Re-expression of HNF4A for mitigating cancer-related cachexia

Abstract

Methods of predicting the clinical outcome of a subject having cancer, methods of detecting non-liver cancer in a subject, and methods of treating or preventing cancer or cancer-related cachexia are provided. Also provided are synthetic lipid nanoparticles encapsulating mRNA encoding HNF4A and compositions comprising the same.

Description

[0001] References to Electronic Sequence Listings

[0002] The contents of the electronic sequence listing (YEDA-TECH-P-025-PCT.xml; size: 55,530 bytes; and creation date: April 30, 2023) are incorporated herein by reference in its entirety.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 337,113 filed on May 1, 2022 and U.S. Provisional Patent Application No. 63 / 440,723 filed on January 24, 2023, both of which are entitled "REEXPRESSION OF HNF4A TO ALLEVIATE CANCER-ASSOCIATED CACHEXIA", the contents of which are all incorporated herein by reference in their entirety. Technical Field

[0005] The present invention belongs to the field of cancer and cancer-related cachexia treatment. Background Art

[0006] The liver communicates with all organs of our body as well as tumors, either directly through messenger molecules or indirectly through the immune system. Notably, the liver is also an immunogenic organ that contains a variety of resident immune cells that respond to systemic or tissue-specific immune-related vulnerabilities by producing acute phase proteins, complement components, cytokines, and chemokines. Although protective in purpose, recent work suggests that the immune system can also exert selective pressures that promote neoplastic features in normal tissue-resident cells. Thus, established hallmarks of carcinogenesis such as systemic inflammation, the tumor microenvironment, and the host may all promote carcinogenesis.

[0007] Unrestrained cancer growth requires a permanent supply of glucose, amino acids, and fatty acids. These nutritional requirements result from cancer-mediated reprogramming of metabolism in the tumor and its microenvironment. Understanding these interactions has led to synergistic therapies that combine chemotherapy with drugs that target metabolic dependencies between the tumor and the microenvironment. In addition to the tumor microenvironment, tumors are connected to the host through a network of nerves, blood, and lymphatic vessels, and extend their influence from the microenvironment to external organs such as the liver. Therefore, tumors can dysregulate tissue-specific metabolism and induce systemic metabolic rewiring, which may lead to cancer manifestations.

[0008] As the liver is a central metabolic organ essential for maintaining body homeostasis, it senses and responds to fluctuations in systemic nutrient levels by promoting tissue-specific adaptations that maintain systemic balance. At the cellular level, hepatocytes play an important role in carbohydrate, protein, amino acid, and lipid metabolism. Some of these metabolic reactions are primarily liver-specific, such as the complete urea cycle (UC), which disposes of excess nitrogen as ammonia by converting it to urea. Reduced UC activity has been reported in the liver of mice bearing 4T1 breast cancer and in the plasma of children with cancer, supporting a potential metabolic link between extrahepatic tumors and the liver.

[0009] The main metabolic phenomenon accompanying cancer is cancer-associated cachexia (CAC). 80% of cancer patients develop CAC in the late stage of the disease, manifested as weight loss, skeletal muscle wasting and atrophy of adipose tissue, and is estimated to be the direct cause of clinical deterioration leading to the death of at least 20% of cancer patients. Specifically, CAC is highly prevalent in pancreatic cancer, affecting more than 70% of patients. CAC is divided into 3 consecutive clinical stages: pre-cachexia, cachexia and refractory cachexia. The definition of pre-cachexia is less than 5% weight loss, while cachectic patients lose more than 5% weight. The refractory cachexia stage is determined by low WHO performance status score and survival of less than 3 months. Currently, there are no specific biomarkers for the identification of pre-cachexia, so most patients are in the cachexia stage or in the incurable refractory cachexia stage at the time of diagnosis. There is a great need to determine the metabolic changes in cancer and diagnostic methods for the onset of cachexia, as well as therapies that target these metabolic changes and prevent the progression of cachexia. Summary of the invention

[0010] The present invention provides methods for predicting clinical outcomes in subjects with cancer, methods for detecting non-liver cancer in subjects, and methods for treating or preventing cancer or cancer-related cachexia. Also provided are synthetic lipid nanoparticles encapsulating mRNA encoding HNF4A and compositions comprising the same.

[0011] According to a first aspect, a method of predicting a clinical outcome in a subject having cancer is provided, wherein the cancer is a non-liver cancer, the method comprising measuring a function of a urea cycle in the subject, wherein a reduced urea cycle function compared to urea cycle function in a healthy control indicates a worse clinical outcome compared to a subject in which the urea cycle function is not reduced, thereby predicting the clinical outcome of the subject.

[0012] According to some embodiments, the non-liver cancer is selected from breast cancer and pancreatic cancer, excluding detectable liver metastases or both.

[0013] According to some embodiments, the functionality of measuring the urea cycle includes at least one of the following:

[0014] a. Measuring the expression of at least one urea cycle enzyme selected from the following in the liver of the subject: argininosuccinate synthetase 1 (ASS1), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL), carbamoyl phosphate synthetase-1 (CPS1), and ornithine translocase (ORNT1 / SLC25A15);

[0015] b. measuring the ratio of urea to glutamine or the ratio of urea to glutamate in the blood or liver of the subject;

[0016] c. measuring the level of glutamate, aspartate or fumarate in the blood or liver of the subject;

[0017] d. measuring ammonia levels in the subject's blood;

[0018] e. measuring the urea level in the subject's urine;

[0019] f. measuring the expression of hepatocyte nuclear factor 4 alpha (HNF4A) in the liver of the subject;

[0020] g. measuring the level of at least one liver enzyme selected from the following in the blood of the subject: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALK-P / ALP) and lactate dehydrogenase (LDH);

[0021] h. measuring the international normalized ratio (INR) of prothrombin time from the subject's blood; and

[0022] i. Measuring albumin in the blood or liver of a subject.

[0023] According to some embodiments,

[0024] a. reduced expression of at least one urea cycle enzyme;

[0025] b. The ratio of urea to glutamine or the ratio of urea to glutamate is reduced;

[0026] c. Increased glutamate or aspartate;

[0027] d. Fumaric acid reduction;

[0028] e. Increased ammonia;

[0029] f. Decrease of urea;

[0030] g.HNF4A decreased;

[0031] h. Elevated levels of at least one liver enzyme;

[0032] i. Decreased INR;

[0033] j. Decreased albumin; or

[0034] k. Any combination thereof

[0035] Indicates decreased urea cycle function in the subject.

[0036] According to some embodiments, measuring function includes generating a liver function score, and wherein a liver function score exceeding a predetermined threshold is indicative of reduced urea cycle function.

[0037] According to some embodiments, the liver function score is a weighted sum of the normalized levels of AST, ALT, ALP, and albumin and the INR in a blood sample from the subject.

[0038] According to some embodiments, the score is normalized from 0 to 1, the predetermined threshold is 0.6, and wherein a score above the predetermined threshold is indicative of reduced urea cycle function.

[0039] According to some embodiments, the clinical outcome is development of cancer-related cachexia, and wherein decreased urea cycle function predicts an increased risk of developing cancer-related cachexia.

[0040] According to some embodiments, the clinical outcome is overall survival, and wherein a liver function score exceeding a predetermined threshold is indicative of a shortened overall survival.

[0041] According to another aspect, a method for detecting a non-liver cancer in a subject in need thereof is provided, the method comprising receiving a blood sample from the subject and measuring a function of a urea cycle in the subject based on the blood sample, wherein a decrease in urea cycle function compared to urea cycle function in a healthy control indicates that the subject has a non-liver cancer, thereby detecting the non-liver cancer in the subject.

[0042] According to some embodiments, the non-liver cancer is selected from breast cancer and pancreatic cancer, excluding detectable liver metastases or both.

[0043] According to some embodiments, the functionality of measuring the urea cycle includes at least one of the following:

[0044] a. measuring the ratio of glutamine to glutamate in a blood sample;

[0045] b. measuring the level of glutamate, aspartate or fumarate in a blood sample;

[0046] c. measuring ammonia levels in blood samples;

[0047] d. measuring aspartate aminotransferase (AST) levels in a blood sample; and

[0048] e. Measure albumin levels in blood samples.

[0049] According to some embodiments,

[0050] a. The ratio of glutamine to glutamate decreases;

[0051] b. Increased glutamate or aspartate;

[0052] c. Fumaric acid reduction;

[0053] d. Increased ammonia;

[0054] e. Elevated AST levels;

[0055] f. Decreased albumin levels; or

[0056] g. Any combination thereof

[0057] Indicates decreased urea cycle function in the subject.

[0058] According to some embodiments, the method further comprises administering to the subject having a worse clinical outcome or determined to have non-liver cancer at least one therapeutic agent selected from the group consisting of an anti-IL6 blocking antibody, an ERK inhibitor, a STAT3 inhibitor, and an agent capable of increasing the expression of HNF4A in the subject's liver.

[0059] According to another aspect, there is provided a synthetic lipid nanoparticle (LNP) comprising mRNA encoding HNF4A encapsulated therein, wherein:

[0060] a. Lipid nanoparticles including SM-102 lipid, cholesterol, DOPE and DMG-PEG;

[0061] b. mRNA comprising or consisting of: a sequence of SEQ ID NO: 10 or 12 or a sequence having at least 85% identity thereto and encoding HNF4A and a poly A tail; or

[0062] c. Both.

[0063] According to some embodiments, the mRNA includes a 5' cap and a poly A tail.

[0064] According to some embodiments, the mRNA encoding HNF4A comprises the mRNA coding sequence of SEQ ID NO: 2 or SEQ ID NO: 5, or a sequence encoding HNF4A having at least 85% identity thereto.

[0065] According to some embodiments, the lipid nanoparticles are targeted to liver cells.

[0066] According to some embodiments, the lipid nanoparticles include about 50 mol % SM-102, 38.5 mol % cholesterol, 10 mol % DOPE, and 1.5 mol % DMG-PEG200.

[0067] According to another aspect, a pharmaceutical composition is provided, which includes the synthetic LNP of the present invention and a pharmaceutically acceptable carrier, excipient or adjuvant.

[0068] According to another aspect, a method of treating a non-liver cancer in a subject in need thereof is provided, the method comprising administering to the subject an agent capable of increasing expression of HNF4A in the subject's liver, thereby treating the non-liver cancer in the subject.

[0069] According to another aspect, a method of treating or preventing cancer-associated cachexia in a subject in need thereof is provided, the method comprising administering to the subject a composition comprising at least one agent selected from the group consisting of an anti-IL6 blocking antibody, an ERK inhibitor, a STAT3 inhibitor, and an agent capable of increasing the expression of HNF4A in the liver of the subject, thereby treating or preventing cancer-associated cachexia in the subject.

[0070] According to some embodiments, the agent comprises a nucleic acid molecule encoding HNF4A.

[0071] According to some embodiments, the nucleic acid molecule is contained within an adeno-associated virus (AAV).

[0072] According to some embodiments, the nucleic acid molecule is mRNA.

[0073] According to some embodiments, the mRNA includes a 5' cap and a poly A tail.

[0074] According to some embodiments, the nucleic acid molecule comprises or consists of SEQ ID NO: 10 or 12 or comprises at least 85% identity thereto and encodes HNF4A.

[0075] According to some embodiments, the agent is a synthetic LNP of the present invention.

[0076] According to some embodiments, the subject has an early stage cancer, a precancerous lesion, or is at risk of developing cancer.

[0077] According to some embodiments, the subject is determined to have non-liver cancer by the methods of the present invention.

[0078] According to some embodiments, the subject is determined to have an increased risk of developing cancer-related cachexia by the methods of the present invention.

[0079] Other embodiments and the entire scope of application of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given by way of illustration only, because through this detailed description, various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1A-1L : Breast cancer induces metabolic changes in the liver during early carcinogenesis. (1A) RT-PCR in the liver of mice bearing breast cancer (BC) showed that the RNA expression of UC enzymes decreased with cancer progression (n=5, Student's T test). Day 4, P=0.003, day 14, P=0.033, 0.01, 0.028 (respectively), day 21, P=0.0002, 0.019, 0.0002, 0.013, 0.007 (respectively). (1B) Left panel - Western blot showed that the protein expression levels of ASS1 and OTC in the liver of BC-bearing mice were reduced compared with WT-PBS-injected mice (n=5, Student's T test). Right panel - Quantification of band intensity, ASS1, P=0.002. OTC, P=0.011. (1C) RT-PCR in livers of MMTV-PyMT cancer-bearing mice at 14 weeks of age showed reduced RNA expression of UC enzymes compared to livers of WT mice (WT n=5, MMTV-PyMT n=3), P=0.033, 3.14E-05, 0.038 (respectively). (1D) Glutamine was infused into 4T1 BC-bearing mice or PBS-injected mice 15 Measurement of UC-related metabolites in plasma and liver after N2. Left: The ratio of urea to glutamine and glutamate m+1 isotopologues was decreased in plasma of 4T1 BC-bearing mice (WT n=5, 4T1 n=7, Student's T test), P=0.019, 0.042 (respectively). Right: Increased glutamate levels (AUC / IS / dry weight) in 4T1 BC-bearing mice and decreased urea to glutamate ratio after infusion in 4T1 BC-bearing mice support UC dysfunction (n=7, Student's T test) P=0.016, 0.048. (1E) Increased aspartate and glutamate levels and decreased fumarate levels in liver (upper panel) and plasma (lower panel) of BC-bearing mice compared with livers of WT-PBS-injected mice. UC intermediate levels were measured by gas chromatography-mass spectrometry (GC-MS), amino acid analyzer (AAA) and liquid chromatography-mass spectrometry (LC-MS) (GC-MS, n=5, AAAWT n=5, 4T1 n=4, LCMS n=5, Student's T test). Liver P values: Asp=0.027, Glu=0.049, Fum=0.045. Plasma P values: Asp=0.004, Glu=0.0005. (1F) Left panel - Ammonia levels in plasma of BC-bearing mice compared to WT-PBS-injected mice were measured using an ammonia assay kit (WT n=4, outlier–IQR method, 4T1 n=5, Student's T test). Right panel - Urea levels in urine of BC-bearing mice compared to WT-PBS-injected mice. P=0.033. (1G) Left panel: Compared to infused15 Levels of m+1 labeled aspartate were reduced in tumors of 4T1 BC bearing mice compared to levels in plasma and liver after N2 glutamine (n=8, plasma n=7, two-way ANOVA) P values: plasma vs. tumor = 0.041, liver vs. tumor = 0.007. Right: The ratio of m+1 uracil to aspartate m+1 isotopologues was increased in tumors of 4T1 BC bearing mice (n=8, plasma n=7, two-way ANOVA) P values: plasma vs. tumor = 0.042, liver vs. tumor = 0.008. (1H) XTT assay of 4T1 cancer cell proliferation after supplementation of the culture medium with UC intermediate (n=3, two-way ANOVA) P values: 24h: Asp–ns, Amn = 0.007, Glu and Fum < 0.0001. All measurements 48 and 72 < 0.0001. Metabolite concentrations were supplemented at the following concentrations: ammonia - 0.75 mM according to published values ​​

[49] , aspartate 0.25 mM, fumarate 0.35 mM, glutamine 0.25 mM. (1I) Ex vivo FACS analysis of cell survival (left) and activation (right) of CD8+ splenocytes after supplementation with 1 mM ammonia

[58] (measured plasma ammonia levels 1.34 mM), (n=5, Student's T test) P=0.011, 0.043 (respectively). (1J) Heat map of differential gene expression in hepatocytes showing unique patterns in livers of 4T1 BC mice at day 21 compared to day 4. (1K) Pathway enrichment analysis of 4T1 d21 vs. 4T1 d4. Each bar shows the fold enrichment of a specific pathway. (1L) Left panel - AST plasma measurements showed significantly elevated levels in BC-bearing mice compared to WT-PBS injected mice (n=5, Student's T test) P=0.011. Right panel - no significant changes in ALT were found.

[0081] Figure 2A-2I: Innate immune cells infiltrate the host's liver during early breast carcinogenesis. (2A) H&E staining of liver sections shows increased immune cell infiltration over time after injection of BC cells. Magnification - 10X and 40X, see figure for details. (2B) Cell types annotated by single-cell RNA sequencing analysis were projected onto 3UMAP, indicating which cells appeared at which time point. Arrows mark neutrophil and monocyte subsets that are absent in the liver of WT mice and gradually accumulate in the liver until day 21. (2C-2E) CyTOF-tSNE analysis (2C, 2E) and (2D) quantification of CD45+ populations in the liver and blood show increased levels of innate immune cells and decreased lymphocytes in the liver (2C) and blood (2E) of BC mice compared with WT-PBS injected mice (WT n=3, 4T1=5, Student's T test). Blood P value <0.0001. Liver P values: B cells = 0.013, T cells = 0.0014, neutrophils = 0.0001. (2F) Multiplex ELISA immunoassays showed that IL-6 levels (left) and TNFα levels (right) increased in BC-bearing mice from day 4 to day 21 after cancer cell injection (n = 5, Student's T test). P values ​​for IL-6: day 4 = 0.024, day 14 < 0.0001, day 21 = 0.0009. P values ​​for TNFα: day 4 = 0.05, day 14 and day 21 < 0.0001 (2G) ELISA measurements of CCL2 levels in plasma, liver, spleen, and lung of BC-bearing mice (n = 5, Student's T test) P values: plasma = 0.0005, liver = 0.017, spleen = 0.04. (2H) ELISA assay showed a significant interaction effect between time after cancer cell injection and CCL2 elevation in the liver of BC mice relative to WT-PBS injected mice (n=5, two-way ANOVA) P=0.0034. (2I) CCR2 expression in immune cells from scRNAseq experiments projected on UMAP showed that CCR2 was mainly expressed in monocyte subsets.

