Patent Description:
Heterotrimeric guanine nucleotide-binding proteins (G proteins) couple to G Protein-couple Receptors (GPCRs), and serve as molecular switches that relay signals from activated GPCRs to a wide variety of intracellular effectors. G proteins represent a large family of heterotrimeric proteins found in mammals composed of alpha (Gα), beta (Gβ), and gamma (Gγ) subunits. In their inactive state, G proteins are heterotrimeric, consisting of one Gα, one Gβ, and one Gγ subunit, and a bound deoxyguanosine diphosphate (GDP). Receptor-catalyzed guanine nucleotide exchange results in deoxyguanosine triphosphate (GTP) binding to the Gα subunit and G protein activation. Gα-GTP dissociates from the Gβ and Gγ subunits, allowing the Gβγ dimer and the Gα-GTP subunit each to activate downstream effectors. Hydrolysis of GTP to GDP deactivates the complex and turns off the cellular response.

Guanine Nucleotide-Binding Protein Alpha-Q (GNAQ) and Guanine Nucleotide-Binding Protein Alpha <NUM> (GNA11) are genes that encode Gαq and Gα<NUM> subunits respectively. The majority of somatic activating mutations found in GNAQ and GNA11 result in constitutively active Gαq or Gα<NUM> proteins, respectively. Uveal melanoma, arising from melanocytes and biologically distinct from cutaneous melanomas, has been shown to have these activating mutations in GNAQ and/or GNA11 genes. GNAQ and GNA11 alterations at Q209 have been characterized as gain of function mutations that confer dependence on downstream Protein Kinase C (PKC) signaling. PKC is a family of multifunctional isoenzymes that plays a vital role in the regulation of signal transduction, cell proliferation and differentiation through positive and negative regulation of the cell cycle. PKC is one of the major downstream effectors in a Gαq (Gα<NUM> or Gαq/<NUM>) signaling pathway. The international patent applications <CIT> and <CIT> disclose the use of PKC inhibitors for the treatment of uveal melanoma carrying GNAQ or GNA11 mutations.

There is an unmet need for effective and safe therapeutic agents that can treat cancers other than uveal melanoma. The clinical significance and frequency of GNAQ and GNA11 mutations in non-uveal melanoma and other cancers is generally unknown and not understood.

Any subject-matter falling outside the scope of the claims is provided for information only. Methods of treatment of the human body by therapy, referred to in this description, are not part of the present invention as such, but are described here in relation to compounds and pharmaceutical compositions for use in said methods for treatment of the human body by therapy, according to the present invention. The present disclosure is based, in part, on the discovery that certain cancers are associated with a GNAQ and/or GNA11 genetic mutation, and for example, once identified, such cancers may be treated by administering a pharmaceutically effective amount of a protein kinase C inhibitor. For example, provided herein is a PKC inhibitor for use in a method of treating a patient having a non-uveal melanoma tumor or cancer, or non-melanocytic tumor of the central nervous system, (e.g. pancreatic cancer, colorectal cancer, lung adenocarcinoma, cutaneous melanoma, stomach cancer, cervical cancer, uterine cancer, bladder cancer, hepatocellular carcinoma, prostate cancer, breast cancer, head and neck cancer, and glioblastoma), the method comprising: determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has a GNAQ or GNA11 genetic mutation that activates the PKC pathway; and orally administering to the patient suffering from the non-uveal melanoma cancer with GNAQ or GNA11 genetic mutation a pharmaceutically effective amount of a protein kinase C inhibitor. Such determining may include determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has a GNAQ Q209, GNA11 Q209, GNAQ R183 or GNA11 R183 genetic mutation, where for example, the non-uveal melanoma cancer or non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system may selected from the group consisting of a pancreatic cancer, colorectal cancer, lung adenocarcinoma, and cutaneous melanoma. Alternatively, such determining may include determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has a mutation other than any of GNAQ Q209, GNA11 Q209, GNAQ R183 or GNA11 R183 genetic mutation, and for example, the tumor or cancer may be selected from the group consisting of uterine, stomach, bladder, cervical, breast, head and neck, hepatocellular carcinoma, and glioblastoma.

For example, the present disclosure relates in a part to a PKC inhibitor for use in a method of treating a carcinoma, e.g., a non-uveal melanoma solid tumor, having a GNAQ or GNA11 genetic mutation (e.g., a GNAQ Q209, GNAQ R183, or a GNA11 Q209 genetic mutation), or a non-Q209 or non-R183 mutation, in a patient in need thereof where the carcinoma for example, is selected from the group consisting of pancreatic adenocarcinoma, stomach adenocarcinoma, cervical carcinoma, lung adenocarcinoma and cutaneous melanoma. In embodiments of the disclosure, such contemplated method may include determining if the patient's carcinoma has a GNAQ or GNA11 genetic mutation; and orally administering to the patient having the carcinoma with the GNAQ or GNA11 genetic mutation a therapeutically effective amount of a protein kinase C inhibitor, for example, a small molecule protein kinase C inhibitor.

Provided herein, for example, is a PKC inhibitor for use in a method of treating a solid tumor cancer in a patient in need thereof, the method comprising: orally administering an effective amount of a protein kinase C inhibitor to the patient, wherein the patient has been determined to have a GNAQ or GNA11 genetic tumor mutation, wherein the solid tumor cancer is selected from pancreatic adenocarcinoma, stomach adenocarcinoma, cervical carcinoma, and lung adenocarcinoma.

For example, described herein is a PKC inhibitor for use in a method of treating a solid tumor cancer having a GNAQ or GNA11 genetic mutation in a patient, for example, a patient in need thereof wherein the method includes determining if the solid tumor cancer has a GNAQ or GNA11 genetic mutation; determining if the patient does not have activating BRAF or NRAS mutations (e.g., the patient may not have a BRAF V600 mutation); and orally administering to the patient without activating BRAF or NRAS mutations a pharmaceutically effective amount of a protein kinase C inhibitor.

In some embodiments, contemplated methods include treating a solid tumor cancer in a patient in need thereof, where the patient is substantially non-responsive to treatment with an immune checkpoint inhibitor, e.g., not responsive to a <NUM>-month course of treatment with an immune checkpoint inhibitor such as one or more of pembrolizumab, ipilimumab, nivolumab, and atezolizumab, comprising orally administering to the patient a pharmaceutically acceptable amount of a protein kinase inhibitor, wherein the patient's solid tumor has been determined to have e.g., a GNAQ or GNA11 mutation, e.g., a Q209L or a GNA11 Q209L genetic mutation.

Contemplated methods of treatment herein may be directed to patients who have a low tumor mutation burden. For example, in some embodiments the patient may have a low tumor load of mutations in one or more of BAP1, SF3B1, EIF1AX, TERT, BRAF, CDKN2A, NRAS, or KRAS.

The present disclosure is directed, in part, to PKC inhibitors for use in methods for treating cancer in patients that have mutations, for example, activating mutations that for example, activate the PKC pathway, in GNAQ or GNA11, wherein the method includes orally administering a PKC inhibitor such as disclosed herein. The disclosure described herein is useful for the treatment of non-uveal melanoma cancers as defined in the claims that have either an activating mutation of GNAQ or GNA11 e.g., heterozygous somatic substitutions of Q209 or R183 of GNAQ or GNA11, or have an activating mutation other than each of a Q209 or R183 mutation, by orally administering to a patient in need thereof a protein kinase C inhibitor.

By "treating" is meant reducing at least one symptom associated with the disease or condition being treated.

A "subject" or "patient" as described herein, refers to any animal at risk for, suffering from or diagnosed for cancer (for example, a carcinoma, a solid tumor cancer, or a non-uveal melanoma tumor or a non-melanocyctic tumor of the central nervous system), including, but not limited to, mammals, primates, and humans. In certain embodiments, the subject may be a non-human mammal such as, for example, a cat, a dog, or a horse. In another embodiment, the subject is a human subject. A subject may be an individual diagnosed with a high risk of developing cancer, someone who has been diagnosed with cancer, someone who previously suffered from cancer, and/or an individual evaluated for symptoms or indications of cancer.

