PRKC FUSIONS

The invention provides PRKC gene fusions, PRKC fusion proteins, and fragments of those genes and polypeptides. The invention further provides methods of diagnosing and treating diseases or disorders associated with PRKC fusions, such as conditions mediated by aberrant PRKC expression or activity, or overexpression of PRKC.

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Description

This application claims the benefit of U.S. Provisional Application No. 62/025,965, filed Jul. 17, 2014, which is incorporated here by reference in its entirety to provide continuity of disclosure.

This invention relates to PRKC (protein kinase C family) gene fusions and PRKC fusion proteins. The invention further relates to methods of diagnosing and treating diseases or disorders associated with PRKC fusions, such as conditions mediated by aberrant PRKC expression or activity, or conditions associated with overexpression of PRKC.

Many forms of cancer are caused by genetic lesions that give rise to tumor initiation and growth. Genetic lesions may include chromosomal aberrations, such as translocations, inversions, deletions, copy number changes, gene expression level changes, and somatic and germline mutations. Indeed, the presence of such genomic aberrations is a hallmark feature of many cancers, including, for example, B cell cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, and colon cancer. In some models, cancer represents the phenotypic end-point of multiple genetic lesions that endow cells with a full range of biological properties required for tumorigenesis.

Recent efforts by The Cancer Genome Atlas (TCGA), the International Cancer Genome Consortium (ICGC), and dozens of other large-scale profiling efforts have generated an enormous amount of new sequencing data for dozens of cancer types—this includes whole-genome DNA, whole-exome DNA, and full-transcriptome RNA sequencing. These efforts have led to the identification of new driver genes and fusion genes within multiple cancer types. Fusions, particularly fusions involving kinases, are of particular interest, as such fusions have been shown to be oncogenic, and have been successfully targeted by new therapeutics.

For example, anaplastic lymphoma kinase (ALK), one of the receptor tyrosine kinases, is known to become oncogenic when fused with various genes. See, e.g., M. Soda et al. “Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer,” Nature 444:561-566 (2007).

A need exists for identifying novel genetic lesions associated with cancer. For example, the presence of fusions involving a kinase in samples collected from more than one source can indicate that the kinase is an oncogenic driver. The identification of such fusions can be an effective approach to diagnosis of cancer, and development of compounds, compositions, methods, and assays for evaluating and treating cancer patients.

In one aspect, the invention provides a method for detecting the presence of a PRKC fusion in a biological sample. The method includes the steps of: (a) obtaining a biological sample from a mammal; and (b) contacting the sample with a reagent that detects a PRKC fusion, to determine whether u PRKC fusion is present in the biological sample. The sample can be from, e.g., a cancer patient. In some embodiments, the cancer can be a lung cancer, such as, e.g., lung squamous cell carcinoma or lung adenocarcinoma. In some embodiments, the fusion can be, e.g., an IGF2BP3:PRKCA fusion, a TANC2:PRKCA fusion, an ADCY9:PRKCB fusion, or an SPNS1:PRKCB fusion. In some embodiments, the IGF2BP3:PRKCA fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively. In some embodiments, the TANC2:PRKCA fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. In some embodiments, the ADCY9:PRKCB fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. In some embodiments, the SPNS1:PRKCB fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively.

In another aspect, the invention provides a method of diagnosing a patient having a disease or disorder associated with aberrant PRKC expression or activity, or overexpression of PRKC; the method includes: (a) obtaining a biological sample from the patient; and (b) contacting the sample with a reagent that detects a PRKC fusion to determine whether a PRKC fusion is present in the biological sample, whereby the detection of the PRKC fusion indicates the presence of a disorder associated with aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC.

The invention also includes a method of determining a therapeutic regimen for treating a cancer in a human subject; a method of identifying a patient likely to respond to treatment with a PRKC inhibitor or a PRKC fusion inhibitor; a method of stratifying a patient population by detecting a PRKC fusion; a method of treating a patient; a method of inhibiting the proliferation of cells containing a PRKC fusion; a method of reducing an activity of a PRKC fusion; a method of treating a condition mediated by aberrant (e.g., constitutive) PRKC activity or expression; a method of treating a condition characterized by overexpression of PRKC; a method of identifying an agent that modulates the activity of a PRKC fusion; and a method of monitoring disease burden in a patient having a condition mediated by PRKC.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the nucleotide sequence of an IGF2BP3:PRKCA gene fusion (SEQ ID NO:1) comprising a portion of the IGF2BP3 gene (NM_006547) up to and including exon 10 and a portion of the PRKCA gene (NM_002737) starting at exon 4. The underlined codons at nucleotides 1201-1203 and 1204-1206 encode the last amino acid of IGF2BP3 and the first amino acid of PRKCA, respectively. The slash after nucleotide 1203 indicates the breakpoint (fusion junction) where translocation and in-franc fusion has occurred.

FIG. 2 depicts the amino acid sequence of an IGF2BP3:PRKCA fusion protein (SEQ ID NO:2). The slash between amino acids 401 and 402 indicates the breakpoint or fusion junction between the IGF2BP3 and PRKCA proteins. Amino acids 401-402 correspond to the codons at nucleotides 1201.1203 and 1204-1206 in SEQ ID NO:1.

FIG. 3 depicts the nucleotide sequence of a TANC2:PRKCA gene fusion (SEQ ID NO:3) comprising a portion of the TANC2 gene (NM_025185) up to and including exon 2 and a portion of the PRKCA gene (NM_002737) starting at exon 6. The underlined codons at nucleotides 136-138 and 142-144 encode the last amino acid of TANC2 and the first amino acid of PRKCA, respectively. The slash after nucleotide 139 indicates the breakpoint (fusion junction) where translocation and in-frame fusion has occurred. The shading at nucleotides 139-141 indicates that nucleotides from both TANC2 and PRKCA are fused in frame to form a codon and encode an amino acid.

FIG. 4 depicts the amino acid sequence of a TANC2:PRKCA fusion protein (SEQ ID NO:4). The shaded amino acid at position 47 corresponds to nucleotides 139-141 in SEQ ID NO:3. This amino acid is encoded by nucleotides from both TANC2 and PRKCA.

FIGS. 5A & 5B depict the nucleotide sequence of an ADCY9:PRKCB gene fusion (SEQ ID NO:5) comprising a ADCY9 gene (NM_001116) up to and including exon 2 and a portion of the PRKCB gene (NM_002738) starting at exon 3. The underlined codons at nucleotides 1690-1692 and 1696-1698 encode the last amino acid of ADCY9 and the first amino acid of PRKCB, respectively. The slash after nucleotide 1693 indicates the breakpoint (fusion junction) where translocation and in-frame fusion has occurred. The shading at nucleotides 1693-1695 indicates that nucleotides from both ADCY9 and PRKCB are fused in frame to form a codon and encode an amino acid.

FIG. 6 depicts the amino acid sequence of an ADCY9:PRKCB fusion protein (SEQ ID NO:6). The shaded amino acid at position 565 corresponds to the codon at nucleotides 1693-1695 in SEQ ID NO:5. This amino acid is encoded by nucleotides from both ADCY9 and PRKCB.

FIG. 7 depicts the nucleotide sequence of a SPNS1:PRKCB gene fusion (SEQ ID NO:7) comprising a SPNS1 gene (NM_001142451) up to and including exon 2 and a portion of the PRKCB gene (NM_(002738) starting at exon 3. The underlined codons at nucleotides 304-306 and 310-312 encode the last amino acid of SPNS1 and the first amino acid of PRKCB, respectively. The slash after nucleotide 307 indicates the breakpoint (fusion junction) where translocation and in-frame fusion has occurred. The shading at nucleotides 307-309 indicates that nucleotides from both SPNS1 and PRKCB are fused in frame to form a codon and encode an amino acid.

FIG. 8 depicts the amino acid sequence of an SPNS1:PRKCB fusion protein (SEQ ID NO:8). The shaded amino acid at position 103 corresponds to the codon at nucleotides 307-309 in SEQ ID NO:7. This amino acid is encoded by nucleotides from both SPNS1 and PRKCA.

 EXEMPLARY EMBODIMENTS OF THE INVENTION

The invention is based, at least in part, on the discovery of novel recombination or translocation events in cancer patients that result in at least a fragment of a PRKC gene linked to a non-homologous promoter via a recombination or translocation event that may result in aberrant expression (e.g., in a location where the kinase is not typically expressed) or overexpression of the kinase domain of the PRKC gene and thus, an increase in kinase activity. In some embodiments, the recombination or translocation may result in constitutive activation of PRKC kinase activity. Thus, a new patient population is identified, which is characterized by the presence of a PRKC fusion, e.g., a PRKC gene fusion or fusion protein. This new patient population suffers from or is susceptible to disorders mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, such as, e.g., a cancer. In another aspect of the invention, a new subtype of cancer is identified, which is characterized by the presence of the PRKC fusions described herein. In some embodiments, the new patient population suffers from or is susceptible to a lung cancer (such as, e.g., lung squamous cell carcinoma or lung adenocarcinoma) characterized by the presence of a PKRC fusion. New methods of diagnosing and treating the patient population and the PRKC fusion cancer subtype are also provided.

The term “PRKC fusion” is used generically herein, and includes any fusion molecule (e.g., gene, gene product (e.g., cDNA, mRNA, or protein), and variants thereof) that includes a fragment of PRKC, particularly the coding region for the kinase domain of PRKC, and a fragment of a second, non-homologous gene, such that the coding sequence for the kinase domain of PRKC is under control of the promoter of the non-homologous gene. In some embodiments, the PRKC fusion removes all or a portion of the coding region of the regulatory domain of PRKC, such as, e.g., the autoinhibitory pseudosubstrate segment. A PRKC fusion protein generally includes the kinase domain of PRKC.