[0082] Figure 3A-Figure 3I: Activated myeloid cells infiltrating the liver perturb liver metabolism through HNF4α depletion. (3A) DE gene pathway enrichment analysis of hepatocytes from BC-bearing mice at day 21 compared with day 4. Bars show Z scores for specific pathways. (3B) Immunofluorescence staining showed elevated pERK levels in CD45+ cells in the livers of BC-bearing mice. (3C) The mean fluorescence intensity (MFI) of pERK in CD45+ cells showed a specific increase in pERK in the livers of BC mice compared with WT-PBS-injected mice (left panel), while there was no significant difference in the staining of CD45+pERK in the blood (middle panel) (n=4, Student's T test). Quantification is shown in the right panel. P<0.0001. (3D) Normalized RNA Seq counts of 3 integrin-binding proteins expressed on hepatocytes at days 4 and 21. P<0.0001, 0.001, 0.027 (respectively). (3E) Normalized RNA expression counts of integrins (left panel – Itga4, middle panel – Itgal, right panel – Itgam) in neutrophils, monocytes, macrophages, and hepatocytes. (3F) Western blots showed increased protein expression levels of pSTAT3 and decreased expression of HNF4α in livers of BC-bearing mice compared to livers of WT-PBS-injected mice (n = 5, Student’s T test). (3G) RT-PCR in livers of BC-bearing mice showed increased levels of miR-24 (n = 5, Student’s T test), P = 0.005. (3H) RT-PCR in livers of BC-bearing mice showed significantly decreased levels of HNF4α (n = 5, Student’s T test), P = 0.005. (3I) RT-PCR in primary hepatocytes showed increased RNA expression of CCL2 after IL-6 supplementation (n = 3, Student’s T test, representative experiment of two independent biological replicates), P = 0.009.

[0083] Figure 4A-4J: IL-6-STAT3-HNF4α signaling induces liver metabolic changes during BC carcinogenesis. (4A) DE pathway enrichment analysis of genes regulated by HNF4α in the liver of BC mice at day 21 compared to day 4. The bars show the Z score of a specific pathway. (4B) Left panel - Plasma albumin measurements showed reduced levels in BC-bearing mice compared to WT-PBS-injected mice (n=5, Student's T test) P=0.002. Right panel - Normalized RNA Seq analysis of albumin expression in hepatocytes at day 4 and day 21. P=0.013. (4C) Primary hepatocyte RT-PCR showed reduced RNA expression levels of OTC after IL-6 supplementation (n=3, Student's T test, representative experiment of two independent biological replicates) P=0.0002. (4D) STAT3 inhibitor HJCO152 restores OTC levels even in the presence of IL-6 (n=3, Student's T test) P<0.0001 - CTRL+DMSO vs IL-6+DMSO and STAT3 inhibitor vs STAT3 inhibitor+IL-6. (4E) Western blot (left panel) shows that the expression level of HNF4α in BC mice treated with ERK inhibitor is increased compared with that in the liver of BC mice treated with vehicle (vehicle n=4, trametinib n=3, Student's T test). The right panel is the quantification of band intensity. P=0.024. (4F) Measurement of tumor weight on days 8 and 14 after treatment with ERK inhibitor (n=5 on day 8, n=4 on day 14, Student's T test) P values: day 8=0.007, day 14=0.014. (4G) RT-PCR in liver of BC-bearing mice treated with AAV8-HNF4α showed increased RNA expression of HNF4α, UC enzyme and albumin compared to AAV8-GFP-treated BC-bearing mice (AAV8-GFP=4, AAV8-HNF4αn=7, Student's T test) P=0.004, 0.004, 0.002, 0.001 (respectively). (4H) 4T1 tumor growth was significantly higher after AAV-GFP injection compared to AAV-HNF4α (AAV8-GFP=12, AAV8-HNF4αn=13, Student's T test) P=0.049. (4I) Left panel - Western blot shows decreased PCNA levels in tumors of BC-bearing mice treated with HNF4α compared to mice treated with GFP. Right panel - Quantification of band intensity relative to actin. P=0.003. (4J) RT-PCR of liver and tumors of BC-bearing mice treated with AAV8-HNF4α showed a significant increase in RNA expression of viral HNF4α (BGH polyadenylation signal) in the liver (n=7, Student's T test) P<0.0001.

[0084] Figure 5A-5M: Innate immune cells induce similar transcriptional and metabolic changes in the liver of KPC mice. (5A) RT-PCR in the liver of PC-bearing mice showed that the RNA expression of OTC enzymes was reduced in the first week after cancer injection (n=6, two-way ANOVA) P=0.01. (5B) Aspartate and glutamate levels were increased and fumarate levels were decreased in the liver of PC-bearing mice. UC intermediate levels were measured by GC-MS and amino acid analyzer (AAA) (n=4, glutamate-outlier-IQR method. Fumarate-WT n=5, KPC n=7, Student's T test) P values: Asp=0.001, Glu=0.019, Fum=0.0001. (5C) H&E staining of liver sections showed increased immune cell infiltration after injection of PC cells. Magnification is 10X, see the figure for details. (5D) Left panel - Quantification of CD45+ populations in liver and blood by FACS analysis showing increased levels of innate immune cells and decreased lymphocytes in the liver and blood of PC-bearing mice compared to WT-PBS injected mice (n=8, liver WT=7, Student's T test) P values: Blood: B cells=0.003, monocytes=0.0002, neutrophils=0.006. Liver: B cells=0.004, T cells=0.05, monocytes<0.0001, neutrophils=0.004. Right panel - MFI=0.0001. Live immune cells in the blood and liver of KPC-bearing PC mice were gated for CD45+, CD11b, CD3, CD19 Ly-6G and Ly-6C. Mean fluorescence intensity was done on Ly-6C high monocytes. (5E) ELISA assay showed that IL-6 levels were increased in PC-bearing mice (n=5, Student's T test) P=0.011, 0.01 (respectively). (5F) Western blot showed that the protein expression level of pSTAT3 in the liver of PC-bearing mice was increased compared with that of WT-PBS-injected mice (n=6). (5G) RT-PCR in the liver of pancreatic cancer-bearing mice showed that the expression level of HNF4α RNA was significantly reduced with tumor progression, while it remained unchanged in WT-PBS-injected mice (WT n=3, KPC n=4, Student's T test) P=0.0012. (5H) RT-PCR in the liver of PC-bearing mice showed that the RNA level of albumin was significantly reduced (WT n=4, KPC n=5, Student's T test), P=0.0009. (5I) Treatment of PC mice with IL-6 antibody maintained the plasma level of albumin (Ctrl IgG=8, IL-6Ab=5, Student's T test) P=0.011. (5J) PC mouse weight measurement showed no weight loss in PC mice compared to WT controls during 22 days of tumor development. (5K) Power analysis of in vivo NMR experiments.The Y-axis shows the Wilcoxon rank sum P value for the difference in weight change between HNF4α-injected vs. control GFP-injected mice, while the X-axis shows the sample size for each condition in each experiment considered for the test. In this experiment, we had two conditions (HNF4α-injected vs. control GFP-injected) and 2 replicates, so the total number of experiments was 4 times the number shown on the X-axis, and the average sample size was 7. For each sample size less than 7, mice were randomly selected for statistical testing, and each box shows the mean and standard deviation of the P value, with whiskers showing the upper and lower 10 percentage points of the P value. (5L) Weight measurements and nuclear magnetic resonance (NMR) body composition analysis of PC AAV-HNF4α-injected mice showed significant reduction in weight loss (left panel), increase in adipose tissue (middle panel), and reduction in free fluid (right panel) compared to AAV-GFP-injected mice (relative to day 0); (n=7). (5M) Immunofluorescence staining of laminin in gastrocnemius muscle sections from KPC-AAV-HNF4α and KPC-AAV-GFP injected mice 4 weeks after cancer cell injection. For each slide, five pictures were taken along different areas of the tissue. Fiber area (μm) was calculated using Fiji-ImageJ. 2 The average fiber area for each mouse was calculated using the average area of ​​five images. 2 The lower cutoff value was used to eliminate artifacts. (AAV-GFP n=9, AAV-HNF4 n=10, two-way ANOVA). P=0.008.

[0085] Figure 6A-6J: Early changes in liver metabolism in CCR2 KO mice and HNF4α rescued PC mice. (6A) H&E staining of livers of CCR2- / - PC and PC mice showed reduced immune cell infiltration in CCR2- / - livers compared to WT PC mouse livers. PC mice are mice injected with KPC. Magnification - 10X, 20X and 40X, see details in the figure. (6B) ELISA assay showed that IL-6 levels in plasma of PC-CCR2- / - mice were reduced relative to PC-WT mice (PC WT n=7, CCR2- / - n=6, Student's T test) P=0.0125. (6C) RT-PCR in livers of PC-bearing CCR2- / - mice showed that RNA expression levels of ASS1, OTC, albumin, and HNF4α were maintained compared to reduced expression levels of these genes in WT PC livers (PC WT n=5, CCR2- / - n=6, Student's T test, Student's T test), P=0.019, 0.011, and 0.002 (respectively). (6D) Aspartate and glutamate levels were reduced in livers of PC-bearing CCR 2- / - mice. UC intermediate levels were measured by gas chromatography-mass spectrometry (GC-MS) (n=7, Student's T test), P=0.011, 0.029 (respectively). (6E) Weight measurements and nuclear magnetic resonance (NMR) body composition analysis of mice bearing CCR2- / - PC showed significant reduction in weight loss (left panel), increase in adipose tissue (middle panel), and decrease in free fluid (right panel) compared to WT PC at day 21 (relative to day 0); (n=7 per group, Student's T test) P=0.014, 0.006, 0.004 (respectively). (6F) Tumor weight measured on the day of sacrifice was significantly lower in pancreatic cancer CCR2- / - mice compared to WT-PC (n=7, Student's T test). P=0.009. (6G) PC tumor growth was significantly more pronounced in mice injected with AAV-GFP compared to AAV-HNF4α (AAV-GFP n=4, AAV-HNF4α n=9, Student's T test) P=0.006. (6H) PC mice injected with AAV-HNF4α showed significantly prolonged survival compared to PC-AAV-GFP mice (Log-rank (Mantel-Cox) test), P = 0.02. (6I) PC mice injected with AAV-HNF4α maintained weight during CAC compared to pancreatic cancer mice injected with AAV-GFP (AAV-GFP n = 8, AAV-HNF4α n = 9, two-way ANOVA), P = 0.05.(6J) NMR body composition analysis of pancreatic cancer mice injected with AAV-HNF4α showed less adipose tissue loss (left panel) and reduced free fluid accumulation (right panel) compared with PC mice injected with AAV-GFP (AAV-GFP n=5, AAV-HNF4α n=6, Student's T test) P=0.007, 0.012 (respectively).

[0086] Figure 7A-Figure 7I: Routine liver tests predict weight loss in PDAC patients. (7A) Analysis of liver biochemical tests from the Clalit Health Care database showed that patients with non-metastatic PDAC disease with shorter survival compared to patients with longer survival had abnormal liver parameters at diagnosis. (PDAC; n=2037-survival <0.5y, n=659-survival 0.5-1y, n=342, survival 1-1.5y; **p<0.01, ***p<0.001. (7B) KM survival curves of PDAC patients from the Clalit database (left) and from Sheba and Souraski Medical Center (right) show that PDAC patients with high liver function scores have shortened survival. P=0.0003 and P<0.0001 (respectively). (7C-7D) Analysis of liver biochemical tests from Sheba and Sourasky (7C) Liver score was not associated with pancreatic cancer disease stage. LA - locally advanced, MTX - metastatic disease. (7D) Weight loss associated with high (red) and low (blue) liver scores during PDAC in PDAC patients from Sheba and Sourasky Medical Center, binned by disease stage at diagnosis. (Linear regression P = 0.02). (7E-7F) Non-metastatic BC patients with shorter survival compared with patients with longer survival had a higher risk of developing pancreatic cancer at diagnosis (7E) and one year before diagnosis (7F). In the case of abnormal liver parameters (BC; n = 4732 - survival < 2y, n = 4086 - survival 2-5y, n = 3984, survival 5-10y; *P < 0.05, ***P < 0.01, ***P < 0.001). (7G) Patients with greater early weight loss (blue) showed shortened survival compared with patients with less weight loss (yellow) (log rank P < 0.0051) - even when controlling for age and sex (cox hazard ratio = 1.21, P < 0.0084). (7H) From the initial (n = 50) data used to train the model KM survival curves of PC patients with high and low liver function scores based on the data. Calculation details are given in the “Methods” section. (7I) KM survival curves of PC patients with high and low liver function scores Top - Sourasky data. For resectable tumors, n = 255, for local and distant spread, n = 362. Bottom - Sheba data, for stage 1-2, n = 82. For stage 3-4, n = 169. (Left - patients with resectable tumors at stage 1-2 at diagnosis, right - patients with local and distant spread at stage 3-4 at diagnosis), P values: resectable = 0.0255, survival LA = 0.0007, survival stage 3-4 = 0.005.

[0087] Figure 8: A graphical summary of these findings suggests a mechanism by which tumor-induced systemic metabolic changes can lead to CAC in the livers of cancer patients and cancer-bearing mice.

[0088] Figure 9A-9H : (9A) Photographs of fluorescence of liver and spleen of mice administered H4-LNP or SM-LNP. Fluorescence was detected only in the liver that received SM-LNP. (9B) Western blot of HNF4A expression in THLE-2 exposed to SM-LNP. Actin was measured as a loading control. (9C-9H) Line graphs of changes in (9C, 9F) weight, (9D, 9G) fat, and (9E, 9H) free fluid percentage in mice given pancreatic cancer cells (day 0) and then administered (9C-9E) a therapeutic dose of HNF4A-LNP on day 21 or (9F-9H) a preventive dose of HNF4A-LNP on day 7. DETAILED DESCRIPTION

[0089] The present invention provides, in some embodiments, a method for predicting a clinical outcome of a subject by measuring the function of the urea cycle in a subject with cancer. Also provided is a method for detecting non-liver cancer in a subject by measuring the function of the urea cycle in a subject. Also provided is a method for treating or preventing cancer or treating or preventing cancer-related cachexia. Also provided is a synthetic lipid nanoparticle encapsulating mRNA encoding HNF4A and a composition comprising the same.

[0090] The present invention is based, at least in part, on the surprising discovery that while exposure to foreign molecules in a healthy liver results in regulated inflammation, following carcinogenesis and immune infiltration, the liver recruits innate immune cells by secreting increased amounts of CCL2. Activated immune cells that are pERK positive and elevated IL-6 levels result in transcriptional changes in the expression of metabolic enzymes in the liver. Tumor-induced IL-6 has been shown to impair the hepatic ketogenic response. Here, we demonstrate that liver metabolism is extensively rewired through the IL-6-pSTAT3 immune-hepatic axis, resulting in depletion of HNF4α, a master regulator of liver metabolism. As a result, there are changes in systemic metabolism that increase the availability of substrates that promote cancer growth and contribute to systemic manifestations such as weight loss and changes in body composition such as cachexia ( Figure 8 ).

[0091] In addition, we also present a biochemical liver score that includes albumin and can predict survival and weight loss regardless of cancer stage. Therapeutically, our data suggest that giving clinically available drugs such as ERK inhibitors, STAT inhibitors, or anti-IL-6 blocking antibodies to identified patients at risk early in the cancer can preserve liver metabolism and limit cancer progression. In addition, exogenous HNF4α can also be used to maintain liver metabolism and limit systemic manifestations. This is achieved by using both HNF4α-AAV and novel liver-targeted LNPs including optimized HNF4A mRNA (Figures 10C-10H), which can not only shrink cancer, but also prevent and treat cachexia.

[0092] In a first aspect, a method of detecting cancer in a subject is provided, the method comprising measuring the function of a urea cycle in the subject, thereby detecting the cancer.

[0093] In a first aspect, a method of predicting a clinical outcome in a subject is provided, the method comprising measuring the function of a urea cycle in the subject, thereby predicting the clinical outcome.

[0094] In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the method is a diagnostic method. In some embodiments, the method is a prognostic method. In some embodiments, the method is a method of treatment. In some embodiments, the method is a method of detecting cachexia. In some embodiments, the method is a method of predicting the development of cachexia. In some embodiments, the method is a method of predicting the risk of developing cachexia. In some embodiments, the method is a method of predicting overall survival. In some embodiments, the method is a method of predicting mortality. In some embodiments, the method is a method of predicting years of survival. In some embodiments, the prediction is at least 1 month before disease manifestation. In some embodiments, the prediction is at least 3 months before disease manifestation. In some embodiments, the prediction is at least 6 months before disease manifestation. In some embodiments, the prediction is at least 1 year before disease manifestation. In some embodiments, the prediction is at least 2 years before disease manifestation. In some embodiments, the disease is cancer. In some embodiments, the disease is cachexia.

[0095] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is in need of the methods of the present invention. In some embodiments, the subject has cancer. In some embodiments, the subject is at risk of developing cancer. In some embodiments, the subject has cachexia. In some embodiments, the subject is at risk of developing cachexia. In some embodiments, risk is determined or predicted by the methods of the present invention. In some embodiments, cancer is detected by the methods of the present invention. In some embodiments, cancer is predicted by the methods of the present invention. In some embodiments, cachexia is detected by the methods of the present invention. In some embodiments, cachexia is predicted by the methods of the present invention.

