Compositions and methods for treating and preventing cancer

Abstract

Provided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6-phosphate (G6PC) (e.g., by administering G6PC mimetic peptides) for treating cancer.

Description

J HU-44479.601COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING CANCERThis application claims priority to provisional patent application 63 / 748,004, filed January 22, 2025; which is herein incorporated by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORTThis invention was made with government support under grant no. DK120309 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELDProvided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6-phosphatc (G6PC) (c.g., by administering G6PC mimetic peptides) for treating cancer.BACKGROUNDCancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal bleeding, prolonged cough, unexplained weight loss, and a change in bowel movements. Over 100 types of cancers affect humans.Tobacco use is the cause of about 22% of cancer deaths. Another 10% are due to obesity, poor diet, lack of physical activity or excessive alcohol consumption. Other factors include certain infections, exposure to ionizing radiation, and environmental pollutants. In the developing world, 15% of cancers are due to infections such as Helicobacter pylori, hepatitis B, hepatitis C, human papillomavirus infection, Epstein-Barr virus and human immunodeficiency virus (HIV). These factors act, at least partly, by changing the genes of a cell. Typically, many genetic changes are required before cancer develops. Approximately 5-10% of cancers are due to inherited genetic defects. Cancer can be detected by certain signs and symptoms or screening tests. It is then typically further investigated by medical imaging and confirmed by biopsy.The risk of developing certain cancers can be reduced by not smoking, maintaining a healthy weight, limiting alcohol intake, eating plenty of vegetables, fruits, and whole grains, vaccination against certain infectious diseases, limiting consumption of processed meat andJ HU-44479.601red meat, and limiting exposure to direct sunlight. Early detection through screening is useful for cervical and colorectal cancer. The benefits of screening for breast cancer are controversial. Cancer is often treated with some combination of surgery, radiation therapy, surgery, chemotherapy and targeted therapy. Pain and symptom management are an important part of care. Palliative care is particularly important in people with advanced disease. The chance of survival depends on the type of cancer and extent of disease at the start of treatment. In children under 15 at diagnosis, the five-year survival rate in the developed world is on average 80%. For cancer in the United States, the average five-year survival rate is 66% for all ages.In 2015, about 90.5 million people worldwide had cancer. In 2019, annual cancer cases grew by 23.6 million people, and there were 10 million deaths worldwide, representing over the previous decade increases of 26% and 21%, respectively.The most common types of cancer in males are lung cancer, prostate cancer, colorectal cancer, and liver cancer. In females, the most common types arc breast cancer, colorectal cancer, lung cancer, and cervical cancer. If skin cancer other than melanoma were included in total new cancer cases each year, it would account for around 40% of cases. In children, acute lymphoblastic leukemia and brain tumors are most common, except in Africa, where non-Hodgkin lymphoma occurs more often. In 2012, about 165,000 children under 15 years of age were diagnosed with cancer. The risk of cancer increases significantly with age, and many cancers occur more commonly in developed countries. Rates are increasing as more people live to an old age and as lifestyle changes occur in the developing world. The global total economic costs of cancer were estimated at US$1.16 trillion (equivalent to $1.56 trillion in 2022) per year as of 2010.New treatments for cancer are needed.SUMMARYProvided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6-phosphate (G6PC) (e.g., by administering G6PC mimetic peptides) for treating cancer.For example, in some embodiments, provided herein is a polypeptide having an amino acid sequence of GQRPYWWVLDTDYYSNTSVPLIKQFPVTCETGPGSPSGIISRIYLAAIIFPII (SEQ ID NO:1) or GDRPFWWVHESGYYSQAPAQVHQFPSSCETGPGSPSGHAHFPHQV (SEQ ID NO: 2). In some embodiments, the polypeptide has a cell-penetration peptide added to theJ HU-44479.601N-terminal end of said polypeptide (e.g., YGRKKRRQRRR (SEQ ID NO:3)). Thus, in some embodiments, the polypeptide is YGRKKRRQRRRGQRPYWWVLDTDYYSNTSVPLIKQFPVTCETGPGSPSGHSRIYLA AHFPH (SEQ ID NO: 4) or YGRKKRRQRRRGDRPFWWVHESGYYSQAPAQVHQFPSSCETGPGSPSGHAHFPHQV (SEQ ID NO: 5).In some embodiments, the polypeptide enzymatically removes the phosphate group from glucose 6-phosphate. In some embodiments, the polypeptide mimics the active site of a glucose-6-phosphatase catalytic subunit (G6PC) (e.g., G6PC-1 or G6PC-3). In some embodiments, the polypeptide inhibits the growth of cancer cells (e.g., by decreasing the oxygen consumption rate (OCR) and decreasing extracellular acidification rate).Also provided is a composition (e.g., pharmaceutical composition) comprising a polypeptide described herein.Further provided is a method of treating cancer in a subject, comprising: administering a polypeptide or composition described herein to the subject.The present disclosure is not limited to particular cancers. Examples include but are not limited to, breast cancer, liver cancer, melanoma, pancreatic cancer, lung cancer, brain cancer, or lymphoma.The present disclosure is not limited to a particular administration method. For examples, in some embodiments, the composition is administered locally to the site of the cancer. In some embodiments, the composition is administered systematically.In some embodiments, the polypeptide inhibits the growth of or kills tumor cells but not normal cells.Further embodiments provide the use of a polypeptide or composition described herein to treat cancer in a subject.Additional embodiments provide a polypeptide or composition described herein for use in treating cancer in a subject.Additional embodiments are described herein.DESCRIPTION OF THE FIGURES FIGs. 1A-B shows the G6PC-1 (Fig. 1A) and G6PC-3 (Fig. IB) active sites. The dashed line shows the omitted amino acids.FIG. 2 shows the enzymatic activity of peptide mimics of G6PC- 1 and G6PC-3 relative to G6PC1 (G6Pase, wild type).J HU-44479.601FIGs. 3A-3E shows breast cancer MCF7 cells treated with indicated concentration of peptide mimics of G6-1PM and G6-3PM for 48 h to inhibit tumor cell growth (n=6) (Fig. 3A), and breast cancer MCF7 cells treated with 20 pM of G6-1PM or 100 mM of G6-3PM for 6 h, oxygen consumption rate (OCR) (Figs. 3B, 3C) and extracellular acidification rate (ECAR) (Figs. 3D, 3E) were determined in Seahorse XF96 Extracellular Flux Analyzer (n=6-8).FIGs. 4A-4E shows hepatoma HepG2 cells treated with indicated concentration of peptide mimics of G6-1PM and G6-3PM for 48 h to inhibit tumor cell growth (n=6) (Fig. 4A), and hepatoma HepG2 cells treated with 50 pM of G6-1PM or 100 mM of G6-3PM for 6 h, oxygen consumption rate (OCR) (Figs. 4B, 4C) and extracellular acidification rate (ECAR) (Figs. 4D, 4E) were determined in Seahorse XF96 Extracellular Flux Analyzer (n=6-8).FIGs. 5A-5C shows inhibition of B-cell lymphoma Ramos cells with 20, 40, and 80 pM of peptide mimics of G6-1PM to inhibit tumor cell growth (Fig. 5 A), and B-cell lymphoma Ramos cells treated with 50 pM of G6-1PM for 6 h significantly decreased the oxygen consumption rate (OCR) (Fig. 5B) and extracellular acidification rate (Fig. 5C) in B-cell lymphoma Ramos cells.FIGs. 6A-6C shows two million HepG2 cells and 2 or 10 nMole / g body weight of G6-1PM subcutaneously implanted in the immunodeficient NSG mice, treatment was repeated at day 3, 5, and 7 after the first treatment (n=8 mice / group). Treatment with 10 nMole / g body of G6-1PM completely suppressed the growth of HepG2 xenografts in eight NSG mice.DEFINITIONSTo facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:As used herein, the term glucose-6-phosphatase catalytic subunit 1 (G6PC1) refers to catalytic subunit one of the G6PC protein (e.g., having accession number NP_000142.2, encoded by the G6PC1 gene (e.g., having accession numbers NM_000151.4 (mRNA) and NG_011808.1 (DNA)).As used herein, the term glucose-6-phosphatase catalytic subunit 2 (G6PC2) refers to catalytic subunit two of the G6PC protein (e.g., having accession number NP_066999.1), encoded by the G6PC2 gene (e.g., having accession numbers NM_021176 (mRNA) and NGJ111682 (DNA)).As used herein, the term glucose-6-phosphatase catalytic subunit 3 (G6PC3) refers to catalytic subunit one of the G6PC protein (e.g., having accession number NP_612396),J HU-44479.601encoded by the G6PC3 gene (e.g., having accession numbers NM_138387.4 (mRNA) and NG_015818 (DNA)).As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.As used herein, the term "subject diagnosed with cancer" refers to a subject who has been tested and found to have cancer. As used herein, the term "initial diagnosis" refers to a test result of initial disease that reveals the presence or absence of disease.As used herein, the term "non-human animals" refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term arc continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.As used herein, the term "eukaryote" refers to organisms distinguishable from "prokaryotes." It is intended that the term encompass all organisms with cells that exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus bounded by a nuclear membrane, within which lie the chromosomes, the presence of membrane-bound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, the term includes, but is not limited to such organisms as fungi, protozoa, and animals (e.g., humans).As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.The terms "test compound" and "candidate compound" refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., cancer). