Patent Application
20230175069
The present disclosure relates to cancer treatment and diagnosis.
Endocrine therapy is the most effective treatment for estrogen-receptor (ER) positive breast cancer. Agents targeting ER, including selective ER modulators (SERMs, such as Tamoxifen), selective ER down-regulators (SERDs, such as Fulvestrant) and aromatase inhibitors (AIs, such as letrozole) are the mainstay of treatment (Comprehensive molecular portraits of human breast tumours (2012); Giltnane JM (2017)). However, the efficacy of endocrine therapy is limited by intrinsic and acquired endocrine resistance (Musgrove EA (2009)). About quarter of patients with primary tumor and almost all patients with metastases will present with or eventually develop endocrine resistance (Ma CX (2015)). Tremendous effort has been made to investigate the mechanism of endocrine resistance, and emerging evidence now suggests that mutations or fusions of the ESR1 gene that encode ERα is one of the most important driving mechanisms (Ma CX (2015); Jeselsohn R (2015); Hartmaier RJ (2018)).
Recurrent gene fusions resulting from chromosome translocations are a critical class of genetic aberrations causing cancer (Mitelman F (2007)), which have fueled modern cancer therapeutics. Recently, several milestone studies have identified recurrent gene fusions in different types of solid tumors with tremendous clinical impact. This is exemplified by an EML4-ALK gene fusion identified in 3-5% of non-small-cell lung cancer patients that has led to a new precision medicine with stunning clinical responses (Koivunen JP (2018)). Most recently, The U.S. Food and Drug Administration granted accelerated approval to larotrectinib as the first pancancer drug for the treatment of solid tumors expressing NTRK gene fusions (Laetsch TW (2018)).
Previously a recurrent gene rearrangement was identified on chromosome 6 between ESR1 and the coiled-coil domain containing 170, CCDC170. The ESR1-CCDC170 fusion join the 5′ untranslated region of ESR1 to the coding region of CCDC170, generating N-terminally truncated CCDC170 proteins (ΔCCDC170) expressed under the ESR1 promoter. When introduced in breast cancer cells, ΔCCDC170 proteins led to increased cell invasiveness and tumorigenesis (Veeraraghavan J (2014)). To date, ESR1-CCDC170 remains the most frequent gene fusion (6%-8%) detected in the more-aggressive and endocrine-resistance form of breast cancer – luminal B breast cancer, and its recurrence has been subsequently supported by several other studies (Giltnane JM (2017); Hartmaier RJ (2018); Matissek KJ (2018); Fimereli D (2018); Lei JT (2018)). In addition, this fusion is also detected as a recurrent event in ovarian cancer, and its presence has been associated with exceptional short-term survival (Yang SYC (2018)). The association of ESR1- CCDC170 fusions with lack of response to neoadjuvant letrozole treatment (Giltnane JM (2017)).
While multiple mechanisms of resistance have been proposed such as loss of ER, dysregulation of ER co-regulators, and crosstalk with growth factor pathways (Britton DJ (2006), deGraffenried LA (2004), Drury SC (2011), Hutcheson IR (2003), Musgrove EA (2009), Osborne CK (2003), Osborne CK (2011), Parisot JP (1999), Zhou W (2014)), the genetic aberration causing endocrine resistance remains to be explored. What is needed are compositions and methods for determining the gene rearrangements associated with resistance to endocrine therapy in breast cancer patients. The compositions and methods disclosed herein address these and other needs.
It is shown herein that ESR1/CCDC170 fusions endow reduced endocrine sensitivity in luminal breast cancer cells in vitro and in vivo, via interactions with HER2, HER3, and SRC, and enhance activation of SRC and PI3K-AKT pathways. Accordingly, provide herein are new diagnostic and therapeutic strategies for breast tumors harboring ESR1/CCDC170 fusions, wherein, in some embodiments, the combination of inhibitors against these kinases and endocrine therapy are administered.
Provided herein are methods of diagnosing a subject with increased resistance to estrogen receptor antagonists (such as increased resistance to estrogen-receptor modulators, estrogen-receptor down-regulators, and/or aromatase inhibitors), comprising: obtaining a biological sample from the subject; and detecting an ESR1/CCDC170 (or ESR1-CCDC170) gene fusion in the sample, wherein the detection indicates the subject has increased resistance to estrogen receptor antagonists (such as increased resistance to estrogen-receptor modulators, estrogen-receptor down-regulators, and/or aromatase inhibitors) and the subject is diagnosed with increased resistance to estrogen receptor antagonists (such as increased resistance to estrogen-receptor modulators, estrogen-receptor down-regulators, and/or aromatase inhibitors). In some embodiments, the ESR1/CCDC170 gene fusion is selected from the group consisting of a E2-E2 fusion, a E2-E4 fusion, a E2-E5 fusion, a E2-E6 fusion, a E2-E7 fusion, a E2-E8 fusion, a E2-E10 fusion, and other fusion variations illustrated in
The method of detection can comprise contacting the biological sample with a reaction mixture comprising a probe specific for a fusion point in one of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41. The method of detection can alternatively or further comprise contacting the biological sample with a reaction mixture comprising two primers, wherein the first primer is complementary to a ESR1 polynucleotide sequence and the second primer is complementary to a CCDC170 polynucleotide sequence, wherein the ESR1/CCDC170 gene fusion is detectable by the presence of an amplicon generated by the first primer and the second primer. The method of detection can also comprise contacting the biological sample with a reaction mixture comprising two primers, wherein the first primer is complementary to a ESR1 polynucleotide sequence and the second primer is complementary to a CCDC 170 polynucleotide sequence, wherein hybridization of the two primers on a ESR1/CCDC170 gene fusion sequence provides a detectable signal, and the ESR1/CCDC170 gene fusion is detectable by the presence of the signal. In some embodiments, a first of the one or more primers is selected from the group consisting of SEQ ID NO: 42, and SEQ ID NO: 44, and a second of the one or more primers is selected from the group consisting of SEQ ID NO: 43 and SEQ ID NO: 45. In some embodiments, the primers are SEQ ID NO: 42 and SEQ ID NO: 43. In some embodiments, the primers are SEQ ID NO: 44 and SEQ ID NO: 45.
The methods described herein can be used to detect an ESR1/CCDC170 gene fusion in a subject that has a cancer, such as a breast cancer, including but not limited to a luminal B or metastatic breast cancer. The methods can further comprise administering to the subject a therapeutically effective amount of a HER inhibitor and/or a SRC inhibitor.
Also included herein are methods of treating a cancer in a subject comprising: detecting a ESR1/CCDC170 gene fusion in a sample obtained from the subject; and administering to the subject a therapeutically effective amount of a HER inhibitor and/or a SRC inhibitor and a therapeutically effective amount of an estrogen-receptor modulator, an estrogen-receptor down-regulator, and/or an aromatase inhibitor. The ESR1/CCDC170 gene fusion can be selected from the group consisting of a E2-E2 fusion, a E2-E4 fusion, a E2-E5 fusion, a E2-E6 fusion, a E2-E7 fusion, a E2-E8 fusion, a E2-E10 fusion, and other fusion variations illustrated in
Further included are methods for detecting an ESR1/CCDC170 gene fusion comprising: obtaining a biological sample from a subject; and detecting the fusion in the sample. In some embodiments, the detection can comprise contacting the biological sample with a reaction mixture comprising a probe specific for a fusion point sequence within one of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41. A detectable moiety can be covalently bonded to the probe, such as in a Nanostring assay. Kits comprising one or more probes are included, wherein each probe specifically hybridizes to a fusion point nucleotide sequence within a sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.
Further included are sequencing based methods such as transcriptome/genome sequencing methods or targeted sequencing for detecting an ESR1/CCDC170 gene fusion comprising: obtaining a biological sample from a subject; and detecting the fusion variants in the sample through transcriptome/genome sequencing methods or targeted sequencing and bioinformatics detection tools.
Further included are genomic DNA based detection methods including but not limit to genomic PCR, fluorescence in situ hybridization, or southern blots for detecting an ESR1/CCDC170 gene fusion comprising: obtaining a biological sample from a subject; and detecting the fusion variants in the sample through genomic PCR, fluorescence in situ hybridization, or southern blots.
