Patent Grant
10828377
This application is a 371 of PCT/JP2016/081738, filed Oct. 26, 2016, which claims benefit of Japanese Patent Application No. 2015-210226 filed on Oct. 26, 2015.
The present invention relates to a method for determining whether a subject suffers from, or likely to suffer from malignant lymphoma or leukemia, an agent for treating and/or preventing malignant lymphoma or leukemia, and the like.
Malignant lymphoma is a malignant neoplasm occurring in lymphoreticular tissue. Malignant lymphoma arises from malignant transformation of cells of the lymphoid lineage and is often found as a swollen lymph node, and infiltration into organs is also observed. Malignant lymphoma is a disease that often develops in elderly people, but also develops commonly in adolescents and young adults. The sites where lymphoma develops are lymph nodes, the spleen, and other sites where lymphoreticular cells locate. Malignant lymphoma is tentatively distinguished from lymphocytic leukemia by the site of occurrence and the tumorigenicity, but both diseases are biologically closely related and it may be difficult to clinically distinguish between the diseases.
Meanwhile, leukemia is a disease in which a hematopoietic cell in the process of differentiation becomes a tumor and clonally proliferates. Leukemia is classified roughly into acute leukemia, which progresses acutely with undifferentiated leukemia blast cells increase, and chronic leukemia, which progresses usually chronically with differentiated leukemia cells increase. Among acute leukemia, in particular, acute lymphoblastic leukemia (ALL) is a disease that develops commonly, regardless of age. While childhood ALL has a relatively good prognosis, adolescent and young adult ALL or elderly ALL responds poorly to chemotherapy and there are almost no effective molecular targeted therapies (Non Patent Literatures 1 and 2).
It is considered that methods of diagnosis and therapies for malignant lymphoma and leukemia have not been sufficiently established and a method of rapid diagnosis and an effective therapy have been eagerly demanded.
An object of the present invention is to provide a method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia and an agent for treating and/or preventing the disease.
The present inventors have found that a fusion mutation of a DUX4 gene and an IGH or IGL gene, overexpression of a DUX4 gene, or a fusion mutation of an MEF2D gene specifically occurs in leukemia patients, by exhaustively analyzing sequences of cDNAs prepared from leukemia cells derived from leukemia patients with a next-generation sequencer. Furthermore, the present inventors have found that an inhibitor of DUX4 suppresses the proliferation of cell lines having the fusion mutation of a DUX4 gene and the overexpression of a DUX4 gene.
The present invention is based on the foregoing findings and encompasses the following aspects.
(1) A method for assisting in determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia, comprising:
a detection step of detecting at least one of: a fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene; an overexpression of a DUX4 gene; and a fusion mutation of an MEF2D gene in a sample obtained from the subject; and
a determination step of determining that the subject suffers from or is likely to suffer from malignant lymphoma or leukemia when at least one of the fusion mutations or the overexpression is detected;
wherein the DUX4 gene encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 and
the fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene is a mutation that forms a fusion gene comprising a part of the DUX4 gene on the 5′ terminal side and a part of the immunoglobulin heavy chain or immunoglobulin light chain gene on the 3′ terminal side.
(2) The method according to (1) described above, wherein the polypeptide encoded by the fusion gene of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene comprises:
(i) an amino acid sequence comprising: an amino acid sequence comprising amino acids at positions 1 to 300 and lacking amino acids at positions 410 to 424 in the amino acid sequence set forth in SEQ ID NO: 2 on the N-terminal side; and 2 to 100 amino acids derived from an immunoglobulin heavy chain or immunoglobulin light chain gene on the C-terminal side;
(ii) an amino acid sequence having an identity of 90% or more to the amino acid sequence of (i); or
(iii) an amino acid sequence modified from the amino acid sequence of (i) by addition, deletion, and/or substitution of one or more amino acids.
(3) The method according to (1) or (2) described above, wherein a gene encoding the amino acid sequence set forth in SEQ ID NO: 2 comprises the nucleotide sequence set forth in SEQ ID NO: 1.
(4) The method according to any one of (1) to (3) described above, wherein the fusion mutation of an MEF2D gene is a mutation that forms a fusion gene comprising a part of the MEF2D gene on the 5′ terminal side and a part of the BCL9 gene on the 3′ terminal side; and
a polypeptide encoded by the fusion gene of an MEF2D gene and a BCL9 gene comprises:
(i) an amino acid sequence comprising amino acids at positions 1 to 200 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426 in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side;
(ii) an amino acid sequence having an identity of 90% or more to the amino acid sequence of (i); or
(iii) an amino acid sequence modified from the amino acid sequence of (i) by addition, deletion, and/or substitution of one or more amino acids.
(5) The method according to any one of (1) to (3) described above, wherein the fusion mutation of an MEF2D gene is a mutation that forms a fusion gene comprising a part of the MEF2D gene on the 5′ terminal side and a part of the HNRNPUL1 gene on the 3′ terminal side; and
a polypeptide encoded by the fusion gene of an MEF2D gene and an HNRNPUL1 gene comprises:
(i) an amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side;
(ii) an amino acid sequence having an identity of 90% or more to the amino acid sequence of (i); or
(iii) an amino acid sequence modified from the amino acid sequence of (i) by addition, deletion, and/or substitution of one or more amino acids.
(6) The method according to any one of (1) to (5) described above, wherein the leukemia is acute lymphoblastic leukemia.
(7) The method according to (6) described above, wherein the acute lymphoblastic leukemia is adolescent and young adult acute lymphoblastic leukemia.
(8) A polypeptide comprising any of amino acid sequences of the following (i) to (iii):
(i) an amino acid sequence comprising: an amino acid sequence comprising amino acids at positions 1 to 300 and lacking amino acids at positions 410 to 424 in the amino acid sequence set forth in SEQ ID NO: 2 encoded by a DUX4 gene on the N-terminal side; and 2 to 100 amino acids derived from an immunoglobulin heavy chain or immunoglobulin light chain gene on the C-terminal side;
(ii) an amino acid sequence having an identity of 90% or more to the amino acid sequence of (i); or
(iii) an amino acid sequence modified from the amino acid sequence of (i) by addition, deletion, and/or substitution of one or more amino acids.
(9) The polypeptide according to (8) described above, wherein the amino acid sequence of (i) comprises an amino acid sequence set forth in any of SEQ ID NOs: 10, 12, 14, 16, and 18.
(10) A polynucleotide encoding the polypeptide according to (8) or (9) described above.
(11) A marker for detecting malignant lymphoma or leukemia, consisting of the polypeptide according to (8) or (9) described above or the polynucleotide according to (10) described above.
(12) A kit for detecting malignant lymphoma or leukemia, comprising a primer set comprising a forward primer and a reverse primer for detecting a gene encoding DUX4 consisting of the amino acid sequence set forth in SEQ ID NO: 2 or an antibody that specifically binds to DUX4.
(13) An agent for detecting malignant lymphoma or leukemia, comprising an antibody that specifically binds to DUX4.
(14) A pharmaceutical composition comprising a DUX4 inhibitor as an active ingredient, for treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene and/or an overexpression of a DUX4 gene, wherein
the DUX4 gene encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 and
the fusion mutation of a DUX4 gene and an immunoglobulin heavy chain or immunoglobulin light chain gene is a mutation that forms a fusion gene comprising a part of the DUX4 gene on the 5′ terminal side and a part of the immunoglobulin heavy chain or immunoglobulin light chain gene on the 3′ terminal side.
(15) The pharmaceutical composition according to (14) described above, wherein the inhibitor of DUX4 is at least one agent selected from the group consisting of an inhibitory nucleic acid to DUX4, a neutralizing antibody to DUX4, and a low molecular weight compound.
(16) The pharmaceutical composition according to (15) described above, wherein the inhibitory nucleic acid is siRNA or shRNA.
