SREB / hmgcr inhibition in cancer with chromosome 3q gain

Abstract

Disclosed are methods of treatment and inhibitors for cancers characterized by the gain of the long arm of chromosome 3 in a subject with an inhibitor of inhibitor of the Sterol Regulatory Element Binding (SREB) / 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR) pathway.

Description

[0001] SREB / HMGCR INHIBITION IN CANCER WITH CHROMOSOME 3Q GAIN

[0002] GOVERNMENT LICENSE RIGHTS

[0003]

[0001] This invention was made with government support under grant numbers CA237733, GM147287, CA273723, and CA197568 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005]

[0002] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos: 63 / 656,833, filed June 6, 2024 and 63 / 763,429, filed on February 26, 2025, each of which is incorporated herein by reference in its entirety.

[0006] BACKGROUND OF THE DISCLOSURE

[0007]

[0003] Aneuploidy, the loss or gain of whole chromosomes or chromosome arms, is rare and poorly tolerated in normal cells, but occurs in about 90% of solid tumors (Taylor et al., Cancer Cell 33 (4): 676-689 (2018)) and 98% of lung squamous cell carcinomas (SCCs); however, the mechanisms through which specific aneuploidies affect cancer development are unclear. Additionally, generating mammalian models of specific chromosome arm alterations is technically difficult, limiting further study. Cancers have tumor, cell, and tissue specific patterns of chromosome arm copy-number alterations that influence tumor evolution and sensitivity to anti-cancer therapies. SCCs affecting lung, head and neck, esophageal, cervical squamous, and bladder squamous tissues have been shown to feature conserved early losses and gains of chromosome 3 short arm (chr3p) and 3 long arm (chr3q), respectively (Taylor et al., Cancer Cell 33(4):676-689 (2018)), and are associated with relatively few other oncogenic drivers and treatment options. These chr3p and chr3q aneuploidies are particularly well established in lung SCCs and have been shown to promote tumorigenesis, metastasis, and poor prognosis. Aneuploidy can promote tumorigenesis by increasing genetic heterogeneity and promoting tumor evolution. Recent attempts have been made to establish genomic signatures shared across cancer subsets (Hoadley et al., Cell 158(4).' 929-944 (2014)). However, the effect of aneuploidy and gene duplication and / or deletion remain poorly known. Therefore, there is a pressing need to better understand and provide effective treatment for cancers exhibiting aneuploidy.

[0008] SUMMARY OF THE DISCLOSURE

[0009]

[0004] The methods of treating cancer provided herein are expected to address the above need in cancers characterized by chr3q gain.

[0010]

[0005] A first aspect of the present disclosure is directed to a method of treating cancer in a subject. The method entails administering to a subject in need thereof one or more inhibitors of the Sterol Regulatory Element Binding (SREB) / 3 -Hydroxy-3 -Methylglutaryl (HMG)-CoA Reductase (HMGCR) pathway to the subject, wherein the cancer comprises a chr3q gain.

[0011]

[0006] A second aspect of the present disclosure is directed to one or more inhibitors of the SREB / HMGCR pathway for use in treating cancer, wherein the cancer comprises a chr3q gain.

[0012]

[0007] A third aspect of the present disclosure is directed to use of one or more inhibitors of the SREB / HMGCR pathway in the manufacture of a medicament for treating cancer.

[0013]

[0008] The inhibitors are generally provided in the form of a pharmaceutical composition comprising the inhibitor and a pharmaceutically acceptable carrier, preferably in a therapeutically effective amount.

[0014]

[0009] The working examples described herein show that chr3q gain is a biomarker of statin response and sensitivity in squamous cancers. The working examples further demonstrate that aneuploidy-induced vulnerabilities will aid in the understanding of how cancers exploit aneuploidies to promote tumorigenesis and to elucidate novel and specific therapeutic targets for cancers with shared copy-number profiles.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016]

[0010] FIG. 1A - FIG. IB are a set of schematic illustrations showing the SREB / HMGCR pathway of cholesterol and fatty acid synthesis. FIG. 1A schematically illustrates the SREB / HMGCR pathway and select inhibitors of the cholesterol and fatty acid synthesis. FIG. IB schematically illustrates the steps of mevalonate metabolism and inhibitors in the SREB / HMGCR pathway. [Oil] FIG. 2A - FIG. 2C are a set of dot and line plots showing the genome-wide CRISPRi screen and the effect of SREBP inhibitors on cell growth. FIG. 2A is a dot plot showing the SREBFF1 hit in the genome-wide CRISPRi screen in AALE cells with or without chr3q gain. FIG. 2B is a line plot showing the effect of Fatostatin on cell growth on cells with two copies of chromosome 3 (WT), cells with chromosome 3p deleted (chr3p del; 3P), and cells with chromosome 3q gain (chr3q gain; 3Q). FIG. 2C is a line plot showing the effect on Betulin on cell growth on WT, chr3p deleted, and chr3q gain cells.

[0017]

[0012] FIG. 3A - FIG. 3G are a set of line plots showing the effects SREB / HMGCR pathway inhibitors on cellular toxicity in cells with and without chromosome 3 aneuploidy. FIG. 3 A is a line plot showing the effect of mevastatin on cell viability. FIG. 3B is a line plot showing the effect of rosuvastatin calcium on cell viability. FIG. 3C is a violin plot showing the effect of lovastatin on viability of 11 lung cancer cell lines. FIG. 3D is a line plot showing the effect of hymeglusin on cell viability. FIG. 3E is a line plot showing the effect of R00488071 (RO 48-8071), an oxidosqualene cyclase inhibitor, on cell viability. FIG. 3F is a bar plot showing the effect of 25- Hydroxycholestoerol on cell viability. FIG. 3G is a set of bar plots showing the effect of the PI3K inhibitors Pl-305 and PKI-179 on cell viability.

[0018]

[0013] FIG. 4A- FIG. 4B are a set of bar plots showing the effect of statins on cellular apoptosis in cells with and without chromosome 3 aneuploidy. FIG. 4Ais a bar plot showing early apoptosis after mevastatin treatment, measured by flow cytometry with propidium iodide and annexin-V staining. FIG. 4B is a bar plot showing caspase activation after mevastatin treatment, measured by Caspase-Gio 3 / 7.

[0019]

[0014] FIG. 5A - FIG. 5C are a set of bar plots showing mevalonate rescue of mevastatin- induced cell death. FIG. 5A is a bar plot showing the effect of mevalonate on cells treated with 31 pM mevastatin. FIG. 5B is a bar plot showing the effect of mevalonate on cells treated with 10 pM mevastatin. FIG. 5C is a bar plot showing the effect of mevalonate on cells treated with 3.1 pM mevastatin.

[0020]

[0015] FIG. 6 is a bar plot showing the synergistic effect of mevastatin and fatostatin on cell viability of chr3q gain cells.

