Ai) was purchased from AdooQ Bioscience.

1.7 antibody

The initiation of TIFA-specific monoclonal antibodies is as described above (20). Anti-olola A, anti-phosphorylated alora A (Thr288), anti-Bcl-2, and anti-Bcl-X _L were purchased from Cell Signalling. Anti-His-tags were purchased from SignalChem. Anti-Myc, anti-Flag, and anti-HA were purchased from Sigma. Anti-IκKα (phosphorylated T23), anti-IκBα (phosphorylated S32+S36), anti-human CD45 PerCP/Cy5.5, anti-human CD33 FITC, anti-β-actin, and anti-Bax were purchased from Abcam. Anti-NF-κB activated p65 subunit (colon 12H11) and wasabi peroxidase (HRP) conjugated anti-mouse or anti-rabbit IgG were purchased from Millipore.

1.8 In vitro kinase assay

The experiment was based on the method previously established by the inventors (20). The indicated 293T cell line was lysed with CHAPS lysis buffer (20 mM PIPES, 1 mM Na ₃ VO ₄ , 1 mM EGTA, 50 mM Tris-HCl, 150 mM NaCl, 50 mM NaF, 1% CHAPS, and 10% glycerol) ), which is supplemented with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor (Sigma). The cell extract was incubated with recombinant His-tagged wild-type or T9A mutant TIFA in reaction buffer (40 mM HEPES pH 7.5, 20 mM MgCl ₂ , and 100 mM ATP) at 1 mCi/mL [g] - ³² P] ATP (Perkin-Elmer) was reacted at 37 ° C for 30-90 minutes. Then, the His-tagged protein was captured with M-280 Dynabeads coated with anti-His mAb. The reaction was terminated by the addition of SDS sample buffer and heating at 95 ° C for 10 minutes, followed by SDS-PAGE. The degree of phosphorylation on the recombinant protein was shown by autoradiography.

1.9 Immunoprecipitation

The immunoprecipitation experiment was carried out as described in the above (28). 5 μg of vector or HA-TIFA was transfected into sub-full 293T cells of p100 culture plates using Jet-PEI (Polyplus Transfection). After 48 hours, cells were treated with or without 10 ng/mL TNF-α (R&D Systems) for 30 min, washed with PBS, and with CHAPS lysis buffer (20 mM PIPES, 1 mM Na ₃ VO ₄ , 1 mM Pyrolysis of EGTA, 50 mM Tris-HCl, 150 mM NaCl, 50 mM NaF, 1% CHAPS, and 10% glycerol was supplemented with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail 3 (Sigma). 3 μg of the indicator antibody was previously coated with 100 mL of M-280 Dynabeads (Invitrogen) overnight at 4 °C. 500 mg of cell extract was incubated in 15 mL of antibody-coated Dynabeads overnight at 4 °C. Subsequently, the protein-bead complex was washed with Tris buffered saline Tween 20 (TBST) buffer and Western blot analysis was performed.

1.10 Western ink point method

To obtain intact cell extract, wash 6 x 10 ⁵ 293T cells or 10 ⁶ suspension cells in ice-cold PBS and in RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5%) Lysate in sodium deoxycholate and 0.1% SDS) supplemented with a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Sigma). After 5 cycles of freeze-thaw cycles, the cell extract was removed by centrifugation at 4 °C. Cell extracts were quantified using the Bradford assay, and equal amounts of soluble protein were mixed with SDS sample buffer, boiled, separated by SDS-PAGE, and spotted onto a PVDF membrane (Millipore). Subsequently, the membrane was blocked with PBST containing 5% dry milk powder (PBS supplemented with 0.1% Tween-20) for 1 hour, and cultured overnight at 4 °C with primary antibody containing 1% bovine serum. Albumin (Sigma) was diluted in PBST. After washing three times with PBST, the membrane was cultured in HRP conjugated anti-mouse or anti-rabbit IgG. The film was washed five times with PBST and the dot layer of the dot protein was presented in an ECL system (Millipore). All Western blot analysis was performed at least three times independently, except for the primary PBMCs.

1.11 Luciferase reporter gene assay

The luciferase-based NF-κB reporter assay was performed as described above (20). All 6×10 ⁵ 293T or 10 ⁶ suspension U937 cells cultured in each well of a 6-well culture plate were subjected to transient transfection for 48 hours with 2 mg of pNF-κB-Luc. Cells were treated with 10 ng/mL TNF-α (R&D Systems) for 30 min and lysed with 1 x passive lysis buffer (Promega). 100 mg of cell lysate was dispensed into each well of a 96-well plate, followed by 100 mL of luciferase assay buffer LARII (Promega) and 100 mL of Stop & Glo reagent (Promega). Chemiluminescence was measured in triplicate using a SpectraMax Paradigm multimode plate reader (Molecular Devices) and normalized to the corresponding amount of β-actin in a cell lysate parallel Western blot analysis. The experimental results are expressed in terms of relative luminescence units (RLU) of three independent experiments.

1.12 WST-1 Cell survival test

All 10 ⁵ suspension cells were seeded in each well of a 96-well culture dish under the indicated conditions, and cell viability was analyzed at the indicated time points after treatment. WST-1 reagent (Roche) was mixed in cells at 10-fold dilution according to the manufacturer's instructions and the absorbance of the three replicates was read with a SpectraMax Paradigm Plate Reader (Molecular Devices), normalized to day 0 or mock control group.

1.13 Patient sample

Sixteen patients diagnosed with neonatal AML at Taichung Veterans General Hospital (VGHTC; AML #1-9) and National Taiwan University Hospital (NTUH; AML #10-16) from December 2007 to March 2014 were included in Western blots. Analysis, WST-1 cell viability, and apoptosis assay. Their patients had an average of blasts of more than 60% ± 18, with 4 exceptions (patients #2, 9, 12, and 16), which took blood samples after chemotherapy to suppress acute conditions. For immunocytochemical staining analysis, 85 adult AML patients who received standard chemotherapy at NTUH between March 2009 and January 2012 and who had cryopreserved BM cells were included (30, 31). Written informed consent was obtained from all participants in accordance with the Helsinki Declaration, and all experimental studies were in accordance with the institutional agreement (CE11166-2, VGHTC, No. 201408027RIND, NTUH, and AS-IRB02-103189 and AS-IRB02-104147, Academia Sinica, Taiwan).

