Compositions and methods for the treatment of KRAS mutant cancers

Abstract

The present disclosure provides methods and compositions for treating KRAS cancers in a subject comprising the use of therapeutic compounds that increase the expression or activity of TUSC2. The present disclosure also provides methods and compositions for treating KRAS mutant cancers in a subject comprising the use of inhibitors of KRAS in combination with therapeutic compounds that increase the expression level or activity of TUSC2.

Description

TITLE OF THE INVENTIONCOMPOSITIONS AND METHODS FOR THE TREATMENT OF KRAS MUTANT CANCERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of U.S. Provisional Appl. Ser. No. 63 / 689,949, filed September 3, 2024, and U.S. Provisional Appl. Ser. No. 63 / 793,838, filed April 24, 2025, the entire disclosure of each of which is incorporated herein by reference.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named “MDCC026WO_ST26.xml” which is 3,102 bytes (measured in MS-Windows®) and created August 15, 2025, and comprises 2 sequences, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of cancer therapy, and more specifically to compositions and methods for the treatment of Kirsten rat sarcoma viral oncogene (KRAS) mutant cancers.BACKGROUND OF THE INVENTION

[0004] The emergence of acquired resistance (AR) to FDA-approved KRAS inhibitors poses a significant challenge in the treatment of KRAS mutant cancers. For example, despite an initial response rate of up to 40% for sotorasib, the first FDA-approved KRAS inhibitor, patients with KRASG12Cmutant non-small cell lung cancer (NSCLC) invariably develop resistance, necessitating alternative therapeutic strategies. The mechanisms underlying acquired resistance include the emergence of additional mutations in the KRAS gene, reactivation of the KRAS pathway, or activation of alternative signaling pathways. Tumor suppressor candidate 2 (TUSC2), a potent tumor suppressor gene with immunogenic properties, exhibits multifunctional activity by directly inhibiting downstream signaling pathways, including MAPK and mTOR, arresting the growth and proliferation of cancer cells, inducing direct tumor cell death, and activating both innate and adaptive immune responses. The present disclosure demonstrates for the first time that1US_ACTIVE\131015922W-1TUSC2 gene therapy effectively overcomes acquired resistance to KRAS inhibitor in KRAS mutant cancer.SUMMARY OF THE INVENTION

[0005] In one aspect, the present disclosure provides a method of treating a subject afflicted with or at risk of developing a cancer comprising a KRAS mutation, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of KRAS and a therapeutically effective amount of a therapy that increases the expression or activity of a TUSC2 polypeptide.

[0006] In another aspect, the present disclosure provides a method of treating a subject afflicted with a cancer comprising a KRAS mutation, wherein the cancer comprises resistance to an inhibitor of KRAS, the method comprising administering to the subject a therapeutically effective amount of a therapy that increases the expression or activity of a TUSC2 polypeptide. In certain embodiments, the methods of the present disclosure may further comprise administering an inhibitor of KRAS or at least a second inhibitor of KRAS to the subject.

[0007] In one embodiment, the KRAS mutation is a missense mutation. In another embodiment, the KRAS mutation is selected from the group consisting of G12D, G12V, G13D, G12C, G12A, Q61H, G12R, A146T, G13C, G12S, Q61L, Q22K, A146V, G12F, Q61R, E63K, L19F, D33E, A59G, T74P, and K117R. In yet another embodiment, the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, choloangiocarcinoma, uterine endometrial carcinoma, testicular germ cell cancer, and cervical squamous cell carcinoma. Non-limiting examples of inhibitors of KRAS that may be used according to embodiments of the present disclosure include sotorasib, adagrasib, MRTX1133, ARS-3248, ARS-853, ARS-1620, opnurasib, GDC-6036, RG6330, D-1553, BP1-421286, GH35, BEBT-607, JAB-21000, Bl-2865, Bl-2493, RMC-6236 and combinations of any thereof.

[0008] The method of the present disclosure may comprise, in one embodiment, administering a TUSC2 polypeptide or a fragment thereof to the subject. In another embodiment, the methods of the present disclosure may comprise administering a polynucleotide molecule encoding a TUSC2 polypeptide or a fragment thereof to the subject. The TUSC2 polypeptide, in yet another2US_ACTIVE\131015922W-1embodiment, comprises a sequence having at least about 85%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1. The polynucleotide molecule encoding the TUSC2 polypeptide, in still yet another embodiment, comprises a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:2. In one embodiment, the methods of the present disclosure may comprise administering an expression vector comprising the polynucleotide molecule encoding the TUSC2 polypeptide or a fragment thereof to the subject. The methods of the present disclosure, in another embodiment, may comprise administering a pharmaceutical composition comprising the expression vector to the subject. In yet another embodiment, the pharmaceutical composition comprises quaratusugene ozeplasmid.

[0009] In one embodiment, the cancer comprises resistance to an inhibitor of KRAS. The resistance, in another embodiment, is an acquired resistance. In yet another embodiment, the subject may have been previously treated with at least one inhibitor of KRAS prior to administering a therapy as described herein. In another embodiment, the methods of the present disclosure may comprise administering a pharmaceutical composition comprising the inhibitor of KRAS or the therapy that increases the expression or activity of the TUSC2 polypeptide to the subject.

[0010] In certain embodiments, administering may comprise local, regional, systemic, or continual administration. The administering, in one embodiment, may comprise administering a single dose to the subject. The administering, in another embodiment, may comprise administering multiple doses to the subject. In yet another embodiment, administering may comprise oral, intravenous, or intramuscular administration. In still yet another embodiment, the methods of the present disclosure may further comprise administering an immunotherapy, a targeted cancer therapy, a chemotherapy, a radiation therapy, or surgery to the subject. In one embodiment, the subject is a mammalian subject. In another embodiment, the subject is a human subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by3US_ACTIVE\131015922W-1reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] FIG. 1A shows the sensitivity or resistance of the TC303 (sensitive), TC303AR (resistant), TC314 (sensitive), and TC314AR (resistant) PDX models to sotorasib (AMG510). FIGs. IB, 1C, and ID show results of an analysis of lobal proteomics and phosphoproteomics of AR PDXs. FIG. IB - Heatmaps show the upregulation of a distinct set of proteins in AR PDXs (TC303AR & TC3 MAR) as compared to their sensitive counterparts. FIG. 1C - Enrichment analysis in global and phosphoproteomics in TC314AR shows that MTORC1 & KRAS and MT0RC1 & PI3K- AKT-MTOR pathways are significantly upregulated, respectively. FIG. ID - Reverse-phase protein array (RPPA) analysis on TC303AR and TC3 MAR PDXs showing upregulation of MAPK and PI3K-AKT-mT0R signaling molecules.

[0013] FIG. 2 shows the sensitivity or resistance of the H23 (sensitive), H23AR (resistant), H358 (sensitive), and H358AR (resistant) cell lines to sotorasib (AMG510), adagrasib, and opnurasib.

[0014] FIG. 3 shows the results of whole genome sequencing of the H23 (sensitive), H23AR (resistant), H358 (sensitive), and H358AR (resistant) cell lines.

[0015] FIG. 4A shows the results of a colony formation assay performed using H23AR and H358AR cells. Briefly, sotorasib acquired resistant cells were co-transfected with TUSC2 / empty vector and pcDNA plasmids. Colonies were selected based on selection markers for several weeks, fixed, and imaged. FIG. 4B shows apoptosis of AR H23AR and H358AR cells after TUSC2 transfection as shown by the increase in annexin- V-positive apoptotic cells.

[0016] FIG. 5 demonstrates the antitumor effect of TUSC2 both alone and in combination with sotorasib (AMG510) in the TC314AR PDX model, including the significant antitumor effect of TUSC2 and combination therapy (top left), the percentage of tumor volume change following treatment at day 50 (top right), and individual mouse responses following treatment (bottom).

[0017] FIG. 6 demonstrates the antitumor effect of TUSC2 both alone and in combination with sotorasib (AMG510) on H23AR xenograft tumors, including the significant antitumor effect of4US_ACTIVE\131015922W-1TUSC2 and combination therapy (left panel) and individual mouse responses following treatment (right panel).

