Patent Application
20150299801
Acute Lymphoblastic Leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of hematopoietic lymphoid progenitors. ALL develops from early forms of lymphocytes. It can start in either early B lymphocytes (B cells) or T lymphocytes (T cells) at different stages of maturity. ALL starting in T-cells is called T-cell ALL and abbreviated T-ALL; similarly, ALL starting in B-cells is called B-cell ALL and abbreviated B-ALL. Conventional chemotherapy treatment includes nucleoside therapy, for example with a combination of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). However, despite intensive chemotherapy, 20% of pediatric and over 50% of adult T-cell acute lymphoblastic leukemia (T-ALL) patients show transient responses to treatment and ultimately die of their disease.
Various embodiments are based on the discovery of certain new somatic single nucleotide substitution mutations of the gene for protein NT5C2 (NT5C2 gene variants) found in some ALL patients. The mutated proteins encoded by these NT5C2 gene variants were discovered to inactivate at least some nucleoside analogs typically used for therapy of ALL. Certain embodiments are directed to determining treatment efficacy based on detecting these newly discovered NT5C2 gene variants, listed below. These new NT5C2 gene variants can be used to diagnose ALL patients who have relapsed or who are at high risk of relapsing, and to determine that a subject whose leukemia cells have the variant will not respond to conventional nucleoside analog therapy, as described below, but benefit from a revised treatment protocol.
In various embodiments, the subject is an animal, typically a mammal, preferably a human, and the biological sample is selected from the group consisting of hair, nails, serum, blood, peripheral blood, plasma, sputum, saliva, mucosal scraping, urine, pleural effusion fluid, cerebrospinal fluid, bone marrow or tissue biopsy. The preferred source is bone marrow or peripheral blood because leukemia cells are primarily recovered from blood and bone marrow aspirates. In various other embodiments, a preferred source is cerebrospinal fluid, pleural effusions and tissue biopsy which are currently often analyzed for leukemia.
In a first set of embodiments, a method includes obtaining a biological sample taken from a subject having ALL, and detecting the presence or absence in the biological sample of a biomarker selected from the group consisting of an NT5C2 gene mutation, an mRNA transcribed from the NT5C2 gene mutation, or a protein encoded by the NT5C2 gene mutation. The method also includes determining that the subject has increased resistance to treatment with 6-mercaptopurine or 6-thioguanine, if the biomarker is detected. In some embodiments, the method still further includes treating the subject with a nucleoside analog other than 6-mercaptopurine or 6-thioguanine if the biomarker is detected.
In a second set of embodiments, a kit is provided for detecting in a biological sample the presence of a biomarker in a biological sample selected from the group consisting of a NT5C2 gene mutation, an mRNA transcribed by the NT5C2 gene mutation, or a protein encoded by the NT5C2 gene mutation. The kit includes reagents for detecting the presence of the NT5C2 gene mutation, the mRNA transcribed from the NT5C2 gene mutation, or the protein encoded by the NT5C2 gene mutation and instructions for use of the kit to determine if a subject having Acute Lymphoblastic Leukemia has reduced sensitivity to treatment with 6-mercaptopurine or 6-thioguanine.
A third set of embodiments is a screening assay for identifying a ligand that binds with high affinity to NT5C2 protein or to a biologically active fragment or variant thereof. The assay includes screening a ligand library against NT5C2 or a biologically active fragment or variant thereof to identify a ligand that binds with high affinity to the NT5C2 protein and isolating the high affinity ligand from the library. The assay further includes combining the high affinity ligand, the NT5C2 protein and the nucleoside analog under conditions that permit the NT5C2 protein to bind to the nucleoside analog, and determining if the NT5C2 protein binds to the nucleoside analog in a mixture forming a NT5C2 protein-nucleoside complex. The assay still further includes selecting and identifying the high affinity ligand as one that prevents NT5C2 protein from inactivating the nucleoside analog, if the NT5C2 protein-nucleoside complex is not formed.
Other embodiments are directed to a pharmaceutical composition comprising a high affinity NT5C2 binding ligand identified using any of the described screening methods.
Other embodiments are directed to a allele-specific PCR assay directed to one of the NT5C2 gene variants.
Still other aspects, features, and advantages of the invention are readily apparent from the following detailed description, simply by illustrating a number of particular embodiments and implementations, including the best mode contemplated for carrying out the invention. The invention is also capable of other and different embodiments, and its several details can be modified in various obvious respects, all without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.
The present invention is illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar elements and in which:
“Biomarker” as used herein means a genetic variant, of any size or type, contained within an individual's genome that is associated with a disease or condition, such as drug response, or any molecule derived therefrom, including a DNA molecule expressing the genetic variant, an RNA molecule transcribing the genetic variant, or a polypeptide that the genetic variant encodes.
“Genetic variation” as used herein means genotypic differences among individuals in a population, at one or more polymorphic loci, excluding common (wild) types, and including an insertion of one or more nucleotides, a deletion of one or more nucleotides, one or more single nucleotide sequence variations (SNVs), such as SNPs, copy number variation, such as one or more duplications, sequence rearrangements, such as one or more inversions, one or more translocations, or one or more repeat sequence expansions or contractions (i.e., changes in microsatellite sequences) at one or more polymorphic loci of interest within a target region of interest as compared to known reference sequences. Gene variants such as SNPs and SNVs can alter gene structure and/or expression level, and thus, in many cases, protein structure and/or expression level.
“Somatic cell mutation” or “somatic mutation” means a mutation in a somatic (non germ line cell) that is acquired. Somatic cells are diploid, containing two copies of each chromosome, whereas the germ cells are haploid, as they only contain one copy of each chromosome. The changes/mutations in NT5C2 occur in the leukemia cells during the process of leukemia formation. They are not present in the population as a whole or in the patient at birth, and they are not present in any other tissues of the patient other than the leukemia cells.
“High affinity” in the context of binding of a ligand to NT5C2 protein refers to affinity, which reflects stability of binding. High affinity binding means that the ligand and the protein bind to each other tightly. Binding affinity can be defined quantitatively by dissociation constant.
“Ligand” as used herein means a substance (usually a small molecule, peptide or antibody or fragment thereof), that forms a complex by binding with high affinity to a target, such as the NT5C2 protein. The binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and van der Waals forces. Actual irreversible covalent binding between a ligand and a protein is rare in biological systems.
“Normal” as used herein, describes what is standard or the usual state. As applied in biology and medicine, a “normal state” or “normal person” is what is usual or most commonly observed. For example, individuals with disease are not typically considered normal. Example usage of the term includes, but is not limited to, “normal subject,” “normal individual,” “normal organism,” “normal cohort,” “normal group,” and “normal population.” A normal gene is also called a wild type (WT).
“NT5C2 protein” as used herein is also known as “cystosolic purine 5′-nucleotodiase” and is a glycoprotein enzyme present in various organs and in many cells. The amino acid sequence is given by NP—036361.1 and NP—001127845.1, which are identical]. The enzyme catalyzes the hydrolysis of a 5′-ribonucleotide to a ribonucleoside and orthophosphate in the presence of water. It is cation-dependent and exists in membrane-bound and soluble forms. Novus Biologicals of Littleton, Colo., offers NT5C2 peptides and NT5C2 proteins for use in common research applications: ELISA, Protein Array, SDS-Page, and Western Blot. NT5C2 Peptides and NT5C2 Proteins can be used in a variety of model species, including human. To distinguish mutations of this protein, the wild type NT5C2 protein is also designated with a suffix “WT,” e.g., “NT5C2 WT.” Various mutations are designated by NT5C2 followed by a different suffix that indicates the amino acid substitution of the mutation. For convenience, in the following, the term NT5C2 suffix is used to refer to the protein or the gene that encodes the protein, as will be clear by the context.
