Patent Description:
One approach to identify new anti-cancer agents is phenotypic screening to discover novel small molecules displaying strong selectivity between cancer cell lines, followed by chemogenomics to identify the cell features associated with drug response. In the <NUM>, Weinstein and colleagues demonstrated that the cytotoxic profile of a compound can be used to identify cellular characteristics, such as gene-expression profiles and DNA copy number that correlate with drug sensitivity. The ability to identify the features of cancer cell lines that mediate their response to small molecules has strongly increased in recent years with automated high-throughput chemosensitivity testing of large panels of cell lines coupled with comprehensive genomic and phenotypic characterization of the cell lines. Phenotypic observations of small-molecule sensitivity can be linked to expression patterns or somatic alterations, as in the case of SLFN11 expression in cancer cell lines sensitive to irinotecan treatment, and an EWS-FLI1 rearrangement in cancer cell lines sensitive to PARP inhibitors, respectively.

Methods of characterizing malignancies at a molecular level are useful for stratifying patients, thereby quickly directing them to effective therapies. Improved methods for characterizing the responsiveness of subjects having cancer are urgently required.

Embodiments not falling within the scope of the appended claims do not form part of the invention. Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy or for diagnosis.

As described below, the present invention features methods of identifying patients having a cancer (e.g., melanoma, adenocarcinoma, lung, cervical, liver, endometrium, lung, hematopoetic / lymphoid, ovarian, cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma, or breast cancer) that is sensitive to treatment with a phosphodiesterase 3A (PDE3A) modulator (i.e. <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one, (5R)-<NUM>-methyl-<NUM>-[<NUM>-(morpholin-<NUM>-yl)phenyl]-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (compound <NUM>), zardaverine, and anagrelide) by detecting co-expression of PDE3A and Schlafen <NUM> (SLPN12) polynucleotides or polypeptides in a cancer cell derived from such patients.

The present disclosure relates to a method of killing or reducing the survival of a cancer cell selected as responsive to a phosphodiesterase 3A (PDE3 A) modulator. The method includes the step of contacting the cell with a PDE3A modulator, where the cell was selected as having an increase in PDE3A and Schlafen <NUM> (SLFN12) polypeptide or polynucleotide relative to a reference, thereby reducing the survival of the cancer cell. In another embodiment, the present disclosure relates to a method of reducing cancer cell proliferation in a subject pre-selected as having a cancer that is responsive to a PDE3A modulator. The method comprises administering to the subject a PDE3A modulator, wherein the subject is pre-selected by detecting an increase in PDE3A and SLFN12 polypeptide or polynucleotide levels relative to a reference, thereby reducing cancer cell proliferation in the subject. In one embodiment, the subject is pre-selected by detecting an increase in PDE3A and SLFN12 polypeptide or polynucleotide levels. In some embodiments, the PDE3A modulator is selected from the group consisting of <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (DNMDP), zardaverine, and anagrelide.

In another embodiment, the present disclosure relates to a method of identifying a subject having a cancer responsive to PDE3A modulation. The method includes the step of detecting an increase in the level of a PDE3A and SLFN12 polypeptide or polynucleotide in a biological sample of the subject relative to a reference, thereby identifying the subject as responsive to PDE3A modulation.

In some embodiments, the increase in the level of PDE3A and SLFN12 polypeptide is detected by a method selected from the group consisting of immunoblotting, mass spectrometry, and immunoprecipitation. In some other embodiments, the increase in the level of PDE3A, and SLFN12 polynucleotide is detected by a method selected from the group consisting of quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. In some embodiments, the activity of PDE3A is reduced. The PDE3A modulator may be administered orally. The PDE3A modulator may be administered by intravenous injection.

In some embodiments, the cancer cell is a melanoma, endometrium, lung, hematopoetic / lymphoid, ovarian, cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma, or breast cancer. In some other embodiments, the cancer cell is not a B-cell proliferative type cancer. In some embodiments, the cancer cell is not multiple myeloma. In some embodiments, the biological sample is a tissue sample.

In another aspect, the present disclosure relates to a kit for identifying a subject having cancer as responsive to PD3A modulation, the kit comprising a capture reagent that binds PDE3A and/or a capture reagent that binds SLFN12. In one embodiment, the kit comprises a capture reagent that binds PDE3A and a capture reagent that binds SLFN12.

In yet another aspect, the present disclosure relates to a kit for decreasing cancer cell proliferation in a subject pre-selected as responsive to a PDE3A modulator, the kit comprising DNMDP, zardaverine, and/or anagrelide.

The present disclosure relates to methods for treating subjects having cancer identified as responsive to treatment with a PDE3A modulator by detecting co-expression of PDE3A and Schlafen <NUM> (SLFN12) polynucleotides or polypeptides in the cancer. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: <NPL>); <NPL>); <NPL>); and <NPL>). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "Anagrelide" (IUPAC Name <NUM>,<NUM>-dichloro-<NUM>,<NUM>-dihydroimidazo (<NUM>,<NUM>-b)quinazolin-<NUM>(<NUM>H)-one) is meant a small molecule phosphodiesterase inhibitor having the following structure:
<CHM>.

By "Cilostamide" (IUPAC Name N-cyclohexyl-N-methyl-<NUM>-[(<NUM>-oxo-<NUM>H-quinolin-<NUM>-yl)oxy]butanamide) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Cilostazol" (IUPAC Name <NUM>-[<NUM>-(<NUM>-cyclohexyl-<NUM>H-tetrazol-<NUM>-yl)butoxy]-<NUM>,<NUM>-dihydro-<NUM>(<NUM>H)-quinolinone) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "DNDMP" (IUPAC Name <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Forskolin" (IUPAC Name (<NUM>R,4aR,<NUM>S,<NUM>S,6aS,<NUM>S,10aR,10bS)-<NUM>,<NUM>,10b-Trihydroxy-<NUM>,4a,<NUM>,<NUM>,10a-pentamethyl-<NUM>-oxo-<NUM>-vinyldodecahydro-<NUM>H-benzo[f]chromen-<NUM>-ylacetate) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Levosimendan" (IUPAC Name (E)-<NUM>-cyano-<NUM>-methyl-<NUM>-(<NUM>-(<NUM>-methyl-<NUM>-oxo-<NUM>,<NUM>,<NUM>,<NUM>-tetrahydropyridazin-<NUM>-yl)phenyl)guanidine) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Milrinone" (IUPAC Name <NUM>-methyl-<NUM>-oxo-<NUM>,<NUM>-dihydro-<NUM>,<NUM>'-bipyridine-<NUM>-carbonitrile) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Papaverine" (IUPAC Name <NUM>-(<NUM>,<NUM>-dimethoxybenzyl)-<NUM>,<NUM>-dimethoxyisoquinoline) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Siguazodan" (IUPAC Name (E)-<NUM>-cyano-<NUM>-methyl-<NUM>-(<NUM>-(<NUM>-methyl-<NUM>-oxo-<NUM>,<NUM>,<NUM>,<NUM>-tetrahydropyridazin-<NUM>-yl)phenyl)guanidine) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Sildenafil" (IUPAC Name <NUM>-[<NUM>-ethoxy-<NUM>-(<NUM>,<NUM>-dihydro-<NUM>-methyl-<NUM>-oxo-<NUM>-propyl-<NUM>H-pyrazolo[<NUM>,<NUM>-d]pyrimidin-<NUM>-yl)phenylsulfonyl]-<NUM>-methylpiperazine) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Trequinsin" (IUPAC Name <NUM>,<NUM>-dimethoxy-<NUM>-methyl-<NUM>-(<NUM>,<NUM>,<NUM>-trimethylphenyl)imino-<NUM>,<NUM>-dihydropyrimido[<NUM>,<NUM>-a]isoquinolin-<NUM>-one) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Vardenafil" (IUPAC Name <NUM>-[<NUM>-ethoxy-<NUM>-(<NUM>-ethylpiperazin-<NUM>-yl)sulfonyl-phenyl]-<NUM>-methyl-<NUM>-propyl-<NUM>,<NUM>,<NUM>,<NUM>-tetrazabicyclo[<NUM>. <NUM>]nona-<NUM>,<NUM>,<NUM>-trien-<NUM>-one) is meant a small molecule inhibitor having the following structure:
<CHM>.

By "Zardaverine (IUPAC Name <NUM>-[<NUM>-(Difluoromethoxy)-<NUM>-methoxyphenyl]-<NUM>H-pyridazin-<NUM>-one)" is meant a small molecule inhibitor having the following structure:
<CHM>.

In some other embodiments, any one of the compounds Cilostamide, Cilostazol, DNDMP, Levosimendan, Milrinone, Papaverine, Siguazodan, Sildenafil, Trequinsin, Vardenifil, and Zardaverine is a small molecule phosphodiesterase inhibitor. In another embodiment, forskolin may be used in a method of the invention.

By "PDE3A polypeptide" is meant a protein or fragment thereof having at least <NUM>% amino acid sequence identity to the sequence provided at NCBI Ref No. NP_000912. <NUM> that catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). An exemplary human full-length PDE3A amino acid sequence is provided below:
<IMG>.

Three PDE3A isoforms are known: PDE3A1, PDE3A2, and PDE3A3. PDE3A1 comprises amino acids <NUM>-<NUM>, PDE3A2 isoform <NUM> comprises amino acids <NUM>-<NUM>, and PDE3A3 comprises amino acids <NUM>-<NUM> of the full-length PDE3A amino acid sequence.

By "PDE3A polynucleotide" is meant any nucleic acid molecule, including DNA and RNA, encoding a PDE3A polypeptide or fragment thereof. An exemplary PDE3A nucleic acid sequence is provided at NCBI Ref: NM_000921. <NUM>:
<IMG>
<IMG>
<IMG>.

By "Schlafen <NUM> (SLFN12) polypeptide" is meant a protein or fragment thereof having at least <NUM>% amino acid sequence identity to the sequence provided at NCBI Ref No. NP_060512. <NUM> that interacts with PDE3A when bound to anagrelide, zardaverine or DNMDP and related compounds. An exemplary human SLFN12 amino acid sequence is provided below:
<IMG>
<IMG>.