[0096] As used herein, "cancer" or "premalignant lesions" refer to diseases associated with uncontrolled cell proliferation. In some embodiments, the cancer is not liver cancer. In some embodiments, the cancer is a non-liver cancer. In some embodiments, the cancer does not include metastasis to the liver. In some embodiments, the metastasis is a detectable metastasis. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a tumor. In some embodiments, the cancer is selected from hepatobiliary cancer, cervical cancer, genitourinary cancer, testicular cancer, prostate cancer, thyroid cancer, ovarian cancer, nervous system cancer, eye cancer, lung cancer, soft tissue cancer, bone cancer, pancreatic cancer, bladder cancer, skin cancer, intestinal cancer, liver cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, gastroesophageal cancer, breast cancer, kidney cancer, skin cancer, head and neck cancer, leukemia and lymphoma. In some embodiments, the cancer is not liver cancer. In some embodiments, the cancer is not leukemia or lymphoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is pancreatic cancer.

[0097] In some embodiments, the cancer is at any stage. In some embodiments, the cancer is early stage cancer. In some embodiments, the cancer is advanced stage cancer. In some embodiments, the cancer is stage I cancer. In some embodiments, the cancer is stage II cancer. In some embodiments, the cancer is stage III cancer. In some embodiments, the cancer is stage IV cancer. In some embodiments, the cancer is a precancerous malignancy. In some embodiments, the malignancy is a malignant lesion. In some embodiments, the methods are not related to cancer or precancerous stage. In some embodiments, the methods are not related to the type of cancer, except for non-liver cancer.

[0098] As used herein, the term "cachexia" refers to a wasting syndrome that causes skeletal muscle and fat loss. In some embodiments, cachexia further includes increased free fluid. In some embodiments, cachexia is determined or diagnosed by measuring fat loss, muscle loss, free fluid increase, or any combination thereof. In some embodiments, cachexia is cancer-related cachexia. In some embodiments, cancer-related cachexia is cachexia caused by cancer. Cachexia includes three consecutive clinical stages: pre-cachexia, cachexia, and refractory cachexia. Pre-cachexia is defined as weight loss of less than 5%. Cachexia is defined as a patient's weight loss of more than 5%. Refractory cachexia is determined by low WHO performance status scores and a survival period of less than 3 months. In some embodiments, cachexia is pre-cachexia. In some embodiments, cachexia is second-stage cachexia. In some embodiments, cachexia is not refractory cachexia. In some embodiments, cachexia is refractory cachexia.

[0099] In some embodiments, the function of the urea cycle in the subject is measured. In some embodiments, the function of the urea cycle in a sample from the subject is measured. In some embodiments, the liver function of the subject is measured. In some embodiments, the liver function in a sample from the subject is measured. In some embodiments, the method further comprises receiving a sample from the subject. In some embodiments, the sample is not a cancer sample. In some embodiments, the measurement is a measurement outside of cancer. In some embodiments, the measurement is in the liver of the subject. In some embodiments, the sample is a liver biopsy. In some embodiments, the measurement is in the blood of the subject. In some embodiments, the sample is a blood sample. In some embodiments, the blood sample is a whole blood sample. In some embodiments, the blood sample is a plasma sample. In some embodiments, the blood sample is a serum sample. In some embodiments, the sample is a urine sample. In some embodiments, the measurement function is based on a blood sample. In some embodiments, the detection or determination is based on data from a sample. In some embodiments, data from a sample is received.

[0100] In some embodiments, measuring urea cycle function includes measuring the expression of at least one urea cycle enzyme. In some embodiments, measuring liver function includes measuring the expression of at least one urea cycle enzyme. In some embodiments, measuring liver function includes measuring the expression of at least one liver enzyme. In some embodiments, expression is protein expression. In some embodiments, expression is mRNA expression. In some embodiments, expression is in the liver of the object. In some embodiments, expression is in a liver sample from the object. In some embodiments, expression is in the hepatocytes of the object. In some embodiments, expression is in the blood of the object. Methods for measuring mRNA and protein levels are well known, and any such method can be used. These methods include, for example, PCR, RT-PCR, qRT-PCR, RNA sequencing, Western blot, ELISA, immunostaining, protein arrays, etc. Commercial reagents and kits for performing this measurement are available.

[0101] In some embodiments, at least one urea cycle enzyme is argininosuccinate synthetase 1 (ASS1). In some embodiments, at least one urea cycle enzyme is ornithine transcarbamylase (OTC). In some embodiments, at least one urea cycle enzyme is argininosuccinate lyase (ASL). In some embodiments, at least one urea cycle enzyme is carbamoyl phosphate synthetase-1 (CPS1). In some embodiments, at least one urea cycle enzyme is ornithine translocase (ORNT1 / SLC25A15). In some embodiments, at least one urea cycle enzyme is selected from ASS1, OTC, ASL, CPS1 and ORNT1. In some embodiments, at least one urea cycle enzyme is selected from ASS1, OTC, ASL and ORNT1. In some embodiments, OTC is measured. In some embodiments, changes in OTC are the earliest biomarkers of urea cycle function. In some embodiments, the mRNA level of at least one urea cycle enzyme is measured. In some embodiments, the protein level of at least one urea cycle enzyme is measured. Exemplary primers for measuring the mRNA expression of these enzymes and others are provided in Table 1. Exemplary antibodies used to detect these targets are as follows: p97 (Thermo Fisher Scientific PA5-22257); ASS1 (Abcam ab124465); OTC (Abcam ab203859); actin (Sigma-Aldrich A5441); TFAM (Cell Signaling #8076); pSTAT3 (Cell Signaling #9145); STAT3 (Cell Signaling #12640); HNF4α (Abcam ab181604); PCNA (Cell Signaling #13110); CAD (Cell Signaling #11933); pCAD (Cell Signaling #12662).

[0102] Table 1: Primers

[0103]

[0104]

[0105] In some embodiments, at least one liver enzyme is aspartate aminotransferase (AST). In some embodiments, at least one liver enzyme is alanine aminotransferase (ALT). In some embodiments, at least one liver enzyme is alkaline phosphatase (ALK-P / ALP). In some embodiments, at least one liver enzyme is lactate dehydrogenase (LDH). In some embodiments, LDH is LDHA. In some embodiments, LDH is LDHB. In some embodiments, LDH is LDHA and LDHB. In some embodiments, at least one liver enzyme is selected from AST, ALT, ALP and LDH. In some embodiments, at least one liver enzyme is selected from ALP and LDH. In some embodiments, at least one liver enzyme is selected from ALP, ALT and AST.

[0106] In some embodiments, measuring urea cycle function comprises measuring a urea cycle substrate. In some embodiments, the substrate is glutamate. In some embodiments, the substrate is aspartic acid. In some embodiments, measuring urea cycle function comprises measuring urea levels. In some embodiments, urea levels are levels in urine. In some embodiments, urea levels are levels in blood. In some embodiments, measuring urea cycle function comprises measuring a ratio of urea to glutamine. In some embodiments, measuring urea cycle function comprises measuring a ratio of urea to glutamine. In some embodiments, measuring urea cycle function comprises measuring a urea cycle metabolite. In some embodiments, a metabolite is a product. In some embodiments, a metabolite is fumarate. In some embodiments, the measurement is in the liver. In some embodiments, the measurement is in the blood.

[0107] In some embodiments, measuring urea cycle function comprises measuring ammonia levels. In some embodiments, ammonia levels are in the blood. In some embodiments, measuring urea cycle function comprises measuring albumin levels. In some embodiments, albumin levels are in the blood.

[0108] In some embodiments, measuring urea cycle function comprises measuring hepatocyte nuclear factor 4 alpha (HNF4A) expression. In some embodiments, the expression is mRNA expression. In some embodiments, the expression is protein expression. In some embodiments, the expression is expression in the liver. In some embodiments, the expression is expression in hepatocytes. Exemplary primers and antibodies for detecting HNF4A are provided above.

[0109] In some embodiments, measuring urea cycle function includes measuring prothrombin. In some embodiments, measuring prothrombin is measuring prothrombin time. In some embodiments, prothrombin time is international normalized ratio (INR). In some embodiments, INR is the INR of blood. In some embodiments, measurement is in blood. In some embodiments, blood is a blood sample. Prothrombin time test is a known clinical determination and its execution is a standard protocol known to those skilled in the art.

[0110] In some embodiments, reduced urea cycle function indicates poor clinical outcome. In some embodiments, reduced urea cycle function indicates the presence of cancer. In some embodiments, reduced urea cycle function indicates that the subject has cancer. In some embodiments, reduced liver function indicates poor clinical outcome. In some embodiments, reduced liver function indicates the presence of cancer. In some embodiments, reduced liver function indicates that the subject has cancer. In some embodiments, reduction is reduced below a predetermined threshold. In some embodiments, reduction is compared to a control. In some embodiments, the predetermined threshold is the level / expression / value in the control. In some embodiments, the healthy control is a healthy control sample. In some embodiments, the sample or control is matched to a sample or subject from a subject. That is, a person skilled in the art will understand that if the sample is a blood sample, the control will also be a blood sample, and if the measurement is in the liver, the control will be measured in the liver. In some embodiments, the control is a healthy control. In some embodiments, a poor clinical outcome is a worse clinical outcome. In some embodiments, worse is compared to a subject whose urea cycle function is not reduced. In some embodiments, worse is compared to a subject whose liver function is not reduced. In some embodiments, worse is compared to a matched control whose function is not reduced. That is, if the subject has cancer, the matched control has the same cancer. And if the subject has a pre-malignancy, the matched control also has a pre-malignancy.

[0111] In some embodiments, the reduction comprises a reduction of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97 99 or 100%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the reduction is a reduction of at least 25%. In some embodiments, the reduction is a reduction of at least 50%. In some embodiments, the reduction is a detectable reduction. In some embodiments, the reduction is a significant reduction. In some embodiments, significant is statistically significant.

[0112] In some embodiments, decreased expression of at least one urea cycle enzyme indicates decreased urea cycle function. In some embodiments, decreased expression of at least one urea cycle enzyme indicates decreased liver function. In some embodiments, decreased expression of at least one liver enzyme indicates decreased liver function. In some embodiments, decreased levels of urea cycle substrates indicate decreased urea cycle function. In some embodiments, decreased levels of urea cycle substrates indicate decreased liver function. In some embodiments, increased levels of urea cycle metabolites indicate decreased urea cycle function. In some embodiments, increased levels of urea cycle metabolites indicate decreased liver function. In some embodiments, decreased levels of urea indicate decreased urea cycle function. In some embodiments, decreased levels of urea indicate decreased liver function. In some embodiments, decreased levels of urea indicate decreased liver function. In some embodiments, decreased ratios of urea to glutamine indicate decreased urea cycle function. In some embodiments, decreased ratios of urea to glutamine indicate decreased liver function. In some embodiments, decreased ratios of urea to glutamate indicate decreased urea cycle function. In some embodiments, decreased ratios of urea to glutamate indicate decreased liver function. In some embodiments, increased levels of fumarate indicate decreased urea cycle function. In some embodiments, an increase in fumarate levels indicates reduced liver function. In some embodiments, an increase in ammonia levels indicates reduced urea cycle function. In some embodiments, an increase in ammonia levels indicates reduced liver function. In some embodiments, a decrease in albumin levels indicates reduced urea cycle function. In some embodiments, a decrease in albumin levels indicates reduced liver function. In some embodiments, a decrease in HNF4A expression indicates reduced urea cycle function. In some embodiments, a decrease in HNF4A expression indicates reduced liver function. In some embodiments, a decrease in INR indicates reduced urea cycle function. In some embodiments, a decrease in INR indicates reduced liver function.

[0113] In some embodiments, measuring liver function includes generating a liver function score. In some embodiments, the liver function score is the sum of the measures of liver function provided above. In some embodiments, the sum is a weighted sum. In some embodiments, the measure is normalized. In some embodiments, the sum is the sum of the levels of at least two of AST, ALT and ALP. In some embodiments, all three levels are summed. In some embodiments, the sum is the sum of the level of at least one of AST, ALT and ALP and the level of albumin. In some embodiments, the sum is the sum of the level of at least two of AST, ALT and ALP and the level of albumin. In some embodiments, the sum is the sum of the level of AST, ALT, ALP and albumin. In some embodiments, the sum is the sum of the level of at least one of AST, ALT and ALP and the measurement of INR. In some embodiments, the sum is the sum of the level of albumin and the measurement of INR. In some embodiments, the sum is the sum of the level of at least one of AST, ALT and ALP, the level of albumin and the measurement of INR. In some embodiments, the sum is the sum of the level of at least one of AST, ALT and ALP, the level of albumin and the measurement of INR. In some embodiments, the sum is the sum of the level of at least two of AST, ALT and ALP, the level of albumin and the measurement of INR. In some embodiments, the sum is the sum of the level of at least two of AST, ALT and ALP, the level of albumin and the measurement of INR. In some embodiments, the sum is the sum of the levels of AST, ALT, ALP, and albumin and the measured INR.

[0114] In some embodiments, at least one is at least two. In some embodiments, at least one is a plurality. In some embodiments, at least one is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Each possibility represents a separate embodiment of the present invention. In some embodiments, at least one is all of them.

[0115] In some embodiments, a score exceeding a predetermined threshold indicates reduced urea cycle function. In some embodiments, a score exceeding a predetermined threshold indicates reduced liver function. In some embodiments, urea cycle function is liver function. In some embodiments, liver function includes urea cycle function. In some embodiments, a high score indicates reduced function, while a low score indicates normal function. In some embodiments, exceeding is above. In some embodiments, exceeding is below. In some embodiments, the standardization of the score is from 0 to 1. In some embodiments, the score is measured on a scale from 0 to 1 or equivalent. In some embodiments, the predetermined threshold is 0.6 on a scale from 0 to 1. In some embodiments, a score above 0.6 indicates reduced urea cycle function. In some embodiments, a score above 0.6 indicates reduced liver function.

[0116] In some embodiments, the clinical outcome is the development of cachexia. In some embodiments, the urea cycle function decreases and predicts the increased risk of developing cachexia. In some embodiments, the urea cycle function decreases and predicts that the object will develop cachexia. In some embodiments, the liver function decreases and predicts the increased risk of developing cachexia. In some embodiments, the liver function decreases and predicts that the object will develop cachexia. In some embodiments, the increase is increased compared to the object that the function is not reduced. In some embodiments, the increase is increased compared to the object whose score does not exceed a predetermined threshold. In some embodiments, the increase is compared to the object with no cancer, precancerous lesions or increased risk of developing cancer. In some embodiments, the increase includes an increase of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 or 500%. Each possibility represents a separate embodiment of the present invention. In some embodiments, the increase is at least a 20% increase. In some embodiments, the increase is at least a 50% increase. In some embodiments, the increase is at least a 100% increase.

[0117] In some embodiments, the clinical outcome is survival. In some embodiments, the survival is overall survival. In some embodiments, reduced urea cycle function predicts shortened survival. In some embodiments, reduced liver function predicts shortened survival. In some embodiments, a score exceeding a predetermined threshold predicts shortened survival. In some embodiments, the shortening is compared to an object with unreduced function. In some embodiments, the shortening is compared to an object with a score within a predetermined threshold. In some embodiments, the shortening of survival is a prediction of less than one year of survival. In some embodiments, the shortening of survival is a prediction of less than 2 years of survival. In some embodiments, the shortening of survival is a prediction of 2-5 years of survival. In some embodiments, the survival is the next 1 year of survival. In some embodiments, the survival is the next 2 years of survival. In some embodiments, the survival is the next 5 years of survival. In some embodiments, the survival is the next 12 years of survival. In some embodiments, the survival is the next 13 years of survival. In some embodiments, the survival is the survival from diagnosis. In some embodiments, the survival is the survival from the execution of the method of the present invention. In some embodiments, the survival period is the survival period when the cancer is resectable at the time of diagnosis. In some embodiments, the survival period is the survival period when the cancer is metastatic at the time of diagnosis. In some embodiments, the survival period is the survival period when the cancer is stage I-II at the time of diagnosis. In some embodiments, the survival period is the survival period when the cancer is stage III-IV at the time of diagnosis.

[0118] In some embodiments, the method further comprises administering anticancer therapy to an object determined to have cancer. Examples of anticancer therapy include, but are not limited to, surgery, radiotherapy, chemotherapy, immunotherapy (e.g., immune checkpoint inhibitors), and targeted antibody therapy. In some embodiments, the method further comprises administering anticancer therapy to an object determined to have a worse clinical outcome. In some embodiments, the anticancer therapy is a therapeutic agent. In some embodiments, the anticancer therapy comprises administering a therapeutic agent.

[0119] In some embodiments, anticancer therapy is anti-IL6 therapy. In some embodiments, the therapeutic agent is anti-IL6 therapy. In some embodiments, anti-IL-6 therapy includes administering IL-6 blocking or neutralizing antibodies. Examples of anti-IL6 antibodies include, but are not limited to, Siltuximab, Olokizumab, Elsilimomab, Clazakizumab, Gerilimzumab, EBI-031, and Sirukumab. In some embodiments, anti-IL6 therapy includes administering IL-6 receptor (IL-6R) blocking or neutralizing antibodies. Examples of anti-IL6R antibodies include, but are not limited to, BCD-089, Tocilizumab, LusiNEX, Sarilumab, and Vobarilizumab. Since anti-IL-6 and IL-6R antibodies are well known in the art, there are a sufficient number of members in this genus to represent the species as a whole.