“NT5C2 gene” means the gene that encodes for the NT5C2 protein and has the official name “5′-nucleotidase, cytosolic II.” The nucleotide sequence is given by D38524.1. This gene encodes a hydrolase that serves as an important role in cellular purine metabolism by acting primarily on inosine 5′-monophosphate and other purine nucleotides. To distinguish mutations of this gene, the wild type gene is also designated with a suffix “WT,” e.g., “NT5C2 WT.” Various mutations are designated by NT5C2 followed by a different suffix that indicates the amino acid substitution resulting from the mutation. For convenience, in the following the term NT5C2 suffix is used to refer to the protein or the gene that encodes the protein, as will be clear by the context.
“Nucleoside analogues” as used herein mean a range of antiviral and anticancer products used to prevent viral replication in infected cells and to treat cancer. Commonly used antitumor nucleoside analogs include 6-mercaptopurine, thioguanine, AraC, nelarabine and fludarabine among others.
The terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to mean a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a modified residue, or a non-naturally occurring residue, such as an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
“Single nucleotide variant” or “SNV” as used herein means a DNA base within an established nucleotide sequence that differs from the known reference sequences. SNVs may be found within a patient sample (e.g., a cancer cell such as ALL), and may or may not be present in unperturbed populations, and include naturally occurring single nucleotide polymorphisms.
“Single nucleotide polymorphism” or “SNP” as used herein means a single nucleotide position in a genomic sequence for which two or more alternative alleles are present at an appreciable frequency (e.g., at least 1%) in a population of organisms.
“Subject” as used herein means an object of a method or material, including mammals, e.g., humans, dogs, cows, horses, kangaroos, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. Synonyms used herein include “patient” and “animal.”
“ALL” as used herein means Acute Lymphoblastic Leukemia, an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors.
By “NT5C2 gene variant” is meant a gene variant described herein and listed in Table 1, of which variants at least some mutations are associated with ALL relapse. A specific gene variant is indicated herein by a suffix that indicates a location in the amino acid sequence for the NT5C2 protein where a genetic mutation has led to an amino acid substitution, as described in more detail below. The normal (wild type) gene is indicated by the suffix “WT.” For convenience, the term NT5C2 and suffix is used to refer to the protein or the gene that encodes the protein, as will be clear by the context.
Treatment of ALL that fails to achieve complete remission, or allows relapse, after intensified chemotherapy is almost invariably associated with therapy resistance, disease progression and death. However, the specific mechanisms responsible for relapse and chemotherapy resistance in ALL was largely unknown before this current work. Using whole exome sequencing, multiple new activating mutations in the gene encoding NT5C2, an enzyme responsible for inactivation of nucleoside analog chemotherapy, have been identified in relapsing pediatric ALL patients at the time of relapse. This discovery supports a prominent role for enhanced nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL. In addition, numerous new genes not previously implicated in the pathogenesis of ALL were identified, including three genes mutated at the time of relapse: TP53 (TP53 R213Q), BANP (BANP H391Y) and RPL11 (RPL11 R18P). Subsequent analysis identified two additional somatic RPL11 mutant alleles; one (RPL11 X178Q) was present both at diagnosis and relapse; and one (RPL11 G30fs) specifically mutated at relapse.
Based on these and other discoveries described below, certain embodiments (also described in more detail in the Summary of the Invention) are directed to the newly identified mutations of NT5C2, and to methods for identifying subjects at risk of relapsing with ALL or who have relapsed, and subjects who are resistant to nucleoside therapy based on the presence of an NT5C2 gene variant in leukemia cells from the subject.
Data concerning survival for patients with ALL in different treatment eras indicates low probability (about 10%) of survival even to 5 years, in the era from 1962 to 1966 and increasing survival in more recent eras 1967-1979, 1979-1983, 1984-1991, 1991-1999, and 2000-2007, respectively. While probability of survival has increased dramatically, there is still about a 20% chance of not surviving 5 years.
NT5C2 is a ubiquitous enzyme responsible for the final dephosphorylation of 6-hydroxypurine nucleotide monophosphates such as IMP, dIMP, GMP, dGMP and XMP before they can be exported out of the cell (Oka, 1994; Spychala, 1988). In addition, and most notably, NT5C2 can also dephosphorylate and inactivate 6-thioinositol monophosphate and 6-thioguanosine monophosphate which mediate the cytotoxic effects of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) (Brouwer, 2005), two nucleoside analogs commonly used in the treatment of ALL.
The normal (wild type) NT5C2 protein dephosphorylates and inactivates the active metabolites of 6-MP and 6-TG, and acts to expel these metabolites TIMP, TXMP and TGMP fueled by available ATP. At sufficient doses of 6-MP and 6-TG, the available NT5C2 and ATP cannot keep pace with the deleterious effects of the nucleoside analogs and the ALL cell line dies out.
The human gene for NT5C2 is located on chromosome 10, starting at 104,845,940 base pairs (bp) from phosphotriesteras (pter) and ending at 104,953,056 bp from pter, comprising 107,117 bases. It is oriented on the minus strand and its sequence is cataloged at EnsEMBL Gene ID: ENSG00000076685. The NTFC2 gene (cloned gene GenBank accession number P49902 for NT5C2 protein Uniprot/Swiss-Prot ID; mRNA GenBank accession numbers NM001134373.2; and protein GenBank accession numbers NP001127845.1 in humans or 5′-nucleotidase, cytosolic II) is also known as GMP, NT5B, PNT5, and cN-11.
Mutations are designated herein, as is standard in the art, by the amino acid change to the NT5C2 WT protein in the format of letter-number-letter, where the number indicates the position of the amino acid in the protein from the end translated by the first exon, the first letter is the one letter symbol for the amino acid in the wild type of the protein at that location, and the second letter is the one letter symbol for the amino acid in the mutated protein at that location. For convenience, the one letter symbols for amino acids are listed in
Each patient in the study is identified by a type of ALL (e.g., T-ALL) and a sequential number within the type. DNA from leukemic ALL blasts at diagnosis and at relapse and matched remission lymphocytes were provided by the Hemato-Oncology Laboratory, Department of Pediatrics, University of Padua, Padua, Italy. All samples were collected in clinical trials with informed consent and under the supervision of local ethics Institutional Review Board (IRB) committees. Consent was obtained from all patients at trial entry according to the Declaration of Helsinki As will be shown in more detail below, every patient with at least one of the NT5C2 mutations relapsed while being treated with conventional nucleoside analog therapy.
All amino acid mutations are based on the NT5C2 wild type protein sequence identified as NP—036361.1. All SNPs are based on the NT5C2 gene wild type sequence identified as D38524.1.
In step 401 a sample from a subject having ALL is obtained. Any method may be used to obtain the sample, from drawing or excising the sample from the subject, to retrieving the sample from a cooler, freezer or other storage vessel, to receiving data about the sample. As described above, in various embodiments, the subject is an animal, typically a mammal, preferably a human, and the biological sample is selected from the group consisting of serum, blood, peripheral blood, plasma, blood cells, pleural effusion, cerebrospinal fluid, bone marrow or tissue biopsy. The preferred source is bone marrow or peripheral blood.