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.J HU-44479.601As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, waler, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.As used herein, the term “effective amount” refers to the amount of a compound (e.g., a polypeptide mimetic described herein) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not limited to or intended to be limited to a particular formulation or administration route.As used herein, the term “co-administration” refers to the administration of at least two agcnt(s) (e.g., polypeptide described herein) or therapies to a subject. In some embodiments, the co-administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents / therapies are coadministered, the respective agents / therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents / therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo, or ex vivo.As used herein, the term “toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. The peptide or polypeptide may be modified by the addition of sugars, lipids or other moieties not included in the amino acid chain. The terms “polypeptide”, “oligopeptide,” and “peptide” are used interchangeably herein. The peptide(s) may be produced by recombinant genetic technology or chemical synthesis. The peptide(s)J HU-44479.601may be isolated and purified by any number of standard methods including, but not limited to, differential solubility (e.g., precipitation), centrifugation, chromatography (e.g., affinity, ion exchange, and size exclusion), or by any other standard techniques known in the art.The recitations “sequence identity,” “percent identity,” “percent homology,” “percent similarity,” or, for example, comprising a “sequence 50% identical to” or “sequence with at least 50% similarity to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman andJ HU-44479.601Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GOG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.The peptide or polypeptide sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et aL, (1990, J. Mol. Biol, 215: 403-10).BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules.BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.The term “amino acid” or “any amino acid” as used here refers to any and all amino acids, including naturally occurring amino acids (e.g., a-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, such as, e.g., the 23 amino acids that combine into peptide chains to form the building-blocks of a vast array of proteins. These are primarily L stereoisomers, although a few D-amino acids occur in bacterial envelopes and some antibiotics. The “non-standard,” natural amino acids include, for example, pyrolysine (found in methanogenic organisms and other eukaryotes), selenocysteine (present in many non-eukaryotes as well as most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). “Unnatural” or “non-natural” aminoJ HU-44479.601acids are non-proteinogenic amino acids (e.g., those not naturally encoded or found in the genetic code) that either occur naturally or are chemically synthesized. Over 140 unnatural amino acids are known and thousands of more combinations are possible. Examples of “unnatural” amino acids include P-amino acids ( 3 and 2), homo-amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally present on the amino acid. Reference to a particular amino acid is meant to encompass that amino acid in both its unbonded and bonded state. For example, the amino acid residue homoSerine (hSer) or homoSerine(Cl) in its unbonded form may take the form of 2-aminobutyric acid (Abu) when participating in an intramolecular bond.For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions suggested by the IUPAC Commission on the Nomenclature of Organic Chemistry and the 1UPAC-IUB Commission on Biochemical Nomenclature as set out in “Nomenclature of a- Amino Acids (Recommendations, 1974)” Biochemistry, 14(2), (1975). To the extent that the names and abbreviations of amino acids and aminoacyl residues employed in this specification and appended claims differ from those suggestions, they will be made clear to the reader.