PRKC Gene Fusions and Fusion proteins

PRKC is a family of protein kinase enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. PRKC in turn is activated by signals such as increases in the cellular concentration of diacylglycerol (DAG) or calcium ions (Ca2+) and plays important roles in several signal transduction cascades. The activity of PRKC itself is also regulated by phosphorylation. The PRKC family contains fifteen isozymes in humans, which are divided into three subfamilies based on their second messenger requirements: conventional (or classical), novel, and atypical. Isoforms a (protein kinase C, alpha (PRKCA)) and β1 and βn (protein kinase C, beta (PRKCB)) belong to the conventional subfamily. The structure of PRKC contains a regulatory domain and a catalytic domain tethered together by a hinge region. The regulatory domain at the N-terminus of PRKC includes the pseudosubstrate segment, which is a small sequence of amino acids that mimic a substrate and bind the substrate-binding cavity in the catalytic domain, keeping the enzyme inactive. Wien Ca2+ and DAG are present in sufficient concentrations, they bind to the regulatory domain and recruit PRKC to the membrane. This interaction with the membrane results in release of the pseudosubstrate segment from the catalytic site and activation of the enzyme. The catalytic region includes the kinase domain of PRKC. Upon activation, PRKC is translocated to the plasma membrane and carries out PRKC-mediated phosphorylation of substrate proteins. PRKC fusions have recently been described in papillary glioneuronal tumors (see Bridge, J. A. et al. Brain Pathol. Zurich Switz. 23: 121-128 (2013)) and benign fibrous histiocytoma (see Plaszczyca, A. et al, Int. J. Biochem. Cell Biol. doi:10.1016/j.biocel.2014.03.027 (2014)).

The invention provides novel PRKC fusions that are associated with different types of disorders. For example, the PRKC fusions disclosed herein can be associated with certain cancers, such as, e.g., a lung cancer. In some embodiments, the lung cancer associated with a PRKC fusion is lung squamous carcinoma or lung adenocarcinoma. In some embodiments, the PRKC fusions disclosed herein can be associated with other disorders mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC.

PRKC gene fusions are generated by a fusion between at least a part of the PRKC gene, such as, e.g., PRKCA or PRKCB, and a part of another gene as a result of a translocation (including inversion) within a chromosome or between chromosomes. As a result of a translocation, the PRKC gene may be placed under the transcriptional control of the partner gene promoter, resulting in aberrant PRKC activity or expression or overexpression of PRKC. Alternatively or additionally, the translocation may result in removal of all or a portion of the regulatory domain of PRKC, such as, e.g., the autoinhibitory pseudosubstrate segment, and cause PRKC to become constitutively activated. The overexpression and/or constitutive activation can lead to certain cancers. As used herein, the 5′-region is upstream of, and the 3′-region is downstream of, a fusion junction or breakpoint in one of the component genes. PRKC and the gene or protein that it is fused to is referred to as “fusion partners.” Alternatively, they may be identified as a “PRKC gene fusion” or a “PRKC fusion protein,” which are collectively termed “PRKC fusions.” The PRKC fusions disclosed herein have a kinase activity. The phrase “having a kinase activity” as used in this application means having an activity as an enzyme phosphorylating the side chain of an amino acid, such as serine or threonine. In some embodiments, the PRKC fusion may include an in-frame fusion of the coding sequences of PRKC and the fusion partner that introduces amino acids into the fusion protein that are not part of PRKC or the fusion partner.

In some exemplary embodiments, the fusion partner is all or a portion of PRKC (insulin-like growth factor 2 mRNA binding protein 3). In other exemplary embodiments, the fusion partner is all or a portion of TANC2 (tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2). In other exemplary embodiments, the fusion partner is all or a portion of ADCY9 (adenylate cyclase type 9). In yet other exemplary embodiments, the fusion partner is all or a portion of SPNS1 (spinster homolog 1). In some embodiments, the PRKC gene is PRKCA. In other embodiments, the PRKC gene is PRKCB.

Reference to “all or a portion” or “all or part” of a PRKC gene fusion or SEQ ID NO: 1, 3, 5, or 7, means that the nucleotide sequence comprises the entire PRKC gene fusion nucleotide sequence or a fragment of that sequence that comprises the fusion junction or breakpoint between PRKC and its fusion partner (such as, e.g., IGF2BP3, TANC2, ADCY9, or SPNS1). The fragment may comprise 7, 8, 9, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 175, 200, 250, 300, or more nucleotide, spanning the fusion junction of the PRKC gene fusion. Reference to “all or a portion” or “all or part” of a PRKC fusion protein or SEQ ID NO:2, 4, 6, or 8, means an amino acid sequence that comprises the entire PRKC fusion protein amino acid sequence or a fragment of that sequence that comprises the fusion junction or breakpoint between PRKC and its fusion partner (such as, e.g., IGF2BP3, TANC2, ADCY9, or SPNS1). The fragment may comprise 8, 10, 12, 14, 15, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more amino acids spanning the fusion junction.

In certain embodiments of the invention, a fusion includes an in-frame fusion of all or a portion of the IGF2BP3 gene (e.g., an IGF2BP3 promotor or a functional fragment thereof, and one or more exons encoding IGF2BP3 or a fragment thereof) and an exon of the PRKCA gene (e.g., one or more exons encoding a PRKCA kinase domain or a functional fragment thereof). Such a fusion can be referred to as an IGF2BP3:PRKCA fusion. In one embodiment, the IGF2BP3:PRKCA fusion comprises sufficient PRKCA sequence to drive expression of a fusion protein that has kinase activity, e.g., has elevated activity as compared with wild type PRKCA in the same tissue or cell. In some embodiments, the IGF2BP3:PRKCA fusion removes all or a portion of the sequence encoding the regulatory domain of PRKCA, such as, e.g., the autoinhibitory pseudosubstrate segment, and results in expression of a fusion protein that has constitutive kinase activity, e.g., has activity without upstream signaling or binding of a ligand or second messenger, such as, e.g., DAG or Ca2+.

In some embodiments, the invention provides an IGF2BP3:PRKC gene fusion comprising the nucleotide sequence depicted in FIG. 1 (SEQ ID NO: 1), or a fragment thereof that includes the fusion junction. SEQ ID NO:1 comprises IGF2BP3 (NM_006547) up to exon 10 fused to PRKCA (NM_002737), beginning at exon 4. In some embodiments, the IGF2BP3:PRKCA gene fusion comprises a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to all or part of SEQ ID NO:1. In some embodiments, the IGF2BP3:PRKCA gene fusion encodes a protein having the sequence depicted in FIG. 2 (SEQ ID NO:2) or a sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to all or part of SEQ ID NO:2.

In other embodiments of the invention, a fusion includes an in-frame fusion of all or a portion of the TANC2 gene (e.g., a TANC2 promotor or a functional fragment thereof, and one or more exons encoding a TANC2 or a fragment thereof) and an exon of the PRKCA gene (e.g., one or more exons encoding a PRKCA kinase domain or a functional fragment thereof). Such a fusion can be referred to as a TANC2:PRKCA fusion. In one embodiment, the TANC2:PRKCA fusion comprises sufficient PRKCA sequence to drive expression of a fusion protein that has kinase activity, e.g., has elevated activity as compared with wild type PRKCA in the same tissue or cell. In some embodiments, the TANC2:PRKCA fusion removes all or a portion of the sequence encoding the regulatory domain of PRKCA, such as, e.g., the autoinhibitory pseudosubstrate segment, and results in expression of a fusion protein that has constitutive kinase activity. e.g., has activity without upstream signaling or binding of a ligand or second messenger, such as, e.g., DAG or Ca2+.

In some embodiments, the invention provides an TANC2:PRKCA gene fusion comprising the nucleotide sequence depicted in FIG. 3 (SEQ ID NO:3), or a fragment thereof that includes the fusion junction. SEQ ID NO:3 comprises TANC2 (NM_025185) up to exon 2 fused to PRKCA (NM_002737), beginning at exon 6. In some embodiments, the TANC2:PRKCA gene fusion comprises a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 95%, or at least 99% identical to all or part of SEQ ID NO:3. In some embodiments, the TANC2:PRKCA gene fusion encodes a protein having the sequence depicted in FIG. 4 (SEQ ID NO:4) or a sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to all or part of SEQ ID NO:4.

In certain embodiments of the invention, a fusion includes an in-frame fusion of all or a portion of the ADCY9 gene (e.g., an ADCY9 promotor or a functional fragment thereof, and one or more exons encoding an ADCY9 or a fragment thereof) and an exon of the PRKCB gene (e.g., one or more exons encoding a PRKCB kinase domain or a functional fragment thereof). Such a fusion can be referred to as an ADCY9:PRKCB fusion. In some embodiments, the ADCY9:PRKCB fusion comprises sufficient PRKCB sequence to drive expression of a fusion protein that has kinase activity, e.g., has elevated activity as compared with wild type PRKCB in the same tissue or cell. In some embodiments, the ADCY9:PRKCB fusion removes all or a portion of the sequence encoding the regulatory domain of PRKCB, such as, e.g., the autoinhibitory pseudosubstrate segment, and results in expression of a fusion protein that has constitutive kinase activity, e.g., has activity without upstream signaling or binding of a ligand or second messenger, such as, e.g., DAG or Ca2.

In some embodiments, the ADCY9:PRKCB fusion has the nucleotide sequence depicted in FIG. 5 (SEQ ID NO:5), or a fragment thereof that includes the fusion junction. SEQ ID NO:5 comprises ADCY9 (NM_001116) up to and including exon 2 fused to PRKCB (NM_002738), beginning at exon 3. In some embodiments, the ADCY9:PRKCB gene fusion comprises a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to all or part of SEQ ID NO:5. In some embodiments, the ADCY9:PRKCB fusion encodes a protein having the sequence depicted in FIG. 6 (SEQ ID NO:6) or a sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to all or part of SEQ ID NO:6.

In certain embodiments of the invention, a fusion includes an in-frame fusion of all or a portion of the SPNS1 gene (e.g., an SPNS1 promotor or a functional fragment thereof, and one or more exons encoding an SPNS1 or a fragment thereof) and an exon of the PRKCB gene (e.g., one or more exons encoding a PRKCB kinase domain or a functional fragment thereof). Such a fusion can be referred to as an SPNS1:PRKCB fusion. In one embodiment, the SPNS1:PRKCB fusion comprises sufficient PRKCB sequence to drive expression of a fusion protein that has kinase activity. e.g., has elevated activity as compared with wild type PRKCB in the same tissue or cell. In some embodiments, the SPNS1:PRKCB fusion removes all or a portion of the sequence encoding the regulatory domain of PRKCB, such as, e.g., the autoinhibitory pseudosubstrate segment, and results in expression of a fusion protein that has constitutive kinase activity, e.g., has activity without upstream signaling or binding of a ligand or second messenger, such as, e.g., DAG or Ca2+.