In step 403, an assay is performed to detect a biomarker based on a mutation of a gene for protein NT5C2. Recall that a biomarker refers to a DNA molecule expressing a genetic variant associated with a particular disease or condition, such as ALL relapse, an RNA molecule transcribing the genetic variant, or a polypeptide that the genetic variant encodes. For example, in various embodiments, an assay is performed for the DNA sequence, or for the messenger RNA, or for the protein for one or more of the mutations indicated in Table 1. In various embodiments, step 403 includes performing at least one of radioactive or fluorescent DNA sequencing, polymerase chain reaction (PCR), reverse transcription PCR(RTPCR), competitive allele-specific TaqMan® (Life Technologies, New York) PCR, allele-specific restriction-endonuclease cleavage, mismatch-repair detection, binding of MutS protein, denaturing-gradient gel electrophoresis, single-strand-conformation polymorphism detection, RNAase cleavage at mismatched base-pairs, chemical or enzymatic cleavage of heteroduplex DNA, methods based on oligonucleotide-specific primer extension, genetic bit analysis, oligonucleotide-ligation assay, oligonucleotide-specific ligation chain reaction (LCR), gap-LCR, peptide nucleic acid (PNA) assays, Southern Blot analyses, single stranded conformational polymorphism analyses (SSCP), deoxyribonucleic acid sequencing, restriction fragment length polymorphism (RFLP) analysis.
In some embodiments, step 403 includes one or more of assaying for mRNA transcribed from the mutated NT5C2 gene detected using a method from the group comprising Northern blot analysis, nuclease protection assays (NPA), in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) mRNA sequencing.
In some embodiments, step 403 includes one or more of assaying for a mutated protein transcribed from the mutated NT5C2 gene detected using a method from the group comprising immunostaining, immunoprecipitation, immunoelectrophoresis, Western blotting (Immunoblotting), and nucleotidase enzymatic assays.
In step 411, it is determined whether the biomarker is detected based on the assay performed in step 403. If not, then there is no indication that the subject is likely to resist conventional treatment and step 413 is performed to apply standard nucleoside analog therapy, such as with a combination of 6-MP and 6-TG. Note that step 411 may be performed initially upon diagnosis of ALL, or after treatment has been applied and suspended because of detection of the biomarker, only to be reinstated when the biomarker is no longer detected. This may occur if the alternative treatment performed in step 413, described below, is effective at killing the cell lines with the NT5C2 mutations that produce the biomarker.
If the biomarker is detected in step 411, then a modified treatment is applied in step 415. For example, a bioactive agent, such as nelarabine, that is not resisted by the NT5C2 mutations, as described in more detail below, is used in addition to or instead of the conventional nucleoside analog therapy agents 6-MP and 6-TG. In some embodiments, the modified treatment includes the application of ligands that bind with at least the mutated NT5C2 proteins to keep them from dephosphorylating the metabolic products of the conventional nucleoside analog therapy. In some embodiments, step 415 includes one or more screening methods to identify ligands that bind with high affinity to NT5C2 protein, which ligands either prevent binding of NT5C2 protein to a nucleoside analog metabolites, or otherwise prevent the NT5C2 protein from inactivating the nucleoside analog agents. For example, an anthraquinone derivative (AdiS) inhibitor of NT5C2 is described in Jordheim et al., 2013.
Following either step 413 or step 415, in step 421 it is determined whether it is again time to test for the biomarker. For example, after initial diagnosis and several months of treatment, it is determined to be time to test for the biomarker to see if the patient remains or has become resistant to conventional nucleoside analog therapy. If so, then steps 401 and following are executed again. If not, then in step 422 it is determined whether the process should end, e.g., due to long time remission, cure or patient death. If so, then the process ends. If not, then after a suitable wait, it is determined in step 421 again whether it is time to re-test.
Thus, an embodiment is directed to early detection of ALL relapse, by (a) identifying a subject that is at risk of developing relapse ALL or has symptoms of relapse ALL, (b) obtaining a biological sample comprising leukemia cells from the subject; (c) determining if the nucleic acid in the leukemia cells comprises a mutated NT5C2 gene variant, and (d) if the gene variant is detected, then determining that the subject is at risk of developing relapsed ALL or has relapsed ALL. In a further embodiment, a subject identified above as having relapsed or being at risk of relapsing ALL, is identified as being resistant to conventional nucleoside therapy, for example with 6-mercaptopurine and 6-thioguanine, and such therapy is either discontinued or is not administered. Other methods of treatment are administered instead of or in addition to the conventional therapy.
In another embodiment, a subject who has been identified as having one of the herein-defined NT5C2 mutations is monitored to see if the mutations disappear so that conventional nucleoside treatment can be continued or initiated or returned. This happens in some embodiments, if the treatment the subject is given kills the leukemia cells generated from a clone having the NT5C2 mutations. In this case, the subject can resume conventional nucleoside treatment.
Other embodiments include methods for determining if a subject will respond favorably to conventional nucleoside analog therapy, by: (a) identifying a subject that has ALL, (b) obtaining a biological sample of leukemia cells comprising nucleic acid from the subject; (c) determining if the nucleic acid comprises a mutated NT5C2 gene variant, and (d) if the mutated gene variant is detected, then determining that the subject will not respond favorably to conventional nucleoside analog therapy.
Furthermore, certain embodiments are directed to screening assays for identifying a ligand that binds with high affinity to NT5C2 protein or to a biologically active fragment or variant thereof, thereby preventing NT5C2 protein from inactivating a nucleoside analog, comprising (a) screening a ligand library against NT5C2 protein (or a biologically active fragment or variant thereof) to identify a ligand that binds with high affinity to the NT5C2 protein, (b) isolating the high affinity ligand from the library; (c) forming a mixture comprising the high affinity ligand, the NT5C2 protein and the nucleoside analog under conditions that permit the NT5C2 protein to bind to the nucleoside analog, (d) determining if the NT5C2 protein binds to the nucleoside analog in the mixture forming an NT5C2 protein-nucleoside complex, and (e) if the NT5C2 protein-nucleoside complex is not formed, selecting and identifying the high affinity ligand as one that prevents NT5C2 protein from inactivating the nucleoside analog (e.g., see Jordheim et al., 2013).
In other similar methods, screening assays identify ligands that bind with high affinity to NT5C2 protein thereby preventing the NT5C2 protein from inactivating a nucleoside analog. In this embodiment, the ligand does not necessarily block binding of the NT5C2 protein to the nucleoside, but does block inactivation of the nucleoside. In some embodiments, the NT5C2 protein is a mutated protein encoded by one of the herein-described NT5C2 gene variants.
Certain screening assays include determining if the binding of the high affinity ligand to the NT5C2 protein reduces the nucleotidase activity of the hyperactive mutant NT5C2 proteins.
Libraries for use in embodiments of the invention include a constrained ligand library or a combination library having constrained and unconstrained ligands. The library of compounds comprises small molecules, peptides or antibodies. The ligands can be bound to a support such as is a bead, a chip, a filter, a dipstick, a membrane, a polymer matrix or a well. Alternatively, the NT5C2 protein or mutant thereof can be so bound while the ligand is in solution.
If it is determined that the subject will not respond favorably to conventional nucleoside analog therapy, the subject can be treated by administering a therapeutically effective amount of a nucleoside analog if it is administered in combination with an amount of a high affinity ligand identified in the above-described screening that prevents inactivation of the nucleoside analog by the mutated NT5C2 protein in the subject.
If it is determined that the subject will not respond favorably to conventional nucleoside analog therapy, the subject can be treated by administering a therapeutically effective amount of a nucleoside analog if it is administered in combination with a nucleoside analog, such as nelarabine, that is not affected by the NT5C2 mutated protein in the subject.