By "Schlafen <NUM> (SLFN12) polynucleotide" is meant any nucleic acid molecule, including DNA and RNA, encoding a SLFN12 polypeptide or fragment thereof. An exemplary SLFN12 nucleic acid sequence is provided at NCBI Ref: NM_018042. <NUM>:
<IMG>
<IMG>.

"Isomers" are different compounds that have the same molecular formula. "Stereoisomers" are isomers that differ only in the way the atoms are arranged in space. As used herein, the term "isomer" includes any and all geometric isomers and stereoisomers. For example, "isomers" include geometric double bond cis- and trans-isomers, also termed E- and Z-isomers; R- and S-enantiomers; diastereomers, (d)-isomers and (l)-isomers, racemic mixtures thereof and other mixtures thereof.

Geometric isomers can be represented by the symbol - - - - - which denotes a bond that can be a single, double or triple bond as described herein. Provided herein are various geometric isomers and mixtures thereof resulting from the arrangement of substituents around a carbon-carbon double bond or arrangement of substituents around a carbocyclic ring. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "E" configuration wherein the terms "Z" and "E" are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the "E" and "Z" isomers.

Substituents around a carbon-carbon double bond alternatively can be referred to as "cis" or "trans," where "cis" represents substituents on the same side of the double bond and "trans" represents substituents on opposite sides of the double bond. The arrangement of substituents around a carbocyclic ring can also be designated as "cis" or "trans. " The term "cis" represents substituents on the same side of the plane of the ring, and the term "trans" represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated "cis/trans.

The term "enantiomers" refers to a pair of stereoisomers that are non-superimposable mirror images of each other. An atom having an asymmetric set of substituents can give rise to an enantiomer. A mixture of a pair of enantiomers in any proportion can be known as a "racemic" mixture. The term "(±)" is used to designate a racemic mixture where appropriate. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn-Ingold-Prelog R-S system. When a compound is an enantiomer, the stereochemistry at each chiral carbon can be specified by either R or S. Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry at each asymmetric atom, as (R)- or (S)-. The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically substantially pure forms and intermediate mixtures.

Optically active (R)- and (S)-isomers can be prepared, for example, using chiral synthons or chiral reagents, or resolved using conventional techniques. Enantiomers can be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC), the formation and crystallization of chiral salts, or prepared by asymmetric syntheses.

Optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, e.g., by formation of diastereoisomeric salts, by treatment with an optically active acid or base. Examples of appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid. The separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts affords separation of the isomers. Another method involves synthesis of covalent diastereoisomeric molecules by reacting disclosed compounds with an optically pure acid in an activated form or an optically pure isocyanate. The synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically enriched compound. Optically active compounds can also be obtained by using active starting materials. In some embodiments, these isomers can be in the form of a free acid, a free base, an ester or a salt.

In certain embodiments, the compound disclosed herein can be a tautomer. As used herein, the term "tautomer" is a type of isomer that includes two or more interconvertible compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa). "Tautomerization" includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. "Prototropic tautomerization" or "proton-shift tautomerization" involves the migration of a proton accompanied by changes in bond order. The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Where tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. Tautomerizations (i.e., the reaction providing a tautomeric pair) can be catalyzed by acid or base, or can occur without the action or presence of an external agent. Exemplary tautomerizations include, but are not limited to, keto-to-enol; amide-to-imide; lactam-to-lactim; enamine-to-imine; and enamine-to-(a different) enamine tautomerizations. A specific example of keto-enol tautomerization is the interconversion of pentane-<NUM>,<NUM>-dione and <NUM>-hydroxypent-<NUM>-en-<NUM>-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-<NUM>-ol and pyridin-<NUM>(<NUM>)-one tautomers.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes an about <NUM>% change in expression levels, preferably an about <NUM>% change, more preferably an about40% change, and most preferably an about <NUM>% or greater change in expression levels.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U. Patent law and can mean " includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected. In particular embodiments, the analyte is a PDE3A or SLFN12 polypeptide.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include melanoma, adenocarcinoma, lung cancer, cervical cancer, liver cancer and breast cancer.

By "effective amount" is meant the amount of a compound described herein required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. In one embodiment, the compound is DNMDP, zardaverine, or anagrelide.

The disclosure provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, or about <NUM>% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, about <NUM>, or about <NUM> nucleotides or amino acids.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "marker" or "biomarker" is meant any protein or polynucleotide having an alteration in expression level or activity (e.g., at the protein or mRNA level) that is associated with a disease or disorder. In particular embodiments, a marker of the invention is PDE3A or SLFN12.

By "modulator" is meant any agent that binds to a polypeptide and alters a biological function or activity of the polypeptide. A modulator includes, without limitation, agents that reduce or eliminate a biological function or activity of a polypeptide (e.g., an "inhibitor"). For example, a modulator may inhibit a catalytic activity of a polypeptide. A modulator includes, without limitation, agents that increase or decrease binding of a polypeptide to another agent. For example, a modulator may promote binding of a polypeptide to another polypeptide. In some embodiments, a modulator of PDE3A polypeptide is DNMDP. In some other embodiments, the modulator of PDE3A polypeptide is anagrelide or zardaverine.

By "reference" is meant a standard or control condition.

Nucleic acid molecules useful in the methods of the disclosure include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be <NUM>% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be <NUM>% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., <NPL>; <NPL>).

For example, stringent salt concentration will ordinarily be less than about <NUM> NaCl and <NUM> trisodium citrate, preferably less than about <NUM> NaCl and <NUM> trisodium citrate, and more preferably less than about <NUM> NaCl and <NUM> trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about <NUM>% formamide, and more preferably at least about <NUM>% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about <NUM>° C, more preferably of at least about <NUM>° C, and most preferably of at least about <NUM>° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at <NUM>° C in <NUM> NaCl, <NUM> trisodium citrate, and <NUM>% SDS. In a more preferred embodiment, hybridization will occur at <NUM>° C in <NUM> NaCl, <NUM> trisodium citrate, <NUM>% SDS, <NUM>% formamide, and <NUM>µg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at <NUM>° C in <NUM> NaCl, <NUM> trisodium citrate, <NUM>% SDS, <NUM>% formamide, and <NUM>µg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about <NUM> NaCl and <NUM> trisodium citrate, and most preferably less than about <NUM> NaCl and <NUM> trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about <NUM>° C, more preferably of at least about <NUM>° C, and even more preferably of at least about <NUM>° C. In a preferred embodiment, wash steps will occur at <NUM>° C in <NUM> NaCl, <NUM> trisodium citrate, and <NUM>% SDS. In a more preferred embodiment, wash steps will occur at <NUM> C in <NUM> mMNaCl, <NUM> trisodium citrate, and <NUM>% SDS. In a more preferred embodiment, wash steps will occur at <NUM>° C in <NUM> NaCl, <NUM> trisodium citrate, and <NUM>% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in <NPL>); <NPL>); <NPL>); <NPL>); and <NPL>.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least <NUM>% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least <NUM>%, more preferably <NUM>% or <NUM>%, and more preferably <NUM>%, <NUM>% or even <NUM>% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, <NUM> University Avenue, Madison, Wis. <NUM>, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-<NUM> and e-<NUM> indicating a closely related sequence.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of <NUM> to <NUM> is understood to include any number, combination of numbers, or sub-range from the group consisting <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within <NUM> standard deviations of the mean. About can be understood as within <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

As described below, the present invention features improved methods of identifying patients having cancer (e.g., melanoma, endometrium, lung, hematopoetic / lymphoid, ovarian, cervical, soft-tissue sarcoma, leiomyosarcoma, urinary tract, pancreas, thyroid, kidney, glioblastoma, or breast cancer)) that is sensitive to treatment with a phosphodiesterase 3A (PDE3A) modulator by detecting co-expression of PDE3A and Schlafen <NUM> (SLFN12) polypeptides or polynucleotides in a cancer cell derived from such patients. The invention is based at least in part on the discovery that sensitivity to phosphodiesterase 3A modulators, such as <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one, or DNMDP, in <NUM> cancer cell lines correlated with expression of the phosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of PDE3A inhibitors kill selected cancer cells while others do not; these cell-sparing PDE3A inhibitors instead block DNMDP induced cytotoxicity. Furthermore, PDE3A depletion leads to DNMDP resistance. DNMDP binding to PDE3A promotes an interaction between PDE3A and Sirtuin <NUM> (SIRT7) and Schlafen <NUM> (SLFN12), suggesting a neomorphic activity, and SLFN12 and PDE3A co-expression correlated with DNMDP sensitivity. These results indicate that PDE3A modulators are promising cancer therapeutic agents and demonstrate the power of chemogenomics in small-molecule discovery and target-identification.

Accordingly, the invention provides methods of selecting a subject as having a cancer that responds to a PDE3A modulator, where the selection method involves detecting co-expression of PDE3A and Schlafen <NUM> (SLFN12) polypeptides or polynucleotides, in a cancer cell derived from such subjects.

The identification of PDE3A modulators was made in connection with a phenotypic screen designed to identify cytotoxic small molecules in a mutant tp53 background. A chemogenomics approach complements target-driven drug development programs, which consists of extensive in vitro and in vivo target validation, and can also be referred to as reverse chemogenomics (<NPL>). Food and Drug Administration (FDA)-approved targeted therapies have been developed this way, among them small-molecule kinase inhibitors that target oncogenic somatic driver mutations (<NPL>). However, the discovery and development of targeted therapies is often hampered by limitations in knowledge of the biological function of the target, its mechanism of action, and the available chemical matter to selectively inhibit the target.

Phenotypic screening can discover novel targets for cancer therapy whose specific molecular mechanism is often elucidated by future studies (<NPL>). In recent years, two classes of anti-cancer drugs found by unbiased phenotypic screening efforts have been approved by the FDA. Lenalidomide and pomalidomide were found to be modulators of an E3-ligase that alter the affinity of its target, leading to degradation of lineage specific transcription factors (<NPL>; <NPL>), whereas romidepsin and vorinostat were later identified as histone deacetylase (HDAC) inhibitors (<NPL>; <NPL>, <NPL>).