1.14 Immunocytochemical staining

Immunocytochemical staining was performed as previously described with minor modifications (32). Cytospin smears of BM leukemia cells from 85 patients were fixed in formalin acetone (3%) for 3 minutes. The specimens were then blocked with peroxidase blocking enzymes (10 min) followed by TIFA monoclonal antibodies (1:200 in blocking solution, overnight at 4oC) (20). Proteins were detected as biotinylated anti-mouse IgG (1 μg/mL, diluted with blocking solution, 30 min; DAKO) and detected by streptomycin-peroxidase complex (DAKO). Specimens were stained with hematoxylin. Each specimen was scored from 0 to 4, based on the intensity of staining intensity (0 = no, 1 = weak, 2 = strong) and the percentage of positive staining of bone marrow cells (0 = 0-25%, 1 = 26-50%, 2 = 51-00%).

1.15 Apoptosis and flow cytometry analysis

On day 2 after transfection of siRNAs, PBMCs were seeded in 6-well plates at 10 ⁶ per well, followed by treatment with different chemotherapeutic drugs. After 48 hours, the cells were washed with ice-cold PBS and assayed for apoptosis using the FITC Annexin V cell apoptosis assay kit according to the manufacturer's instructions (BD Biosciences). Subsequently, according to the aforementioned method (33), the bone marrow lineage of the cell is gated by light scattering, and the apoptotic rate, such as double staining with propidium iodide and FITC annexin V, which is BD FACSCalibur Flow Cytometer (BD FACSCalibur) analysis, as described previously (27). To analyze U937 cells in xenograft mode, BM cells or mouse PBMCs were stained with anti-human CD33 FITC and CD45 PerCP/Cy5.5 for 1 hour on ice, respectively. The cells were washed with ice-cold PBS and subjected to flow cytometry as described above.

1.16 Evaluation of interleukin and chemokine secretion ( Interleukin array and ELISA)

At 72 hours after the indicated treatment, conditioned medium of the same amount of U937 cell culture was collected and evaluated using human interleukin array C1 according to the manufacturer's instructions (AAH-CYT-1, RayBiotech). Data was taken from ImageJ software and normalized to the positive control group. When specified, the amount of interleukin and chemoattractant secreted in the conditioned medium was determined by enzyme binding immunosorbent assay (ELISA) test kit according to the manufacturer's instructions (R&D Systems). The reflectance of the three replicate samples was read using a SpectraMax Paradigm Plate Reader (Molecular Devices). All raw data were normalized to the PBS control group and the results were expressed as relative secretion of interleukins from three independent experiments.

1.17 Xenograft of human leukemia cells and chemotherapy in vivo

Six to eight-week-old athymic nude mice purchased from the National Laboratory Animal Center (Taiwan) were raised at the Chang Gung Memorial Hospital's pathogen-free facility (Agreement No. 2014092305). For the myeloma model, mice were subcutaneously inoculated with 5×10 ⁶ U937 stable cells, which were dissolved in 50% Matrigel (Corning), followed by intraperitoneal injection of PBS on days 6, 8, and 11 after inoculation. Or cytarabine (Ara-C, 50 mg/kg). The tumor size was recorded twice a week, and the calculation was ² × length × 0.52 (34). For the in situ leukemia model, mice were surgically injected with 1.5×10 ⁵ U937 stable cells per infusion via the inferior vena cava (IVC), followed by 5 days daily for the same treatment on the 5th day after transplantation. . Mice were sacrificed on day 60 and analyzed by flow cytometry with femoral BM cells.

1.18 Statistical Analysis

All data are expressed as the mean ± standard deviation of three independent experiments. Statistical significance is expressed as a t-test of independent samples, such as *P < 0.05, **P < 0.01, and ***P < 0.001 unless otherwise indicated. For immunocytochemical staining, the correlation between variables was assessed by Spearman rank correlation. The Mann–Whitney U assay was used to analyze differences in TIFA performance in AML patients. Overall survival (OS) was measured from the first day of diagnosis until death for any cause, and disease-free survival (DFS) was calculated from complete remission (CR) until recurrence, death, or end of study for any reason. Kaplan-Meier estimates were used to plot survival curves and test the differences between groups by logarithmic scale. Risk ratios and 95% confidence interval (CI) were estimated using the Cox proportional hazard model to determine independent risk factors associated with OS and DFS in multivariate analysis. Bilateral P values <0.05 were considered statistically significant.

2. result

2.1 TIFA Participate in Alora A Kinase dependence NF-κB Communication

The inventors previously suggested that phosphorylation of TIFA in threonine 9 may involve the signaling pathway of Ser/Thr kinase in PI3K/AKT (20). To determine the most direct kinase for Thr9 phosphorylation by TIFA, the inventors screened for kinase inhibitors (20, 23) via previously established methods and found that Alorora A inhibitors are most effective against in vitro phosphorylation. It is produced by treating 293T cell lysate with TNF-α. The inventors further verified that Aurora A is required for the phosphorylation of Thr9 of TIFA, which is based on three experiments: First, cell lysates treated with aolola A-specific siRNA and two different olora A inhibitors The ability of TIFA Thr9 phosphorylation was shown to be diminished, similar to the results of the unphosphorylated T9A mutant (Fig. 1A, lines 5-7). Second, immunofluorescence staining showed that endogenous TIFA spots (20) coexisted with activated aolola A (phosphorylated-Thr288) when treated with TNF-[alpha] (data not shown). Furthermore, immunoprecipitation experiments showed a direct interaction between Alora A and TIFA, which was enhanced by TNF-α stimulation (Fig. 1B).

Aurora A has been reported to regulate NF-κB signaling (17), and increased expression of aurora A is consistent with increased TNF-α, NF-κB, and chronic inflammation in gastric cancer (35). The inventors also observed that activated NF-κB, phosphorylated IκK and IκB, and NF-κB-driven luciferase activity were significantly increased in response to excessive expression of aurora A or TNF-α treatment (Fig. 1C-D, line 2), and additionally shows that this rise disappears when TIFA is silent (line 4). The results overall show that the role of Aurora A in the regulation of NF-κB signaling requires TIFA.