Analysis of 5 pediatric ALL patients, all of whom had relapsed at least once, showed there was a mean mutation load of 13 somatic mutations per sample. Whole exome sequencing of matched diagnosis, remission and relapse DNA samples from these 5 human pediatric ALL patients with T-cell immunophenotype identified a mean mutation load of 13 somatic mutations per sample (range 5-17). Out of 60 somatic mutations identified in total, 17 mutations were present at diagnosis and at relapse, 24 genes were selectively mutated in relapsed ALL samples and 19 mutations were present only at diagnosis. Moreover, 4 of the 5 relapsed cases analyzed showed the presence of at least one somatic mutation present also at diagnosis, in addition to secondary mutations specifically acquired at the time of relapse. In addition, 4 out of the 5 cases showed loss of at least one mutation marker present at diagnosis during disease progression leading to relapse. Single nucleotide polymorphism analysis of exome sequencing results ruled out that loss of these markers was due to loss of heterozygosity at relapse This result is consistent with previous studies based on copy number alteration analyses (Bunz, 1999; McCubrey, 2007; Jordheim, 2003) and supports that relapsed ALLs can originate as derivates of ancestral subclones related to, but distinct from the main leukemic population present at diagnosis.
New genes not previously implicated in the pathogenesis of T-ALL were identified. Three new genes were identified at the time of relapse encoding proteins involved in positive regulation of TP53 signaling, including TP53 itself (TP53 R213Q), BANP (BANP H391 Y) and RPL11 (RPL11 R18P). Although TP53 R213 has been described in cancer before, it was not associated with T-ALL. Two new additional somatic RPL11 mutant alleles were identified; one present both at diagnosis and relapse (RPL11 X178Q); and the other one (RPL11 G30fs) specifically mutated at relapse.
Two diagnostic and relapse sample pairs harboring a prototypical NRAS G12S activating allele and a third patient with a heterozygous activating NRAS G12R mutation present at diagnosis, which showed loss of heterozygosity at the time of relapse were identified.
One remarkable finding in the exome sequence analysis was the presence of a relapse-associated heterozygous mutation in the cytosolic 5′-nucleotidase II gene (NT5C2 K359Q), which encodes an enzyme responsible for the inactivation of nucleoside analog antitumor drugs (Jordheim, 2003). Extended mutation analysis of NT5C2 in diagnosis and relapse ALL patients identified 23 additional patients harboring NT5C2 mutations (NT5C2 gene variants) acquired at the time of relapse, bringing the frequency of relapse-associated NT5C2 mutations to 16% (23/138) at the time. Strikingly, 15 of the 23 relapse associated NT5C2 mutations in independent relapsed patients harbored the same NT5C2 R367Q mutation. The R367Q mutation is the most frequent and the most important mutation which suggests it should be among the variants screened to determine relapse or risk of relapse or nucleoside therapy resistance, if the number of NT5C2 gene variants tested is limited.
Continued data collection identified the 138 relapsed ALL patients mentioned above. Twenty-four (24) of the relapsed patient samples in the study are listed in Table 2. All listed show NT5C2 mutations.
Comparison between the distances of electrostatic interactions in the wild type and mutant NT5C2 structures suggest a retained active site pocket that is capable of binding nucleoside monophosphate substrates and nucleoside analogs (
Given the described role of NT5C2 in the metabolism and inactivation of nucleoside analog drugs (Brouwer, 2005; Hunsucker, 2005; Galmarini, 2003a); the recurrent finding of certain alleles including the NT5C2 R367Q, NT5C2 R238W and NT5C2 L375F alleles; and the reported association of increased levels of nucleotidase activity with thiopurine-resistance and worse clinical outcome (Pieters, 1992); it was hypothesized that relapse-associated NT5C2 mutations may represent gain of function alleles with increased enzymatic activity. Structural analysis using the Site Directed Mutator (SDM) statistical potential energy function algorithm (Hof, 2011) showed that the NT5C2 K359Q mutation in the Helix A region of the protein mimics the effect of these positive allosteric regulators. Thus, the mutant NT5C2 K359Q protein has increased helix stability and reduced solvent accessibility.
In addition, and despite the absence of clear structural cues suggesting a role of other mutations in NT5C2 activation, analysis of the NT5C2 R367Q and NT5C2 D407A mutant proteins in this assay revealed an 18-fold and a 16-fold increase in their 5′-IMP nucleotidase activity compared with wild type NT5C2, respectively.
To formally test the role of NT5C2 mutations in chemotherapy resistance, the effects of wild type and relapse-associated mutant NT5C2 expression were analyzed in the response of CCRF-CEM T-ALL cells to 6-mercaptopurine (6-MP) and 6-thyogunanine (6-TG). Expression of NT5C2 mutations in ALL cells induces resistance to chemotherapy with 6-MP and 6-TG. Viability assays were performed in CCRF-CEM and CUTLL1 T-ALL cells expressing wild type NT5C2, relapse-associated mutant NT5C2 alleles, or a red fluorescent protein (RFP) as control, treated with increased concentrations of the nucleoside analog chemotherapeutic drugs.
Graphs 740, 750, 760 show viability of the cell line CUTLL1 expressing one of three mutant proteins, NT5C2 R367Q, NT5C2 R359Q, and NT5C2 D407A, respectively, relative to RFP (open circles) trace 746a and NT5C2 WT (closed squares) trace 746b plotted on each graph. CUTLL1 cells expressing the NT5C2 wild type show greater viability than those expressing the RFP control. In graph 740 the viability of CUTLL1 cells expressing mutation R367C is given by trace 748 (down solid triangles), and is greater than control and wild cells for some log concentrations about −5. The log concentration at which viability falls to 50% is not included in the data for the mutation, and is about −5.2 for the control or wild proteins. That is, a higher concentration of 6-MP is needed to reduce viability of CUTLL1 cells expressing the mutation than needed to reduce by the same amount the viability of cells expressing the control or wild type proteins. Similar results are shown for the other mutations, R359Q trace 758 (up solid triangles) in graph 750 and D407A trace 768 (solid circles) in graph 760.
Graphs 840, 850, 860 show viability of the cell line CUTLL1 expressing one of three mutant proteins, NT5C2 R367Q, NT5C2 R359Q, and NT5C2 D407A, respectively, relative to RFP (open circles) trace 846a and NT5C2 WT (closed squares) trace 846b plotted on each graph. CUTLL1 cells expressing the NT5C2 wild type show greater viability than those expressing the RFP control. In graph 840 the viability of CUTLL1 cells expressing mutation R367C is given by trace 848 (down solid triangles) and is greater than control and wild cells for some log concentrations below about −5. The log concentration at which viability falls to 50% is about −5.5 for the mutation and about −6 for the control or wild proteins. That is, a higher concentration of 6-TG is needed to reduce viability of CUTLL1 cells expressing the mutation than needed to reduce by the same amount the viability of cells expressing the control or wild type proteins. Similar results are shown for the other mutations, R359Q trace 858 (up solid triangles) in graph 850 and D407A trace 868 (solid circles) in graph 860.
Cell viability analysis in the presence of increased drug concentrations demonstrated increased resistance to 6-MP and 6-TG therapy in cells expressing NT5C2 K359Q, NT5C2 R367Q and NT5C2 D407A compared with empty vector and wild type NT5C2 controls. Similar results were obtained in the CUTLL1 T-ALL cell line.