[0018] FIG. 7A demonstrates the antitumor effect of TUSC2 both alone and in combination with sotorasib (AMG510) on TC314AR PDX tumors in a humanized mouse model, including the significant antitumor effect of TUSC2 and combination therapy (right panel) and individual mouse responses following treatment (bottom left panel). Humanization status was confirmed for each humanized mouse before implantation of the TC314AR PDXs (top left panel). FIG. 7B shows the results from a tumor microenvironment analysis in humanized mice: (upper panels) effect on human CD45, CD3 T, CD4 T, CD8 T, Treg; (middle panels) effect on human NK, PD1+CD3 T, PD1+CD8 T, PD1+NK, Effector memory CD3 & CD8 T cells; (bottom panels) effect on human MDSC, DC, Ml & M2 MQ, Residential memory CD3 T and residential memory NK cells. * = p < 0.05, ** = p<0.005, and = p < 0.0005.

[0019] FIGS. 8A, 8B, 8C, and 8D show that TUSC2 is cytotoxic for AR PDXOs. FIG. 8A - Sotorasib-resistant PDX-derived organoids (PDXOs) were generated. FIG. 8B - Sotorasib sensitivity of two isogeneic AR PDXOs is shown. FIG. 8C - Evaluation of TUSC2 transfection on PDXOs by fluorescence imaging (left) and RT PCR (right). FIG. 8D. Cell viability as assessed by Glow 3D assay of AR PDXOs after TUSC2 transfection.

[0020] FIGS. 9A, 9B, 9C, 9D, 9E, 9F 9G, 9H, 91, 9J, and 9K illustrate the TUSC2 antitumor immune response in H23AR xenografts in humanized mice. FIG. 9A - Experimental strategy. FIG. 9B - Antitumor effect of TUSC2 in combination with sotorasib. FIG. 9C - Individual mouse responses. FIG. 9D, FIG. 9E. FIG. 9F, FIG. 9G, and FIG. 9H - Tumor microenvironment analysis for lymphoid cells in humanized mice after treatment. FIG. 91, FIG. 9J, and FIG. 9K - TME analysis for myeloid cells in humanized mice after treatment. * = p < 0.05, ** = p<0.005.BRIEF DESCRIPTION OF THE SEQUENCES

[0021] SEQ ID NO:1 is a representative sequence of a TUSC2 polypeptide.

[0022] SEQ ID NO:2 is a representative nucleotide sequence that encodes a TUSC2 polypeptide.5US_ACTIVE\131015922W-1DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides methods and compositions for the treatment of KRAS mutant cancers. The emergence of acquired resistance to FDA-approved KRAS inhibitors poses a significant challenge in the treatment of KRAS mutant cancers. Despite a relatively high initial response rate, patients invariably develop resistance, necessitating alternative therapeutic strategies. Thus, there is a continuing need for new therapies and therapeutic strategies for the treatment of KRAS mutant cancers. The present disclosure describes such therapeutic strategies.

[0024] The mechanisms underlying acquired resistance to KRAS inhibitors include the emergence of additional mutations in the KRAS gene, reactivation of the KRAS pathway, or activation of alternative signaling pathways. TUSC2, a potent tumor suppressor gene with immunogenic properties, exhibits multifunctional activity by directly inhibiting downstream signaling pathways, including MAPK and mTOR, arresting the growth and proliferation of cancer cells, inducing direct tumor cell death, and activating both innate and adaptive immune responses. The present disclosure demonstrates for the first time that TUSC2 gene therapy effectively overcomes acquired resistance to inhibitors of KRAS in KRAS mutant cancers.A. Therapeutic Compounds or Compositions and Administration Thereof

[0025] In accordance with the present disclosure, any inhibitor of KRAS may be used to treat or prevent KRAS mutant cancer. Non-limiting examples of inhibitors of KRAS include sotorasib, adagrasib, MRTX1133, ARS-3248, ARS-853, ARS-1620, opnurasib, GDC-6036, RG6330, D- 1553, BPI-421286, GH35, BEBT-607, JAB-21000, and BI-2865, BI-2493, RMC-6236. According to the present disclosure, any therapeutic molecule may be used to treat or prevent a KRAS mutant cancer by decreasing mutant KRAS expression or inhibiting its function. Nonlimiting examples of such therapeutic molecules include a protein, a peptide, a polypeptide, an RNA molecule, a peptidomimetic, an siRNA molecule, a gRNA molecule, or a small molecule, and the like. In some embodiments, such therapeutic compounds target KRAS mRNA or protein to reduce the activity of KRAS in a cell or individual subject. In one embodiment, the inhibitor of KRAS function is a small molecule inhibitor that covalently binds to mutant KRAS and effectively locks KRAS in its inactive form.

[0026] In another embodiment, liposomes or vectors may be used to provide therapeutic molecules of interest to a subject for targeting KRAS or mutant KRAS. In further embodiments, a therapeutic 6US_ACTIVE\131015922W-1compound described herein may be useful for targeting cells in the subject. In some embodiments, an siRNA for use according to the present disclosure may be complementary to a portion of the mRNA sequence encoded by the KRAS gene. In certain embodiments, a gRNA for use according to the present disclosure may be complementary to at least a portion the KRAS gene. In an embodiment, the siRNA molecule or gRNA molecule functions to inhibit KRAS or mutant KRAS expression or function in a subject.

[0027] As used herein “mutation” refers to one or more nucleotide deletions, insertions, substitutions, inversions, or duplications. As used herein the term “missense mutation” refers to a DNA change that results in a different amino acid being encoded at a particular position in the encoded polypeptide. Non-limiting examples of KRAS mutations include G12D, G12V, G13D, G12C, G12A, Q61H, G12R, A146T, G13C, G12S, Q61L, Q22K, A146V, G12F, Q61R, E63K, L19F, D33E, A59G, T74P, and K117R.

[0028] The term “inhibitor” as used herein refers to an inhibitory molecule identified using an in vitro or in vivo assay. Inhibitors are compounds that bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of KRAS. In some embodiments, assays for inhibitors may include expressing mutant KRAS in vitro, applying putative inhibitor compounds, and then determining the functional effects on activity, as described herein. Test samples or assays that are treated with a potential inhibitor may be compared to a control sample lacking the inhibitor in order to determine the extent of inhibition. Control samples to which a test sample or assay is compared may be assigned a relative activity value of 100%. Inhibition is achieved, in certain embodiments, when the activity value of the test sample relative to the control sample is less than about 90%, including about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, and about 0%.

[0029] Any composition or method known in the art for interfering with KRAS or mutant KRAS activity in a subject may be useful according to the compositions and methods provided by the present disclosure. Additionally, any composition or method known in the art for increasing the expression or activity of TUSC2 in a subject may be useful according to the compositions and methods provided by the present disclosure. See, for example, U.S. Pat. No. 7,902,441 and U.S.7US_ACTIVE\131015922W-1Pat. Pub. No. 2012 / 0244209, which are incorporated herein by reference. In certain embodiments, a therapeutic molecule may be combined with a non-naturally occurring pharmaceutically acceptable carrier such as one described herein. In some embodiments, treatment methods of the present disclosure may involve delivery of a therapeutic molecule, nucleic acid, polypeptide, or compound. In other embodiments, treatment methods of the present disclosure may involve delivery of, for example, a vector expressing a functional copy of a therapeutic compound or molecule, such as a coding polynucleotide, an siRNA, or a gRNA molecule. For example, a vector expressing an siRNA or gRNA molecule to reduce or eliminate the function of KRAS or mutant KRAS may be used according to certain embodiments of the present disclosure. A vector expressing TUSC2 may also be used according to particular embodiments of the present disclosure. In further embodiments, treatment may comprise any combination of therapeutic compounds described herein or a combination of these therapeutic compounds with any known treatment for KRAS mutant cancers, including but not limited to administering an immunotherapy, a targeted cancer therapy, a chemotherapy, a radiation therapy, or surgery.

[0030] In certain embodiments, the methods of the present disclosure may comprise administering a TUSC2 polypeptide or a polynucleotide molecule encoding a TUSC2 polypeptide to a subject. In one embodiment, the TUSC2 polypeptide may comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1 or fragment thereof, including all ranges and values derivable therebetween. In particular embodiments, the methods of the present disclosure may comprise administering a polynucleotide molecule encoding a TUSC2 polypeptide to a subject. In another embodiment, the polynucleotide molecule may comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:2 or a fragment thereof, including all ranges and values derivable therebetween. In some embodiments, a polypeptide fragment of the present disclosure may comprise at least one activity of TUSC2. In one embodiment, a fragment may retain at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% of at least one activity of SEQ ID NO:2, including all ranges and values derivable therebetween. The activities or functions of TUSC2 is known in the ail, non-limiting examples of which include, regulating8US_ACTIVE\131015922W-1normal mitochondrial calcium movement and homeostasis, decreasing cell proliferation, decreasing cell sternness, decreasing tumor growth, increasing apoptosis, and activating adaptive and innate immune responses.