Further included are protein-based methods known in the art, such as Mass spectrometry, immunohistochemistry, or western blot for detecting an ESR1-CCDC170 protein product comprising: obtaining a biological sample from a subject; and detecting the fusion variant proteins in the sample through Mass spectrometry, immunohistochemistry, or western blot.
The findings disclosed herein include that ESR1-CCDC170 gene fusions result in increased resistance to estrogen receptor antagonists, and that patients harboring the ESR1-CCDC170 gene fusions are more effectively treated with HER2, HER3 and/or SRC inhibitors.
Accordingly, disclosed herein is a method for detecting ESR1/CCDC170 gene fusion. The fusion can be detected by contacting the sample with one or more primers specific for ESR1-CCDC170 fusion transcript or rearranged genomic DNA, performing an amplification reaction, and detecting an amplification product or amplicon. The fusion can also be detected by transcriptome or genome sequencing, or targeted sequencing, or Nanostring assay, or Fluorescence In Situ Hybridization, or Southern Blot. In some examples, the detection of the fusion indicates an increased resistance to tamoxifen in the subject. In some embodiments, the subject having a detected ESR1/CCDC170 gene fusion is administered with a therapeutically effective amount of a HER2, HER3 and/or SRC inhibitor. The subject can also be administered a therapeutically effective amount of tamoxifen or other estrogen receptor antagonist.
Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicants desire that the following terms be given the particular definition as provided below.
As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof.
The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.
“Amplifying,” “amplification,” and grammatical equivalents thereof refers to any method by which at least a part of a target nucleic acid sequence is reproduced in a template-dependent manner, including without limitation, a broad range of techniques for amplifying nucleic acid sequences, either linearly or exponentially. Exemplary means for performing an amplifying step include ligase chain reaction (LCR), ligase detection reaction (LDR), ligation followed by Qreplicase amplification, PCR, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplexed amplifications, rolling circle amplification (RCA), recombinase-polymerase amplification (RPA)(TwistDx, Cambridg, UK), and self-sustained sequence replication (3SR), including multiplex versions or combinations thereof, for example but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combined chain reaction-CCR), and the like. Descriptions of such techniques can be found in, among other places, Sambrook et al. Molecular Cloning, 3rd Edition; Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002), Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002).
“Administration” of “administering” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, or via a transdermal patch, and the like. Administration includes self-administration and the administration by another.
The term “biological sample” as used herein means a sample of biological tissue or fluid. Such samples include, but are not limited to, tissue isolated from animals. Biological samples can also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A biological sample can be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods as disclosed herein in vivo. Archival tissues, such as those having treatment or outcome history can also be used.
As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
“Complementary” or “substantially complementary” refers to the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid. Complementary nucleotides are, generally, A and T/U, or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%. Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary. See Kanehisa (1984) Nucl. Acids Res. 12:203.
“Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be “positive” or “negative.”
“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Accordingly, it should be understood that “encode” or “encoding”
The “fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as regulating the transcription of the target gene.
The term “gene” or “gene sequence” refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a “gene” as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term “gene”, or may include any coding sequence (i.e., exon), non-coding sequence (e.g., intron), or control sequence, fragments thereof, and combinations thereof. The term “gene” or “gene sequence” includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) nucleotide sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.
“Inhibit”, “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
“Inhibitors” or “antagonist” of expression or of activity are used to refer to inhibitory molecules, respectively, identified using in vitro and in vivo assays for expression or activity of a described target protein, e.g., ligands, antagonists, and their homologs and mimetics. Inhibitors are agents that, e.g., inhibit expression or bind to, partially or totally block stimulation or activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, e.g., antagonists. Control samples (untreated with inhibitors) are assigned a relative activity value of 100%. Inhibition of a described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5%, or 1% or less.
The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides (DNA) or ribonucleotides (RNA). The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides. The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.
Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
“Pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
“Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms “carrier” or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington’s Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.
The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.
The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
The term “primer” or “amplification primer” refers to an oligonucleotide that is capable of acting as a point of initiation for the 5′ to 3′ synthesis of a primer extension product that is complementary to a nucleic acid strand. The primer extension product is synthesized in the presence of appropriate nucleotides and an agent for polymerization such as a DNA polymerase in an appropriate buffer and at a suitable temperature. The most widely used target amplification procedure is PCR, first described for the amplification of DNA by Muliis et al. in U.S. Pat. No. 4,683,195 and Mullis in U.S. Pat. No. 4,683,202 and is well known to those of ordinary skill in the art.
A “primer” or “primer sequence” hybridizes to a target nucleic acid sequence (for example, a DNA template to be amplified) to prime a nucleic acid synthesis reaction. The primer may be a DNA oligonucleotide, a RNA oligonucleotide, or a chimeric sequence. The primer may contain natural, synthetic, or modified nucleotides. Both the upper and lower limits of the length of the primer are empirically determined. The lower limit on primer length is the minimum length that is required to form a stable duplex upon hybridization with the target nucleic acid under nucleic acid amplification reaction conditions. Very short primers (usually less than 3-4 nucleotides long) do not form thermodynamically stable duplexes with target nucleic acids under such hybridization conditions. The upper limit is often determined by the possibility of having a duplex formation in a region other than the pre-determined nucleic acid sequence in the target nucleic acid. Generally, suitable primer lengths are in the range of about 10 to about 40 nucleotides long. In certain embodiments, for example, a primer can be 10-40, 15-30, or 10-20 nucleotides long. A primer is capable of acting as a point of initiation of synthesis on a polynucleotide sequence when placed under appropriate conditions. The primer will be completely or substantially complementary to a region of the target polynucleotide sequence to be copied. Therefore, under conditions conducive to hybridization, the primer will anneal to the complementary region of the target sequence. Upon addition of suitable reactants, including, but not limited to, a polymerase, nucleotide triphosphates, etc., the primer is extended by the polymerizing agent to form a copy of the target sequence. The primer may be single-stranded or alternatively may be partially double-stranded.
The term “primer pair” as used herein means a pair of oligonucleotide primers that are complementary to the sequences flanking a target sequence. The primer pair consists of a forward primer and a reverse primer. The forward primer has a nucleic acid sequence that is complementary to a sequence upstream, i.e., 5′ of the target sequence. The reverse primer has a nucleic acid sequence that is complementary to a sequence downstream, i.e., 3′ of the target sequence.
The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In some embodiments, administering an increased amount of an estrogen receptor antagonist is an administration that is increased by a percentage or fold listed above as compared to an amount previously administered to the subject or to an amount that is used in a general or study population under the same or similar circumstances.
The term “reduced”, “reduce”, “reduction”, “decrease”, or “decreased” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
“Reporter probe” refers to a molecule used in an amplification reaction, typically for quantitative or real-time PCR analysis, as well as end-point analysis. Such reporter probes can be used to monitor the amplification of the target nucleic acid sequence. In some embodiments, reporter probes present in an amplification reaction are suitable for monitoring the amount of amplicon(s) produced as a function of time. Such reporter probes include, but are not limited to, the 5′-exonuclease assay (e.g., U.S. Pat. No. 5,538,848) various stem-loop molecular beacons (see for example, U.S. Pat. Nos. 6,103,476 and 5,925,517), stemless or linear beacons (see, e.g., WO 99/21881), PNA MOLECULAR BEACONS (see, e.g., U.S. Pat. Nos. 6,355,421 and 6,593,091), linear PNA beacons, non-FRET probes (see, for example, U.S. Pat. No. 6,150,097), SUNRISE/AMPLIFLUOR probes (U.S. Pat. No. 6,548,250), stem-loop and duplex Scorpion probes (U.S. Pat. No. 6,589,743), bulge loop probes (U.S. Pat. No. 6,590,091), pseudo knot probes (U.S. Pat. No. 6,589,250), cyclicons (U.S. Pat. No. 6,383,752), MGB ECLIPSE probe (Epoch Biosciences), hairpin probes (U.S. Pat. No. 6,596,490), peptide nucleic acid (PNA) light-up probes, self-assembled nanoparticle probes, and ferrocene-modified probes described, for example, in U.S. Pat. No. 6,485,901. Reporter probes can also include quenchers, including without limitation black hole quenchers (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate/carboxylate Quenchers (Epoch).