In some embodiments, the invention provides an SPNS1:PRKCB gene fusion comprising the nucleotide sequence depicted in FIG. 7 (SEQ ID NO:7), or a fragment of thereof that includes the fusion junction. SEQ ID NO:7 comprises SPNS1 (NM_001142451) up to exon 2 fused to PRKCB (NM_002738), beginning at exon 3. In some embodiments, the SPNS1:PRKCB gene fusion comprises a nucleotide sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to all or part of SEQ ID NO:7. In some embodiments, the SPNS1:PRKCB gene fusion encodes a protein having the sequence depicted in FIG. 8 (SEQ ID NO:8) or a sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to all or part of SEQ ID NO:8.

The nucleic acid sequences of PRKC gene fusions may be used as probes, primers, or bait to identify nucleotides from a biological sample that include, flank, or hybridize to, PRKC fusions, such as IGF2BP3:PRKCA (e.g., all or part of SEQ ID NO: 1). TANC2:PRKCA (e.g., all or part of SEQ ID NO:3), ADCV9:PRKCB (e.g., all or part of SEQ ID NO:5), or SPNS1:PRKCB (e.g., all or part of SEQ ID NO:7), at, e.g., the fusion junctions. In certain embodiments, the probe, primer, or bait molecule is an oligonucleotide that allows capture, detection, and/or isolation of a PRKC gene fusion in a biological sample. In certain embodiments, the probes or primers derived from the nucleic acid sequences of PRKC gene fusions (e.g., from the fusion junctions) may be used, for example, for polymerase chain reaction (PCR) amplification. The oligonucleotide can comprise a nucleotide sequence substantially complementary to a fragment of the PRKC gene fusion nucleic acid molecules described herein. The sequence identity between the nucleic acid fragment. e.g., the oligonucleotide and the target PRKC gene fusion sequence, need not be exact, so long as the sequences are sufficiently complementary to allow the capture, detection or isolation of the target sequence. In one embodiment, the nucleic acid fragment is a probe or primer that includes an oligonucleotide between about 5 and 25, e.g., between 10 and 20, or 10 and 15 nucleotides in length that includes the fusion junction of a PRKC fusion, such as, e.g., IGF2BP3:PRKCA (e.g., all or part of SEQ ID NO: 1), TANC2:PRKCA (e.g., all or part of SEQ ID NO:3), ADCY9:PRKCB (e.g., all or part of SEQ ID NO:5), or SPNS1:PRKCB (e.g., all or part of SEQ ID NO:7). In other embodiments, the nucleic acid fragment is a bait that includes an oligonucleotide between about 100 to 300 nucleotides, 130 and 230 nucleotides, or 150 and 200 nucleotides in length that includes the fusion junction of a PRKC fusion, such as, e.g., IGF2BP3:PRKCA (e.g., all or part of SEQ ID NO: 1). TANC2:PRKCA (e.g., all or part of SEQ ID NO: 3). ADCY9:PRKCB (e.g., all or part of SEQ ID NO:5), or SPNS1:PRKCB (e.g., all or part of SEQ ID NO:7).

In certain embodiments, the nucleic acid fragments hybridize to a nucleotide sequence that includes a breakpoint or fusion junction, e.g., a breakpoint or fusion junction as identified by a slash (“/”) in FIG. 1, 3, 5, or 7. For example, the nucleic acid fragment can hybridize to a nucleotide sequence that includes the fusion junction between the IGF2BP3 transcript and the PRKCA transcript (e.g., nucleotides 1201-1206 of SEQ ID NO: 1), between the TANC2 transcript and the PRKCA transcript (e.g., nucleotides 136-144 of SEQ ID NO:3), between the ADCY9 transcript and the PRKCB transcript (e.g., nucleotides 1690-1698 of SEQ ID NO:5), or between the SPNS1 transcript and the PRKCB transcript (e.g., nucleotides 304-312 of SEQ ID NO:7), i.e., a nucleotide sequence that includes a portion of SEQ ID NO: 1, 3, 5, or 7.

In other embodiments, the nucleic acid fragment includes a bait that comprises a nucleotide sequence that hybridizes to a PRKC gene fusion nucleic acid molecule described herein, and thereby allows the detection, capture, and/or isolation of the nucleic acid molecule. In one embodiment, a bait is suitable for solution phase hybridization. In other embodiments, a bait includes a binding entity or detection entity, e.g., an affinity tag or fluorescent label, that allows detection, capture, and/or separation, e.g., by binding to a binding entity, of a hybrid formed by a bait and a nucleic acid hybridized to the bait.

In exemplary embodiments, the nucleic acid fragments hybridize to a nucleotide sequence that includes a fusion junction between the IGF2BP3 transcript and the PRKCA transcript, e.g., a nucleotide sequence within SEQ ID NO:1 comprising nucleotides 1201-1206 (such as, e.g., a sequence comprising nucleotides 1199-1208, 1194-1213, 1179-1228, or 1154-1253 of SEQ ID NO:1).

In other exemplary embodiments, the nucleic acid fragments hybridize to a nucleotide sequence that includes a fusion junction between the TANC2 transcript and the PRKCA transcript, e.g., a nucleotide sequence within SEQ ID NO:3 comprising nucleotides 136-144 (such as, e.g., a sequence comprising nucleotides 134-146, 129-151, 114-166, or 89-191 of SEQ ID NO:3).

In other exemplary embodiments, the nucleic acid fragments hybridize to a nucleotide sequence that includes a fusion junction between the ADCY9 transcript and the PRKCB transcript. e.g., a nucleotide sequence within SEQ ID NO:5 comprising nucleotides 1690-1698 (such as, e.g., a sequence comprising nucleotides 1688-1700, 1683-1705, 1668-1720, or 1643-1745 of SEQ ID NO:5).

In other exemplary embodiments, the nucleic acid fragments hybridize to a nucleotide sequences that includes a fusion junction between the SPNS1 transcript and the PRKCB transcript, e.g., a nucleotide sequence within SEQ ID NO:7 comprising nucleotides 304-312 (such as, e.g., a sequence comprising nucleotides 302-314, 297-319, 282-334, or 257-359 of SEQ ID NO:7).

Another aspect of the invention provides PRKC fusion proteins (such as, e.g., a purified or isolated IGF2BP3:PRKCA. TANC2:PRKCA. ADCY9:PRKCB, or SPNS1:PRKCB fusion protein), biologically active or antigenic fragments thereof, and use of those polypeptides for detecting and/or modulating the biological activity (such as tumorigenic activity) of a PRKC fusion protein. Exemplary embodiments of the PRKC fusion proteins comprise the amino acid sequence set forth in SEQ ID NO:2, 4, 6, or 8, and fragments of those sequences.

In some embodiments, the PRKC fusion protein of the invention can include a fragment of an IGF2BP3 protein, a TANC2 protein, an ADCV9 protein, or an SPNS1 protein, and a fragment of a PRKC protein, such as, e.g., a PRKCA protein or a PRKCB protein. In one embodiment, the PRKC fusion protein is an IGF2BP3:PRKCA fusion protein having the amino acid sequence of SEQ ID NO:2 or a fragment thereof, such as, e.g., amino acids 397-406, 392-411, or 377-426 of SEQ ID NO:2. In other embodiments, the PRKC fusion protein is a TANC2:PRKCA fusion protein having the amino acid sequence of SEQ ID NO-4 or a fragment thereof, such as, e.g., amino acids 42-51, 37-56, or 22-71 of SEQ ID NO:4. In some embodiments, the PRKC fusion protein is an ADCY9:PRKCB fusion protein having the amino acid sequence of SEQ ID NO:6 or a fragment thereof, such as, e.g., amino acids 560-569, 555-574, or 540-589 of SEQ ID NO:6. In other embodiments, the PRKC fusion protein is an SPNS1:PRKCB fusion protein having the amino acid sequence of SEQ ID NO:8, or a fragment thereof, such as, e.g., amino acids 98-107, 93-112, or 78-127 of SEQ ID NO:8.

In some embodiments, the PRKC fusion protein is an IGF2BP3:PRKCA fusion protein comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 or a fragment thereof (e.g., amino acids 397-406, 392-411, or 377-426 of SEQ ID NO:2). In other embodiments, the PRKC fusion protein is a TANC2:PRKCA fusion protein comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:4 or a fragment thereof (e.g., amino acids 42-51, 37-56, or 22-71 of SEQ ID NO:4). In yet other embodiments, the PRKC fusion protein is an ADCY9:PRKCB fusion protein comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6 or a fragment thereof (e.g., amino acids 560-569, 555-574, or 540-589 of SEQ ID NO:6). In some embodiments the PRKC fusion protein is an SPNS1:PRKCB fusion protein comprising an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:8 or a fragment thereof (e.g., amino acids 98-107, 93-112, or 78-127 of SEQ ID NO:8).

In certain embodiments, the PRKC fusion protein includes a functional kinase domain. In some embodiments, the PRKC fusion protein comprises elevated PRKC activity as compared with wild type PRKC activity (e.g., in a cancer cell, a non-cancer cell adjacent to the cancer cell, or a non-cancer cell from a control sample, such as a cancer free subject). In some embodiments, the PRKC fusion protein removes all or a portion of the regulatory domain of PRKC, such as, e.g., the autoinhibitory pseudosubstrate segment. In such embodiments, the PRKC fusion protein comprises constitutive kinase activity. e.g., has activity without upstream signaling or binding of a ligand or second messenger, such as, e.g., DAG or Ca2+. In one exemplary embodiment, the PRKC fusion protein is an IGF2BP3:PRKCA fusion and includes a PRKC serine/threonine kinase domain or a functional fragment thereof. In other exemplary embodiments, the PRKC fusion protein is a TANC2:PRKCA fusion and includes a PRKC serine/threonine kinase domain or a functional fragment thereof. In some exemplary embodiments, the PRKC fusion protein is an ADCY9:PRKCB fusion and includes a PRKC serine/threonine kinase domain or a functional fragment thereof. In yet other exemplary embodiments, the PRKC fusion protein is an SPNS1:PRKCB fusion and includes a PRKC serine/threonine kinase domain or a functional fragment thereof.