[0031] In certain embodiments, administering a polynucleotide molecule encoding a TUSC2 polypeptide may comprises administration of a TUSC2 expression vector or expression construct. In one embodiment, the methods of the present disclosure may comprise administering a DNA plasmid encoding TUSC2. An expression vector or an expression construct for use according to certain embodiments of the present disclosure may comprise, for example, control elements for the expression of the TUSC2 coding sequence. In certain embodiments, a vector may comprise a promoter or an enhancer element for effective expression in cancer cell of interest. In particular embodiments, TUSC2 expression may be provided by a CMV promoter or recombinant version thereof, such as the CMV promoter construct described in U.S. Patent Publication No. 2007 / 0092968, which is incorporated herein by reference. In certain embodiments, a vector or a construct provided by the present disclosure may comprise a modified CMV promoter or a mini-CMV promoter. Additional expression control elements, in certain embodiments, may be included in a vector or construct of the present disclosure. Non-limiting examples of which include an intron, a drug response element, an RNA stabilizing or destabilizing sequence, a cellular localization signal, a polyadenylation signal sequence, and / or an optimized translation start codon. Plasmid DNA vectors or constructs, in certain embodiments, may also comprise sequences that help facilitate DNA production, such as for example, a bacterial origin of replication and / or a drug resistance marker. In one embodiment, the TUSC2 expression vector or construct may be the pLJ143 / KGB2 / FUSl plasmid described in U.S. Patent No. 9,675,663, which is incorporated herein by reference.

[0032] In vivo methods for delivery of an expression vector or construct to cells are well known in the art and any such method may be used according to certain embodiments of the present disclosure. Non-limiting examples of such methods include delivery using nanoparticles (e.g., liposome nanoparticles), lipid conjugates, and viral vectors. In certain aspects, a TUSC2 expression vector may be administered in a nanoparticle, such as a N-[l-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP):cholesterol liposome nanoparticle. A skilled artisan will recognize that various properties of liposomes can be adjusted to optimize vector delivery. For example, the liposomes may be modified to have a certain size range and / or a9US_ACTIVE\131015922W-1particular ratio of DNA to lipid, DNA to cholesterol, and / or lipid to cholesterol. In certain embodiments and in the case of a DOTAP:cholcstcrol liposome, the DOTAP:cholcstcrol ratio can may defined as between about 1.5:1 and 1:1.5, such as about 10:9. In further embodiments, a TUSC2 expression vector may be provided in a liposome nanoparticle, wherein the nanoparticle comprises an average particle size of about 50 nm to about 500 nm or about 200 nm to about 500 nm, including all ranges and values derivable therebetween. In still further embodiments, a TUSC2-nanoparticle formulation can be defined by its optical density (OD), such as having OD400 of about 0.65 to about 0.95, including all ranges and values derivable therebetween. In one embodiment, the TUSC2 expression construct or vector may be administered as a pharmaceutical composition. The pharmaceutical compositions, in another embodiment, may be quaratusugene ozeplasmid or REQORSA™, a lipoplex gene therapy comprising the TUSC2 gene.

[0033] In further aspects, the methods of the present disclosure may comprise administration of a TUSC2 polypeptide or an active fragment thereof. Any method for administration of a TUSC2 polypeptide known in the art may be used according to certain embodiments of the present disclosure. Methods for administration of a TUSC2 polypeptide are described, for example, in U.S. Pat. Pub. Nos. 2006 / 0251726 and 2009 / 0023207, which are incorporated herein by reference. In particular embodiments, a TUSC2 polypeptide may be modified to enhance its activity and / or ability to enter cancer cells. A TUSC2 polypeptide, in one embodiment, may be modified with a lipid moiety (e.g., myristoylated). A TUSC2 polypeptide in another embodiment, may be provided as pail of a nanoparticle (e.g., a lipid-based nanoparticle) such as, a superparamagnetic nanoparticle, a nanoshell, a semiconductor nanocrystal, a quantum dot, a polymer-based nanoparticle, a silicon-based nanoparticle, a silica-based nanoparticle, a metal-based nanoparticle, a fullerene, or a nanotube. In some embodiments, a nanoparticle-polynucleotide or a nanoparticle- polypeptide complex may be used to deliver a polypeptide or polynucleotide molecule to a cell of interest. In one embodiment, a nanoparticle-polynucleotide or a polynucleotide complex may comprise a TUSC2 polypeptide or a TUSC2 polynucleotide in association with a nanoparticle. As used herein the phrase “in association with” refers to a physical association and / or a chemical association. In certain embodiments, an association may include a covalent bond, a hydrophobic interaction, encapsulation, and / or surface adsorption. In certain embodiments, nanoparticles of the present disclosure may exploit the increased vascular permeability of tumor vasculature to enhance nanoparticle concentration in a tumor. A nanoparticle complex, in certain embodiments, may be10US_ACTIVE\131015922W-1targeted to specific tissues or cells using a cell targeting molecule. Non-limiting examples of celltargeting molecules include a polypeptide, a peptide, a lipid, a steroid, a sugar, a carbohydrate, and a synthetic compound, an antigen binding polypeptide, and an antibody. Targeted nanoparticle complexes, in particular embodiments, may enhance delivery specificity and / or increase the amount of therapeutic agent that enters a target cell. Non-limiting examples of proteins or peptides that can be used to target nanoparticles include transferin, lactoferrin, TGF-a, nerve growth factor, albumin, the HIV Tat peptide, RGD peptide, and insulin.

[0034] Therapeutic compounds or compositions within the scope of the present disclosure may also contain other compounds, which may be biologically active or inactive. For example, one or more small molecule inhibitors, siRNAs, or gRNAs described herein may be present, within a composition according to the disclosure. Therapeutic compounds or compositions may generally be used for prophylactic and / or therapeutic purposes. Embodiments of the disclosure provide therapeutic compounds or compositions for treatment of cancer comprising a KRAS mutation, non-limiting examples of which include lung cancer, non-small cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, choloangiocarcinoma, uterine endometrial carcinoma, testicular germ cell cancer, and cervical squamous cell carcinoma. In certain embodiments, the KRAS mutation may be a missense mutation. In one embodiment, the KRAS mutation may be selected from the group consisting of G12D, G12V, G13D, G12C, G12A, Q61H, G12R, A146T, G13C, G12S, Q61L, Q22K, A146V, G12F, Q61R, E63K, L19F, D33E, A59G, T74P, and K117R.

[0035] Therapeutic compounds or compositions may be provided to a subject in a single dose or multiple doses and as such provided in single-dose or multi-dose containers, such as sealed ampules or vials. Such containers may be sealed to preserve sterility of the composition until use. In general, compositions as described herein may be stored as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, such a composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use. In some embodiments the therapeutic compounds or compositions may be stored in tablet form.

[0036] As described herein, a therapeutic composition may be combined with a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutically acceptable carrier as described herein is non-naturally occurring. The selection of a suitable carrier may be determined in part by11US_ACTIVE\131015922W-1the particular composition being administered (e.g., small molecule inhibitors, protein, modulatory compounds, siRNA molecules, gRNA molecules, or the like), as well as by the particular method used to administer the composition. Accordingly, a wide variety of suitable formulations of therapeutic compositions are available that may be of use in the present disclosure. Administration may be in any convenient manner, e.g., by injection, oral administration, inhalation, transdermal application, or rectal administration.

[0037] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended subject, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this disclosure, compositions may be administered, for example, by intravenous infusion into the circulation, orally, topically, or intraperitoneally.

[0038] Such compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a subject, suspending agents, thickening agents, and / or preservatives. Alternatively, compositions of the present disclosure may be formulated as a lyophilizate. Compounds may also be encapsulated within liposomes using methods known in the art.

[0039] Injection solutions and suspensions may be prepared from sterile powders, granules, and tablets as described herein. An injection as described herein may involve a suspension of one or more of a purified or non-purified solution of a protein, nucleic acid, or other type of molecule as described herein. An injection solution may also contain a pharmaceutically acceptable carrier as described herein.