The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is a non-natural chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified amino acid residues, and non-naturally occurring amino acid polymer.

As used herein, the term "pharmaceutically acceptable salts" refers to the nontoxic acid or alkaline earth metal salts of compounds described herein, for example, compounds of Formula (II). These salts can be prepared in situ during the final isolation and purification of the compounds, for example, the compounds of Formula (II), or by separately reacting a base or acid functional group of compounds described herein, for example, compounds of Formula (II), with a suitable organic or inorganic acid or base, respectively. Representative salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, <NUM>-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, <NUM>-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, <NUM>-phenylproionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or dispersible products are thereby obtained.

As used herein, the term "GNAQ" refers to Guanine Nucleotide-Binding Protein Alpha-Q gene that encodes the Gq alpha subunit (Gαq) and the term "GNA11" refers to Guanine Nucleotide-Binding Protein Alpha <NUM> genes that encodes the G11alpha subunit (Gα11) subunit. The term encompasses nucleic acid and polymorphic variants, alleles, mutants, and fragments of GNAQ and GNA11. GNAQ and GNA11 sequences are well known in the art. Examples of human GNAQ sequences are available under the reference sequences NM_002072 in the NCBI nucleotide database (nucleotide sequence) and accession number NP_002063. <NUM> (polypeptide sequence). Human GNAQ has been localized to chromosome 9q21. Examples of human GNA11 sequences are available under the reference sequences NM_002067 in the NCBI nucleotide database (nucleotide sequence) and accession number NP_002058. <NUM> (polypeptide sequence). Human GNA11 is localized to chromosome region 19p13.

As used herein, "mutations" can refer to changes in a polynucleotide sequence that result in changes to protein activity. Mutations can be nucleotide substitutions, such as single nucleotide substitutions, insertions, or deletions. GNAQ and GNA11 mutations are typically activating mutations that lead to constitutive activation of the α subunit. Without being bound to a theory, it is believed that the constitutive activity results from a lack of the GTP-hydrolase activity in the mutant GNAQ or GNA11 protein. Activating mutations can also refer to mutations that result in a loss or decrease of GTP hydrolyzing activity of a Gα subunit. Mutations in GNAQ and GNA11 include a substitution of arginine in codon R183 or substitution of glutamine in codon Q209, or may be other mutations. In an embodiments, mutations in GNAQ and/or GNA11 can be selected from group comprising of: Q209P, Q209L, Q209H, Q209K, Q209Y, Q209R, Q209H, R183Q, R183, for example, GNAQ Q209 may be mutated to either P or L as well as to R or H; GNAQ R183 may be mutated to Q; GNA11 Q209 may be mutated to L as well as to P or K; GNAQ R183 may mutate to C or H.

GNA11 Q209 can be mutated to L as well as rarely to P or K; also GNAQ R183 is most often mutate to C and more rarely to H.

As used herein, the term "PKC" refers to protein kinase C. The PKC family of serine/threonine kinases is composed of at least ten isoforms, pivotal in various cellular differentiation processes with distinctive means of regulation and tissue distribution Five isozymes are known to be present in human neutrophils (see <NPL>).

As used herein, the term "PKC inhibitor" refers to a protein kinase C inhibitor that may be pan (multi-subtype) or selective to one or more PKC isozymes. The term PKC generally refers to the entire family of isoforms: conventional isoforms; alpha, beta, and gamma, novel isoforms; delta, epsilon, eta, and theta, and atypical isoforms; zeta, and iota. The term "inhibitor" refers to modulatory molecules or compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of a e.g., a protein, e.g., PKC.

The phrase "alkyl" refers to alkyl groups that do not contain heteroatoms. Thus, the phrase includes straight chain alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following which are provided by way of example: -CH(CH<NUM>)<NUM>, -CH(CH<NUM>)(CH<NUM>CH<NUM>), - CH(CH<NUM>CH<NUM>)<NUM>, -C(CH<NUM>)<NUM>, -C(CH<NUM>CH<NUM>)<NUM>, -CH<NUM>CH(CH<NUM>)<NUM>, -CH<NUM>CH(CH<NUM>)(CH<NUM>CH<NUM>), - CH<NUM>CH(CH<NUM>CH<NUM>)<NUM>, -CH<NUM>C(CH<NUM>)<NUM>,-CH<NUM>C(CH<NUM>CH<NUM>)<NUM>, -CH(CH<NUM>)- CH(CH<NUM>)(CH<NUM>CH<NUM>), - CH<NUM>CH<NUM>-CH(CH<NUM>)<NUM>, -CH<NUM>CH<NUM>CH(CH<NUM>)(CH<NUM>CH<NUM>), -CH<NUM>CH<NUM>CH(CH<NUM>CH<NUM>)<NUM>, -CH<NUM>CH<NUM> C(CH<NUM>)<NUM>, -CH<NUM>CH<NUM>C(CH<NUM>CH<NUM>)<NUM>, -CH(CH<NUM>)CH<NUM>-CH(CH<NUM>)<NUM>, - CH(CH<NUM>)CH(CH<NUM>)CH(CH<NUM>)<NUM>, -CH(CH<NUM>CH<NUM>)CH(CH<NUM>) CH(CH<NUM>)(CH<NUM>CH<NUM>), and others. The phrase also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, and such rings substituted with straight and branched chain alkyl groups as defined above. Thus, the term "C<NUM>-<NUM> alkyl group" includes primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. Alkyl groups include straight and branched chain alkyl groups and cyclic alkyl groups having <NUM> to <NUM> carbon atoms.

As used herein, "C<NUM>-<NUM> alkyl" includes both substituted or unsubstituted straight or branched chain alkyl groups having from <NUM> to <NUM> carbon atoms. Representative C<NUM>-<NUM> alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, neopentyl, trifluoromethyl, pentafluoroethyl and the like. Unless stated otherwise, C<NUM>-<NUM> alkyl groups may be substituted, such as with halo, hydroxy, amino, nitro and/or cyano groups, and the like. Representative C<NUM>-<NUM> haloalkyl and C<NUM>-<NUM> hydroxyalkyl include chloromethyl, trichloromethyl, trifluoromethyl, fluoromethyl, fluoroethyl, chloroethyl, hydroxymethyl, hydroxyethyl, and the like. Other suitable substituted C<NUM>-<NUM> alkyl moieties include, for example, aralkyl, aminoalkyl, aminoaralkyl, carbonylaminoalkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl, aralkylcarbonylaminoalkyl, aminoalkoxyalkyl and arylaminoalkyl, unless stated otherwise.

As used herein, "C<NUM>-<NUM> alkoxy" as used herein refers to the radical RO-, wherein R is C<NUM>-<NUM> alkyl. Representative examples of C<NUM>-<NUM> alkoxy groups include methoxy, ethoxy, t-butoxy, trifluoromethoxy and the like.

As used herein, the term "halogen" or "halo" refers to chloro, bromo, fluoro and iodo groups.

"Haloalkyl" refers to a C<NUM>-<NUM> alkyl radical substituted with one or more halogen atoms.

The term "haloalkoxy" refers to a C<NUM>-<NUM> alkoxy radical substituted with one or more halogen atoms.

"Amino" refers herein to the group -NH<NUM>.

The term "C<NUM>-<NUM> alkylamino" refers herein to the group -NRR' where R and R' are each independently selected from hydrogen or a C<NUM>-<NUM> alkyl provided at least one of R and R' is C<NUM>-<NUM> alkyl.

The term "arylamino" refers herein to the group -NRR' where R is C<NUM>-<NUM> aryl, including phenyl, and R' is hydrogen, a C<NUM>-<NUM> alkyl, or C<NUM>-<NUM> aryl, including phenyl.