(17) A polypeptide comprising any of amino acid sequences of the following (i) to (iii):
(i) an amino acid sequence comprising amino acids at positions 1 to 200 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426 in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side;
(ii) an amino acid sequence having an identity of 90% or more to the amino acid sequence of (i); or
(iii) an amino acid sequence modified from the amino acid sequence of (i) by addition, deletion, and/or substitution of one or more amino acids.
(18) The polypeptide according to (17) described above, wherein the amino acid sequence of (i) comprises an amino acid sequence set forth in any of SEQ ID NOs: 20, 22, 24, and 26.
(19) A polypeptide comprising any of amino acid sequences of the following (i) to (iii):
(i) an amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side;
(ii) an amino acid sequence having an identity of 90% or more to the amino acid sequence of (i); or
(iii) an amino acid sequence modified from the amino acid sequence of (i) by addition, deletion, and/or substitution of one or more amino acids.
(20) The polypeptide according to (19) described above, wherein the amino acid sequence of (i) comprises an amino acid sequence set forth in SEQ ID NO: 28.
(21) A polynucleotide encoding the polypeptide according to any of (17) to (20) described above.
(22) A marker for detecting malignant lymphoma or leukemia, consisting of the polypeptide according to any of (17) to (20) described above or the polynucleotide according to (21) described above.
(23) A kit for detecting malignant lymphoma or leukemia, comprising a primer set comprising a forward primer and a reverse primer for specifically detecting the polynucleotide according to (21) described above and/or an antibody that specifically binds to the polypeptide according to any of (17) to (20) described above.
(24) A primer set comprising a forward primer and a reverse primer for specifically detecting a gene encoding a fusion protein of MEF2D and BCL9L, wherein
(1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in SEQ ID NO: 30 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in a sequence complementary to the sequence set forth in SEQ ID NO: 31; or
(2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of a sequence complementary to the sequence set forth in SEQ ID NO: 30 under stringent conditions and the reverse primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of the sequence set forth in SEQ ID NO: 31 under stringent conditions.
(25) A primer set comprising a forward primer and a reverse primer for specifically detecting a gene encoding a fusion protein of MEF2D and HNRNPUL1, wherein
(1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in SEQ ID NO: 32 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides in a sequence complementary to the sequence set forth in SEQ ID NO: 33; or
(2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of a sequence complementary to the sequence set forth in SEQ ID NO: 32 under stringent conditions and the reverse primer consists of nucleotides comprising a 14 to 30 nucleotide sequence that hybridizes to a nucleic acid consisting of the sequence set forth in SEQ ID NO: 33 under stringent conditions.
The specification of the present application encompasses the contents disclosed in JP Patent Application No. 2015-210226, to which the present application claims priority.
A method according to the present invention makes it possible to determine whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia rapidly and easily by detecting a novel marker gene or protein. Moreover, the present invention may provide a pharmaceutical composition for effectively treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation or an overexpression of a DUX4 gene.
1. Method for Determining Disease Based on Mutation or Overexpression of DUX4 Gene
In one aspect, the present invention relates to a method for assisting in determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia, comprising: a detection step of detecting a fusion mutation of a DUX4 (Double homeobox 4) gene and an IGH (immunoglobulin heavy chain) or IGL (immunoglobulin light chain) gene and/or an overexpression of a DUX4 gene in a sample obtained from the subject; and a determination step of determining that the subject suffers from or is likely to suffer from malignant lymphoma or leukemia when the fusion mutation or the overexpression is detected.
As used herein, the malignant lymphoma or leukemia is not limited to a particular kind. Examples of the malignant lymphoma include Hodgkin's lymphoma and non-Hodgkin's lymphoma and preferably a malignant lymphoma is B-cell lymphoma. Examples of the leukemia include acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), etc. and the leukemia is preferably ALL, particularly preferably child or adolescent and young adult (AYA) acute lymphoblastic leukemia, and more preferably adolescent and young adult acute lymphoblastic leukemia (AYA-ALL). As used herein, childhood means subjects aged 0 to 14 years and adolescent and young adult means subjects aged 15 to 39 years.
As used herein, the “subject” may be either of an individual that is suspected to suffering from malignant lymphoma or leukemia, or a normal individual. By applying a method according to the present invention to an individual that is suspected to suffering from malignant lymphoma or leukemia, whether a subject suffers from malignant lymphoma or leukemia can be determined more accurately and, by applying a method according to the present invention to a normal individual, whether a subject suffers from malignant lymphoma or leukemia, or malignant lymphoma or leukemia, or the likelihood that the subject would suffer from malignant lymphoma or leukemia in the future can be determined.
As used herein, the “sample” means a biological sample used in a method according to the present invention. The sample which can be used in the present invention includes, but not limited to, cells or tissues isolated from a living body. Examples of the cells include, for example, peripheral blood cells, lymph and tissue fluids containing cells, hair matrix cells, buccal cells, nasal cavity cells, intestinal tract cells, intravaginal cells, mucosal cells, and expectoration (that may contain alveolus cells or tracheal cells). Examples of the tissues include lesion sites, for example, the lymph node, the bone marrow, the spleen, the thymus, and the liver and, for example, biopsy samples of these tissues may be used.
As used herein, the “genes” encompass, unless otherwise specified, both coding regions that are transcribed into protein and structural genes (cistrons) encompassing coding regions plus untranslated regions that are not transcribed into protein and intron regions. A schematic diagram of a gene herein is illustrated in
The detection step of the method according to the present invention involves detecting a fusion mutation of a DUX4 gene and an IGH or IGL gene, preferably a fusion mutation of a DUX4 gene and an IGH gene, and/or an overexpression of a DUX4 gene. The fusion mutations and overexpression detected in the detection step of the method according to the present invention and detection step will be described in detail below.
<Fusion Mutation>
As used herein, the “DUX4 gene” may be a gene encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence set forth in SEQ ID NO: 2. Moreover, the DUX4 gene may be a gene encoding a polypeptide containing an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 2 by addition, deletion, and/or substitution of one or more amino acids. As used herein, the value of the identity is expressed as a value calculated using a software (for example, FASTA, DANASYS, and BLAST) performing the operation for identity between a plurality of sequences with a default setting. For details of the method for determining the identity, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. Mol. Biol. 215, 403-410, 1990. Moreover, as used herein, the range of “1 or more” is 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3 or 1 or 2. The gene encoding the amino acid sequence set forth in SEQ ID NO: 2 preferably contains the nucleotide sequence set forth in SEQ ID NO: 1.
As used herein, the “fusion mutation of a DUX4 gene and an IGH or IGL gene” means a fusion gene containing a part (preferably 5′ terminal side) of the DUX4 gene on the 5′ terminal side and a part of the IGH or IGL gene on the 3′ terminal side. The fusion mutation of a DUX4 gene and an IGH or IGL gene according to the present invention is generated preferably by the translocation of a partial region of the DUX4 gene to the IGH or IGL locus on genomic DNA as illustrated in
The polypeptide encoded by the fusion gene of a DUX4 gene and an IGH or IGL gene comprises preferably (i) an amino acid sequence comprising amino acids at positions 1 to 300, preferably positions 1 to 316, in the amino acid sequence set forth in SEQ ID NO: 2 and lacking amino acids at positions 410 to 424, preferably positions 409 to 424, on the N-terminal side and further comprising an IGH or IGL gene, preferably 2 to 100 amino acids, preferably 2 to 32 amino acids, derived from the IGH gene on the C-terminal side. In relation with this, since the IGH or IGL gene is composed of a variety of sequences based on the gene rearrangement, the sequences thereof cannot be determined univocally. As used herein, “comprising an amino acid sequence derived from an IGH or IGL gene” means comprising a sequence encoding a protein of the IGH or IGL gene; or a sequence derived from an intron part of the IGH or IGL gene. The fusion polypeptide “comprising a sequence derived from an intron part of the IGH or IGL gene” means, when the DUX4 gene or a part thereof is inserted in an intron part of the IGH or IGL gene and there is a stop codon in the intron part in an open reading frame following from DUX4 to the intron of IGH or IGL, the polypeptide comprising an amino acid sequence derived from a nucleotide sequence from a fusion part between DUX4 and the intron to the stop codon. An example when a polypeptide encoded by a fusion gene of a DUX4 gene and an IGH or IGL gene contains amino acids derived from an intron part of an IGH or IGL gene is illustrated in
The polypeptide encoded by the fusion gene may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above, or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
The polypeptide encoded by the fusion gene has tumorigenic activity. The presence or absence of tumorigenic activity of a polypeptide can be specified by a known method in the art and determined, for example, by introducing a fusion gene into suitable cells (for example, mammalian cells), growing the cells, and observing the presence or absence of the focus formation. For details of an assay for the presence or absence of tumorigenic activity, see, for example, Example 2 herein.