Tumor suppressor alterations are suitable targets for phenotypic screening as they are not directly targetable with small molecules, although synthetic lethal approaches such as olaparib treatment of BRCA1/BRCA2 mutant cancers have proven to be effective. According to current knowledge, the tp53 tumor suppressor gene is the most frequently mutated across human cancer, with somatic mutations detected in <NUM>% of <NUM> cancers subjected to whole exome sequencing. Despite many attempts, no compounds that selectively kill tp53 mutant cells have been identified.

A phenotypic screen developed to identify small molecules causing synthetic lethality in tp53 mutant cancer cells enabled the serendipitous discovery of a class of cancer-selective cytotoxic agents which act as modulators of phosphodiesterase 3A (PDE3A), as described herein below. Cyclic nucleotide phosphodiesterases catalyze the hydrolysis of second messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), and are important in many physiological processes. Several phosphodiesterase inhibitors have been approved for clinical treatment, including PDE3 inhibitors milrinone, cilostazol, and levosimendan for cardiovascular indications and inhibition of platelet coagulation, as well as the PDE3 inhibitor anagrelide for thrombocythemia. PDE5 inhibitors, e.g. vardenafil, are used for smooth muscle disorders including erectile dysfunction and pulmonary arterial hypertension, and the PDE4 inhibitor roflumilast reduces exacerbations from chronic obstructive pulmonary disease (COPD).

Phosphodiesterase inhibitors act by direct inhibition of their targets or by allosteric modulation; for example, structural analysis of PDE4 has led to the design of PDE4D and PDE4B allosteric modulators (<NPL>; <NPL>). The data provided herein below indicates that the cancer cytotoxic phosphodiesterase modulator DNMDP likely acts through a similar allosteric mechanism.

Accordingly, the invention provides methods for identifying subjects that have a malignancy that is likely to respond to PDE3A modulator treatment based on the level of PDE3A and SLFN12 expression in a subject biological sample comprising a cancer cell. In some embodiments, the PDE3A modulator is DNMDP. In some other embodiments, the PDE3A modulator is anagrelide or zardaverine.

The compounds of the present invention include the compounds themselves, as well as their salts, if applicable. A salt, for example, can be formed between an anion and a positively charged substituent (e.g., amino) on a compound described herein. Suitable anions include chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt can also be formed between a cation and a negatively charged substituent (e.g., carboxylate) on a compound described herein. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion. Examples of prodrugs include C<NUM>-<NUM> alkyl esters of carboxylic acid groups, which, upon administration to a subject, are capable of providing active compounds.

Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from pharmaceutically acceptable inorganic and organic acids and bases. As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by the addition of a pharmaceutically acceptable acid or base to a compound disclosed herein. As used herein, the phrase "pharmaceutically acceptable" refers to a substance that is acceptable for use in pharmaceutical applications from a toxicological perspective and does not adversely interact with the active ingredient.

Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, <NUM>-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, <NUM>-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, <NUM>-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)<NUM>+ salts. The present invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. Salt forms of the compounds of any of the formulae herein can be amino acid salts of carboxyl groups (e.g., L-arginine, -lysine, -histidine salts).

Lists of suitable salts are found in <NPL>; <NPL>); and "<NPL>] each of which is incorporated herein by reference in their entireties.

The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.

The present invention also includes various hydrate and solvate forms of the compounds.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (<NUM>H), iodine-<NUM> (<NUM>I) or carbon-<NUM> (<NUM>C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The present invention features diagnostic assays for the characterization of cancer. In one embodiment, levels of PDE3A and/or Schlafen <NUM> (SLFN12) polynucleotides or polypeptides are measured in a subject sample and used as an indicator of cancer that is responsive to treatment with a PDE3A modulator. Levels of PDE3A and/or Schlafen <NUM> polynucleotides may be measured by standard methods, such as quantitative PCR, Northern Blot, microarray, mass spectrometry, and in situ hybridization. Standard methods may be used to measure levels of PDE3A and/or Schlafen <NUM>, polypeptides in a biological sample derived from a tumor. Such methods include immunoassay, ELISA, western blotting using an antibody that binds PDE3A and/or Schlafen <NUM> and radioimmunoassay. Elevated levels of PDE3A and Schlafen <NUM> polynucleotides or polypeptides relative to a reference are considered a positive indicator of cancer that is responsive to treatment with a PDE3A modulator.

In characterizing the responsiveness of a malignancy in a subject to PDE3A modulator treatment, the level of PDE3A and/or SLFN12 expression is measured in different types of biologic samples. In one embodiment, the biologic sample is a tumor sample.

PDE3A and/or SLFN12 expression is higher in a sample obtained from a subject that is responsive to PDE3A modulator treatment than the level of expression in a non-responsive subject. In another embodiment, PDE3A and/or SLFN12 is at least about <NUM>, <NUM>, <NUM>, or <NUM>-fold higher in a subject with a malignancy than in a healthy control. Fold change values are determined using any method known in the art. In one embodiment, change is determined by calculating the difference in expression of PDE3A and/or SLFN12 in a cancer cell vs the level present in a non-responsive cancer cell or the level present in a corresponding healthy control cell.

As reported herein below, subjects suffering from a malignancy may be tested for PDE3A and SLFN12 expression in the course of selecting a treatment method. Patients characterized as having increased PDE3A and SLFN12 relative to a reference level are identified as responsive to PDE3A modulator treatment.

The invention provides kits for characterizing the responsiveness or resistance of a subject to PDE3A modulator treatment.

Also provided herein are kits that can include a therapeutic composition containing an effective amount of a PDE3A modulator in, e.g., unit dosage form.

In one embodiment, a diagnostic kit of the invention provides a reagent for measuring relative expression of PDE3A and SLFN12. Such reagents include capture molecules (e.g., antibodies that recognize PDE3A and SLFN12 polypeptides or nucleic acid probes that hybridize with PDE3A and SLFN12 polynucleotides).

In some embodiments, the kit comprises a sterile container which includes a therapeutic or diagnostic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

In one embodiment, a kit of the invention comprises reagents for measuring PDE3A and/or SLFN12 levels. If desired, the kit further comprises instructions for measuring PDE3A and/or SLFN12 and/or instructions for administering the PDE3A modulator to a subject having a malignancy, e.g., a malignancy selected as responsive to PDE3A modulator treatment. In particular embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of malignancy or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "<NPL>); "<NPL>); "<NPL>); "<NPL>); "<NPL>); "<NPL>); "<NPL>); "<NPL>). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention.

To identify anti-cancer compounds with cell-selective cytotoxic activity, an unbiased chemical screen was performed in two lung adenocarcinoma cell lines, A549 and NCI-H1734, both of which harbor oncogenic KRAS mutations and truncating STK11 mutations, and which were TP53 wild type and mutant (R273L), respectively. <NUM>,<NUM> compounds were screened from the Molecular Libraries Small-Molecule Repository validation set in the A549 and NCI-H1734 cell lines at a single concentration of <NUM> in <NUM>-well format in duplicate. As a proxy for cellular viability, ATP content was measured after <NUM> hours of compound treatment.

Three compounds showed a selective reduction in cell viability for the NCI-H1734 cell line compared to the A549 cell line, with an approximately <NUM>% reduction in the NCI-H1734 cell line, which is > <NUM> median absolute deviations from the median in the negative direction, compared to a minimal change of < <NUM> median absolute deviations from the median in the A549 cell line (<FIG>). Retesting the three compounds in a dose-response analysis validated that one compound, <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>H)-one, or DNMDP, was specifically toxic to the NCI-H1734 cell line (<FIG>).

Testing of additional cell lines with DNMDP showed clear cell-selective cytotoxicity, with an EC<NUM> between <NUM> and <NUM> for two additional lung adenocarcinoma cell lines, NCI-H1563 and NCI-H2122, and for HeLa cervical carcinoma cells, but an EC<NUM> greater than <NUM> for A549, MCF7, and PC3 cells (<FIG>). Caspase activity was detected by a caspase-sensitive luciferase assay and by poly ADP ribose polymerase (PARP) cleavage in HeLa cells upon DNMDP treatment, indicating that sensitive cells undergo apoptosis after DNMDP exposure (<FIG>). To characterize cellular sensitivity to DNMDP further, <NUM> genomically characterized cancer cell lines were screened for DMNDP sensitivity at concentrations ranging from <NUM> to <NUM> in <NUM>-fold dilution steps for <NUM> hours. From these cell lines, <NUM> cell lines were categorized as sensitive with a robust Z-score lower than -<NUM>, which represented multiple lineages including multiple melanoma cell lines, amongst others (Table <NUM>).

Next, the DNMDP enantiomers were separated by chiral super-critical fluid (SCF) chromatography. One enantiomer was <NUM>-fold more potent in HeLa cells than the other (<FIG> and <FIG>). The (R)-enantiomer was synthesized from commercially available starting materials (<FIG>). This synthesized enantiomer had similar activity to the more potent separated material and was identical by chiral SCF chromatography, confirming stereochemistry of the active enantiomer (<FIG>). Two (R)-des-nitro analogues of DNMDP were synthesized, both of which tested similarly to (R)-DNMDP (<FIG>). <FIG> show super-critical fluid (SCF) chromatographs of <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (DNMDP) (top to bottom: ES+, diode array, ES- traces). <FIG> shows Peak <NUM> (CRO separation); <FIG> shows Peak <NUM> (CRO separation); and <FIG> shows synthesized (R)-DNMDP (<NUM>:<NUM> ratio peaks <NUM>:<NUM> by uv).