[0040] Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of a small molecule inhibitor or other type of molecule suspended in diluents, such as water, saline or PEG 400; (b) capsules or tablets, each containing a predetermined amount12US_ACTIVE\131015922W-1of the active ingredient, as liquids, solids, granules, or gelatin; (c) suspensions in an appropriate liquid; or (d) suitable emulsions. Tablet forms may include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms may comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

[0041] The compound of choice, alone or in combination with other suitable components, may be made into aerosol formulations to be administered via inhalation. Aerosol formulations may be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0042] The dose administered to a subject in the context of the present disclosure should be sufficient to affect a beneficial therapeutic response in the subject over time. The dose will be determined by the efficacy of the particular molecule employed and the condition of the subject, as well as the body weight and / or surface area of the patient to be treated. The size of the dose also may be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular molecule or therapeutic compound in a particular subject.

[0043] For administration, compounds of the present disclosure can be administered at a rate determined by the LD50 of the molecule or therapeutic compound, and the side-effects thereof at various concentrations, as applied to the mass and overall health of the subject. Administration may be accomplished via single, multiple, or divided doses.

[0044] As used herein, a “therapeutic compound” or “therapeutic composition” refers to a molecule, such as a small molecule inhibitor, an RNA molecule, a protein, a peptide, a polypeptide, an siRNA molecule, a gRNA molecule, a polynucleotide molecule, a cDNA molecule, a mRNA molecule, a DNA molecule, or a vector or that inhibits the expression or function of KRAS or mutant KRAS or increases the expression or activity of TUSC2. Such a compound or composition is meant to encompass a composition suitable for administration to a subject, such as a mammal,13US_ACTIVE\131015922W-1or particularly a human subject. In general, a therapeutic composition is sterile, and preferably free of contaminants that arc capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the immunogenic composition is pharmaceutical grade). Therapeutic compositions may be designed for administration to subjects in need thereof via a number of different routes of administration including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalational, and the like.

[0045] In certain embodiments, a TUSC2 expression vector complexed with DOTAP:cholesterol liposome can be administered via intravenous infusion. In particular embodiments, a therapy that increases the expression or activity of TUSC2 may be administered intravenously at a dose of about 0.01 mg / kg to about 0.10 mg / kg, about 0.02 mg / kg, about 0.03, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, or about 0.10 mg / kg, including all ranges and values derivable therebetween. In embodiments, a therapy that increases the expression or activity of TUSC2 may be administered at least one, two, three, four, five, six, seven, eight, nine, or ten times, including all ranges and values derivable therebetween. The timing between multiple doses of a therapy, in some embodiments, can be varied and can include about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 3 weeks, about 1 month, or more in between doses, including all ranges and values derivable therebetween.

[0046] In particular embodiments, a small molecule inhibitor of KRAS may be administered at a dose of about 25 mg to about 1500 mg, about 50 mg to about 1400 mg, about 75 mg to about 1300 mg, about 100 mg to about 1000 mg, about 150 mg to about 1000 mg, about 200 mg to about 1000 mg, about 300 mg to about 1000 mg, about 400 mg to about 1000 mg, about 500 mg to about 1000 mg, about 600 mg to about 1000 mg, about 700 mg to about 1000 mg, about 800 mg to about 1000 mg, about 900 mg to about 1000 mg, about 200 mg to about 300 mg, about 240 mg, or about 960 mg per day, including all ranges and values derivable therebetween. In some embodiments, a small molecule inhibitor of KRAS may be administered orally. In certain embodiments, the daily dose of the inhibitor of KRAS may be administered in one, two, three, or more doses per day.

[0047] Provided herein, in certain embodiments, is a method of treating a subject afflicted with a cancer comprising a KRAS mutation, wherein the cancer displays resistance to an inhibitor of KRAS, the method comprising administering to the subject a therapeutically effective amount of14US_ACTIVE\131015922W-1a therapy that increases the expression or activity of a TUSC2 polypeptide. Provided herein, in particular embodiments, is a method of treating a subject afflicted with a cancer comprising a KRAS mutation, wherein the cancer comprises resistance to an inhibitor of KRAS, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid encoding a TUSC2 protein or a pharmaceutical composition comprising a nucleic acid encoding a TUSC2 protein. Provided herein, in some embodiments, is a method for activating the innate immunity in a subject afflicted with a cancer comprising a KRAS mutation, wherein the cancer comprises resistance to an inhibitor of KRAS, the method comprising administering to the subject a therapeutically effective amount of a therapy that increases the expression or the activity of a TUSC2 polypeptide. Provided herein, in certain embodiments, is a method for reducing tumor size or tumor growth in a subject afflicted with a cancer comprising a KRAS mutation, wherein the cancer comprises resistance to an inhibitor of KRAS, the method comprising administering to the subject a therapeutically effective amount of a therapy that increases the expression or the activity of a TUSC2 polypeptide. Provided herein, in particular embodiments, is a method for treating cancer in a subject in need thereof, the method comprising: (a) determining that the subject has mutation in the gene encoding for KRAS; and (b) administering to the subject a nucleic acid comprising a sequence encoding a TUSC2 protein. The subject, in one embodiment, may have acquired resistance to an inhibitor of KRAS. In another embodiment, the subject has acquired resistance to an inhibitor of KRASG12C. In some embodiments, the inhibitor of KRASG12Cis sotorasib or adagrasib.

[0048] In certain aspects of the present disclosure an inhibitor of KRAS may be administered before, after, or approximately simultaneously with administration of a therapy that increases the expression or activity of TUSC2. In certain embodiments, the present disclosure provides a method of treating cancer which may include administering an inhibitor of KRAS and a therapy that increases the expression or activity of TUSC2. In one embodiment, the inhibitor of KRAS and the therapy that increases the expression or activity of TUSC2 may be administered along with any other anti-cancer therapy. As used herein the term ’‘anti-cancer therapy” refers to any therapy that is capable of negatively affecting cancer in a subject. In some embodiments, an anti-cancer therapy may kill cancer cells, increase apoptosis of cancer cells, promote an anti-cancer immune response, reduce the incidence or number of metastases, reduce tumor size, inhibit tumor growth, inhibit, or prevent tumor progression, or increase the lifespan of a subject with cancer. In some15US_ACTIVE\131015922W-1embodiments, the present disclosure may comprise contacting a cancer cell with one or more anticancer therapies. This may be achieved, in certain embodiments, by contacting the cancer cell with a single composition comprising two or more of the anti-cancer therapies or by contacting the cell with multiple compositions, each of which comprises at least one anti-cancer therapy.

[0049] In particular embodiments, the methods of the present disclosure may comprise administering a first anti-cancer agent and a second anti-cancer agent, wherein the first anti-cancer agent is administered before, after, or before and after administration of the second anti-cancer agent. The first anti-cancer agent and the second anti-cancer agent may be administered, in some embodiments, within about 12 to about 24 hours or about 6 to about 12 hours, including all ranges and values derivable therebetween. In additional embodiments, the first anti-cancer agent and the second anti-cancer agent may be administered within about 1 day to about 8 weeks of each other, including all ranges and values derivable therebetween. In further embodiments, the first anticancer agent and the second anti-cancer agent may be administered within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks of each other, including all ranges and values derivable therebetween.

[0050] Provided herein, in one embodiment, is a method of inducing apoptosis in a cancer cell, wherein the cancer cell comprises a KRAS mutation and wherein the cancer cell is resistant to an inhibitor of KRAS, the method comprising contacting the cancer cell with a composition disclosed herein that increases the expression or activity of a TUSC2 polypeptide.

[0051] As used herein, “subject” or “patient” refers to animals, including humans, who are treated with the therapeutic compounds or compositions or in accordance with the methods described herein. For diagnostic or research applications, a wide variety of mammals may be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine, such as inbred pigs and the like. In certain embodiments, a subject may be afflicted with or at risk of developing a cancer comprising a KRAS mutation, such as primary or metastatic tumor. The cancer may be an early-stage cancer or may be a metastatic or late-stage cancer. In certain aspects, the cancer is an oral cancer, oropharyngeal cancer, nasopharyngeal cancer, respiratory cancer, a urogenital cancer, a gastrointestinal cancer, a central or peripheral nervous system tissue cancer, an endocrine or neuroendocrine cancer, a hematopoietic cancer, a glioma, a sarcoma, a carcinoma, a lymphoma,16US_ACTIVE\131015922W-1a melanoma, a fibroma, a meningioma, brain cancer, oropharyngeal cancer, nasopharyngeal cancer, renal cancer, biliary cancer, prostatic cancer, pheochromocytoma, pancreatic islet cell cancer, a Li-Fraumeni tumor, thyroid cancer, parathyroid cancer, pituitary tumors, adrenal gland tumors, osteogenic sarcoma tumors, multiple neuroendocrine type I and type II tumors, breast cancer, lung cancer, a non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC)), head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, skin cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, or skin cancer. In further aspects, a cancer of the present disclosure may be defined as a cancer that is resistant to one or more anticancer therapies, such a chemotherapy resistant cancer or a cancer resistant to an inhibitor of KRAS.