Without wishing to be bound by theory, in one embodiment, the gene fusion of DUX4 can be involved in the induction of malignant lymphoma or leukemia via at least one of the following three mechanisms: (i) increase of the DUX4 transcriptional activity caused by the translocation of DUX4 to the vicinity of the enhancer of IGH or IGL, (ii) reduction of apoptotic activity by deletion of the carboxy terminal of the encoded DUX4 protein, and (iii) translational activation of the DUX4 gene by a polyadenylation signal derived from IGH or IGL.
<Overexpression>
As used herein, the “overexpression” means expression that is statistically significantly higher than a reference. Examples of the reference include the amount of expression of the gene or protein obtained from a plurality (for example, 3 or more, 4 or more, or 5 or more) of normal subjects known not to suffer from malignant lymphoma or leukemia. The overexpression of DUX4 gene or protein means expression, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, for example, 10 times or more as high as the reference.
<Detection Step>
The step of detecting a fusion mutation of a DUX4 gene and an IGH or IGL gene in the method according to the present invention comprises for example, a step of detecting the fusion gene in the genome, mRNA (or cDNA derived from mRNA) expressed from the fusion gene, or a polypeptide encoded by the fusion gene. Similarly, the step of detecting overexpression of a DUX4 gene comprises, for example, a step of detecting mRNA (or cDNA derived therefrom) expressed from the gene or a polypeptide encoded by the gene.
The detection of a gene in the genome may be performed using genomic DNA extracted, for example, by a known method. The extraction of genomic DNA may be performed using a commercially available DNA extraction kit. Moreover, the step of detecting mRNA expressed from the fusion gene may be performed by using mRNA or by using cDNA reverse-transcribed from mRNA extracted from the sample. The extraction and the reverse transcription of mRNA may be performed by using, for example, commercially available mRNA extraction and reverse transcription kits.
The step of detecting the fusion gene in the genome, or mRNA expressed from the fusion gene, or mRNA expressed from a DUX4 gene may be performed according to a known method for gene analysis (for example, PCR assay commonly used as a method for gene detection, RT-PCR, Real-time PCR, LCR (Ligase chain reaction), LAMP (Loop-mediated isothermal amplification), a microarray method, Northern hybridization, dot blotting, in situ hybridization, and an analysis by a next-generation sequencer, and the like). For example, a gene amplification technique using a suitable primer or a hybridization technique with a suitable probe, or the like is used, using a nucleic acid extracted from a sample derived from a subject, for example, mRNA. The detection of a DNA or mRNA in the genome is performed qualitatively or quantitatively and, when performed quantitatively, the amount of DNA or mRNA may be measured, for example, by a method for the gene amplification reaction.
The primer used for detecting the fusion gene or DUX4 gene in this detection step is not particularly limited, as long as they can be used to specifically amplify the fusion gene or DUX4 gene, and can be designed based on a nucleotide sequence of the fusion gene or DUX4 gene or mRNA expressed therefrom. Similarly, the probe used for detecting the fusion gene or DUX4 gene in this detection step is not particularly limited, as long as they can specifically hybridize to the fusion gene or DUX4 gene, and can be designed based on a nucleotide sequence of the fusion gene or DUX4 gene or mRNA expressed therefrom. Examples of the primer and probe that can be used for detecting the fusion gene or DUX4 gene include the primer sets and probes described in “3. Primer, probe, and antibody for detecting mutation or overexpression of DUX4 gene” described below.
The presence or absence of the fusion gene of a DUX4 gene and an IGH or IGL gene can be confirmed by a method suitable for each technology when the sequence of the fusion gene is known. For example, in the PCR method, the presence or absence of the mutation may be detected by analyzing the PCR product by agarose gel electrophoresis and confirming by ethidium bromide staining and the like whether an aimed size of amplification fragment has been obtained. For example, if an amplification fragment having a size corresponding to the fusion gene or mRNA expressed from the fusion gene is obtained, then it is presumed that the fusion gene is present in the sample obtained from the subject. As needed, the amplified fragment may be further sequenced. In this way, the presence or absence of the fusion gene can be detected. Moreover, when the sequence of the fusion gene is not known, the presence or absence of the fusion gene of a DUX4 gene and an IGH or IGL gene can be specified by sequencing the terminal of the DUX4 gene by 3′-RACE in candidate specimens that may contain the fusion of a DUX4 gene and an IGH or IGL gene or exhaustive analysis of cDNAs from cells in the candidate specimens by a next-generation sequencer. The candidate specimens can be specified by, for example, the step of detecting overexpression of the fusion genes described below.
The step of detecting a polypeptide encoded by the fusion gene may be performed by, for example, preparing a lysate derived from a sample obtained from a subject and conducting an immunological assay or an enzyme activity assay and the like for a polypeptide encoded by the fusion gene contained therein using an anti-DUX4 antibody or using an antibody to a polypeptide encoded by the fusion gene. In particular, use of the anti-DUX antibodies described in “3. Primers, probes, and antibodies for detecting mutation or overexpression of DUX4 gene” described below is preferred. Preferably, techniques such as enzyme immunoassay, two-antibody sandwich ELISA, fluorescent immunoassay, radioimmunoassay, Western blotting, and the like using a monoclonal antibody or polyclonal antibody specific to a polypeptide encoded by the fusion gene may be used.
Similarly, the presence or absence of overexpression of the DUX4 gene can be confirmed by a method suitable for each technology. The overexpression of the DUX4 gene can be detected, for example, in Real-time PCR by amplifying a sample obtained from a subject with a fluorescence-labeled primer and comparing the fluorescence intensity with the value of a reference. The overexpression of DUX4 can be detected by, for example, immunostaining cells or tissue with an antibody to DUX4 and detecting the presence or absence of the fluorescence intensity or difference from a reference. Examples of the reference include samples obtained from a plurality (for example, 3 or more, 4 or more, or 5 or more) of normal subjects known not to suffer from malignant lymphoma or leukemia.
The method according to the present invention may be used in combination with other methods (for example, X-ray radiography, endoscopy, and the like). By combining the method with another method of determining a disease or a method of diagnosis, the accuracy of the method of determination according to the present invention can be increased.
2. DUX4 Fusion Polypeptide and Polynucleotide
In one aspect, the present invention relates to a polypeptide comprising (i) an amino acid sequence comprising: an amino acid sequence comprising amino acids at positions 1 to 300, preferably positions 1 to 316, in the amino acid sequence set forth in SEQ ID NO: 2 coded by the DUX4 gene and lacking amino acids at positions 410 to 424, preferably positions 409 to 424 on the N-terminal side; and comprising 2 to 100 amino acids, preferably 2 to 32 amino acids, derived from an IGH or IGL gene on the C-terminal side. Examples of the amino acid sequence of (i) include amino acid sequences set forth in any of SEQ ID NOs: 10, 12, 14, 16, and 18.
In one embodiment, the present invention relates to a polypeptide comprising (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
The polypeptide according to the present invention has tumorigenic activity. The presence or absence of the tumorigenic activity of the polypeptide can be specified by a known method in the art and determined, for example, by introducing a fusion gene into suitable cells (for example, mammalian cells), growing the cells, and observing the presence or absence of the focus formation.