Given the potent cell-selective growth inhibition by <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (DNMDP), its mechanism of action was examined in more detail. To determine the molecular target of DNMDP, chemogenomic analysis was performed of the <NUM> tested cell lines, previously characterized for mutations, copy number, and gene expression features as part of the <NPL>), to look for correlation between these genomic features and DNMDP sensitivity. Analysis of Pearson correlations between DNMDP sensitivity and expression of individual genes across the cell line set showed a strong correlation with expression of the PDE3A gene, encoding phosphodiesterase 3A (<FIG>). The correlation between DNMDP sensitivity and PDE3A expression is not perfect (<FIG>), and it is possible that some errors are introduced due to the high-throughput nature of the cell line sensitivity characterization, as manual validation for all <NUM> cell lines was not logistically feasible. Mutation and copy number features, in contrast, did not correlate with DNMDP sensitivity. Conversely, of <NUM> compounds tested, DNMDP sensitivity was the closest correlate of PDE3A expression (<FIG>), indicating that cancer cell lines with high PDE3A expression were more distinctly sensitive to DNMDP than to any other tested compound. In contrast to the motivation of the initial screen, there was no correlation between TP53 mutation, or other measures of p53 function, and DNMDP sensitivity.

Given these results and the clear structural similarity of DNMDP to known PDE3 inhibitors, e.g., levosimendan and siguazodan (<FIG>), biochemical analysis of DNMDP against <NUM> phosphodiesterases representing <NUM> PDE super families was performed. At a concentration of <NUM>, DNMDP specifically inhibited both PDE3A and PDE3B, weakly inhibited PDE10, and had little or no detectable effect on other phosphodiesterases (Table <NUM>).

Because of the cellular correlation between PDE3A expression and DNMDP sensitivity, the in vitro inhibition of PDE3A and PDE3B by DNMDP, and the structural similarity of DNMDP to known PDE3 inhibitors, it was analyzed whether all PDE3 inhibitors would exhibit a similar cytotoxic profile to DNMDP. Surprisingly, there was almost no correlation between IC<NUM> for in vitro enzymatic PDE3A inhibition and HeLa cell cytotoxicity across a series of tested compounds (<FIG> and <FIG>). Indeed, the potent PDE3 inhibitor trequinsin (PDE3 IC<NUM> = <NUM>, <NPL>) did not affect HeLa cell viability in any detectable way. Despite their differential effects on HeLa cell viability, the non-cytotoxic PDE3 inhibitor trequinsin and the potent cytotoxic compound DNMDP had similar effects on intracellular cAMP levels in forskolin-treated HeLa cells (<FIG>). This result indicates that inhibition of the cAMP and cGMP hydrolysis functions of PDE3A was not sufficient for the cytotoxic activity of DNMDP.

The complex relationship between phosphodiesterase 3A (PDE3A) inhibition and cell killing, in which <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (DNMDP) and some PDE3 inhibitors kill HeLa and other DNMDP-sensitive cells, whereas others PDE3 inhibitors do not affect cell viability, indicated several possible interpretations including: <NUM>) the cytotoxic activity might be PDE3-independent and due to action on a different protein though screening <NUM> kinases found no kinase inhibition by <NUM> DNMDP; <NUM>) cytotoxic and non-cytotoxic PDE3 inhibitors might bind to different sites within the protein and exert distinct activities; or <NUM>) the cytotoxic and non-cytotoxic PDE3 inhibitors might bind to the PDE3 active sites but have different effects on the conformation and activity of the protein. This third possibility might be unexpected, but allosteric modulators of PDE4 have been shown to bind the PDE4 active site and interact with upstream (UCR2), and downstream (CR3) regulatory domains and thereby stabilize specific inactive conformations (<NPL>). Most importantly, PDE4 competitive inhibitors and PDE4 allosteric modulators with similar IC<NUM>s for cAMP hydrolysis in vitro had different cellular activities and safety profiles in animal studies (<NPL>). To evaluate whether PDE inhibitors or other small molecules compete with DNMDP, the PHARMAKON <NUM> collection of <NUM> bioactive compounds (PHARMAKON <NUM> is a unique collection of <NUM> known drugs from US and International Pharmacopeia) was screened to identify compounds that were able to rescue cell death induced by DNMDP. HeLa cells were co-treated with <NUM> DNMDP (the EC<NUM> concentration) and <NUM> of each bioactive compound. Cell viability after <NUM>-hour treatment was assessed by ATP consumption as described earlier. The five most potent compounds that rescued cell death induced by DNMDP were all PDE inhibitors, and the three most potent compounds, levosimendan, milrinone, and cilostazol, were all selective PDE3 inhibitors (<FIG>).

In follow-up experiments, it was confirmed that cilostamide, levosimendan, milrinone, and several other non-cytotoxic selective PDE3 inhibitors were able to rescue DNMDP cytotoxicity in a dose-dependent manner (<FIG>). The most potent DNMDP competitor was trequinsin, with an "RC<NUM>" (the concentration at which it achieved <NUM>% rescue) of < <NUM>; in contrast, PDE5 inhibitors such as sildenafil and vardenafil, as well as the pan-PDE inhibitors idubulast and dipyridamole, were not effective competitors up to <NUM> concentrations in this assay (<FIG>). This indicated that non-cytotoxic PDE3 inhibitors and DNMDP compete for binding to the same molecular target that is mediating the cytotoxic phenotype.

To identify the molecular target of DNMDP, an affinity purification was performed using an (R)-des-nitro-DNMDP solid-phase tethered linker analogue (<FIG>) incubated with HeLa cell lysate. This linker analogue had the same DNMDP cytotoxicity rescue phenotype as non-cytotoxic PDE3 inhibitors described above (<FIG>), indicating that it too bound to the same molecular target. It was competed for the molecular target by adding either an excess of trequinsin or separate enantiomers of DNMDP, where only the (R)-enantiomer was cytotoxic. Immunoblotting for PDE3A of the affinity purified material showed that PDE3A indeed binds to the linker analogue. Binding of PDE3A to the linker analogue was blocked by both trequinsin and (R)-DNMDP, but not by the non-cytotoxic enantiomer (S)-DNMDP (<FIG>). Thus both trequinsin and (R)-DNMDP prevented the binding of PDE3A to the tethered DNMDP analogue, and it was concluded that both molecules bind PDE3A directly.

Based on the observations that DNMDP-sensitive cells expressed high levels of PDE3A, and that DNMDP competed with non-cytotoxic inhibitors for PDE3A binding, it was hypothesized that DNMDP mediated its cytotoxic phenotype through the interaction with PDE3A and that PDE3A abundance was a direct cellular determinant of DNMDP sensitivity. To validate this hypothesis, the effect of reducing levels of PDE3A on the response to DNMDP was tested. A clustered regularly interspaced short palindromic (CRISPR)-associated CAS9 enzyme that was targeted with three guide RNAs (sgRNA) targeting three different sites in the PDE3A locus led to complete loss of PDE3A expression (<NPL>) sgRNA2 and sgRNA3 almost completely reduced PDE3A protein levels, whereas sgRNA1 had a moderate effect on PDE3A expression (<FIG>). Importantly, both sgRNA2 and sgRNA3 led to significant rescue of toxicity by an active cytotoxic DNMDP analog, <NUM> (<FIG> and <FIG> and <FIG>). Both sgRNA2 and sgRNA3 led to significant rescue of toxicity by DNMDP (<FIG>). Changes in proliferation rate or morphology in HeLa cells with reduced PDE3A expression were not observed, indicating that PDE3A was not required for cell survival. In an independent approach using an siRNA smart-pool containing four different siRNAs targeting PDE3A, PDE3A expression was reduced in HeLa cell line with a maximum efficiency of <NUM>% between <NUM> and <NUM> hours after transfection. HeLa cells treated with siPDE3A had a higher EC<NUM> to a DNMDP analog compared to the control siRNA condition (<FIG>). Without being bound by theory it was concluded that DNMDP cytotoxicity requires PDE3A, and that DNMDP likely modulates the function of PDE3A.

The dependence of <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (DNMDP) cytotoxicity on phosphodiesterase 3A (PDE3A) protein abundance indicated a possible mechanism similar to that recently observed for lenalidomide, which acts by a neomorphic or hypermorphic mechanism by stabilizing an interaction between cereblon and IKAROS Family Zinc Finger <NUM> (IKZF1) and IKZF3 (<NPL>; <NPL>). In addition, PDE4 allosteric modulators, but not competitive inhibitors, have been shown to bind and stabilize a "closed" protein conformation that has independently been shown to uniquely bind the PDE4-partner protein DISC1 (<NPL>). The protein complexes in which PDE3A resides were characterized under normal conditions, and it was examined how these complexes change when PDE3A is bound to DNMDP or the non-cytotoxic PDE3 inhibitor trequinsin. PDE3A and interacting proteins from Hela cells were immunoprecipitated in the presence of DNMDP and trequinsin followed by labeling with isobaric stable isotope tags for relative abundance and quantitation by mass spectrometry (iTRAQ/MS, <FIG>). PDE3A immunoprecipitates from HeLa cells were enriched for multiple protein phosphatase subunits including protein phosphatase <NUM> subunits (PPP2CA, PPP2R1A, PPP2R1B, PPP2R2A, PPP2R2D), calcineurin (PPP3R1, PPP3CA, <NPL>), <NUM>-<NUM>-<NUM> (YWHAB, YWHAQ, YWHAG, YWHAZ, <NPL>), and tubulin (TUBA1C, TUBA1B) family members (<FIG> and <FIG>). In addition, it was found that PDE3A and PDE3B reside in the same protein complex, which has been previously reported (<NPL>).

Binding of DNMDP altered the composition of interacting proteins that were co-immunoprecipitated with PDE3A. Proteins that were specifically enriched in PDE3A immunoprecipitates after treatment with DNMDP included Sirtuin <NUM> (SIRT7) and Schlafen <NUM> (SLFN12) (<FIG> and <FIG>). These proteins specifically interacted with PDE3A in the presence of DNMDP, and were not observed in the trequinsin treated control, whereas a known PDE3B interactor, abhydrolase domain-containing protein <NUM> (ABHD15, <NPL>), was enriched in the immunoprecipitate from trequinsin-treated cells (<FIG> and <FIG>). The interaction promoted by DNMDP between PDE3A and both SIRT7 and SLFN12 was validated with affinity reagents. Immunoprecipitation of endogenous PDE3A in HeLa cells treated with DNMDP, but not DMSO or trequinsin, enhanced complex formation of ectopically expressed V5-tagged SIRT7 and SLFN12 with PDE3A, as evidenced by coimmunoprecipitation (<FIG> further shows that DNMDP and (weakly) anagrelide, but not trequinsin, induced PDE3A and SFLN12 complex formation.