[0120] As used herein, the term "antibody" refers to a polypeptide or polypeptide group comprising at least one binding domain formed by the folding of a polypeptide chain having a three-dimensional binding space having an internal surface shape and charge distribution that are complementary to the characteristics of the antigenic determinant of the antigen. Antibodies generally have a tetrameric form, comprising two pairs of identical polypeptide chains, each pair having one "light" chain and one "heavy" chain. The variable region of each light / heavy chain pair forms an antibody binding site. Antibodies may be oligoclonal antibodies, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, camelid antibodies, CDR-grafted antibodies, multispecific antibodies, bispecific antibodies, catalytic antibodies, humanized antibodies, fully human antibodies, anti-idiotypic antibodies, and antibodies and fragments that can be labeled in soluble form or in a bound form, including epitope binding fragments, variants or derivatives thereof, alone or in combination with other amino acid sequences. Antibodies may be from any species. The term antibody also includes binding fragments, including but not limited to Fv, Fab, Fab', F(ab')2 single-chain antibodies (svFC), dimer variable regions (diabodies) and disulphide-linked variable regions (dsFv). Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. Antibody fragments may or may not be fused with another immunoglobulin domain, which includes but is not limited to the Fc region or a fragment thereof. The technician will further understand that other fusion products can be generated, including but not limited to scFv-Fc fusions, variable regions (e.g., VL and VH) ~ Fc fusions and scFv-scFv-Fc fusions.

[0121] The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0122] In some embodiments, anticancer therapy is ERK inhibition. In some embodiments, therapeutic agent is ERK inhibitor. In some embodiments, anticancer therapy includes administration of ERK inhibitor. Examples of ERK inhibitors include but are not limited to CAS1049738-54-6 (ERK inhibitor), trametinib, PD98059, SCH772984, tauroursodeoxycholate, patritumab, ulixertinib, reavoxertinib, astragaloside IV, sodium tauroursodeoxycholate, piperamide, temuterkib, lidocaine, BIX02189, FR180204, XMD8-92 and MK-8353ERK, etc. Other examples of inhibitors can be found at medchemexpress.com / Targets / ERK, the contents of which are hereby incorporated by reference in their entirety.

[0123] In some embodiments, anticancer therapy is STAT inhibition. In some embodiments, the therapeutic agent is a STAT inhibitor. In some embodiments, STAT is signal transducer and activator of transcription 3 (signal transducer and activator of transcription 3, STAT3). In some embodiments, anticancer therapy includes administering a STAT inhibitor. Examples of STAT3 inhibitors include, but are not limited to, Stattic, AG490, artesunate, niclosamide, cilengitide, STX-0119, STAT3-IN-15, homoharringtonine, C188-9, TPCA-1, napabucasin, cryptotanshinone, WP1066, NSC74859, SD-36, baicalein, astaxanthin and pimozide, etc. Other examples of STAT3 inhibitors can be found at medchemexpress.com / Targets / STAT / stat3, the contents of which are hereby incorporated by reference in their entirety.

[0124] In some embodiments, the inhibitor is a specific inhibitor. In some embodiments, the specific inhibitor does not substantially inhibit any protein other than the target (e.g., ERK or STAT). In some embodiments, substantially is significantly. In some embodiments, substantially is detectably. In some embodiments, the inhibitor or antibody is provided in a pharmaceutical composition. In some embodiments, the composition includes a therapeutically effective carrier, excipient, or adjuvant.

[0125] In some embodiments, the anticancer therapy comprises administering an agent that increases the expression of HNF4A. In some embodiments, the therapeutic agent increases the expression of HNF4A. In some embodiments, the increase is in the liver of the subject. In some embodiments, the in the liver is in liver cells. In some embodiments, the in the liver is in hepatocytes. In some embodiments, the agent is a lipid nanoparticle (LNP) of the present invention.

[0126] In some embodiments, the agent comprises a nucleic acid molecule. In some embodiments, the agent is a nucleic acid molecule. In some embodiments, the nucleic acid molecule encodes HNF4A. In some embodiments, the nucleic acid molecule comprises a coding region encoding HNF4A. In some embodiments, the nucleic acid molecule comprises an open reading frame encoding HNF4A.

[0127] The term "nucleic acid" is well known in the art. As used herein, "nucleic acid" will generally refer to a molecule (i.e., a strand) of DNA, RNA, or derivatives or analogs thereof that include a nucleobase. Nucleobases include, for example, naturally occurring purine or pyrimidine bases present in DNA (e.g., adenine "A", guanine "G", thymine "T", or cytosine "C") or RNA (e.g., A, G, uracil "U", or C).

[0128] The term "nucleic acid molecule" includes, but is not limited to, single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), small RNA (such as miRNA, siRNA and other short interfering nucleic acids), snoRNA, snRNA, tRNA, piRNA, tnRNA, small rRNA, hnRNA, circulating nucleic acids, genomic DNA or RNA fragments, degraded nucleic acids, ribozymes, viral RNA or DNA, infectious nucleic acids, amplification products, modified nucleic acids, cytoplasmic or organellar nucleic acids, and artificial nucleic acids (such as oligonucleotides).

[0129] Hepatocyte nuclear factor 4 alpha (HNF4A) is also known as NR2A1, HNF4, TCF14, Nuclear Receptor Subfamily 2 group A member 1, transcription factor HNF-4, and transcription factor 14, as well as other names known in the art. In some embodiments, HNF4A is mammalian HNF4A. In some embodiments, HNF4A is rodent HNF4A. In some embodiments, the rodent is a mouse. In some embodiments, HNF4A is human HNF4A. The mouse HNF4A gene can be found at Entrez #15378. The mouse HNF4A protein sequence can be found at Uniprot IDP49698. The RefSeq mRNA sequence of mouse HNF4A can be found at NM_008261, NM_001312906, and NM_001312907. RefSeq protein sequences for mouse HNF4A can be found at NP_032287, NP_001299835, and NP_001299836. The human HNF4A gene can be found at Entrez #3172. The human HNF4A protein sequence can be found at UniprotID P41235. The RefSeq mRNA sequences for human HNF4A can be found at NM_000457, NM_001030003, NM_001030004, NM_001258355, and NM_001287182. The RefSeq protein sequences for human HNF4A can be found at NP_000448, NP_001025174, NP_001025175, NP_001245284, and NP_001274111.

[0130]

[0131] In some embodiments, the mouse HNF4A protein comprises MRLSKTLAGMDMADYSAALDPAYTTLEFENVQVLTMGNDTSPSEGANLNSSNSLGVSALCAICGDRATGKHYGASSCDGCKGFFRRSVRKNHMYSCRFSRQCVVDKDKRNQCRYCRLKKCFRAGMKKEAVQNERDRISTRRSSYEDSSLPSINALLQAEVLSQQITSPISGINGDIRAKKIANITDVCESMKEQLLVLVEWAKYIPAFCELLLDDQVALLRAHAGEHLL LGATKRSMVFKDVLLLGNDYIVPRHCPELAEMSRVSIRILDELVLPFQELQIDDNEYACLKAIIFFDPDAKGLSDPGKIKRLRSQVQVSLEDYINDRQYDSRGRFGELLLLLPTLQSITWQMIEQIQFIKLFGMAKIDNLLQEMLLGGSASDAPHTHHPLHPHLMQEHMGTNVIVANTMPSHLSNGQMCEWPRPRGQAATPETPQPSPPSGSGSESYKLLPGAITTIVKPPSAIPQPTITKQEAI (SEQ ID NO: 3). In some embodiments, the mouse HNF4A protein consists of SEQ ID NO: 3. In some embodiments, the coding region encodes SEQ ID NO: 3. In some embodiments, the HNF4A protein is a protein having at least 85% homology to SEQ ID NO: 3.

[0132]

[0133] In some embodiments, the human HNF4A protein comprises MRLSKTLVDMDMADYSAALDPAYTTLEFENVQVLTMGNDTSPSEGTNLNAPNSLGVSALCAICGDRATGKHYGASSCDGCKGFFRRSVRKNHMYSCRFSRQCVVDKDKRNQCRYCRLKKCFRAGMKKEAVQNERDRISTRRSSYEDSSLPSINALLQAEVLSRQITSPVSGINGDIRAKKIASIADVCESMKEQLLVLVEWAKYIPAFCELPLDDQVALLRAHA GEHLLLGATKRSMVFKDVLLLGNDYIVPRHCPELAEMSRVSIRILDELVLPFQELQIDDNEYAYLKAIIFFDPDAKGLSDPGKIKRLRSQVQVSLEDYINDRQYDSRGRFGELLLLLPTLQSITWQMIEQIQFIKLFGMAKIDNLLQEMLLGGSPSDAPHAHHPLHPHLMQEHMGTNVIVANTMPTHLSNGQMSTPETPQPSPPGGSGSEPYKLLPGAVATIVKPLSAIPQPTITKQEVI (SEQ ID NO: 6). In some embodiments, the human HNF4A protein consists of SEQ ID NO: 6. In some embodiments, the coding region encodes SEQ ID NO: 6. In some embodiments, the HNF4A protein is a protein having at least 85% homology to SEQ ID NO: 6.

[0134] In some embodiments, the nucleic acid molecule is a vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector includes at least one regulatory element operably connected to the nucleic acid molecule of the present invention. In some embodiments, the vector includes at least one regulatory element operably connected to an open reading frame encoding an antigen binding molecule of the present invention. In some embodiments, at least one regulatory element is a promoter.

[0135] The terms "operably linked" and "operatively linked" are used interchangeably herein and mean that a nucleotide sequence of interest is linked to one or more regulatory elements in a manner that permits expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0136] As used herein, the term "expression" refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. Thus, the expression of a nucleic acid molecule may refer to the transcription of a nucleic acid fragment (e.g., the transcription of an mRNA or other functional RNA) and / or the translation of an RNA into a precursor or mature protein (polypeptide).

[0137] The expression of genes in cells is well known to those skilled in the art. It can be implemented by various methods such as transfection, viral infection or directly changing the genome of the cell. In some embodiments, the gene is in an expression vector such as a plasmid or a viral vector.

[0138] Vector nucleic acid sequences generally contain at least one origin of replication for propagation in cells and optionally additional elements such as heterologous polynucleotide sequences, expression control elements (e.g., promoters, enhancers), selectable markers (e.g., antibiotic resistance), polyadenine sequences.

[0139] The vector can be a DNA plasmid delivered via a non-viral method or via a viral method. The viral vector can be a retroviral vector, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector or a poxvirus vector. In some embodiments, the vector is an AAV vector. The promoter can be active in mammalian cells. The promoter can be a viral promoter. In some embodiments, the promoter is a human promoter. In some embodiments, the promoter is a hepatocyte promoter.

[0140] In some embodiments, the HNF4A coding region is operably linked to a promoter. The term "operably linked" means that the nucleotide sequence of interest is linked to one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (eg, in a host cell when the vector is introduced into the host cell).

[0141] In some embodiments, the vector is introduced into the cell by standard methods, including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection with a viral vector, high-speed ballistic penetration of small particles having nucleic acids within or on the surface of a bead or particle array (Klein et al., Nature 327. 70-73 (1987)), and / or the like.

[0142] As used herein, the term "promoter" refers to a group of transcription control modules that are clustered around the start site of RNA polymerase (i.e., RNA polymerase II). The promoter is composed of discrete functional modules, each of which is composed of approximately 7-20 bp of DNA and contains one or more recognition sites for transcriptional activators or repressors.

[0143] In some embodiments, the nucleic acid sequence is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme present in eukaryotic cells. It catalyzes the transcription of DNA to synthesize precursors of mRNA and most snRNAs and microRNAs.

[0144] In some embodiments, mammalian expression vectors include but are not limited to: pcDNA3, pcDNA3.1(±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, which can be purchased from Invitrogen; pCI, which can be purchased from Promega; pMbac, pPbac, pBK-RSV and pBK-CMV, which can be purchased from Strategene; pTRES, which can be purchased from Clontech, and derivatives thereof.

[0145] In some embodiments, the present invention uses expression vectors containing regulatory elements from eukaryotic viruses (such as retroviruses). SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and vectors that allow protein expression under the guidance of SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters that show effective expression in eukaryotic cells.

[0146] In some embodiments, the recombinant viral vector of the advantage such as lateral infection and target specificity is provided for in vivo expression.In one embodiment, lateral infection is intrinsic in the life cycle of for example retrovirus, and is the process that single infected cell produces a plurality of progeny virions, and this progeny virion sprouts and infects adjacent cell.In one embodiment, the result is that large tracts of land are infected rapidly, and wherein major part is not infected by original virus particle at first.In one embodiment, produce the viral vector that can not spread laterally.In one embodiment, if the purpose of expectation is only to import specific gene into the target cell of local quantity, then this characteristic can be useful.

[0147] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6):504-512, 1986], and include, for example, stable or transient transfection, lipofection, electroporation, and infection with recombinant viral vectors. In addition, see US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0148] It will be appreciated that, in addition to containing the necessary elements for transcription and translation of the inserted coding sequence (which encodes a polypeptide), the expression constructs of the invention may also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0149] In some embodiments, the nucleic acid molecule is an adeno-associated virus (AAV) vector. In some embodiments, the nucleic acid molecule is contained within an AAV. In some embodiments, the agent is an AAV comprising a nucleic acid molecule.

[0150] In some embodiments, the nucleic acid molecule is mRNA. In some embodiments, the mRNA includes a 5' cap. In some embodiments, the mRNA includes a 5' untranslated region (UTR). In some embodiments, the 5'UTR includes a ribosome binding site. In some embodiments, the 5'UTR includes the 5' end of the Kozak sequence (ACC). In some embodiments, the 5'UTR includes the 5' end of the Kozak sequence (GCCACC). In some embodiments, ACC is the 3' end of the 5'UTR. In some embodiments, the 5'UTR includes a T7 RNA promoter. In some embodiments, the T7 RNA promoter includes the nucleotide sequence UAAUACGACUCACUAUA (SEQ ID NO: 46). In some embodiments, the T7 RNA promoter consists of SEQ ID NO: 46. In some embodiments, the 5' end of the mRNA is a T7 RNA promoter. The T7 RNA promoter is commonly used in in vitro transcription reactions. In some embodiments, the 5'UTR includes human alpha globin mRNA 5'UTR. In some embodiments, the human alpha globin 5'UTR comprises the nucleotide sequence GAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 47). In some embodiments, the 5'UTR comprises the sequence UAAUACGACUCACUAUAAGGGAGACCCAAGCUGGCUAGCGUUUAAACUUAAGCUUGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAAGGGAGACUCUUCUGGUCCCCACAGACUCAGAGAGAACCCACC (SEQ ID NO: 7). In some embodiments, the 5'UTR consists of SEQ ID NO: 7. In some embodiments, the 5'UTR comprises a sequence having at least 85% homology to SEQ ID NO: 7. In some embodiments, the sequence retains a ribosome binding site. In some embodiments, the sequence retains a 5' end of a Kozak sequence at its 3' end.

[0151] In some embodiments, the mRNA includes a 3'UTR. In some embodiments, the 3'UTR enhances the stability of the mRNA. In some embodiments, the 3'UTR is derived from a mitochondrial rRNA 3'UTR sequence. In some embodiments, the 3'UTR produces a thermodynamically stable secondary structure at a temperature for LNP formation. In some embodiments, the temperature is about 37 degrees Celsius. In some embodiments, the temperature is between 50 and 70 degrees Celsius. In some embodiments, the 3'UTR includes the sequence GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO:8). In some embodiments, the 3'UTR consists of SEQ ID NO:8. In some embodiments, the 3'UTR includes a sequence having at least 85% homology to SEQ ID NO:8. In some embodiments, the nucleic acid molecule includes a stop codon at the 3' end of the coding region. In some embodiments, the region encoding HNF4A includes a stop codon at its 3' end. In some embodiments, the stop codon separates the coding region from the 3' UTR. In some embodiments, the mRNA is polyadenylated. In some embodiments, the mRNA includes a poly A tail. In some embodiments, the poly A tail is 3' to 3' UTR.

[0152] In some embodiments, the mRNA comprises a sequence encoding mouse HNF4A and comprises SEQ ID NO: 9. In some embodiments, the mRNA comprises a sequence encoding mouse HNF4A and consists of SEQ ID NO: 9. SEQ ID NO: 9 provides the DNA counterpart of the mRNA sequence. In some embodiments, the mRNA comprises a sequence encoding mouse HNF4A and comprises SEQ ID NO: 10. In some embodiments, the mRNA comprises a sequence encoding mouse HNF4A and consists of SEQ ID NO: 10. In some embodiments, the mRNA comprises a sequence encoding human HNF4A and comprises SEQ ID NO: 11. In some embodiments, the mRNA comprises a sequence encoding human HNF4A and consists of SEQ ID NO: 11. SEQ ID NO: 11 provides the DNA counterpart of the mRNA sequence. In some embodiments, the mRNA comprises a sequence encoding human HNF4A and comprises SEQ ID NO: 12. In some embodiments, the mRNA comprises a sequence encoding human HNF4A and consists of SEQ ID NO: 12.

[0153] In some embodiments, the nucleic acid molecule comprises a chemically modified backbone. In some embodiments, the RNA comprises a chemically modified backbone. Chemical modifications of the backbone are known to enhance half-life and stability. In some embodiments, the chemically modified backbone comprises: a phosphate-ribose backbone, a phosphate-deoxyribose backbone, a thiophosphate-deoxyribose backbone, a 2'-O-methylthiophosphate backbone, a phosphorodiamidate morpholino backbone, a peptide nucleic acid backbone, a 2-methoxyethylthiophosphate backbone, an alternating locked nucleic acid backbone, a thiophosphate backbone, an N3'-P5' aminophosphoric acid, a 2'-deoxy-2'-fluoro-β-d-arabino nucleic acid, a cyclohexene nucleic acid backbone nucleic acid, a tricyclic DNA (tcDNA) nucleic acid backbone, and any combination thereof.

[0154] In some embodiments, the nucleic acid molecule is encapsulated in a nanoparticle. In some embodiments, the nanoparticle is a nanoparticle of the present invention.

[0155] In another aspect, a nucleic acid molecule is provided that encodes HNF4A.

[0156] In another aspect, nanoparticles are provided that are targeted to the liver of a subject.