The polypeptide according to the present invention can be prepared from, for example, a sample derived from a subject determined to have the fusion gene or from a host cell into which a vector containing the fusion gene or fragment thereof is introduced. The purification of the peptide can be accomplished by performing a known method, for example, ammonium sulfate precipitation, separation by precipitation with an organic solvent (ethanol, methanol, acetone, etc.), chromatography such as ion exchange chromatography, isoelectric chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using a substrate, an antibody, or the like, reverse-phase column chromatography, HPLC, and the like, filtration treatment such as microfiltration, ultrafiltration, reverse osmosis, and the like alone or in combination.
In one embodiment, the present invention relates to a polynucleotide encoding a polypeptide according to the present invention. Examples of such a polynucleotide include a polynucleotide comprising: (i) a nucleotide sequence set forth in any of SEQ ID NO: 9, 11, 13, 15, and 17; (ii) a nucleotide sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the nucleotide sequence of (i) described above; or (iii) a nucleotide sequence modified from the nucleotide sequence of (i) described above by addition, deletion, and/or substitution of one or more nucleotides.
Examples of a method for preparing a polynucleotide according to the present invention include (1) a method using polymerase chain reaction (PCR); (2) a method involving selecting a transformant containing a desired polynucleotide from transformants transformed with a cDNA library; and (3) a method of chemical synthesis. For example, by performing RT-PCR with mRNA extracted from a sample obtained from a subject determined to have the fusion gene, a polynucleotide encoding a fusion polypeptide according to the present invention can be prepared.
Moreover, in one embodiment, the present invention relates to a marker for detecting malignant lymphoma or leukemia, consisting of the polypeptide or polynucleotide.
3. Primers, Probes, and Antibodies for Detecting Mutation or Overexpression of DUX4 Gene
In one aspect, the present invention relates to a kit for detecting malignant lymphoma or leukemia. The kit according to the present invention comprises at least one of a primer set comprising a forward primer and a reverse primer for detecting a gene encoding DUX4 consisting of the amino acid sequence set forth in SEQ ID NO: 2, a probe for detecting the gene, and an antibody that specifically binds to DUX4. The kit according to the present invention may comprise 2 or more of the at least one of the primer set, the probe, and the antibody.
The primer set for detecting a gene encoding DUX4 is not particularly limited, as long as it can be used to specifically detect the DUX4 gene and examples thereof include a forward primer and a reverse primer wherein (1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in SEQ ID NO: 29 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in a sequence complementary to the sequence set forth in SEQ ID NO: 29; or (2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes to a nucleic acid consisting of a complementary sequence set forth in SEQ ID NO: 29 under stringent conditions and the reverse primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes to a nucleic acid consisting of the sequence set forth in SEQ ID NO: 29 under stringent conditions. As used herein, the term “stringent conditions” means conditions in which so-called specific hybrid is formed and no nonspecific hybrid is formed. The stringent conditions can be determined as appropriate, for example, with reference to Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions may be set according to a temperature and a salt concentration contained in a solution in Southern hybridization and a temperature and a salt concentration contained in a solution in the washing step in Southern hybridization. More particularly, examples of the stringent conditions include a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68° C., preferably 42 to 65° C. More specifically, examples include 5×SSC (83 mM NaCl, 83 mM sodium citrate) at a temperature of 42° C.
The probe for detecting the gene encoding DUX4 is not particularly limited, as long as it can be used to detect the DUX4 gene, and preferably composed of, for example, (1) a polynucleotide that hybridizes to a polynucleotide consisting of at least 14, for example 20, preferably 30, consecutive nucleotide sequence in SEQ ID NO: 29 under stringent conditions, or (2) a polynucleotide that hybridizes to a polynucleotide consisting of a sequence complementary to at least 14, for example 20, preferably 30, consecutive nucleotide sequence in SEQ ID NO: 29 under stringent conditions.
The primer and probe can be prepared by a known method known those skilled in the art, for example but not limited to, a method of chemical synthesis.
The antibody that specifically binds to DUX4 may be a commercially available product or may be prepared by a method known to those skilled in the art. Examples of the antibody include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies (scFVs), humanized antibodies, fully human antibodies, and antibody fragments such as Fab, Fab′, F(ab′)2, Fc, Fv, and the like. Preferable examples include polyclonal or monoclonal antibodies.
The antibody according to the present invention, for example, a polyclonal antibody and a monoclonal antibody can be prepared using a target protein or a partial fragment thereof, for example, peptide containing SEQ ID NO: 41 or a cell expressing them, as a sensitized antigen in a method well known to those skilled in the art (E. Harlow et al. (Ed.), “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, 1988).
The polyclonal antibody can be obtained, for example, by administering an antigen such as a DUX4 protein or a fragment thereof and the like to a mammal such as a mouse or a rabbit, collecting blood from the mammal, and purifying the antibody. The method of immunization is well known to those skilled in the art and can be performed, for example, by administering an antigen one or more times. Moreover, the antigen (or a partial fragment thereof) may be dissolved in a suitable buffer solution containing a commonly used adjuvant such as the complete Freund's adjuvant and used to increase the immunogenicity. The method for purifying an antibody is also known for those skilled in the art. For example, the polyclonal antibody may be purified by conducting one or more of centrifugation, precipitation using ammonium sulfate and the like and chromatography, affinity chromatography using, for example, an antigen peptide, and the like.
A method for producing a monoclonal antibody includes the hybridoma method. In the hybridoma method, first, a mammal is immunized in a way similar to the production of a polyclonal antibody. The spleen is subsequently extracted from the immunized animal that has been sensitized to obtain B cells. Then, the B cells were fused with myeloma cells according to a conventional method to generate antibody-producing hybridoma. As a method for fusing cells, any method known for those skilled in the art may be selected and used. The selection of the hybridoma may be performed by culturing cells for a suitable period of time in HAT medium (hypoxanthine, aminopterin, and thymidine-containing medium) according to a conventional method. Next, screening for and cloning of antibody-producing hybridoma of interest are conducted. The screening can be performed by a known method for detecting an antibody such as ELISA and the cloning may be performed by a method known to those skilled in the art such as limiting dilution. Subsequently, obtained hybridoma may be cultured in a suitable culture liquid and a monoclonal antibody can be purified from the culture liquid by salt precipitation, gel filtration, ion exchange chromatography, affinity chromatography, or the like.
The primer set, probe, and antibody described above may have a label detectable by chemical or physical detection means to further facilitate the detection. Examples of the substance used as a label include fluorescent substances, enzymes, radioisotopes, luminescent substances, and the like. Examples of the fluorescent substances include fluorescamine, fluorescein isothiocyanate, and the like, examples of the enzymes include peroxidase, alkaline phosphatase, and the like, examples of the radioisotopes include 125I, 131I, 3H, and the like, and examples of the luminescent substances include luciferin, luminol, derivatives thereof, and the like.
The kit according to the present invention may comprise, for example, a buffer, an enzyme, an instruction, and the like, in addition to the primer set or probe described above.
In one aspect, the present invention relates to an agent for detecting malignant lymphoma or leukemia, comprising an antibody that specifically binds to DUX4. The agent for detecting malignant lymphoma or leukemia according to the present invention may be used as a reagent for sample analysis and the like or for the diagnosis of malignant lymphoma or leukemia.
4. Pharmaceutical Composition Comprising Inhibitor of DUX4
In one aspect, the present invention relates to a pharmaceutical composition comprising a DUX4 inhibitor as an active ingredient, for treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation of a DUX4 gene and an IGH or IGL gene and/or an overexpression of a DUX4 gene.
The inhibitor of DUX4 is not particularly limited, as long as it can inhibit the activity of DUX4, and examples thereof include inhibitory nucleic acids to DUX4, neutralization antibodies to DUX4, and low molecular weight compounds. These inhibitors may be used alone or in combination.
Examples of the inhibitory nucleic acids to DUX4 include nucleic acid aptamers to DUX4 and siRNAs or shRNA to DUX4, preferably siRNAs or shRNAs to DUX4.