[0021]

[0016] FIG. 7A- FIG. 7E is set of heatmaps, bar, and dot plots showing gene expression after mevastatin or fatostatin treatment. FIG. 7Ais a heatmap showing differential expression of SREBF1 transcriptional targets after mevastatin or fatostatin treatment in chr3 WT and chr3q gain cells. FIG. 7B is a bar plot showing pathways enriched in gene expression in DMSO-treated chr3 WT vs. chr3q gain cells. FIG. 7C is a bar plot showing pathways enriched in gene expression in mevastatin-treated chr3q gain cells as compared to DMSO-treated chr3q gain cells. FIG. 7D is a dot plot showing the top differentially expressed genes in mevastatin-treated chr3q gain cells. FIG. 7E is a bar plot showing pathways enriched in gene expression in mevastatin-treated chr3q gain cells as compared to mevastatin chr3 WT cells.

[0022]

[0017] FIG. 8A- FIG. 8C is a set of bar plots and heatmaps showing lipidomic profiling of chr3 WT and chr3q gain cells with and without mevastatin. FIG. 8A is a bar plot that shows cholesterol uptake in chr3 WT and chr3q gain cells. FIG. 8B is a bar plot and heatmap that shows negative ion profiling of select lipid classes (left) and lipids (right) in chr3 WT and chr3q gain cells. FIG. 8C is a heatmap that shows lipidomic profiling results of top differential lipids from an independent experiment in chr3 WT and chr3q gain cells treated with mevastatin or DMSO.

[0023]

[0018] FIG. 9A - FIG. 9B is a set of dose curve plots showing the effects of mevastatin and fatostatin on cell viability on an esophageal squamous cancer cell line with chr3p loss and chr3q gain as compared to esophageal squamous cancer cell line with only chr3p loss. FIG. 9A is a dose curve plot showing the effect of mevastatin on cell viability. FIG. 9B is a dose curve plot showing the effect of fatostatin on cell viability.

[0024]

[0019] FIG. 10A- FIG. 1 OB is a set of bar plots showing the effects of mevastatin and fatostatin on chr3 WT and chr3q gain engineered isogenic organoids. FIG. lOA is a bar plot that shows the effects of mevastatin on chr3 WT and chr3q gain engineered isogenic organoids. FIG. lOA is a bar plot that shows the effects of fatostatin on chr3 WT and chr3q gain engineered isogenic organoids.

[0020] FIG. 11 is a dot plot showing guide enrichment in mevastatin-treated chr3q gain cells.

[0025]

[0021] FIG. 12A- FIG. 12B is a set of bar plots showing cholesterol in WT and chr3q gain cells as measured by cholesterol-glo. FIG. 12Ais a bar plot that shows total intracellular cholesterol. FIG. 12B is a bar plot that shows total extracellular cholesterol.

[0026]

[0022] FIG. 13A - FIG. 13B is a set of dot and bar plots showing cell viability in CRISPR knockdown in SREB / HMGCR pathway genes. FIG. 13 A is a dot plot showing cell viability by CellTiter-Glo on WT, chr3g gain, and chr3p deletion cells that have SREBF1 knockdown in a CRISPRi. FIG. 13B is a is a bar graph showing cell death measured by TrypanBlue on cancer cell lines with and without chr3q gain; CRISPR guides targeted controls, SREBF1, SREBF2, or HMGCR.

[0027] DETAILED DESCRIPTION OF THE DISCLOSURE

[0028]

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary, the following terms have the meaning indicated to facilitate the understanding of the present disclosure.

[0024] As used in the description and the appended claims, the singular forms “a”, “an”, and “the” mean “one or more” and therefore include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0029]

[0025] Unless stated otherwise, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.”

[0030]

[0026] The term “approximately” as used herein refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0031]

[0027] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element or method step not specified in the claim (or the specific element or method step with which the phrase “consisting of’ is associated). The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified elements and method or steps and “unrecited elements and method steps that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.

[0032] Methods of Treating Cancer

[0033]

[0028] In one aspect, the disclosure provides a method of treating cancer in a subject. The method entails administering to a subject in need thereof one or more inhibitors of the Sterol Regulatory Element Binding (SREB) / 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR) pathway to the subject, wherein the cancer comprises a chromosome 3q gain.

[0034]

[0029] A second aspect of the present disclosure is directed to one or more inhibitors of the SREB / HMGCR pathway for use in treating cancer, wherein the cancer comprises a chr3q gain.

[0035]

[0030] A third aspect of the present disclosure is directed to use of one or more inhibitors of the SREB / HMGCR pathway in the manufacture of a medicament for treating cancer.

[0036]

[0031] The inhibitors are generally provided in the form of a pharmaceutical composition comprising the inhibitor and a pharmaceutically acceptable carrier, preferably in a therapeutically effective amount.

[0037]

[0032] The disclosure generally refers to the method of treatment of the first aspect, but applies equally to the inhibitors for use and use of inhibitors described in the second and third aspects. One or more inhibitors of the SREB / HMGCR pathway may be combined, as described below.

[0038]

[0033] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone (or disposed) to or suffering from a chr3q gain cancer. In some embodiments, the subject is a human. Therefore, a subject “having cancer” or “in need of’ treatment according to the present disclosure broadly embraces subjects who have been positively diagnosed with a chr3q gain cancer, including subjects having a diagnosed chr3q gain cancer who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g., in remission) but who might still be at risk of cancer, and subjects who have not been positively diagnosed but who are predisposed to having cancer (e.g., on account of the basis of prior medical history and / or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to cancer).

[0039]

[0034] The terms “treat”, “treating”, and “treatment” as used herein refer to any type of intervention, process performed on, or the administration of an active agent to the subject in need thereof (e.g., a subject suspected of having a chr3q gain cancer or who has been diagnosed with a chr3q gain cancer and who may be undergoing treatment) with the therapeutic objective (“therapeutic effect”) of reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, or preventing or delaying the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a chr3q gain cancer.

[0040]

[0035] The term “inhibitor of the SREB / HMGCR pathway” relates to an inhibitor of the expression or function of one or more protein expressed by the genes shown in FIG. 1 A - FIG. IB including specific examples described below.