The term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.
The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating or mitigating one or more causes of a disorder or condition. Treatments according to the invention may be applied pallatively or remedially.
Prophylactic administrations are given to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a cancer.
“Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
“Therapeutically effective amount” or “therapeutically effective dose” of a composition refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is a reduction of tumor size. In some embodiments, a desired therapeutic result is a reduction of cancer metastasis. In some embodiments, a desired therapeutic result is a reduction of a breast cancer, or a symptom of a breast cancer. In some embodiments, a desired therapeutic result is a reduction of a luminal B breast cancer, or a symptom thereof. In some embodiments, a desired therapeutic result is the prevention of cancer relapse. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as control of tumor growth. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.
Disclosed herein are methods of detecting an ESR1/CCDC170 gene fusion, said methods comprising obtaining a sample from a subject, and detecting whether the fusion is present in the sample. In some embodiments, an ESR1/CCDC170 gene fusion is detected in a sample derived from a subject having breast cancer and the detection indicates that the breast cancer has decreased sensitivity to an estrogen receptor antagonist (such as an estrogen-receptor modulator, an estrogen-receptor down-regulator, or an aromatase inhibitor). Accordingly, the present invention includes methods of diagnosing a breast cancer in a subject having decreased sensitivity to an estrogen receptor antagonist (such as an estrogen-receptor modulator, an estrogen-receptor down-regulator, or an aromatase inhibitor). In some examples, the estrogen-receptor modulator is tamoxifen, clomifene, raloxifene, or exemestane. In some examples, the estrogen-receptor down-modulator is fulvestrant. In some examples, the aromatase inhibitor is letrozole.
Also disclosed herein is a method of treating a breast cancer in a subject, said method comprising detecting an ESR1/CCDC170 gene fusion in a breast tissue sample obtained from the subject, and administering to the subject a therapeutically effective amount of a HER inhibitor and/or a SRC inhibitor. In some embodiments, an estrogen receptor antagonist is also administered to the subject, including administering an increased amount of an estrogen receptor antagonist.
As used herein, “gene fusion” refers to a chimeric genomic DNA resulting from the fusion of at least a portion of a first gene to a portion of a second gene. The point of transition between the sequence from the first gene in the fusion to the sequence from the second gene in the fusion is referred to as the “fusion point.” Transcription of the gene fusion results in a chimeric mRNA. Methods for detecting a gene fusion include detection of the chimeric genomic DNA, including by detection of the fusion point sequence within the chimeric genomic DNA, detection of the resultant chimeric mRNA, and detection of the resultant chimeric protein.
“ESR1” or “Estrogen Receptor 1” refers herein to a polypeptide that is involved in transducing signals following the activation of estrogen, and in humans, is encoded by the ESR1 gene. In some embodiments, the ESR1 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 3467, Entrez Gene: 2099, Ensembl: ENSG00000091831, OMIM: 133430, UniProtKB: P03372. In some embodiments, the ESR1 polypeptide comprises the sequence of SEQ ID NO: 1, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1. The ESR1 polypeptide of SEQ ID NO: 1 may represent an immature or pre-processed form of mature ESR1, and accordingly, included herein are mature or processed portions of the ESR1polypeptide in SEQ ID NO: 1.
The term “ESR1 polynucleotide” refers to a polynucleotide that encodes a ESR1 polypeptide, or any fragment thereof. In some embodiments, the ESR1 polynucleotide comprises an ESR1 exon 1 polynucleotide having a sequence of SEQ ID NO: 2, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 2, or a polynucleotide comprising a portion of SEQ ID NO: 2. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 2 polynucleotide having a sequence of SEQ ID NO: 3, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 3, or a polynucleotide comprising a portion of SEQ ID NO: 3. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 3 polynucleotide having a sequence of SEQ ID NO: 4, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 4, or a polynucleotide comprising a portion of SEQ ID NO: 4. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 4 polynucleotide having a sequence of SEQ ID NO: 5, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 5, or a polynucleotide comprising a portion of SEQ ID NO: 5. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 5 polynucleotide having a sequence of SEQ ID NO: 6, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 6, or a polynucleotide comprising a portion of SEQ ID NO: 6. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 6 polynucleotide having a sequence of SEQ ID NO: 7, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 7, or a polynucleotide comprising a portion of SEQ ID NO: 7. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 7 polynucleotide having a sequence of SEQ ID NO: 8, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 8, or a polynucleotide comprising a portion of SEQ ID NO: 8. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 8 polynucleotide having a sequence of SEQ ID NO: 9, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 9, or a polynucleotide comprising a portion of SEQ ID NO: 9. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 9 polynucleotide having a sequence of SEQ ID NO: 10, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 10, or a polynucleotide comprising a portion of SEQ ID NO: 10. In some embodiments, the ESR1 polynucleotide comprises a ESR1 exon 10 polynucleotide having a sequence of SEQ ID NO: 11, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 11, or a polynucleotide comprising a portion of SEQ ID NO: 11.
“CCDC170” or “Coiled-Coil Domain Containing 170” refers herein to a polypeptide that is encoded by the CCDC170 gene in human. In some embodiments, the CCDC170 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 21177, Entrez Gene: 80129, Ensembl: ENSG00000120262, UniProtKB: Q8IYT3. In some embodiments, the CCDC170 polypeptide comprises the sequence of SEQ ID NO: 12 or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 12, or a polypeptide comprising a portion of SEQ ID NO: 3. The CCDC170 polypeptide of SEQ ID NO: 12 may represent an immature or pre-processed form of mature CCDC170, and accordingly, included herein are mature or processed portions of the CCDC170 polypeptide in SEQ ID NO: 12.
The term “CCDC 170 polynucleotide” refers to a polynucleotide that encodes a CCDC170 polypeptide, or any fragment thereof. In some embodiments, the CCDC170 polynucleotide comprises an CCDC170 exon 1 polynucleotide having a sequence of SEQ ID NO: 13, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 13, or a polynucleotide comprising a portion of SEQ ID NO: 13. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 2 polynucleotide having a sequence of SEQ ID NO: 14, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 14, or a polynucleotide comprising a portion of SEQ ID NO: 14. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 3 polynucleotide having a sequence of SEQ ID NO: 15, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 15, or a polynucleotide comprising a portion of SEQ ID NO: 15. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 4 polynucleotide having a sequence of SEQ ID NO: 16, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 16, or a polynucleotide comprising a portion of SEQ ID NO: 16. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 5 polynucleotide having a sequence of SEQ ID NO: 17, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 17, or a polynucleotide comprising a portion of SEQ ID NO: 17. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 6 polynucleotide having a sequence of SEQ ID NO: 18, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 18, or a polynucleotide comprising a portion of SEQ ID NO: 18. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 7 polynucleotide having a sequence of SEQ ID NO: 19, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 19, or a polynucleotide comprising a portion of SEQ ID NO: 19. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 8 polynucleotide having a sequence of SEQ ID NO: 20, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 20, or a polynucleotide comprising a portion of SEQ ID NO: 20. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 9 polynucleotide having a sequence of SEQ ID NO: 21, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 21, or a polynucleotide comprising a portion of SEQ ID NO: 21. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 10 polynucleotide having a sequence of SEQ ID NO: 22, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 22, or a polynucleotide comprising a portion of SEQ ID NO: 22. In some embodiments, the CCDC170 polynucleotide comprises a CCDC170 exon 11 polynucleotide having a sequence of SEQ ID NO: 23, or a polynucleotide having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 23, or a polynucleotide comprising a portion of SEQ ID NO: 23.
It should be understood that the term “fusion” as used herein refers to a polynucleotide or polypeptide made by joining parts of two previously independent polynucleotides or polypeptides of ESR1 and CCDC170. In some embodiments, a fusion is formed by joining parts of two previously independent genes through translocation, interstitial deletion, or chromosomal inversion. Accordingly, “a fusion of a ESR1 polynucleotide sequence and a CCDC170 polynucleotide sequence” refers herein to a fusion of a ESR1 DNA sequence and a CCDC170 DNA sequence or a fusion mRNA transcribed from the fusion DNA. “ESR1/CCDC170 polynucleotide fusion” is used interchangeably herein with “fusion of a ESR1 polynucleotide sequence and a CCDC170 polynucleotide sequence.” “ESR1/CCDC170 fusion” refers to a “ESR1/CCDC170 polynucleotide fusion” and/or a “ESR1/CCDC170 polypeptide fusion.”