2.2 TIFA Olora A Survival factor, and AML Poor prognosis

Aurora A was found to be highly representative of AML (18). Since the inventors have shown that TIFA is required for NF-κB activation in A937 cell lines U937 and THP-1 (Fig. 1C-D), the inventors further explored whether the amount of TIFA protein is related to Aurora A during leukemia. Associated with NF-κB signaling factors. Due to the cytogenetic cluster heterogeneity of AML (36) and the limited volume of blood samples from patients, the inventors chose to use Western TI-specific monoclonal antibodies for Western blot analysis (20) and compared 16 neonatal AML diseases. Freshly isolated PBMCs with 16 normal individuals. The inventors found that in all AML-derived PBMCs, the amount of TIFA protein was significantly increased, but remained unchanged in normal donors. In addition, TIFA elevation is accompanied by up-regulation of the extent of Aurora A, activated alora A, phosphorylated IκK/IκB, activated NF-κB, and pro-survival factor Bcl-2/Bcl-X _L (Fig. 2A). In all PBMCs, the pairwise comparison of all tested protein levels showed a strong correlation, indicating that these factors are functionally related to AML (Pearl correlation coefficient = 0.862-0.991).

Immunocytochemical staining of cryopreserved BM leukemia cells from 85 newborn AML patients was used to further enhance the prognosis of TIFA protein levels observed by non-fractionated PBMCs. According to the results of immunocytochemistry, patients who received conventional intensive chemotherapy (31) were divided into low (score 0-2, n = 40) and high (score 3-4, n = 45) performance groups. Patients with AML with higher TIFA protein content appear to have higher WBCs and burst counts than those with lower TIFA doses. In addition, higher TIFA protein performance was closely related to FAB M4 subtype (P = 0.0315), but negatively correlated with CD7 expression in leukemia cells (P = 0.0454).

Of all AML patients examined, 54 (63.5%) achieved CR. Higher TIFA performance was associated with lower response rates (CR rate, 51.1% vs. 77.5%, P = 0.0139). After tracking an intermediate period of 57.8 months (range, 0.3 to 79.1 months), patients with higher TIFA performance showed significantly lower OS and DFS than those with lower TIFA. In subgroups of non-M3 subtypes, moderate risk cytogenetics, and normal karyotypes, the difference was still significant, but the level of DFS was lower. In the multivariate analysis, higher TIFAs were independent of OS prognosis, regardless of age, WBC count at diagnosis, cytogenetics, and NPM1/FLT3 -ITD (Table 2). Table 2 Analysis of multiple mutations between OS and DFS (Cox) Abbreviations: RR, relative risk; CI, confidence interval, * statistically significant (P < 0.05) ^† age > 50 vs. age £50 (reference volume) ^§ WBC greater than 50,000/mL vs. 50,000/mL ^z NPM1 ^mut / FLT3-ITD ^neg compared to other subtypes ^‡ higher TIFA contrast lower TIFA performance ^Y unfavorable cytogenetics comparison other

Together, these results show that TIFA performance is not only associated with Aurora A-dependent NF-κB signaling, but also with poor prognosis for AML.

2.3 TIFA Silent promotion AML Chemical toxicity with other cancer cell lines

To verify the association of TIFA-induced tumorigenesis with Alora A signaling, the inventors targeted TIFA-dependent signaling via RNA interference and tested the proliferation of AML cell lines via the WST-1 survival assay in a time-dependent manner. The results showed that TIFA silencing attenuated the growth of AML cell lines HL-60, KG-1, THP-1, and U937 (Fig. 3A), which is similar to the VX680 treatment result (Fig. 3B) (18). In addition, cell proliferation of both acute lymphocytic leukemia (ALL) cell line Jurkat and CML cell line K562 can also be inhibited in response to TIFA silencing and VX680 treatment.

Inhibition of aolala A kinase promotes chemosensitivity in leukemia and is considered an anti-cancer strategy (14, 37). To test whether targeting TIFA can also alleviate the invasiveness and chemical resistance of hematological malignancies, such as inhibition of aolola A, the inventors investigated the survival rate of leukemia cells at each treatment under TIFA silence. The WST-1 based survival test showed that IC50 for all four AML cell lines tested was reduced by approximately 50% for the chemotherapy of three traditional AML chemotherapeutic drugs, etoposide, idarubicin, and cytarabine. Similar to the effect of the VX680 (Figure 3C). Similar results were obtained with silent, atypical drug therapy, and ALL and CML cell lines from Aurora A.

The inventors then tested the effect of TIFA inhibition with freshly isolated AML PBMCs. In addition to growth retardation (Figure 4A, right on the right side of Figure 4B), it can be seen from the AML PBMCs consumed by TIFA that elevated NF-κB signaling factors have a significant reversal, even in the presence and activation of Apolla A kinase. (Fig. 4C). Interestingly, the effect of TIFA silencing on normal PBMCs survival appears to be less effective than treatment of VX680, which is comparable to comparable chemical toxicity in AML counterparts (compare the right and left portions of Figure 4B), showing targeted TIFA versus The cytotoxicity of leukemia cells may be small. More importantly, silent TIFAs continue to promote the chemosensitivity of patient-derived PBMCs, which is similar to inhibition of aolola A by VX680 (Fig. 4D), showing that TIFA is required for leukemia chemoresistance, similar to Oro Pull A (38). The chemical sensitivities promoted by them are most likely due to increased apoptosis in TIFA silence (Fig. 4E) and downregulation of NF-κB signaling factors (Fig. 4C). The inventors' research shows that TIFA is required to maintain leukemia cell growth and chemical resistance, and TIFA can be used as a potential therapeutic target.

Other cancer cell lines have also been found to inhibit the cytotoxic effects of anti-cancer treatment by TIFA inhibition. Each cancer cell line was irradiated with sorafenib, cisplatin, etoposide, or X-ray radiation in the presence or absence of TIFA silence, and the WST-1 test was performed. Day 4 cytotoxicity of the cells was normalized and presented in the no TIFA silent treatment group. Table 3 summarizes the cytotoxic fold changes of TIFA silenced by anticancer treatment of different cancer cell lines. * Cytotoxicity (1-% survival) was calculated and the fold change in cytotoxicity was determined using (TIFA silent cytotoxicity/non-silent cytotoxicity).

2.4 Dominant negative (DN) Inhibition targeting TIFA

To explore the molecular mechanism of targeting TIFAs, the inventors used phenotypes to rescue TIFA depleted AML cell lines using wild-type TIFA and T9A mutants via a silent complementation strategy (27). In all AML cell lines tested, wild-type TIFAs, but not T9A mutants, were shown to be effective in relieving silencing-dependent chemosensitivity (Fig. 5A), showing that pThr9-directed TIFA oligomerization is critical for AML chemoresistance. step.