[0041]

[0036] The term “chromosome 3q gain” relates to an aneuploidy of part (e.g., 3q26 or 3q26-28) or all of the 3 long arm of chromosome 3 (chr3q) in some or all of the tumor cells forming the cancer (Taylor et al., Cancer Cell 33(4 / 676-689 (2018)). Detection of chr3q gain may be achieved by identification of increased expression of part or all of chr3q using techniques such as single nucleotide polymorphism (SNP) arrays (Taylor et al., Cancer Cell 33(4 / 676-689 (2018)), immunohistochemistry, comparative genomic hybridization (CGH) or fluorescence in situ hybridization (FISH) (Qian and Massion, J. Thorac. Oncol. 3(3 / 212-5 (2008)). Additionally, chr3q gain can be identified from panel sequencing data (Spurr et al., Bioinformatics 37(76 / 2461-2463 (2021)). Copy number variation may be quantified for individual genes within this region (Schwaederle et al., Cell Cycle 77(77 / 2355-61 (2015); Jeon et al., Thorac. Cancer 74(26 / 2635- 2641 (2023)) such ^ SOX2, KLHL6,MCF2L2, LAMP3,MCCC1, ABCC5,ATP11B, TP63, YEATS2, TERC, TFG, or PIK3CA. Apreferred quantitative definition of “chromosome 3q gain” is that >80% of cells in a tumor or biopsy contain at least one extra copy of one gene or SNP from chr3q using methods described in Taylor et al., Cancer Cell 33(4 / 676-689 (2018).

[0042]

[0037] The present disclosure is based, in part, on the surprising discovery of that the SREB / HMGCR cholesterol synthesis pathway is a novel, druggable target of cancers characterized by the gain of the long arm of chromosome 3 (chr3q). The SREB / HMGCR pathway is schematically illustrated in FIG. 1A and FIG. IB. SREB proteins (SREBPs) are master transcription factors that play a crucial role in regulating genes involved in the biogenesis of cholesterol, fatty acids (FAs), and triglycerides (TGs). SREBPs are encoded by the genes SREB Transcription Factor (SREBF)1 and SREBF2. REBP1 expresses two isoforms, SREBPla and SREBPlc, which have different transcriptional start sites and therefore different first exons.

[0038] SREBPs are expressed as precursors that are cleaved to release the N-terminal transcriptional activation domain, which then enters the nucleus to stimulate the transcription of target genes. SREBP cleavage-activating protein (SCAP) acts as an escort protein to transport SREBP precursors to the Golgi apparatus, where the active forms are generated by two proteases (site 1 protease (S IP) and site 2 protease(S2P)) and an anchoring protein. ER membrane proteins insulin-induced genes (INSIGs) bind the sterol-sensing domains of SCAP, causing the retention of the SCAP-SREBP complex in the ER based on sterol levels. (Li et al., Biomedicines 77(72 / 3280 (2023)). This disclosure identifies that disruption of SREBF1 genetically or with small molecule inhibitors (“chemically”) reduced cell growth and increased apoptosis of chr3q gain cancer cells.

[0043]

[0039] Active Agents useful in the practice of the present disclosure include SREB inhibitors, HMGCR inhibitors, SCAP inhibitors, and lanosterol synthase (LSS) inhibitors. The term “inhibitor” is used in its broadest sense and includes any agent such as a small molecule, nucleotide-based antisense inhibitors, or a combination thereof that acts to disrupt, directly or indirectly, and reduce or even eliminate the function of the target. In some embodiments, protein inhibition (i.e., disruption) may include blockage of a protein active site, a cleavage site, or a protein-protein interaction.

[0044] SREB inhibitors

[0045]

[0040] In some embodiments, the inhibitor is a SREB inhibitor. A SREB inhibitor may be an inhibitor that acts to disrupt the function or expression of a SREB gene (i.e., SREBF1, SREBF2), a SREB protein (e g., SREBP la, SREBP 1c, SREBP2), or a SREBP complex. NCBI Accession numbers for the SREBF1 and SREBF2 genes, and their respective mRNA and proteins are set forth in Table 1 and are incorporated herein by reference.

[0046] Table 1

[0047]

[0048]

[0041] In some embodiments, the SREB inhibitor is a small molecule. Representative SREB small molecule inhibitors include, for example, fatostatin and betulin. The preceding small molecule inhibitors have the following structures:

[0049]

[0043] In some embodiments, the SREB inhibitor is a nucleotide-based antisense inhibitor targeted against SREBF1 and / or SREBF2.

[0050]

[0044] The term “antisense inhibitor” as used herein refers to a non-naturally occurring polymer of nucleotides (oligomer) capable of binding a target RNA molecule. The term “antisense inhibitor” as used herein embraces antisense oligonucleotides (ASOs) and RNA interference (RNAi) inhibitors, including short interfering RNAs (siRNAs), synthetic mircoRNAs (miRNAs), short hairpin RNAs (shRNAs), guide RNAs (gRNAs), and single-guide RNAs (sgRNAs).

[0051]

[0045] ASOs may be made up of ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides, or a combination thereof. ASOs modulate target RNAs by hybridization through at least partial complementary region(s). ASO modulation mechanisms include transcriptional arrest, RNA synthesis disruption (e.g., at various stages including capping, splicing, and / or transport from nucleus to cytoplasm), ribosome attachment, ribonuclease (RNAse) H recruitment, degradation of mRNA, translational arrest (e.g., ASO binding to a target RNA blocking translation), or steric blocking of target RNA by ASO hybridization.

[0052]

[0046] RNAi by antisense inhibitors is a phenomenon in which the introduction of RNA, typically double-stranded RNA (dsRNA), into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent 6,506,559; Fire et al., 1998, Nature 391 (19):306-311 ; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, P A (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2003). Soutschek et al. (2004, Nature 432: 173-178) describes a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G / C content, C / T content at the termini, melting temperature (Tm) and the nucleotide content of the 3' overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209- 216. Therefore, the present disclosure also includes methods of using antisense inhibitors to decrease levels of SREBF1, SREBF2, or other target proteins.

[0053]

[0047] In some embodiments of the present methods, the subject is treated with a SREB inhibitor alone, by way of monotherapy.

[0054] Other Active Agents

[0055]

[0048] In other embodiments, the subject is treated by way of combination therapy, with at least one additional active agent. In some embodiments, the additional active agent is a HMGCR inhibitor, a SCAP inhibitor, a LSS inhibitor, a PI3K inhibitor, or a combination thereof.

[0056] HMGCS inhibitors

[0057]

[0049] HGM-Co-A synthase (HMGCS) is a SREB-activated gene and catalyzes the reaction in which acetyl-CoA condenses with acetoacetyl-CoA to form 3 -hydroxy-3 -methylglutaryl -Co A, the second step in the mevalonate-dependent isoprenoid biosynthesis pathway. Blocking HMGCS may provide an additional means of inhibiting the SREB / HMGCR pathway or enhancing the therapeutic effect of a SREB inhibitor. NCBI Accession numbers for the HMGCS gene, mRNA, and proteins are set forth in Table 1 and are incorporated herein by reference.

[0058]

[0050] A HMGCS inhibitor may be an inhibitor that acts to disrupt the function or expression of the HMGCS gene or the HMGCS protein. In some embodiments, the HMGCS inhibitor is a small molecule. Representative HMGCS small molecule inhibitors include, for example, hymeglusin

[0059] (1233A; F244; L-659-699), acetoacetyl -Co A, (S)-3-hydroxy-3-methylglutaryl-CoA and combinations thereof. The preceding small molecule inhibitors have the following structures: y y methylglutaryl-CoA).