In some embodiments, the phrase “a fusion of a ESR1 polynucleotide sequence and a CCDC170 polynucleotide sequence” herein refers to a fusion of any ESR1 exon and any CCDC170 exon. In some embodiments, the fusion described herein is a fusion containing a fusion exon junction of any of the exons of ESR1 polynucleotide with any of the exons 2-11 (having at least a portion of exon 11) of a CCDC170 polynucleotide. In some embodiments, the fusion is: a fusion of exons 1-2 of a ESR polynucleotide with exons 2-11 of a CCDC170 polynucleotide (having at least a portion of exon 11) of a CCDC170 polynucleotide (referred to herein as an “E2-E2 fusion”); a fusion of exons 1-2 of a ESR1 polynucleotide with exons 4-11 (having at least a portion of exon 11) of a CCDC170 polynucleotide (referred to herein as an “E2-E6 fusion”); a fusion of exons 1-2 of a ESR1 polynucleotide with exons 5-11 (having at least a portion of exon 11) of a CCDC170 polynucleotide (referred to herein as an “E2-E6 fusion”); a fusion of exons 1-2 of a ESR1 polynucleotide with exons 6-11 (having at least a portion of exon 11) of a CCDC170 polynucleotide (referred to herein as an “E2-E6 fusion”); a fusion of exons 1-2 of a ESR1 polynucleotide with exons 8-11 (having at least a portion of exon 11) of a CCDC170 polynucleotide (referred to herein as an “E2-E8 fusion”); a fusion of exons 1-2 of a ESR1 polynucleotide with exons 10-11 (having at least a portion of exon 11) of a CCDC170 polynucleotide (referred to herein as an “E2-E10 fusion”).
The fusions described herein can be detected by contacting the sample with one or more primers specific for the fusion, performing an amplification reaction, and detecting an amplification product or amplicon. It should be understood and herein contemplated that the term “amplification reaction” of polynucleotide as used herein means the use of an amplification reaction (e.g., PCR) to increase the concentration of a particular nucleic acid sequence within a mixture of nucleic acid sequences. The term “PCR” as used herein refers to the polymerase chain reaction, a laboratory technique used to make multiple copies of a segment of a polynucleotide, as is well- known in the art. The term “PCR” includes all forms of PCR, such as real-time PCR, quantitative reverse transcription PCR (qRT-PCR), multiplex PCR, nested PCR, hot start PCR, or GC-Rich PCR. In some embodiments, the amplification reaction is real-time PCR. Exemplary procedures for real-time PCR can be found in “Quantitation of DNA/RNA Using Real-Time PCR Detection” published by Perkin Elmer Applied Biosystems (1999) and to PCR Protocols (Academic Press New York, 1989), incorporated by reference herein in their entireties. The amplification reaction can also be a loop-mediated isothermal amplification (LAMP), a reaction at a constant temperature using primers recognizing the distinct regions of target DNA for a highly specific amplification reaction. In some embodiments, the ESR1-CCDC170 polynucleotide fusion disclosed herein is detected by methods such as the Nanostring nCounter assay which directly measures target molecules without PCR amplification using ghost probes against one fusion partner gene, and reporter probes against the other fusion partner gene. In some embodiments, a fusion protein encoded by the fusion polynucleotide disclosed herein is detected by one or more protein detection assays including, for example, Western blotting, immunoblotting, ELISA, immunohistochemistry, or an electrophoresis method (e.g., SDS-PAGE).
The fusion can also be detected by any RNA or DNA, or protein-based methods known in the art, such as Nanostring assay or whole transcriptome, whole genome or targeted transcriptome or genome sequencing, or fluorescence in situ hybridization, or southern blot, or immunohistochemistry, or western blot.
In some embodiments, the one or more primers or Nanostring probes comprise a sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, or a polynucleotide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with a sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, or a polynucleotide comprising a portion of a sequence selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45. In some embodiments, a first primer or Nanostring probe comprises a sequence selected from the group consisting of SEQ ID NO: 42 and SEQ ID NO:44, or a polynucleotide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% homology with the sequence selected from SEQ ID NO: 42 and SEQ ID NO:44, or a polynucleotide comprising a portion of with the sequence selected from SEQ ID NO: 42 and SEQ ID NO:44, and second primer or Nanostring probe comprises a sequence selected from the group consisting of SEQ ID NO: 43 and SEQ ID NO:45, or a polynucleotide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% homology with the sequence selected from SEQ ID NO: 43 and SEQ ID NO:45, or a polynucleotide comprising a portion of with the sequence selected from SEQ ID NO: 43 and SEQ ID NO:45.
As used herein, the term “detecting” refers to detection of a level of a fusion (e.g., the fusion of a ESR1 polynucleotide sequence and a CCDC170 polynucleotide) that is at least about 5% (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, at least about 2000%, at least about 3000%, or at least about 5000%) or at least about 5 times (e.g., at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, or at least about 100 times) higher as compared to a sample from a subject in general or a study population (e.g., healthy control).
In certain embodiments, the primers are used in DNA amplification reactions. Typically, the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and/or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, regular PCR, real-time PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, and reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments, the primers are used for the DNA or RNA amplification reactions, such as PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. In some embodiments, the primers are used for gene array analysis. Typically, the disclosed primers hybridize with a region of the disclosed nucleic acids (e.g., ESR1 or CCDC170) or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids.
In some embodiments, subject has a cancer. The cancer can be any of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, and lung cancer. In certain aspects, the cancer is a breast cancer. In certain aspects, the cancer is a luminal A breast cancer. In certain aspects, the cancer is a luminal B breast cancer. It should be understood and herein contemplated that luminal A breast cancer refers to breast tumors that are estrogen receptor (ER) positive, progesterone receptor (PR) positive, and HER2 negative. Luminal B breast cancer refers to breast tumors that are estrogen receptor (ER) positive, progesterone receptor (PR) negative, and HER2 positive.
“ER” or “estrogen receptor” refers herein to receptors that are activated by estrogen. The two types of ERs include ERα and ERβ. ERα is encoded by the ESR1 gene, while ERβ is encoded by ESR2 gene. In some embodiments, the ESR2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 3468, Entrez Gene: 2100, Ensembl: ENSG00000140009, OMIM: 601663, UniProtKB: Q92731. In some embodiments, the ESR2 polypeptide comprises the sequence of SEQ ID NO: 24 or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 24, or a polypeptide comprising a portion of SEQ ID NO: 24. The ESR2 polypeptide of SEQ ID NO: 24 may represent an immature or pre-processed form of mature ESR2, and accordingly, included herein are mature or processed portions of the ESR2 polypeptide in SEQ ID NO: 24.
“Progesterone receptor” or “PR” refers herein to a polypeptide is encoded by the PGR gene in human. In some embodiments, the PR polypeptide is that identified in one or more publicly available databases as follows: HGNC: 8910, Entrez Gene: 5241, Ensembl: ENSG00000082175, OMIM: 607311, UniProtKB: P06401. In some embodiments, the PR polypeptide comprises the sequence of SEQ ID NO: 25 or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 25, or a polypeptide comprising a portion of SEQ ID NO: 25. The PR polypeptide of SEQ ID NO: 25 may represent an immature or pre-processed form of mature PR, and accordingly, included herein are mature or processed portions of the PR polypeptide in SEQ ID NO: 25.
The “sample” referred to herein is a fluid or tissue sample. In some embodiments, the sample is a breast tissue sample. In some embodiments, the breast tissue is cancerous. Included herein are methods that comprise detection of an increased amount of the ESR1/CCDC170 fusion in a breast tissue sample as compared to a control, wherein the control can be a normal breast tissue or any normal tissue other than testis tissue, and wherein the control can be obtained from the same subject or a different subject. In some embodiments, the control is a level or amount of the ESR1/CCDC170 fusion in a general or study population. In some embodiments, the cancerous breast tissue exhibits an increased amount of the fusion of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a control, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold, at least about a 20-fold, at least about a 50-fold, at least about a 100-fold, at least about a 500-fold, or at least about a 1000-fold as compared to a control.