Similar to PDE3A, overexpression of SLFN12 appears to have a cytotoxic effect in DNMDP sensitive cell lines, contributing to the difficulty of detecting SLFN12 in whole cell lysates.

The enhanced interaction of PDE3A with SIRT7 and SLFN12 indicated the possibility that one or more of these interacting proteins might contribute to DNMDP sensitivity. SIRT7 mRNA expression was relatively constant among all cells tested, but the co-expression of SLFN12 and PDE3A mRNA showed a strong correlation with DNMDP sensitivity; almost all DNMDP-sensitive cell lines expressed high levels of SLFN12 (<FIG>). Importantly, almost half of sensitive cell lines expressing high levels of SLFN12 and PDE3A were found to be melanoma cell lines (<FIG>). SLFN12 expression alone was also one of the top genes correlating with sensitivity to DNMDP, corroborating the hypothesis that SLFN12 could be functionally involved in DNMDP-induced cytotoxicity (<FIG>). Moreover, when correcting for PDE3A expression, SLFN12 expression was the top correlating gene with DNMDP sensitivity (<FIG>). To assess whether SLFN12 is required for the cytotoxic phenotype of DMNDP, we reduced SLFN12 mRNA expression by <NUM>% by knockdown with two shRNAs in HeLa cells (<FIG>). Similar to reduction in PDE3A expression, reduction of SLFN12 expression did not result in cytotoxicity, and in fact decreased sensitivity to DNMDP (<FIG>). These results show that SLFN12, like PDE3A, is required for the cytotoxic phenotype of DMNDP. Characterization of normal expression of SLFN12 and PDE3A by the GTEX consortium (<NPL>)) shows low expression of SLFN12 in normal tissues, while high co-expression of both PDE3A and SLFN12 is rarely observed (Table <NUM>). This could suggest that on-target toxicity of DNMDP and related compounds may be potentially limited.

<FIG> shows that SLFN12 is lost in cells that have acquired resistance to DNMDP. Cell lines initially sensitive to DNMDP were made resistant by persistent exposure to DNMDP and subsequently analyzed by RNA-seq. One gene was downregulated in both HeLa and H2122: SLFN <NUM>. Accordingly, a reduction in levels of SLFN <NUM> indicates that cells have become resistant to DNMDP and other PDE3A modulators.

<FIG> shows sensitization of a DNMDP-resistant cell line by expression of SLFN12 or expression of SFLN12 and PDE3A. Expression of SLFN12 was sufficient to confer DNMDP sensitivity to A549 cells. Adding PDE3A expression led to further sensitization.

Leiomyosarcomas are malignant smooth muscle tumors. Patient tumor samples from leiomyosarcomas were analyzed for PDE3A and SLFN12 expression to characterize sensitivity of leiomyosarcomas (LMS) to DNMDP. Leiomyosarcomas are thought to be sensitive to DNMDP due to prevalence among high purity TCGA samples expressing elevated levels of PDE3A and SLFN12 (<FIG>, Table <NUM>). P value for association of biomarker expression with leiomyosarcoma lineage: <NUM>.

Differential scanning fluorimetry (DSF) was used to demonstrate binding of DNMDP to purified PDE3A catalytic domain, PDE3A(<NUM>-<NUM>). In this experiment, <NUM> hsPDE3A(<NUM>-<NUM>) was incubated in the absence or presence of <NUM> compounds, as indicated in Table <NUM>. Binding buffer: <NUM> Hepes pH <NUM>, <NUM> TCEP, <NUM> MgCl<NUM>, <NUM> NaCl.

Using chemogenomics, a class of compounds was discovered, exemplified by DNMDP, that targeted a novel cancer dependency by small-molecule modulation of PDE3A. These compounds bound PDE3A in a mutually exclusive manner with non-cytotoxic PDE3 inhibitors and exerted a neomorphic or hypermorphic effect on the function of PDE3A, leading to a change in its protein-protein interactions. One unique protein-interaction partner, SLFN12, was highly expressed in DNMDP-sensitive cell lines, indicating a functional role in the pathway through which the cytotoxic signal was relayed. As a result, DNMDP was both selective and potent across a large panel of cancer cell lines.

Here, a novel cytotoxic compound was identified with great selectivity and low-nM potency against cancer cell lines across multiple lineages. Using gene-expression correlates for chemogenomics, PDE3A was identified as the putative target of this small molecule, DNMDP. Interestingly, loss of PDE3A expression resulted in resistance to DNMDP. Moreover, PDE3A immunoprecipitation followed by isobaric stable isotope tags for relative abundance and quantitation by mass spectrometry (iTRAQ/MS) identified SLFN12 and SIRT7 as novel protein-protein interaction partners of PDE3A upon DNMDP binding, possibly due to allosteric modulation of the function of PDE3A. Importantly, SLFN12 expression was the top correlating gene with DNMDP sensitivity when corrected for PDE3A expression. Single gene or multi-gene expression correlations have shown to help elucidate the mechanism of action and relevant signaling pathways of small molecules. A novel biochemical target for cancer treatment was identified that is unlikely to have been found by target identification approaches such as loss-of-function screens or genomic analysis.

PDE3A belongs to the superfamily of phosphodiesterases and together with PDE3B forms the PDE3 family. The PDE3 family has dual substrate affinity and hydrolyses both cAMP and cGMP. Expression of PDE3A is highest in the cardiovascular system, platelets, kidney, and oocytes (<NPL>). The clinical PDE3 inhibitor cilostazol has been developed to treat intermittent claudication, as PDE3A inhibition in platelets impairs activation and platelet coagulation (<NPL>). Other PDE3 inhibitors, such as milrinone, amrinone, and levosimendan, are indicated to treat congestive heart failure, where the combination of vasodilation and elevated cardiac cAMP levels increases cardiac contractility (<NPL>). None of these clinical inhibitors were able to replicate the cytotoxic phenotype of DNMDP, indicating that cyclic nucleotide hydrolysis was not sufficient to induce cell death in DNMDP-sensitive cell lines.

Interestingly however, other PDE3 inhibitors such as zardaverine, anagrelide, and quazinone have been reported previously to have cell cytotoxic characteristics in a select number of cancer cell lines (<NPL>; <NPL>). In concordance with the present findings, other PDE3 and PDE4 inhibitors were found not to replicate the cytotoxic phenotype of zardaverine where retinoblastoma protein retinoblastoma <NUM> (RB1) expression was reported to separate zardaverine sensitive cell lines from non-sensitive cell lines (<NPL>). This finding was in contrast to the present data where a correlation between cytotoxic activities of DNMDP and copy-number or mRNA expression of RB1 was not identified. Another PDE3 inhibitor, anagrelide, uniquely inhibited megakaryocyte differentiation, resulting in apoptosis. Other PDE3 inhibitors tested did not have this activity (<NPL>; <NPL>). It was hypothesized that the reported effects of zardaverine on cell viability and anagrelide on megakaryocyte differentiation are mediated through the same PDE3A modulation as described in this study.

Multiple PDE3 inhibitors were competitive inhibitors and have been shown to occupy the catalytic binding site of cAMP and cGMP (<NPL>; <NPL>). In addition, zardaverine has been co-crystalized in a complex with PDE4D, where it occupies the cAMP-binding site, and has been modeled to bind PDE3B in a similar manner (<NPL>). Given the structural similarity of DNMDP to zardaverine and that DNMDP inhibited both PDE3A and PDE3B, it was hypothesized that the binding mode of DNMDP is very similar to that of zardaverine. This indicated that in addition to acting as a cAMP/cGMP-competitive inhibitor, DNMDP allosterically induces a conformation that is responsible for its cytotoxic phenotype. Allosteric modulation of phosphodiesterases has been described previously for PDE4, where small molecules bound in the active site and simultaneously interacted with regulatory domains that came across the PDE4 active site. As a result, allosteric modulators stabilized a protein conformation that has been shown to differentially bind different PDE4 partner proteins (<NPL>).

The study of proteins associated with PDE3A might illuminate both its normal function and the way in which PDE3A modulators such as DNMDP kill cancer cells. PDE3A interacted with protein phosphatase <NUM> subunits, which are implicated in oncogenic viral transformation and are mutated in human cancers (<NPL>; <NPL>; <NPL>), indicating a role for PDE3A in cancer cell signaling. Even though these interactions were not induced by DNMDP binding, the importance of the protein phosphatases in cancer biology would warrant further research.

The enhanced interaction between PDE3A and SLFN12, facilitated by DNMDP binding to PDE3A, and the correlation between sensitivity to DNMDP with SLFN12 expression strongly indicated that it is necessary to understand the functional impact of the PDE3A-SLFN12 interaction. However, little is known at this time about the functional role of SLFN12 in human physiology and cancer biology. SLFN12 is part of the schlafen gene family that diverges largely between humans and rodents. The large difference is due to rapid gene evolution and positive selection (<NPL>). Therefore, SLFN12 has no murine orthologue, preventing the study of SLFN12 in a well-understood model organism. The single publication on SLFN12 showed modulation of prostate cancer cell lines after ectopic expression of SLFN12 (<NPL>). Additional studies into the function ofSLFN12 and its interaction with PDE3A could elucidate the mechanism of DNMDP cytotoxicity. Two observations indicated that DNMDP acted as a neomorph or hypermorph on PDE3A function: <NUM>) DNMDP-sensitive cancer cell lines did not depend on PDE3A expression for survival, but rather PDE3A knock-down led to DNMDP resistance; and <NUM>) DNMDP induced or enhanced protein-protein interactions upon binding to PDE3A. Lenalidomide was an example of a small molecule that acted as a neomorph or hypermorph rather than as an enzymatic inhibitor. Lenalidomide modulated a specific protein-protein interaction between the cereblon ubiquitin ligase and Ikaros transcription factors, which were then subsequently targeted for degradation (<NPL>; <NPL>). By analogy, DNMDP might directly stabilize a PDE3A-SLFN12 interaction, or DNMDP could allosterically stabilize a PDE3 conformation that binds SLFN12. Either of these mechanisms could result in a neo- or hypermorphic phenotype. Further characterization of the neomorphic phenotype induced by DNMDP might facilitate synthesis of small molecules that will not inhibit cyclic nucleotide hydrolysis by PDE3A. Toxicity profiles of such small molecules should differ from PDE3 inhibitors prescribed for cardiovascular indications.