[0052] As used herein, a “biological sample” or “sample” may include blood and blood pails including, but not limited to serum, plasma, platelets, or red blood cells; sputum, mucosa, tissue, cultured cells, including primary cultures, and transformed cells; biological fluids, stool, and urine. A biological sample may also include sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. A biological sample may be obtained from a eukaryotic organism, such as a human. Any tissue appropriate for use in accordance with the disclosure may be used, for instance, tumor tissue, skin, brain, spinal cord, adrenals, pectoral muscle, lung, heart, liver, duodenum, small intestine, large intestine, kidney, spleen, pancreas, adrenal gland, bone marrow, lumbosacral spinal cord, or blood.

[0053] As used herein, a “pharmaceutically acceptable carrier,” “pharmaceutically acceptable adjuvant,” or “adjuvant” refers to refers to reagents, cells, compounds, materials, compositions, and / or dosage forms that are not only compatible with the agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit / risk ratio. Also included may be an agent that modifies the effect of other agents and is useful in preparing a therapeutic compound or composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable. Such an agent may be added to a therapeutic composition to modify the immune response of a subject by boosting the response and providing longer-lasting protection. Such an agent may include an excipient, diluent, carrier, or adjuvant that is acceptable for pharmaceutical17US_ACTIVE\131015922W-1use. Such an agent may be non-naturally occurring, or may be naturally occurring, but not naturally found in combination with other agents in the immunogenic composition.

[0054] The term “isolated compound” means a compound which has been substantially separated from, or enriched relative to, other compounds with which it occurs in nature. Isolated compounds are usually at least about 80%, at least 90% pure, at least 98% pure, or at least about 99% pure, by weight.

[0055] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for animal subjects, each unit containing a predetermined quantity of a compound calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for unit dosage forms depend on the particular compound employed, the route and frequency of administration, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0056] As used herein, “introducing,” “delivering,” and “administering” refer to the therapeutic introduction of a therapeutic compound or composition as described herein to a subject. Administration may take place by any route that provides the therapeutic compound to the circulation of the subject in accordance with the disclosure.

[0057] The phrase “effective amount” refers to a concentration or amount of a therapeutic compound or composition as described herein, reagent, or other agent, which is effective for producing an intended result, including treatment of KRAS mutant cancers as described herein. With respect to the administration of a therapeutic compound as disclosed herein, an effective amount may be any effective range or concentration. The exact dose will depend on the purpose of the treatment, and one of skill in the art will be able to determine such a dose using techniques known in the ail.

[0058] As used herein, “expression” refers to the combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide or functional nucleic acid (e.g., an RNAi, gRNA, antisense molecule, ribozyme, aptamer, etc.).18US_ACTIVE\131015922W-1B. Nanoparticles

[0059] As used herein, the term “nanoparticle” refers to any material having a size of about 1 nm to about 1000 nm or about 50 nm to about 500 nm, including all ranges and values derivable therebetween. Non-limiting examples of nanoparticles include lipid-based nanoparticles, superparamagnetic nanoparticles, nanoshells, semiconductor nanocrystals, quantum dots, polymer-based nanoparticles, silicon-based nanoparticles, silica-based nanoparticles, metal-based nanoparticles, fullerenes, and nanotubes. Conjugation of a polypeptide or nucleic acid to a nanoparticle produces a structure that may be used, for example, for targeted delivery, controlled release, enhanced cellular uptake, enhanced intracellular trafficking, and molecular imaging of therapeutic peptides in vitro and in vivo.

[0060] Lipid-based nanoparticles include liposomes, lipid preparations, and lipid-based vesicles, such as DOTAP:cholesterol vesicles. Lipid-based nanoparticles may be positively charged, negatively charged, or neutral. In certain embodiments, a lipid-based nanoparticle is neutrally charged, such as a DOPC liposome.

[0061] A “liposome” as used herein refers to a single or multilamellar lipid vehicle formed by the generation of enclosed lipid bilayers or aggregates. Liposomes, in some embodiments, may be characterized as having vesicular structures with a bilayer membrane. In certain embodiments, a liposome may comprise a phospholipid and an inner medium comprising an aqueous composition. Liposomes, include but are not limited to, unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. In certain embodiments of the present disclosure, liposomes may be positively charged, negatively charged, or neutral in charge.

[0062] A multilamellar liposome, in certain embodiments, may comprise multiple lipid layers separated by aqueous medium. A multilamellar liposome may, in some embodiments, form spontaneously lipid compositions comprising phospholipids are suspended in an excess of an aqueous solution. In certain embodiments, the lipid components may undergo self-rearrangement before forming closed structures that entrap water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

[0063] Methods of producing nanoparticles and liposomes arc known in the art and any such method may be used according to the embodiments of the present disclosure. In certain19US_ACTIVE\131015922W-1embodiments, a polypeptide or polynucleotide molecule of the present disclosure may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilaycr of a liposome, or conjugated to the liposome using a linking molecule associated with both the liposome and the polypeptide or polynucleotide molecule. For example, a phospholipid, such as for example the neutral phospholipid dioleoylphosphatidylcholine (DOPC), may dissolved in tertbutanol. The lipid may then be mixed with a polypeptide, a polynucleotide molecule, and / or one or more additional components. Tween-20 is then added to the lipid mixture until Tween-20 is about 5% of the total weight of the composition. Excess tert-butanol is added to this mixture until the volume of tert-butanol is at least 95%. The mixture is then vortexed, frozen in a dry ice / acetone bath, and lyophilized overnight. The lyophilized preparation is stored at -20 °C. and can be used for up to three months. When required for use, the lyophilized liposomes are reconstituted in 0.9% saline.

[0064] Alternatively, a liposome can be prepared by mixing lipids in a solvent in a container and the solvent removed at approximately 40 °C using a rotary evaporator under negative pressure. The composition can then be dried further in a desiccator under vacuum. Dried lipids can be hydrated in sterile, pyrogen-free water by shaking until all the lipid film is resuspended. The aqueous liposomes can be then separated into aliquots, placed in a vial, lyophilized, and sealed under vacuum.

[0065] Dried lipids or lyophilized liposomes may be reconstituted in a solution of a polypeptide or polynucleotide molecule and diluted to an appropriate concentration with a suitable solvent. Following vigorous shaking in a vortex mixer, unencapsulated materials such as hormones, drugs, polynucleotide molecules, or polypeptide molecules may be removed by centrifugation and the liposomes washed. Washed liposomes may be resuspended at an appropriate total phospholipid concentration. In some embodiments, an appropriate phospholipid concentration may be, for example, about 50 to about 200 mM, including all ranges and values derivable therebetween. The amount of material encapsulated can be determined in accordance with standard methods. Liposomes may be diluted, if needed, to an appropriate concentration and stored at 4° C until use.

[0066] Liposome size may vary depending on the method of synthesis, and liposomes of the present disclosure may also vary in size. In some embodiments, a liposome of the present disclosure may less than about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or20US_ACTIVE\131015922W-1less than about 50 nm in external diameter. Tn one embodiment, a DOTAP:cholesterol liposome for use according to the methods and compositions of the present disclosure may be about 50 nm to about 500 nm in external diameter, including all ranges and values derivable therebetween prior to the incorporation of polypeptide or polynucleotide molecule. Liposome formulations, in particular embodiments, may also be defined by particle charge (zeta potential) and / or optical density (OD). A DOTAP:cholesterol liposome formulation, in another embodiment, may comprise an OD400 of less than about 0.45 prior to incorporation of a polynucleotide or polypeptide molecule. The overall charge of such particles in solution, in yet another embodiment, may be defined by a zeta potential of about 50 mV to about 80 mV, including all ranges and values derivable therebetween.