The term "C<NUM>-<NUM> cycloalkyl" refers to a mono- or polycyclic, heterocyclic or carbocyclic C<NUM>-<NUM> alkyl substituent. Typical cycloalkyl substituents have from <NUM> to <NUM> backbone (i.e., ring) atoms in which each backbone atom is either carbon or a heteroatom. The term "heterocycloalkyl" refers herein to cycloalkyl substituents that have from <NUM> to <NUM>, and more typically from <NUM> to <NUM> heteroatoms in the ring structure. Suitable heteroatoms employed in compounds of the present disclosure are nitrogen, oxygen, and sulfur. Representative heterocycloalkyl moieties include, for example, morpholino, piperazinyl, piperidinyl and the like. Carbocycloalkyl groups are cycloalkyl groups in which all ring atoms are carbon. When used in connection with cycloalkyl substituents, the term "polycyclic" refers herein to fused and non-fused cyclic alkyl structures. The term "carbobicyclic or carbobicyclyl" refers to a saturated, or partially unsaturated carbocyclic ring fused to another carbocyclic ring, aryl ring, heterocyclic ring or heteroaryl ring. The cycloalkyl group is unsubstituted or substituted.

The term "heterocycle" or "heterocyclyl" includes rings in which nitrogen is the heteroatom as well as partially and fully-saturated rings. Exemplary heterocycles include but are not limited to, for example: piperidinyl, piperazinyl, <NUM>,<NUM>-oxazinane, <NUM>-oxopiperazinyl, <NUM>-oxopiperidinyl, N-methyl piperazinyl, and morpholinyl, each optionally substituted.

Heterocyclic moieties can be unsubstituted or monosubstituted or disubstituted with various substituents independently selected from hydroxy, halo, oxo (C=O), alkylimino (RN=, wherein R is a C<NUM>-<NUM> alkyl or C<NUM>-<NUM> alkoxy group), amino, C<NUM>-<NUM> alkylamino, C<NUM>-<NUM> dialkylamino, acylaminoalkyl, C<NUM>-<NUM> alkoxy, C<NUM>-<NUM> alkyl, cycloalkyl or C<NUM>-<NUM> haloalkyl. Heterocyclic groups (heterocyclyl) may be attached at various positions as will be apparent to those having skill in the organic and medicinal chemistry arts in conjunction with the herein.

The term "heteroaryl" refers to <NUM>-<NUM> membered carbocyclic ring system, including fused ring systems, having <NUM> to <NUM> heteroatoms each independently selected from the group consisting of: O, N and S. Said heteroaryl may be optionally substituted with one or two substituents. The term "heteroaryl" also refers herein to C<NUM>-<NUM> aryl groups having from <NUM> to <NUM> heteroatoms as ring atoms in an aromatic ring with the remainder of the ring atoms being carbon atoms. Exemplary substituents include, but are not limited to: halo, CN, C<NUM>-<NUM> alkyl, C <NUM>-<NUM> alkoxy, C<NUM>-<NUM> haloalkyl, C<NUM>-<NUM> haloalkoxy, C<NUM>-<NUM> cycloalkyl, and <NUM>-<NUM> membered heterocyclyl having <NUM> or <NUM> heteroatoms selected from N, O and S, said heterocyclyl optionally substituted with <NUM> to <NUM> substituents each independently selected from the group consisting of: halo, CN, C<NUM>-<NUM> alkyl, C<NUM>-<NUM> alkoxy, C<NUM>-<NUM> haloalkyl, and C<NUM>-<NUM> haloalkoxy. Representative heteroaryl groups include, for example, those shown below. Representative heteroaryls include, for example, imidazolyl, pyridinyl (also referred to aspyridyl), pyrazinyl, azetidinyl, thiazolyl, triazolyl, benzimidazolyl, benzothiazolyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, azetidinyl, N-methylazetidinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoazolidinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, triazolyl, benzothienyl diazapinyl, pyrryl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazoyl, imidazolinyl, imidazolidinyl and benzoxazolyl. The heteroaryl is unsubstituted or substituted with <NUM> to <NUM> substituents each independently selected from the group consisting of: H, <NUM>H, halo, C<NUM>-<NUM> alkynyl, C<NUM>-<NUM> alkenyl, CN, C<NUM>-<NUM> alkyl, C<NUM>-<NUM> alkoxy, C<NUM>-<NUM> haloalkyl, C<NUM>-<NUM> haloalkoxy, C<NUM>-<NUM> cycloalkyl, CONH<NUM>, CONHC<NUM>-<NUM> alkyl, CONHC<NUM>-<NUM> aryl, SO<NUM>NH<NUM>, SO<NUM>NHC<NUM>-<NUM> alkyl, SO<NUM>NHC<NUM>-<NUM> aryl and <NUM>-<NUM> membered heterocyclyl having <NUM> to <NUM> heteroatoms selected from N, O and S, said heterocyclyl optionally substituted one or two substituents each independently selected from the group consisting of: H, <NUM>H, halo, CN, C<NUM>-<NUM> alkyl, C<NUM>-<NUM> alkoxy, C<NUM>-<NUM> haloalkyl, and C<NUM>-<NUM> haloalkoxy.

The term "<NUM>H" refers to a heavy isotope of hydrogen that is also referred to as deuterium (D). It is understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with five fluoro groups or a halogen atom substituted with another halogen atom).

As used herein, the term "MEK inhibitor" refers to a mitogen-activated protein kinase (MEK) inhibitor. The term "inhibitor" refers to modulatory molecules or compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of MEK, mTOR, or CDKi. Representative MEK inhibitors include trametinib, cobimetinib, binimetinib, and selumetinib as well as TAK733, PD-<NUM>, CI-<NUM>, PD <NUM>, and PD035901. A representative mTor inhibitor includes rapamycin. CDKi (cyclin dependent kinase inhibitors) include ribociclib, palbociclib, abemaciclib, abernaciclib, ribociclib, and trilaciclib.

As used herein, the term "HDM2-p53 inhibitor" refers to human double minute <NUM> protein inhibitors, regulators or modulators, pharmaceutical compositions containing the compounds and potential methods of treatment using the compounds and compositions to potentially treat diseases such as, for example, cancer, diseases involving abnormal cell proliferation, and include small molecule inhibitors HDM201 (siremaldin), and JNJ-<NUM>.

As used herein, the term "mutational load" refers to the level, e.g., number, of an alteration (e.g., one or more alterations, e.g., one or more somatic alterations) per a preselected unit (e.g., per megabase) in a predetermined set of genes or all analyzed genes (e.g., in the coding regions of the predetermined set of genes). Mutation load can be measured, e.g., on a whole genome or exome basis, or on the basis of a subset of genome or exome. In certain embodiments, the mutation load measured on the basis of a subset of genome or exome can be extrapolated to determine a whole genome or exome mutation load. The terms "mutation load," "mutational load," "mutation burden," and "mutational burden" are used interchangeably herein. In the context of a tumor, a mutational load is also referred to herein as "tumor mutational burden," "tumor mutation burden," or "TMB.

As used herein, the term "BAP1" refers to BRCA1-associated protein-<NUM> gene (ubiquitin carboxy-terminal hydrolase; BAP1). The nucleic acid and amino acid sequences of BAP1 are known and publicly available (Genbank NM_004656. <NUM>, Genbank NP_004647. BAP1 has been functionally implicated in the DNA damage response as well as in the regulation of apoptosis, senescence and the cell cycle. Deletions and inactivating mutations in BAP1 have been previously associated with tumors of the breast and lung and, consistent with BAP1's role as tumor suppressor, restoration of BAP1 function has been shown to suppress cell growth and tumorigenicity in a BAP1-mutant lung cancer cell line.

As used herein, "SF3B1" refers to a gene that encodes Splicing Factor 3b Subunit <NUM>. The nucleic acid and amino acid sequences of SF3B1 are known and publicly available (NM_012433. <NUM>, Genbank NP_036565. Subunit <NUM> of the splicing factor 3b protein complex plays a number of critical roles in the splicing mechanism of the cell. Mutations in SF3B1 affect the ability of a cell to convert pre-mRNA, which contains intronic sequence, into mature mRNA.