As used herein, “siRNA” (small interference RNA) is small double strand RNA consisting of a sense strand (passenger strand) having a nucleotide sequence corresponding to a part of the target gene and an antisense strand thereof (guide strand). As used herein, “shRNA” (short hairpin RNA) refers to a single strand RNA in which the siRNA described above or a sense strand and an antisense strand of a mature double strand miRNA were connected with a short spacer sequence having a suitable sequence. In other words, shRNA forms a hairpin-shaped stem-loop structure as a whole molecule by a sense region and an antisense region forming base-pairing between each other in a molecule to form a stem structure, and a spacer sequence simultaneously forming a loop structure.
Those skilled in the art can design siRNA or shRNA to DUX as appropriate based on the sequence of DUX4. Examples of such a sequence include shRNA containing the sequence set forth in SEQ ID NO: 40. The inhibitory nucleic acid is preferably introduced into a vector and administered to a subject and examples of vectors that can be used for the delivery of the inhibitory nucleic acid include the pLMN vector (Transomic).
The pharmaceutical composition according to the present invention may comprise an active ingredient, for example, an anticancer agent, such as 5-FU (fluorouracil), methotrexate, leucovorin, trastuzumab, and the like, other than the inhibitor of DUX4.
The pharmaceutical composition according to the present invention can be formulated by a method known in the field as a general rule. For example, it can be formulated using a method described in Remington's Pharmaceutical Sciences (Merck Publishing Co. Easton, Pa.). Specific methods of formulation vary depending on the mode of administration. Modes of the administration are roughly classified into the oral administration and the parenteral administration and the mode of administration can be selected as appropriate.
When the pharmaceutical composition according to the present invention is administered orally, a pharmaceutically acceptable carrier may be added.
The “pharmaceutically acceptable carrier” refers to a substance that facilitates the formulation of an agent and the application to the living body is added in a range that does not inhibit or suppress the effect of the active ingredient. Examples thereof include excipients, binders, disintegrators, fillers, emulsifiers, flow additives and modifiers or lubricants.
Examples of the “excipients” include sugar such as monosaccharides, disaccharides, cyclodextrin, and polysaccharides (specifically including, but are not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (for example, sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate), citric acid, tartaric acid, glycine, low, medium, high molecular weight polyethyleneglycol (PEG), Pluronic, or a combination thereof.
Examples of the “binders” include starch paste using starch of corn, wheat, rice, or potato, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone and the like.
Examples of the “disintegrators” include the starch or carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate, or a salt thereof.
Examples of the “fillers” include the sugar and/or calcium phosphate (for example, tricalcium phosphate or calcium hydrogen phosphate).
Examples of the “emulsifiers” include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.
Examples of the “flow additives and modifiers” and “lubricants” include silicates, talc, stearates, or polyethyleneglycol.
Examples of the dosage form of an oral formulation include solid preparations (including tablets, pills, lingusorbs, capsules, and drops), granules, powdered medicines, powders, solutions, and the like. Furthermore, the solid preparations can be formulated into a dosage form with a coating known in the field, for example, sugar coated tablets, gelatin coated tablets, enteric coated tablets, film coated tablets, double-coated tablets, multilayered tablets, as needed. Either of the specific shape and size of the dosage form is not particularly limited as long as it is in the range of dosage forms known in the field for each dosage form.
When the pharmaceutical composition according to the present invention is parenterally administered, specific examples thereof include administration by injection. When the pharmaceutical composition according to the present invention is administered by injection, the pharmaceutical composition may be prepared as a suspension in which the inhibitor is mixed with a pharmaceutically acceptable solvent and a pharmaceutically acceptable carrier was added to the mixture, as needed.
The “pharmaceutically acceptable solvent” may be any of water or another pharmaceutically acceptable aqueous solution or an oily liquid. Examples of the aqueous solution include physiological saline and isotonic solutions containing glucose or another pharmaceutic aid. Example of the pharmaceutic aid include D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and low-concentrations of non-ionic surfactants (for example, polysorbate 80 (TM), HCO-60), polyoxyethylene sorbitan fatty acid esters, and the like. Example of the oily liquid include sesame oil and soybean oil and may be used in combination with for example, benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, a buffer, for example, a phosphate buffer solution, a sodium acetate buffer solution, a soothing agent, for example, hydrochloric acid procaine, a stabilizer, for example, benzyl alcohol, phenol, an antioxidant may be blended.
The injections may be formulated by mixing the inhibitor in combination with a pharmaceutically acceptable excipient, emulsifier, suspension, surfactant, stabilizer, pH regulator, or the like, as appropriate in a unit dose form required in generally accepted pharmaceutical practices.
Examples of injection include intravascular injection, intralymphatic injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like and injection is preferably cardiovascular administration such as intravascular injection or intralymphatic injection, which is systemic administration, but may be a topical administration involving the direct administration into lymphoma.
The content of the active ingredient in the pharmaceutical composition according to the present invention may be an amount that may result in the delivery of the active ingredient to the target site by one time of administration in principle and that has almost or completely no adverse side effects to the subject to which the composition is administered. Such content varies depending on the stage of the disease, the kind of the DUX4 inhibitor, the dosage form of the pharmaceutical composition, the mode of administration, and the like, but is determined as appropriate by a person skilled in the art.
The fusion mutations of the DUX4 gene and overexpression of the DUX4 gene that a subject to which the pharmaceutical composition according to the present invention is administered (subject to which the pharmaceutical composition according to the present invention is to be applied) has are as described in “1. Method for determining disease based on mutation or overexpression of DUX4 gene” described above and therefore not described here.
Examples of the kind of malignant lymphoma and leukemia to be treated and/or prevented with the pharmaceutical composition according to the present invention include the aforementioned Hodgkin's lymphoma and non-Hodgkin's lymphoma for malignant lymphoma and the malignant lymphoma is preferably B-cell lymphoma. Examples thereof for leukemia include the aforementioned acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia and the leukemia is preferably ALL, particularly preferably childhood or adolescent and young adult (AYA) acute lymphoblastic leukemia, and more preferably adolescent and young adult acute lymphoblastic leukemia (AYA-ALL).
The pharmaceutical composition according to the present invention may be administered to, for example, a subject to which the method according to the present invention, that is, the method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia has been applied. Administration of the pharmaceutical composition according to the present invention to a subject suffering from malignant lymphoma or leukemia is expected to have a therapeutic effect and administration of the pharmaceutical composition according to the present invention to a subject who is likely to suffer from malignant lymphoma or leukemia is expected to have a preventive effect.
5. Method for Treating and/or Preventing Malignant Lymphoma or Leukemia Using DUX4 Inhibitor or Pharmaceutical Composition
In one aspect, the present invention relates to a method for treating and/or preventing malignant lymphoma or leukemia in a subject having a fusion mutation of a DUX4 gene and an IGH or IGL gene and/or an overexpression of a DUX4 gene, the method comprising administering the aforementioned DUX4 inhibitor or the aforementioned pharmaceutical composition as an active ingredient.
A method for treatment and/or prevention according to the present invention may preferably be performed to a subject to which the method for determination according to the present invention, that is, the method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia has been applied. That is, administration of the DUX4 inhibitor or the aforementioned pharmaceutical composition to a subject determined to suffer from malignant lymphoma or leukemia by the aforementioned method for determination is expected to have a therapeutic effect and administration of the DUX4 inhibitor or the aforementioned pharmaceutical composition to a subject determined to be likely to suffer from malignant lymphoma or leukemia is expected to have a preventive effect.
The kind of malignant lymphoma and leukemia to be treated and/or prevented by the method according to the present invention is as described above.
6. Method for Determining Disease Based on Mutation of MEF2D Gene
In one aspect, the present invention relates to a method for assisting in determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia, comprising: a detection step of detecting a fusion mutation of an MEF2D (Monocyte-specific enhancer factor 2) gene in a sample obtained from a subject, and a determination step of determining that the subject suffers from or is likely to suffer from malignant lymphoma or leukemia when the fusion mutation is detected. The fusion mutations detected in this aspect will be described in detail below.