[0060]

[0052] In some embodiments, the HMGCS inhibitor is an antisense inhibitor targeted against the HMGCS gene.

[0061] HMGCR inhibitors

[0053] HMGCR is a SREB-activated gene and the rate-limiting enzyme for cholesterol synthesis. Blocking HMGCR may provide an additional means of inhibiting the SREB / HMGCR pathway or enhancing the therapeutic effect of a SREB inhibitor. NCBI Accession numbers for the

[0062] HMGCR gene, mRNA, and proteins are set forth in Table 1 and are incorporated herein by reference.

[0054] A HMGCR inhibitor may be an inhibitor that acts to disrupt the function or expression of the HMGCR gene or the HMGCR protein. In some embodiments, the HMGCR inhibitor is a small molecule. Representative HMGCR small molecule inhibitors include, for example, mevastatin, rosuvastatin (Crestor®), lovastatin (Altoprev®, Mevacor®), pitavistatin (Livalo®), simvastatin (FloLipid®, Zocor®), atorvastatin (Lipitor®), 25-hydroxcholesterol, and combinations thereof. The preceding small molecule inhibitors have the following structures:

[0063]

[0064]

[0056] In some embodiments, the HMGCR inhibitor is an antisense inhibitor targeted against the HMGCR gene.

[0065] SCAP inhibitors

[0066]

[0057] SCAP is the SREB escort protein which escorts SREB to the nucleus. Blocking SCAP may provide an additional means of inhibiting the SREB / HMGCR pathway or enhancing the therapeutic effect of a SREB inhibitor, a HMGCR inhibitor, or a combination therapy of a SREB inhibitor and a HMGCR inhibitor. NCBI Accession numbers for the SCAP gene, mRNA, and proteins are set forth in Table 1 and are incorporated herein by reference.

[0067]

[0058] A SCAP inhibitor may be an inhibitor that acts to disrupt the function or expression of the SCAP gene, the SCAP protein, or the SCAP / SREB complex.

[0068]

[0059] In some embodiments, the SCAP inhibitor is an antisense inhibitor targeted against the SCAP gene.

[0069] LSS inhibitors

[0070]

[0060] LSS (also known as 2,3-Oxidosqualene cyclase (OSC)) catalyzes the first step in the biosynthesis of cholesterol; the conversion of (S)-2,3 oxidosqualene to lanosterol. Blocking LSS may provide an additional means of inhibiting the SREB / HMGCR pathway or enhancing the therapeutic effect of a SREB inhibitor, a HMGCR inhibitor, a SCAP inhibitor, or a combination therapy of any two or more thereof. NCBI Accession numbers for the LSS gene, mRNA, and proteins are set forth in Table 1 and are incorporated herein by reference.

[0061] ALSS inhibitor may be an inhibitor that acts to disrupt the function or expression of the LSS gene or the LSS protein. In some embodiments, the LSS inhibitor is a small molecule. Representative LSS small molecule inhibitors include, for example, 24(S),25-epoxycholesterol,

[0071] MM0299 (Nguyen et al., Cell Chem. Biol. 30(2) :214-229 (2023)), R00488071 ({4-[6- (allylmethylamino)hexyloxy]-2-fluorophenyl}-(4-bromophenyl)m ethanone fumarate) (Morand et al., J. Lipid Res. 33(2 / 373-390 (1997)), RO0613479 (allyl-{6-[3-(4- bromophenyl)benzo[c / ]isothiazol-6-yloxy]hexyl (methylamine fumarate) (Dehmlow et al., J. Med.

[0072] Chem. 46(75 / 3354-70 (2003)), RO0717625 ( / raw.s-methyl-{4-[5-(methylpropylamino)pent-l- ynyl]cyclohexyl(carbamic acid 4-chlorophenyl ester fumarate) (Staedler et al., J. Med. Chem. 55(77 / 4990-5002 (2012)), RO0713852 (Zraws-{4-[5-

[0073] (allylmethylamino)pentyl]cy cl ohexyl (methyl carbamic acid 4-(trifluoromethyl)phenyl ester citrate), and combinations thereof. The preceding small molecule inhibitors have the following structures:

[0074]

[0063] In some embodiments, the LSS inhibitor is an antisense inhibitor targeted against the LSS gene.

[0075] PI3K inhibitors

[0064] Phosphatidylinositol 3 -kinases (PI3Ks) are lipid kinases that regulate a diverse set of cellular processes including cell proliferation, adhesion, survival, and motility. The PI3K and mechanistic target of rapamycin kinase (mTOR) in its role as part of mTOR complex 1 (mTORCl) are upstream of the SREB / HMGCR pathway. Blocking PI3K and / or mTORCl may provide an additional means of inhibiting the SREB / HMGCR pathway or enhancing the therapeutic effect of a SREB inhibitor, a HMGCR inhibitor, or a combination therapy of a SREB inhibitor and a HMGCR inhibitor. NCBI Accession numbers for the PI3K and mTOR genes, mRNAs, and proteins are set forth in Table 1 and are incorporated herein by reference.

[0076]

[0065] API3K inhibitor may be an inhibitor that acts to disrupt the function or expression of the PIK3CA gene, the PIK3CB gene, the PIK3CD gene, the PIK3CG gene, or the MTOR gene, the PIK3CA protein, the PIK3CB protein, the PIK3CD protein, the PIK3CG protein, or the MTOR protein, or the mTORCl or the mTOR complex 2 (mT0RC2).

[0077]

[0066] A PI3K inhibitor may be an inhibitor that acts to disrupt the function or expression of a PI3K gene or a PI3K protein. In some embodiments, the PI3K inhibitor is a small molecule. Representative PI3K small molecule inhibitors include, for example, PI- 103, PKI-179, alpelisib (Piqray®; Vijoice®), buparlisib, idelalisib (Zydelig®), leniolisib (Joenja®), copanlisib, umbralisib (Ukoniq®), pictilisib, PX-866, pilaralisib, inavolisib (Itovebi®), duvelisib (Copiktra®), parsaclisib, apitolisib, bimiralisib, dactolisib, everolimus (Afinitor®; Zortress ®), gedatolisib, olcorolimus, omipalisib, onatasertib, paxalisib, rapamycin , ridaforolimus, samotolisib, sirolimus (Rapamune ®), temsirolimus (Torisel ®), voxtalisib, zotarolimus, BEZ235, GSK2126458, PQR309, taselisib, MLN1117, GSK2636771, AZD8186, SAR260301, and IPI-549.