The inventors then attempted to disrupt TIFA oligomerization by ectopically expressing a DN (dominant negative) fragment of TIFA in AML cells. The inventors designed a minimally efficient region for the targeted localization of TIFAs in AML cells by retroviral transduction (Fig. 5B). The immunoprecipitation of the transduced U937 cell lysate showed that the fragments F1F2 and F2F3, both containing FHA dimerization cores with additional C-terminal and N-terminal extensions, were able to capture endogenous TIFAs (Fig. 5C), showing The two fragments can interfere with the self-binding of endogenous TIFAs (20). As a result, the F1F2 or F2F3 fragment of U937 cells attenuated TNF-α-dependent NF-κB activation and NF-κB signaling factor elevation (Fig. 5D-E). More importantly, the di-DN fragment also significantly promoted the chemosensitivity of AML cell lines with IC50 reduced by more than 50% (Fig. 5F). Together, these observations indicate the potential of molecularly targeted therapy of TIFAs in AML.

2.5 Targeting TIFA Enhance the clearance rate of leukemia myeloid cells

Abnormal secretion of interleukins contributes to the pathogenesis of leukemia (39) and is considered a prognostic indicator of AML recurrence (39, 40). The inventors investigated whether molecular targeting of TIFAs can disrupt NF-κB-dependent leukemia interleukin secretion and achieve better therapeutic efficacy. The inventors performed an array of interleukin antibodies to analyze the interleukin secreted by U937 cells. The results showed that treatment with cytarabine promoted the secretion of TNF-α, CXCL-1, and IL-8, but inhibited IL-6, while the expression of TIFA DN fragment showed the opposite effect (Fig. 6A, detailed ELISA analysis 6B). Regarding the treatment of cytarabine to promote TNF-α secretion, supplementation with TNF-α enhanced U937 cell survival in response to cytarabine treatment, while TIFA DN fragment significantly antagonized TNF-α (Fig. 6C). Inhibition of TNF-α and downstream NF-κB by enalapril (Enbrel, TNF-α inhibitor), bortezomib (Velcade, NF-κB inhibitor), or ABT-263 (Navitoclax, BCL-2 inhibitor) Survival messaging enhances the cytotoxicity of cytarabine-treated U937 cells, which is similar to the effect of the TIFA DN fragment (Fig. 6D). These results show that the TIFA DN fragment is therapeutically effective in blocking inflammatory interleukin secretion, resulting in the deactivation of NF-κB survival factor in AML.

Therefore, the inventors assessed the in vivo therapeutic potential of TIFA DN fragments. Although transplanted hematopoietic tissue proved to be difficult to propagate on nude mice compared to NOD-SCID or NSG/NOG mice (41), the inventors chose this more difficult mode to verify the effect of targeting TIFAs. This is a complete inflammatory cytokine required by proliferating bone marrow precursor cells to mimic the microenvironment to support chemical resistance (42). The ectopic xenograft model showed that, in addition to cytarabine treatment, retroviral transduction of TIFA DN fragments significantly prevented myelosarcoma expansion compared to cytarabine alone (Fig. 6E). This prevention of AML amplification in vivo can also be observed by TNF-α, NF-κB, or BCL-2 inhibition (Fig. 6F), supporting the chemical toxicity enhanced by targeting TIFA, possibly due to blocking TNF-α Dependent NF-κB survival pathway. In addition, the inventors also directly injected U937 stable cells into nude mice via IVC to establish an in situ xenograft mode without immunosuppression by chemical or sublethal radiation. Flow cytometric analysis of the recipient mouse BM cells showed that targeting TIFA significantly promoted clearance of transplanted human CD45 ⁺ CD33 ⁺ myelocytes after cytarabine treatment (Fig. 6G). Constrained leukemia myeloid cells together with fluctuating interleukins show that molecular targeting of TIFAs can disrupt positive feedback between NF-κB and TNF-α, which is required for AML progression (43).

TIFA is a relatively novel member of the NF-κB signaling pathway. Early studies by the inventors revealed that TIFA oligomerization plays a key role in promoting the NF-κB signaling pathway under TNF-α stimulation, and the mechanism is through the intermolecular binding between the phosphorylated Thr9 and FHA domains of TIFA dimers ( 20). A recent study showed that the phosphorylation-dependent oligomerization of this TIFA can also be driven by the monosaccharide heptose-1,7-diphosphate (HBP) derived from Gram-negative bacteria, resulting in the activation of innate immunity (23 ). In another document (44), the inventors further show that TIFAs mediate innate immune responses via assembly of NLRP3 inflammatory bodies. In the present study, the inventors found that TIFA is a functional link between the Aurora A signaling axis in AML and the pro-survival factor of NF-κB regulation. In addition, the inventors demonstrated that TIFA inhibition interferes with leukemic interleukin secretion, confirming the therapeutic potential of TIFA targeting in the treatment of AML, which significantly enhances the chemical toxicity of AML cells and enhances the clearance of leukemia myeloid cells in a xenograft mode. rate.

The inventors analyzed the freshly collected patient samples by western blots to directly assess the amount of TIFA protein, rather than the conventional real-time PCR-based prognostic factor transcription level detection. The inventors observed that the amount of TIFA protein in AML patients was elevated compared to normal donors, similar to its kinase olola A, and confirmed a close correlation between TIFA and NF-κB-driven pro-survival factors. Using immunocytochemical staining of bone marrow cells from cryopreserved patients, the inventors also speculated that the increase in TIFA protein in BM patients was an independent factor for poor prognosis of OS, and with age, WBC count, karyotype, and Other genetic markers are irrelevant. Their observations confirmed that patients with AML with higher TIFA protein content were difficult to cure with chemotherapy, and the CR rate was lower and the OS was poorer. Based on their findings, higher TIFA protein levels in BM can also serve as novel biomarkers for predicting clinical outcomes in AML patients. The expression of TIFA silent or dominant negative TIFA fragments can reduce NF-κB activation, leading to leukemia cell proliferation arrest and increased chemical toxicity.

Ectopic manifestations of DN proteins can antagonize signaling and trigger therapeutic potential in cancer therapy. Specifically, IκB super-inhibitor, which is a DN IκB mutant and ultimately inhibits NF-κB activity, has been shown to selectively increase ALL sensitivity to vincristine treatment, which is regulated by NF-κB. Dead (45). The inventors also observed that the chemosensitivity of leukemia cells is enhanced in the ectopic presentation of the DN fragment of TIFA. The inventors believe that the minimal DN fragment must include the FHA dimerization core plus the N-terminal Thr9 motif to disrupt endogenous TIFA oligomerization or C-terminal TRAF6/TRAF2 interaction motifs to disrupt signaling (20, 22). Interestingly, both transcriptional repression (via TIFA siRNA; Figure 3 and Figure 4) and molecular inhibition (via DN fragment; Figure 5 and Figure 6) showed almost identical targeting efficacy for TIFA, showing TIFA in their cells. An irreplaceable and non-redundant role in the process.