This study has uncovered a previously unknown role for PDE3A in cancer maintenance, in which its function can be modified by a subset of PDE3 inhibitors, resulting in toxicity to a subset of cancer cell lines. These data indicated that DNMDP and its analogs had a hyper- or neomorphic effect on PDE3A, leading to cellular toxicity, which was corroborated by cells becoming less sensitive to DNMDP with decreasing levels of cellular PDE3A. These observations are comparable with other reports of allosteric modulation of phosphodiesterases (<NPL>), indicating that DNMDP and analogues may have similar effects on PDE3A. The exact mechanism of cell-selective cytotoxicity remains unknown for now; however, further studies into the novel interactions with SLFN12, and perhaps SIRT7, might be informative.

In summary, the study herein used differential cytotoxicity screening to discover a cancer cell cytotoxic small molecule, DNMDP. Profiling of DNMDP in <NUM> genomically-characterized cancer cell lines revealed stereospecific nanomolar efficacy in about <NUM>% of cell lines tested. A search for genomic features that indicated sensitivity revealed that elevated PDE3A expression strongly correlated with DNMDP response. DNMDP inhibited PDE3A and PDE3B, with little or no activity towards other PDEs. However, unexpectedly, most other PDE3A inhibitors tested did not phenocopy DNMDP, including the potent and selective PDE3A inhibitor, trequinsin. Co-treatment of DNMDP-sensitive cells with trequinsin competed away the cancer cell cytotoxic activity of DNMDP, and knockout of PDE3A rescued the otherwise sensitive cells from DNMDP-induced cytotoxicity, leading us to hypothesize that PDE3A is required for cancer cell killing by DNMDP, which induces a neomorphic alteration of PDE3A. Mass spectrometric analysis of PDE3A immunoprecipitates alone or in the presence of DNMDP or trequinsin revealed differential binding of SLFN12 and SIRT7 only in the presence of DNMDP. Similar to PDE3A, SLFN <NUM> expression levels were elevated in DNMDP-sensitive cell lines, and knock down of SLFN12 with shRNA decreased sensitivity of cells to DNMDP, indicating that DNMDP-induced complex formation of PDE3A with SLFN12 is critical to the cancer cell cytotoxic phenotype. Results herein therefore implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of chemogenomics in small molecule discovery.

The experiments above were performed with the following methods and materials.

<NUM> NCI-H1734 or <NUM> A549 cells were plated in a <NUM>-well plate in <NUM>µl of RPMI supplemented with <NUM>% Fetal Bovine Serum and <NUM>% Pen/Strep. <NUM> hours after plating, a compound-library of <NUM> small molecules was added at a concentration of <NUM>. Staurosporine was used a positive control for cytotoxicity at a concentration of <NUM>, and DMSO was used a negative control at a concentration of <NUM>%. All compounds were incubated for <NUM> hours with indicated small molecules. After <NUM> hours, <NUM>-well plates were removed from the incubator and allowed to cool to room temperature for <NUM> minutes. Cell viability was assessed by adding <NUM>µl of a <NUM>% CELLTITERGLO® (Promega) in PBS with a THERMO COMBI™ or multichannel-pipette and incubated for <NUM> minutes. The luminescence signal was read using a Perkin-Elmer EnVision. Viability percentage was calculated by normalizing to DMSO controls.

<NUM> HeLa (DMEM), <NUM> A549 (RPMI), <NUM> MCF-<NUM> (DMEM), <NUM> PC3 (F12-K), <NUM> NCI-H2122 (RPMI) or <NUM> NCI-H1563 (RPMI) cells were plated in a <NUM>-well plate in <NUM>µl of corresponding growth media supplemented with <NUM>% Fetal Bovine Serum. <NUM> hours after plating, indicated compounds were added at indicated concentrations and incubated for <NUM> hours. Cell viability was assessed as described in Compound library screening in NCI-H1734 and A549 cell lines.

<NUM> HeLa cells were plated in <NUM>-well plate in <NUM>µl of corresponding growth media supplemented with <NUM>% Fetal Bovine Serum. <NUM> hours after plating, indicated compounds were added at indicated concentrations and incubated for <NUM> hours. Caspase-Glo from Promega was added according to the manufacturers recommendations and luminescence was determined as described in Compound library screening in NCI-H1734 and A549 cell lines.

The sensitivity of <NUM> cancer cell lines (CCLs) was measured drawn from <NUM> different lineages to DNMDP. Each cell line was plated in its preferred media in white opaque <NUM>-plates at a density of <NUM> cells/well. After incubating overnight, DNMDP was added by acoustic transfer at <NUM> concentrations ranging from <NUM> - <NUM> in <NUM>-fold steps in duplicate (Labcyte Echo <NUM>, Labcyte Inc. , Sunnyvale, CA). After <NUM> hours treatment, cellular ATP levels were measured as a surrogate for viability (CELLTITERGLO®, Promega Corporation, Madison, WI) according to manufacturer's protocols using a ViewLux Microplate Imager (PerkinElmer, Waltham, MA) and normalized to background (media-only) and vehicle (DMSO)-treated control wells.

Concentration response curves were fit using nonlinear fits to <NUM>- or <NUM>-parameter sigmoid functions through all <NUM> concentrations with the low-concentration asymptote set to the DMSO-normalized value, and an optimal <NUM>-point dose curve spanning the range of compound-sensitivity was identified. The area under the <NUM>-point dose curve (AUC) was computed by numeric integration as a metric for sensitivity for further analysis. Similar sensitivity measurements have been obtained for a collection of <NUM> other compounds, enabling analyses that identify cell lines responding uniquely to DNMDP (see Broad Institute Cancer Therapeutics Response Portal, a dataset to identify comprehensively relationships between genetic and lineage features of human cancer cell lines and small-molecule sensitivities for complete list of compounds).

Gene-centric robust multichip average (RMA)-normalized basal mRNA gene expression data measured on the Affymetrix GeneChip Human Genome U133 Plus <NUM> Array were downloaded from the Cancer Cell Line Encyclopedia (CCLE, a detailed genetic characterization of a large panel of human cancer cell lines; <NPL>). Pearson correlation coefficients were calculated between gene expression (<NUM>,<NUM> transcripts) and areas under the curve (AUCs) across <NUM> overlapping CCLs. For comparisons across small molecules exposed to differing numbers of CCLs, correlation coefficients were transformed using Fisher's transformation.

All reactions were carried out under nitrogen (N2) atmosphere. All reagents and solvents were purchased from commercial vendors and used as received. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker (<NUM> or <NUM> <NUM>H, <NUM> or <NUM> <NUM>C) spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) referenced to the NMR solvent. Data are reported as follows: chemical shifts, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet; coupling constant(s) in Hz). Flash chromatography was performed using <NUM>-<NUM> Silica Gel (<NUM>Å mesh) on a Teledyne Isco Combiflash Rf. Tandem Liquid Chromatography/Mass Spectrometry (LC/MS) was performed on a Waters <NUM> separations module and <NUM> mass detector with a Waters Symmetry C18 column (<NUM>, <NUM> × <NUM>) with a gradient of <NUM>-<NUM>% CH3CN in water over <NUM> with constant <NUM>% formic acid. Analytical thin layer chromatography (TLC) was performed on EM Reagent <NUM> silica gel <NUM>-F plates. Elemental analysis was performed by Robertson Microlit Laboratories, Ledgewood NJ.

In <NUM> of acetic anhydride, <NUM> (<NUM> mmol) of (R)-<NUM>-(<NUM>-aminophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>H)-one (A, Toronto Research Chemicals) was stirred <NUM> hour before addition of <NUM> water, filtration, rinsing the solids with water and drying to yield <NUM> of product B (<NUM>%). <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, DMSO-d<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>,<NUM>,<NUM>, <NUM>. HPLC: Rt <NUM>, purity > <NUM>%. MS: <NUM> (M + <NUM>).

To <NUM> of B (<NUM> mmol) dissolved in <NUM> of sulfuric acid and cooled in an ice bath was added <NUM> of <NUM>% nitric acid (<NUM> mmol) in <NUM> sulfuric acid via an addition funnel over <NUM> minutes. After stirring <NUM> hour the mixture was poured onto ice. The yellow solid was filtered off and the water was rinsed several times with EtOAc before drying and combining with the yellow solid. Chromatography with <NUM>-<NUM>% EtOAc in hexane yielded <NUM> (<NUM>%) of product as a yellow solid which was recrystallized from EtOAc. <NUM>H NMR (<NUM>, DMSO-d<NUM>) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, DMSO-d6) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. TLC: Rf <NUM> (<NUM>:<NUM> EtOAc:hexane). HPLC: Rt <NUM>, purity > <NUM>%. MS: <NUM> (M + <NUM>). HRMS Exact Mass (M + <NUM>): <NUM>. Found: <NUM>.

To <NUM> of C (<NUM> mmol) dissolved in <NUM> of MeOH was added a solution of <NUM> NaOH (<NUM> mmol) in <NUM> water. After <NUM> hour the reaction was concentrated, water was added and rinsed with EtOAc, the EtOAc was dried and concentrated to give <NUM> (<NUM>%) of product D. <NUM>H NMR (<NUM>, DMSO-d6) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, DMSO-d<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. MS: <NUM> (M+ <NUM>).

To <NUM> of amine D (<NUM> mmol) dissolved in <NUM> Dimethylformamide (DMF) was added <NUM> of acetaldehyde (<NUM> mmol) and <NUM> of NaBH(OAc)<NUM> (<NUM> mmol) and <NUM>µL, (<NUM> mmol) of HOAc. After stirring <NUM> hours, water and EtOAc were added, the EtOAc separated, dried, concentrated and chromatographed with <NUM>-<NUM>% EtOAc in hexane to isolate <NUM> of the (R)-DNMDP (<NUM>%). The synthesized material was identical to purchased racemic material by TLC, HPLC and <NUM>H NMR. <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>). TLC: Rf <NUM> (<NUM>:<NUM> EtOAc:hexane). HPLC: Rt <NUM>, purity > <NUM>%. MS: <NUM> (M + <NUM>). Exact Mass (M + <NUM>): <NUM> Found: <NUM>. <NUM>C NMR (<NUM>, CDCl<NUM>, purchased material) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>.