It should be understood and herein contemplated that detection of the ESR1/CCDC170 fusion or an increase in the amount of the ESR1/CCDC170 fusion as compared to a control indicates a decreased sensitivity of the tissue sample, cancer cell or tumor to an estrogen receptor antagonist (such as an estrogen-receptor modulator, an estrogen-receptor down-regulator, or an aromatase inhibitor).
It should be understood that the term “estrogen-receptor modulator” herein refers to a class of drugs that bind with estrogen receptors. In some examples, the estrogen-receptor modulator acts as an agonist to enhance estrogen activity. In some examples, the estrogen-receptor modulator acts as an antagonist to inhibit estrogen activity. In some examples, the estrogen-receptor modulator is selected from tamoxifen, clomifene, raloxifene, and exemestane. In some embodiments, the estrogen-receptor modulator is tamoxifen.
As used herein, “tamoxifen” refers to a composition having the below chemical structure.
As used herein, “clomifene” refers to a composition having the below chemical structure.
As used herein, “raloxifene” refers to a composition having the below chemical structure.
As used herein, “exemestane” refers to a composition having the below chemical structure.
The term “estrogen-receptor down-modulator” or “estrogen-receptor down-regulator” herein refers to a class of drugs that bind to estrogen receptors and cause the receptor to be degraded. In some examples, the estrogen-receptor down-modulator used herein is fulvestrant.
As used herein, “fulvestrant” refers to a composition having the below chemical structure.
Aromatase inhibitors work by blocking the enzyme aromatase, which turns the hormone androgen into estrogen in the body. In some examples, the aromatase inhibitor is letrozole. As used herein, “letrozole” refers to a composition having the below chemical structure.
The ESR1/CCDC170 gene fusion can be detected using any method described herein. In some embodiments, the decreased sensitivity of a cancer cell or tumor refers to a more significant increase in tumor growth, a larger increase in tumor volume or size, a slower clearance of tumor, a decrease in cancer cell death, an increase in cell migration, metastasis, and/or proliferation as compared to a control cancer cell or tumor, wherein the control tumor or cancer cell does not have the ESR1/CCDC170 fusion disclosed herein. In some embodiments, the tumor or cancer cell comprising the ESR1/CCDC170 fusion exhibits a decreased sensitivity to an estrogen receptor antagonist (such as an estrogen-receptor modulator, an estrogen-receptor down-regulator, or an aromatase inhibitor) of at least about at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or at least about 100%, or a decreased sensitivity to an estrogen receptor antagonist (such as an estrogen-receptor modulator, an estrogen-receptor down-regulator, or an aromatase inhibitor) of at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold, at least about a 20-fold, at least about a 50-fold, at least about a 100-fold, or at least about a 500-fold as compared to a control.
In some embodiments, detection of the ESR1/CCDC170 fusion or an increase in the amount of the ESR1/CCDC170 fusion as compared to a control indicates a decreased sensitivity of the tissue sample, cancer cell or tumor to an estrogen receptor antagonist.
Since detection of a ESR1/CCDC170 fusion indicates an increased resistance to an estrogen receptor antagonist (such as an estrogen-receptor modulator, an estrogen-receptor down-regulator, or an aromatase inhibitor), or a decrease in the effectiveness of an estrogen receptor antagonist (such as an estrogen-receptor modulator, an estrogen-receptor down-regulator, or an aromatase inhibitor) in the subject, certain embodiments further include treating the subject with a therapeutically effective amount of a HER inhibitor (e.g., a HER2 inhibitor) and/or a SRC inhibitor. The administration of the HER inhibitor and/or the SRC inhibitor can be in addition to administration of an estrogen receptor antagonist or an increased amount of an estrogen receptor antagonist.
“HER” refers to any of ErbB1/EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4, which are members of the ErbB receptor tyrosine kinase family. As used herein, “HER inhibitor” refers to a composition that inhibits the tyrosine kinase activity and/or intracellular signaling of a HER molecule. In some embodiments, the HER molecule is a HER2, a HER3, or a HER2/HER3 heterodimer. “HER2” “Erb-B2 Receptor Tyrosine Kinase 2” refers herein to a polypeptide encoded by the ERBB2 gene in humans. In some embodiments, the HER2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 3430, Entrez Gene: 2064, Ensembl: ENSG00000141736, OMIM: 164870, UniProtKB: P04626. In some embodiments, the HER2 polypeptide comprises the sequence of SEQ ID NO: 26 or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 26, or a polypeptide comprising a portion of SEQ ID NO: 26. The HER2 polypeptide of SEQ ID NO: 26 may represent an immature or pre-processed form of mature HER2, and accordingly, included herein are mature or processed portions of the HER2 polypeptide in SEQ ID NO: 26. “HER3” or “Erb-B2 Receptor Tyrosine Kinase 3” refers herein to a polypeptide encoded by the ERBB3 gene in humans. In some embodiments, the HER2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 3431, Entrez Gene: 2065, Ensembl: ENSG00000065361, OMIM: 190151, UniProtKB: P21860. In some embodiments, the HER3 polypeptide comprises the sequence of SEQ ID NO: 27 or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 27, or a polypeptide comprising a portion of SEQ ID NO: 27. The HER3 polypeptide of SEQ ID NO: 27 may represent an immature or pre-processed form of mature HER3, and accordingly, included herein are mature or processed portions of the HER3 polypeptide in SEQ ID NO: 27.
In some embodiments, the HER2 inhibitor is lapatinib. The term “lapatinib” refers to a composition having the below chemical structure.
In some embodiments, the HER2 inhibitor is neratinib. The term “neratinib” refers to a composition having the below chemical structure.
In some embodiments, the HER2 inhibitor is trasuzumab. In some embodiments, the HER2 inhibitor is persuzumab. In some emobidments, the HER2 inhibitor is selected from the group consisting of lapatinib, neratinib, trasuzumab and persuzumab.
In some embodiments, the HER3 inhibitor is Patritumab, Seribantumab, Elgemtumab, KTN3379, AV-203, or GSK2849330.
“SRC” of “SRC Proto-Oncogene, Non-Receptor Tyrosine Kinase” refers herein to a polypeptide that is encoded by the SRC gene in humans. In some embodiments, the SRC polypeptide is that identified in one or more publicly available databases as follows: HGNC: 11283, Entrez Gene: 6714, Ensembl: ENSG00000197122, OMIM: 190090, UniProtKB: P12931. In some embodiments, the SRC polypeptide comprises the sequence of SEQ ID NO: 28 or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% homology with SEQ ID NO: 28, or a polypeptide comprising a portion of SEQ ID NO: 28. The SRC polypeptide of SEQ ID NO: 28 may represent an immature or pre-processed form of mature SRC, and accordingly, included herein are mature or processed portions of the SRC polypeptide in SEQ ID NO: 28. As used herein, “SRC inhibitor” refers to a composition that inhibits the tyrosine kinase activity and/or intracellular signaling of a SRC molecule.
In some embodiments, the SRC inhibitor is dasatinib. The term “dasatinib” refers to a composition having the below chemical structure.
In other embodiments, the SRC inhibitor is bosutinib, ponatinib, vandetanib, or saracatninib.
As the timing of a cancer can often not be predicted, it should be understood the disclosed methods of treating, preventing, reducing, and/or inhibiting the disease or disorder described herein can be used prior to or following the onset of the disease or disorder, to treat, prevent, inhibit, and/or reduce the disease or disorder or symptoms thereof. In one aspect, the disclosed methods can be employed 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 years, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 months, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 days, 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 hours, 60, 45, 30, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute prior to onset of the disease or disorder; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more years after onset of the disease or disorder.
Dosing frequency for the composition of any preceding aspects, includes, but is not limited to, at least once every year, once every two years, once every three years, once every four years, once every five years, once every six years, once every seven years, once every eight years, once every nine years, once every ten year, at least once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, at least once every month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, daily, two times per day, three times per day, four times per day, five times per day, six times per day, eight times per day, nine times per day, ten times per day, eleven times per day, twelve times per day, once every 12 hours, once every 10 hours, once every 8 hours, once every 6 hours, once every 5 hours, once every 4 hours, once every 3 hours, once every 2 hours, once every hour, once every 40 min, once every 30 min, once every 20 min, or once every 10 min. Administration can also be continuous and adjusted to maintaining a level of the compound within any desired and specified range.