The inventors have consistently confirmed the efficacy of targeting TIFA in the bone marrow and leukemia lymphocyte lineage using AML, ALL, and CML cell lines, indicating that the TIFA-driven NF-κB signaling axis plays an important role in the development of leukemia. In addition, positive feedback between NF-κB and TNF-α has also been described recently for leukemic initiation cells (LICs) that promote leukemia (43). LIC-enriched leukemia BM cells exhibit constitutively activated NF-κB signaling, which is considered to be the cause of recurrence after AML remission (43). The inventors' Figure 6C results show that treatment of TNF-α does attenuate the cytotoxicity of cytarabine on undifferentiated U937 monocytes, whereas molecular inhibition of TIFA blocks this effect by perturbing NF-κB-dependent survival signaling. (Fig. 5D-E). Leukemia cells have been shown to be difficult to propagate in athymic nude mice due to residual innate immunity (41). However, the inventors observed that the homing of their undifferentiated monocytes in the mouse femur was inhibited by treatment with cytarabine (Fig. 6E), and TIFA inhibition significantly promoted this effect. Although this xenograft mode may not indicate the true dryness of LICs, it may mean that TIFA is required for the maintenance of undifferentiated leukemia cells under chemical drug therapy, and long-term expansion and self-renewal of LICs can be attenuated by targeting TIFAs. ability.

Deregulation of innate immunity and inflammatory mediation can lead to myelodysplastic syndrome (MDS), a cluster of hematological malignancies that overexpress BM and progress to AML (46). The formation of NF-κB signaling may cause pathogenic activation of inflammatory mediators and pro-survival factors, leading to dysregulation of MDS BM precursor cells, whereas inhibition of NF-κB activity by normal and MDS BM precursor cells may induce cell apoptosis. Dead (47). Therefore, Bcl-2 and Bcl-X _L can be regarded as indicators of chemical resistance of bone marrow malignancies (including AML) (9, 12, 48, 49). Since TIFA regulates NF-κB-driven innate immunity (23), the role of TIFA in leukemia observed by the inventors in AML is more likely to be via the NF-κB-dependent anti-apoptotic/pro-survival pathway. The inventors used the results of leukemia cell lines consistent with PBMCs derived from AML patients to show that TIFA is required for cell survival during chemotherapy. TIFA of PBMCs in silent patients significantly blocked NF-κB-dependent pro-survival factors, although over-expressing Olola A (Fig. 4C) and treating chemotherapy drugs, the inventors showed in Figure 3C, Figure 4D, Figure 5A, and Figure 5F. The observed loss of chemoresistance was due to disturbed anti-apoptotic/promoting factors Bcl-2 and Bcl-X _L .

The tumor microenvironment is a pre-inflammatory response environment composed of a number of inflammatory factors, including interleukins and chemokines, and this tumor-promoting response essentially contributes to cancer development (50). An increasing number of inhibitors targeting members of the pro-inflammatory TNF superfamily have been shown to be therapeutically effective (51). In view of the important role of TIFA in the signaling chain between TNF-α and NF-κB, its inhibition should attenuate the inflammatory factors regulated by NF-κB transcription. The inventors observed that attenuating CXCL-1 and IL-8 secretions when molecularly targeting TIFAs is associated with delaying leukemia cell growth and reducing chemoresistance (Fig. 6A-B), supporting the two previously suggested inflammatory factors. The tumorigenic function (52, 53).

It has been determined that Aurora A can be overexpressed in AML (16, 18, 54) and its inhibition is considered to be the target treatment for several hematopoietic malignancies (55-57). Although many types of solid tumors often observe gene amplification of Alora A, leading to overexpression and tumor progression (58), it is still unknown whether or not Alora A is upregulated in this way in AML. The inventors determined that TIFAs bind to TNF-α-dependent NF-κB survival signals via functionally required site-specific phosphorylation of aolala A kinase activity, thereby maintaining a positive feedback loop to drive inflammatory responses and support leukemia Chemical resistance of cells (40). The inventors observed enhanced chemical toxicity of AML cells treated with TNF-α, NF-κB, BCL-2, and aolola A inhibitors, which are similar to TIFA transcriptional inhibition and molecular targeting (Fig. 3, 4, 5, 6C). The translational efficacy was further enhanced by the combination of cytarabine in vivo with inhibitors of TNF-α, NF-κB, or BCL-2 (Fig. 6F).

Overall, the inventors' findings confirm the functional role of TIFA in supporting Alora A-dependent NF-κB survival and inflammatory pathways, and serve as a molecular basis for leukemia cell growth and chemoresistance in AML. Since the initial treatment with anthracyclines or cytarabine remains unchanged for decades, and due to drug resistance, CR is still insufficient, and future AML treatment with TIFA targeting strategy can provide reduced chemical resistance. The therapeutic advantage of the rate increases the efficacy of conventional chemotherapy and improves long-term clinical outcomes. Sequence information table 4

no

The above summary of the present invention, as well as the following detailed description, To illustrate the invention, the drawings show the preferred embodiments of the present invention. However, it should be understood that the invention is not limited to the precise arrangements and arrangements shown.

The figure below:

Figures 1A, 1B, 1C, and 1D show that TIFA is required for Aurora A-dependent NF-κB signaling. (Fig. 1A) In vitro kinase assay for TIFA phosphorylation. On the upper side, transfected with Flag-labeled Aurora A (Flag-Aola A) or Aurora A siRNA (siAurora A), or treated with Aurora A inhibitor (VX-680 and MK-5108) Protein Western blot analysis of 293T cell lysates. Aurora A pT288 represents an activated kinase (18). On the left, an in vitro kinase assay using recombinant TIFA wild-type (WT) and non-phosphorylated T9A mutant proteins, treated with anolase A inhibitor and TNF-α in the presence of [γ- ³² P]ATP 293T cell lysate culture. WCE, whole cell extract; Si, Aurora A siRNA; VX, VX680; MK, MK5108; PPTase, alkaline dephosphatase. On the right side, quantitative and statistical analysis of three independent experiments. (Fig. 1B) The upper side, immunoprecipitation of olaline A of 293T cell lysate, which was transfected with HA-labeled TIFA under different time TNF-α stimulation. Lower side, Western blot analysis for all lysates on the upper side. S, supernatant; P, precipitate. (Fig. 1C) Western blot analysis of 293T cell lysates on the left side, transfected with Flag-tagged Alora A and TIFA siRNA. Right, as on the left, except for U937 cells transfected with TIFA siRNA and treated with 10 ng/mL TNF-α for 30 min. (Fig. 1D) As in (Fig. 1C), NF-κB activity was analyzed using a luciferase reporter gene assay in addition to transfection of cells treated with pNF-κB-Luc plastids. The representations represent the relative brightness units (RLU) normalized to the control group that were either non-transfected or non-TNF-[alpha] stimulated.