[0039] Throughout the present specification, unless naturally occurring amino acids are referred to by their full name (e.g., alanine, arginine, etc.), they are designated by their conventional three-letter or single-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term “L-amino acid,” as used herein, refers to the “L” isomeric form of a peptide, and conversely the term “D-amino acid” refers to the “D” isomeric form of a peptide (e.g., Dphe, (D)Phe, D-Phe, or DF for the D isomeric form of Phenylalanine). Amino acid residues in the D isomeric form can be substituted for any L-amino acid residue, as long as the desired function is retained by the polypeptide.In the case of less common or non-naturally occurring amino acids, unless they are referred to by their full name (e.g. sarcosine, ornithine, etc.), frequently employed three- or four-character codes are employed for residues thereof, including, Sar or Sarc (sarcosine, i.e. N-methylglycine), Aib (a-aminoisobutyric acid), Dab (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), y-Glu ( / -glutamic acid), Gaba (y-aminobutanoic acid), 0-Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-aminoJ HU-44479.601butyric acid), phPro (P-homoproline), phPhe (P-homophenylalanine) and Bip (P,P diphenylalanine), and Ida (Iminodiacetic acid).The term “pharmaceutically acceptable salt” in the context of the present disclosure (pharmaceutically acceptable salt of a peptide, mimetic, or polypeptide described herein) refers to a salt which is not harmful to a patient or subject to which the salt in question is administered. It may suitably be a salt chosen, e.g., among acid addition salts and basic salts. Representative salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bi sulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3 -phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, trichloroacetate, trifluoroacetate, glutamate, para-toluenesulfonate, undecanoate, hydrochloric, hydrobromic, sulfuric, phosphoric and the like. The amino groups of the peptides may also be quatcmizcd with alkyl chlorides, bromides, and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like. Other examples of pharmaceutically acceptable salts are described in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977).DETAILED DESCRIPTION OF THE DISCLOSUREProvided herein are compositions and methods for treating cancer. In particular, provided herein are compositions, methods, and uses of increasing the level of glucose-6-phosphate (G6PC) (e.g., by administering G6PC mimetic peptides) for treating cancer.A century ago, Otto Warburg discovered that tumor cells utilized tremendously more glucose and produced large amounts of lactate than normal differentiated cells even in the presence of sufficient oxygen to support mitochondrial oxidative phosphorylation (Warburg O. On the metabolism of cancer cells. Naturwissenschaften. 1924; 12:1131-7). This phenomenon is referred to as aerobic glycolysis, and also termed “the Warburg effect”. All proliferating cells have great bioenergetic and biosynthetic demand to produce new cells, imposing a large requirement for ATP, ribose 5-phosphate, and intermediates that are needed for the synthesis of nucleotides, lipids, and amino acids (Ortega AD, Sanchez- Arago M, Giner-Sanchez D, Sanchez-Cenizo L, Willers I, and Cuezva JM. Glucose avidity ofJ HU-44479.601carcinomas. Cancer Lett. 2009;276:125-35; Koppenol WH, Bounds PL, and Dang CV. Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer.2011; 11:325-37; Vander Heiden MG, and DeBerardinis RJ. Understanding the Intersections between Metabolism and Cancer Biology. Cell. 2017;168:657-69; Ghergurovich JM, Lang JD, Levin MK, Briones N, Facista SJ, Mueller C, et al. Local production of lactate, ribose phosphate, and amino acids within human triple-negative breast cancer. Med (N Y).2021;2:736-54). Tumor cells exhibit active anabolic metabolism to generate requisite building blocks, and thus, the complete metabolism of glucose to CO2 and to maximize ATP production through oxidative phosphorylation is against the needs of the proliferating tumor cells. Glucose can be used to generate NADPH, ribose 5-phosphate, and 3 or 6 carbons (glyceraldehyde 3-phosphate or fructose 6-phosphate) via pentose phosphate pathway. While through glycolysis, one molecule glucose will generate 2 molecules of pyruvate, and 2 ATPs. Pyruvate can be converted to acetyl-CoA or oxaloacetate by pyruvate dehydrogenase or pyruvate carboxylase respectively. Acetyl-CoA and oxaloacetate can enter the mitochondrial tricarboxylic acid (TCA) cycle to replenish the intermediates that can be used to make amino acids. Acetyl-CoA can also be used to synthesize the lipids. Therefore, glucose metabolism can provide ATP, NADPH, carbon skeleton for the synthesis of the needed building blocks (nucleotides, lipids, and amino acids) in proliferating tumor cells. However, excessive pyruvate will be converted to lactate to regenerate the NAD+, which is a limiting factor for glycolysis. Based on the critical role of aerobic glycolysis in tumor cells’ proliferation, targeting glycolysis is an attractive therapeutic intervention for tumor treatment (Amer EN, and Rathmell JC. Metabolic programming and immune suppression in the tumor microenvironment. Cancer Cell. 2023;41(3):421-33; Stine ZE, Schug ZT, Salvino JM, and Dang CV. Targeting cancer metabolism in the era of precision oncology. Nat Rev Drug Discov. 