As used herein, the “MEF2D gene” may be a gene encoding a polypeptide containing the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence set forth in SEQ ID NO: 4. Moreover, the MEF2D gene may be a gene encoding a polypeptide containing an amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 4 by addition, deletion, and/or substitution of one or more amino acids. The gene encoding the amino acid sequence set forth in SEQ ID NO: 4 preferably contains the nucleotide sequence set forth in SEQ ID NO: 3.
As used herein, the fusion mutations of an MEF2D gene are fusion mutations of an MEF2D gene and a BCL9 gene or an MEF2D gene and an HNRNPUL1 gene. As used herein, the “fusion mutations of an MEF2D gene and a BCL9 gene” mean fusion genes containing a part of the MEF2D gene (preferably 5′ terminal side) on the 5′ terminal side and a part of the BCL9 (B-cell Lymphoma 9) gene (preferably 3′ terminal side) on the 3′ terminal side. The polypeptides encoded by the fusion genes comprise (i) an amino acid sequence comprising amino acids at positions 1 to 200, preferably positions 1 to 202, in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426, preferably positions 1055 to 1426, in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side. Examples of the amino acid sequence of (i) include any of SEQ ID NOs: 20, 22, 24, and 26, preferably the amino acid sequence set forth in SEQ ID NO: 20 or 22. The polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
As used herein, the “fusion mutations of an MEF2D gene and an HNRNPUL1 gene” mean fusion genes containing a part of the MEF2D gene (preferably 5′ terminal side) on the 5′ terminal side and a part of the HNRNPUL1 (Heterogeneous Nuclear Ribonucleoprotein U-Like 1) gene (preferably 3′ terminal side) on the 3′ terminal side. The polypeptides encoded by the fusion genes comprise (i) an amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side. Examples of the amino acid sequence of (i) include the amino acid sequence set forth in any of SEQ ID NO: 28. The polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
The polypeptides encoded by the MEF2D fusion genes have tumorigenic activity. Methods for determining the presence or absence of the tumorigenic activity of the polypeptide and a detection step of detecting a fusion mutation of a MEF2D gene are as described in “1. Method for determining disease based on mutation or overexpression of DUX4 gene” described above and therefore not described here.
7. MEF2D Fusion Polypeptide and Polynucleotide
In one aspect, the present invention relates to a fusion polypeptide of MEF2D and BCL9. The fusion polypeptides comprise (i) an amino acid sequence comprising amino acids at positions 1 to 200, preferably positions 1 to 202, in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 1100 to 1426, preferably positions 1055 to 1426, in the amino acid sequence set forth in SEQ ID NO: 6 on the C-terminal side. Examples of the amino acid sequence of (i) include any of SEQ ID NOs: 20, 22, 24, and 26, preferably the amino acid sequence set forth in SEQ ID NO: 20 or 22. The polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
In one aspect, the present invention relates to a fusion polypeptide of MEF2D and HNRNPUL1. The fusion polypeptide comprises (i) an amino acid sequence comprising amino acids at positions 1 to 335 in the amino acid sequence set forth in SEQ ID NO: 4 on the N-terminal side and amino acids at positions 563 to 856 in the amino acid sequence set forth in SEQ ID NO: 8 on the C-terminal side. Examples of the amino acid sequence of (i) include the amino acid sequence set forth in any of SEQ ID NO: 28. The polypeptides encoded by the fusion genes may comprise (ii) an amino acid sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of (i) described above or (iii) an amino acid sequence modified from the amino acid sequence of (i) described above by addition, deletion, and/or substitution of one or more amino acids.
The fusion polypeptide has tumorigenic activity. Methods for measuring the presence or absence of the tumorigenic activity of the polypeptide are as described in “1. Method for determining disease based on mutation or overexpression of DUX4 gene” described above and therefore not described here.
In one embodiment, the present invention relates to a polynucleotide encoding the fusion polypeptide. Examples of such a polynucleotide include a polynucleotide comprising: (i) a nucleotide sequence set forth in any of SEQ ID NOs: 19, 21, 23, 25, and 27; (ii) a nucleotide sequence having, for example, 70% or more, 80% or more, preferably, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the nucleotide sequence of (i) described above; or (iii) a nucleotide sequence modified from the nucleotide sequence of (i) described above by addition, deletion, and/or substitution of one or more nucleotides.
Methods for preparing the fusion polypeptide and polynucleotide are similar to the method described in “2. DUX4 fusion polypeptide and polynucleotide” and therefore not described here.
In one embodiment, the present invention relates to a marker for detecting malignant lymphoma or leukemia consisting of the polypeptide or polynucleotide.
Since the present inventors have shown that subjects who are positive for a fusion mutation of a MEF2D gene have relatively bad prognosis, an MEF2D fusion polypeptide or polynucleotide can be used as a prognosis marker. Since it is expected that prognosis would be bad, for example, when a fusion mutation of an MEF2D gene is detected, the likelihood of recurrence of malignant lymphoma or leukemia can be reduced by conducting treating or preventing measures such as bone marrow transplantation beforehand.
8. Primers, Probes, and Antibodies for Detecting Mutation of MEF2D Gene
In one aspect, the present invention relates to a primer set comprising a forward primer and a reverse primer for detecting a fusion gene of an MEF2D gene and a BCL9 gene. The primer set is not particularly limited, as long as it can be used to specifically detect the fusion gene and examples thereof include a forward primer and a reverse primer wherein (1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in SEQ ID NO: 30 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in a sequence complementary to the sequence set forth in SEQ ID NO: 31; or (2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes to a nucleic acid consisting of a complementary sequence set forth in SEQ ID NO: 30 under stringent conditions and the reverse primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes to a nucleic acid consisting of the sequence set forth in SEQ ID NO: 31 under stringent conditions.
In one aspect, the present invention relates to a probe for detecting a fusion gene of an MEF2D gene and a BCL9 gene. The probe is not particularly limited, as long as it can be used to detect the fusion gene, and preferably composed of, for example, (1) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 30 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 31 under stringent conditions; or (2) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 30 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 31 under stringent conditions.
In one aspect, the present invention relates to a primer set comprising a forward primer and a reverse primer for detecting a fusion gene of an MEF2D gene and an HNRNPUL1 gene. The primer set is not particularly limited, as long as it can be used to specifically detect the fusion gene and examples thereof include a forward primer and a reverse primer wherein (1) the forward primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in SEQ ID NO: 32 and the reverse primer consists of nucleotides comprising consecutive 14 to 30 nucleotides, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, in a sequence complementary to the sequence set forth in SEQ ID NO: 33; or (2) the forward primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes to a nucleic acid consisting of a complementary sequence set forth in SEQ ID NO: 32 under stringent conditions and the reverse primer consists of nucleotides comprising a 14 to 30 nucleotide sequence, for example 16 to 28 nucleotides, preferably 18 to 26 nucleotides, that hybridizes to a nucleic acid consisting of the sequence set forth in SEQ ID NO: 33 under stringent conditions.
In one aspect, the present invention relates to a probe for detecting a fusion gene of an MEF2D gene and an HNRNPUL1 gene. The probe is not particularly limited, as long as it can be used to detect the fusion gene, and preferably composed of, for example, (1) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 32 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 33 under stringent conditions; or (2) a polynucleotide that hybridizes to a polynucleotide consisting of at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 32 and at least 7, for example 10, preferably 15 consecutive nucleotide sequence in SEQ ID NO: 33 under stringent conditions.
The primer and probe can be prepared by a method known to those skilled in the art, for example but not limited to, by a method of chemical synthesis.