[0078]

[0067] In some embodiments, the PI3K inhibitor is an antisense inhibitor targeted against the PIK3CA gene, the PIK3CB gene, the PIK3CD gene, the PIK3CG gene, or the MTOR gene.

[0079] Chr3q Gain Cancers

[0080]

[0068] The present disclosure is directed at least in part to the discovery that SREB / HMGCR pathway inhibitors display increased toxicity to chr3q gain cancer cells.

[0081]

[0069] The vast majority of squamous cell cancers present with a chr3q gain (Taylor el cd., Cancer Cell 33(4) :676-689 (2018)). Representative chr3q gain squamous cell cancers that may be treated with the methods disclosed herein include, for example, squamous cell lung carcinoma, head and neck squamous cell carcinoma (HNSCC), oral squamous cell carcinoma, esophageal squamous cell carcinoma (ESCC), cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC). In some embodiments, the cancer is squamous cell lung cancer.

[0082] Compositions and Formulations

[0083]

[0070] The active agent or agents described herein may be formulated into pharmaceutical compositions in accordance with known techniques. The pharmaceutical compositions may include a pharmaceutically acceptable carrier.

[0084]

[0071] Broadly, the active agent(s) disclosed herein and their pharmaceutically acceptable salts may be formulated together, in combinations of two or more, or individually, into a given type of composition in accordance with conventional pharmaceutical practice such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (2C)11' ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). The type of formulation depends on the mode of administration which may include enteral (e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (s.c.), intravenous (i.vf, intramuscular (i.mf, and intrastemal injection, or infusion techniques, intra-ocular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, interdermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation, and inhalation) and topical (e.g., transdermal). In general, the most appropriate route of administration will depend upon a variety of factors including, for example, the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). For example, parenteral (e.g., intravenous) administration may also be advantageous in that the inhibitor may be administered relatively quickly such as in the case of a single-dose treatment and / or an acute condition.

[0085]

[0072] In some embodiments, the active agents are formulated for oral, intramuscular, subcutaneous, or intravenous administration (e.g., systemic intravenous injection).

[0086]

[0073] Accordingly, active agents may be formulated into solid compositions (e.g., powders, tablets, dispersible granules, capsules, cachets, and suppositories), liquid compositions (e.g., solutions in which the inhibitor is dissolved, suspensions in which solid particles of the inhibitor are dispersed, emulsions, and solutions containing liposomes, micelles, or nanoparticles, syrups and elixirs), semi-solid compositions (e.g., gels, suspensions and creams), and gases (e.g., propellants for aerosol compositions). Inhibitors may also be formulated for rapid, intermediate or extended release.

[0087] Methods of Administration

[0088]

[0074] As used herein, the terms, “effective amount” and “therapeutically effective amount” refers to an amount of an active agent disclosed herein (e.g., a SREB inhibitor, a HMGCR inhibitor, a SCAP inhibitor, or a LSS inhibitor) or a pharmaceutically acceptable salt thereof, effective in producing the desired therapeutic response in a subject. Therefore, the term “effective amount” embraces amounts of an active agent, that when administered, induces a positive modification in the chr3q gain cancer, or is sufficient to inhibit or even prevent development or progression of a chr3q gain cancer, or alleviate to some extent, one or more of the symptoms of chr3q gain cancer, or which simply kills or inhibits the growth of chr3q gain cancer or otherwise blocks or reduces the activity of the SREB / HMGCR pathway in diseased cells. The effective amount of an active agent may vary depending on several factors among which may include the severity and stage of the chr3q gain cancer, the mode of administration, the age, body weight, and general health of the subject, and like factors well known in the medical arts (see, for example, Goodman and Gilman s’ , The Pharmacological Basis of Therapeutics, 10thEdition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001). Ultimately, an attending physician or veterinarian will decide upon the appropriate amount and dosage regimen.

[0089]

[0075] The active agents disclosed herein may be administered to a subject as a monotherapy or by way of combination therapy. Therapy may be “front / first-line”, i.e., as an initial treatment in subj ects who have undergone no prior anti-cancer treatment regimens, either alone or in combination with other treatments; or “second-line”, as a treatment in patients who have undergone a prior anticancer treatment regimen, either alone or in combination with other treatments; or as “third-line”, “fourth-line”, etc. treatments, either alone or in combination with other treatments. Therapy may also be given to patients who have had previous treatments which were unsuccessful or partially successful but who became intolerant to the particular treatment. Therapy may also be given as an adjuvant treatment, i.e., to prevent reoccurrence of cancer in patients with no currently detectable disease or after surgical removal of a tumor. Thus, in some embodiments, the active agents may be administered to a patient who has received, will receive, or is receiving another therapy, such as chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy, radiation therapy, targeted immunotherapy, cryotherapy, hormone therapy, stem cell transplant, or any combination thereof.

[0090]

[0076] The terms “in combination” and “concurrently” in this context mean that the active agents are co-administered, which includes substantially contemporaneous administration, by way of the same or separate dosage forms, and by the same or different modes of administration, or sequentially, e.g., as part of the same treatment regimen, or by way of successive treatment regimens. Thus, if given sequentially, at the onset of administration of the second inhibitor, the first active agent is in some cases still detectable at effective concentrations at the site of treatment. The sequence and time interval may be determined such that they can act together (e.g., synergistically) to provide an increased benefit than if they were administered otherwise. For example, the active agents may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion. Thus, the terms are not limited to the administration of the active agents at exactly the same time.

[0091]

[0077] These and other aspects of the present application will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain embodiments of the application but are not intended to limit its scope, as defined by the claims.

[0092] EXAMPLES

[0093] Example 1: Chr3q gain cells exhibit increased cellular toxicity to SREBP genetic and chemical inhibition

[0094]

[0078] To study cancer vulnerabilities associated with chromosome 3 arm aneuploidies, CRISPR-Cas9 was used to delete a 3p copy in human immortalized lung epithelial cell lines (AALE), the lung SCC cell -of-ori gin (Taylor et al., Cancer Cell 33( 676-689 (2018)). A subset of clones duplicated a wildtype chromosome 3 copy, transitioning to 3q gain and simultaneously providing independent models for chr3 WT, chr3p loss (deleted), and Chr3q gain. Next, a CRISPRi screen was performed with two replicates of each of two clones for a total of 12 pooled screens (Sanson et al., Nat. Commun. 9(7 5416 (2018)) in the chr3 WT, chr3p deleted, and chr3q gain AALE cells. Results were deconvoluted and analyzed using MAGeCK (Li et al., Genome Biol. 75(72 / 554 (2014)), a computational method which identifies positively and negatively selected genes from CRISPR screening data. Knockdown of genes with negative scores is more toxic to the aneuploid cells. A positive score represents enrichment of the CRISPR guide in aneuploid cells in comparison to wildtype cells, and knockdown of these genes is less toxic to the aneuploid cells.