Mutation analysis performed of an extended panel of 103 relapse T-ALL and 35 relapse B-precursor ALL samples identified 22 additional T-ALL patients and one B-precursor ALL patient harboring NT5C2 mutations in first relapse. Strikingly, 13 of these samples harbored the same NT5C2 R367Q mutation, 4 cases showed a recurrent NT5C2 R238W mutation and 2 samples harbored a L375F single amino acid substitution. In each of the 9 cases for which original diagnostic DNA was available for analysis, NT5C2 mutations showed to be specifically acquired at the time of relapse. No NT5C2 mutations were identified in 23 T-ALL and 27 B-precursor ALL additional diagnostic samples, further supporting the specific association of NT5C2 mutations with relapsed disease Analysis of clinical and molecular features associated with NT5C2 mutant relapsed T-ALLs treated in Berlin Frankfurt Munster (BFM) group based clinical trials (ALL-BFM 95, ALL-BFM 2000, COALL 06-97, NHL-BFM 95 and Euro-LB 02) showed an association of NT5C2 mutations with early disease recurrence (very early or early relapse vs. late relapse, P<0.05) and relapse under treatment (P=0.002).
The effects of relapse-associated NT5C2 mutations in the response to nelarabine were tested. Nelarabine is a mixture of an AraG precursor highly active in relapsed T-ALL—and AraG (Sanford, 2008; Berg, 2005; Gokbuget, 2011; DeAngelo, 2007). Strikingly, both nelarabine and AraG showed to be equally active in cells expressing relapse-associated NT5C2 mutations compared to controls. This means that treatment with nelarabine can be continued in relapse or at risk subjects having the herein-described NT5C2 gene variants.
For purposes of illustration an example model of tumor development is presented here; however, embodiments of the invention are not limited by the accuracy or completeness of this model.
For purposes of illustration, a proposed mechanism for the resistance to 6-MP is illustrated in the enlarged cell at the bottom of
Prolonged maintenance treatment with 6-mercaptopurine is advantageous to obtain durable remissions in the treatment of ALL (Clappier, 2011; Thompson, 2007). Indeed, low-adherence to 6-mercaptopurine treatment, defined as less than 95% compliance, results in increased relapse rates and may account for as much as 59% of all ALL relapses (Bhatia, 2012). In this context, the results described herein highlight the prominent role of relapse-specific mutations in NT5C2 as a mechanism of resistance to 6-MP and a genetic driver of relapse in ALL. In addition, and most notably, the lack of nelarabine cross-resistance in cells expressing activating NT5C2 alleles analyzed here, suggests that these mutations may not impair the effectiveness of nelarabine-based salvage therapies in relapse T-ALL.
Overall, these results demonstrate that chemotherapy-driven clonal selection is a common driver mechanism of leukemia relapse and highlight the prominent role of relapse-specific mutations in NT5C2 in disease progression and chemotherapy resistance in ALL. This finding supports the embodiments directed to methods of diagnosis and screening assays to develop specific NT5C2 inhibitors or chemotherapeutic alternatives to be administered therapeutically to avoid nucleoside-analog chemotherapy resistance in the treatment of relapsed ALL.
DNAs from leukemic ALL blasts at diagnosis and relapse and matched remission lymphocytes were provided by the Hemato-Oncology Laboratory, Department of Pediatrics, University of Padua, Padua, Italy. All samples were collected in clinical trials with informed consent and under the supervision of local IRB committees. Consent was obtained from all patients at trial entry according to the Declaration of Helsinki.
Matched diagnostic remission and relapsed DNA samples from 5 ALL patients were used for exome capture with the SureSelect 50 Mb All Exon kit (Agilent Technologies, Santa Clara, Calif.) following standard protocols. Paired-end sequencing (2×100 bp) was carried out by using HiSeq2000 sequencing instruments at Centrillion Biosciences (Mountain View, Calif.). Illumina HiSeq analysis (Illumina, San Diego, Calif.) produced between 60 and 120 million paired-end reads per sample. Reads were mapped to the reference genome hg19 using the Burrows-Wheeler Aligner (BWA) alignment tool version 0.5.9. Mean depth (defined as mean number of reads covering the captured coding sequence of a haploid reference) was 50× with 80% of the genome covered more than 10× (review here) and 57% covered more than 30×. Sites that differ from reference (called here variants) were identified in each sample independently. Empirical priors were constructed for the distribution of variant frequencies for each sample. Credibility intervals were obtained for the change in frequency between tumor and normal samples, using the SAVI algorithm (Statistical Algorithm for Variant Identification) developed at Columbia University (Tiacci, 2011; Pasqualucci, 2011). The number of germline SNPs in the coding region were 18,000 comparable with previous reports (Tiacci, 2011). Most of the candidate germline SNPs (16,000, or −90% of germline variants) were reported in dbSNP database. Candidate somatic variants were identified using the following criteria: variant total depth in tumor and normal larger than 10× and smaller than 300×, variant frequency larger than 15% in tumor and less than 3% in normal, lower bound of the change in frequency in tumor versus normal larger or equal to 1%. Also to remove systematic errors all variants that are present in any of the 23 normal cases were removed. In addition, a 40 base sequence context around the variant were mapped to hg19 genome using BLAST algorithm to confirm unique mappability (to eliminate ambiguous mapping from captured pseudogenes, and regions of low complexity).
Primers flanking exons containing candidate somatic variants were designed using Primer3 (see file primer3 in subdomainfrodo of subdomain wi at domain mit in category edu), and used for PCR amplification from whole-genome-amplified (WGA) tumor, relapse and matched normal (remission) DNAs. The resulting amplicons were analyzed by direct bidirectional dideoxynucleotide sequencing. Mutation recurrence in TP53, RPL11, BANP genes and NT5C2 was analyzed in an independent cohort of 23 relapsed T-ALL patient samples via PCR amplification and bidirectional dideoxinucleotide sequencing. Similarly, the frequency of hotspot NRAS activating mutations was analyzed by direct sequencing of PCR products encompassing exons 1 and 2 of the NRAS gene.
Structural coverage of the NT5C2 protein was identified through use of the PSI-Blast and SKAN algorithms; viable structures were subsequently mapped to all NT5C2 isoforms, and analyzed with the use of Chimera Suite 3,4. Protein database (PDB) structures 2XCW, 2XCX, 2J2C, 2XCB, 2XCV, 2XJB, 2XJC, 2XJD, 2XJE, 2XJF, 2J2C, and 2JC9 were structurally aligned and the resulting composite structure was subsequently analyzed to assess conformational flexibilities. The identified mutations were structurally modeled utilizing the I-TASSER software suite, and subsequently refined and analyzed via minimization and rotamer library analysis in Chimera3,5. Protein stability changes upon mutation were predicted through use of the SDM potential energy statistical algorithm and associated software6. All structural images were created using UCSF Chimera3.
Biochemical nucleotidase assays measuring release of phosphate upon incubation of NT5C2 with phosphonucleodides were used, including a commercial kit that measures enzymatic activity. (See file DZ123A.php in subfolder reagents of folder products in the World Wide Web at domain diazyme in category corn.)
Table 2 lists mutations identified by exome sequencing in diagnosis and relapse T-ALL. Genomic regions of LOH are available in diagnostic and Libraries of Small Molecules, Peptides and Antibodies. Table 1 lists NT5C2 mutations in ALL. The correlative clinical data on NT5C2 mutated relapsed T-ALLs treated in AIEOP clinical trials, and correlative clinical and molecular data on NT5C2 mutated relapsed T-ALLs in BFM based protocols, and correlative clinical data on rescue treatment for NT5C2 mutated relapsed T-ALLs in BFM based protocols, are all available.
The NT5C2 mutations at residues R291 W, K359Q, R367Q and D407A were generated by site directed mutagenesis onto the mammalian expression pLOC-NT5C2 vector (Open Biosystems/Thermo Scientific of Pittsburgh, Pa.) using the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene/Agilent Technologies of Santa Clara, Calif.) according to the manufacturer's instructions.