In one aspect, the present invention relates to an antibody that specifically binds to a fusion protein of MEF2D and BCL9 or a fusion protein of MEF2D and HNRNPUL1. The kind of and the method for preparing the antibody are similar to those described in “3. Primers, probes, and antibodies for detecting mutation or overexpression of DUX4 gene” described above and therefore not described here. For example, the antibody to the aforementioned fusion protein may be prepared by a method known to those skilled in the art. For example, a mammal is immunized with a protein containing a fusion part and an antibody can be obtained from the serum by any method known to those skilled in the art.
The primer set, probe, and antibody described above may have a label detectable by chemical or physical detection means to further facilitate the detection. Examples of the substance used as a label include fluorescent substances, enzymes, radioisotopes, luminescent substances, and the like. Examples of the fluorescent substances include fluorescamine, fluorescein isothiocyanate, and the like, examples of the enzymes include peroxidase, alkaline phosphatase, and the like, examples of the radioisotopes include 125I, 131I, 3H, and the like, and examples of the luminescent substances include luciferin, luminol, derivatives thereof, and the like.
In one embodiment, the present invention relates to a kit for detecting malignant lymphoma or leukemia comprising at least one of the aforementioned primer set, probe, and antibody. The kit according to the present invention may comprise, for example, a buffer, an enzyme, an instruction, and the like, in addition to the probe or antibody.
In one aspect, the present invention relates to an agent for detecting malignant lymphoma or leukemia, comprising an antibody that specifically binds to a fusion protein of MEF2D and BCL9 or a fusion protein of MEF2D and HNRNPUL1. The agent for detecting malignant lymphoma or leukemia according to the present invention may be used as a reagent for sample analysis and the like or for the diagnosis of malignant lymphoma or leukemia.
9. Pharmaceutical Composition Containing MEF2D Inhibitor
In one aspect, the present invention relates to a pharmaceutical composition for treating and/or preventing malignant lymphoma or leukemia, comprising an MEF2D inhibitor as an active ingredient in a subject having the aforementioned fusion mutation of the MEF2D gene.
The MEF2D inhibitor is not particularly limited, as long as it can inhibit the activity of MEF2D, and examples thereof include inhibitory nucleic acids to MEF2D, neutralizing antibodies to MEF2D, and low molecular weight compounds. These inhibitors may be used alone or in combination.
The constitution other than comprising the MEF2D inhibitor as an active ingredient is similar to “4. Pharmaceutical composition comprising inhibitor of DUX4” described above and therefore not described here.
10. Method for Treating and/or Preventing Malignant Lymphoma or Leukemia Using MEF2D Inhibitor or Pharmaceutical Composition
In one aspect, the present invention relates to a method for treating and/or preventing malignant lymphoma or leukemia in a subject having the aforementioned fusion mutation of the MEF2D gene, comprising administering the aforementioned MEF2D inhibitor or the aforementioned pharmaceutical composition to the subject.
A method for treatment and/or prevention according to the present invention may preferably be performed to a subject to which the method for determination according to the present invention, that is, the method for determining whether a subject suffers from, or is likely to suffer from malignant lymphoma or leukemia has been applied.
<Materials and Methods>
Cell Line
Human embryonic kidney (HEK) 293 cells and murine 3T3 fibroblasts were obtained from American type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium-F12 (DMEM-F12) (Thermofisher) containing 10% fetal bovine serum (FBS) (Thermofisher). B-cell lines (Kasumi-7, Kasumi-9, NALM6, NAGL-1, and CCRF-SB) were purchased from the JCRB cell bank and maintained in RPMI1640 medium (Thermofisher) containing 10% FBS. Unless otherwise specified, cell culturing was conducted similarly in all Examples.
Clinical Specimen
Subjects for screening cohorts or with Ph-positive ALL were used for the study of JALSG. CD19+ B-cell and T-cell fractions were selected from the peripheral blood of normal volunteers by a magnetic beads-purification system (Miltenyi Biotech). Bone marrow CD34+ cells were purchased from Takara Bio. The CD10+ fraction was concentrated from bone marrow mononuclear cells purchased from Takara Bio by a magnetic beads purification system. The validation cohort consisted of 62 patients with B-cell ALL including 5 recurrences. The median age was 24 year old (in a range of 0 to 81 year old). The samples of complete remission were obtained from 34 patients. In addition, this study has obtained informed consents from all patients and been approved by the Ethical Review Board of all participated research institutes including University of Tokyo.
RNA-seq
Complementary RNA was prepared from RNA using NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) and subjected to NGS sequencing from the both ends in HiSeq2000/2500 platform (Illumina).
In RNA-seq data, the known fusion genes exhibited the following parameter values in the deFuse analysis; Probability >0.9 and split read count >100. Therefore, Round 1 of screening for fusion genes was conducted with the same parameters as those that give yes for the inframe option (Algorithm A). Subsequently, in the next round of screening, fusion genes were searched for using the probe genes specified with Algorithm A as described above (set forth in the Table below) or already known to be involved in gene fusions, by the following standards; Probability >0.9 or split read count >100 (Algorithm B).
The fusion gene candidates specified with Algorithm A or Algorithm B were confirmed by RT-PCR and subsequent sequencing.
For expression profiling by RNA-seq data, paired-end reads were aligned to hg19 human genome assembly using TopHat2. The expression levels of respective RefSeq genes were calculated from read counts mapped using HTSeq and standardized by DESeq2 pipeline. For the clustering analysis, the standardized read counts were further converted by dispersion visualization conversion in DESeq2 and subjected to the hierarchical clustering analysis by the Ward's method. The genes used for the clustering have been defined in Roberts et al., N. Engl. J. Med., 371, pp. 1005-1015 (2014), but slightly modified according to the recent Refseq database.
Reads were mapped onto the DUX4 locus (chr4: 191,007,101 to 191,011,800) using TopHat2 and the read cover range was calculated by SAMtools to make
Identification of nonsynonymous SNV
The sequence reads with Q value for each base ≥20 were selected from the data set and mapped onto the RNAseq database using the Bowtie2 algorithm. Mismatches were removed when (i) the obtained read contains ≥3 independent mismatches; (ii) they are already listed as a genome change in normal humans in the “1000 genomes” database or the in-house database of the present inventors; or (iii) they are supported by only one strand in the genome. Genetic mutations were annotated by SnpEff. Since no normal cells to be paired were obtained in most subjects, the present inventors examined only the nonsynonymous SNVs that had been already reported in public databases, for example, Catalogue of Somatic Mutations in Cancer, International Cancer Genome Consortium, The Cancer Genome Atlas, and Cancer Cell Line Encyclopedia.
Genome PCR for Amplifying Fusion Points
Genomic DNA was subjected to PCR amplification using the following primers:
<Result>
RNA was isolated from bone marrow mononuclear cells from Ph (Philadelphia chromosome)-negative AYA (adolescent and young adult)-ALL (acute lymphoblastic leukemia) treated with Japan Adult Leukemia Study Group (JALSG) ALL202-U protocol (Hayakawa, F. et al., Blood Cancer J., 4, e252 (2014)) (the screening cohort: 54 individuals with B cells ALL, 18 individuals with T cells ALL, and 1 individual with ALL of unknown lineage). By screening for known fusion genes in ALL based on reverse transcription (RT) polymerase chain reaction (PCR), TCF3-PBX1 was identified in 4 individuals, STIL-TAL1 in 4 individuals, ETV6-RUNX1 in 1 individual, KMT2A-MLLT1 in 1 individual, and KMT2A-AFF3 in 1 individual (STIL-TAL1 was identified in T-cell ALL and the others were identified in B-cell ALL). Furthermore, RNA was isolated from Ph-positive ALL (n=3) and normal volunteers (n=8). These RNA was subjected to RNA-seq and 40.8±4.0 Gbp per individual (mean±SD) of sequence data was obtained. The sequences were also mapped onto 16170±471 genes per sample.