2022;21(2): 141-62).In the first step of glycolysis, glucose is required to be phosphorylated to yield glucose 6-phosphate for subsequent reactions. In a reversible reaction, glucose 6-phosphatase catalytic subunit (G6PC) catalyzes the conversion of glucose 6-phosphate to glucose. Several solid tumors, including hepatocellular carcinoma (HCC), have low G6PC expression (Tian L, and Liao Y. Identification of G6PC as a potential prognostic biomarker in hepatocellular carcinoma based on bioinformatics analysis. Medicine (Baltimore). 2022;101(33):e29548; Xu WH, Xu Y, Tian X, Anwaier A, Liu WR, Wang J, et al. Large-scale transcriptome profiles reveal robust 20-signatures metabolic prediction models and novel role of G6PC in clear cell renal cell carcinoma. J Cell Mol Med. 2020;24(16):9012-27) and the low G6PC expression isJ HU-44479.601correlated to poor survival rate of these patients (Tian L, and Liao Y. Identification of G6PC as a potential prognostic biomarker in hepatocellular carcinoma based on bioinformatics analysis. Medicine (Baltimore). 2022;101(33):e29548; Xu WH, Xu Y, Tian X, Anwaier A, Liu WR, Wang J, et al. Large-scale transcriptome profiles reveal robust 20-signatures metabolic prediction models and novel role of G6PC in clear cell renal cell carcinoma. J Cell Mol Med. 2020;24(16):9012-27). Moreover, low G6PC expression can promote liver tumor formation through glycogen accumulation (Liu Q, Li J, Zhang W, Xiao C, Zhang S, Nian C, et al. Glycogen accumulation and phase separation drives liver tumor initiation. Cell.2021;184(22):5559-76 el9). In this respect, increasing G6PC expression provides a therapeutic strategy for tumor treatment through reduction of the use of glucose. In particular, AMPK can regulate the expression of G6PC (He L, Sabet A, Djedjos S, Miller R, Sun X, Hussain MA, et al. Metformin and insulin suppress hepatic gluconeogenesis through phosphorylation of CREB binding protein. Cell. 2009;137(4):635-46; Wang Y, An H, Liu T, Qin C, Scsaki H, Guo S, ct al. Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK. Cell Rep. 2019;29(6): 1511-23 e5). Experiments described herein investigated the antitumor effects of G6PC in inhibiting glucose utilization in tumor cells and resulted in the identification of agents that inhibit the growth of tumors (e.g., by inhibiting glucose utilization).Accordingly, provided herein are G6PC polypeptide mimetics for use in research, screening and therapeutic (e.g., treating cancer) applications.The present disclosure contemplates G6PC1 mimetic polypeptides (e.g., thoseshown in NOs:l and 4) or polypeptides with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 1 and 4.The present disclosure further contemplates G6PC3 mimetic polypeptides ((e.g., those shown in SEQ ID NO:2 and 5) or polypeptides with at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to SEQ ID NOs: 2 and 5.In some embodiments, polypeptides comprise one or more additions (e.g., cell penetrating peptides such as those shown in SEQ ID NO:3). Exemplary peptides with cellpenetrating peptides are described by SEQ ID NOs: 4 and 5.In some embodiments, polypeptides comprises one or more substitutions relative to SEQ ID NOs: 1, 2, 4, or 5. In some embodiments, polypeptides comprise one or more (e.g., I, 2,34, 5, or more) insertions or additions to the C or N terminus of amino acids to the peptides of SEQ ID NOs: 1, 2, 4, or 5.J HU-44479.601In some embodiments, the polypeptide comprises, consisting essentially of, or consists of a polypeptide described by SEQ ID NOs: 1, 2, 4, or 4.In some embodiments, polypeptides comprise one or more additional components useful in aiding the polypeptide in entering a cell. For example, in some embodiments, polypeptides are attached to a nanomaterial such as a nanoparticle (See e.g., ((Harish, V. et al. Review on Nanoparticles and Nanostructured Materials: Bioimaging, Biosensing, Drug Delivery, Tissue Engineering, Antimicrobial, and Agro-Food Applications. Nanomaterials (Basel) 12, doi:10.3390 / nanol 2030457 (2022); herein incorporated by reference in its entirety).Variants or analogs can differ from the polypeptides described herein by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d cd.), CSH Press, 1989, or Current Protocols in Molecular Biology" (Ausubel, 1987). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., P or y amino acids.Amino acids include naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxyglutamate, and O- phosphoserine, phosphothreonine. An amino acid analog is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium), but that contains some alteration not found in a naturally occurring amino acid (e.g., a modified side chain); the term "amino acid mimetic" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acid analogs may have modified R groups (for example, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In one embodiment, an amino acid analog is a D-amino acid, a P-amino acid, or an N-methyl amino acid.Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB BiochemicalJ HU-44479.601Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Non-protein analogs having a chemical structure designed to mimic functional activity of the peptides described herein can be administered according to methods of the disclosure. Variants and analogs may exceed the physiological activity of the original peptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs exhibit the activity of a reference polypeptide. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. Preferably, the polypeptide or peptide analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.In some embodiments, candidate polypeptides are screened for activity (e.g., using the methods described in the experimental section below or another suitable assay).In some embodiments, nucleic acids encoding the G6PC mimetics are provided. In order to express a gene encoding a G6PC polypeptide mimetic, one introduces a nucleic acid encoding the gene of interest (e.g., on a plasmid or other vector or as a naked nucleic acid) into a subject. In such instance, a vector suitable for in vivo administration would be utilized, including but not limited to a virus such as retroviruses, adenoviruses, adeno-associated viruses, herpes simplex virus, and the like. See Lundstrom, Viral Vectors in Gene Therapy, Diseases, 2018, 6(2):42. Alternatively, components of the system are administered to a subject via naked polynucleotides (e.g. naked DNA), or physical vehicles such as liposomes and nanopaiticles.Vectors may comprise a nucleic acid sequence into which a foreign nucleic acid sequence is inserted. A common way to insert one segment of nucleic acid sequence into another segment of a nucleic acid sequence involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double- stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily be introduced into a suitable cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA.J HU-44479.601Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of vectors, including plasmid and fungal vectors which replicate or exist episomally, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.The present disclosure further provides pharmaceutical compositions (e.g., comprising the nucleic acids or polypeptides described above). The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical earners, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.J HU-44479.601Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.The compositions of the present disclosure may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers.In one embodiment of the present disclosure the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and / orJ HU-44479.601aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the polypeptide is administered in maintenance doses, ranging from 0.1 to 5 mg per kg of body weight, once or more daily, to once every 20 years.Methods of treating cancerIn some embodiments, the polypeptides described herein find use in the treatment of cancers.The present disclosure is not limited to particular cancers. Examples include but are not limited to, breast cancer, liver cancer, melanoma, pancreatic cancer, lung cancer, brain cancer, or lymphomas (e.g., B-cell lymphoma, T-cell lymphoma, or Hodgkin’s lymphoma).In some embodiments, the agents described herein are administered in combination with one or more additional agents, treatment, or interventions (e.g., agents, treatments, or interventions useful in the treatment of cancer.Examples of anti-cancer therapies include targeting cancer therapy (e.g., targeting the cancer markers described herein), surgery, chemotherapy, radiation therapy, immunotherapy / biological therapy, and photodynamic therapy.Chemotherapeutic agents may also be used for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, anti-metabolites, plant alkaloids and terpenoids, vinca alkaloids, podophyllotoxin, taxanes, topoisomerase inhibitors, and cytotoxic antibiotics. Cisplatin, carboplatin, and oxaliplatin are examples of alkylating agents. OtherJ HU-44479.601alkylating agents include mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide. Alkylating agents may impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules.Alternatively, alkylating