[0157] In some embodiments, when administered systemically to a subject, the nanoparticles are targeted to the liver. In some embodiments, systemically is intravenously. In some embodiments, targeting the liver comprises targeting hepatocytes. In some embodiments, the nanoparticles are targeted to mammalian livers. In some embodiments, the nanoparticles are targeted to mouse livers. In some embodiments, the nanoparticles are targeted to human livers.

[0158] In some embodiments, the nanoparticle comprises an agent. In some embodiments, the nanoparticle comprises a nucleic acid molecule. In some embodiments, the nanoparticle comprises an mRNA. In some embodiments, comprising is encapsulated. In some embodiments, the nanoparticle comprises an aqueous core. In some embodiments, the agent / nucleic acid molecule / mRNA is in the aqueous core. In some embodiments, being therein is dissolved therein.

[0159] As used herein, "nanoparticle" refers to a nanoscale carrier capable of transporting nucleic acid molecules. In some embodiments, the nanoparticle comprises an average diameter of at most 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 175, 150, 140, 135, 130, 125, 120, 115, 110, 105, 100, 90, 80, 75, 70, 60 or 50 nanometers (nm). Each possibility represents a separate embodiment of the present invention. In some embodiments, the nanoparticle comprises a diameter of at most 250nm. In some embodiments, the nanoparticle comprises a diameter of at most 140nm. In some embodiments, the nanoparticle comprises a diameter of at most 125nm. In some embodiments, the nanoparticle comprises a diameter of at most 100nm. In some embodiments, the nanoparticle comprises a diameter of at most 50nm. In some embodiments, the average diameter is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300nm. Each possibility represents a separate embodiment of the present invention. In some embodiments, the average diameter is at least 50nm. In some embodiments, the average diameter is at least 90nm. In some embodiments, the average diameter is at least 100nm.In some embodiments, the average diameter is between 50-700, 50-650, 50-600, 50-550, 50-500, 50-450, 50-400, 50-350, 50-300, 50-250, 50-200, 50-150, 50-140, 50-130, 50-120, 50-110, 50-100, 90-700, 90-650, 90-600, 90-550, 90-500, 90-450, 90-400, 90-350, 90-300, 90-250, 90-200, 90-150, 90-140, 90-130, 90-120, 90-110, 90-100, 100-700, 100-650, 100-600, 100-550, 100-500, 100-450, 100-400, 10 0-350, 100-300, 100-250, 100-200, 100-150, 100-140, 100-130, 100-120, 100-110, 150-700, 150-650, 150-600, 150-550, 150-500, 150-450, 150-400, 150-350, 150-300, 150-250, 150-20 In some embodiments, the average diameter is between 150 and 500 nm. In some embodiments, the average diameter is between 50 and 500 nm.

[0160] In some embodiments, the nanoparticle is a lipid nanoparticle (LNP). As used herein, the phrase "lipid nanoparticle" refers to a transfer medium comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). In some embodiments, the LNP is a liposome. In some embodiments, the LNP is a micelle. In some embodiments, the nanoparticle is a synthetic nanoparticle. In some embodiments, the nanoparticle is an artificial nanoparticle. In some embodiments, the nanoparticle is not a naturally occurring nanoparticle. In some embodiments, the LNP is not an exosome. In some embodiments, the LNP is not a naturally secreted vesicle.

[0161] Preferably, lipid nanoparticles are formulated to deliver one or more agents (i.e., nucleic acid molecules) to liver / hepatocytes. The example of suitable lipids includes, for example, phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides). It is also contemplated that polymers are used as transfer media, either alone or in combination with other transfer media. Suitable polymers may include, for example, polyacrylates, polycyanoacrylate alkyl esters, polylactides, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrin, dendrimers and polyethyleneimine. In one embodiment, the selection of transfer media is based on its ability to promote nucleic acid transfection to target cells.

[0162] The present invention contemplates the use of lipid nanoparticles as transfer vehicles, which include cationic lipids to encapsulate and / or enhance the delivery of nucleic acids to target cells. As used herein, phrase "cationic lipids" refers to any of the various lipid species (species) carrying a net positive charge under selected pH (such as physiological pH). Lipid nanoparticles can be prepared by including a multi-component lipid mixture of the different proportions of one or more cationic lipids, non-cationic lipids and PEG-modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available.

[0163] Suitable cationic lipids for the compositions and methods of the present invention include those described in International Patent Publication WO 2010 / 053572, which is incorporated herein by reference. In certain embodiments, the compositions and methods of the present invention employ lipid nanoparticles comprising ionizable cationic lipids. In some embodiments, cationic lipid N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride or "DOTMA" (U.S. Patent No. 4,897,355) is used. DOTMA can be formulated alone, or it can be combined with neutral lipids, i.e., dioleoylphosphatidylethanolamine or "DOPE" or other cationic or non-cationic lipids to form liposome transfer vehicles or lipid nanoparticles, and such liposomes can be used to enhance delivery of nucleic acids into target cells. Other suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS", 2,3-dioleyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-pr-opanaminium or "DOSPA" (U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761), 1,2-dioleoyl-3-dimethylammonium-propane or "DODAP", 1,2-dioleoyl-3-trimethylammonium-propane or "DOTAP". Cationic lipids contemplated also include 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane or "DSDMA", 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyloxy-N,N-dimethyl-3-aminopropane or "DLenDMA", N-dioleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", 1,2-dilin ...inDMA", 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane Dimethylammonium chloride or "DODAC", N,N-distearoyl-N,N-dimethylammonium bromide or "DDAB", N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide or "DMRIE", 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane1-2-octadecadienoxy)propane or "CLinDMA", 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethylll-(cis,cis-9',l-2'-octadecadienoxy)propane or "CpLinDMA", N,N-dimethyl-3,4-dioleyloxybenzylamine or "DMOBA", 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane or "DOcarbDAP" , 2,3-dilinoleoyloxy-N,N-dimethylpropylamine or "DLinDAP", 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane or "DLincarbDAP", 1,2-dilinoleylcarbamoyl-3-dimethylaminopropane or "DLinCDAP", 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or "DLin-K-DMA", 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or "DLin-K-XTC2-DMA" and 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-di-methylethylamine (DLin-KC2-DMA)) (see WO 2010 / 042877; Semple et al., Nature Biotech. 28: 172-176 (2010)), or a mixture thereof (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol. 23 (8): 1003-1007 (2005); PCT Publication WO 2005 / 121348 A1). ,

[0164] The present invention also contemplates the use of cholesterol-based cationic lipids. Such cholesterol-based cationic lipids can be used alone or in combination with other cations or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, DC-Chol (N, N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis (3-N-oleylamino-propyl) piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Patent number 5,744,335) or ICE. In some embodiments, LNP includes cholesterol.

[0165] The present invention also contemplates the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), either alone or preferably in combination with other lipids, including lipid nanoparticles, including N-octanoyl-sphingosine-1-[succinyl (methoxypolyethylene glycol)-2000] (C8 PEG-2000 ceramide). The addition of such components can prevent complex aggregation and can also provide a means of increasing circulation life and increasing delivery of lipid-nucleic acid compositions to target cells (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they can be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides with shorter acyl chains (e.g., C14 or C18). The PEG-modified phospholipids and derivatized lipids of the present invention can account for a molar ratio of about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipids present in the liposome transfer vehicle. In some embodiments, the LNP includes a PEGylated lipid. In some embodiments, the PEGylated lipid is a PEGylated myristoyl diglyceride (DMG-PEG).

[0166] The present invention also contemplates the use of non-cationic lipids. As used herein, the phrase "non-cationic lipid" refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase "anionic lipid" refers to any of a variety of lipid species that carry a net negative charge at a selected pH (such as physiological pH). Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. Such non-cationic lipids can be used alone, but are preferably used in combination with other excipients (e.g., cationic lipids). When used in combination with cationic lipids, the non-cationic lipids can account for 5% to about 90% of the total lipid present in the transfer vehicle, or preferably a molar ratio of about 10% to about 70%.

[0167] In some embodiments, non-cationic lipid is ionizable lipid. In some embodiments, ionizable lipid is synthetic amino lipid. The example of ionizable lipid includes but is not limited to ALC-0315, SM-102, lipid 5, DLin-DMA, D-Lin-MC3-DMA, DLin-KC2-DMA, YSK05, AA3-DLin, SSPAlmM, SSPamO-Phe, lipid A9, L319, CL4H6, DODMA, CL1, BP lipid 308, ATX-100, 80-O16B, 93-O17S, (3-O17O and NT1 etc. In some embodiments, LNP comprises SM-102.

[0168] In some embodiments, lipid nanoparticles are prepared by combining multiple lipids and / or polymer components. The selection of cationic lipids, non-cationic lipids and / or PEG-modified lipids constituting lipid nanoparticles and the relative molar ratio of such lipids to each other are based on the characteristic of the lipid (one or more) selected and the ability of nucleic acid molecules (that is, mRNA) targeting liver thereof. Other considerations include, for example, the size, charge, pH, pKa, fusogenicity and toxicity of the saturation of alkyl chains and the lipid (one or more) selected.

[0169] The LNPs used in the compositions of the present invention can be prepared by various techniques currently known in the art. Multilamellar vesicles (MLVs) can be prepared by conventional techniques, for example, by depositing the selected lipids on the inner side of the wall of a suitable container or vessel - by dissolving the lipids in a suitable solvent, then evaporating the solvent to leave a thin film on the inner side of the vessel, or by spray drying. The aqueous phase can then be added to the vessel using a vortex motion (which results in the formation of MLVs). Unilamellar vesicles (ULVs) can then be formed by homogenizing, ultrasonic treatment or extruding multilamellar vesicles. In addition, unilamellar vesicles can also be formed by detergent removal techniques. In some embodiments, LNPs are produced by ethanol injection.

[0170] In some embodiments, LNP comprises SM-102 and cholesterol. In some embodiments, LNP comprises SM-102 and PEGylated lipid. In some embodiments, PEGylated lipid is DMG-PEG. In some embodiments, LNP comprises SM-102 and cationic lipid. In some embodiments, cationic lipid DOPE. In some embodiments, LNP comprises SM-102, cholesterol and DOPE. In some embodiments, LNP comprises SM-102, cholesterol and DMG-PEG. In some embodiments, LNP comprises SM-102, DOPE and DMG-PEG. In some embodiments, LNP comprises SM-102, cholesterol, DOPE and DMG-PEG. In some embodiments, PEG is PEG200. In some embodiments, PEG is low molecular weight PEG.

[0171] In some embodiments, the LNP comprises 40-60 mol% SM-102. In some embodiments, the LNP comprises 45-55 mol% SM-102. In some embodiments, the LNP comprises about 50 mol% SM-102. In some embodiments, the LNP comprises 30-50 mol% cholesterol. In some embodiments, the LNP comprises 35-45 mol% cholesterol. In some embodiments, the LNP comprises 33.5-43.5 mol% cholesterol. In some embodiments, the LNP comprises about 40 mol% cholesterol. In some embodiments, the LNP comprises about 38.5 mol% cholesterol. In some embodiments, the LNP comprises 5-15 mol% DOPE. In some embodiments, the LNP comprises 7.5-12.5 mol% DOPE. In some embodiments, the LNP comprises about 10% DOPE. In some embodiments, the LNP comprises 0.5-2.5 mol% DMG-PEG. In some embodiments, the LNP comprises 1-2 mol% DMG-PEG. In some embodiments, the LNP comprises about 1.5 mol% DMG-PEG.In some embodiments, the LNP comprises about 50 mol% SM-102, 38.5 mol% cholesterol, 10 mol% DOPE, and 1.5 mol% DMG-PEG200.

[0172] As used herein and in the art, molar percentage ("% mol") refers to the percentage of a particular component or compound based on the total moles of the components or compounds that make up the nanoparticle. For example, if the nanoparticle contains 3 moles of compound A and 1 mole of compound B, then compound A accounts for 75 mol% of the mixture and compound B accounts for 25 mol%.

[0173] In another aspect, a composition is provided comprising the nanoparticles of the present invention.

[0174] In some embodiments, the composition is a therapeutic composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient or adjuvant.

[0175] As used herein, the term "carrier", "excipient" or "adjuvant" refers to any component of a pharmaceutical composition that is not an active agent. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic inert solid, semi-solid, liquid filler, diluent, encapsulating material, any type of formulation auxiliary, or simply a sterile aqueous medium, such as saline. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol solutions and phosphate buffered saline solutions, and other nontoxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of materials that can serve as carriers herein include sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffered solution, cocoa butter (suppository base), emulsifiers, and other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as colorants, flavoring agents, excipients, stabilizers, antioxidants and preservatives may also be present. Any non-toxic, inert and effective carrier can be used to formulate the compositions contemplated herein. In this regard, suitable pharmaceutically acceptable carriers, excipients and diluents are well known to those skilled in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, NJ (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the "Inactive Ingredient Guide," US Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management (the entire contents of which are hereby incorporated by reference in their entirety).Examples of pharmaceutically acceptable excipients, carriers, and diluents for use in the present compositions include distilled water, saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO. These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks such as Goodman and Gillman's: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005) (each of which is incorporated herein by reference in its entirety). The compositions described herein may also be contained in artificially created structures such as liposomes, ISCOMS, slow-release particles, and other vehicles that increase the half-life of peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. The liposomes used with the peptides described herein are formed by standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids generally depends on considerations such as liposome size and stability in blood. For example, Coligan, JE et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, summarizes various methods that can be used to prepare liposomes, and also see U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

[0176] Carriers may comprise in total from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0177] In some embodiments, the composition is formulated for systemic administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for administration to the liver. In some embodiments, the composition is formulated for liver administration. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the composition is formulated for administration to a human.

[0178] As used herein, the terms "administering," "administration," and similar terms refer to any method of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect in reasonable medical practice. One aspect of the present subject matter provides for intravenous administration of a therapeutically effective amount of the present subject matter composition to a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, intrahepatic, intramuscular, or intraperitoneal.

[0179] The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

[0180] In another aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject an agent capable of increasing expression of HNF4A in the subject, thereby treating the cancer in the subject.

[0181] In another aspect, a method of treating cachexia in a subject in need thereof is provided, the method comprising administering to the subject a composition comprising at least one agent selected from the group consisting of an anti-IL6 blocking antibody, an ERK inhibitor, a STAT3 inhibitor, and an agent capable of increasing the expression of HNF4A in the subject, thereby treating cachexia in the subject.

[0182] In another aspect, a method of preventing cachexia in a subject in need thereof is provided, the method comprising administering to the subject a composition comprising at least one agent selected from the group consisting of an anti-IL6 blocking antibody, an ERK inhibitor, a STAT3 inhibitor, and an agent capable of increasing the expression of HNF4A in the subject, thereby preventing cachexia in the subject.

[0183] In some embodiments, the agent increases the expression of HNF4A in the liver of the subject. In some embodiments, the agent increases the expression of HNF4A in the hepatocytes of the subject. In some embodiments, the expression is protein expression. In some embodiments, increasing expression comprises delivering HNF4A to the liver or hepatocytes. In some embodiments, the agent is a nanoparticle of the invention.

[0184] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a composition of the present invention. In some embodiments, the method comprises administering a nanoparticle of the present invention. In some embodiments, the method comprises administering an LNP of the present invention. In some embodiments, the method comprises administering a composition of the present invention.

[0185] In another aspect, there is provided a nanoparticle of the invention for use in treating cancer. In another aspect, there is provided a nanoparticle of the invention for use in the manufacture of a medicament for treating cancer.

[0186] In another aspect, there is provided a nanoparticle of the invention for use in treating cachexia. In another aspect, there is provided a nanoparticle of the invention for use in the manufacture of a medicament for treating cachexia.

[0187] In some embodiments, the cancer is a non-liver cancer. In some embodiments, the cachexia is cancer-related cachexia. In some embodiments, the subject has cancer. In some embodiments, the cancer is an early stage cancer. In some embodiments, the subject has a precancerous lesion. In some embodiments, the subject is at risk of developing cancer. In some embodiments, the subject is at risk of developing cachexia. In some embodiments, the subject has been determined to have cancer by the methods of the present invention. In some embodiments, the subject has been diagnosed with cancer by the methods of the present invention. In some embodiments, the subject is determined to have cachexia by the methods of the present invention. In some embodiments, the subject is determined to be at risk of developing cachexia by the methods of the present invention. In some embodiments, the method further comprises determining the presence of reduced urea cycle function in the subject. In some embodiments, treatment is performed in a subject in which reduced urea cycle function is confirmed. In some embodiments, the method further comprises determining the presence of reduced liver function in the subject. In some embodiments, treatment is performed in a subject in which reduced liver function is confirmed. In some embodiments, reduced function is determined by the methods of the present invention.

[0188] As used herein, the term "treatment" or "treating" a disease, disorder, or condition encompasses alleviating at least one symptom thereof, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. To be effective, the compositions or methods useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of the symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

[0189] In some embodiments, treatment comprises reducing the size of a tumor. In some embodiments, treatment comprises reducing the growth of a tumor. In some embodiments, reduction is cessation. In some embodiments, treatment comprises reducing at least one symptom of cachexia. In some embodiments, treatment comprises reducing muscle loss in cachexia. In some embodiments, treatment comprises reducing fat loss in cachexia. In some embodiments, treatment comprises reducing free fluid in cachexia. In some embodiments, treatment comprises at least one of increasing fat, increasing muscle, and reducing free fluid. In some embodiments, treatment comprises extending survival. In some embodiments, treatment comprises extending the time before developing cachexia. In some embodiments, treatment comprises extending survival by more than 1 year. In some embodiments, treatment comprises extending survival by more than 2 years. In some embodiments, treatment comprises extending survival by more than 5 years. In some embodiments, treatment comprises extending survival by more than 10 years.

[0190] As used herein, the term "about" when combined with a value refers to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm +- 100 nm.