As used herein, "E1F1AX" refers to a gene that encodes for the protein X-linked eukaryotic translation initiation factor 1A, which plays a role in protein synthesis. The nucleic acid and amino acid sequences of E1F1AX are known and publicly available (NM_001412. <NUM>, Genbank NP_001403. E1F1AX is commonly mutated in uveal melanoma.

As used herein, "TERT" can refer to either the gene encoding the enzyme Telomerase Reverse Transcriptase (TERT) or to the enzyme (i.e., protein) itself. TERT refers to the nucleoprotein, or enzyme, portion of telomerase. TERT genes have also been called "Ever Shorter Telomeres" or "EST" genes. Mutations in the promoter region of TERT have been associated with cancers including, but not limited to, thyroid cancer, bladder cancer and glioblastoma. The nucleic acid and amino acid sequences of TERT are known and publicly available (NM_198253. <NUM>, Genbank NP_937983.

As used herein, "NRAS" or "neuroblastoma RAS viral oncogene homolog" refer to a small GTPase Ras family protein encoded on chromosome <NUM>. The nucleic acid and amino acid sequences of NRAS are known and publicly available (NM_002524, Genbank NP_002515).

As used herein, "BRAF" or "v-Raf murine sarcoma viral oncogene homolog B" refer to a Raf kinase family serine/threonine-specific protein kinase that interacts with AKT1; CRaf, HRAS, and YWHAB. The sequences of BRAF are well known in the art for a number of species, e.g., human BRAF (NM_004333, Genbank NP_004324). the NRAS and/or BRAF sequence variation(s) can be point mutations. In some embodiments, a NRAS point mutation can be a point mutation resulting in one of the following amino acid residue changes: G12D; G12S; G13A; G13C; G13D; G12R; G13V; Q61H1; Q61K; Q61L; Q61R1; and Q61R2. In some embodiments, a BRAF point mutation can be a point mutation resulting in one of the following amino acid residue changes: V600D TG/AT; V600E T/A; V600E TG/AA; and V600K GT/AA.

As used herein, the terms 'solid tumors' and 'solid tumor cancers' may be used interchangeably. Provided herein, for example, is a method of treating a patient having a non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system, comprising: determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has a GNAQ or GNA11 genetic mutation that activates the PKC pathway; and orally administering to the patient suffering from the non-uveal melanoma cancer with GNAQ or GNA11 genetic mutation a pharmaceutically effective amount of a protein kinase C inhibitor, such as one described herein. Determining may include determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has a GNAQ Q209, GNA11 Q209, GNAQ R183 or GNA11 R183 genetic mutation Such mutations may be present for example, in a contemplated method of treating non-uveal melanoma cancer or non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system selected from the group consisting of a pancreatic cancer tumor, colorectal cancer tumor, lung adenocarcinoma, and cutaneous melanoma, in a patient in need thereof.

Alternatively, determining may include determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has a mutation other than any of GNAQ Q209, GNA11 Q209, GNAQ R183 or GNA11 R183 genetic mutation, such as another mutation provided herein. Such mutations may be present in a non-uveal melanoma cancer or non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system selected from the group consisting of uterine, stomach, cervical, bladder, hepatocellular carcinoma, prostate, breast, head and neck, or glioblastoma cancers/tumors. For example, contemplated methods may be used to treat patients having a non-uveal tumor that may have a different mutation in GNAQ or GNA11 other a Q209 or R183 mutation that e.g., activates the PKC pathway in a specific cancer such as e.g., uterine, stomach, cervical, bladder, hepatocellular carcinoma, prostate, breast, head and neck, or glioblastoma cancer.

Provided herein, in one embodiment, is a PKC inhibitor for use in a method of treating a patient having a non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system, the method comprising determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has a GNAQ or GNA11 mutation, e.g., a GNAQ Q209L or GNA11 Q209L genetic mutation; and orally administering to the patient suffering from the non-uveal melanoma cancer with the GNAQ or GNA11 genetic mutation a pharmaceutically effective amount of a protein kinase C inhibitor. Such non-uveal melanoma cancer or non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system may include pancreatic cancer tumor, stomach cancer tumor, colorectal cancer tumor, cervical cancer tumor, lung adenocarcinoma, and cutaneous melanoma, uterine, stomach, cervical, bladder, hepatocellular carcinoma, prostate, breast, head and neck, or glioblastoma cancer/tumors.

For example, a PKC inhibitor for use in a method of treating a carcinoma having a GNAQ or GNA11 genetic mutation in a patient in need thereof is provided, wherein the carcinoma is selected from the group consisting of pancreatic adenocarcinoma, stomach adenocarcinoma, cervical carcinoma, and lung adenocarcinoma, the method comprising determining if the carcinoma has a GNAQ or GNA11 genetic mutation; and orally administering to the patient having the carcinoma with the GNAQ or GNA11 genetic mutation a therapeutically effective amount of a protein kinase C inhibitor.

In another embodiment, a PKC inhibitor for use in a method of treating a solid tumor cancer in a patient in need thereof is provided the method, comprising orally administering an effective amount of a protein kinase C inhibitor to the patient, wherein the patient has been determined to have a GNAQ or GNA11 genetic tumor mutation, wherein the solid tumor cancer is selected from pancreatic adenocarcinoma, stomach adenocarcinoma, cervical carcinoma, and lung adenocarcinoma.

In some embodiments of the methods disclosed herein, the GNAQ or GNA11 mutation can be any one of a number of mutations, including a substitution mutation, an insertion mutation, and/or a deletion mutation. In some embodiments, the GNAQ or GNA11 mutation is a gain of function mutation. In some embodiments, the GNAQ or GNA11 mutation is the substitution of glutamine in codon <NUM> (Q209) and/or a substitution of arginine in codon R183. In some embodiments, the GNAQ mutation is one of Q209P, Q209L, Q209H, Q209K, or Q209Y. Also as noted above, in some embodiments the carcinoma, the cancer, the solid tumor cancer, or the non-uveal melanoma tumor or non-melanocyctic tumor of the central nervous system has a GNA11 mutation. For example, in embodiments described herein, the GNA11 mutation is one of Q209P, Q209L, Q209K or Q209H. For example, in particular embodiments, the GNAQ or GNA11 mutation is Q209L. In some embodiments, the GNAQ or GNA11 mutation is the substitution of arginine in codon R183. For example, in particular embodiments, the GNAQ mutation is R183Q, or the GNA11 mutation is R183C or R183H. Alternatively, the GNAQ or GNA11 mutation is at R256, L279, R166, A168, R210, R213, R166, A231, A342, D333, G171, R147, R73, T47, E191, E221, R149, T175, T379, T85, A86, E163, D195, E319, E191, E280, E49, P293, R300, R338, R60, D155, D205, D321, I226, R37, or V240.

Determining if a patient (e.g., the carcinoma or tumor) has a GNAQ or GNA11 genetic mutation may include identifying GNAQ or GNA11 mutations in DNA extracted from a tumor sample and/or in circulating tumor or tumor cell DNA.

For example, provided herein is a PKC inhibitor for use in a method of treating a non-uveal solid tumor cancer having a GNAQ or GNA11 genetic mutation in a patient in need thereof, the method comprising: determining if the solid tumor cancer has a GNAQ or GNA11 genetic mutation; determining if the patient does not have activating BRAF or NRAS mutations (e.g. does not have a BRAF V600 mutation); and orally administering to the patient without activating BRAF or NRAS mutations a pharmaceutically effective amount of a protein kinase C inhibitor. Such solid tumor cancers may be selected from the group consisting of pancreatic cancer, stomach cancer, colorectal cancer, cervical cancer, lung adenocarcinoma, cutaneous melanoma, uterine, bladder, hepatocellular carcinoma, prostate, breast, head and neck, and glioblastoma.

In another embodiment, a PKC inhibitor for use in a method of treating a non-uveal solid tumor cancer in a patient in need thereof is provided, wherein the patient is substantially non-responsive to treatment with an immune checkpoint inhibitor, comprising orally administering to the patient a pharmaceutically effective amount of a protein kinase C inhibitor, wherein the patient's solid tumor has been determined to have a GNAQ or GNA11 genetic mutation.