Figure 2 shows that overexpression of TIFA protein is associated with pro-survival factors and poor prognosis in AML patients. Representative protein expression of TIFA and pro-survival factors in the normal group and the AML PBMCs group (n=16 in each group). Western blot analysis results (Fig. 4C) were quantified and normalized to the corresponding amount of b-actin, while in the normal control group, the relative intensities of the various signaling factors were normalized to intermediate values. Horizontal strip, intermediate value.

Figures 3A, 3B, and 3C show that TIFA silently enhances chemical toxicity to AML cell lines. (Fig. 3A) The cell survival time course of four AML cell lines at the time of TIFA silencing was examined by the WST-1 assay. The graph represents the relative cell viability normalized on day 0 in three independent experiments. (Fig. 3B) graphical representation of the relative cell viability normalized by the simulated control on day 4 in the corresponding experiment of experiment (Fig. 3A) and treatment of VX680. (Fig. 3C) AML cell lines for (Fig. 3A) and (Fig. 3B) were analyzed for cell viability under chemotherapeutic drug treatment using the WST-1 assay. The figure represents the calculated chemotherapeutic drug IC50. siCon, control siRNA; siTIFA, TIFA siRNA.

Figures 4A, 4B, 4C, 4D, and 4E show that TIFA silently enhances chemical toxicity to AML PBMCs. (Fig. 4A) The WST-1 assay was used to detect the cell survival time of eight AML PBMCs at TIFA silence. The graph represents the relative cell viability normalized on day 0. (Fig. 4B) The graph represents the relative cell viability normalized (Fig. 4A) in the corresponding experiment with normal PBMCs and the VX680 treated group (n=8 in each group) normalized on day 4 of the mock control group. (Fig. 4C) Western blot analysis of 8 AML PBMCs at TIFA silence and 8 normal PBMCs. (Fig. 4D) AML PBMCs for (Fig. 4A) and (Fig. 4B) were analyzed for cell viability under chemotherapeutic drug treatment by WST-1 assay. The figure represents the calculated chemotherapeutic drug IC50. (Fig. 4E) Four AML PBMCs (AML #9, 10, 13, and 15) were treated with TIFA silently treating 10 μM etoposide, 10 nM idarubicin, or 10 μM cytarabine (cytarabine), or treated with VX680, and analyzed by flow cytometry. The graph represents the percentage of cells stained positive for Annexin V and propidium iodide. Horizontal strip, intermediate value. siCon, control siRNA; siTIFA, TIFA siRNA.

Figures 5A, 5B, 5C, 5D, 5E, and 5F show that the molecular targets of TIFA inhibit TIFA-mediated NF-κB activation. (FIG. 5A) The graph represents the calculated chemotherapeutic drug IC50 of the AML cell line under TIFA silence and siRNA-resistant (siR) TIFA wild type (WT) or T9A mutant overexpression. (Fig. 5B) Schematic diagram of the TIFA dominant negative structure. AML cell lines are each infected with a pseudotyped retrovirus carrying their constructs to produce stable cells. 2Myc, two consecutive Myc-tags. (Fig. 5C) U937 cell lysate stably expressing the TIFA fragment as shown by anti-Myc immunobead immunoprecipitation and Western blot analysis (Fig. 5B). (Fig. 5D) The cell line used for (Fig. 5C) was additionally transfected with pNF-κB-Luc plastid, treated with 10 ng/mL TNF-α for 30 min, and analyzed for NF-κB by luciferase reporter gene assay. active. Luciferase activity was normalized to a non-TNF-α stimulated control group. The graph represents the relative luminance units (RLU) obtained from three independent experiments. (Fig. 5E) The amount of NF-κB signaling factor in the lysate was analyzed by Western blotting (Fig. 5D). (Fig. 5F) AML cell strains stably expressing TIFA fragments as described in a dose-dependent manner of chemotherapeutic drug treatment (Fig. 5B), and cell viability were analyzed using the WST-1 assay. The figure represents the calculated IC50 化疗 of the calculated chemotherapeutic drug obtained from three independent experiments. siCon, control siRNA; siTIFA, TIFA siRNA.

Figures 6A, 6B, 6C, 6D, 6E, 6F, and 6G show that targeting TIFA enhances the clearance of leukemia myeloid cells. (Fig. 6A) The interleukin secretion profile of the conditioned medium collected by 10 μM cytarabine-treated U937-stabilized cells in Fig. 5F was analyzed using an interleukin-antibody array. The circled interleukins were quantified and displayed on the right. (Fig. 6B) The leukemia interleukin line secreted by the cells (Fig. 6A) was quantitatively analyzed by ELISA. The amount of interleukin in the conditioned medium was normalized to the PBS control group, and the graph represents the relative amount of interleukin obtained by three independent experiments. siTIFA, TIFA siRNA transfection. (Fig. 6C) U937 cell line for (Fig. 6A) Cell viability in the presence of 10 ng/mL TNF-α and 10 μM cytarabine was analyzed by WST-1 assay. The experimental results were normalized on day 0 and the graph represents the time course of relative cell viability. (Fig. 6D) U937 cell line for (Fig. 6A) was analyzed by WST-1 assay at 5 mg/mL etanercept, 50 nM bortezomib, or 1 mM ABT-263. Cell viability in the presence of μM cytarabine. The experimental results were normalized on day 0 and the graph represents the time course of relative cell viability obtained from three independent experiments. (Fig. 6E) Upper side, experimental procedure for growth of osteomyeloma of nude mice. The retrovirus-transduced U937 cells used in the mice were injected subcutaneously (Fig. 6A) and treated with cytarabine. The time course of tumor volume. Figure 6 (F) Upper side, experimental procedure for growth of bone marrow sarcoma in nude mice. The retrovirus-transduced U937 cells used in the mouse subcutaneous injection (Fig. 6A) were injected intraperitoneally with 5 mg/kg etanercept, 0.5 mg/kg bortezomib, or 50 mg/kg ABT- 263 binds 50 mg/kg cytarabine. Time course of tumor volume (n = 4 for each group). (Fig. 6G) As in (Fig. 6E), U937 stable cells were injected via IVC and analyzed for BM implantation. On the left side, BM cells of treated mice were evaluated by flow cytometry using anti-human CD45 and CD33 antibodies. On the right side, plot the percentage of human CD45 ⁺ CD33 ⁺ cells analyzed on the left side (n = 7 for each group). Horizontal strip, intermediate value. SC, subcutaneous injection; IP, intraperitoneal injection; IVC, injection of cells via the inferior vena cava.