agents may chemically modify a cell's DNA.Biological therapy (sometimes called immunotherapy, biotherapy, or biological response modifier (BRM) therapy) uses the body’s immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments. Biological therapies include interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents. In some embodiments, the biological therapy is immune checkpoint therapy. Immune checkpoint inhibitors target CTLA-4, PD-1, or PD-L1. Examples include but are not limited to, ipilimumab, nivolumab, pembrolizumab, spartalizumab, and atezolizumab.EXPERIMENTALThe following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.Example 1Peptides that mimic the active site of G6PC-1 and G6PC-3 and that have the enzymatic activity to remove the phosphate group from glucose 6-phosphate were designed (FIG. 1) (Ghosh, et al. The Journal of Biological Chemistry, 2002, 277 32837-328420;Ghosh, et al. The Journal of Biological Chemistry, 2004, 279 12479-12483).The following peptide mimics were designed:1. Peptide mimic of G6PC- 1 :GQRPYWWVLDTDYYSNTSVPLIKQFPVTCETGPGSPSGHSRIYLAAHFPH (SEQ ID NO:1)2. Peptide mimic of G6PC-3:GDRPFWWVHESGYYSQAPAQVHQFPSSCETGPGSPSGHAHFPHQV (SEQ ID NO:2)J HU-44479.601To test whether these peptide mimics impact cellular glucose metabolism, a cell-penetration peptide was added to the N-terminal of these peptide mimics to generate the following peptide mimetics:1. G6-1PM:YGRKKRRQRRRGQRPYWWVLDTDYYSNTSVPLIKQFPVTCETGPGSPSGHSRIYLA AHFPH (SEQ ID NO: 4)2. G6-3PM:YGRKKRRQRRRGDRPFWWVHESGYYSQAPAQVHQFPSSCETGPGSPSGHAHFPHQV (SEQ ID NO:5)Peptide mimics G6-1PM and G6-3PM exhibited 33.1% and 28.3% of the enzymatic activity of native G6PC-1 protein in an in vitro assay using a Glucose-6-Phosphatase Assay Kit (Biomedical Research Service, University at Buffalo) (FIG. 2).Since G6-1PM exhibited relatively higher enzymatic activity, its effects on inhibiting the growth of breast cancer MCF7 cells, Hepatoma HepG2 cells, and B-lymphoma Ramos cells was assayed. Treatment with 20, 40, or 80 pM of both peptide mimics of G6-1PM and G6-3PM significantly inhibited the growth of breast cancer MCF7 cells in a concentrationdependent manner (FIG. 3A).Next, experiments were performed to assess the effects of the peptide mimics on glucose uptake and utilization in a Seahorse assay (Pearah, et al. Cell Chemical Biology, 2023, 30, 1585-1600). Treatment with either G6-1PM or G6-3PM for 6 h significantly decreased the oxygen consumption rate (OCR) in breast cancer MCF7 cells (FIG. 3B, C). Furthermore, these treatments also significantly decreased the extracellular acidification rate (FIG. 3D, E), which reflects decreased glycolytic flux and lactate efflux (Divakaruni, et al. Methods Enzymology 2014, 547, 309-354).Treatment with 20, 40, or 80 pM of both peptide mimics of G6-1PM and G6-3PM also significantly inhibited the growth of hepatoma HepG2 cells in a concentration-dependent manner (FIG. 4A).In Hepatoma HepG2 cells, treatment with either G6-1PM or G6-3PM for 6 h also significantly decreased the oxygen consumption rate (OCR) (FIG. 4B, C). These treatments also significantly decreased the extracellular acidification rate, which reflects decrease glycolytic flux and lactate efflux (FIG. 4D, E) (Divakaruni, et al. Methods Enzymology 2014, 547, 309-354).J HU-44479.601Treatment with 20, 40, 80 jiM of peptide mimic G6-1PM significantly inhibited the growth of B-lymphoma Ramos cells in a concentration-dependent manner (Fig. 5A).Next, experiments were performed to assess the effect of peptide mimic G6-1PM on glucose uptake and utilization in a Seahorse assay (Pearah, et al. Cell Chemical Biology, 2023, 30, 1585-1600). Treatment with G6-1PM for 6 h significantly decreased the oxygen consumption rate (OCR) in B-cell lymphoma Ramos cells. This treatment also significantly decreased the extracellular acidification rate, which reflects a decrease in glycolytic flux and lactate efflux (Divakaruni, et al. Methods Enzymology 2014, 547, 309-354) (FIG. 5B, C).Collectively, these results indicate that G6PC peptide mimics can significantly decrease glucose uptake and utilization.An in vivo animal experiment was performed to test the antitumor effect of G6-1PM. Two million HepG2 cells and 2 or 10 nMole / g body weight of G6-1PM with Vitrogel were subcutaneously implanted in immunodeficient NSG mice. The treatment was repeated at day 3, 5, and 7. Strikingly, treatment with 10 nMolc / g body of G6-1PM completely suppressed the growth of HepG2 xenografts in eight NSG mice (Fig. 6A-C). In addition, in another group, NSG mice treated with 5 times lower dosage of G6-1PM (2 nMole / g body weight), there was only one mouse in this group (seven mice in total) that had a smaller tumor.All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled relevant fields are intended to be within the scope of the following claims.