For example, the GNAQ or GNA11 mutation may be the substitution of glutamine in codon <NUM> (Q209) or wherein the GNAQ or GNA11 mutation is the substitution of arginine in codon R183, and for example, the solid tumor is one of cutaneous melanoma, colorectal, lung adenocarcinoma, or pancreatic. Alternatively, the GNAQ or GNA11 mutation is other than a substitution of glutamine in codon <NUM> (Q209) and other than a substitution of arginine in codon R183, and the solid tumor may be, e.g., one of uterine, stomach, cervical, hepatocellular carcinoma, prostate, breast, head and neck or glioblastoma.

The disclosed methods may include treating patients with low tumor mutational burden of less than <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb, <NUM> mut/Mb or <NUM> mut/Mb, for example, a low tumor mutational load of mutations in one or more of BAP1, SF3B1, EIF1AX, TERT, BRAF, KRAS, and/or NRAS. For example, the patient may have a low tumor mutational burden of less than <NUM> mut/Mb or of less than <NUM> mut/Mb, for example, the patient may have a low tumor mutational load of mutations across the genome, e.g., one or more of BAP1, SF3B1, EIF1AX and TERT. In certain embodiments, contemplated patients may not have activating BRAF or NRAS mutations. In certain embodiments, disclosed methods are directed to patients that do not have a BRAF V600 mutation.

In other aspects, the present disclosure provides a PKC inhibitor for use in a method for treating cancers with mutations in GNAQ and/or GNA11, for example, activating GNAQ or GNA11 mutations, in a human or animal subject in recognized need of such treatment comprising administering to said subject an amount of a compound, for example, a compound of formula (II), or e.g., a compound represented by Formula III effective to inhibit PKC activity in the subject.

Provided herein, in an embodiment, is a PKC inhibitor for use in a method of treating a patient having a non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system, the method comprising: determining if the non-uveal melanoma or non-melanocytic tumor of the central nervous system has Q209L genetic mutation or a non Q209 mutation, and orally administering to the patient suffering from the non-uveal melanoma cancer with GNAQ Q209L or GNA11 Q209L genetic mutation, or non Q209 mutation, a pharmaceutically effective amount of a protein kinase C inhibitor.

In other aspects, the present disclosure provides a PKC inhibitor for use in a method for treating non-uveal melanoma tumor or non-melanocytic tumor of the central nervous system harboring GNAQ or GNA11 mutations in a human or animal subject in recognized need of such treatment. The method can include orally administering to the subject a therapeutically effective amount of a PKC inhibitor compound, for example, a compound of formula (II) or e.g., a compound represented by Formula III.

In another aspect, the present disclosure provides a PKC inhibitor for use in a method for treating a patient that is or has been substantially non-responsive to treatment with an immune checkpoint inhibitor. The method can include orally administering to such a patient a therapeutically effective amount of a PKC inhibitor compound, for example, a compound of Formula (II), Immune checkpoint inhibitors to which a patient is or has been nonresponsive can include pembrolizumab, ipilimumab, nivolumab, cemiplimab, avelumab, durvalumab, and atezolizumab. The aforementioned patient can be a patient that is or has been non-responsive to a <NUM> month, <NUM> month, <NUM> month, <NUM> month, <NUM> month, <NUM> month, <NUM> year, or longer than <NUM> year course of treatment with an immune checkpoint inhibitor. For example, such a patient is not and/or has not been responsive to a <NUM>-month course of treatment with an immune checkpoint inhibitor such as pembrolizumab, ipilimumab, nivolumab, and/or atezolizumab.

For example, described herein is a PKC inhibitor for use in a method of treating a cancer in patient in need thereof wherein the method includes: identifying the patient as having a tumor with a mutation that includes a substitution of arginine in codon R183 in GNAQ or GNA11; and orally administering a composition comprising a compound represented by Formula III, or a pharmaceutically acceptable salt thereof, to the patient; where the cancer is selected from the group consisting of pancreatic cancer tumor, stomach cancer tumor, colorectal cancer tumor, cervical cancer tumor, lung adenocarcinoma, and cutaneous melanoma.

Disclosed methods may include administering the protein kinase C inhibitor as a monotherapy or may further include administering a therapeutically effective amount of one or more additional therapeutic agents such as a mitogen-activated protein kinase (MEK) inhibitor, a mTOR inhibitor, a CDKi inhibitor, an immune checkpoint inhibitor, or a HDM2-p53 inhibitor. MEK inhibitors include trametinib, cobimetinib, binimetinib, and selumetinib. In some embodiments, methods described herein further include administering a therapeutically effective amount of a HDM2-p53 inhibitor.

Also provided herein is a PKC inhibitor for use in a method of treating a cancer in patient in need thereof, said method comprising: identifying the patient as having a tumor with a mutation of GNAQ or GNA11; orally administering a composition comprising a small molecule PKC inhibitor such as represented in Formula III to the patient; wherein the cancer is selected from the group consisting of pancreatic cancer tumor, stomach cancer tumor, colorectal cancer tumor, cervical cancer tumor, lung adenocarcinoma, cutaneous melanoma, colorectal cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, prostate cancer, breast cancer, head and neck cancer, and glioblastoma.

PKC inhibitors useful in the practice of the present disclosure may inhibit several isoforms of PKC, e.g., may inhibit one or more of the isoforms α, β-<NUM>, β2, γ,δ, ε, η, θ, ζ, <IMG>. In particular embodiments described herein, contemplated PKC inhibitors may inhibit a inhibit PKC α and θ isoforms, and/or the δ and/or ε isoforms. Suitable PKC inhibitors include those disclosed herein, or may include maleimide derivatives such as bisindoylmaleimide, enzastaurin, staurospoine, , roboxistaurin, sotrastaurin, rottlerin, and/midostaurin. In some embodiments, a contemplated PKC inhibitor is a small molecular protein kinase C inhibitor, which may for example, be orally administrable.

For example, protein kinase C (PKC) inhibitors contemplated herein can have potency against one or multiple PKC isoforms including, but not limited to, one or more of δ, ε, η, θ, and/or α PKC isoforms. For example, a contemplated protein kinase C inhibitor for use in the disclosed methods may be a small molecule protein kinase C inhibitor that has potency against multiple protein kinase C informs. For example, a contemplated protein kinase C inhibitor of the present disclosure has potency against one or more (e.g.; one, two, three, four, five, six, seven or more) of δ, ε, η, θ, β, γ, α protein kinase C isoforms. For example, the protein kinase C inhibitor of the present disclosure may have a potency against two or more of δ, ε, η, θ, β, γ, or α protein kinase C isoforms. The protein kinase C inhibitor, of the present disclosure may, in an embodiment, may have a potency against each of δ, ε, η, θ, β, γ, or α protein kinase C isoforms. In yet another embodiment, the protein kinase C inhibitor has an IC<NUM> with respect to PKC θ/α and/or PKC δ and/or PKC ε isoforms of less than <NUM> For example, in some embodiments the PKC inhibitor has an IC<NUM> with respect to each of PKC θ/α, PKC δ, and/or PKC ε isoforms of independently selected, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, less than <NUM>, or in some embodiments, less than <NUM>.

Examples of PKC inhibitors suitable for use with the present disclosure include those contemplated herein, and may also include include staurosporine, the staurosporine analogue CPG41251, bryostatin-<NUM>, KAI-<NUM>, <NUM>-hydroxystaurosporine, L-threo-dihydrosphingosine (safingol), AHT956 and AEB071, the non-selective PKC inhibitor (PKC412), ilmofosine (BM <NUM><NUM>), indolcarbazole Gö6796 which is a more specific inhibitor of the classical PKC isoforms including PKCµ, the PKC-alpha antisense inhibitor LY900003, and the PKC-beta inhibitors LY333531, LY317615 (Enzastaurin). An example of an antisense molecule suitable for use in depleting PKC-alpha mRNA is <NUM>'-GTTCTCGCTGGTGAGTTTCA-<NUM>'.