[0067] In certain embodiments of the present disclosure a lipid-based nanoparticle may be a neutral liposome, for example, a DOPC liposome). As used herein the term “neutral liposome” or “non-charged liposome,” refers to a liposome having one or more lipid components that yield an essentially neutral, net charge (substantially non-charged). As used herein the term “essentially neutral” or “essentially non-charged,” refers to a state where few, if any, lipid components within a given population of liposomes include a charge that is not canceled by an opposite charge of another component. In some embodiments, a neutral or non-charged liposome may have fewer than about 10%, fewer than about 5%, or fewer than about 1% of components that include a noncanceled charge. A neutral liposome, in one embodiment, may include mostly lipids and / or phospholipids that neutral under physiological conditions, such as at a pH of about 7.

[0068] In some embodiments of the present disclosure a liposome or nanoparticle of the present disclosure may comprise a single type of phospholipid or a number of different types of phospholipids. Phospholipids may include, but are not limited to, phosphatidylcholines, phosphatidylglycerols, phosphatidylethanolamines glycerophospholipids, and sphingolipids. Phosphatidylethanolamines and phosphatidylcholines are non-charged under physiological conditions, and therefore, in some embodiments, may be particularly useful for generating neutral liposomes. In one embodiment, the phospholipid DOPC may be used to produce non-charged liposomes. In another embodiment, a lipid that is not a phospholipid, such as cholesterol, may be used to produce liposomes.21US_ACTIVE\131015922W-1

[0069] Non-limiting examples of phospholipids that may be used according to certain embodiments of the present disclosure include diolcoylphosphatidylycholinc (DOPC), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), l-myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1- palmitoyl-2-myristoyl phosphatidylcholine (PMPC), l-palmitoyl-2- stearoyl phosphatidylcholine (PSPC), l-stearoyl-2-palmitoyl phosphatidylcholine (SPPC), dilauryloylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), di stearoylphosphatidylglycerol (“DSPG”), distearoyl sphingomyelin (“DSSP”), distearoylphophatidylethanolamine (DSPE), dioleoylphosphatidylglycerol (DOPG), dimyristoyl phosphatidic acid (DMPA), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylserine (DMPS), dipalmitoyl phosphatidylserine (DPPS), brain phosphatidylserine (BPS), brain sphingomyelin (BSP), dipalmitoyl sphingomyelin (DPSP), dimyristyl phosphatidylcholine (DMPC), l,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), 1,2- diarachidoyl-sn-glycero-3-phosphocholine (DBPC), l,2-dieicosenoyl-sn-glycero-3- phosphocholine (DEPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloeoyl phosphatidylcholine (POPC), palmitoyloeoyl phosphatidylethanolamine (POPE), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine. In some embodiments, phospholipids from natural and / or synthetic sources may be used according to the embodiments of the present disclosure.

[0070] A DOTAP:cholesterol nanoparticle may be used according to particular embodiments of the present disclosure. Methods for the preparation of such nanopaiticles are known in the art and any such method may be used according to the embodiments of the present disclosure. In some embodiments, DOTAP:cholesterol nanoparticles may be prepared by mixing the cationic lipid DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)-propane) with cholesterol. Nanoparticles prepared with a polynucleotide molecule or polypeptide molecule may form a structure, in some embodiments, where the polynucleotide molecule is condensed between two lipid bilayers as described in U.S. Pat. No. 6,770,291 and U.S. Pat. No. 6,413,544.

[0071] In some embodiments, a DOTAP:cholesterol-polynucleotide molecule complex can be prepared by mixing 420 mg of DOTAP and 208 mg of cholesterol with 30 ml of chloroform as 22US_ACTIVE\131015922W-1described below. The mixture may then be allowed to dry on a rotary evaporator for 30 minutes and then freeze dried for 15 minutes. The dried mixture may be reconstituted in 30 ml of dextrose 5% in water by swirling at 50 °C for 45 minutes and 37 °C for 10 minutes. The mixture may then be sonicated at low frequency for 5 minutes to form liposomes. The DOTAP:cholesterol liposomes are then heated to 50 °C and sequentially filtered through sterile Whatman filters. The synthesized nanoparticles are stored at 4 °C and prior to forming nanoparticle complexes. The formulated DOTAP:cholesterol liposome should be evenly dispersed with a particle size of about 50 to about 250 nm, have an OD400 of less than 0.45, and have a zeta potential of about 50 mV to about 80 mV. Residual CHC13 levels should be less than 60 ppm. To prepare DOTAP:cholesterol- polynucleotide molecule nanoparticles, 240 pl of liposomes may be diluted in 360 pl dextrose 5% in water at room temperature. DNA ( about 5 mg / ml) may be added to the mixture to a total volume of 600 pl. Pipet to mix. Once settled the mixture should have an OD400 of between about 0.65 and about 0.95, a particle size of about 200 nm to about 500 nm and be confirmed gram stain negative. The liposome complexes may be stored at between about 3 °C. and about 28 °C without agitation.C. Vectors

[0072] As used herein the term “vector” or “construct” refers to a polynucleotide molecule used as a vehicle to carry another polynucleotide molecule. In some embodiments, a vector may be introduced into a cell. Once introduced into a cell, in certain embodiments, a vector may be replicated. Non-limiting examples of vectors for use according to the embodiments for the present disclosure include plasmid vectors, retroviral vectors, lentiviral vectors, cosmids, viral vectors, adenoviral vectors, adeno-associated viral vectors, bacteriophages, animal viruses, plant viruses, and artificial chromosomes. As used herein, the term “recombinant” refers to a polynucleotide molecule, protein, or cell that is not naturally present, or is not naturally present in the same form or structure and was created by human intervention. In one embodiment, a recombinant polynucleotide may be a DNA molecule or may be an RNA molecule. A recombinant polynucleotide molecule or a recombinant polypeptide molecule or protein may comprise, in certain embodiments, a combination of two or more polynucleotide or polypeptide sequences that do not naturally occur together in the same manner, such as a polynucleotide molecule or protein that comprises at least two polynucleotide or protein sequences that are operably linked but heterologous with respect to each other. As used herein the term “heterologous” refers to a 23US_ACTIVE\131015922W-1polynucleotide molecule or protein that is not naturally present or is not naturally present in the same form or structure and was created by human intervention. For example, a heterologous polynucleotide molecule or protein may not naturally occur in the cell being transformed or may be expressed in a manner or genomic context that differs from the natural expression pattern or genomic context found in the cell being transformed. The heterologous polynucleotide molecule or protein, in some embodiments, may be overexpressed in the cell being transformed. In certain embodiments, a recombinant polynucleotide molecule, protein, construct, or vector may comprise any combination of two or more polynucleotide or protein sequences in the same molecule which are heterologous to one another, such that the combination is man-made and not normally found in nature. As used herein, the phrase “not normally found in nature” means not found in nature without human intervention. A recombinant polynucleotide or protein molecule, may comprise, for example, polynucleotide or protein sequences that are separated from other polynucleotide or protein sequences that exist in proximity to each other in nature. A recombinant polynucleotide or protein molecule may also comprise, for example, polynucleotide or protein sequences that are adjacent to or contiguous with other polynucleotide or protein sequences that are not naturally in proximity with each other. Such a recombinant polynucleotide molecule, protein, or expression construct may also refer to a polynucleotide or protein molecule or sequence that has been genetically engineered or constructed outside of a cell. For example, a recombinant polynucleotide molecule may comprise any engineered or man-made plasmid, vector, or expression construct, and may include a linear or circular DNA molecule. Such plasmids, vectors, and expression constructs may comprise, for example, various maintenance elements including, but not limited to, a heterologous promoter sequence, a prokaryotic origin of replication, or a selectable marker.

[0073] As used herein the term “expression vector” refers to a vector comprising a polynucleotide molecule encoding an RNA molecule capable of being transcribed. In some embodiments, encoded RNA molecules may be translated into a protein, polypeptide, or peptide. In other embodiments, encoded RNA may not be translated but instead may serve as a siRNA, gRNA, or ribozyme.

[0074] In certain embodiments of the present disclosure, a vector of the present disclosure may include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector. As used herein the term “restriction enzyme digestion” refers to24US_ACTIVE\131015922W-1catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of t restriction enzymes arc commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. As used here the term “ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology and any such technique may be used according to the embodiments of the present disclosure.

[0075] The term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. When used in conjunction with the word "comprising" or other open language in the claims, the words "a" and "an" denote "one or more," unless specifically noted otherwise. The terms "comprise," "have," and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any system or method that "comprises," "has," or "includes" one or more components is not limited to possessing only those components and covers other unlisted components.