The optical purity of (R)-DNMDP was determined using chiral SCF chromatography and comparison to commercially available racemic material: Column: ChiralPak AS-H, <NUM> × <NUM>, <NUM>, Mobile Phase Modifier: <NUM>% Methanol, Gradient: <NUM> to <NUM>% Methanol over <NUM> minutes, Flow Rate: <NUM>/min, Back Pressure: <NUM> bar, Column Temperature: <NUM> C. UV detection was from <NUM>-<NUM>. Retention times of separated isomers: <NUM>, <NUM> minutes; retention time of (R)-DNMDP, <NUM> minutes, <NUM>:<NUM> ratio of enantiomers detected.

To <NUM> (<NUM> mmol) of A dissolved in <NUM> of MeOH was added <NUM> of acetaldehyde (<NUM> mmol), <NUM> uL ofHOAc (<NUM> mmol) and <NUM> (<NUM> mmol) of NaBH<NUM>CN and the reaction was stirred overnight at room temperature. The next day the same quantity of reagents were added and the reaction stirred another <NUM> hours. The mixture was concentrated and partitioned between CH<NUM>Cl<NUM> and water, the CH<NUM>Cl<NUM> was separated, dried, and concentrated before chromatography with <NUM>-<NUM>% EtOAc in hexane isolated <NUM> of product as a white solid (<NUM>%). <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>). <NUM>C NMR (<NUM>, CDCl<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. TLC: Rf <NUM> (<NUM>:<NUM> EtOAc:hexane). HPLC: Rt <NUM>, purity > <NUM>%. MS: <NUM> (M + <NUM>). HRMS Exact Mass (M + <NUM>): <NUM>. Found: <NUM>
<CHM>.

To <NUM> (<NUM> mmol) of A dissolved in <NUM> of Dimethylformamide (DMF) was added <NUM>µL (<NUM> mmol) of bis (<NUM>-bromoethyl) ether and <NUM> of K2CO3 and the mixture was stirred overnight at <NUM>. The next day another <NUM>µL of bis (<NUM>-bromoethyl) ether and <NUM> of K2CO3 were added. After <NUM> hours, EtOAc and water were added, the water was rinsed with EtOAc, the combined EtOAc washes were dried and concentrated. Chromatography with <NUM>-<NUM>% MeOH in CH<NUM>Cl22 yielded <NUM> of product (<NUM>%). <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CDCl<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. TLC: Rf <NUM> (<NUM>:<NUM> MeOH:CH<NUM>Cl<NUM>). HPLC: Rt <NUM>, purity > <NUM>%. MS: <NUM> (M + <NUM>). HRMS: calcd. <NUM> (M + <NUM>); found <NUM>. for C<NUM>H<NUM>N<NUM>O<NUM>: C, <NUM>; H, <NUM>; N, <NUM>; Found. <NUM>, H, <NUM>, N, <NUM>.

DNMDP-<NUM>. To <NUM> of A (<NUM> mmol) dissolved in <NUM> of Dimethylformamide (DMF) was added <NUM> of tert-butyl <NUM>-(<NUM>-(<NUM>-bromoethoxy)ethoxy)-ethylcarbamate (Toronto Research Chemical, <NUM> mmol) and <NUM> of K<NUM>CO<NUM> (<NUM> mmol) and the mixture was stirred at <NUM> overnight. After cooling, water was added and rinsed several times with EtOAc. The combined EtOAc layers were dried, concentrated, and chromatographed with <NUM>-<NUM>% EtOAc to yield <NUM> of product (<NUM>%). <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CDCl<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>; HPLC: Rt <NUM> (<NUM> run), purity > <NUM>%. MS: <NUM> (M + <NUM>). This product (<NUM> mmol) was dissolved in <NUM> MeOH and to the solution was added acetaldehyde (<NUM> uL, <NUM> mmol), <NUM> uL HOAc (<NUM> mmol) and <NUM> NaBH<NUM>CN (<NUM> mmol). After <NUM> hour, NaHCO<NUM>(aq) and CH<NUM>Cl<NUM> were added, the CH<NUM>Cl<NUM> was separated and the water washed twice with CH<NUM>Cl<NUM>. The combined CH<NUM>Cl<NUM> was dried, concentrated, and chromatography with <NUM>-<NUM>% EtOAc in hexane yielded <NUM> of product as a clear oil (<NUM>%). <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dt, J = <NUM>, <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>). <NUM>C NMR (<NUM>, CDCl<NUM>) δ <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. Rt <NUM> (<NUM> run), purity > <NUM>%. MS: <NUM> (M + <NUM>).

To a solution of <NUM> of DNMDP-<NUM> (<NUM> mmol) in <NUM> of CH<NUM>Cl<NUM> was added <NUM> of trifluoroacetic acid (TFA) and the solution was stirred <NUM> before concentration and dissolution in <NUM> DMSO. To this was added <NUM> uL of Et<NUM>N (<NUM> mmol) and <NUM> of N,N'-disuccinimidyl carbonate (DSC) (<NUM> mmol) and the solution was stirred overnight. LC analysis indicated the reaction was not complete, another <NUM> of N,N'-disuccinimidyl carbonate (<NUM> mmol) was added. LC analysis after <NUM> hours showed ca. <NUM>:<NUM> ratio of DSC product:amine. A <NUM> sample of Affi-Gel <NUM> resin was rinsed five times with DMSO with a centrifuge, then suspended in <NUM> DMSO. To the resin was added <NUM> uL of the DSC product solution and <NUM> uL Et3N and the mixture was swirled. After <NUM> days, LC analysis of the DMSO solution showed complete disappearance of the DCS adduct; the underivatized amine was still present. The DMSO was removed by centrifuge and decanted and the resin was rinsed several times with DMSO and stored in PBS buffer.

<NUM> HeLa cells were plated in a <NUM>-well plate in <NUM>µl of DMEM supplemented with <NUM>% Fetal Bovine Serum and <NUM>% Pen/Strep. <NUM> hours after plating, a compound-library of <NUM> bioactive molecules (Pharmacon) was added at a concentration of <NUM>. In parallel to bioactive compound incubation, DNMDP was added to a final concentration of <NUM> and incubated for <NUM> hours. Cell viability was assessed as described in Compound library screening in NCI-H1734 and A549 cell lines.

HeLa cells were washed with ice-cold PBS before lysed with NP-<NUM> lysis buffer (<NUM> NaCl, <NUM>% glycerol, <NUM> Tris-Cl pH <NUM>, <NUM> MgCl<NUM>, <NUM>% NP-<NUM>) supplemented with EDTA-free protease inhibitors (Roche) and phosphatase inhibitor mixtures I and II (Calbiochem). Cell lysates were incubated on ice for at least <NUM> minutes and subsequently centrifuged for <NUM> minutes at <NUM>° C at <NUM>,<NUM> x g after which the supernatant was quantified using BCA protein assay kit (Pierce). <NUM>µg total HeLa cell lysate was incubated with <NUM>µl Affi-Gel <NUM> resin (BioRad) coupled to affinity linker DNMDP-<NUM> in a total volume of <NUM>µl for four hours. Prior to incubation, indicated compounds were added to affinity purifications at a final concentration of <NUM>. Samples were washed three times with lysis buffer containing corresponding compound concentrations of <NUM>. Proteins bound to Affi-Gel <NUM> resin were reduced, denatured, and separated using Tris-Glycine gels (Novex) and transferred to nitrocellulose membranes using the iBlot transfer system (Novex). Membranes were incubated overnight at <NUM>° C with primary antibodies against PDE3A (<NUM>: <NUM>, Bethyl). Incubation with secondary antibodies (<NUM>:<NUM>,<NUM>, LI-COR Biosciences) for two hours at room temperature and subsequent detection (Odyssey Imaging System, LI-COR Biosciences) were performed according to manufacturer's recommendations.

HeLa cells were treated with indicated concentration of DNMDP and staurosporine for <NUM> hours. HeLa cells were lysed and processed as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting. Membranes were incubated with an antibody against PARP (<NUM>: <NUM>, Cell Signaling #<NUM>) and actin and subsequently imaged as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting.

CRISPR target sites were identified using the MIT CRISPR Design Tool (online MIT CRISPR design portal). For cloning of sgRNAs, forward and reverse oligos were annealed, phosphorylated and ligated into BsmBI-digested pXPR_BRD001. Oligo sequences are as follows:.

To produce lentivirus, 293T cells were co-transfected with pXPR_BRD001, psPAX2 and pMD2. G using calcium phosphate. Infected HeLa cells were selected with 2ug/ml of puromycin.

HeLa cells were plated in <NUM>-well plates and transfected after <NUM> hours with PDE3A and Non-Targeting siRNA smartpools (On Target Plus, Thermo Scientific) according to the manufacturers recommendations. HeLa cell lysate was obtained <NUM> hours and <NUM> hours after transfection and immunoblotted for PDE3A and Actin (<NUM>:<NUM>,<NUM>, Cell Signaling) as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting. HeLa cells were treated for <NUM> hours with indicated concentrations of Compound <NUM>. Cell viability was assessed as described in Compound library screening in NCI-H1734 and A549 cell lines.

<NUM> HeLa cells were plated in <NUM>-well plates. <NUM> hours after plating, HeLa cells were incubated for one hour with indicated compounds at indicated concentrations. cAMP levels were determined with the CAMP-GLO™ assay (Promega) according to the manufacturers recommendations. Cellular concentrations of cAMP were determined by normalizing to a standard curve generated according to the manufacturers recommendations.