CCRF-CEM and CUTLL1 cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, 100U/ml penicillin G and 100 ug/ml streptomycin at 37 C in a humidified atmosphere under 5% CO2. HEK293T were maintained under similar conditions in DMEM media.
The lentiviral constructs pLOC-NT5C2, pLOC-NT5C2 359, pLOC-I 367, pLOC-I 407 and the pLOC-RFP control plasmid were transfected with gag-pol and V-SVG expressing vectors into HEK293T cells using JetPEI transfection reagent (Polyplus, of Illkirch, France). Viral supernatants were collected after 48 h and used for infection of CCRF-CEM and CUTLL1 cells by spinoculation. After infection, cells were selected for 5 days in blasticidin and ficolled the day before experiments.
Cell viability was determined by measurement of the metabolic reduction of the tetrazolium salt MTT using the Cell Proliferation Kit I (Roche, of Indianapolis, Ind.) following the manufacturer instructions. Experiments were done in triplicates. Viability was analyzed after 48 or 72 hours of initiation of treatment with 6-mercaptopurine, 6-thioguanine, nelarabine and Ara-G.
Full length cDNA constructs encoding wild type NT5C2, NT5C2 K359Q, NT5C2 R367Q and NT5C2 D407A were cloned with an N-terminal His6 tag in the pET28a-LIC expression vector using the In-Fusion HD PCR cloning system (Clontech, of Mountain View, Calif.), as per manufacturer's instructions. Recombinant proteins were expressed from Rosetta 2(DE3) E. coli cells by induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside for 3 hours at 37° C. Harvested cells were lysed in lysis buffer (50 mM sodium phosphate pH7.4, 100 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 1% Triton X-100, 0.5 mg/ml lysozyme, 20 mM imidazole) supplemented with Complete EDTA-free protease inhibitor (Roche, of Indianapolis, Ind.). His-tagged NT5C2 proteins were bound to and purified with Nickel-sepharose beads and eluted with 50 mM sodium phosphate pH7.4, 100 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 300 mM imidazole Imidazole was removed by buffer exchange using PD-10 desalting columns (GE Healthcare of Waukesha, Wis.). Protein expression and purity was assessed y SDS-PAGE and Coomassie staining.
5′-Nucleotidase activity of purified recombinant wild type and mutant NT5C2 proteins was assessed using the 5′-nucleotidase (5′-NT) Enzymatic Test Kit (Diazyme, of Poway, Calif.), according to the manufacturer's instructions. The assay measures the enzymatic hydrolysis of inosine 5′-monophosphate to inosine, which is reacted further to hypoxanthine by purine nucleoside phosphorylase, and then to uric acid and hydrogen peroxide by xanthine oxidase. H2O2 is quantified using a Trinder reaction. 5′-Nucleotidase activity levels are calculated using a calibrator of known 5′-NT activity as standard. Assays were performed in triplicate in an Infinite M200 Tecan (of Mannedorf, Switzerland) plate reader.
Differences in the percentages of NT5C2 wild type and mutant ALL patients in different relapsed categories were evaluated using the Fisher's exact test. Equality of categorical and continuous variables was analyzed by Fisher's exact test and by Mann-Whitney U test, respectively.
In various embodiments, determining whether the sample includes the biomarker further comprises performing at least one of radioactive or fluorescent DNA sequencing, polymerase chain reaction (PCR), reverse transcription PCR(RTPCR), allele-specific restriction-endonuclease cleavage, allele-specific PCR, allele-specific PCR with suppression of wild type, mismatch-repair detection, binding of MutS protein, denaturing-gradient gel electrophoresis, single-strand-conformation polymorphism detection, RNAase cleavage at mismatched base-pairs, chemical or enzymatic cleavage of heteroduplex DNA, methods based on oligonucleotide-specific primer extension, genetic bit analysis, oligonucleotide-ligation assay, oligonucleotide-specific ligation chain reaction (LCR), gap-LCR, peptide nucleic acid (PNA) assays, Southern Blot analyses, or single stranded conformational polymorphism analyses (SSCP). Assays based on any of these methods are anticipated.
Mutant allele detection is based on a modified allele-specific primer, while the wild-type background is suppressed by a proprietary MGB blocker oligonucleotide. Assays can detect less than 0.1% mutant molecules in a background of wild type gDNA, as demonstrated in spiking experiments. Assays demonstrate 7 logs of dynamic range and an average efficiency of 100%±10%. A sample is run with one or more assays that target the mutant allele of a given mutation and the corresponding wild type allele assay(s). Three sample replicates are run. This approach is recommended for highly accurate quantitation of the percent mutation in a sample. For data analysis, Mutation Detector™ Software v1.0 allows users to determine the mutation status of their samples from TaqMan®Mutation Detection Assay data collected on Applied Biosystems® ViiA™ Real-Time PCR Systems.
For example, to prepare the primers, probes and blockers for the NT5C2 R367Q mutation, the gene name as indicated above and the SNP location in the cDNA sequence and nucleotide change, as listed in Table 1, were provided to the vendor. (e.g., Life Technologies of Grand Island, N.Y.)), which generated the various components for this assay including the ASB. In addition, a DNA sequence of 200 bp flanking each side of the SNP in genomic DNA with the nucleotide change indicated in brackets was also sent. Thus, NT5C2 R367Q castPCR design: Single nucleotide change with cDNA location Ref. Seq NM—012229 1100 G>A. The assay is designed for detection in genomic DNA samples. Genomic region encompassing the mutation is: >Human_NT5C2_R367Q, (SEQ. ID NO. 9) atgaatcatagtatttaatggttaaatactattggaatgctgattttaatatagggacagttttctcattgttcactctgttggttcagGTTC TTCTGATACGATCTGTGACCTGTTGGGAGCCAAGGGAAAAGACATTTTGTATATT GGAGATCACATTTTTGGGGACATTTTAAAATCAAAGAAACGGCAAGGGTGGC[G/A]AACTTTTTTGGTGATTCCTGAACTCGCACAGGAGCTACATGTCTGGACTGACA AGAGTTgtaagccattcataaatctttggtgtaatttatatctaaatgaccagattgaatcagtcgtattgataaaccattactttgtat tcaggaagtcagaggtggtcaaaaggcaggccttgatatttggatacttccttcc in which intron sequences are shown in lower case and exon sequences in upper case.
Northern analysis remains the standard for detection and quantitation of mRNA levels despite the advent of more sensitive techniques. Northern analysis presents several advantages over the other techniques. The most compelling of these is that it is the easiest method for determining transcript size, and for identifying alternatively spliced transcripts and multigene family members. It can also be used to directly compare the relative abundance of a given message between all the samples on a blot. The Northern blotting procedure is straightforward and provides opportunities to evaluate progress at various points (e.g., intactness of the RNA sample and how efficiently it has transferred to the membrane). RNA samples are first separated by size via electrophoresis in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane, crosslinked and hybridized with a labeled probe. Nonisotopic or high specific activity radiolabeled probes can be used including random-primed, nick-translated, PCR-generated DNA probes, in vitro transcribed RNA probes, and oligonucleotides. Additionally, sequences with only partial homology (e.g., cDNA from a different species or genomic DNA fragments that might contain an exon) may be used as probes. Despite these advantages, there are limitations associated with Northern analysis. First, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. Second, a standard Northern procedure is, in general, the least sensitive of several popular techniques. However, substantial improvements can be made to increase detection of most mRNA species (see below). Another limitation of Northern blotting has been the difficulty associated with multiple probe analysis. To detect more than one message, it is usually necessary to strip the initial probe before hybridizing with a second probe. This process can be time consuming and problematic.