In another embodiment, the PRKC fusion protein or fragment is a peptide, e.g., an immunogenic peptide or protein, that contains a fusion junction with a heterologous protein as described herein. Such immunogenic peptides or proteins can be used for vaccine preparation for use in the treatment or prevention of cancers caused by or exacerbated by PRKC gene fusions and PRKC fusion proteins. In other embodiments, such immunogenic peptides or proteins can be used to raise antibodies specific to the fusion protein. In some embodiments, the PRKC fusion protein is present in combination with or is further conjugated to one or more adjuvant(s) or immunogen(s), e.g., a protein capable of enhancing an immune response to the PRKC fusion protein (e.g., a hapten, a toxoid, etc.). In some embodiments, the PRKC fusion protein is an IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB fusion protein. In some embodiments, the PRKC fusion protein comprises the fusion junction of SEQ ID NO:2, 4, 6, or 8.

Thus, another aspect of the invention provides an antibody that binds to a PRKC fusion protein (such as, e.g., an IGF2BP3:PRKCA, a TANC2:PRKCA, an ADCY9:PRKCB, or an SPNS1:PRKCB fusion protein) or a fragment thereof. In certain embodiments, the antibody recognizes a PRKC fusion protein but does not recognize wild type PRKC or the wild type fusion partner (such as, e.g., IGF2BP3, TANC2, ADCY9, or SPNS1). In some embodiments, the antibody binds to an epitope comprising the junction between PRKC and the fusion partner (e.g., the junction of IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB). In one embodiment, the antibody binds to an IGF2BP3:PRKCA fusion protein having the amino acid sequence of SEQ ID NO:2 or a fragment thereof, such as, e.g., amino acids 397-406, 392-411, or 377-426 of SEQ ID NO:2. In other embodiments, the antibody binds to a TANC2:PRKCA fusion protein having the amino acid sequence of SEQ ID NO:4 or a fragment thereof, such as, e.g., amino acids 42-51, 37-56, or 22-71 of SEQ ID NO:4. In some embodiments, the antibody binds to an ADCY9:PRKCB fusion protein having the amino acid sequence of SEQ ID NO:6 or a fragment thereof, such as, e.g., amino acids 560-569, 555-574, or 540-589 of SEQ ID NO:6. In other embodiments, the antibody binds to an SPNS1:PRKCB fusion protein having the amino acid sequence of SEQ ID NO:8 or a fragment thereof, such as, e.g., amino acids 98-107, 93-112, or 78-127 of SEQ ID NO:8.

In certain embodiments, the antibodies of the invention inhibit and/or neutralize the biological activity of the PRKC fusion protein, and more specifically, in some embodiments, the kinase activity of the PRKC fusion protein. In other embodiments, the antibodies may be used to detect a PRKC fusion protein or to diagnose a patient suffering from a disease or disorder associated with the expression of a PRKC fusion protein.

Detection and Diagnostic Methods

In another aspect, the invention provides a method of detecting the presence of a PRKC gene fusion or fusion protein, such as, e.g., an IGF2BP3:PRKCA, a TANC2:PRKCA, an ADCY9:PRKCB, or an SPNS1:PRKCB fusion as described herein. The presence of a PRKC fusion indicates that the mammal providing the biological sample suffers from or is at risk of developing a disorder mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, such as, e.g., a cancer. The presence of a PRKC fusion may also indicate that the disorder is treatable with a PRKC inhibitor, such as, e.g., a kinase inhibitor or an antibody specific to PRKC, or a PRKC fusion inhibitor. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is lung squamous cell carcinoma. In some embodiments, the cancer is lung adenocarcinoma. In some embodiments, the PRKC fusion present in the sample is IGF2BP3:PRKCA, TANC2:PRKCA, or ADCY9:PRKCB and the cancer to be treated is lung squamous cell carcinoma. In other embodiments, the PRKC fusion present in the sample is SPNS1:PRKCB and the cancer to be treated is lung adenocarcinoma.

In one embodiment, the PRKC fusion detected is a nucleic acid molecule or a polypeptide. The method includes detecting whether a PRKC fusion nucleic acid molecule or polypeptide is present in a cell (e.g., a circulating cell or a cancer cell), a tissue (e.g., a tumor), or a sample, e.g., a tumor sample, from a subject. In one embodiment, the sample is a nucleic acid sample. In one embodiment, the nucleic acid sample comprises DNA, e.g., genomic DNA or cDNA, or RNA, e.g., mRNA. In other embodiments, the sample is a protein sample.

The sample can be chosen from one or more sample types, such as, for example, tissue, e.g., cancerous tissue (e.g., a tissue biopsy), whole blood, serum, plasma, buccal scrape, sputum, saliva, cerebrospinal fluid, urine, stool, circulating tumor cells, circulating nucleic acids, or bone marrow.

I. Methods for Detecting Gene Fusions

In certain embodiments, the sample is acquired from a subject having or at risk of having a cancer (e.g., a patient), or alternatively, the method further includes acquiring a sample from the subject. In some embodiments, the PRKC fusion is detected in a nucleic acid molecule by a method chosen from one or more of: e.g., nucleic acid hybridization assay (e.g. in situ hybridization, comparative genomic hybridization, microarray, Southern blot, northern blot), amplification-based assays (e.g., PCR, PCR-RFLP assay, or real-time PCR), sequencing and genotyping (e.g. sequence-specific primers, high-performance liquid chromatography, or mass-spectrometric genotyping), and screening analysis (including metaphase cytogenetic analysis by karyotype methods.

(1) Hybridization Methods

In some embodiments, the reagent hybridizes to a PRKC gene fusion, such as, e.g., nucleotides 1201-1206, 1199-1208, 1194-1213, 1179-1228, or 1154-1253 of SEQ ID NO:1. In alternate embodiments, the reagent detects the presence of nucleotides 136-144, 134-146, 129-151, 114-166, or 89-191 of SEQ ID NO:3, nucleotides 1690-1698, 1688-1700, 1683-1705, 1668-1720, or 1643-1745 of SEQ ID NO:5, or nucleotides 304-312, 302-314, 297-319, 282-334, or 257-359 of SEQ ID NO:7. In one embodiment, the method includes: contacting a nucleic acid sample, e.g., a genomic DNA sample (e.g., a chromosomal sample or a fractionated, enriched or otherwise pre-treated sample) or a gene product (e.g., mRNA, or cDNA), obtained from the subject, with a nucleic acid fragment e.g., a probe or primer as described herein (e.g., an exon-specific or a breakpoint-specific probe or primer), under conditions suitable for hybridization, and determining the presence or absence of the PRKC gene fusion, such as, e.g. IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB. In an alternate embodiment, the method includes the steps of obtaining a sample; exposing the sample to a nucleic acid probe which hybridizes to an mRNA or cDNA encoding a PRKC fusion protein that comprises amino acids 397-406, 392-411, or 377-426 of SEQ ID NO:2, amino acids 42-51, 37-56, or 22-71 of SEQ ID NO:4, amino acids 560-569, 555-574, or 540-589 of SEQ ID NO:6, or amino acids 98-107, 93-112, or 78-127 of SEQ ID NO:8.

Hybridization, as described throughout the specification, may be carried out under stringent conditions, e.g., medium or high stringency. See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Pr; 2nd edition (1989); T. Brown, Hybridization Analysis of DNA Blots, Current Protocols in Molecular Biology at 21:2.10.1-2.10.16 (2001). High stringency conditions for hybridization refer to conditions under which two nucleic acids must possess a high degree of base pair homology to each other in order to hybridize. Examples of highly stringent conditions for hybridization include hybridization in 4× sodium chloride/sodium citrate (SSC), at 65 or 70° C., or hybridization in 4×SSC plus 50% formamide at about 42 or 50° C., followed by at least one, at least two, or at least three washes in 1×SSC, at 65 or 70° C. Another example of highly stringent conditions includes hybridization in 2×SSC; 10×Denhardt solution (Fikoll 400+PEG+BSA; ratio 1:1:1): 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 μg/ml of herring sperm DNA: 50 μg/ml of tRNA; or 0.25 M of sodium phosphate buffer, pH 7.2; 1 mM EDTA 7% SDS at 60° C.; followed by washing 2×SSC, 0.1% SDS at 60° C.

The nucleic acid fragments can be detectably labeled with, e.g., a radiolabel, a fluorescent label, a bioluminescent label, a chemiluminescent label, an enzyme label, a binding pair label (e.g., biotin/streptavidin), an antigen label, or can include an affinity tag, or identifier (e.g., an adaptor, barcode or other sequence identifier). Labeled or unlabeled nucleic acids and/or nucleic acid fragments may be used in reagents for detecting, capturing, or isolating PRKC gene fusions. Labeled or unlabeled nucleic acids and/or nucleic acid fragments may be used in reagents for detecting, capturing, and/or isolating PRKC gene fusions, such as, e.g., IGF2BP3:PRKCA (e.g., all or part of SEQ ID NO: 1). TANC2:PRKCA (e.g., all or part of SEQ ID NO:3), ADCY9:PRKCB (e.g., all or part of SEQ ID NO:5), or SPNS1:PRKCB (e.g., all or part of SEQ ID NO:7). In some embodiments, the labeled reagent can be detected using. e.g., autoradiography, microscopy (e.g., brightfield, fluorescence, or electron microscopy), enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry.

In some embodiments, the method comprises performing chromosome in situ hybridization with chromosomal DNA from a biological sample to detect the presence of a PRKC gene fusion (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein). In some embodiments, the chromosome in situ hybridization comprises the steps of: providing a chromosome (e.g., interphase or metaphase chromosome) preparation (e.g., by attaching the chromosomes to a substrate (e.g., glass)); denaturing the chromosomal DNA (e.g., by exposure to formamide) to separate the double strands of the polynucleotides from each other; exposing the nucleic acid probe to the chromosomes under conditions to allow hybridization of the probe to the target DNA; removing unhybridized or non-specifically hybridized probes by washing; and detecting the hybridization of the probe with the target DNA. In some embodiments, the chromosome in situ hybridization is fluorescence in situ hybridization (FISH). In some embodiments, the probe is labeled directly by a fluorescent label, or indirectly by incorporation of a nucleotide containing a tag or reporter molecule (e.g., biotin, digoxigenin, or hapten) which after hybridization to the target DNA is then bound by fluorescently labeled affinity molecule (e.g., an antibody or streptavidin). In some embodiments, the hybridization of the probe with the target DNA in FISH can be visualized using a fluorescence microscope.