Claims

1. J HU-44479.601CLAIMSWe claim:

1. A polypeptide having an amino acid sequence of GQRPYWWVLDTDYYSNTSVPLIKQFPVTCETGPGSPSGHSRIYLAAHFPH (SEQ ID NO:1).

2. A polypeptide having an amino acid sequence of:GDRPFWWVHESGYYSQAPAQVHQFPSSCETGPGSPSGHAHFPHQV (SEQ ID NO: 2).

3. The polypeptide of claim 1 or 2, wherein said polypeptide has a cellpenetration peptide added to the N-terminal end of said polypeptide.

4. The polypeptide of claim 3, wherein said cell-penetration peptide has an amino acid sequence of YGRKKRRQRRR (SEQ ID NO:3).

5. The polypeptide of any one of the preceding claims, wherein said polypeptide enzymatically removes the phosphate group from glucose 6-phosphate.

6. The polypeptide of any one of the preceding claims, wherein said polypeptide mimics the active site of a glucose-6-phosphatase catalytic subunit (G6PC).

7. The polypeptide of claim 6, wherein said G6PC is G6PC-1 or G6PC-3.

8. The polypeptide of any one of the preceding claims, wherein said polypeptide inhibits the growth of cancer cells.

9. A composition comprising the polypeptide of any one of the preceding claims.

10. The composition of claim 9, wherein said composition is a pharmaceutical composition.J HU-44479.60111. A method of treating cancer in a subject, comprising:administering the polypeptide or composition of any one of the preceding claims to said subject.

12. The method of claim 11, wherein said cancer is selected from the group consisting of breast cancer, liver cancer, melanoma, pancreatic cancer, lung cancer, brain cancer, and lymphoma.

13. The method of claim 11 or 12, wherein said polypeptide or composition is administered locally to the site of said cancer.

14. The method of claim 11 or 12, wherein said polypeptide or composition is administered systematically.

15. fhe use of the polypeptide or composition of any one of the preceding claims to treat cancer in a subject.

16. A polypeptide or composition of any one of the preceding claims for use in treating cancer in a subject.

17. The composition of claim 16, wherein said composition is a pharmaceutical composition.

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