[0191] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes a plurality of such polynucleotides, and reference to a "polypeptide" includes reference to one or more polypeptides and their equivalents known to those skilled in the art, and so on. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely", "only", etc. in conjunction with limitations of claim elements, or use of a "negative" limitation.

[0192] In those cases where a convention similar to "at least one of A, B, and C, etc." is used, such a construction is generally intended to be in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, having A and B together, having A and C together, having B and C together, and / or having A, B, and C together, etc.). One skilled in the art would further understand that virtually any antonymous conjunction and / or phrase presenting two or more optional terms, whether in the specification, claims, or drawings, would be understood to contemplate the possibility of including one of these terms, any one of these terms, or both of these terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0193] It should be understood that, for the sake of clarity, certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for the sake of brevity, various features of the present invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. All combinations of embodiments related to the present invention are specifically encompassed by the present invention and disclosed herein, just as each combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically encompassed by the present invention and disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0194] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. The term "a" (or "one") and the terms "one or more" and "at least one" are used interchangeably.

[0195] Furthermore, “and / or” should be considered to specifically disclose each of the two specified features or components - in the presence or absence of the other. Thus, the term “and / or” used in the phrase “A and / or B” is intended to include A and B, A or B, A (alone) and B (alone). Similarly, the term “and / or” used in phrases such as “A, B and / or C” is intended to include A, B and C; A, B or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0196] Whenever an embodiment is described with the term "comprising," similar embodiments described with respect to "consisting of" and / or "consisting essentially of are also included.

[0197] After examining the following examples, other objects, advantages and novel features of the present invention will become apparent to those skilled in the art, and these examples are not intended to be limiting. In addition, as described above and as claimed in the following claims, each of the various embodiments and aspects of the present invention is experimentally supported in the following examples.

[0198] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0199] Example

[0200] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully described in the literature.See, for example, "Molecular Cloning: Alaboratory Manual," Sambrook et al., (1989); "Current Protocols in Molecular Biology," Vol. I-III, Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology," John Wiley and Sons, Baltimore, Maryland (1989); to Molecular Cloning", John Wiley&Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: ALaboratory Manual Series", Volumes 1-4, ColdSpring Harbor Laboratory Press, New York (1998); the methods listed in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Culture of Animal Cells-A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), third edition; "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference.Other general references are provided throughout this document.

[0201] Materials and Methods

[0202] In vivo animal studies Animal experiments were performed by the Weizmann Institute Animal Care and Use Committee in accordance with the US National Institute of Health, European Commission, and Israeli guidelines. To generate syngeneic mouse cancer models, 8-12 week old C57BL / 6 or BALB / c male and female mice were purchased from Envigo and randomly assigned to experimental groups. For the BC model, 8-week old BALB / c female mice were injected with 1x10 6 For the PC model, 12-week-old C57BL / 6 male mice and CCR2-RFP knockout mice were injected with 0.3-0.4x10 4T1 BC cells in the tail of the pancreas. 6 KPC PC cells (in DMEM 50% matrigel). After sacrifice, the liver, spleen, bone marrow and lungs were removed from the mice, and blood was collected for further analysis by quantitative PCR, Western blot and immunohistochemistry.

[0203] Cell lines : 4T1-luciferase cells derived from mouse breast cancer cells were provided by Professor Yossi Yarden of the Department of Biological Regulation, Weizmann Institute of Science. KrasG12D / Trp53R172H / Pdx-1-Cre (KPC)-luciferase cells derived from mouse solid PDAC were provided by Professor Avigdor Scherz of the Department of Plant and Environmental Sciences, Weizmann Institute of Science. All cells were routinely tested for mycoplasma using the Mycoplasma EZ-PCR Test Kit (#20-700-20, Biological Industries, Kibbutz Beit Ha'emek).

[0204] Western blotting: Tissues were ground and lysed in RIPA (Sigma-Aldrich) and 1% protease inhibitor cocktail (Calbiochem), 1% phosphatase inhibitor cocktail (P5726, Sigma-Aldrich). After centrifugation, the supernatant was collected and the protein content was assessed by Bradford assay or BCA protein assay kit (ThermoFisher Scientific, cat#23225). 20-50 μg of each sample under reducing conditions was loaded into each lane and separated by electrophoresis on a 10% SDS polyacrylamide gel. After electrophoresis, the protein was transferred to a nitrocellulose membrane (Tamar, Israel). Nonspecific binding was blocked by incubation with TBST (10 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20) containing 5% skim milk for 1 h at room temperature. The membrane was then incubated with antibodies (WB antibody list).

[0205] Antibodies were detected using peroxidase-conjugated AffiniPure goat anti-rabbit IgG or goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) and enhanced chemiluminescent western blot detection reagent (EZ-Gel, Biological Industries). Gels were quantified by Gel Doc XR+ (BioRad) and analyzed by ImageLab 5.1 software (BioRad). The relative intensity of each band was calculated by dividing the specific band intensity by the value obtained from the load control.

[0206] Liver perfusion and hepatocyte dissociation: The liver of anesthetized mice was perfused and specifically adjusted. A 27G syringe was inserted into the vena cava and fixed on the perfusion line. 10 ml of PPML buffer pre-warmed to 42°C was perfused through the vena cava, followed by 25 ml of PM buffer pre-warmed to 42°C containing Liberase (Roche, cat#05401127001). At the beginning of perfusion, the portal vein was immediately cut off. After perfusion of 25 ml of PM buffer and Liberase solution, the liver was harvested into a culture dish containing 10 ml of pre-warmed PM buffer and minced with tweezers. The dissociated liver cells were collected and filtered through a tilted 100um cell strainer. The cells were rotated at 30g for 3 minutes at 4°C to obtain a precipitate enriched in hepatocytes. The precipitate was resuspended in 25ul of cold PM buffer. To enrich live hepatocytes, add 21.6 ml of cold Percoll (GE Healthcare #17-0891-01) mixed with +2.4 ml DPBSX10+CaCl2+MgCl2 to the cells. Centrifuge the cells at 600 rpm for 10 minutes at 4°C. Aspirate the supernatant containing dead cells and resuspend the cells in 25 ml of cold Williams E+Glutamx-TM-1 (Gibco, cat#32551, 1% penicillin-streptomycin, 10 FBS, 1% L-glutamine). Centrifuge the cells at 600 rpm for 5 minutes at 4°C. Aspirate the supernatant and resuspend the cells in 3 ml of cold Williams E+Glutamx-TM-1.

[0207] Primary hepatocyte culture : After perfusion, 1x10 6 Isolated hepatocytes were seeded in 3 ml Williams E + Glutamx-TM-1 in a 6-well plate. After four hours, the medium was aspirated, and the cells were washed with PBS and incubated overnight with 2 ml Williams E + Glutamx-TM-1. The cells were treated with 10 μM HJC0152 STAT3 inhibitor (Selleckchem # S8561) for 30 minutes, and then treated with 10 ng / ml recombinant mouse IL-6 (R & D # 406-ML-005) for 48 hours.

[0208] Histopathological and immunohistochemical staining analysis : PFA-fixed liver and lung tissues were embedded in paraffin blocks 4, 14, and 21 days after injection of 4T1 BC cells and 7, 14, and 21 days after injection of KPC PC cells. Blocks were cut into 4 μm and tissue sections were placed at 37°C overnight. H&E staining was performed according to a standard protocol, including the following steps: de-paraffinization, rehydration, hematoxylin and eosin staining, followed by dehydration. Slides were washed with xylene and mounted.

[0209] Immunofluorescence : 21 days after injection of 4T1 BC cells, livers were collected, fixed in 4% paraformaldehyde, embedded in paraffin and made into 4 μm sections. Slides were dewaxed and antigen retrieval was completed using citric acid pH=6. Nonspecific binding was blocked using 20% ​​normal horse serum (Normal Horse Serum, NHS), 0.1% Triton in PBS. Rat anti-CD45 (BioRad # MCA1031G) and mouse anti-pERK 1:100 (Sigma # M8159) were diluted in 2% NHS and 0.1% Triton and incubated overnight. The slides were then incubated for 1.5 hr with biotinylated donkey anti-rat 1:100 (Jackson Immunoresearch # 712-065-153) and HRP-conjugated goat anti-mouse 1:100 (Perkin Elmer # NEF822001EA) diluted in 2% NHS. The slides were then incubated with 1:500 OPAL 690 (Akoya Biosciences #FP1497001KT) and streptavidin Cy3 (016-160-084-Jackson immunoresearch). The slides were imaged using a Leica Mi8 microscope equipped with a motorized stage and a Leica DFC365FX camera. Single images at x20 magnification were tiled to receive a complete scan of the tumor sections. Liver sections stained with pERK were quantified using ImageJ.

[0210] RNA processing and quantitative PCR : Use QIAzol lysis reagent (according to Manual) or via Direct-zol TMRNA was extracted from liver tissue using the MiniPrep Plus kit (Zymo Research ZR-R2070). For hepatocyte RNA sequencing, RNA was extracted from dissociated hepatocytes. After liver perfusion as described above, hepatocytes were resuspended in QIAzol and frozen at -80°C. RNA was extracted using QIAzol lysis reagent. RNA was extracted from cultured primary hepatocytes using the RNeasy Mini kit (QIAGENe#74104). To assess mtDNA copy number, total DNA was isolated using a DNA purification kit (DNeasy Blood&Tissue Kits Qiagen#69504). cDNA was synthesized from 1 μg RNA using the qScript cDNA synthesis kit (Quanta#95749). cDNA detection was performed using Syber Green FAST mix Perfect CT (Quantabio#95073) and the required primers (qPCR primer list - Table 1).

[0211] Perfusion for CyTOF and FACS assays :For blood collection, after anesthesia, 10 μ l of heparin was injected into the left ventricle of mice, and about 700 μ l of blood was extracted by puncturing the right ventricle using the 27G needle of the 1 ml syringe coated with heparin, or blood was extracted via the retroorbital blood collection procedure using heparinized microhematocrit capillary tubes for immune cell separation. Ice-cold PBS was perfused into the mouse heart. These assays were carried out 14 days after 4T1 BC cells and control PBS injection and 21 days after KPC PC cells and control PBS injection.

[0212] Blood immune cell isolation : After perfusion, blood was drawn and transferred into a 15 ml tube. 5 ml of red blood cell lysis buffer (ThermoFisher, ACK lysis buffer, A104920) was added and incubated for 5-10 min at RT. The blood was then centrifuged at 300 g for 5 minutes at 4 ° C. The supernatant was aspirated and the pellet was resuspended in the remaining volume. The cells were washed with 5 ml of ice-cold FACS buffer (PBS + 2 mM EDTA + 0.5% BSA or 5% FCS without Ca / Mg2) and centrifuged at 300 g for 5 minutes at 4 ° C. The supernatant was aspirated and the pellet was resuspended in the remaining volume.

[0213] Isolation of liver immune cells: After perfusion, remove the liver and transfer it to a culture dish. Then chop the liver into approximately 1 mm pieces. Add 3 ml of DMEM-F12 (ThermoFisher, 31330038) to a 15 ml tube on ice. Add 3 ml of collagenase (Worthington, LS004188) mixture (1 mg / ml collagenase IV + 0.2 mg / ml DNAse I + 20% FBS in DMEM / gF12) and incubate the tissue homogenate at 37°C for 60 minutes - shake at 250 rpm and vortex briefly every 15 minutes. Filter the cell suspension into a 50 ml tube with a 40um filter and wash with 20 ml of ice-cold FACS buffer. Then centrifuge the cells at 600 g for 5 minutes at 4°C. Aspirate the supernatant and resuspend the pellet in 5 ml of red blood cell lysis buffer. At the end of the incubation, add 15 ml of ice-cold FACS buffer and centrifuge the sample at 600 g for 5 minutes at 4°C. Leukocyte enrichment was performed according to a Percoll gradient. Isotonic Percoll (9 parts Percoll, 1 part sterile 10x PBS), 80% Percoll (8 parts isotonic Percoll, 2 parts 1x PBS) and 40% Percoll (5 parts 80% Percoll, 5 parts DMEM-F12) solutions were prepared. The pellet was resuspended in 8 ml of 40% Percoll solution and carefully transferred into 15 ml of a solution containing 5 ml 80% Percoll. The cells were centrifuged at 1500 g for 30 minutes at 4°C (acceleration 5 / brake 0). The middle layer containing immune cells was collected and transferred into a new 15 ml tube containing 5 ml of ice-cold PBS buffer. The volume was uniformly made up to 10 ml with ice-cold PBS and the cells were centrifuged at 600 g for 5 minutes at 4°C. The supernatant was aspirated and the pellet was resuspended in the remaining volume.

[0214] Isolation of spleen immune cells : After perfusion, the spleen was squeezed with a 70um filter and filtered with 10 ml FACS buffer. The cells were centrifuged at 400g for 5 minutes at 4°C. The supernatant was aspirated and the cells were resuspended in 1 ml of red blood cell lysis buffer. After incubation at RT for 5 minutes, 10 ml of ice-cold PBS was added and the cells were centrifuged at 300g for 5 minutes at 4°C. The supernatant was aspirated and the pellet was resuspended in the remaining volume.

[0215] Bone marrow immune cell isolation: After perfusion, the femur was dislocated and moved into a culture dish containing ice-cold PBS. The condyle, patella and epiphysis were removed to expose the metaphysis. The bone marrow (BM) was flushed with 2 ml of culture medium on a 70um filter. The BM was broken up with a syringe plunger and the filter was washed with 10 ml of RPMI+10% FBS+2mM EDTA. The cells were centrifuged at 400g for 5 minutes at 4°C. The supernatant was aspirated and the pellet was resuspended in 1 ml of red blood cell lysis buffer and placed at RT for 5 minutes. 10 ml of RPMI+10% FBS+2mM EDTA was added and centrifuged at 400g for 5 minutes at RT.

[0216] Flow cytometry Immune cells were washed with ice-cold PBS and then assayed with LIVE / DEAD according to the manufacturer's instructions. TM Fixable Aqua Dead (Thermo Fisher) staining. After Fc blocking (Biolegend, BLG-101320), cells were stained for surface antigens. Flow cytometry data were acquired on CytoFLEX (Beckman Coulter) and analyzed using FlowJo software. In each experiment, relevant negative, single staining and fluorescence minus one controls were used to identify the population of interest.

[0217] Mass cytometry : 3x 10 per liver sample 6 Cells, as well as all blood-derived immune cells, were stained for mass cytometry analysis. Cisplatin viability staining was used prior to barcoding samples with palladium metal isotopes. Briefly, individual samples were incubated with TruStain FcX TM (BioLegend) for 30 min, followed by staining with a panel of antibodies (CyTOF antibody list - Supplementary Table S6) at room temperature (RT) with 5 ml of The cells were washed with the cell staining buffer, fixed with Fix I buffer and permeabilized with Barcode Perm buffer. The samples were then incubated with their respective barcodes at RT for 45 min, after which they were The samples were washed with Barcode Perm buffer and combined into a composite sample. After washing, the mixed samples were incubated with 4% formaldehyde at 4°C overnight. TM II Prior to acquisition in the system, samples were stained with iridium to detect cells and washed with cell staining buffer and mass cytometry grade water. The multidimensional datasets were analyzed using the Cytobank cloud-based platform, FlowJo software (Tree Star, Inc) and R (R Core Team, 2017).

[0218] Algorithm-based high-dimensional analysis :Mass spectrometry flow cytometry data were normalized and debarcoded using Fluidigm CyTOF software version 6.7. Single samples were manually gated using Cytobank to exclude normalization beads, cell debris, and dead cells. Only CD45+ cells were used for downstream analysis. All analyses of CyTOF data were performed after inverse hyperbolic sine (cofactor = 5) transformation of marker expression. Clustering, data visualization, and dimensionality reduction (UMAP) were performed using the CyTOF workflow package. All graphs were drawn using ggplot2 or GraphPad Prism (version 8.0.1).

[0219] L-Glutamine-15N2 Infusion : Isotope infusion experiments were performed x weeks after orthotropic tumor inoculation. Mice were fasted for 4 hours and then infused with L-glutamine-15N2 (Sigma) for 5 hours. An infusion solution containing 1.725 grams of L-glutamine-15N2 per kg body weight was prepared in saline. Mice were anesthetized on a heating pad and a catheter connected to the infusion solution was inserted into the caudal vein. Initially, each mouse was infused with a bolus of 150 μl / min for 1 min, followed by a continuous infusion of 2.5 μl / min for 5 hours. Throughout the infusion, mice were kept awake in separate infusion cages. At the end of the infusion, mice were anesthetized and blood was collected into heparin tubes. Organs were harvested and snap-frozen in liquid nitrogen.

[0220] Gas chromatography-mass spectrometry (GC-MS) : Plasma was collected from blood samples by centrifugation at 1000 rcf for 15 minutes at 4°C. 20 μl of plasma was resuspended in an ice-cold MeOH / H2O mixture (mixed with Ribitol at 8:1), incubated on ice for 20 minutes and centrifuged at 15,000 rpm for 10 minutes. The supernatant was vacuum dried overnight, and the dried sample was incubated with 20 μl of methoxyamine hydrochloride solution (20 mg ml-1, dissolved in pyridine) at 37°C for 90 min, followed by incubation at 60°C for 60 minutes with 40 ul of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (Sigma) containing 1% tert-butyldimethylchlorosilane.

[0221] About 5-25 mg of tissue samples were vacuum dried and powdered using two 3.2 mm stainless steel balls on a cryomill (Retscht). The powdered tissue was resuspended and sonicated for 20 minutes with methanol and ribitol as internal standards. Polar metabolites were extracted after adding 1 volume of water and 0.5 volume of chloroform. The mixture was vortexed and centrifuged at 15,000 RPM for 15 minutes at 4°C. The supernatant of the sample was dried overnight, then derivatized (dervitization) with 40 ul methoxyamine hydrochloride solution (20 mg ml-1, dissolved in pyridine) at 37°C for 90 min while shaking, and then incubated with 70 ml N, O-bis (trimethylsilyl) trifluoroacetamide (Sigma) at 37°C for another 30 min.