Figure 7 shows an alignment of TIFA amino acid sequences of different species in a particular embodiment, including SEQ ID NO: 1 from Homo sapiens, SEQ ID NO: 2 from rabbits, SEQ ID NO: 3 from canines SEQ ID NO: 4 is derived from mouse, SEQ ID NO: 5 is derived from rat, SEQ ID NO: 6 is derived from gray hamster, SEQ ID NO: 7 is derived from American beaver, SEQ ID NO: 8 is derived from Guinea pig, SEQ ID NO: 9 derived from European cattle, SQE ID NO: 10 derived from goat, SQE ID NO: 11 derived from pig, and SEQ ID NO: 12 derived from horse, wherein F1 fragment is indicated (position 1-45, Including an N-terminal phosphorylation/oligopolymerization motif and a b1-b2 chain), an F2 fragment (positions 46-103, comprising a b3-b8 chain), and an F3 fragment (positions 104-184, comprising a b9-b12 chain), And a pThr-recognition motif located in the F2 domain (eg, a residue located at about position 51-89 of SEQ ID NO: 1, the bottom line) and a C-terminal loop sequence located in the F3 domain (eg, located in SEQ ID NO: 1) About position 144-184).

Figure 8 shows the topological profile of the b-sandwich structure of TIFA, which consists of twelve b-sheets.

no

Claims (31)