[0095]

[0079] The genome-wide CRISPRi library is described by Sanson et al., Nat. Commun. 9(7 / 5416 (2018) and found that chr3q gain cells are more sensitive to knockdown of sterol regulatory element-binding factor 1 (SREBF1), a master regulator of cholesterol and fatty acid biosynthesis, its co-factor SREBP cleavage activating protein (SCAP), and downstream target HMG-CoA reductase (HMGCR), the rate-limiting enzyme for cholesterol synthesis (FIG. 1 A - FIG. IB). SREBF1 was identified as depleted from chr3q gain cells with a p-value < 10'6, suggesting a stronger effect on viability (FIG. 2A). Chr3q gain cells also showed up-regulated fatty acid metabolites, ceramides, triglycerides, and phosphatidylinositols. A confirmatory CRISPR inhibition (CRISPRi) of SREBF1 confirmed that chr3q gain cells had decreased cell viability as measured by CellTiter-Glo, while chr3p deletion cells (as well as cells with chr3 disomy) were unaffected (FIG. 13 A). CRISPR knockout of SREBFJ, SREBF2, or HMGCR confirmed that cancer cells with chr3q gain (KYSE30) had increased cell death as measured by Trypan blue staining, compared to cancer cells without chr3q gain (KYSE520) (FIG. 13B). A chemical screen, using a chemical library described by Corsello etal., Nat. Med. 23(4 / 405-408 (2017), found that engineered chr3q gain cells also had reduced proliferation, increased apoptosis, and DNA damage in response to statins (HMGCR inhibitors). These findings demonstrate increased toxicity in cells with chr3q gain using different targets in the pathway as well as different mechanisms of inhibition (genetic knockdown, genetic knockout, and chemical).

[0096]

[0080] The genetic screen hit of SREBF1 knockdown for chr3q gain cells was reproduced with the SREBF1 inhibitor fatostatin. Chr3q gain cells had significantly lower cell viability after 72-hour treatment in small airway growth medium (SAGM) with increasing amounts of fatostatin (0.1 to 100 pM), as compared to chr3 WT cells (FIG. 2B). Viability is relative to DMSO treatment in each cell type and measured by cell titer gio luminescence assay. The difference between chr3 WT and chr3q gain cells was significantly different (p < 0.001) when treated with 1 pM fatostatin. 72 hours of treatment with a second SREBF1 inhibitor, betulin, also caused lower cell viability in chr3q gain cells as compared to chr3 WT cells (FIG. 2C).

[0081] Surprisingly, CRISPR genetic ablation of fatty acid synthesis genes did not have increased cellular toxicity in chr3q gain cells as compared to cells without chr3q gain (FIG. 1A). This result was surprising because both cholesterol and fatty acid synthesis genes are activated by SREBP

[0097] Example 2: Chr3q gain cells exhibit increased cellular toxicity to SREB-activated gene HMGCR chemical inhibition

[0098]

[0082] An overview of the SREB / HMGCR pathway is schematically illustrated in FIG. 1A - FIG. IB. The mevalonate pathway, which produces sterols and isoprene, and the inhibition of the pathway by Statins, is schematically illustrated in FIG. IB. Chr3 WT, WT chr3p deleted, and chr3q gain AALE cells were treated with increasing amounts of HMGCR inhibitors for 72 hours in SAGM. Mevastatin-treated chr3q gain cells had lower cell viability as compared to chr3 WT cells (FIG. 3A). Rosuvastatin (Crestor®)-treated chr3q gain cells also had lower cell viability as compared to chr3 WT cells (FIG. 3B). The Y-axis of FIG. 3A and FIG. 3B reflects cell viability normalized to DMSO and measured by cell titer gio luminescence assay. The p-value significance of mevastatin-treated chr3q gain cell’s viability as compared to chr3 WT cells is given in Table 2.

[0099] Table 2: Effects of Mevastatin on cell viability of chr3q cells

[0100]

[0083] Additional HMGCR inhibitors that reduced cell viability of chr3q gain cells as compared to chr3 WT cells included lovastatin (Altoprev®, Mevacor®), pitavistatin (Livalo), simvastatin (FloLipid®, Zocor®), and atorvastatin (Lipitor®). Furthermore, post hoc analysis of large-scale screen databases (e.g., Dependency Map (Tsherniak etal., Cell 170(3) .564-576 (2017))) containing chr3q cancer cell lines revealed that chr3q gain lung cancer cells were significantly more sensitive to the HMGCS inhibitor lovastatin, * is p < 0.05 (FIG. 3C). The post hoc analysis included data from 11 lung cancer cell lines, of which 11 cell lines contained chr3p deletion, 3 cell lines contained chr3q gain, and 8 cell lines were chr3 WT.

[0101]

[0084] Differential viability on chr3q gain cells was seen throughout the SREBP / HMGCR pathway. The non-statin HMGCS inhibitor hymeglusin resulted in decreased cell viability in chr3q gain cells as compared to chr3 WT and chr3p deletion cells from 0.1 pM to 3.16 pM (FIG. 3D); p < 0.001 between chr3q gain and chr3 WT cells at 0.316 pM. The cholesterol biosynthesis inhibitor RO 48-8071 resulted in decreased cell viability in chr3q gain and chr3p deletion cells as compared to chr3 WT cells (FIG. 3E). The HMG-CoA reductase inhibitor 25-Hydroxycholesterol resulted in decreased cell viability in chr3q gain as compared to chr3 WT cells when used at 0.1 pM, p < 0.0002 (FIG. 3E). The PI3K inhibitors PI-103 (0.1 pM) and PKI-179 (10 pM) also resulted in decreased cell viability in chr3q gain cells as compared to chr3 WT cells, p < 0.01 and p < 0.05, respectively (FIG. 3G).

[0102]

[0085] The decrease in chr3q gain cell viability seen after mevastatin treatment is due to increased apoptosis. Chr3 WT and chr3q gain AAEE cells were treated with 10 pM mevastatin for 72 hours and measured for early apoptosis by propidium iodide (PI) and annexin V staining by flow cytometry. The early apoptotic cell population was identified as PE-Annexin V double positive cells and plotted as frequency of parent and normalized to DMSO. More chr3q gain cells were early apoptotic positive after mevastatin treatment as compared to chr3 WT cells, * is p < 0.05 (FIG. 4A). Chr3q gain cells had significantly more caspase-3 / caspase-7 cleavage after treatment with 10 pM mevastatin for 72 hours, * is p < 0.05 (FIG. 4B), as measured by the caspase-glo kit (Promega), demonstrating that these cells progress to a fully apoptotic state after mevastatin treatment.