The NPA (including both ribonuclease protection assays and S1 nuclease assays) is an extremely sensitive method for the detection and quantitation of specific mRNAs. The basis of the NPA is solution hybridization of an antisense probe (radiolabeled or nonisotopic) to an RNA sample. After hybridization, single-stranded, unhybridized probe and RNA are degraded by nucleases. The remaining protected fragments are separated on an acrylamide gel. Solution hybridization is typically more efficient than membrane-based hybridization, and it can accommodate up to 100 μg of sample RNA, compared with the 20-30 μg maximum of blot hybridizations. NPAs are also less sensitive to RNA sample degradation than Northern analysis since cleavage is only detected in the region of overlap with the probe (probes are usually about 100-400 bases in length). NPAs are the method of choice for the simultaneous detection of several RNA species. At least 6 different probes and an internal control can be used together for detection of mRNA transcripts each within 4 different RNA samples. During solution hybridization and subsequent analysis, individual probe/target interactions are completely independent of one another. Thus, several RNA targets and appropriate controls can be assayed simultaneously (up to twelve have been used in the same reaction), provided that the individual probes are of different lengths. NPAs are also commonly used to precisely map mRNA termini and intron/exon junctions. The primary limitation of NPAs is the lack of information on transcript size. The portion of probe homologous to target RNA determines the size of the protected fragment. Another drawback to NPAs is the lack of probe flexibility. The most common type of NPA, the ribonuclease protection assay, requires the use of RNA probes. Oligonucleotides and other single-stranded DNA probes can only be used in assays containing S1 nuclease. The single-stranded, antisense probe must typically be completely homologous to target RNA to prevent cleavage of the probe:target hybrid by nuclease. This means that partially related sequences (i.e., cross species) usually cannot be used.
Ambion (of Austin, Tex.) released the first commercially available Ribonuclease Protection Assay Kit in 1990 and has since continued to refine and improve the procedure resulting in a number of kits with different advantages. The RPA III™ Kit is recommended for all new users and consists of a single-tube protocol. The HybSpeed™ RPA Kit utilizes a 10-minute hybridization step so that the entire assay can be completed in a single day.
In situ hybridization (ISH) is a powerful and versatile tool for the localization of specific mRNAs in cells or tissues. Unlike Northern analysis and nuclease protection assays, ISH does not require the isolation or electrophoretic separation of RNA. Hybridization of the probe takes place within the cell or tissue. Since cellular structure is maintained throughout the procedure, ISH provides information about the location of mRNA within the tissue sample. The procedure begins by fixing samples in neutral-buffered formalin, and embedding the tissue in paraffin. The samples are then sliced into thin sections and mounted onto microscope slides. (Alternatively, tissue can be sectioned frozen and post-fixed in paraformaldehyde.) After a series of washes to dewax and rehydrate the sections, a Proteinase K digestion is performed to increase probe accessibility, and a labeled probe is then hybridized to the sample sections. Radiolabeled probes are visualized with liquid film dried onto the slides, while nonisotopically labeled probes are conveniently detected with colorimetric or fluorescent reagents. The major drawback to ISH is the procedure itself. Standard protocols are time-consuming, laborious and may require specialized equipment for preparing samples and visualizing results of the experiment. Additionally, quantitation of gene expression is not as straightforward as with the other techniques.
RT-PCR has revolutionized the study of gene expression. It is now theoretically possible to detect the RNA transcript of any gene, regardless of the scarcity of the starting material or relative abundance of the specific mRNA. In RT-PCR, an RNA template is copied into a complementary DNA (cDNA) using a retroviral reverse transcriptase. The cDNA is then amplified exponentially by PCR. As with NPAs, RT-PCR is somewhat tolerant of degraded RNA. As long as the RNA is intact within the region spanned by the primers, the target will be amplified. Although RT-PCR is the most sensitive method of mRNA detection available, it does have drawbacks. It can be the most technically challenging method of detection and quantitation, often requiring substantial pre-experimental planning and design. Additionally, because of its extreme sensitivity, even minute amounts of contamination by genomic DNA or previously amplified PCR products can lead to aberrant results, so steps must be taken to avoid this pitfall. Relative quantitative RT-PCR involves amplifying an internal control simultaneously with the gene of interest. The internal control is used to normalize the samples. Once normalized, direct comparisons of relative abundance of a specific mRNA can be made across the samples. It is crucial to choose an internal control with a constant level of expression across all experimental samples (i.e., not affected by experimental treatment). Commonly used internal controls (e.g., GAPDH, -actin, cyclophilin) often vary in expression and, therefore, may not be appropriate internal controls. Additionally, most common internal controls are expressed at much higher levels than the mRNA being studied. For relative RT-PCR results to be meaningful, all products of the PCR reaction must be analyzed in the linear range of amplification. This becomes difficult for transcripts of widely different levels of abundance.
This is used for absolute quantitation. This technique involves designing, synthesizing, and accurately quantitating a competitor RNA that can be distinguished from the endogenous target by a small difference in size or sequence. Known amounts of the competitor RNA are added to experimental samples and RT-PCR is performed. Signals from the endogenous target are compared with signals from the competitor to determine the amount of target present in the sample. Ambion (Austin, Tex.) has a complete line of products to simplify RT-PCR. The RETROscript™ Kit provides a convenient and flexible system for synthesizing first-strand cDNA. The QuantumRNA™ 18S Internal Standards Kits utilize Ambion's Competimer™ technology (patent pending) to allow the use of the best internal control, 18S rRNA, for relative RT-PCR (
Some embodiments are directed to a method for screening and identifying test compounds (ligands) that bind to a preselected target protein. Direct, non-competitive binding assays are advantageously used to screen libraries of compounds (such as bead-based libraries) for those that selectively bind to a preselected target. Binding of target molecules to a particular test compound is detected using any physical method known in the art for detecting high affinity binding of the ligand to the target protein. The structure of the test compound (ligand) that binds to the target can also be determined, if it is not known beforehand. The methods used will depend, in part, on the nature of the library screened. The methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify ligands that bind with high affinity to the target NT5C2 protein.
For example, in some embodiments, a screening assay for identifying a ligand that binds with high affinity to NT5C2 protein or to a biologically active fragment or variant thereof, includes screening a ligand library against NT5C2 or a biologically active fragment or variant thereof to identify a ligand that binds with high affinity to the NT5C2 protein. The assay includes isolating the high affinity ligand from the library. Then the high affinity ligand is combined with the NT5C2 protein and the nucleoside analog under conditions that permit the NT5C2 protein to bind to the nucleoside analog. The assay includes determining whether the NT5C2 protein binds to the nucleoside analog in a mixture forming a NT5C2 protein-nucleoside complex. If not, then the assay includes selecting and identifying the high affinity ligand as one that prevents NT5C2 protein from inactivating the nucleoside analog.
In some embodiments, the assay also includes combining the high affinity ligand, the NT5C2 protein and the nucleoside analog under conditions that permit the nucleoside analog to produce active metabolites in a cell. This assay also includes measuring for the presence or absence of the active metabolites in the cell if the NT5C2 protein-nucleoside complex is not formed. If the active metabolites of nucleoside analogs are present in normal amounts in the cell, then it is determined that the high affinity ligand prevents the expulsion of metabolites of conventional nucleoside analog therapy and is suitable for including in a treatment for a patient with the NT5C2 mutations. In some of these embodiments, the metabolites are selected from the group consisting of TIMP, TXMP and TGMP.