In other embodiments, the method comprises performing Southern blot with DNA polynucleotides from a biological sample to detect the presence of a PRKC gene fusion (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein). In some embodiments, the Southern blot comprises the steps of: optionally fragmenting the polynucleotides into smaller sizes by restriction endonucleases; separating the polynucleotides by gel electrophoresis; denaturing the polynucleotides (e.g., by heat or alkali treatment) to separate the double strands of the polynucleotides from each other; transferring the polynucleotides from the gel to a membrane (e.g., a nylon or nitrocellulose membrane); immobilizing the polynucleotides to the membrane (e.g., by UV light or heat); exposing the nucleic acid probe to the polynucleotides under conditions to allow hybridization of the probe to the target DNA; removing unhybridized or non-specifically hybridized probes by washing; and detecting the hybridization of the probe with the target DNA.

(2) Amplification-Based Assays

In certain embodiments, the method of detecting the presence of a PRKC gene fusion, comprises (a) performing a PCR amplification reaction with polynucleotides from a biological sample, wherein the amplification reaction utilizes a pair of primers which will amplify at least a fragment of the PRKC gene fusion, wherein the fragment comprises the fusion junction, wherein the first primer is in sense orientation and the second primer is in antisense orientation; and (b) detecting an amplification product, wherein the presence of the amplification product is indicative of the presence of a PRKC fusion polynucleotide in the sample. In specific exemplary embodiments, the PRKC gene fusion is IGF2BP3:PRKCA, such as, e.g., the gene fusion of SEQ ID NO: 1 or a fragment thereof comprising nucleotides 1201-1206, 1199-1208, 1194-1213, 1179-1228, or 1154-1253 of SEQ ID NO:1 In other exemplary embodiments, the gene fusion is TANC2:PRKCA such as, e.g. the gene fusion of SEQ ID NO:3 or a fragment thereof comprising nucleotides 136-144, 134-146, 129-151, 114-166, or 89-191 of SEQ ID NO:3. In other exemplary embodiments, the gene fusion is ADCY9:PRKCB such as, e.g. the gene fusion of SEQ ID NO:5 or a fragment thereof comprising nucleotides 1690-1698, 1688-1700, 1683-1705, 1668-1720, or 1643-1745 of SEQ ID NO:5. In certain exemplary embodiments, the gene fusion is SPNS1:PRKCB such as, e.g. the gene fusion of SEQ ID NO:7 or a fragment thereof comprising nucleotides 304-312, 302-314, 297-319, 282-334, or 257-359 of SEQ ID NO:7.

In some embodiments, step (a) of performing a PCR amplification reaction comprises: ti) providing a reaction mixture comprising the polynucleotides (e.g., DNA or cDNA) from the biological sample, the pair of primers which will amplify at least a fragment of the PRKC gene fusion wherein the first primer is complementary to a sequence on the first strand of the polynucleotides and the second primer is complementary to a sequence on the second strand of the polynucleotides, a DNA polymerase, and a plurality of free nucleotides comprising adenine, thymine, cytosine, and guanine (dNTPs); (ii) heating the reaction mixture to a first predetermined temperature for a first predetermined time to separate the double strands of the polynucleotides from each other; (iii) cooling the reaction mixture to a second predetermined temperature for a second predetermined time under conditions to allow the first and second primers to hybridize with their complementary sequences on the first and second strands of the polynucleotides, and to allow the DNA polymerase to extend the primers; and (iv) repeating steps (ii) and (iii) for a predetermined number of cycles (e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50 cycles).

In some embodiments, the polynucleotides from the biological sample comprise RNA, and the method further comprises performing a RT-PCR amplification reaction with the RNA to synthesize cDNA as the template for subsequent or simultaneous PCR reactions. In some embodiments, the RT-PCR amplification reaction comprises providing a reaction mixture comprising the RNA, a primer which will amplify the RNA (e.g., a sequence-specific primer, a random primer, or oligo(dT)s), a reverse transcriptase, and dNTPs, and heating the reaction mixture to a third predetermined temperature for a third predetermined time under conditions to allow the reverse transcriptase to extend the primer.

(3) Sequencing and Genotyping

Another method for determining the presence of a PRKC gene fusion molecule (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein) includes: sequencing a portion of the nucleic acid molecule (e.g., sequencing the portion of the nucleic acid molecule that comprises the fusion junction of a PRKC gene fusion), thereby determining that the PRKC gene fusion is present in the nucleic acid molecule. Optionally, the sequence acquired is compared to a reference sequence, or a wild type reference sequence. In one embodiment, the sequence is determined by a next generation sequencing method. In some embodiments, the sequencing is automated and/or high-throughput sequencing. The method can further include acquiring, e.g., directly or indirectly acquiring, a sample, e.g., a tumor or cancer sample, from a patient.

In some embodiments, the sequencing comprises chain terminator sequencing (Sanger sequencing), comprising: providing a reaction mixture comprising a nucleic acid molecule from a biological sample, a primer complementary to a region of the template nucleic acid molecule, a DNA polymerase, a plurality of free nucleotides comprising adenine, thymine, cytosine, and guanine (dNTPs), and at least one chain terminating nucleotide (e.g., at least one di-deoxynucleotide (ddNTPs) chosen from ddATP, ddTTP, ddCTP, and ddGTP), wherein the at least one chain terminating nucleotide is present in a low concentration so that chain termination occurs randomly at any one of the positions containing the corresponding base on the DNA strand; annealing the primer to a single strand of the nucleic acid molecule; extending the primer to allow incorporation of the chain terminating nucleotide by the DNA polymerase to produce a series of DNA fragments that are terminated at positions where that particular nucleotide is used; separating the polynucleotides by electrophoresis (e.g., gel or capillary electrophoresis); and determining the nucleotide order of the template nucleic acid molecule based on the positions of chain termination on the DNA fragments. In some embodiments, the sequencing is carried out with four separate base-specific reactions, wherein the primer or the chain terminating nucleotide in each reaction is labeled with a separate fluorescent label. In other embodiments, the sequencing is carried out in a single reaction, wherein the four chain terminating nucleotides mixed in the single reaction are each labeled with a separate fluorescent label.

In some embodiments, the sequencing comprises pyrosequencing (sequencing by synthesis), comprising: (i) providing a reaction mixture comprising a nucleic acid molecule from a biological sample, a primer complementary to a region of the template nucleic acid molecule, a DNA polymerase, a first enzyme capable of converting pyrophosphate into ATP, and a second enzyme capable using ATP to generates a detectable signal (e.g., a chemiluminescent signal, such as light) in an amount that is proportional to the amount of ATP; (ii) annealing the primer to a single strand of the nucleic acid molecule; (iii) adding one of the four free nucleotides (dNTPs) to allow incorporation of the correct, complementary dNTP onto the template by the DNA polymerase and release of pyrophosphate stoichiometrically; (iv) converting the released pyrophosphate to ATP by the first enzyme; (v) generating a detectable signal by the second enzyme using the ATP; (vi) detecting the generated signal and analyzing the amount of signal generated in a program; (vii) removing the unincorporated nucleotides; and (viii) repeating steps (iii) to (vii). The method allows sequencing of a single strand of DNA, one base pair at a time, and detecting which base was actually added at each step. The solutions of each type of nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base of the template. The order of solutions which produce detectable signals allows the determination of the sequence of the template.

In some embodiments, the method of determining the presence of a PRKC fusion (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein) comprises analyzing a nucleic acid sample (e.g., DNA, cDNA, or RNA, or an amplification product thereof) by HPLC. The method may comprise: passing a pressurized liquid solution containing the sample through a column filled with a sorbent, wherein the nucleic acid or protein components in the sample interact differently with the sorbent, causing different flow rates for the different components; separating the components as they flow out the column at different flow rates. In some embodiments, the HPLC is chosen from, e.g., reverse-phase HPLC, size exclusion HPLC, ion-exchange HPLC, and bioaffinity HPLC.

In some embodiments, the method of determining the presence of a PRKC fusion (such as, e.g., IGF2BP3:PRKCA. TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein) comprises analyzing a nucleic acid sample (e.g., DNA, cDNA, or RNA, or an amplification product thereof) by mass spectrometry. The method may comprise: ionizing the components in the sample (e.g., by chemical or electron ionization); accelerating and subjecting the ionized components to an electric or magnetic field: separating the ionized components based on their mass-to-charge ratios; and detecting the separated components by a detector capable of detecting charged particles (e.g., by an electron multiplier).

II. Methods for Detecting Fusion Proteins

Another aspect of the invention provides a method of detecting the presence of a PRKC fusion protein in a mammal. The method comprises the steps of obtaining a biological sample of a mammal (such as, e.g., a human cancer), and exposing that sample to at least one reagent that detects a PRKC fusion protein (e.g., an antibody that recognizes the PRKC fusion but does not recognize the wild type PRKC or the wild type fusion partner) to determine whether a PRKC fusion protein is present in the biological sample. The detection of a PRKC fusion protein indicates the presence of a mutant PRKC in the mammal (such as, e.g., in the human cancer). In some embodiments, the PRKC fusion protein comprises an amino acid sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with an amino acid sequence of all or part of any one of SEQ ID NOs 2, 4, 6, or 8 or fragments thereof that include the fusion junction. In some embodiments the cancer is lung cancer. In some embodiments, the cancer is lung squamous cell carcinoma. In some embodiments, the cancer is lung adenocarcinoma.

In some embodiments, the reagent that detects a PRKC fusion protein can be detectably labeled with, e.g., a radiolabel, a fluorescent label, a bioluminescent label, a chemiluminescent label, an enzyme label, a binding pair label (e.g., biotin/streptavidin), an antigen label, or can include an affinity tag or identifier (e.g., an adaptor, barcode or other sequence identifier). In some embodiments, the labeled reagent can be detected using, e.g., autoradiography, microscopy (e.g., brightfield, fluorescence, or electron microscopy). ELISA, or immunohistochemistry. In some embodiments, the PRKC fusion protein is detected in a biological sample by a method chosen from one or more of: antibody-based detection (e.g., western blot, ELISA, immunohistochemistry), size-based detection methods (e.g., HPLC or mass spectrometry), or protein sequencing.