A TIFA inhibitor comprising an isolated TIFA peptide fragment comprising a dimeric core peptide segment, the N-terminus binding to a Thr9 peptide segment or the C-terminus binding to a TRAF6/TRAF2 interaction peptide segment, wherein The Thr9 peptide segment comprises an N-terminal phosphorylation/oligopolymerization motif MX ₁ X ₂ FEDX ₃ DTX ₄ EX ₅ X ₆ T, the sequence of which is set forth in SEQ ID NO: 13, wherein X ₁ is silk Aminic acid (S) or threonine (T), X ₂ is serine (S), threonine (T), aspartic acid (N), X ₃ is alanine (A) or guanamine Acid (V), X ₄ is glutamic acid (E) or glutamine proline (Q), X ₅ is threonine (T) or methionine (M), and X ₆ is proline ( V) or leucine (L); (ii) the dimeric core peptide segment comprises six b chains comprising (a) a b3 chain having a b3 motif VKFG; (b) a b4 chain, It has a b4 motif YX ₁ F, wherein X ₁ is threonine (T) or isoleucine (I); (c) a b5 chain having a b5 motif QFX ₁ LX ₂ X ₃ F, wherein X ₁ is serine (S), proline (V), or alanine (A), X ₂ is glutamine proline (Q) or histidine (H), and X ₃ is leucine ( L), proline (P), or valine (V); (d) a b6 chain with b6 phantom SFEIKN; (e) a chain b7, b7 having LIV motif; and (f) a chain b8, b8 die body having a X ₁ X ₂ L, wherein X ₁ is arginine (R), bran An amine proline (Q), or an amide acid (K), and X ₂ is glutamic acid (E) or threonine (T); wherein each of the b3 to b8 chains utilizes a plurality of internal rings The sequence is sequentially linked from the N-terminus to the C-terminus to the next; and (iii) the TRAF6/TRAF2 interaction peptide segment comprises four b-chains comprising (a) a b9 chain having a b9 motif LX ₁ KX ₂ D, wherein X ₁ is aspartic acid (N) or histidine (H), and X ₂ is methionine (M), leucine (L), proline (V) Or isoleucine (I); (b) a b10 chain having b10 motif X ₁ CX ₂ X ₃ RF, wherein X ₁ is arginine (R) or lysine (K), X ₂ Is methionine (M) or leucine (L), and X ₃ is valine (V), leucine (L), or isoleucine (I); (c) a b11 chain, It has b11 motif YQX ₁ LX ₂ X ₃ X ₄ E, wherein X ₁ is phenylalanine (F) or isoleucine (I), X ₂ is methionine (M), leucine (L) , valine (V), or isoleucine (I), X ₃ is glutamic acid (E) or bran amine acid amide (Q), and X ₄ is from amine (K) or arginine (R &lt); and (d) a chain b12, b12 die body having _{X 1 FX 2 X 3 X 4} FX 5 X 6, wherein X ₁ is phenylalanine (F) or serine (S), X ₂ is glutamic acid (E) or glutamine proline (Q), X ₃ is threonine (T) or isoleucine (I), and X ₄ is glutamine proline ( Q), histidine (H), or glutamic acid (E), X ₅ is isoleucine (I), valine (V), serine (S), or phenylalanine (F), And X ₆ is leucine (L), methionine (M), or phenylalanine (F); wherein each of the b9 to b12 chains utilizes a plurality of internal loop sequences from N to C a sequence linked to the next; and a C-terminal loop sequence linked to the C-terminus of the b12 chain; wherein the isolated TIFA peptide fragment does not contain the Thr9 peptide segment and the TRAF6/TRAF2 interaction peptide segment By. The TIFA inhibitor of claim 1, wherein the N-terminal Thr9-phosphorylation motif is located at positions 1-14 corresponding to SEQ ID NO: 1; the b3 motif is located at a position corresponding to SEQ ID NO: 1. 47-50 where; the b4 modular system is located at a position corresponding to positions 58-60 of SEQ ID NO: 1; the b5 modular system is located at a position corresponding to positions 69-75 of SEQ ID NO: 1: the b6 modular system Located at position 84-89 corresponding to SEQ ID NO: 1; the b7 motif is located at position 96-98 corresponding to SEQ ID NO: 1; the b8 motif is located at a position corresponding to SEQ ID NO: 1. 101-103; the b9 mode system is located corresponding to position 106-110 of SEQ ID NO: 1; the b10 mode system is located corresponding to position 114-119 of SEQ ID NO: 1; the b11 mode system Located at a position corresponding to positions 122-129 of SEQ ID NO: 1; the b12 motif system is located at a position corresponding to positions 136-143 of SEQ ID NO: 1; and/or the C-terminal loop sequence is located corresponding to SEQ ID NO: 1 position 144-184. The TIFA inhibitor of claim 1, wherein the Thr9 peptide segment further comprises two b-chains comprising (a) a b1 chain having a b1 motif X ₁ LX ₂ X ₃ TX ₄ Y, wherein X ₁ Is cysteine (C) or serine (S), X ₂ is glutamine proline (Q) or histidine (H), X ₃ is methionine (M), isoleucine (I), leucine (L) or valine (V), and X ₄ is valine (V), isoleucine (I) or leucine (L); and (b) a b2 a chain having a b2 motif at position X ₁ X ₂ X ₃ P, wherein X ₁ is glutamic acid (E) or aspartic acid (D), and X ₂ is lysine (K) or sulphate ( T), and X ₃ is leucine (L) or phenylalanine (F), wherein the b1 chain is sequentially linked from the N-terminus to the C-terminus to the b2 chain using an internal loop sequence. The TIFA inhibitor of claim 3, wherein the b1 modular system is located at a position corresponding to position 15-21 of SEQ ID NO: 1; and/or the b2 modular system is located at a position corresponding to SEQ ID NO: 1 39-42 Where. The TIFA inhibitor of claim 1, wherein the Thr9 peptide segment comprises an amino acid sequence corresponding to positions 1-45 of SEQ ID NO: 1, or the amino acid sequence has at least 70 % identical; the dimeric core peptide segment comprises an amino acid sequence corresponding to positions 46-103 of SEQ ID NO: 1, or the amino acid sequence is at least 70% identical to the sequence; And/or the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence corresponding to positions 104-184 of SEQ ID NO: 1, or the amino acid sequence is at least 70% identical to the sequence . The TIFA inhibitor of claim 1, wherein the Thr9 peptide segment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-34; the dimeric core peptide segment comprises a An amino acid sequence selected from the group consisting of SEQ ID NOs: 35-46; and/or the TRAF6/TRAF2 interaction peptide segment comprises an amino acid sequence selected from SEQ ID NOs : Group of 47-58. The TIFA inhibitor of claim 1, which is an F1-F2 peptide fragment comprising the Thr9 peptide segment and the dimeric core peptide segment, wherein the Thr9 peptide segment C-terminus is linked to the dimerization The N-terminus of the core peptide segment. The TIFA inhibitor of claim 7, which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 71-82. The TIFA inhibitor of claim 1, which is an F2-F3 peptide fragment comprising the dimeric core peptide segment and the TRAF 6 interaction peptide segment, wherein the C-terminal of the dimeric core peptide segment The line is linked to the N-terminus of the TRAF 6 interaction peptide segment. A TIFA inhibitor according to claim 9 which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83-94. A recombinant nucleic acid comprising a nucleotide sequence encoding a TIFA peptide fragment of claim 1. A recombinant nucleic acid according to claim 11, which is a vector, in particular a viral vector. A composition comprising the TIFA peptide fragment of claim 1 and a physiologically acceptable carrier. A use of the TIFA peptide fragment of claim 1, the nucleic acid encoding the same, or a composition comprising the peptide or the nucleic acid, for use in the manufacture of a medicament for treating a disease associated with TIFA activation in an individual in need thereof or Condition. The use of claim 14, wherein the disease or condition is a cancer or inflammatory disease associated with TIFA activation, or a cancer or inflammatory disease associated with interleukin-stimulated NF-κB activation. The use of claim 14, wherein the disease or condition is cancer, selected from the group consisting of leukemia, liver cancer, and gastric cancer. The use of claim 14, wherein the disease or condition is an inflammatory disease selected from the group consisting of hepatitis, atherosclerosis, pulmonary hypertension, cardiomyopathy, rheumatoid arthritis, inflammatory bowel disease, and Fabry disease. (Fabry disease) group. The use of claim 14, wherein the individual has cancer, and the peptide fragment, the encoding nucleic acid, and the composition comprising the peptide or the encoding nucleic acid are administered to the individual in combination with an anti-cancer therapy. The use of claim 18, wherein the anticancer treatment comprises administering an anticancer agent selected from the group consisting of etoposide, idarubicin, cytarabine, sorafenib ( Group of sorafenib), regorafenib, bleomycin, bortezomib, busulfan, and obatoclax, or radiation Irradiation. The use of claim 19, wherein the peptide fragment, the encoding nucleic acid, or the composition is administered in an amount effective to increase the cytotoxicity of the anti-cancer treatment. A method for predicting the prognosis of leukemia comprising determining the amount of expression of a TRAF-interacting protein (TIFA) having a FHA domain from a leukemia patient sample, and determining the prognosis of the leukemia of the patient based on the amount of TIFA in the sample. The increase in the amount of TIFA in this sample represents a poor prognosis. A use of a TIFA antagonist for the manufacture of a medicament for inhibiting the activation of interleukin-stimulated NF-κB in an individual. The use of claim 22, wherein the TIFA antagonist is an interfering nucleic acid or a TIFA dominant negative inhibitor that targets TIFA. The use of claim 23, wherein the TIFA dominant negative inhibitor is a TIFA peptide fragment as claimed in claim 1. The use of claim 22, wherein the individual is a patient having a disease or condition associated with interleukin-stimulated NF-κB activation. The use of claim 22, wherein the individual is a patient suffering from an inflammatory disease selected from the group consisting of hepatitis, atherosclerosis, pulmonary hypertension, cardiomyopathy, rheumatoid arthritis, inflammatory bowel disease, and Fabry. A group consisting of Fabry disease. The use of claim 22, wherein the individual is a patient having cancer. The use of claim 27, wherein the TIFA antagonist is administered to the patient in combination with an anti-cancer therapy. The use of claim 28, wherein the TIFA antagonist is administered in a single dose, which is effective to increase the cytotoxicity of the anticancer treatment to cancer cells as compared to anti-cancer treatment without the TIFA antagonist. The use of claim 29, wherein the cancer cell line is selected from the group consisting of lung cancer cells, liver cancer cells, leukemia cells, breast cancer cells, testicular embryonic cancer cells, and osteosarcoma cells. The use of claim 28, wherein the patient is resistant to the anti-cancer treatment.