Administration of any combination of a HER inhibitor, a SRC inhibitor and an estrogen receptor antagonist can occur simultaneously, or non-simultaneously in an order. In some embodiments, a HER inhibitor and/or a SRC inhibitor are administered simultaneously with an estrogen receptor antagonist. In other embodiments, a HER inhibitor and/or a SRC inhibitor are administered before an estrogen receptor antagonist. In other embodiments, a HER inhibitor and/or a SRC inhibitor are administered after an estrogen receptor antagonist.
Included herein are kits comprising a probe or a set of probes, for example, a detectable probe or a set of amplification primers that specifically recognize a nucleic acid comprising a fusion point or break point. The kit can further include, in the same vessel, or in a separate vessel, a component from an amplification reaction mixture, such as a polymerase, typically not from human origin, dNTPs, and/or UDG. In some embodiments, the amplification primers are selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45. In some embodiments, the detectable probe is selected from polynucleotide sequence that specifically hybridizes to a fusion point nucleotide sequence within SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41. In some embodiments, the kit comprises a detectable moiety that is covalently bonded to the probe. Furthermore, the kit can include a control nucleic acid. For example, the control nucleic acid can include a sequence that includes a fusion point sequence within a sequence selected from the group of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.
All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes.
The following examples are set forth below to illustrate the compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Emerging evidence shows that ESR1 mutations or fusions constitute the most common genetic mechanisms for endocrine resistance, accounting for approximately 20-50% of advanced breast cancers (Ma CX (2015), Jeselsohn R (2015), Hartmaier RJ (2018), Lei JT (2019), Giltnane JM (2017), Robinson DR (2013)). A previous study discovered the first and most prevalent form of ESR1 fusions generated by localized rearrangements between ESR1 and its immediate centromeric neighbor gene coiled-coil domain containing 170 (CCDC170), a majority of which are tandem duplications (
The previous data show that, ESR1-CCDC170 fusions endow ligand-independent growth factor signaling, leading to increased cell motility, invasion, anchorage-independent growth, and reduced endocrine sensitivity in vitro, as well as enhanced tumor formation in vivo (Veeraraghavan J, 2014). To date, ESR1-CCDC170 remains the most frequent pathological gene fusion detected in luminal breast cancer, and its recurrence has been subsequently supported by several recent studies (Hartmaier RJ (2018), Giltnane JM (2017), Matissek KJ (2018), Fimereli D (2018), Lei JT (2018)). In addition to breast cancer, a recent publication reported ESR1-CCDC170 as a recurrent event in ovarian cancer associated with exceptional short-term survival (Yang SYC (2018)). Nonetheless, detailed functional evidence demonstrating the causal role of ESR1-CCDC170 in endocrine resistance especially in the in vivo and clinical context, and the precise mechanism for this fusion to endow ligand-independent growth factor signaling has been lacking. Here the data show that ESR1-CCDC170 fusions endow breast cancer cell survival and reduced endocrine sensitivity in vitro and in vivo via physically binding to the HER2/HER3/SRC complex and activate their downstream signaling. Furthermore, the RNAseq data from four metastatic breast cancer patient cohorts were analyzed including University of Michigan cohort (MET500) (Robinson DR, 2017), Count me in Metastatic Breast Cancer Project (MBC) (Nikhil Wagle CP, 2018), UPMC cohort, and the BCRF AURORA project (Zardavas D, 2014). These analyses detected ESR1-CCDC170 fusions in ~8% of metastatic luminal breast cancers (
An independent report in Science Translational Medicine assessed ESR1 fusions in surgical samples from stage I-III ER positive and HER2 negative breast cancer patients following neoadjuvant letrozole treatment (Giltnane JM, 2017). This study detected ESR1-CCDC170 in three out of 27 endocrine resistant or intermediate resistant tumors (11%), among which two tumors harbor E2-E6 variants, and one tumor harbors E2-E8 variant. In addition, they also detected ESR1-CCDC170 in one out of 29 endocrine sensitive tumors. This case however, express a E4-E5 variant encoding a c-terminally truncated ESR1 protein that disrupts the DNA binding domain, thus is likely nonfunctional. This shows that fusion variants V2 and V4 are associated with lack of response to neoadjuvant letrozole treatment, and that truncation of CCDC170 that removes CC1 and CC2 domains are sufficient to confer reduced endocrine sensitivity. Furthermore, another clinical study reported in 2019 SABCS meeting detected ESR1-CCDC170 in the primary tumors of HER2-negative luminal breast cancer patients that experienced metastatic relapse. ESR1-CCDC170 fusions are detected in 29 out of 307 patients (9.4%) which significantly correlate with worse disease-free survival following initial surgery (AM Sieuwerts SV, 2019). The most relevant fusion variants involve ESR1 E2 and CCDC170 E4-10 (V2-5).
Recurrent gene fusions have laid the foundation for modern cancer therapeutics (Mitelman F, 2007). In addition to leukemia, several milestone studies have identified recurrent fusions in multiple solid tumors that led to therapeutic breakthroughs. The most prominent examples are EML4-ALK and ROS1 fusions in 3%-5% and 1%-2% of non-small cell lung cancer, respectively, FGFR-TACC fusion in approximately3% of glioblastomas, and NTRK fusions detected in approximately 0.3% of tumors, all of which are matched with stunningly effective targeted therapies (Koivunen JP (2008), Singh D (2012)). While low in percentages, therapies against these fusions have generated tremendous clinical impacts and are considered as recent milestones in cancer treatments. In particular, FDA granted accelerated approval to the first pan-cancer drug for the treatment of solid tumors, larotrectinib targeting NTRK fusions (Laetsch TW (2018)). Breast cancer accounts for 2.1 million new cancer cases world-wide annually. These results indicate the uses of HER2/HER3/SRC inhibitors to treat ESR1-CCDC170 positive patients. Based on the frequency observed in luminal B, endocrine resistant, and metastatic breast cancers, ESR1-CCDC170 can affect a patient population comparable to that of EML4-ALK positive lung cancer or NTRK positive adult cancer patients.
ESR1-CCDC170 fusions endow reduced endocrine sensitivity in vitro and in vivo. To explore their role in endocrine resistance, the consequences of ESR1-CCDC170 ectopic expression on the outcome of tamoxifen treatment in vitro and in vivo was assessed. First, the engineered T47D cells (
Next, the consequences of ESR1-CCDC170 ectopic expression on the outcome of tamoxifen treatment in vivo was assessed. T47D xenograft tumors expressing vector, E2-E7, or E2-E10 fusion variants were developed in female athymic nude mice. When the tumors reached 150-200 mm3, the mice were randomized into tamoxifen treatment (25 ug/kg) and untreated groups. When treated with tamoxifen, the vector group showed true tumor regression with 51% decrease in size, whereas the E2-E7 expressing tumors sustained steady tumor volumes with only 14% decrease in size. The E2-E10 expressing tumors continued to grow and then stabilized at higher tumor burdens, with almost two folds increase in tumor volumes (
It is notable that, ER-positive breast cancer patients are treated with endocrine therapy with a curable intent aimed to achieve long-term survival. Thus, cancer cell survival factor (i.e., E2-E7) is equally important as resistant factor (i.e., E2-E10). The relative tamoxifen-resistance of E2-E7 expressing xenograft tumors is comparable to that of the xenograft tumors expressing ESR1 mutations as previously reported (Toy W, (2017)). While mutations of ESR1 ligand binding domain constitute one of the most important driving mechanisms of acquired endocrine resistance (Ma CX (2015), Jeselsohn R (2015), Lei JT (2019)), the growth of the xenograft tumor models ectopically expressing ESR1 mutations can be effectively inhibited by endocrine treatment (Toy W, (2017)). The relative endocrine resistance of the xenograft tumors expressing ESR1 mutations was only evident when the different tumor models within the endocrine treated group are compared (Toy W, (2017)). Clinically the tumors expressing ESR1-CCDC170 fusions can respond to endocrine therapy initially but suffer from residue cancer cells that eventually can lead to metastatic relapse, a typical response pattern of endocrine-therapy naïve luminal B breast cancer.