[0222] 1ul sample was injected in splitless or 1:25 split mode, the inlet temperature was 270°C, the GC oven was kept at 100°C for 3min, then heated to 300°C at a gradient of 3°C min-1, and then operated at 315°C for 5min. The MS system was operated at 70eV electron impact ionization and scanned the mass range of 100–650amu. The resulting chromatograms were analyzed with MassHunter software (Agilent Technologies). IsoCor software was used to correct the isotopologue distribution of metabolites for naturally occurring isotopes.

[0223] Amino Acid Analyzer Frozen liver samples were lyophilized and ground into powder, extracted with 50% methanol, homogenized in a bullet blunder after sonication in ice-cold water for 10 minutes. The samples were centrifuged at 15,000×g for 15 min and lyophilized again as described in the GCMS section. The samples were resuspended in lithium loading buffer (Biochrom) and proteins were precipitated by adding (v / v) cold 5% 5-sulfosalicylic acid (SSA) solution - supplemented with 500μmol / L norleucine as internal standard. After centrifugation at 15,000×g for 15 min, the mixture was incubated at 4°C for 30 min. The supernatant was filtered through a 0.22μm size filter and then injected into a Biochrom 30 series amino acid analyzer (Biochrom Ltd., Cambridge Science Park, England) with a lithium accelerated cation exchange column (200X 4.6). A mixture of amino acids of known concentrations (Calibration standards, Biochrom) was supplemented with glutamine and used as standards. Amino acids were post-column derivatized with ninhydrin reagent and absorbance was measured at 440 nm (proline and hydroxyproline) or 570 nm (all other amino acids).

[0224] Extraction of polar metabolites from urine and plasma : To extract polar metabolites from urine (20-100uL) samples, 1mL of methanol (with labeled amino acids as internal standards) was added to each Eppendorf tube containing the biological sample. The resulting mixture was then vortexed and sonicated for 15min, vortexed again, and centrifuged at 14000rpm for 10min. The liquid phase was transferred to a new tube and lyophilized. The precipitate was then dissolved using 150uL DDW-methanol (1:1), centrifuged twice to remove possible precipitates, and injected into the LC-MS system.

[0225] LC-MS polar metabolite analysis : Briefly, the analysis was performed using an Acquity I class UPLC system in conjunction with a mass spectrometer (Thermo Exactive Plus Orbitrap) operating in negative ionization mode. LC separation was performed using SeQuant Zic-pHilic (150 mm × 2.1 mm) (Merck) with a SeQuant guard column (20 mm × 2.1 mm). Mobile phase A: acetonitrile and mobile phase B: 20 ​​mM ammonium carbonate + 0.1% aqueous ammonium hydroxide. The flow rate was maintained at 200 μl min-1 and the gradient was as follows: 0-2 min 75% B, 17 min 12.5% ​​B, 17.1 min 25% B, 19 min 25% B, 19.1 min 75% B, 19 min 75% B.

[0226] Polar metabolite data analysis Data processing was performed using TraceFinder Thermo Fisher software. Detected compounds were identified by retention time and fragmentation and validated using an in-house mass spectral library. Urine metabolites were normalized to creatinine peak area.

[0227] Cytokine detection : The ProcartaPlex immunoassay (ThermoFisherProcartaPlex TM Cytokine levels were measured using IL-6 ELISA kit (ThermoFisher 88-7064-22).

[0228] CCL2 and ammonia levels : CCL2 and ammonia levels were measured by CCL2 ELISA kit (R&D systems #MJE00B) and ammonia assay kit (Abcam ab83360), respectively, according to the manufacturer's instructions.

[0229] Activation of mouse spleen T cells10-week-old WT female Balb / c mice were sacrificed and spleens were harvested into cold PBS on ice. The spleens were homogenized using a syringe plunger through a 70uM filter and washed with PBS. After centrifugation at 1200rpm for 5min, the pellet was treated with RBC lysis buffer according to the manufacturer's instructions. 2x10 6 Cells were resuspended in spleen cell culture medium (complete RPMI medium supplemented with 50 M β-mercaptoethanol, 10% sodium pyruvate and non-essential amino acids) supplemented with 6,000 IU / mL IL-2 (Chiron, rhIL2) and seeded in 24-well plates pre-coated with CD3 (BLG#100302). After 72 h, cells were collected, centrifuged at 1200 rpm for 5 min, washed with spleen cell culture medium X2, and analyzed using a CytoFLEX (Beckman Coulter) FACS analyzer.

[0230] Cancer cell proliferation : 20x10 4 4T1 cells were seeded in 100ul complete RPMI medium. The next day, cells were washed with PBS and the medium was replaced with DMEM medium without glutamine (Biological Industries #01-057-1A) supplemented with ammonia (0.75mM), aspartic acid (0.25mM), fumaric acid (0.35mM) or glutamine (0.25mM). Proliferation assays (XTT cell proliferation kit Biological Industries #20-300-1000) were used according to the manufacturer's instructions 24, 48 and 72hr after metabolite supplementation.

[0231] Measurement of respiratory chain complex activity : Enzyme activities of respiratory chain complexes were measured by standard spectrophotometric methods at 37°C. Briefly, complex I was measured as a rotenone-sensitive NADH-CoQ reductase monitoring the oxidation of NADH at 340 nm in the presence of coenzyme Q1. Complex II was measured as succinate dehydrogenase (SDH) based on succinate-mediated phenazine methylsulfate reduction of p-dichloroindophenol at 600 nm. Complex II+III was measured as succinate cytochrome c reductase following the reduction of oxidized cytochrome c at 550 nm. Complex IV (cytochrome c oxidase) was measured by following the oxidation of reduced cytochrome c at 550 nm. Citrate synthase (CS) is a ubiquitous mitochondrial matrix enzyme measured in the presence of acetyl-CoA and oxaloacetate by monitoring the release of coenzyme ASH coupled to 5′,5′-dithiobis(2-nitrobenzoic acid) at 412 nm. Protein concentration was determined by the Lowry method and calculated based on a bovine serum albumin (BSA) standard curve.

[0232] In vivo Erk inhibition : 24h after injection of 4T1 BC cells, mice were injected IP with 1 mg / kg ERK inhibitor trametinib GSK1120212 (Selleckchem #S2673) in 4% DMSO corn oil or injected with 4% DMSO corn oil alone, 6 times a week. Mice were sacrificed 8 or 14 days after tumor injection.

[0233] In vivo IL-6 inhibition : 4 days after KPC cell injection, mice were injected IP with 200 μg / mouse of IL-6Ab (InVivoMab anti-mouse IL-6 (Bio X Cell) #BE0046) or control IgG (InVivoMab rat IgG1 isotype control (anti-HRP) (Bio X Cell) #BE0088) every 2 days. Mice were sacrificed 21 days after tumor injection.

[0234] rAAV-HNF4α : Cells - Low-passage HEK293T were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2. Production of rAAV - To produce rAAV8, a triple co-transfection procedure was used to introduce rAAV vector plasmids (pAAV-CMV-mHNF4α or pAAV-CMV-GFP) together with pXR8, an AAV8 helper plasmid carrying AAV rep and cap genes, and pXX6-80, an Ad helper plasmid, at a molar ratio of 1:1:1.

[0235] In brief, HEK293T cells (linear; molecular weight [MW], 25,000) (Poly-sciences, Inc., Warrington, PA) were transfected with polyethyleneimine (PEI), and the culture medium was changed 18 h after transfection. Cells were harvested 72 h after transfection, 3 rounds of freeze-thaw were performed, and then digested with 100 U / ml Benzonase (EMD Millipore, Billerica, MA) at 37 ° C for 1 h. Viral vectors were purified by iodixanol (Serumwerk Bernburg AG, Germany) gradient ultracentrifugation, and then further concentrated and washed with phosphate buffered saline (PBS- / -) using Amicon ultra-15 100K (100,000 molecular weight cutoff, Merck Millipore, Ireland). The final concentration of rAAV8 particles was 2.78E+10 vg / μl (AAV-CMV-mHNF4α) and 2.35E+10 vg / μl (pAAV-CMV-GFP). 48 hr after cancer cell inoculation, mice were injected with 5E11 vg via the tail vein.

[0236] RNA-seq Total RNA was fragmented, followed by reverse transcription and second-strand cDNA synthesis. Double-stranded cDNA was subjected to end repair, base addition, adapter ligation, and PCR amplification to create libraries. Libraries were evaluated by Qubit and TapeStation. Sequencing libraries were constructed using barcodes to enable multiplexing of 12 samples on one lane of an Illumina HiSeq 2500V4 instrument, generating approximately 23 million single-end 60 bp reads per sample. Bioinformatics: Poly A / T stretches and Illumina adapters were trimmed from reads using cutadapt [doi: 10.14806 / ej.17.1.200]; reads shorter than 30 bp were discarded. Reads were mapped to the M. musculus reference genome GRCm38 using STAR, which was provided with gene annotations downloaded from Ensembl (options EndToEnd and outFilterMismatchNoverLmax set to 0.04). Using the above gtf, htseq-count was used to quantify the expression level of each gene. Differentially expressed genes were identified using DESeq2 with betaPrior, cooksCutoff, and independentFiltering parameters set to False. Raw P values ​​were adjusted for multiple testing using the Benjamini and Hochberg procedure. This pipeline was built using snakemake.

[0237] Principal component analysis (using R Stats package) was performed on the variance-stabilized transformed values ​​of DESeq2 for the 1,000 most variable genes. The figure depicts the first principal component vs. the second principal component in a scatter plot. The first PC explained 73% and the second PC explained 9% of the variance in the data. A total of 2,829 genes that were significant in any comparison (4T1 mammary mice and CTRL WT mice at day 4 and day 21) were analyzed and shown in heat maps. A gene was considered significant if its absolute fold change was above 1.5, the FDR was below 0.05, and the gene accounted for at least 30 in at least one of the samples. The log 2 normalized counts were normalized so that each gene had a zero mean and unit standard deviation. The number of clusters was estimated using Gap Statistics. K-means clustering was performed on the normalized values. The expression profiles are accompanied by colored bars indicating the normalized log 2 normalized counts. For pathway enrichment analysis, we used QIAGEN's Pathway analysis. Identification of genes regulated by HNF4α-Differentially expressed genes between day 21 and day 4 4T1 hepatocytes were calculated and normalized to control mice at both time points (|logFC|>=1.5 and FDR<0.05). 1914 genes were upregulated at the day 21 time point compared to day 4, and 514 genes were downregulated at this time point. The list of downregulated genes was crossed with the list of target genes of the HNF4α transcription factor from the Harmonizome tool (https: / / maayanlab.cloud / Harmonizome / gene_set / HNF4A / ENCODE+Transcription+Factor+Targets), which was constructed using a ChIP-seq dataset from the ENCODE transcription factor target dataset. The final list contained 149 genes. The RNA-Seq data discussed in this disclosure have been deposited in NCBI's Gene Expression Omnibus and can be accessed through GEO Series Accession No. GSE 212113 (ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE212113).

[0238] Single-cell RNA-seq using the Chromium10x Genomics Platform: For NPC enrichment of the liver, the livers of mice injected with PBS or 4T1 BC cells 4 and 21 days after perfusion were as described above. After centrifugation for 3 min, 30 g of supernatant was collected and centrifuged at 300 g for 5 min. Cell pellets were treated with red blood cell lysis buffer (ThermoFisher, ACK Lysing Buffer # A104920) according to the manufacturer's instructions. Single cell RNA-seq libraries were prepared using the Chromium Single Cell RNA-seq platform (10x Genomics). Cells were counted and diluted to a final concentration in PBS supplemented with 0.04% BSA. The cell suspension was loaded onto the Next GEM Chip G targeting liver non-parenchymal cells and then run on the Chromium Controller to generate GEM emulsions (10x Genomics). Single cell 3'RNA-seq libraries were generated according to the manufacturer's protocol (10x Genomics Chromium Single Cell 3'Reagent Kit User Guide v3Chemistry). The final library was quantified using the NEB Next Library Quant Kit for Illumina (NEB) and the high-sensitivity D1000 TapeStation (Agilent). The libraries were merged according to the number of cells targeted, with a goal of approximately 50,000 reads per cell. The merged libraries were sequenced on a NovaSeq 6000 instrument using the SP100 cycle kit (Illumina). The scRNA-Seq data discussed in this disclosure have been deposited in the NCBI's gene expression database and can be accessed through the GEO series accession number GSE223835 (ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE223835).

[0239] Single-cell RNA analysis: Metacell pipeline - The metacell pipeline 36 was used to derive informative genes and calculate inter-cell similarities, calculate k-NN graph coverage and derive the distribution of RNA in cohesive groups of cells (or metacells), and derive strongly separated clusters using self-citation analysis and graph coverage calculations on resampled data. We removed specific mitochondrial genes, immunoglobulin genes, and genes associated with poorly supported transcriptional models (annotated with the prefix "Rp-"). Gene features were selected using a normalized variable / mean (var / mean) threshold (Tvm) = 0.3 and a minimum total UMI count > 50. We then hierarchically clustered the correlation matrix between metacells and grouped them into clusters representing cell types and states. We used K = 100,500 self-citation iterations and other standard parameters. Neutrophil subpopulation analysis - Subpopulations of neutrophils were identified based on maturation scores and chemotaxis scores as well as granule identification. Pathway analysis – GO - To assess DEG enriched pathways, we used GO gene ontology for genes regulated by HNF4α in the differentially upregulated genes at day 21 compared with day 0 in macrophages and in the differentially downregulated genes between day 21 and day 4 in hepatocytes37,38,39. Statistical analysis - Differential gene expression analysis was performed using the Mann-Whitney U test with FDR correction on UMIs divided by the median UMI count.

[0240] Human Data from Clalit Healthcare: The analysis of data from the Clalit Healthcare environment was approved by Helsinki No. 195-17COM2. Breast Cancer Cohort Definition-Female patients diagnosed with breast cancer between 2002 and 2019 (all stages, right and left breast, all quadrants, all histological subtypes) were included in the breast cancer cohort. Cohort 1: Breast cancer patients with a survival of less than 2 years from diagnosis, N=4732. Cohort 2: Breast cancer patients with a survival of 2 to 5 years from diagnosis, N=4086. Cohort 3: Breast cancer patients with a survival of 5 to 10 years from diagnosis, N=3984. Cohort 4: Breast cancer patients with a CBC test indicating a neutrophil percentage higher than 80% or a lymphocyte percentage lower than 10% within one year of diagnosis (-365 days before diagnosis to 365 days after diagnosis). N=10556. Cohort 5: Breast cancer patients with CBC test indicating neutrophil percentage less than 80% and lymphocyte percentage greater than 10% within one year of diagnosis (-365 days before diagnosis to 365 days after diagnosis), N=35723. Pancreatic cancer cohort definition-Male patients diagnosed with pancreatic cancer between 2002 and 2019 (all stages, all locations in the pancreas, all histological subtypes) were included in the pancreatic cancer cohort. Cohort 1: Pancreatic cancer patients with a survival of less than half a year since diagnosis, N=2037. Cohort 2: Pancreatic cancer patients with a survival of half a year to 1 year since diagnosis, N=659. Cohort 3: Pancreatic cancer patients with a survival of 1-1.5 years since diagnosis, N=342. Cohort 4: Pancreatic cancer patients with CBC test indicating neutrophil percentage greater than 80% or lymphocyte percentage less than 10% within one year of diagnosis (-365 days before diagnosis to 365 days after diagnosis). N=4238. Cohort 5: Pancreatic cancer patients with CBC test indicating neutrophil percentage less than 80% and lymphocyte percentage greater than 10% within one year after diagnosis (-365 days before diagnosis to 365 days after diagnosis), N=4218. Liver function laboratory data - For cohorts 1-3 of each cancer type (if available), laboratory test results of selected variables at 2 time points (one year before diagnosis and at diagnosis) were obtained for each patient aged 60-70 years. Liver function variables: AST (aspartate aminotransferase), ALT (alanine aminotransferase), albumin, ALK-P (alkaline phosphatase), LDH (lactate dehydrogenase), PT-SEC. Survival analysis - For cohorts 4-5 of each cancer type, the pct of patients alive at the end of each year was calculated (0=diagnosis time) and survival curves were created - that is, XY plots of the % of patients alive each year.

[0241] Liver function scores in pancreatic patient data: Data on participant demographics, previous surgical procedures, blood test values, and survival at Sheba and Souraski Medical Center were extracted from IRB-approved patient medical records (4474&5073-18&0551-17-TLV). Written informed consent was obtained from all patients before study enrollment. The protocol was approved by the Institutional Review Board of Sheba and Sourasky Medical Center, and the study was conducted in accordance with the Good Clinical Practice guidelines and the Declaration of Helsinki. Pancreatic cancer patients with liver metastases or ascites were excluded for non-liver cancer data analysis. Souraski: N=732 (N=255 in the resectable group, N=362 in the LA group and MTX group). Sheba: N=252 (N=82 in the 1+2 stage, N=170 in the 3+4 stage). Then, for each sample, we determined its liver function score, which is the weighted sum of the normalized expression of 5 liver enzymes and function-based molecules, normalized to the mean and std in pancreatic cancer patients (derived from this cohort study), and weights defined based on the correlation of each value with survival in a small random cohort (50 patients), i.e., 2*|ALT(IU / I)-25.5|19.5+2*|AST(IU / I)-25|15+5*|INR-1.025|0.175+|ALKP(IU / I)-79.5|35.5+7*|4.46-Albumin(g / dL)|0.8617, where the names of the genes represent their laboratory result values. The cutoff value separating high scores from low scores was set to cutoff value = 0.6, with approximately 66% of patients achieving high scores and 33% of patients achieving low scores. For patients with high and low scores, Kaplan-Meier survival curves were drawn for patients of all cancer stages (stage at diagnosis) and stratified by stage.