[0103] Example 3 ; Mevalonate rescues mevastatin toxicity

[0104]

[0086] Exogenous metabolites of the SREBP / HMGCR pathway are known to rescue statin- induced phenotypes. Therefore, to confirm the on-target effects seen with mevastatin, chr3 gain AALE cells were treated with mevastatin with and without mevalonate for 72 hours (FIG. 5 A - FIG. 5C) and were measured for cell viability by the Cell Titer-Gio® kit (Promega). 2.5 mM mevalonate reduced mevastatin-induced chr3q cell death when treated with 31 pM mevastatin (FIG. 5 A), 10 pM mevastatin (FIG. 5B), and 3.1 pM mevastatin (FIG. 5C); * is p < 0.05. These data demonstrate that the mevastatin phenotype is on-target.

[0105] Example 4: Chr3q gain cells exhibit increased cellular toxicity to combination treatment of SREB and statin treatment.

[0106]

[0087] The combinational effects of two inhibitors in the SREBP / HMGCR pathway were next investigated. Co-treatment of chr3q gain cells with the HMGCR inhibitor mevastatin and the SREBP inhibitor fatostatin resulted in synergistic reduction of chr3q gain cell viability (FIG. 6). Chr3 WT and chr3q gain AALE cells were treated with 10 pM mevastatin with and without fatostatin for 72 hours; * is P < 0.05; ** is p < 0.01, reported as relative luminescence units (RLU) normalized to cells treated with DMSO. These data highlight the importance of inhibition of the SREBP / HMGCR pathway in human airway epithelial cells with chr3q gain and their use for treating chr3q gain cancer. Example 5: Effects of mevastatin and fatostatin on gene expression in chr3q disomic and chr3q gain cells.

[0107]

[0088] To elucidate the genetic pathways involved in the observed decreased cell viability, the genetic effects of the two SREBP / HMGCR pathway inhibitors were investigated. RNA-sequencing was performed on chr3 WT and chr3q gain AALE cells treated with (1) vehicle control (DMSO), (2) mevastatin low dose (10 pM), (3) mevastatin high dose (31 pM), and (4) fatostatin (3 pM). In chr3 WT AALE cells (chr3q diploid), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA a chr3q gene, is lowly expressed after DMSO treatment and neither mevastatin nor fatostatin treatment increased gene expression. In chr3 WT cells, fatostatin treatment increased SREBF1 gene expression and mevastatin treatment increased HMGCR, MHGCS1, and acyl-CoA synthetase short chain family member 2 (ACSS2) gene expression (FIG. 7A).

[0108]

[0089] Conversely, fatostatin treated chr3q gain cells had a smaller increase of SREBF1 gene expression as compared to chr3 WT cells and mevastatin treated chr3q gain cells had a smaller increase of HMGCR, MHGCS1, and ACSS2 gene expression as compared to chr3 WT cells (FIG. 7A). PIK3CA gene expression was also higher in mevastatin-treated chr3q gain cells as compared to chr3 WT cells. However, PIK3CA gene expression was also elevated in DMSO-treated chr3q gain cells.

[0109]

[0090] Pathway analysis showed that DMSO-treated chr3q gain cells had enriched gene expression of genes in the phosphatidylinositol 3 phosphate biosynthetic process (FIG. 7B). DMSO- treated chr3 WT cells had enriched gene expression of genes in 11 processes (FIG. 7B). Mevastatin- treated chr3q gain cells had enriched gene expression of genes in 14 processes as compared to DMSO-treated chr3q gain cells (FIG. 7B) and 12 processes as compared to Mevastatin-treated chr3 WT cells (FIGs. 7C-7D). Chr3q gain cells did upregulate cholesterol and fatty acid biosynthetic pathways upon mevastatin treatment (FIG. 7C), but chr3q gain cells did show reduced upregulation compared to chr3 disomy (FIGs. 7D-7E). Overall, these findings demonstrate that chr3q gain correlates with decreased cholesterol and fatty acid biosynthesis pathways in cells with chr3q gain.

[0091] Furthermore, chr3q gain cells had a similar reduction of total intracellular cholesterol after treatment with mevastatin as compared to WT chr3 cells (FIG. 12A). However, chr3q gain cells had significantly more total extracellular cholesterol after treatment with mevastatin as compared to WT chr3 cells (FIG. 12B). This suggests a cholesterol trafficking issue in cells with chr3q gain.

[0110]

[0092] Additional elucidation of the pathway mediating the increased sensitivity to SREB pathway inhibitors was conducted. A chr3q gene driver screen was conducted and compared genetic dependency in chr3q cells treated with mevastatin and DMSO. A library of CRISPRi guides targeting all genes on chr3q, along with some controls, was used for this pooled screen. Guides against TFG (Trafficking from ER to Golgi Regulator) were significantly enriched in mevastatin- treated chr3q gain cells (FIG. 11). TFG is important in endoplasmic reticulum (ER)-Golgi transport. FIG. 11 plots hits of a chr3q gene driver screen in the isogenic cells. FIG. 11 shows log fold change of CRISPRi guide level between mevastatin-treated and DMSO-treated chr3q gain cells.

[0111] Example 6: Lipidomic profiling on mevastatin treated on chr3q gain cells.

[0112]

[0093] Next lipid and cholesterol differences between chr3 WT and chr3q gain cells were investigated. RNA-sequencing was performed on chr3 WT and chr3q gain AALE cells and pathway analyses (e.g., KEGG, Reactome, GO, and Hallmark) identified up-regulated pathways (T3q) or down-regulated pathways 3q) in chr3q gain cells as compared to chr3 WT cells (Table 3). Cholesterol update was also measured using a luminescence kit (Cayman chemical). Chr3q gain cells had decreased cholesterol update as compared to chr3 WT cells, with *** is p < 0.001 (FIG. 8A). Table 3 : Pathway analyses of RNA Seq Data

[0113]

[0094] Phosphatidylinositols (Pls) are phosphorylated forms of phosphatidylinositol (PI) and play important roles in lipid signaling, cell signaling, and membrane trafficking. Chr3q gain cells and chr3 WT cells were characterized for their constituent lipid molecular species. N- hexadecanoyl sphingosine 1-phosphate (CerP), PI, and phosphatidylinositol 4,5-bisphosphate (PIP2) were enriched in chr3q gain cells as compared to chr3 WT cells (FIG. 8B).

[0114]

[0095] Further lipidomic profiling in chr3 WT and chr3q gain AALE cells with and without mevastatin treatment revealed additional lipid differences. Mevastatin treatment (10 pM), as compared to vehicle control (DMSO), resulted in significantly decreased levels of free cholesterol and cholesterol ester (CE 18: 1) in both chr3 WT and chr3q gain cells (FIG. 8C, “1” top two rows). The levels of triglyceride (TG) lipids and ceramides (Cer) increased significantly in mevastatin treated chr3 WT and chr3q gain cells (FIG. 8C, “2”). Interestingly, the level of several phospholipids, including phosphatidylethanolamines (PEs), Pls, phosphatidylcholines (PCs), and diacylglycerides (DGs) increased in chr3q gain cells (FIG. 8C, “3”). The working examples described herein show that cells containing the chr3q gain aneuploidy, a common mutation found in SCCs, have different gene expression patterns, lipid profiles, and viability to SREBP / HMGCR pathway inhibitors. These SREBP inhibitors therefore present ideal therapeutics for SCCs.