(1) Antibody-Based Detection

In some embodiments, the method comprises performing a western blot with polypeptides from a biological sample to detect the presence of a PRKC fusion protein (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein). In some embodiments, the western blot comprises the steps of: separating the polypeptides by gel electrophoresis; transferring the polypeptides from the gel to a membrane (e.g., a nitrocellulose or polyvinylidene difluoride (PVDF) membrane); blocking the membrane to prevent nonspecific binding by incubating the membrane in a dilute solution of protein (e.g., 3-5% bovine serum albumin (BSA) or non-fat dry milk in Tris-Buffered Saline (TBS) or I-Block, with a minute percentage (e.g., 0.1%) of detergent, such as, e.g., Tween 20 or Triton X-100); exposing the polypeptides to at least one reagent that detects a PRKC fusion protein (e.g., an antibody that recognizes the PRKC fusion but does not recognize the wild type PRKC or the wild type fusion partner); removing unbound or non-specifically bound reagent by washing; and detecting the binding of the reagent with the target protein. In some embodiments, the method comprises two-step detection: exposing the polypeptides to a primary antibody that specifically binds to a PRKC fusion protein; removing unbound or non-specifically bound primary antibody by washing; exposing the polypeptides to a secondary antibody that recognizes the primary antibody; removing unbound or non-specifically bound secondary antibody by washing; and detecting the binding of the secondary antibody. In some embodiments, the reagent that detects a PRKC fusion protein (e.g., the fusion specific antibody, or the secondary antibody) is directly labeled for detection. In other embodiments, the reagent is linked to an enzyme, and the method further comprises adding a substrate of the enzyme to the membrane; and developing the membrane by detecting a detectable signal produced by the reaction between the enzyme and the substrate. For example, the reagent may be linked with horseradish peroxidase to cleave a chemiluminescent agent as a substrate, producing luminescence in proportion to the amount of the target protein for detection.

In some embodiments, the method comprises performing ELISA with polypeptides from a biological sample to detect the presence of a PRKC fusion protein (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein). In some embodiments, the ELISA is chosen from, e.g., direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA.

In one embodiment, the direct ELISA comprises the steps of: attaching polypeptides from a biological sample to a surface; blocking the surface to prevent nonspecific binding by incubating the surface in a dilute solution of protein; exposing the polypeptides to an antibody that specifically binds to a PRKC fusion protein (e.g., an antibody that recognizes the PRKC fusion (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein) but does not recognize the wild type PRKC or the wild type fusion partner); removing unbound or non-specifically bound antibody by washing; and detecting the binding of the antibody with the target protein. In some embodiments, the antibody is directly labeled for detection. In other embodiments, the antibody is linked to an enzyme, and the method further comprises adding a substrate of the enzyme; and detecting a detectable signal produced by the reaction between the enzyme and the substrate.

In another embodiment, the indirect ELISA comprises the steps of: attaching polypeptides from a biological sample to a surface; blocking the surface to prevent nonspecific binding by incubating the surface in a dilute solution of protein; exposing the polypeptides to a primary antibody that specifically binds to a PRKC fusion protein (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein); removing unbound or non-specifically bound primary antibody by washing; exposing the polypeptides to a secondary antibody that recognizes the primary antibody; removing unbound or non-specifically bound secondary antibody by washing; and detecting the binding of the secondary antibody. In some embodiments, the secondary antibody is directly labeled for detection. In other embodiments, the secondary antibody is linked to an enzyme, and the method further comprises adding a substrate of the enzyme; and detecting a detectable signal produced by the reaction between the enzyme and the substrate.

In some embodiments, the method comprises performing immunohistochemistry with polypeptides from a biological sample to detect the presence of a PRKC fusion protein (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein). In some embodiments, the immunohistochemistry comprises the steps of: fixing a cell or a tissue section (e.g., by paraformaldehyde or formalin treatment); permeabilizing the cell or tissue section to allow target accessibility; blocking the cell or tissue section to prevent nonspecific binding; exposing the cell or tissue section to at least one reagent that detects a PRKC fusion protein (e.g., an antibody that recognizes the PRKC fusion but does not recognize the wild type PRKC or the wild type fusion partner); removing unbound or non-specifically bound reagent by washing; and detecting the binding of the reagent with the target protein. In some embodiments, the reagent is directly labeled for detection. In other embodiments, the reagent is linked to an enzyme, and the method further comprises adding a substrate of the enzyme; and detecting a detectable signal produced by the reaction between the enzyme and the substrate. In some embodiments, the immunohistochemistry may comprise the two-step detection as in the indirect ELISA.

(2) Size-Based Detection Methods

In some embodiments, the method of determining the presence of a PRKC fusion (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein) comprises analyzing a protein sample by HPLC. The method may comprise: passing a pressurized liquid solution containing the sample through a column filled with a sorbent, wherein the nucleic acid or protein components in the sample interact differently with the sorbent, causing different flow rates for the different components; separating the components as they flow out the column at different flow rates. In some embodiments, the HPLC is chosen from, e.g., reverse-phase HPLC, size exclusion HPLC, ion-exchange HPLC, and bioaffinity HPLC.

In some embodiments, the method of determining the presence of a PRKC fusion (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA. ADCY9:PRKCB, or SPNS1:PRKCB, as disclosed herein) comprises analyzing a protein sample by mass spectrometry. The method may comprise: ionizing the components in the sample (e.g., by chemical or electron ionization); accelerating and subjecting the ionized components to an electric or magnetic field; separating the ionized components based on their mass-to-charge ratios; and detecting the separated components by a detector capable of detecting charged particles (e.g., by an electron multiplier).

Detection of a PRKC gene fusion or a PRKC fusion protein in a patient can lead to assignment of the patient to the newly identified patient population that bears the PRKC fusion. Because this patient population can suffer from or be susceptible to a disorder associated with an aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, detection of the PRKC fusion can also lead to diagnosis of such disorder. Thus, a further aspect of the invention provides a method of stratifying a patient population (e.g., assigning a patient, to a group or class) and/or diagnosing a patient, comprising: obtaining a biological sample from the patient, contacting the sample with at least one reagent that detects a PRKC gene fusion or a PRKC fusion protein to determine whether a PRKC fusion is present in the biological sample. The detection of a PRKC fusion indicates that the patient belongs to the newly identified patient population that bears the PRKC fusion, and/or the presence of a disorder associated with aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, such as, e.g., a cancer. The detection of a PRKC fusion also identifies a new subtype of cancer, which is characterized by the presence of the PRKC fusion.

In some embodiments, the cancer is lung cancer. In some embodiments, the caner is lung squamous cell carcinoma or lung adenocarcinoma. In certain embodiments, the PRKC fusion is IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB. In some embodiments, the IGF2BP3:PRKCA fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively. In some embodiments, the TANC2:PRKCA fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. In some embodiments, the ADCY9:PRKCB fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. In some embodiments, the SPNS1:PRKCB fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:7 and SEQ ID NO:8, respectively. In some embodiments, the PRKC gene fusion or PRKC fusion protein is detected prior to initiating, during, or after, a treatment of a patient with, e.g., a PRKC inhibitor or a PRKC fusion inhibitor. In one embodiment, the PRKC gene fusion or PRKC fusion protein is detected at the time the patient is diagnosed with a cancer. In other embodiment, the PRKC fusion is detected at a pre-determined interval, e.g., a first point in time and at least at a subsequent point in time. In certain embodiments, in response to detection of a PRKC fusion, such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, the method further includes one or more of:

(1) stratifying a patient population (e.g., assigning a patient, to a group or class):

(2) identifying or selecting the patient as likely or unlikely to respond to a treatment, e.g., a PRKC inhibitor treatment (e.g., a kinase inhibitor treatment or an anti-PRKC antibody treatment), or a PRKC fusion inhibitor treatment as described herein;

(3) selecting a treatment regimen, e.g., administering or not administering a preselected therapeutic agent, such as, e.g., a PRKC inhibitor, or a PRKC fusion inhibitor;

(4) prognosticating the time course of the disease in the patient (e.g., evaluating the likelihood of increased or decreased patient survival); or

(5) monitoring the effectiveness of treatment (e.g., by detecting a reduction in the level of PRKC gene fusion or fusion protein in a patient sample).

In certain embodiments, upon detection of a PRKC gene fusion or PRKC fusion protein in a patient's biological sample, the patient is identified as likely to respond to a treatment that comprises a PRKC inhibitor, or a PRKC fusion inhibitor. In some embodiments, the PRKC fusion detected is an IGF2BP3:PRKCA fusion. In alternate embodiments, the PRKC fusion detected is a TANC2:PRKCA fusion. In some embodiments, the PRKC fusion detected is an ADCY9:PRKCB fusion. In some embodiments the PRKC fusion detected is an SPNS1:PRKCB fusion.

A further aspect of the invention provides a method of selecting a treatment option by detecting a PRKC fusion. The method comprises obtaining a biological sample from a patient and exposing the sample to at least one reagent that detects a PRKC gene fusion or fusion protein to determine whether a PRKC fusion is present in the biological sample. The detection of the PRKC fusion indicates the likelihood of the patient responding to treatment with a PRKC inhibitor, or a PRKC fusion inhibitor. The method may be augmented or personalized by evaluating the effect of a variety of PRKC, or PRKC fusion inhibitors on the biological sample shown to contain a PRKC fusion to determine the most appropriate inhibitor to administer. In certain embodiments, the PRKC fusion is IGF2BP3:PRKCA, TANC2:PRKCA. ADCY9:PRKCB, or SPNS1:PRKCB. In some embodiments, the IGF2BP3:PRKCA fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively. In some embodiments, the TANC2:PRKCA fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. In some embodiments, the ADCY9:PRKCB fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. In some embodiments, the SPNS1:PRKCB fusion has all or part of the nucleotide and/or amino acid sequence (such as, e.g., the fusion junction) set forth in SEQ ID NO:7 and SEQ ID NO:5, respectively.

Methods of Treatment

Alternatively, or in combination with the detection and diagnostic methods described herein, the invention provides method for treating the newly identified patient population and the new PRKC fusion cancer subtype, which are characterized by the presence of a PRKC fusion. The patient population and cancer subtype can be associated with or predict the onset of a condition mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, such as, e.g., a cancer or a tumor harboring a PR K C fusion. The methods comprise administering a therapeutic agent, e.g., a PRKC inhibitor, such as e.g., a kinase inhibitor or an antibody specific to PRKC; or a PRKC fusion inhibitor, i.e., an inhibitor that blocks the activity of the PRKC fusion but not wild type PRKC or wild type fusion partner (such as, e.g., IGF2BP3. TANC2, ADCY9, or SPNS1), such as, e.g., an antibody specific to an IGF2BP3:PRKCA, a TANC2:PRKCA, an ADCY9:PRKCB, or an SPNS1:PRKCB fusion protein, e.g., any one of the antibodies described above; an agent that mimics the pseudosubstrate segment and binds the substrate-binding cavity in the catalytic domain of PRKC, keeping the enzyme inactive; or an RNA inhibitor that recognizes PRKC or the fusion junction of a PRKC gene fusion, including but not limited to siRNA, dsRNA, shRNA, or any other antisense nucleic acid inhibitor, alone or in combination with e.g., other chemotherapeutic agents or procedures, in an amount sufficient to treat a condition mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC by one or more of the following: e.g., impeding growth of a cancer, causing a cancer to shrink by weight or volume, extending the expected survival time of the patient, inhibiting tumor growth, reducing tumor mass, reducing size or number of metastatic lesions, inhibiting the development of new metastatic lesions, prolonging survival, prolonging progression-free survival, prolonging time to progression, and/or enhancing quality of life.