[0076] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the ail from this detailed description. Any embodiment or aspect of the present disclosure may be used in combination with any other embodiment or aspect described herein.

[0077] All references herein are incorporated herein by reference in their entirety.25US_ACTIVE\131015922W-1EXAMPLES

[0078] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.EXAMPLE 1Development of Sotorasib Acquired Resistant NSCLC Patient-Derived Xenografts and Cell Lines

[0079] Twelve well-characterized and annotated NSCLC patient-derived xenograft (PDX) models harboring the KRASG12Cmutation were identified from an extensive NSCLC PDX bank. Among these, the TC303 and TC314 PDX models were determined to be the most sensitive to sotorasib treatment. These two models were selected for the development of isogenic sotorasib-resistant PDX models. Through prolonged in vivo sotorasib (AMG510) treatment and subsequent in vivo passaging, TC3O3AR and TC314AR resistant models were successfully developed. Both resistant PDX models exhibited significant resistance, with no observable antitumor activity when treated with sotorasib (FIG. 1A).

[0080] Next, the global proteomics and phosphoproteomics were examined for AR PDXs. Distinct sets of proteins were upregulated in AR PDXs (TC303AR & TC314AR) as compared with the sensitive PDXs (FIG. IB). Enrichment analysis of global and phosphoproteomics in TC314AR showed that the MTORC1 & KRAS, and the MTORC1 & PI3K-AKT-MTOR pathways, respectively, were significantly upregulated in AR PDXs (FIG. 1C). Reverse-phase26US_ACTIVE\131015922W-1protein array (RPPA) analysis of TC303AR and TC314AR PDXs showed upregulation of MAPK and PI3K-AKT-mT0R signaling molecules (FIG. ID).

[0081] Two sotorasib-acquired resistant isogenic cell lines, H23AR and H358AR, were developed through prolonged treatment with incrementally increasing doses of sotorasib until they exhibited complete resistance to the drug. The H23AR and H358AR cells demonstrated over 600-fold and 200-fold resistance, respectively, compared to their sensitive counterparts, H23 and H358. Additionally, both resistant cell lines showed cross-resistance to other KRASG12Cinhibitors, including adagrasib and opnurasib (FIG. 2).

[0082] Whole exome sequencing (WES) was performed on the H23 (sensitive), H23AR (resistant), H358 (sensitive), and H358AR (resistant) cell lines. The KRASG12Cmutation was retained and no additional mutations in KRAS were identified in either H23AR or H358AR. Some exonic changes were found in H23AR and H358AR compared to their respective sensitive counterpart models (H23 and H358) (FIG. 3).EXAMPLE 2TUSC2 Inhibits Colony Formation and Induces Apoptosis of H23AR and H358AR Cells

[0083] A colony formation assay was conducted following the transient transfection of TUSC2 and an empty vector into the acquired resistant H23AR and H358AR cells. Briefly, sotorasib acquired resistant cells were co-transfcctcd with TUSC2 / cmpty vector and pcDNA plasmids. Colonies were selected based on selection markers for several weeks, fixed, and imaged. The introduction of TUSC2 significantly reduced colony formation in both H23AR and H358AR cells (FIG. 4A). Additionally, TUSC2 transfection induced apoptosis in both H23AR and H358AR cells (FIG. 4B).EXAMPLE 3Antitumor Effect of TUSC2 on Sotorasib Resistant TC314AR PDX Tumors

[0084] TC314AR PDX models were developed in NSG mice, and once the tumors reached approximately 200 mm3, the mice were randomized and treated with sotorasib (AMG510), TUSC2 (also known as quaratusugene ozeplasmid, a lipoplex gene therapy comprising the TUSC2 gene), or a combination of both. Surprisingly, TUSC2 alone demonstrated a strong antitumor effect in27US_ACTIVE\131015922W-1the TC314AR PDX model, which was significantly superior to the effect of sotorasih alone. Sotorasib alone showed no significant antitumor activity in these models. However, a synergistic antitumor effect was observed when the combination of quaratusugene ozeplasmid and sotorasib was administered (FIG. 5).EXAMPLE 4Antitumor Effect of TUSC2 on Sotorasib Resistant H23AR Xenograft Tumors

[0085] H23AR xenograft tumors were established in NSG mice, which were then randomized and treated with TUSC2 (quaratusugene ozeplasmid), sotorasib (AMG510), or a combination of both. The H23AR tumors exhibited significantly reduced sensitivity to sotorasib compared to their parental counterparts. Treatment with quaratusugene ozeplasmid, however, either alone or in combination with sotorasib, was highly effective in controlling tumor growth, significantly outperforming both the sotorasib alone and control groups (FIG. 6).EXAMPLE 5Antitumor Effect of TUSC2 on Sotorasib Resistant TC314AR PDX Tumors in a Humanized Mouse Model

[0086] Humanized mice were generated by engrafting fresh human umbilical cord-derived hematopoietic stem cells (CD34+stem cells) into sub-lcthally irradiated NSG mice. The humanization of these mice was confirmed by the presence of over 25% human CD45+cells in the mouse system prior to implanting the TC314AR PDX tumors. Once the tumors reached approximately 200 mm3, the PDX-bearing mice were randomized and treated with TUSC2 (quaratusugene ozeplasmid), sotorasib (AMG510), or a combination of both. Similar to the results observed in non-humanized NSG mice, sotorasib alone did not produce a significant antitumor effect in the humanized mice. In contrast, TUSC2 gene therapy demonstrated pronounced control of tumor growth, which was significantly greater than that of sotorasib alone. The combination of TUSC2 and sotorasib also showed a very significant antitumor effect, which was comparable to the effect of TUSC2 alone (FIG. 7A).

[0087] Tumor microenvironment (TME) analysis was performed for the humanized mice. Specifically, the effect of the indicated therapy on human CD45, CD3 T, CD4 T, CD8 T, Treg, NK, PD1+CD3 T, PD1+CD8 T, PD1+NK, effector memory CD3 T cells, effector memory CD8 28US_ACTIVE\131015922W-1T cells, MDSC, DC, Ml & M2 MQ, residential memory CD3 T and residential memory NK cells was assessed (FIG. 7B). The TUSC2 antitumor effect was associated with enhanced antitumor immune mice, which was characterized by the significantly increased infiltration of human CD3, CD4, cytotoxic T, and NK cells, and inhibition of human regulatory T cells. PD1 -expressing T and NK cells were significantly downregulated, whereas effector memory CD3 and CD8 T cells were markedly increased by TUSC2 treatment. TUSC2 activated innate immunity by enhanced infiltration of DC and Ml macrophages, with significant inhibition of MDSC and M2 macrophages.EXAMPLE 6TUSC2 Sensitizes Sotorasib-Acquired Resistant PDX-Derived Organoids

[0088] Sotorasib-resistant organoids (PDXO) were derived from AR patient-derived xenografts (PDX) (PDXO303AR and PDXO314AR) (FIG. 8A). Resistance of the two isogeneic AR PDXOs to sotorasib was confirmed (FIG. 8B). TUSC2 transfection on PDXOs was evaluated by fluorescence imaging (FIG. 8C left) and RT PCR (FIG. 8C right). Expression of TUSC2 in AR PDXOs significantly decreased the viability of organoids (FIG. 8D).EXAMPLE 7Antitumor Immune Response of TUSC2 for H23AR Xenograft Tumors in a Humanized Mouse Model

[0089] To evaluate the antitumor immune responses, immune-competent humanized-NSG mice were generated by transplanting fresh human cord blood-derived CD34+stem cells into sub- lethally irradiated NSG mice to reconstitute a human immune system. The experimental strategy is shown in FIG. 9A.

[0090] A robust antitumor effect of TUSC2 was observed in xenograft humanized mice (FIG. 9B and FIG. 9C). When sotorasib was combined with TUSC2, synergistic antitumor activity was found.

[0091] Tumor microenvironment analysis for lymphoid cells (FIG. 9D, FIG. 9E, FIG. 9F, and FIG. 9H) and for myeloid cells (FIG. 91, FIG. 9J, and FIG. 9K) in the humanized mice was performed after treatment. The observed antitumor effect correlated with significant infiltration by human CD8 T, NK, DC, and Ml MQ, and downregulation of MDSCs and exhausted T cells.29US_ACTIVE\131015922W-1

[0092] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments or aspects, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims30US_ACTIVE\131015922W-1

Claims

CLAIMS1. A method of treating a subject afflicted with or at risk of developing a cancer comprising a KRAS mutation, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of KRAS and a therapeutically effective amount of a therapy that increases the expression or activity of a TUSC2 polypeptide.