HeLa cells were treated for four hours prior to lysis with <NUM> of indicated compounds: DMSO, DNMDP and trequinsin. HeLa cells were lysed with ModRipa lysis buffer (<NUM>%NP-<NUM>: <NUM> Tris-HCl, pH <NUM>, <NUM> NaCl, <NUM>% sodium deoxycholate, <NUM> EDTA) supplemented with protease and phosphatase inhibitors as described in Linker-affinity purification of molecular target of DNMDP and immunoblotting, and indicated compounds as described above to a final concentration of <NUM>. <NUM> of HeLa total cell lysate was incubated with <NUM>% PDE3A antibody (Bethyl) and incubated overnight. Blocking peptide (Bethyl) against the PDE3A antibody was added simultaneously with the PDE3A antibody in the corresponding condition. Total cell lysate and antibody mixture was then incubated with <NUM>µl Protein A Plus Agarose (Fisher Scientific) for <NUM> minutes at <NUM>° C. Protein A Plus Agarose was then washed two times with lysis buffer containing indicated compounds at a concentration of <NUM>. Finally, Protein A Plus Agarose was washed once with lysis buffer containing no NP-<NUM> and indicated compounds at a concentration of <NUM>.

The beads from immunopurification were washed once with IP lysis buffer, then three times with PBS, the three different lysates of each replicate were resuspended in <NUM> uL digestion buffer (<NUM> Urea, <NUM> Tris HCl), <NUM> ug of sequencing grade trypsin added, <NUM> hour shaking at <NUM> rpm. The supernatant was removed and placed in a fresh tube. The beads were then washed twice with <NUM> uL digestion buffer and combined with the supernatant. The combined supernatants were reduced (<NUM> uL <NUM> DTT, <NUM> minutes, room temperature), alkylated (<NUM> uL <NUM> IAA, <NUM> minutes, dark) and a longer overnight digestion performed: <NUM> ug (<NUM> uL) trypsin, shake overnight. The samples were then quenched with <NUM> uL <NUM>% folic acid (FA) and desalted on <NUM> SEP-PAK® columns.

Desalted peptides were labeled with isobaric tags for relative and absolute quantification (iTRAQ)- reagents according to the manufacturer's instructions (AB Sciex, Foster City, CA). Peptides were dissolved in <NUM>µl of <NUM> TEAB pH <NUM> solution and labeling reagent was added in <NUM> ul of ethanol. After <NUM> hour incubation the reaction was stopped with <NUM> Tris/HCl pH <NUM>. Differentially labeled peptides were mixed and subsequently desalted on <NUM> SEP-PAK® columns.

SCX fractionation of the differentially labelled and combined peptides was done as described in Rappsilber et al. (<NPL>), with <NUM> pH steps (buffers- all contain <NUM>% acetonitrile) as below:.

Empore SCX disk used to make stop-and-go-extraction-tips (StageTips) as described in the paper.

Reconstituted peptides were separated on an online nanoflow EASY-NLC™ <NUM> UHPLC system (Thermo Fisher Scientific) and analyzed on a benchtop Orbitrap Q EXACTIVE™ mass spectrometer (Thermo Fisher Scientific). The peptide samples were injected onto a capillary column (PICOFRIT® with <NUM> tip opening/ <NUM> diameter, New Objective, PF360-<NUM>-<NUM>-N-<NUM>) packed in-house with <NUM> C18 silica material (<NUM> REPROSIL-PUR® C18-AQ medium, Dr. Maisch GmbH, rl <NUM>. The UHPLC setup was connected with a custom-fit microadapting tee (<NUM>, IDEX Health & Science, UH-<NUM>), and capillary columns were heated to <NUM> in column heater sleeves (Phoenix-ST) to reduce backpressure during UHPLC separation. Injected peptides were separated at a flow rate of <NUM> nL/min with a linear <NUM> gradient from <NUM>% solvent A (<NUM>% acetonitrile, <NUM>% formic acid) to <NUM>% solvent B (<NUM>% acetonitrile, <NUM>% formic acid), followed by a linear <NUM> gradient from <NUM>% solvent B to <NUM>% solvent B. Each sample was run for <NUM> minutes, including sample loading and column equilibration times. The Q EXACTIVE™ instrument was operated in the data-dependent mode acquiring high-energy collisional dissociation (HCD) MS/MS scans (R=<NUM>,<NUM>) after each MS1 scan (R=<NUM>,<NUM>) on the <NUM> top most abundant ions using an MS1 ion target of <NUM>× <NUM> ions and an MS2 target of <NUM>×<NUM> ions. The maximum ion time utilized for the MS/MS scans was <NUM>; the HCD-normalized collision energy was set to <NUM>; the dynamic exclusion time was set to <NUM>, and the peptide match and isotope exclusion functions were enabled.

All mass spectra were processed using the Spectrum Mill software package v4. <NUM> beta (Agilent Technologies) which includes modules developed by Applicants for isobaric tags for relative and absolute quantification (iTRAQ)-based quantification. Precursor ion quantification was done using extracted ion chromatograms (XIC' s) for each precursor ion. The peak area for the XIC of each precursor ion subjected to MS/MS was calculated automatically by the Spectrum Mill software in the intervening high-resolution MS1 scans of the liquid chromatography (LC)-MS/MS runs using narrow windows around each individual member of the isotope cluster. Peak widths in both the time and m/z domains were dynamically determined based on MS scan resolution, precursor charge and m/z, subject to quality metrics on the relative distribution of the peaks in the isotope cluster vs theoretical. Similar MS/MS spectra acquired on the same precursor m/z in the same dissociation mode within +/-<NUM> seconds were merged. MS/MS spectra with precursor charge ><NUM> and poor quality MS/MS spectra, which failed the quality filter by not having a sequence tag length > <NUM> (i.e., minimum of <NUM> masses separated by the in-chain mass of an amino acid) were excluded from searching.

For peptide identification MS/MS spectra were searched against human Universal Protein Resource (Uniprot) database to which a set of common laboratory contaminant proteins was appended. Search parameters included: ESI-Q EXACTIVE™-HCD scoring parameters, trypsin enzyme specificity with a maximum of two missed cleavages, <NUM>% minimum matched peak intensity, +/- <NUM> ppm precursor mass tolerance, +/- <NUM> ppm product mass tolerance, and carbamidomethylation of cysteines and iTRAQ labeling of lysines and peptide n-termini as fixed modifications. Allowed variable modifications were oxidation of methionine, N-terminal acetylation, Pyroglutamic acid (N-termQ),Deamidated (N),Pyro Carbamidomethyl Cys (N-termC),with a precursor MH+ shift range of - <NUM> to <NUM> Da. Identities interpreted for individual spectra were automatically designated as valid by optimizing score and delta rankl-rank2 score thresholds separately for each precursor charge state in each liquid chromatography (LC)-MS/MS while allowing a maximum target-decoy-based false-discovery rate (FDR) of <NUM>% at the spectrum level.

In calculating scores at the protein level and reporting the identified proteins, redundancy is addressed in the following manner: the protein score is the sum of the scores of distinct peptides. A distinct peptide is the single highest scoring instance of a peptide detected through an MS/MS spectrum. MS/MS spectra for a particular peptide may have been recorded multiple times, (i.e. as different precursor charge states, isolated from adjacent SCX fractions, modified by oxidation of Met) but are still counted as a single distinct peptide. When a peptide sequence ><NUM> residues long is contained in multiple protein entries in the sequence database, the proteins are grouped together and the highest scoring one and its accession number are reported. In some cases when the protein sequences are grouped in this manner there are distinct peptides which uniquely represent a lower scoring member of the group (isoforms or family members). Each of these instances spawns a subgroup and multiple subgroups are reported and counted towards the total number of proteins. iTRAQ ratios were obtained from the protein-comparisons export table in Spectrum Mill. To obtain iTRAQ protein ratios the median was calculated over all distinct peptides assigned to a protein subgroup in each replicate. To assign interacting proteins the Limma package in the R environment was used to calculate moderated t-test p, as described previously and added Blandt-Altman testing to filter out proteins for which the CI for reproducibility was below <NUM>% (<NPL>).

HeLa cells were transfected with ORF overexpression constructs expressing V5-tagged SIRT7, V5-tagged SLFN12, or V5-tagged GFP. ORF expression constructs were obtained from the TRC (clone IDs: TRCN0000468231, TRCN0000476272, ccsbBroad304_99997). At <NUM> hours post transfection, cells were treated with <NUM> DNMDP or trequinsin for <NUM> hours followed by lysis using the ModRipa lysis buffer and immunoprecipitation of PDE3A. For each condition, <NUM> total protein lysate was incubated with <NUM>µg of anti-PDE3A antibody at <NUM>° C overnight, after which <NUM>µl each of Protein A- and Protein G- Dynabeads (Life Technologies 10001D and 10003D) were added and incubated for another <NUM> hour. Beads were washed and bound proteins were eluted with <NUM>µl of LDS PAGE gel loading buffer. Input (~<NUM>µg total protein lysate) and IP products were resolved on <NUM>-<NUM>% Tris-Glycine PAGE gels and immunoblotted with an anti-V5 antibody (Life Technologies R96205, <NUM>:<NUM>), the Bethyl anti-PDE3A antibody (<NUM>:<NUM>), and secondary antibodies from LiCOR Biosciences (Cat. # <NUM>-<NUM> and <NUM>, each at <NUM>:<NUM>,<NUM>). Blots were washed and imaged using a LiCOR Odyssey infrared imager.

Claim 1:
A PDE3A modulator selected from the group consisting of <NUM>-(<NUM>-(diethylamino)-<NUM>-nitrophenyl)-<NUM>-methyl-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (DNMDP), (5R)-<NUM>-methyl-<NUM>-[<NUM>-(morpholin-<NUM>-yl)phenyl]-<NUM>,<NUM>-dihydropyridazin-<NUM>(<NUM>)-one (compound <NUM>), zardaverine and anagrelide, or a pharmaceutically acceptable salt thereof for use in a method of treating cancer by killing or reducing the survival of a cancer cell selected as responsive to said compound, the method comprising contacting the cell with said compound, wherein the cell was selected by having an increase in the level of a phosphodiesterase 3A (PDE3A) and a Schlafen <NUM> (SLFN12) polypeptide or polynucleotide relative to the level present in a corresponding healthy control cell, thereby reducing the survival of the cancer cell.