Fusion gene candidates were searched for from the data set using a computer pipeline by the deFuse algorithm (McPherson, A. et al., PLoS Comput. Biol., 7, e1001138 (2011)). In addition to BCR-ABL1, the fused transcription products which were already identified in PCR were successfully detected in correspondent specimens. Furthermore, in 48 AYA-ALL patients, 26 independent fusion genes in total were found and 11 of them were novel (
Since novel chromosome rearrangement was frequently found in B-cell ALL, further 61 individuals with B-cell ALL in all age groups were analyzed (validation cohort: children (0 to 14 year old) 23 individuals, AYA 19 individuals, ≥40 year old 19 individuals). As illustrated in
Based on the gene expression profiling, B-cell ALL can be classified into Ph-like or non-Ph-like and the tyrosine kinase pathway is often activated in Ph-like B-cell ALL. The clustering analysis with no objective variable of the screening cohort including cases of Ph-positive ALL was conducted using RNA-seq data and it was revealed that 7 patients with Ph-negative ALL as well as 3 patients with Ph-positive ALL were classified into Ph-like ALL. The clinical result (data not shown) of genetic mutation profiling of the screening cohort has indicated that the gene fusions are mutually exclusive. The DUX4- and MEF2D-fusions were all classified into the non-Ph-like group. However, the effects of these mutations on prognosis of patients are different. Whereas long disease-free survival after complete remission is predicted with ALL having DUX4-gene fusions, the prognosis of the patients having MEF2D-gene fusions was bad similarly to the prognosis of Ph-like ALL (
The D4Z4 repeat, in which the DUX4 gene locates, was found to be mainly translocated to the IGH locus in chromosome 14 in the AYA-ALL subset (
In all cases where DUX4 was translocated, the 3′ terminal of the coding region of DUX4 was substituted with an IGH fragment or the like, resulting in DUX4 proteins having a mutant carboxy terminal (
<Materials and Method>
To identify the 3′ terminal of DUX4-IGH mRNA, rapid amplification of cDNA ends (RACE) was performed by SMARTer RACE 5′/3′ kit (Takara Bio) according to the instructions of the manufacturer. Full length cDNAs encoding DUX4-IGH, MEF2D-BCL9, or MEF2D-HNRNPUL1 were amplified by PCR from specimens from patients and confirmed by Sanger sequencing. They were then ligated with the retroviral vector pMXS (Cell Biolabs), the expression vector pcDNA3.1 (Thermofisher), or the vector pMSCV-ires-GFP and the cDNAs and GFP were expressed simultaneously. To express the wildtype DUX4, the vector pMSCV-GFP-ires, with which the translation of DUX4 is controlled by the ires fragment, was used (Tsuzuki, S. & Seto, M., Stem Cells 31, 236-247 (2013)).
The MEF2 reporter plasmid was purchased from Qiagen. A human MMP7 promoter region (Corveleyn, A. et al., J. Cell. Biochem. 94, 1112-1125 (2005)) was amplified by PCR from human genomic DNA and ligated to the luciferase vector pGL3 (Promega). 100 ng of the reporter plasmid, 200 ng of the expression vector, and 4 ng of pGL-TK (Promega) were transfected into HEK293T cells using Lipofectamine LTX reagent (Thermofisher). 48 hours later, the cells were lysed and the luciferase activity was measured. The reporter activity based on the firefly luciferase was standardized with the activity of Renilla luciferase.
For the focus formation assay, recombinant retroviruses were produced by introducing a pMXS- or pMSCV-based expression vector and ecotropic packaging plasmid (Takara Bio) into HEK293T cells. 3T3 cells were infected with the recombinant retroviruses and the cells were then cultured for 2 weeks in DMEM-F12 (Thermofisher) supplemented with 5% bovine serum (Thermofisher).
Expression vectors for various cDNAs were transiently transduced into HEK293T cells using Lipofectamine LTX and the cells were lysed 2 days later. The immunoblot analysis was performed using antibodies to DUX4 (ab124699) or MEF2D (ab93257) (both from Abcam). The cell lysates were also analyzed by immunoblotting with an antibody to ACTB (#4967, Cell Signaling Technology).
A shRNA expression vector was constructed using the vector pLMN (Transomic). The sequence of shRNA to DUX4-IGH is 5′-ACCCUGUGUGUCUCAGUUCAUA-3′ (SEQ ID NO: 40) and this targets an IGH region in the transcript of a DUX4-IGH fusion gene in NALM6.
<Result>
To examine the relation of DUX4-IGH fusion genes with cancer, the wildtype DUX4 or DUX4 fusion genes were expressed in murine 3T3 fibroblasts and the focus formation assay was performed. As illustrated in
RNA-seq of total 5 cell lines with B-cell ALL revealed high expression of DUX4 in NALM6 established from a male aged 19 years (
MEF2D-BCL9 and MEF2D-HNRNPUL1 were found in 2 individuals and 3 individuals respectively in the AYA-ALL cohort of the present invention (
Both MEF2D-BCL9 and MEF2D-HNRNPUL1 proteins had high transcriptional activity relative to the corresponding wild type protein (
<Materials and Methods>
As described before (Tsuzuki, S. et al., Stem Cells, 2013, 31, 236-247), B220+c-kit+pro-B cells were induced from fetus hepatocytes cultured on OP9 cells in Iscove's modification Dulbecco medium (Thermofisher) supplemented with 15% FBS, Stem cell factor, flt3 ligand, interleukin-7, and 2-mercaptoethanol.
These cells were infected with the recombinant retroviruses that express a fusion gene and improved GFP. The obtained GFP-positive cells were inoculated with NSG mice (Jackson Laboratory) exposed to a sublethal dose (2 grays) of radiation. 28 days after the transplant, the expression of CD19, CD43, c-kit, CD25, IL7Ra, or IgM on the cell surface of B220+ GFP+ cells among the (primary) bone marrow cells was evaluated with a flow cytometer. For the flow cytometry analysis, the following antibodies: anti-B220 (RA3-6B2), anti-CD19 (1d3), anti-c-kit (2B8), anti-CD43 (S7), anti-CD25 (PC61.5), and anti-IgM (II/41) antibodies were used. All antibodies except S7, which was purchased from BD Biosciences, were obtained from eBioscience. All animal experiments were conducted following the approved protocols by Institutional Animal Care and Use Committee at Aichi Cancer Center and Nagoya University.
<Result>
DUX4-IGH fusion cDNA was introduced into murine pro-B cells with retrovirus and the cells were injected into immunodeficiency mice. As a result, the DUX4-IGH expression pro-B cells were proliferated in vivo, but maintained at an immature stage (CD19+CD43+c-kit+ CD25− IL7Ra+ IgM−) (
When MEF2D-BCL9-expressing B cells were grown in vivo using the same transplant assay, MEF2D-BCL9 does not provide the in vivo proliferation superiority to the pro-B cells, but both MEF2D-BCL9 and MEF2D-HNRNPUL1 prevented the differentiation of the B cell at the stage of the pro-B cell in 4 of 17 mice (
As a result of the DUX-IGH expression injection experiment, the mice finally developed leukemia with a median incubation period of 157 days (
Anti-DUX antibodies were produced using 1 to 13 amino acid residues (MALPTPSDSTLPA: SEQ ID NO: 41) of the human DUX4 protein according to a conventional method in a rabbit and an anti-DUX4 polyclonal antibody was affinity purified with the aforementioned peptide. Subsequently, the obtained antibody (1.2 μg/ml) was contacted with bone marrow cells derived from leukemia patients for 32 minutes at room temperature and then detection was made by BenchMark XT system (Ventana Medical Systems Inc) according to instructions of the manufacturer.
As a result, the bone marrow cell samples derived from leukemia patients were strongly stained with the anti-DUX4 polyclonal antibody (
All publications, patents and patent applications cited herein shall be entirely incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2015-210226 | Oct 2015 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2016/081738 | 10/26/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/073619 | 5/4/2017 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20120225034 | Belayew et al. | Sep 2012 | A1 |
| 20140105873 | Belayew et al. | Apr 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| 2426203 | Mar 2012 | EP |
| 2005082933 | Sep 2005 | WO |
| Entry |
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| Number | Date | Country | |
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| 20180318448 A1 | Nov 2018 | US |