In accordance with some aspects of the present disclosure, a contemplated PKC inhibitor may be represented by formula (II):
<CHM>
wherein:.

For example, contemplated PKC inhibitors may be represented by Formula II wherein X is CR; R<NUM>, R<NUM> and R<NUM> are each H; R<NUM> is independently H, CH<NUM>, CH<NUM>F, CHF<NUM>, CF<NUM>, CH<NUM>OH, and CH<NUM>-O-C<NUM>-<NUM> alkyl; R5a and R5b are each H; R5c and R5d are each independently H, F, C<NUM>-<NUM> alkyl, or C<NUM>-<NUM> alkoxy or R5c and R5d are joined together forming a methylene, ethylene or -CH<NUM>-O- bridging group; and R<NUM> and R<NUM> are each independently selected from H, halo, C<NUM>-<NUM> haloalkyl, C<NUM>-<NUM>haloalkoxy, C<NUM>-<NUM> cycloalkyl, morpholino, piperinyl and piperazinyl.

For example, a contemplated PKC inhibitor may be represented by Formula III:
<CHM>
or a pharmaceutically acceptable salt thereof.

It can be appreciated that the disclosed methods may include administering the PKC inhibitor as part of a pharmaceutical compositions, for example, may include administering a PKC inhibitor compound, such as those disclosed herein, in a dosage unit form. A pharmaceutical composition should be formulated to be compatible with its intended route of administration, i.e. the oral route.

Determining whether a patient has a specific mutation, for example, a GNAQ or GNA11 genetic mutation, can include collecting and/or analyzing a patient sample. Patient samples of interest include, for example, carcinoma tissue, cancer tissue, solid tumor tissue, tumor tissue, body tissue, blood, serum, plasma, or body fluid, for example, circulating blood containing tumor DNA, obtained from the patient. Patient samples used in a method described herein can also include tissue samples such as, but not limited to, gastrointestinal, mucosal, submucosal, intestinal, esophageal, ileal, rectal, cervical, colonic, epidermal, lung, thymus, pancreatic, stomach, rectal, cutaneous, subcutaneous, or lymphatic samples. Samples may also include cellular samples, for examples, cutaneous cell samples. The presence of a genetic mutation of interest in a sample from a patient may be determined using various assays. For example, in methods of the disclosure , a genetic mutation in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF NRAS, and/or another gene or DNA sequence may be determined by nucleotide analysis, for example, genetic sequencing, Southern blotting, FISH, high-throughput sequencing, phage display, shotgun sequencing, or PCR, for example, by RT-PCR. Sequencing methods described herein may employ the use of DNA primer sequences targeted to specific gene sequences, for example, a gene sequence of GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS.

The presence of a genetic mutation in a patient, for example a genetic mutation in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS may be analyzed by analyzing gene products of a gene of interest, including mRNA and protein levels of the GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS genes. For example, in some embodiments, the presence of one or more genetic mutations is analyzed by analyzing GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS protein or mRNA. The presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS identified at the protein or mRNA level may be determined using various detection methods. For example, in some embodiments, the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS identified at the protein or mRNA level is determined by immunohistochemistry or by nucleotide analysis.

In addition, methods of the disclosure may also include a determining if a genetic mutation is associated with a gene other than GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS.

Methods of determining the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS include, but are not limited to, methods of analyzing analyte mRNA transcripts such as polymerase chain reaction methods, for example, quantitative polymerase chain reaction methods. Nucleotide analysis may be performed using an oligonucleotide probe that binds an analyte nucleotide sequence (e.g., a GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS nucleotide sequence) or a pair of oligonucleotide primers capable of amplifying an analyte nucleotide sequence via a polymerase chain reaction, for example, by a quantitative polymerase chain reaction. Oligonucleotide probes and oligonucleotide primers may be linked to a detectable tag, such as, for example, a fluorescent tag. In determining the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS by nucleotide analysis, the practitioner may evaluate a particular gene's mRNA transcript makeup or concentration in a sample.

The presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS in a patient may be determined by obtaining a sample from the patient. For example, in some embodiments, the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS in the patient is determined in a sample obtained from the patient. In some embodiments, the sample is a blood, serum, plasma, tumor, or tissue sample. According to the methods described herein, a sample may be a tissue sample (e.g., an intestinal tissue sample, a duodenal tissue sample, a jejunal tissue sample, a stomach sample, a pancreatic tissue sample, a lung tissue sample, a gastrointestinal tissue sample, a rectal tissue sample, a colonic tissue sample, or a cervical tissue sample), a tumor sample (for example, a carcinoma sample, a solid tumor sample, or a bodily fluid sample that includes tumor DNA), or a bodily fluid sample (e.g., a saliva sample, a stool, or a urine sample). A sample can be a sample obtained from a patient tissue biopsy, for example, a mucosal tissue biopsy, for example, an intestinal mucosal tissue biopsy, for example a small intestinal mucosal tissue biopsy. Furthermore, the sample may be a blood, serum, or plasma sample. A blood sample from a subject may be obtained using techniques well-known in the art. Blood samples may include peripheral blood mononuclear cells (PBMCs), RBC-depleted whole blood, or blood serum. PBMCs can be separated from whole blood samples using different density gradient (e.g., Ficoll density gradient) centrifugation procedures. For example, whole blood (e.g., anticoagulated whole blood) is layered over the separating medium and centrifuged. At the end of the centrifugation step, the following layers are visually observed from top to bottom: plasma/platelets, PBMC, separating medium and erythrocytes/granulocytes.

Methods of the claimed disclosure include steps that may be carried out in vitro. For instance, it is contemplated that the steps of the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS in the subject may involve determining the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS in a sample. For example, the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS in a sample may be determined by performing nucleotide analysis on the sample in vitro. Alternatively, in some embodiments of the disclosure, the steps of determining and analyzing the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS in a patient may be carried out in vivo.

The methods described herein contemplate identifying the presence of one or more genetic mutations in e.g., GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS by analyzing a patient or a sample from a patient, for example, a tumor, blood, or tissue sample from a patient. Analyzing a patient or a sample from a patient, for example, a tumor, blood, or tissue sample from a patient, can include collecting, purifying, and/or extracting DNA from the patient or the sample. DNA collection, purification, and extraction techniques are well-known to those skilled in the art. In general, DNA extraction requires collecting cells that contain DNA for analysis, and breaking down cell membranes to expose DNA. DNA extraction may entail steps of concentrated salt solution treatment, centrifugation to separate DNA from other cellular components, and DNA purification using, for example, ethanol precipitation, phenol-chloroform extraction, and/or minicolumn purification. Common DNA extraction methods include organic extraction, Chelex extraction, and solid phase extraction. DNA purification can include collecting a blood sample to analyze for the presence of circulating tumor cells or free DNA from tumor cells. Kits available for collecting cell-free tumor DNA include MagNA Pure Compact (MPC) Nucleic Acid Isolation Kit I, Maxwell® RSC (MR) ccfDNA Plasma Kit, and the QIAamp Circulating Nucleid Acid (QCNA) Kit.

The presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS can be accomplished for example, using any method known in the art. In general, presence is detected in a sample taken from the patient. Identification of the presence of a genetic mutation in a patient can be accomplished using standard techniques, including hybridization-based techniques, sequencing techniques, and array-based techniques.

The determination of the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS can be detected in any biological sample and can be any specimen obtained from a patient or test subject that contains a nucleic acid (e.g., genomic DNA or RNA) that encodes GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS. Exemplary samples include a tissue biopsy, cell, bodily fluid (e.g., blood, serum, plasma, semen, urine, saliva, amniotic fluid, or cerebrospinal fluid).