As used herein, and unless otherwise specified, a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a condition mediated by aberrant PRKC expression or activity, or overexpression of PRKC, such as, delaying or minimizing one or more symptoms associated with a cancer or a tumor harboring a PRKC fusion (such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as described herein). A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the condition mediated by aberrant PRKC expression or activity or overexpression of PRKC, or enhances the therapeutic efficacy of another therapeutic agent.

In certain embodiments, the therapeutic agent is a PRKC inhibitor, or a PRKC fusion inhibitor. In some embodiments, the therapeutic agent is a PRKC inhibitor, e.g., a compound, biological or chemical, which inhibits, directly or indirectly, the expression and/or activity of PRKC. For example, the PRKC inhibitors may be an antibody (such as, e.g., antibodies specific to PRKC, e.g., PRKCA or PRKCB) or a small molecule inhibitor. In some embodiments, the inhibitors may act directly on PRKC itself, modify the activity of PRKC, or inhibit the expression of PRKC. In other embodiments, the inhibitors may indirectly inhibit PRKC activity by inhibiting the activity of proteins or molecules other than PRKC itself. For example, the inhibitors may modulate the activity of regulatory kinases that phosphorylate or dephosphorylate PRKC, interfere with binding of ligands, or inhibit the activity of interacting or downstream proteins or molecules. Exemplary PRKC; inhibitors also include kinase inhibitor, which may be a pan-kinase inhibitor with activity against several different kinases or a kinase-specific inhibitor (e.g., a serine/threonine kinase inhibitor, or an inhibitor specific to the kinase activity of PRKC). In some embodiments, the PRKC inhibitor is a PRKC-specific inhibitor.

In other embodiments, the therapeutic agent is a PRKC fusion inhibitor, e.g., a PRKC gene fusion inhibitor or a PRKC protein fusion inhibitor. In some embodiments, the PRKC fusion inhibitor inhibits the expression of nucleic acid encoding a PRKC fusion. Examples of PRKC fusion inhibitors include nucleic acid molecules, for example, antisense molecules, ribozymes, RNAi, or triple helix molecules, that hybridize to a nucleic acid encoding a PRKC fusion (e.g., genomic DNA, or mRNA), or a transcription and/or translation regulatory region, and blocks or reduces mRNA and/or protein expression of a PRKC fusion. In some embodiments, the antisense molecule recognizes the PRKC fusion junction, or an RNA inhibitor, including but not limited to siRNA, dsRNA, and short-hairpin RNA specific to the PRKC fusion junction. In some embodiments, the PRKC fusion is IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB. Exemplary PRKC fusion inhibitors also include an antibody that recognizes the fusion protein, and/or does not recognize the wild type fusion partners. In some embodiments, the PRKC fusion inhibitors include an antibody that recognizes IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB fusion protein, and/or does not recognize wild type PRKC, e.g., PRKCA or PRKCB, IGF2BP3, TANC2, ADCY9, or SPNS1. In some embodiments, the PRKC fusion protein (such as, e.g., an IGF2BP3:PRKCA fusion protein, a TANC2:PRKCA fusion protein, an ADCY9:PRKCB fusion protein, or an SPNS1:PRKCB fusion protein) is inhibited by an agent that mimics the pseudosubstrate segment and binds the substrate-binding cavity in the catalytic domain of PRKC, keeping the enzyme inactive.

In some embodiments, the patient to be treated is suffering from lung squamous cell carcinoma or lung adenocarcinoma and the method for treating the condition comprises administering to the patient a therapeutically effective amount of a PRKC inhibitor or a PRKC fusion inhibitor. In some embodiments, the patient to be treated is suffering from lung squamous cell carcinoma, and the method for treating the condition comprises administering to the patient a therapeutically effective amount of a PRKC inhibitor, or a PRKC gene fusion inhibitor or a PRKC fusion protein inhibitor as described above. In some embodiments, the patient to be treated is suffering from lung adenocarcinoma, and the method for treating the condition comprises administering to the patient a therapeutically effective amount of a PRKC inhibitor, or a PRKC gene fusion inhibitor or a PRKC fusion protein inhibitor.

Screening Methods

Therapeutic agents, such as, e.g., PRKC inhibitors, and PRKC fusion inhibitors (gene fusion and fusion protein), used in the therapeutic methods of the invention can be evaluated using the screening assays described herein. Thus, the invention provides a method of identifying an agent useful for treating a condition mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, such as, e.g., a cancer or a tumor harboring a PRKC fusion, such as, e.g., lung squamous cell carcinoma or lung adenocarcinoma, comprising contacting a cell expressing a PRKC gene fusion or PRKC fusion protein with a candidate agent and using one of the detection methods referenced above to determine whether the expression level of the fusion is decreased or a biological function associated with the fusion is altered. In one embodiment, therapeutic agents can be evaluated in a cell-free system, e.g., a cell lysate or in a reconstituted system. In other embodiments, the therapeutic agents are evaluated in u cell in culture. e.g., a cell expressing u PRKC fusion (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell). In yet other embodiments, the therapeutic agents are evaluated in a cell in vivo (e.g., a PRKC fusion-expressing cell present in a subject, e.g., an animal subject (e.g., an in vivo animal model).

Exemplary parameters to evaluate in determining the efficacy of a therapeutic agent for treating a condition mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, such as, e.g., a cancer or a tumor harboring a PRKC fusion include one or more of:

    • (i) a change in binding activity. e.g., direct binding of the candidate agent to a PRKC fusion protein or a binding competition between a known ligand and the candidate agent to a PRKC fusion protein;
    • (ii) a change in kinase activity, e.g., phosphorylation levels of a PRKC fusion protein (e.g., an increased or decreased phosphorylation or autophosphorylation) or a change in phosphorylation of a target of a PRKC kinase—in certain embodiments, a change in kinase activity, e.g., phosphorylation, is detected by any of Western blot (e.g., using an anti-PRKC antibody; or a phosphor-specific antibody, detecting a shift in the molecular weight of a PRKC fusion protein), mass spectrometry, immunoprecipitation, immunohistochemistry, immunomagnetic beads, among others;
    • (iii) a change in an activity of a cell containing a PRKC fusion (e.g., a tumor cell or a recombinant cell), e.g., a change in proliferation, morphology, or tumorigenicity of the cell;
    • (iv) a change in tumor present in an animal subject, e.g., size, appearance, or proliferation of the tumor;
    • (v) a change in the level, e.g., expression level (transcription and/or translation), of a PRKC fusion protein or nucleic acid molecule; or
    • (vi) a change in an activity of a signaling pathway involving PRKC, e.g., phosphorylation or activity of an interacting or downstream target, or expression level of a target gene.

In some embodiments, the PRKC fusion is an IGF2BP3:PRKCA fusion, a TANC2:PRKCA fusion, an ADCY9:PRKCB fusion, or an SPNS1:PRKCB fusion.

In one embodiment, a change in the activity of a PRKC fusion, or interaction of a PRKC fusion with a downstream ligand detected in a cell free assay in the presence of a candidate agent indicates that the candidate agent will be effective as a therapeutic agent for treatment of a condition mediated by aberrant (e.g., constitutive) PRKC activity or expression, or overexpression of PRKC, such as, e.g., a cancer or a tumor harboring a PRKC fusion.

In other embodiments, a change in an activity of a cell expressing a PRKC fusion, such as, e.g., IGF2BP3:PRKCA, TANC2:PRKCA, ADCY9:PRKCB, or SPNS1:PRKCB, as described herein, (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell) is detected in a cell in culture. In one embodiment, the cell is a recombinant cell that is modified to express a PRKC fusion nucleic acid. e.g., is a recombinant cell transfected with a PRKC fusion nucleic acid. The transfected cell can show a change in response to the expressed PRKC fusion. e.g., increased proliferation, changes in morphology, increased tumorigenicity, and/or acquired a transformed phenotype. A change in any of the activities of the cell, e.g., the recombinant cell, in the presence of the candidate agent can be detected. For example, a decrease in one or more of: proliferation, tumorigenicity, or transformed morphology, in the presence of the candidate agent can be indicative of an inhibitor of a PRKC fusion. In other embodiments, a change in binding activity or phosphorylation of PRKC or its interacting or downstream proteins or molecules as described herein is detected.

In yet other embodiment, a change in a tumor present in an animal subject (e.g., an in vivo animal model) is detected. Tim one embodiment, a tumor containing animal or a xenograft comprising cells expressing a PRKC fusion (e.g., tumorigenic cells expressing a PRKC fusion) is employed. The therapeutic agents can be administered to the animal subject and a change in the tumor is evaluated. In one embodiment, the change in the tumor includes one or more of a tumor growth, tumor size, tumor burden, or survival is evaluated. A decrease in one or more of tumor growth, tumor size, tumor burden, or an increased survival is indicative that the candidate agent is an inhibitor or modulator.

In another aspect of the invention provides a method or assay for screening for agents that modulate (e.g., inhibit) the expression or activity of a PRKC fusion as described herein. The method includes contacting e.g., a PRKC fusion, or a cell expressing a PRKC fusion, with a candidate agent; and detecting a change in a parameter associated with a PRKC fusion, e.g., a change in the expression or an activity of the PRKC fusion. The method can, optionally, include comparing the treated parameter to a reference value, e.g., a control sample (e.g., comparing a parameter obtained from a sample with the candidate agent to a parameter obtained from a sample without the candidate agent). In one embodiment, if a decrease in expression or activity of the PRKC fusion is detected, the candidate agent is identified as an inhibitor. In another embodiment, if an increase in expression or activity of the PRKC fusion is detected, the candidate agent is identified as an activator. In certain embodiments, the PRKC fusion is a PRKC gene fusion or PRKC fusion protein, such as, e.g., an IGF2BP3:PRKCA fusion, a TANC2:PRKCA fusion, an ADCY9:PRKCB fusion, or an SPNS1:PRKCB fusion.