As used herein, a “library” refers to a plurality of test compounds (protein, antibody, small molecules) with which a target protein is contacted. A library can be a combinatorial library, e.g., a collection of test compounds synthesized using combinatorial chemistry techniques, or a collection of unique chemicals of low molecular weight (less than 1000 daltons) that each occupy a unique three-dimensional space. As used herein, a “label” or “detectable label” is a composition that is detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
Methods are described in which a preselected target protein having a detectable label is used to screen a library of test compounds. Any complexes formed between the target and a member of the library are identified using methods that detect the target bound to a test compound. In an embodiment, the present invention relates to methods for using a target (optionally having a detectable label) to screen a library of test compounds. Compounds in the library that bind to the target will form a complex, which can be separated from the unbound target. The complex can then be identified and removed from the uncomplexed target and uncomplexed compounds.
In an embodiment, the protein is bound to a support and the test compound is free. In some embodiments the target is labeled, for example with a fluorescent dye, phosphorescent dye, ultraviolet dye, infrared dye, visible radiolabel, enzyme, spectroscopic colorimetric label, affinity tag, or nanoparticle. In an embodiment, a combinatorial library of test compounds is used comprising peptoids; polypeptides; nonpeptidal peptidominetics; oligocarbamates; antibody libraries; carbohydrate libraries; and organic molecule libraries.
Libraries screened using the methods of the present invention can comprise a variety of types of test compounds, optionally bound to solid supports. In all of the embodiments described below, all of the libraries can be synthesized on solid supports or the compounds of the library can be attached to solid supports by linkers, or they can be in solution.
In some embodiments, the test compounds are peptide molecules that can exist in a phage display library. In other embodiments, types of test compounds include, but are not limited to, peptide analogs including peptides comprising non-naturally occurring amino acids, e.g., D-amino acids, phosphorous analogs of amino acids, such as α-amino phosphoric acids anda-amino phosphoric acids, or amino acids having non-peptide linkages, nucleic acid analogs such as phosphorothioates and PNAs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic molecules, pheromones, adenosine, sucrose, glucose, lactose and galactose. Libraries of polypeptides or proteins can also be used.
In a preferred embodiment, the combinatorial libraries are small organic molecule libraries, such as, but not limited to, benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, and diazepindiones. In another embodiment, the combinatorial libraries comprise peptoids; random bio-oligomers; benzodiazepines; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; or carbohydrate libraries. Combinatorial libraries are themselves commercially available (see, e.g., Advanced ChemTech Europe Ltd., Cambridgeshire, UK; ASINEX, Moscow, Russia; Bifocals plc, Sittingbourne, UK; Bionet Research [a division of Key Organics Limited], Camelford, UK; ChemBridge Corporation, San Diego, Calif.; ChemDiv Inc, San Diego, Calif.; ChemRx Advanced Technologies, South San Francisco, Calif.; ComGenex Inc., Budapest, Hungary; Evotec OAI Ltd, Abingdon, UK; IF LAB Ltd., Kiev, Ukraine; Maybridge plc, Cornwall, UK; PharmaCore, Inc., High Point, N.C.; SIDDCO Inc, Tucson, Ariz.; TimTec Inc, Newark, Del.; Tripos Receptor Research Ltd, Bude, UK; Toslab, Ekaterinburg, Russia).
In one embodiment, the combinatorial compound library for the methods of the present invention may be synthesized. Combinatorial compound libraries useful for the methods of the present invention can be synthesized on solid supports. As used herein, the term “solid support” is not limited to a specific type of solid support. Rather a large number of supports are available and are known to one skilled in the art. Solid supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, polystyrene beads, doped polystyrene beads (as described by Fenniri, 2001), alumina gels, and polysaccharides. A suitable solid support may be selected on the basis of desired end use and suitability for various synthetic protocols. In some embodiments of the present invention, compounds can be attached to solid supports via linkers.
Screening comprises contacting a target with an individual or small group, of the components of the compound library. Preferably, the contacting occurs in an aqueous solution, and most preferably, under physiologic conditions. The aqueous solution preferably stabilizes the target and prevents denaturation or degradation without interfering with binding of the test compounds.
The methods of some embodiments for screening a library of test compounds preferably comprise contacting a test compound with a target in the presence of an aqueous solution, the aqueous solution comprising a buffer and a combination of salts, preferably approximating or mimicking physiologic conditions.
As used herein, a “dye” refers to a molecule that, when exposed to radiation, emits radiation at a level that is detectable visually or via conventional spectroscopic means. As used herein, a “visible dye” refers to a molecule having a chromophore that absorbs radiation in the visible region of the spectrum (i.e., having a wavelength of between about 400 nm and about 700 nm) such that the transmitted radiation is in the visible region and can be detected either visually or by conventional spectroscopic means. As used herein, an “ultraviolet dye” refers to a molecule having a chromophore that absorbs radiation in the ultraviolet region of the spectrum (i.e., having a wavelength of between about 30 nm and about 400 nm). As used herein, an “infrared dye” refers to a molecule having a chromophore that absorbs radiation in the infrared region of the spectrum (i.e., having a wavelength between about 700 nm and about 3,000 nm). A “chromophore” is the network of atoms of the dye that, when exposed to radiation, emits radiation at a level that is detectable visually or via conventional spectroscopic means. One of skill in the art will readily appreciate that although a dye absorbs radiation in one region of the spectrum, it may emit radiation in another region of the spectrum. For example, an ultraviolet dye may emit radiation in the visible region of the spectrum. One of skill in the art will also readily appreciate that a dye can transmit radiation or can emit radiation via fluorescence or phosphorescence.
As used herein, a “label” or “detectable label” is a composition that is detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes (e.g., 32P, 35S, and 3H), dyes, fluorescent dyes, electron-dense reagents, enzymes and their substrates (e.g., as commonly used in enzyme-linked immunoassays, e g, alkaline phosphatase and horseradish peroxidase), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. Moreover, a label or detectable moiety can include an “affinity tag” that, when coupled with the target nucleic acid and incubated with a test compound or compound library, allows for the affinity capture of the target nucleic acid along with molecules bound to the target nucleic acid. One skilled in the art will appreciate that a affinity tag bound to the target nucleic acids has, by definition, a complimentary ligand coupled to a solid support that allows for its capture. For example, useful affinity tags and complimentary ligands include, but are not limited to, biotin-streptavidin, complimentary nucleic acid fragments (e.g., oligo dot-oligo da, oligo T-oligo A, oligo dig-oligo do, oligo G-oligo C), aptamer complexes, or haptens and proteins for which antisera or monoclonal antibodies are available. The label or detectable moiety is typically bound, either covalently, through a linker or chemical bound, or through ionic, van der Waals or hydrogen bonds to the molecule to be detected.
Some embodiments comprise a kit for detecting the presence of a biomarker in a biological sample selected from the group consisting of a NT5C2 gene mutation, an mRNA transcribed from the NT5C2 gene mutation, or a protein encoded by the NT5C2 gene mutation in a biological sample. The kit includes reagents for detecting the presence of the NT5C2 gene mutation, the mRNA transcribed from the NT5C2 gene mutation, or the protein encoded by the NT5C2 gene mutation, and instructions for use of the kit to determine if a subject has increased resistance to treatment with 6-mercaptopurine or 6-thioguanine. In some embodiments the kit includes one or more control samples. Examples of negative control samples are: wild type DNA, RNA or NT5C2 protein. Examples of positive control samples include: mutant NT5C2 DNA, RNA and proteins. Example, calibration and sensitivity controls include: mutant NT5C2 DNA, RNA and protein diluted in wild type NT5C2 DNA, RNA and proteins at known ratios.
In the present specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference as if set forth herein in their entirety, except where terminology is not consistent with the definitions herein. Although specific terms are employed, they are used as in the art unless otherwise indicated.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US13/68821 | 11/6/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61723315 | Nov 2012 | US |