Once obtained, the presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS can be detected using any appropriate method. In some embodiments, a hybridization approach is used. Hybridization approaches include dynamic allele-specific hybridization (<NPL>). This approach relies on differential melting temperatures between the sequence containing the polymorphism as compared to the sequence without the polymorphism. Briefly, a DNA region of interest is amplified by PCR using a biotinylated primer. The resulting PCR product is attached to a streptavidin support and is hybridized to an allele-specific probe in the presence of a DNA duplex-binding fluorescent molecule. The duplex is heated, and the temperature at which the duplex denatures is determined based on loss of fluorescence. The denaturation temperature is determinative of the presence or absence of the polymorphism or mutation.

Another hybridization approach for identifying the presence of SNPs is the use of nucleic acid arrays designed for this purpose. For example, arrays designed to detect one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, and/or NRAS can be used to detect the presence of genetic mutations in samples taken from a patient.

In some embodiments, presence of one or more genetic mutations in GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS may be determined by performing a "nucleotide analysis. " A nucleotide analysis may include analysis of analyte nucleotide transcript levels (e.g., GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS mRNA transcript levels) in a sample, for example, a blood sample. Analyte transcript levels may be determined by Northern blot, for example, a quantitative Northern blot; or polymerase chain reaction, for example, a quantitative polymerase chain reaction. Reagents necessary to perform Northern blot include oligonucleotide probes, for example, oligonucleotide probes linked to a detectable label. A nucleotide analysis may include analysis to determine the gene and/or mRNA sequence of a gene of interest (e.g., GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS). Methods of determining genetic sequence include PCR, RNA sequencing, RNA-seq, ChIP-sequencing, Massively parallel signature sequencing (MPSS), Nanopore DNA sequencing, Polony sequencing, <NUM> pyrosequencing (<NUM> Life Sciences), Illumina (Solexa) sequencing, Single molecule real time (SMRT) sequencing (Pacific Biosciences), Combinatorial probe anchor synthesis (cPAS), SOLiD sequencing (Applied Biosystems), Ion Torrent semiconductor sequencing (Ion Torrent Sequencing; Life Technologies), DNA nanoball sequencing, Heliscope single molecule sequencing, phage display, de novo sequencing, bridge PCR, Southern blotting, shotgun DNA sequencing, and high throughput DNA sequencing. Detectable labels may include fluorescent labels or enzymes capable of reacting with a specific substrate. Reagents necessary to perform polymerase chain reaction include oligonucleotide primers capable of specifically binding to a particular analyte mRNA transcript and amplifying the number of analyte mRNA transcripts by polymerase chain reaction. Oligonucleotide primers may be linked to a detectable label to enable, for example, quantitative polymerase chain reaction. Other reagents necessary to perform quantitative polymerase chain reaction include, but are not limited to, primers capable of amplifying a control transcript signal, for instance, a beta tubulin transcript signal. Buffers, reagents (including oligonucleotide primers and probes), techniques, and equipment necessary for performing nucleotide sequencing methods described herein are readily available and are well-known in the art.

The methods described herein include, in some embodiments, determining if a cancer, tumor, or carcinoma has a GNAQ or GNA11 genetic mutation and/or a GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS genetic mutation, methods wherein the patient has been determined to have a GNAQ or GNA11 genetic tumor mutation and/or a GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS genetic mutation, methods of treating a cancer, for example, a solid tumor cancer having a GNAQ or GNA11 genetic mutation and/or a GNAQ, GNA11, BAP1, SF3B1, EIF1AX, CDKN2A, TERT, BRAF, or NRAS genetic mutation in a patient, and identifying a patient as having a tumor with a mutation that includes a specific genetic mutation, for example, a substitution of arginine in codon R183 in GNAQ or GNA11. Genetic mutations can include any of the following: insertion mutations, substitution mutations, deletion mutations, gain of function mutations, loss of function mutations, and non-synonymous mutations. An insertion mutation is the addition of one or more nucleotide base pairs into a DNA sequence. A substitution mutation can be caused by chemicals or malfunction of DNA replication, and include the exchange of a single nucleotide for another. A deletion mutation removes one or more nucleotides from the DNA, and can alter the reading frame of the gene. A gain of function mutation results in an altered gene product that possesses a new molecular function or a new pattern of gene expression. A loss of function mutation produces an altered gene product that lacks the molecular function of the equivalent wild-type gene. A non-synonymous is a genetic mutation that alters the resulting amino acid encoded by the gene.

The disclosure now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure , and are not intended to limit the disclosure in any way.

The following examples are merely illustrative and are not intended to limit the scope or content of the disclosure in any way.

The frequency of all alterations on GNAQ and GNA11 across tumor types was determined using three cancer /tumor based datasets: The Cancer Genome Atlas (TCGA), Genomics Evidence Neoplasia Information Exchange (Genie), and Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-Impact). The number of tumor samples evaluated in each dataset included <NUM> tumor samples from TCGA, <NUM> tumor samples from Genie, and <NUM> tumor samples from MSK-Impact. Many GNAQ and GNA11 alterations were identified including the activating mutations at Q209 and R183 - gain of function mutations that confer dependence on downstream PKC signaling. In order to rank these mutations for downstream validation and to assess prevalence of amino acid recurrence which resulted from missense mutations across all samples, mutation type ratio analysis was performed. The analysis of synonymous vs non-synonymous ratios was performed using the TCGA and Genie datasets.

This ranking analysis led to the identification of <NUM> mutations other than mutations at Q209 or R183, in either GNAQ or GNA11 and with a combined prevalence in tumor samples across all three datasets ranging between <NUM> to <NUM> total samples per mutation. While <NUM> mutations (other than Q209 or R183 mutations) were identified for GNAQ, a total of <NUM> non-Q209/R183 mutations were identified for GNA11, at least some of which may be activating mutations that confer dependence on downstream PKC signaling.

Following further analysis, <NUM> other, non-Q209/R183, mutations were identified in either GNAQ or GNA11 with a prevalence of <NUM> or more samples across all three datasets. These other mutations show enrichment in colorectal, lung, endometrial, and bladder cancers along with melanoma and glioma. These mutations have been listed in Table <NUM>. These results demonstrate the enrichment of other mutations in GNAQ or GNA11 in certain non-uveal forms of cancer, e.g., colorectal, non-small cell lung cancer, pancreatic cancer.

An analysis was conducted using cBioPortal (Combined Studies including a total of <NUM> samples). Table <NUM> shows the number of patients with a cancer type and mutation type (e.g., GNAQ Q209 mutation or a non Q209 mutation).

Three-dimensional coordinates were downloaded for murine Galpha-q (R183C) protein in complex with G protein signaling <NUM> (RGS2) from the RCSB protein data bank. The protein was prepared by adding hydrogens and adjusting the protonation states of ionizable groups using the protein preparation tool and epik within Maestro <NUM>-<NUM>. This was followed by a restrained minimization to converge the heavy atoms to an RMSD of <NUM>Å. Activating mutations that confer dependence on downstream PKC signaling were mapped onto the protein and visual inspection revealed residues in contact with the GDP binding site as well as secondary residues directly interacting with structural motifs involved in binding to GTP. A homology model of human GNA11 (GenBank: CAG33285. <NUM>) was built using a crystal structure of human regulator of G protein signaling <NUM> (RGS2) in complex with murine Galpha-q(R183C) (4EKC) as the template protein. Prime <NUM> was employed to build a single template, single chain model with the ligand GDP from 4EKD. Sidechain positions were predicted for non-conserved residues by mutating the template protein residue to the desired identity. Subsequently, the first low energy rotomer to not produce a clash was retained. This was followed by optimization of all non-template atoms using a Ca-Cb bond angle sampling and minimization using OPLS3e forcefield in a VSGB solvation model for all residues. A restrained minimization using a Broyden-Fletcher-Goldfarb-Shanno method to converge the heavy atoms to an RMSD (root-mean-square deviation, and a measure to assess the structural similarity between two macromolecules)of <NUM>Å. Activating mutations that confer dependence on downstream PKC signaling were mapped onto the protein as shown in <FIG> and <FIG> and visual inspection revealed residues in contact with the GDP binding site as well as secondary residues directly interacting with structural motifs involved in binding to GTP. This data indicates that mutations (including mutations other than Q209 or R183), associated with GNAQ and GNA11 in non-uveal forms of cancer affect G protein function.