In one embodiment, the contacting step is detected in a cell-free system, e.g., a cell lysate or in a reconstituted system. In other embodiments, the contacting step is detected in a cell in culture, e.g., a cell expressing a PRKC fusion (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell). In yet other embodiments, the contacting step is detected in a cell in vivo (e.g., a PRKC expressing cell present in a subject, e.g., an animal subject (e.g., an in vivo animal model)).

Exemplary parameters evaluated in identifying an agent that modulates the activity of a PRKC fusion. e.g., a PRKC fusion (e.g., an IGF2BP3:PRKCA fusion, a TANC2:PRKCA fusion, an ADCY9:PRKCB fusion, or an SPNS1:PRKCB fusion) include one or more of:

    • (i) a change in binding activity, e.g., direct binding of the candidate agent to a PRKC fusion protein; a binding competition between a known ligand and the candidate agent to a PRKC fusion protein;
    • (ii) a change in kinase activity, e.g., phosphorylation levels of a PRKC fusion protein (e.g., an increased or decreased phosphorylation or autophosphorylation) or a change in phosphorylation of a target of a PRKC kinase—in certain embodiments, a change in kinase activity, e.g., phosphorylation, is detected by any of Western blot (e.g., using an anti-PRKC antibody; or a phosphor-specific antibody, detecting a shift in the molecular weight of a PRKC fusion protein), mass spectrometry, immunoprecipitation, immunohistochemistry, immunomagnetic beads, among others;
    • (iii) a change in an activity of a cell containing a PRKC fusion (e.g., a tumor cell or a recombinant cell), e.g., a change in proliferation, morphology or tumorigenicity of the cell;
    • (iv) a change in tumor present in an animal subject, e.g., size, appearance, proliferation, of the tumor;
    • (v) a change in the level, e.g., expression level (transcription and/or translation), of a PRKC fusion protein or nucleic acid molecule; or
    • (vi) a change in an activity of a signaling pathway involving PRKC, e.g., phosphorylation or activity of an interacting or downstream target, or expression level of a target gene.

Methods for Validating PRKC Fusions

PRKC gene fusions, such as, e.g., PRKC gene fusions (e.g., IGF2BP3:PRKCA gene fusion, TANC2:PRKCA gene fusion, ADCY9:PRKCB gene fusion, or SPNS1:PRKCB gene fusion) may be evaluated to ensure that the breakpoints are in-frame and can produce a protein product containing the full kinase domain, i.e., that the breakpoint occurs such that complete triplet codons are intact, and that the RNA sequence will produce a viable protein. The PRKC gene fusion can be transfected into cells to confirm that the protein is functionally active with respect to kinase activity and oncogenic activity. cDNA encoding the PRKC fusion protein can be produced by standard solid-phase DNA synthesis. Alternatively the PRKC fusion cDNA can be produced by RT-PCR using tumor mRNA extracted from samples containing the gene fusion. The DNA amplified can be subcloned into an appropriate vector and characterized by DNA sequence analysis or in vitro/in vivo expression analyses.

Expression vectors containing the PRKC gene fusion (such as, e.g., a PRKC gene fusion, e.g., an IGF2BP3:PRKCA gene fusion, a TANC2:PRKCA gene fusion, an ADCY9:PRKCB gene fusion, or an SPNS1:PRKCB gene fusion) can be introduced into host cells to thereby produce a PRKC fusion protein (such as, e.g., a PRKC fusion protein, e.g., an IGF2BP3:PRKCA fusion protein, a TANC2:PRKCA fusion protein, an ADCY9:PRKCB fusion protein, or an SPNS1:PRKCB fusion protein). The PRKC fusion protein expression vector can be a yeast expression vector, a vector for expression in insect cells, e.g., a baculovirus expression vector, or a vector suitable for expression in mammalian cells. Vector DNA can be introduced into host cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell.

Cells harboring the expression vector carrying the recombinant PRKC gene fusion can then be tested for production of the unique fusion protein via standard Western blotting using either an antibody probe that detects the gene product itself or that recognizes a tag peptide (e.g., FLAG tag) that can be added to the gene product via the expression vector (using standard, commercially available reagents). Western blotting can be used to confirm the ectopic expression of the encoded PRKC fusion protein by comparing the samples from cells transfected with the vector containing the PRKC gene fusion cDNA to cells transfected with the empty expression vector. The functional activity can be assessed by measuring the level of phosphorylation on the kinase or substrate. Comparison of the level of phosphorylation activity between the wild type (normal) form of PRKC and the PRKC fusion protein can indicate if the PRKC fusion protein has elevated activity that could drive oncogenic activity. Whether the PRKC gene fusion is oncogenic can be assessed by measuring capacity of the expressed PRKC fusion protein to transform cells, that is, to enable cells to grow and proliferate under conditions which are not permissive for growth of normal cells. One commonly used method of measuring the transforming activity of a kinase is by assessing if expression of the gene product can allow BaF3 cells to grow in the absence of the growth factor IL3, which is required for the survival and growth of BaF3 cells. Another assay for measuring transforming activity is a soft agar growth assay. This is another standard method which tests the capacity of an introduced gene product to confer the ability to grow in a soft agar matrix, or anchorage-independent conditions. These methods and others can be used to test the oncogenic activity of a PRKC gene fusion (such as, e.g., an IGF2BP3:PRKCA gene fusion, a TANC2:PRKCA gene fusion, an ADCY9:PRKCB gene fusion, or an SPNS1:PRKCB gene fusion) and provide a level of validation of a PRKC fusion protein (such as, e.g., an IGF2BP3:PRKCA fusion protein, a TANC2:PRKCA fusion protein, an ADCY9:PRKCB fusion protein, or an SPNS1:PRKCB fusion protein) as a potential target for treating patients that harbor these fusions.

A change in an activity of a cell can be detected in a cell in culture. e.g., a cell expressing a fusion (e.g., a mammalian cell, a tumor cell or cell line, a recombinant cell). The transfected cell can show a change in response to the expressed fusion, e.g., increased proliferation, changes in morphology, increased tumorigenicity, and/or an acquired transformed phenotype.

To further validate the biological implication of the gene fusion, a change in any of the activities of the cell, e.g., the recombinant cell, in the presence of a known inhibitor of one of the fusion partners. e.g., a PRKC inhibitor, can be detected. For example, a decrease in one or more of: proliferation, tumorigenicity, and transformed morphology, in the presence of the PRKC inhibitor can be indicative of an inhibitor of a fusion. In other embodiments, a change in binding activity or phosphorylation of PRKC or its interacting or downstream proteins or molecules is detected.

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification will supersede any contradictory material. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. All ranges given in the application encompass the endpoints unless stated otherwise.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1-81. (canceled)

82. A compound capable of specifically inhibiting a TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2 fusion, wherein the fusion results in aberrant activity or expression of NTRK2, or overexpression NTRK2, and wherein the compound is an antibody that specifically binds to a TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2 fusion protein or an RNA inhibitor that hybridizes under stringent conditions to a TRIM24:NTRK2, AFAP1:NTRK2, or PAN3:NTRK2 gene fusion.

83. The compound of claim 82, wherein

(a) the antibody specifically binds to: (i) a fragment of SEQ ID NO:3 comprising at least amino acids 665-674 of SEQ ID NO:3; (ii) a fragment of SEQ ID NO:4 comprising at least amino acids 665-674 of SEQ ID NO:4; (iii) a fragment of SEQ ID NO:6 comprising at least amino acids 544-553 of SEQ ID NO:6; or (iv) a fragment of SEQ ID NO:8 comprising at least amino acids 138-147 of SEQ ID NO:8; and
(b) the RNA inhibitor hybridizes under stringent conditions to: (i) a fragment of SEQ ID NO:1 comprising at least nucleotides 2010-2019 of SEQ ID NO:1; (ii) a fragment of SEQ ID NO:2 comprising at least nucleotides 2000-2009 of SEQ ID NO:2; (iii) a fragment of SEQ ID NO:5 comprising at least nucleotides 1641-1650 of SEQ ID NO:5; or (iv) a fragment of SEQ ID NO:7 comprising at least nucleotides 426-435 of SEQ ID NO:7.

84. A method for detecting the presence of a PAN3:NTRK2 fusion in a patient, said method comprising:

a) contacting a biological sample from the patient with an oligonucleotide that specifically binds to the fusion junction of the PAN3:NTRK2 fusion, and detecting binding between the PAN3:NTRK2 fusion and the olignucleotide; or
b) amplifying or sequencing a portion of a nucleic acid from the patient, and detecting the presence of a nucleotide sequence comprising at least the fusion junction of the PAN3:NTRK2 fusion.

85. The method of claim 84, wherein the PAN3:NTRK2 fusion to be detected is a PAN3:NTRK2 gene fusion that comprises SEQ ID NO: 7, or a portion of SEQ ID NO: 7, wherein the portion encodes a nucleotide sequence comprising the fusion junction.

86. The method of claim 85, wherein the oligonucleotide hybridizes to the fusion junction of the NTRK2 gene fusion under stringent conditions.

87. The method of claim 86, wherein the oligonucleotide hybridizes under stringent conditions to

a fragment of SEQ ID NO:7 comprising at least nucleotides 426-435 of SEQ ID NO:7.

88-90. (canceled)

91. The method of claim 84, further comprising administering to the patient a therapeutically effective amount of an NTRK2 inhibitor or an NTRK2 fusion inhibitor.

92. The method of claim 84, wherein the patient is suffering from or susceptible to a cancer.

93. The method of claim 92, wherein the cancer is lung adenocarcinoma, low grade glioma, squamous cell carcinoma, or head and neck squamous cell carcinoma.

Patent History
Publication number: 20220213553
Type: Application
Filed: Jan 21, 2022
Publication Date: Jul 7, 2022
Applicant: BLUEPRINT MEDICINES CORPORATION (Cambridge, MA)
Inventors: Nicolas STRANSKY (Charlestown, MA), Joseph L. Kim (Wayland, MA)
Application Number: 17/581,669
Classifications
International Classification: C12Q 1/6886 (20060101); C07K 16/30 (20060101); C12N 15/113 (20060101); G01N 33/574 (20060101);