ESR1-CCDC170 fusions augment HER2/HER3/SRC/AKT pathway under endocrine treatment. Next, signaling alterations were examined in T47D cells ectopically expressing the ESR1-CCDC170 fusion variants or wtCCDC170. Under estrogen deprived condition, the phosphorylation levels of pHER3-Y1289 and pAKT-S473 were upregulated in only E2-E8 or E2-E10 variant, respectively (
Interestingly, estrogen deprivation induced activation of pSRC-Y416 and pAKT-S473, which was more potent in T47D expressing ESR1-CCDC170 variants compared to wtCCDC170 or vector control. In addition, the activation of pHER3-Y1289 was induced by estrogen deprivation in the E2-E8 expressing T47D cells (
To gain insights into ESR1-CCDC170 driven mechanisms, reverse phase protein array (RPPA) analysis was performed in the HCC1428 cells with or without ESR1-CCDC170 silencing, using about 200 validated antibodies against an array of cancer related signaling molecules. ESR1-CCDC170 silencing leads to substantial repression of ERα, BCL2, HER3, c-SRC protein levels as well as total/phospho-HER2 (
ESRI-CCDC170 localizes to cytoplasm, associates with HER2/HER3/SRC, and forms homodimers. HER2/HER3 heterodimer functions as an oncogenic unit and is known to engage SRC and PI3K/AKT pathway to drive breast cancer (Holbro T (2003); Yarden Y (2012)). Next experiment assessed whether ESR1-CCDC170 fusions modulate the HER2/HER3/SRC pathway by forming complex with them. To test this, immunoprecipitations were performed using HER2, HER3, or SRC antibodies on the T47D cells ectopically expressing E2-E10 or wtCCDC170, and performed western blots using anti-CCDC170 antibody. The ΔCCDC170 protein encoded by E2-E10, but not wtCCDC170, co-immunoprecipitated with endogenous HER2, HER3 and SRC (
Wild type CCDC170 contains a structural maintenance of chromosomes (SMC) domain. The SMC proteins contains highly conserved ATP-binding cassette (ABC) that drives its dimerization (Hirano T (2006)). The ΔCCDC170 proteins encoded by the ESR1-CCDC170 fusions have different degrees of deletion of the N-terminal region of the SMC domain, but retain a putative high-affinity ATP-binding pocket at its C-terminus (
SRC and HER2 inhibitors increase endocrine sensitivity of luminal breast cancer cells expressing ESRI-CCDC170. Next, whether HER2/SRC inhibitors can revert the tamoxifen resistance driven by ESR1-CCDC170 was tested. Two drugs were selected for this test, lapatinib, a dual-specificity inhibitor targeting EGFR and HER-2 (Yarden Y, (2012)), and the SRC inhibitor dasatinib, both of which are FDA-approved (Schenone S (2010), Araujo J (2010)). The T47D cells ectopically expressing ESR1-CCDC170 variants were cultured in estrogen-deprived condition and treated either with tamoxifen alone or combined with lapatinib or dasatinib. Clonogenic assay results showed that the colony formations were significantly reduced when the cells were treated with tamoxifen plus lapatinib (
The therapeutic effect of lapatinib and dasatinib was then assessed in ZR-75-1 cells harboring the E2-E6 fusion (Veeraraghavan J, (2014)). ZR-75-1 is an ER+/HER2- endocrine therapy-naive cell line derived from ascitic effusion of a 63 year-old metastatic breast cancer patient who was subsequently treated with tamoxifen without apparent benefit (Engel LW, (1978)). This cell line was excluded from genetic silencing study as the fusion variant expressed in this cell line is not amendable to design siRNAs against its fusion junction due to their general toxicity to the cells. However, this cell line can be useful to provide additional insights into the therapeutic values of HER2/SRC inhibition in fusion positive cancer cells. The ZR-75-1 cells were treated with increasing doses of tamoxifen or fulvestrant, in combination with lapatinib, dasatinib, or both (
ESR1-CCDC170 positive luminal breast cancer cells show increased sensitivity to lapatinib in the context of endocrine therapy. To test the accountability of ESR1-CCDC170 on the Tamoxifen-sensitizing effects of lapatinib, the ESR1-CCDC170 positive cell lines HCC1428 (E2-E10 variant) and ZR-75-1 (E2-E6 variant), and fusion-negative cell lines MDA-MB-415 and CAMA-1 were treated with titrated dosages of Lapatinib in combination with 0.5 µM tamoxifen. All these cell lines are ER+/HER2- luminal breast cancer cell lines. The clonogenic viabilities following the respective treatments were assessed by clonogenic assays. The results showed that HCC1428 and ZR-75-1 cells exhibited substantially increased sensitivity to lapatinib than MDA-MB-415 and CAMA-1 cells in the context of tamoxifen treatment (
ESRI-CCDC170 proteins can facilitate HER2, HER3, SRC interactions, leading to uncontrolled activation of their downstream signaling. HER2 are amplified in approximately 15-20% of breast cancer, for which HER2-targeted therapy is the standard-of-care. While modest HER2 protein expression (IHC 1+ or 2+) can be detected in 60% of luminal breast cancer (Zhang H, (2018)), HER2 inhibitors are not indicated in these tumors. A recent clinical trial indicates that adding lapatinib to endocrine therapy in unselected advanced ER-positive breast cancer does not improve patient survival (Burstein HJ, (2014)). HER2/HER3 forms heterodimer when bind to HER3 ligand NRG½, which phosphorylates HER3 C-terminal tail thus activates its kinase activity (Lee-Hoeflich ST, (2008)). HER3 plays a central role in HER2 positive breast cancer and is the most potent activator of AKT/SRC (Lee-Hoeflich ST (2008), Lyu H (2018)). SRC is broadly overexpressed in luminal breast cancer (Anbalagan M (2012)) and can crosstalk with HER2 when facilitated by other molecules such as CDCP1, leading to phosphorylation of HER2 at the Y877 (Yarden Y (2012), Holbro T (2003), Alajati A (2015)). While SRC plays a key role in breast cancer bone metastasis and hormonal therapy resistance (Kennedy LC (2019), Vallabhaneni S (2010), Zhang XH (2009)), the clinical trials of SRC inhibitors in unselected metastatic luminal breast cancer patients have been disappointing (Kennedy LC, (2018)). SMC proteins are formed by two long coiled-coil domains connected by a non-helical sequence called “hinge”, which presumably corresponds to the low complexity region of CCDC170 (
Based on the above, it is possible that the N-terminal truncations of CCDC170 that delete the CC1 and CC2 domains can expose the CC4 coiled-coil domain via reducing antiparallel coiled-coil interactions and thus alter protein interaction and localization properties. Through ATP-driven autonomous homodimerization, ESR1-CCDC170 proteins can facilitate interactions between HER2, HER3, or SRC, leading to uncontrolled activation of their downstream signaling (
Revealing an Achilles’ heel of the deadly forms of luminal breast cancers harboring ESRI-CCDC170 fusions. Endocrine resistant and metastatic tumors account for most of devastating deaths from luminal breast cancers. A recent study indicated that ESR1-CCDC170 significantly correlates with poor disease-free survival of breast cancer patients following surgery, accounting for 9% of distant relapse (AM Sieuwerts SV, (2019)). These patients’ lives could be saved if they can be appropriately treated and followed up after surgery. ESR1-CCDC170 engenders a special exploitable vulnerability in these deadly tumors by conferring sensitivity to HER2/HER3/SRC inhibitors. While HER2 and SRC have been implicated in endocrine resistance and metastasis, HER2/SRC inhibitors are not clinically indicated for ESR1-CCDC170 positive patients and their clinical trials in unselected luminal breast cancer patients have been disappointing. Thus, the finding shown herein can benefit a substantial population of ESR1-CCDC170 positive patients that otherwise can have a devastating clinical outcome.