[0115] Example 7: Increased cellular toxicity to HMGCR and SREBP inhibitors remains in cells with both Chr3p loss and Chr3q gain

[0096] Chromosome 3p deletion and chromosome 3q gain frequently co-occur in SCC, including lung, head and neck, and esophagus SCC. A panel of SCC cancer cell lines of various tissues was compiled. To determine their chromosome 3 aneuploidy status, the cell lines were sequenced using low-pass whole genome sequencing prior to drug treatment. In these analyses, a pair of esophageal SCC cell lines were identified, one with concurrent chr3p del and chr3q gain (KYSE30; 3p del, 3q gain) and one with only chromosome 3p deletion (KYSE520; 3p del). These two cell lines were plated in 96-well plates, with drug (mevastatin or fatostatin) added one day after plating. Drugs were added at a range of doses from 0.01 to 31.6 pM (FIG. 9A - FIG. 9B), and viability was measured after 3 days of treatment using cell-titer-glo. The line with chr3q gain (KYSE30) was more sensitive to three days of inhibition of HMGCR (mevastatin) and inhibition of SREBP1 (fatostatin), as compared to cells with only chr3p deletion (KYSE520). This experiment suggests that aneuploidy-associated differential drug response to statins is not specific to the engineered isogenic cells and is also observed in cancer cell lines.

[0116] Example 8: Chr3q gain organoids exhibit increased cellular toxicity to SREB-activated gene HMGCR chemical inhibition

[0117]

[0097] Organoids are 3D cellular structures that can maintain long-term in vitro viability and a more physiologically relevant microenvironment. Organoids containing the chr3q gain cells were maintained in vitro and treated with mevastatin, fatostatin, or DMSO for 72 hours. Proliferation was measured by CellTitler-Glo 3D. Similar to the isogenic cell culture results above, organoids of chr3q gain cells showed increased cellular toxicity after treatment mevastatin (FIG. 10A) and fatostatin (FIG. 10B) as compared to treatment with DMSO, *** is p-value < 0.001, **** is p-value < 0.0001. These data demonstrate that HMGCR inhibitors are also effective at halting cell proliferation in the more physiologically relevant microenvironment of an organoid.

[0118]

[0098] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0119]

[0099] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

What is claimed is:

1. A method of treating cancer in a subject, comprising: administering to a subject in need thereof one or more inhibitors of the Sterol Regulatory Element Binding (SREB) / 3-Hydroxy-3-Methylglutaryl (HGM)-CoA Reductase (HMGCR) pathway; wherein the cancer comprises a chromosome 3q gain.

2. The method of claim 1, wherein the inhibitor of the SREB / HMGCR pathway is a small molecule or an antisense inhibitor (ASO).

3. The method of claim 1 or 2, wherein the inhibitor of the SREB / HMGCR pathway is a SREB inhibitor, a SREB protein cleavage-activating protein (SCAP) inhibitor, an HMG-CoA synthase (HMGCS) inhibitor, a HMGCR inhibitor, a lanosterol synthase (LSS) inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, a statin, or a combination thereof.

4. The method of any one of claims 1-3, wherein the inhibitor of the SREB / HMGCR pathway is a SREB inhibitor.

5. The method of claim 4, wherein the SREB inhibitor is fatostatin, botulin, or a combination thereof.

6. The method of any one of claims 1-3, wherein the inhibitor of the SREB / HMGCR pathway is a statin.

7. The method of claim 6, wherein the statin is mevastatin, rosuvastatin, lovastatin, pitavistatin, simvastatin, atorvastatin, hymeglusin, or a combination thereof.

8. The method of any one of claims 3-7, wherein the HMGCR inhibitor is25-hydroxcholesterol.

9. The method of any one of claims 3-8, wherein the HMGCS inhibitor is hymeglusin.

10. The method of any one of claims 6-9, further comprising administering to the subject a SREB inhibitor alongside the statin.

11. The method of claim 10, wherein the SREB inhibitor is fatostatin, botulin, or a combination thereof.

12. The method of any one of claims 1-3, wherein the inhibitor of the SREB / HMGCR pathway is an antisense inhibitor to SREB transcription factor 1 (SREBF1), SCAP, HMGCS, HMGCR, or a combination thereof.

13. The method of any one of claims 1-3, wherein the inhibitor of the SREB / HMGCR pathway is a LSS inhibitor.

14. The method of claim 13, wherein the LSS inhibitor is 24(S),25-epoxycholesterol, MM0299, R00488071, RO0613479, RO0717625, RO0713852, or a combination thereof.

15. The method of claim 13 or 14, wherein the LSS inhibitor is R00488071.

16. The method of any one of claims 1-15, wherein the PI3K inhibitor is PL103, PKI-179, alpelisib, buparlisib, idelalisib, leniolisib, copanlisib, umbralisib, pictilisib, PX-866, pilaralisib, inavolisib, duvelisib, parsaclisib, apitolisib, bimiralisib, dactolisib, everolimus, gedatolisib, olcorolimus, omipalisib, onatasertib, paxalisib, rapamycin , ridaforolimus, samotolisib, sirolimus, temsirolimus, voxtalisib, zotarolimus, BEZ235, GSK2126458, PQR309, taselisib, MLN1117, GSK2636771, AZD8186, SAR260301, IPL549, or a combination thereof.

17. The method of claim 16, wherein the PI3K inhibitor is PI-103, PKI-179, or a combination thereof.

18. The method of any one of claims 1-17, wherein the inhibitor is administered orally, intramuscularly, subcutaneously, or intravenously.

19. The method of any one of claims 1-18, wherein the cancer comprises a chromosome 3p deletion.

20. The method of any one of claims 1-19, wherein the cancer is a squamous cell cancer.

21. The method of claim 20, wherein the squamous cell cancer is squamous cell lung carcinoma, head and neck squamous cell carcinoma (HNSCC), oral squamous cell carcinoma, esophageal squamous cell carcinoma (ESCC), or cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC).

22. The method of claim 20 or 21, wherein the cancer is squamous cell lung carcinoma.

23. The method of any one of claims 1-22, wherein the subject is a human.

24. The method of any one of claims 1-23, further comprising administering to the subject a chemotherapeutic agent, radiation therapy, cryotherapy, hormone therapy, immunotherapy, or stem cell transplant.

25. The method of any one of claims 1-24, wherein the inhibitor of the SREB / HMGCR pathway is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

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