This example describes the phase <NUM>/<NUM> study of compound of Formula (III) above, ("IDE196"), in patients with solid tumors harboring GNAQ/<NUM> mutations. The study consists of two phases: a dose escalation phase, followed by a dose expansion phase.

This is a phase <NUM>/<NUM>, multi-center, open-label, dose-escalation, and expansion study of IDE196 in patients with solid tumors harboring GNAQ/<NUM> mutations. A schematic diagram of the study is described in <FIG>.

Phase I Dose Escalation: A standard <NUM>+<NUM> methodology is used to commence the IDE196 dose escalation portion of the study. For dose escalation, there are estimated three to five cohorts, with a minimum of three patients with metastatic uveal melanoma (MUM), and up to <NUM>, enrolled in each cohort. Once a dose level has satisfied the DLT observation criteria (discussed in Section <NUM>), patients with non-MUM tumors harboring GNAQ/<NUM> hotspot mutations are enrolled at that dose level, contributing to the safety and efficacy evaluations, but not to the DLT assessment. Each treatment cycle comprises continuous dosing over <NUM> days. All dose escalations are based on assessment of DLTs, overall safety and tolerability, and PK that occur with each cohort, and are agreed upon between the investigators and sponsor. Dose escalation may proceed if <NUM> of <NUM> or at most <NUM> of <NUM> patients experience a DLT during the DLT assessment period in Cycle <NUM> Days <NUM> - <NUM>.

Recommended phase <NUM> dose (RP2D) may be selected based on one or more of the following: (<NUM>) the maximum tolerated dose (MTD), defined as the maximum daily oral dose at which no more than <NUM> in <NUM> patients (<<NUM>%) experience a DLT during Cycle <NUM> (safety and PK assessment period). If a DLT is observed in one of three patients, then three additional patients are enrolled at that same dose level. Dose escalation are continued until two or more of the three to six patients treated at a dose level experience a DLT. The next lower dose is then considered the MTD; (<NUM>) maximum administered dose (MAD), if no MTD is identified; (<NUM>) anti-tumor, PK and/or PD results; and/or (<NUM>) the occurrence, nature and severity of toxicities occurring after Cycle <NUM>.

A dose-limiting toxicity (DLT) is defined as a drug-related (or at least possibly related) event grade <NUM> or higher adverse event or abnormal laboratory value assessed as unrelated to disease, disease progression, inter-current illness, or concomitant medications that occurs within the first cycle of study drug with IDE196 and meets any of the criteria included below:.

For patients with normal baseline aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and total bilirubin values:.

General Exclusion Criteria for Phase I and Phase <NUM>: The presence of any of the following would exclude a patient from being eligible for the study:.

No waivers of inclusion or exclusion criteria are granted by the investigator and the sponsor or its designee for any patient enrolled into the study.

The starting dose in dose escalation is <NUM> twice daily (BID), which has been deemed safe and tolerable in an ongoing Phase I study CLXS196X2010 (EudraCT <NUM>-<NUM>-<NUM>, NCT02601378). In certain exemplary embodiments, the subsequent planned dose levels are <NUM> BID. In certain exemplary embodiments, subsequent planned dose levels are <NUM> BID. There are no intra-patient dose escalation in the dose escalation cohorts.

Concomitant antineoplastic therapies or investigational therapies other than IDE196 are prohibited. In addition, the following classes of medications are prohibited:.

The use of any concomitant non-cancer medication/therapy, including over-the-counter medications deemed necessary for the care of the patient or to treat AEs is permitted except for those specified as prohibited.

Study drug may be discontinued due to any of the following: adverse event, lost to follow-up, physician's decision, progressive disease, study terminated by the Sponsor, patient/guardian decision, protocol deviation, or non-compliance with the protocol. Study drug is discontinued if death or pregnancy occurs.

Pharmacokinetic Assessments: PK sampling is taken at various timepoints after continuous twice daily dosing. For Phase I patients, on Cycle <NUM> Day <NUM> and Cycle <NUM> Day <NUM>, the dose is followed by frequent PK sampling for <NUM> hours to determine the PK properties of IDE196 per the Schedule of Events. For Cycles <NUM> and beyond, only single trough-level PK samples are collected during each treatment cycle.

PK evaluation is based on the determination of the following parameters for IDE196 including, but not limited to: AUC<NUM>-t, AUCO-∞, AUCtau, Cmax, Tmax, T½, Vss/F, CL/F. PK assay is conducted in a central laboratory.

Efficacy Assessments: Efficacy measures include tumor assessments, consisting of clinical examination and digital photography with calipers and rulers for cutaneous lesions, and appropriate imaging techniques (preferably computed tomography (CT) scans of the chest, abdomen, and pelvis with appropriate slice thickness per RECIST v1. <NUM>); other studies (magnetic resonance imaging [MRI], X-ray, positron emission tomography [PET], and ultrasound) may be performed if required.

Patients are evaluated for disease response per RECIST v1. <NUM> with the first assessment being performed at screening, and then every <NUM> weeks (± <NUM> days) starting from the first dose for the first <NUM> cycles and every <NUM> weeks (± <NUM> days) thereafter.

Safety Assessments: Safety is assessed through AEs, changes in laboratory tests, and changes in vital signs. The incidence and duration of toxicities per the NCI-CTCAE v5. <NUM> are recorded. AEs are coded using the Medical Dictionary for Regulatory Activities (MedDRA) dictionary.

All treatment-emergent AEs are summarized as follows:.

All AE, SAE, and DLT data is listed. Change from baseline for selected laboratory parameters is summarized by treatment group and scheduled visits. All laboratory test data are listed with abnormal values flagged.

Change from baseline for vital sign parameters is summarized by group and scheduled visits; all vital signs data are listed.

The MTD and RP2D for IDE196 is determined as a function of observed toxicity. PK and PD analyses and other parameters may be considered with determining RP2D.

Archival metastatic tumor tissue samples is mandatory.

Expansion cohorts: a minimum of <NUM> paired fresh tumor biopsies in non-MUM patients enrolled in the expansion cohorts are required at both screening and between Cycle <NUM> Day <NUM> - Cycle <NUM> Day <NUM>. Tumor tissue biopsies at time of disease progression/discontinuation are optional for all patients.

For the non-MUM expansion cohorts, the Simon <NUM>-stage design is used to explore preliminary efficacy and to further characterize the safety profile of IDE196 therapy at the recommended phase <NUM> dose (R2PD).

For each of the non-MUM expansion GNAQ/<NUM> hotspot mutation cohorts, an ORR of <NUM>% is of no clinical interest (null) whereas ORR of <NUM>% is of interest. With a <NUM>% type I error and <NUM>% power, <NUM> patients are accrued in the first stage for each cohort. If zero responses are observed in the <NUM> patients for a cohort, no additional patients are enrolled to that cohort. Otherwise, <NUM> additional patients are accrued for a total of <NUM> in that cohort. Three or more responders are required from the <NUM> patients in a cohort to warrant further investigation.

Exploratory cohorts cover other GNAQ/<NUM>-hotspot mutated tumors. Approximately <NUM> patients with other GNAQ/<NUM>-hotspot mutated tumors are enrolled to assess ease of identification of these tumors and explore preliminary activity of IDE196 in this setting.

Analyses of safety, efficacy, PK, and PD is conducted. The analysis populations include:.

Claim 1:
A protein kinase C inhibitor for use in a method of treating a carcinoma having a GNAQ or GNA11 genetic mutation in a patient in need thereof, wherein the carcinoma is selected from the group consisting of pancreatic adenocarcinoma, stomach adenocarcinoma, cervical carcinoma, and lung adenocarcinoma, the method comprising:
determining if the carcinoma has a GNAQ or GNA11 genetic mutation; and
orally administering to the patient having the carcinoma with the GNAQ or GNA11 genetic mutation a therapeutically effective amount of the protein kinase C inhibitor.