2. The method of claim 1, wherein the KRAS mutation is a missense mutation.

3. The method of claim 1, wherein the KRAS mutation is selected from the group consisting of G12D, G12V, G13D, G12C, G12A, Q61H, G12R, A146T, G13C, G12S, Q61L, Q22K, A146V, G12F, Q61R, E63K, L19F, D33E, A59G, T74P, and K117R.

4. The method of claim 1, wherein the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, choloangiocarcinoma, uterine endometrial carcinoma, testicular germ cell cancer, and cervical squamous cell carcinoma.

5. The method of claim 1 , wherein the inhibitor of KRAS is selected from the group consisting of sotorasib, adagrasib, MRTX1133, ARS-3248, ARS-853, ARS-1620, opnurasib, GDC-6036, RG6330, D-1553, BPI-421286, GH35, BEBT-607, JAB-21000, BI-2865, BI-2493, RMC-6236, and combinations of any thereof.

6. The method of claim 1, wherein the method comprises: a) administering a TUSC2 polypeptide or a fragment thereof to said subject; or b) administering a polynucleotide molecule encoding a TUSC2 polypeptide or a fragment thereof to said subject.

7. The method of claim 6, wherein the TUSC2 polypeptide comprises a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1.

8. The method of claim 6, wherein the polynucleotide molecule encoding the TUSC2 polypeptide comprises a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:2.31US_ACTIVE\131015922W-19. The method of claim 6, wherein the method comprises administering an expression vector comprising the polynucleotide molecule encoding the TUSC2 polypeptide or the fragment thereof to said subject.

10. The method of claim 9, the method comprises administering a pharmaceutical composition comprising the expression vector to said subject.

11. The method of claim 10, wherein the pharmaceutical composition comprises quaratusugene ozeplasmid.

12. The method of claim 1, wherein the cancer comprises resistance to the inhibitor of KRAS or to at least a second inhibitor of KRAS.

13. The method of claim 12, wherein the resistance is an acquired resistance.

14. The method of claim 1, wherein the subject was previously treated with at least one inhibitor of KRAS prior to said administering.

15. The method of claim 12, wherein the inhibitor of KRAS or the second inhibitor of KRAS is selected from the group consisting of sotorasib, adagrasib, MRTX1133, ARS-3248, ARS-853, ARS-1620, opnurasib, GDC-6036, RG6330, D-1553, BPI-421286, GH35, BEBT-607, JAB- 21000, BI-2865, BI- 2493, RMC-6236, and combinations of any thereof.

16. The method of claim 1, wherein the administering comprises local, regional, systemic, or continual administration.

17. The method of claim 1, wherein the administering comprises administering a single dose to said subject.

18. The method of claim 1, wherein the administering comprises administering multiple doses to said subject.

19. The method of claim 1, wherein the administering comprises oral, intravenous, or intramuscular administration.

20. The method of claim 1, wherein the administering comprises administering a pharmaceutical composition comprising the inhibitor of KRAS or the therapy that increases the expression or activity of the TUSC2 polypeptide to said subject.32US_ACTIVE\131015922W-121. The method of claim 1 , the method further comprising administering an immunotherapy, a targeted cancer therapy, a chemotherapy, a radiation therapy, or surgery to said subject.

22. The method of claim 1, wherein the subject is a mammalian subject.

23. The method of claim 1, wherein the subject is a human subject.

24. A method of treating a subject afflicted with a cancer comprising a KRAS mutation, wherein the cancer comprises resistance to an inhibitor of KRAS, the method comprising administering to the subject a therapeutically effective amount of a therapy that increases the expression or activity of a TUSC2 polypeptide.

25. The method of claim 24, wherein the resistance is an acquired resistance.

26. The method of claim 24, wherein the inhibitor of KRAS is selected from the group consisting of sotorasib, adagrasib, MRTX1133, ARS-3248, ARS-853, ARS-1620, opnurasib, GDC-6036, RG6330, D-1553, BPI-421286, GH35, BEBT-607, JAB-21000, BI-2865, BI-2493, RMC-6236, and combinations of any thereof.

27. The method of claim 24, wherein the subject was previously treated with at least one inhibitor of KRAS prior to said administering.

28. The method of claim 27, wherein the at least one inhibitor of KRAS is selected from the group consisting of sotorasib, adagrasib, MRTX1133, ARS-3248, ARS-853, ARS-1620, opnurasib, GDC-6036, RG6330, D-1553, BPI-421286, GH35, BEBT-607, JAB-21000, BI-2865, BI-2493, RMC-6236, and combinations of any thereof.

29. The method of claim 24, wherein the KRAS mutation is a missense mutation.

30. The method of claim 24, wherein the KRAS mutation is selected from the group consisting of G12D, G12V, G13D, G12C, G12A, Q61H, G12R, A146T, G13C, G12S, Q61L, Q22K, A146V, G12F, Q61R, E63K, L19F, D33E, A59G, T74P, and K117R.

31. The method of claim 24, wherein the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, colorectal cancer, choloangiocarcinoma, uterine endometrial carcinoma, testicular germ cell cancer, and cervical squamous cell carcinoma.

32. The method of claim 24, wherein the method comprises:33US_ACTIVE\131015922W-1a) administering a TUSC2 polypeptide or a fragment thereof to said subject; or b) administering a polynucleotide molecule encoding a TUSC2 polypeptide or a fragment thereof to said subject.

33. The method of claim 32, wherein the TUSC2 polypeptide comprises a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:1.

34. The method of claim 32, wherein the polynucleotide molecule encoding the TUSC2 polypeptide comprises a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity to SEQ ID NO:2.

35. The method of claim 32, wherein the method comprises administering an expression vector comprising the polynucleotide molecule encoding the TUSC2 polypeptide or the fragment thereof to said subject.

36. The method of claim 35, wherein the method comprises administering a pharmaceutical composition comprising the expression vector to said subject.

37. The method of claim 36, wherein the pharmaceutical composition comprises quaratusugene ozeplasmid.

38. The method of claim 24, wherein the administering comprises local, regional, systemic, or continual administration.

39. The method of claim 24, wherein the administering comprises administering a single dose to said subject.

40. The method of claim 24, wherein the administering comprises administering multiple doses to said subject.

41. The method of claim 24, wherein the administering comprises oral, intravenous, or intramuscular administration.

42. The method of claim 24, wherein the administering comprises administering a pharmaceutical composition comprising the therapy that increases the expression or activity of a TUSC2 polypeptide to said subject.34US_ACTIVE\131015922W-143. The method of claim 24, the method further comprising administering an immunotherapy, a targeted cancer therapy, a chemotherapy, a radiation therapy, or surgery to said subject.

44. The method of claim 24, the method further comprising administering the inhibitor of KRAS or at least a second inhibitor of KRAS to said subject.

45. The method of claim 44, wherein the inhibitor of KRAS or the second inhibitor of KRAS is selected from the group consisting of sotorasib, adagrasib, MRTX1133, ARS-3248, ARS-853, ARS-1620, opnurasib, GDC-6036, RG6330, D-1553, BPI-421286, GH35, BEBT-607, JAB- 21000, BI-2865, BI- 2493, RMC-6236, and combinations of any thereof.

46. The method of claim 44, wherein administering the inhibitor of KRAS or the second inhibitor of KRAS comprises local, regional, systemic, or continual administration.

47. The method of claim 44, wherein administering the inhibitor of KRAS or the second inhibitor of KRAS comprises administering a single dose to said subject.

48. The method of claim 44, wherein administering the inhibitor of KRAS or the second inhibitor of KRAS comprises administering multiple doses to said subject.

49. The method of claim 44, wherein administering the inhibitor of KRAS or the second inhibitor of KRAS comprises oral, intravenous, or intramuscular administration.

50. The method of claim 44, wherein administering the inhibitor of KRAS or the second inhibitor of KRAS comprises administering a pharmaceutical composition comprising the inhibitor of KRAS or the second inhibitor of KRAS to the subject.

51. The method of claim 24, wherein the subject is a mammalian subject.

52. The method of claim 24, wherein the subject is a human subject.35US_ACTIVE\131015922W-1

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