A novel genotype-directed therapy for saving the lives and improving the life quality of ESRI-CCDC170+ patients. Luminal B and metastatic breast tumors are routinely treated with hormone therapy and chemotherapy that have life-threatening toxicity. This innovation can lead to a new paradigm to treat ESR1-CCDC170 positive patients with more potent therapeutic effect and much less toxicity to drastically improve their life quality. While HER2/HER3/SRC inhibitors are not novel, their indications in unselected luminal breast cancer patients are not supported by recent clinical trials that lack the guidance of predictive biomarkers: adding lapatinib to endocrine therapy in advanced ER-positive breast cancer does not improve patient survival (Burstein HJ, (2014)), and clinical trials of dasatinib in metastatic luminal breast cancers have been disappointing (Kennedy LC, (2019)). The data shown herein repurposes these drugs to treat genotypically selected luminal breast cancer patients based on ESR1-CCDC170 positivity.
Cell culture. ER+ breast cancer cell lines T47D cells and HCC1428 were purchased from American Type Culture Collection, cultured in RPMI-1640 (Corning) supplemented with 10% fetal bovine serum (Hyclone). For estrogen deprivation, cells were cultured in phenol red-free RPMI 1640 (Invitrogen) supplemented with 5% charcoaldextran- stripped serum (Sigma).
Engineering ectopic overexpression models. Lentiviral constructs and the lentivirus for ectopic expression of ESR1-CCDC170 fusion variants and wtCCDC170 were from our previous study (Veeraraghavan J (2014)). The T47D cells were infected by lentivirus containing these constructs in medium containing 4 µg/ml polybrene. Medium was replaced after overnight incubation. The cells with high GFP reporter expression were selected using flow cytometry two days later.
Antibodies and Reagents. Primary antibodies against pEGFR-Y1068 (D7A5), EGFR, pHER2Y877), pHER2-Y1221/1222 (6B12), pHER2- Y1248, HER2/ErbB2 (D8F12), pHER3-Y1289 (D1B5), HER3 (D22C5), pMet-Y1234/1235 (D26), Met (D1C2), pER-S118α (16J4), pERα-S167 (D1A3), ERα (D8H8), pAKT-S473 (D9E), AKT (C67E7), pERK (137F5), ERK (D13E1), pSrc-S416 (D49G4), Src (32G6), Integrin β1(D2E5) and Bcl-2 were purchased from Cell Signaling Technology. HER2/neu Ab-8 (Clone e2-4001) mouse monoclonal antibody used for IP is from Thermo. C6orf97 antibody is from Gene Tex. V5 antibody MA5-15253 is from Thermo. (Z)-4-Hydroxytamoxifen is from sigma. Fulvestrant (ICI-182780, ZD 9238), Lapatinib (GW572016), and Dasatinib were purchased from selleckchem, Drugs were resolved in DMSO, diluted in culture medium before use.
In vivo xenograft experiments. All animal experiments were performed in accordance with the institutional guidelines and regulations, and the animal protocol was approved by the BCM Institutional Animal Care and Use Committee (Approval # AN-6123). Briefly, 1×107 T47D cells ectopically expressing the empty vector, or the ΔCCDC170 fusion variants E2-E7 and E2-E10 were resuspended in 20% Matrigel solution and injected bilaterally to 4-6 week-old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 60-day-release 170-estradiol pellets. Xenograft tumors of the T47D models were successfully engrafted in 6-9 mice per group. Growth of the xenograft tumors was monitored twice per week and tumor volume was measured using the formula ½ (length × width2). When the tumors reached 200 mm3, mice with tumors expressing the empty vector, E2-E7, and E2-E10 variants were each randomized to +/- tamoxifen treatment. Tamoxifen (25 µg/Kg body weight) was injected subcutaneously for 5 days/week. Growth of the xenograft tumors was monitored twice per week until the end of the experiment.
siRNA and transfection. The E2-E10-specific siRNA (5′-CAUCACUGAGAUUAAAACU-3′) (SEQ ID NO: 29), ERα-specific siRNA (5′-AGGCUCAUUCCAGCCACAGTT-3′) (SEQ ID NO: 30), HER2-specific siRNA-1 (L-003126-00, ON-TARGETplus Human ERBB2 siRNA SMARTPool), HER2-specific siRNA-2 (5′-CACGUUUGAGUCCAUGCCCAA-3′) (SEQ ID NO: 31), Src-specific siRNA-1 (J-003175-15, 5′-CCAAGGGCCUCAACGUGAA-3′) (SEQ ID NO: 32), and Src-specific siRNA-2 (J-003175-16, 5′-GGGAGAACCUCUAGGCACA-3′) (SEQ ID NO: 33) and control siRNA (D-001810-10-50, ON-TARGETplus Non-targeting Pool) were purchased from Dharmacon. All siRNAs were transfected using Lipofectamine RNAi MAX Reagent (Invitrogen) according to manufacturer’s instructions..
Reverse phase protein array analysis. Reverse phase protein array assay was performed as described previously (Kim JA (2016)). Briefly, protein lysates were prepared from HCC1428 cells with control or E2-E10 siRNA knockdown using modified Tissue Protein Extraction Reagent (TPER) (Pierce) and a cocktail of protease and phosphatase inhibitors (Roche Life Science). The protein lysates were then diluted into 0.5 mg/ml of total protein in SDS sample buffer and denatured on the same day. For each spot on the array, the-background-subtracted foreground signal intensity was subtracted by the corresponding signal intensity of the negative control slide (omission of primary antibody) and then normalized to the corresponding signal intensity of total protein for that spot. The median of the triplicate experimental values (normalized signal intensity) is taken for each sample for subsequent statistical analysis.
Protein subcellular localization. Fractionation of the nuclear and cytoplasmic proteins from T47D cells ectopically overexpressing the empty vector, wtCCDC170 or the ΔCCDC170 fusion variants E2-E7 and E2-E10 was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo) according to manufacturer’s instructions. The fractionated protein samples (30 µg protein) were then analyzed by western blot, as previously described (Veeraraghavan J (2014)).
Immunoprecipitation. For IP with HER2/HER3/Src antibody. As described previously (Yan K (2015)), cells were harvested and lysed in NETN- 400 buffer (50 mM Tris-HCl, pH 8.0, 400 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40), for 25 min on ice. The samples were centrifuged at 13,000 rpm for 15 min, and the supernatants were diluted with the same buffer without NaCl (NETN-0) to obtain a final concentration of NaCl at 150 mM. The samples were incubated with the appropriate antibodies at 4° C. with rocking for 2 h. Protein G agarose was then added, and the incubation was continued for an additional 2 h. Beads were then washed three times using the NETN-150 buffer. The bound proteins were eluted with 100 mM glycine, pH 2.5, and then neutralized by adding ⅒ vol of 1 M Tris-Cl, pH 8.0. Eluted proteins were separated on 4-12% SDS-PAGE and blotted with the corresponding antibodies as indicated.
Clonogenic assay. For T47D, cells cultured in normal medium, RPMI1640 with phenol red and 10% FBS, were seeded at a density of 5000 cells per well in 24/48-well plate, 24 hours later normal medium was changed to ED medium, phenol red-free RPMI1640 supplemented with 5% charcoal-stripped serum (CSS), with or without indicated drugs, for 14 days. For HCC1428, cells were seeded at a density of 5000 cells per well in 24-well plate, added the indicated drugs 24 hours later, continue to culture for 18 days. Then the cells were fixed and stained by crystal violet, the colonies formed were calculated as intensity by using Image J with Colony Area Plug In.
Statistical analysis. The results of all in vitro experiments were compared by Student’s t-tests, and all data are shown as mean ± standard deviation. For the in vivo study, statistical comparison of tumor volumes was performed using one-way analysis of variance. Long-term outcomes were evaluated by survival analysis methods. ‘Events’ were defined to mimic clinically relevant outcomes; time to tumor regression (tumor-volume-halving) was analyzed using Kaplan-Meier survival curves and compared by the generalized Wilcoxon test.
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This application claims the benefit of U.S. Provisional Application No. 63/017,312, filed Apr. 29, 2020, which is expressly incorporated herein by reference in its entirety.
This invention was made with government support under grant numbers CA181368 and CA183976 awarded by the National Institutes of Health; and grant numbers W81XWH-12-1-0166, W81XWH-12-1-0167 and W81XWH-13-1-0431 awarded by United States Army Medical Research and Materiel Command. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/029086 | 4/26/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63017312 | Apr 2020 | US |