[0242] Weight reduction analysis : For weight loss analysis, we excluded patients who underwent Whipple or distal pancreatectomy and followed up patients who had at least 2 weight measurements during the course of their disease. N = 369. The correlation between liver enzyme scores and BMI change was evaluated by linear regression analysis controlling for tumor stage and patient age. BMI change, ΔBMI was defined as (second BMI-first BMI) / first BMI. Linear regression followed the following form, ΔBMI = score + stage + age, where score represents liver enzyme score.

[0243] Statistical analysis:Unless otherwise specified, all statistical analyses were performed using two-way ANOVA, Student's t test or multiple or two-group Wilcoxon rank sum test, with Dunnett correction when necessary. Sample size was pre-selected according to the common practice of the described experiment and each experiment was mentioned. Unless otherwise specified, each experiment was performed using biological replicates and technical replicates and repeated at least three times. Results are presented in the form of mean ± standard deviation. All error bars represent standard deviation. All analyses considered p<0.05 to be significant (* represents p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001).

[0244] Data availability : Due to privacy regulations, all data analysis was performed on a secure, authenticated, dedicated server within the Clalit Healthcare environment.

[0245] Example 1: Non-liver cancers induce early metabolic changes in the liver during carcinogenesis

[0246] To assess changes in liver metabolism during carcinogenesis in non-liver cancers, we used an orthotopic 4T1-luciferase breast cancer (BC) model that rarely metastasizes to the liver. Using this approach, we were able to distinguish changes in liver metabolism induced by the primary tumor from those triggered by metastasis. To control for potential side effects resulting from orthotopic injection of cancer cells, we injected mice with sham saline and, in addition, used MMTV-PyMT mice, an intrinsic BC genetic model. We analyzed UC enzymes and intermediates as a readout of host liver-specific metabolic changes during the first 3 weeks of carcinogenesis. We excluded arginase I from our analysis to accurately distinguish UC from NO metabolism. In addition, a board-certified pathologist analyzed and excluded the presence of liver metastases at all experimental time points.

[0247] In the 4T1 BC model, we found reduced expression of UC enzymes in the host liver starting as early as day 4 after orthotopic injection. The reduction in UC enzyme expression was dynamic over the course of 3 weeks, with both the magnitude of the reduction and the number of enzymes involved increasing ( Figure 1A-1B Interestingly, the genetic MMTV-PyMT BC model also had reduced expression levels of UC enzymes compared to controls, thus ruling out the possibility that injections led to changes in UC expression levels ( Figure 1C To verify the significance of UC expression results on UC function, we used 1 15N2-labeled glutamine was infused in vivo and the ratio of labeled urea to glutamine and glutamate was measured. We found that both ratios were decreased in the plasma of mice bearing breast cancer and, in addition, in the liver, when total glutamate was elevated, the ratio of labeled urea to glutamate was decreased in 4T1 mice, confirming UC dysfunction ( Figure 1D ). In addition, we measured UC-related upstream substrates, glutamate and aspartate, and downstream metabolites, fumarate, in plasma and liver of 4T1. As expected for dysfunctional UC, we found elevated levels of UC substrates and decreased levels of the UC product fumarate ( Figure 1E We further found elevated plasma ammonia levels and decreased urine urea levels, confirming UC failing ( Figure 1F ). It is worth noting that although 15 The level of N2-labeled aspartate was lower than that in plasma and liver, but the proportion of downstream metabolites synthesized from aspartate, namely labeled uracil, was higher in tumors, indicating that aspartate is used to synthesize nucleotides ( Figure 1G Indeed, high levels of UC substrates and reduced levels of UC products resulting from UC failure in the host liver directly enhance cancer cell proliferation ( Figure 1H Interestingly, ex vivo ammonia supplementation reduced the survival and activation of T cell splenocytes isolated from wild-type (WT) mice ( Fig. 1I ). Therefore, during carcinogenesis, rewiring of liver UC may benefit cancer growth by increasing substrate availability for tumor growth and by reducing systemic immune responses.

[0248] To broadly assess whether cancer in vivo affects liver-specific metabolic pathways other than UC, we performed RNA sequencing on hepatocytes isolated from perfused livers of WT and 4T1 cancer-bearing mice at days 4 and 21 after cancer cell injection. Confirming our previous findings, we found reduced expression of the UC enzymes argininosuccinate synthetase (ASS1), ornithine transcarbamylase (OTC), and carbamyl phosphate synthetase I (CPS1) at day 21, with a significant and unique disease-specific gene expression signature ( Figure 1J Within this signature, we found reduced expression of multiple other enzymes involved in central metabolic pathways, supporting a general reprogramming of hepatocyte metabolism during carcinogenesis ( Figure 1K ). Further analysis of hepatocyte RNAseq showed that not all liver genes had altered expression, supporting the specificity of metabolic gene changes. Interestingly, widespread changes in liver metabolism were associated with significant increases in AST levels ( Figure 1L ).

[0249] Since several of the metabolic pathways we found to be altered, including UC, depend on fully functioning mitochondria, we evaluated whether mitochondrial function or number is reduced after carcinogenesis. In isolated mitochondria, we found that the activity of respiratory chain complexes, including subunits (I, II-III, IV) encoded by mitochondrial DNA (mtDNA), was reduced relative to the fully nuclear-encoded succinate dehydrogenase (SDH, complex II). This finding was corroborated by the reduced mtDNA levels observed in liver of BC-bearing mice. To further understand the perturbations in mitochondrial metabolism, we analyzed the protein and RNA levels of mitochondrial transcription factor A (TFAM), which regulates mtDNA levels and transcription, and found that it was significantly downregulated in the liver of BC-bearing mice. However, the total mitochondrial population, as estimated by citrate synthase activity in liver homogenates, was unchanged. Therefore, it is likely that transcriptional changes, rather than mtDNA depletion, account for the observed metabolic changes.

[0250] Together, these data suggest that in the BC mouse model, there are early transcriptional alterations in the expression of metabolic enzymes that induce global changes in liver metabolism at both the pathway and organelle levels.

[0251] Example 2: Innate immune cells infiltrate the host liver during early carcinogenesis

[0252] To understand the potential causes of this early and extensive metabolic rewiring, we first examined morphological changes in the liver after orthotopic injection of BC cells. Interestingly, we found evidence of immune cell infiltration of the liver—beginning as early as day 4 after BC cell injection and increasing along the course of disease ( Figure 2A ). FACS analysis further confirmed that the population of liver immune cells (CD45+) in the livers of BC-bearing mice gradually increased from day 0 to day 21. Notably, liver histological sections of the MMTV-PyMT genetic BC model also showed immune cell infiltration compared with the livers of WT mice, which supports that the liver immune infiltration was not caused by the injection reaction.

[0253] To identify which immune cells infiltrate the liver, we performed single-cell RNA sequencing (scRNA) analysis of non-hepatic parenchymal cells in the liver. We complemented these results at the protein level by Cytometry Time Of Flight (CyTOF). Using both techniques, we found a significant decrease in lymphocyte infiltration and an increase in innate immune cells infiltrating the liver, namely neutrophils and monocytes ( Figure 2B-Figure 2D ). Due to high sensitivity to elevated ammonia levels ( Figure 2E ), hepatic lymphopenia may be caused by blood lymphopenia, which may be sensitive to elevated ammonia levels ( Fig. 1I). In contrast, increased infiltration of innate immune cells into the liver has been described during the formation of pre-metastatic niches and could also be part of extramedullary hematopoiesis of inflammatory origin. We exclude the option of extramedullary hematopoiesis because we did not find erythrocytes, megakaryocytes, and young granulocytes in the liver. Instead, we found a significant increase in the number of mature neutrophils (CXCR2+) and a decrease in CXCR2- neutrophils. Moreover, FACS analysis excluded bone marrow failure and demonstrated extensive maturation of neutrophils, associated with a significant increase in plasma levels of granulocyte colony-stimulating factor (G-CSF).

[0254] It is possible that liver infiltration of immune cells during carcinogenesis increases the susceptibility of the liver to late metastasis formation. Nevertheless, our data suggest that liver involvement at this early stage is more likely part of a systemic inflammatory response.

[0255] Our scRNA data demonstrated that neutrophils in the liver could be clustered into four subgroups based on significant differences in gene expression along the time course of carcinogenesis. Based on high expression levels of maturation and chemotaxis genes, granule genes, and elevated inflammatory marker IL1β, neutrophil subpopulation 1 present in the liver at day 4 likely represents immature neutrophils. Three weeks after carcinogenesis, we found a large accumulation of more mature neutrophils (subpopulations 2-4) in the liver. In addition, measurement of cytokine levels in the plasma of BC-bearing mice showed a significant increase in IL-6 and TNF-α, supporting the notion that tumorigenesis induces a systemic inflammatory response involving the liver ( Figure 2F ). Since it is often challenging to detect direct increases in RNA levels of secreted cytokines in scRNA from in vivo samples, we analyzed the data for RNA increases of their downstream targets. Encouragingly, we found that ANXA1 expression in macrophages, known to be driven by IL-6, was significantly elevated at day 21.

[0256] The chemokine CCL2 and its major receptor CCR2 have been implicated in the pathogenesis of inflammation and cancer. Indeed, we found that CCL2 levels were elevated in the liver, plasma, and spleen of BC mice during the first week after cancer cell injection, whereas levels were decreased in the lung ( Figure 2G ). Notably, although the elevated plasma CCL2 levels in BC-bearing mice remained unchanged compared with healthy mice, the relative elevation of CCL2 in the liver of BC-bearing mice continued to increase during cancer progression ( Figure 2H In addition, we found an increase in CCR2+ cells in the monocytes infiltrating the liver and upregulation of genes involved in migration pathways ( Fig.2I ).

[0257] Thus, following carcinogenesis, there is an early induction of a systemic immune response, in which immune tissues such as the liver secrete elevated levels of CCL2, leading to the infiltration of immune cells into different organs.

[0258] Example 3: pERK activation in myeloid cells leads to HNF4α depletion from hepatocytes

[0259] In addition to the alterations we found in metabolic pathways, our bulk RNA sequencing analysis of isolated hepatocytes from BC mice also revealed upregulation of signaling pathways ( Figure 3A ). IL-6, NFκβ, and ERK—which we found elevated in our scRNA pathway analysis—have been described previously as participating together as signaling pathways. In addition, IL-6 has also been shown to downregulate TFAM, and we found its levels to be elevated in the plasma of 4T1 mice ( Figure 2F ). Therefore, we further evaluated the activation status of the ERK-IL-6 pathway in the liver. Immunostaining and FACS analysis of phosphorylated ERK (pERK) and CD45+ cells in the liver of BC-bearing mice revealed increased co-staining of pERK and CD45+ in infiltrating liver cells ( Figure 3B-Figure 3C ). Notably, we did not find pERK activation in blood cells of BC mice, suggesting that activation occurs in the liver ( Figure 3C ).

[0260] After integrins interact with other cells, they can induce pERK activation. Therefore, we performed ligand-receptor interaction analysis on scRNA data of immune cells infiltrating the liver, while bulk RNA sequencing of hepatocytes was performed. We found support for the intercellular crosstalk between immune cells and hepatocytes through integrins and their receptors that stabilize their liver localization ( Figure 3D-Figure 3E ). Activated ERK has been shown to lead to IL-6 secretion from myeloid cells, with multiple effects on other cells, one of which is the concomitant increase in pSTAT3 levels. Elevated pSTAT3 by mir-24 leads to downregulation of HNF4α, a master regulator of liver metabolism. Indeed, we found significantly elevated levels of pSTAT3 and miR-24 in the livers of BC tumor-bearing mice, along with complete depletion of HNF4α at both the RNA and protein levels ( Figure 3F-3H ). Notably, supplementation of primary hepatocytes with IL-6 increased CCL2 RNA levels ( Fig. 3I ). Thus, cancer-induced inflammation recruits innate immune cells to the liver, at least in part, via CCL2. Subsequent immune activation and IL-6 secretion activate pSTAT3 in hepatocytes, leading to HNF4α depletion and liver metabolic perturbations.

[0261] Example 4: HNF4α depletion disrupts liver metabolism during extrahepatic carcinogenesis

[0262] Further analysis of RNA sequencing data from BC mouse livers confirmed that many of the genes we found to be dysregulated and contributing to the perturbed metabolic and signaling pathways are indeed regulated by HNF4α ( Figure 4A Since albumin is an established downstream target of HNF4α, we confirmed that it was decreased in the liver and plasma of 4T1 mice at both the RNA and protein levels ( Figure 4B ).

[0263] To verify the causal relationship between the signaling cascade initiated by immune cells and the subsequent metabolic changes in the liver of cancer-bearing mice, we measured the expression levels of the UC enzyme OTC, a known HNF4α target gene, in isolated primary hepatocytes. We found that IL-6 supplementation reduced OTC expression levels, while the STAT3 inhibitor HJCO152 was able to rescue this effect ( Figure 4C-4D Despite the potential oncolytic effects of trametinib (a MEK-ERK inhibitor), treatment of 4T1 mice with trametinib maintained expression levels of HNF4α and hepatic UC enzymes and reduced tumor size, further supporting the proposed signaling cascade ( Figure 4E-4F ).

[0264] Finally, re-expression of HNF4α in BC mice by viral transduction increased hepatic HNF4α levels, reversed changes in UC enzyme expression, and limited BC tumor growth ( Figure 4G-4H ). Furthermore, tumors from BC mice treated with HNF4α had lower PCNA levels and lower pCAD, suggesting that preserving liver metabolism by re-expressing HNF4α reduces tumor growth by limiting proliferation ( Fig. 4I ). HNF4α injection reduced glutamate and aspartate levels in the liver, plasma, and tumors, suggesting that preserved hepatic UC metabolism limits tumor growth. Notably, HNF4α-AAV injection increased viral HNF4α expression in the liver but did not elevate viral HNF4α levels in tumors, supporting the liver specificity of AAV-HNF4 ( Figure 4J ). Thus, pERK-activated immune cells infiltrating the liver secrete IL-6, which activates pSTAT3 in hepatocytes, leading to HNF4α depletion and subsequent metabolic perturbations in the liver of BC-bearing mice. Importantly, re-expression of HNF4α limited changes in liver metabolism.

[0265] Example 5: Similar liver metabolic changes in pancreatic cancer (PC) mice via HNF4α

[0266] To evaluate our results in another mouse model of cancer, we used the KrasG12D / Trp53R172H / Pdx-1-Cre pancreatic cancer (KPC) orthotopic mouse. This mouse is also a known model of cancer-induced cachexia. Encouragingly, we found that the early metabolic findings demonstrated in the 4T1 BC model occurred in the pancreatic cancer (PC) model. Indeed, we found that in the PC model, the levels of OTC, a direct target of HNF4α, were significantly reduced already in the first week after injection of KPC cells, as well as increased UC substrates and decreased UC product levels at day 21 ( Figure 5A-Figure 5B In addition, we found that in PC mice, the infiltration of immune cells into the liver gradually increased during the 3 weeks before tumorigenesis ( Figure 5C ). FACS analysis confirmed that the immune cells infiltrating the liver were mainly innate immune and were associated with an increase in CCR2+ monocytes and lymphocyte depletion ( Figure 5D ). Consistent with our findings in the BC model, our results in the KPC model were associated with increased IL-6 plasma levels, increased pSTAT3 protein levels, decreased HNF4α RNA levels, and a consequent decrease in albumin levels ( Figure 5E-Figure 5H In further support, treatment of KPC mice with anti-IL-6 antibodies rescued albumin levels ( Fig.5I ). Notably, while the weight of KPC mice did not change ( Figure 5J ) and when liver metastases were excluded by a certified pathologist. Thus, the KPC mouse model demonstrates changes in liver metabolism similar to those we describe in the BC mouse model.

[0267] Example 6: CCR2 KO and HNF4α re-expression rescue early metabolic changes in the liver during carcinogenesis in PC mice

[0268] To further confirm that CCL2 drives immune infiltration into the liver and causes the metabolic changes we found, we orthotopically injected KPC cells into C57 / Bl6 wild-type and CCR2 - / - Knockout mice do not express the receptor for CCL2 and are unable to recruit CCL2+ immune cells. + / + Compared with mice, we found that KPC CCR2 - / - Mice developed PC but did not show infiltration of the liver by immune cells ( Fig. 6A ). Therefore, we found that PC CCR2 - / - The plasma IL-6 level in mice was reduced ( Figure 6B ), while the expression levels of UC enzyme, albumin and HNF4α were preserved ( Figure 6C). It is worth noting that CCR2 - / - The mice had reduced levels of UC substrates in the liver and did not develop systemic metabolic manifestations associated with carcinogenesis, such as weight loss and changes in body composition, associated with preserved albumin levels. Importantly, tumor growth was significantly reduced in CCR2- / - mice ( Figure 6D-6F ).

[0269] Finally, to evaluate the potential therapeutic relevance of our findings, we injected AAV8-HNF4α virus into KPC mice. AAV8-HNF4α virus significantly restricted PC tumor growth ( Figure 6G ) and improve survival ( Figure 6H ).

[0270] Notably, overexpression of HNF4α also reduced CAC phenotypes such as weight loss ( Fig.6I ), reduced fat loss ( Figure 6J ) and limit the accumulation of body fluids ( Figure 6J )——This was confirmed by body composition analysis conducted by NMR (5K-5M).

[0271] Thus, maintaining HNF4α levels in the liver by preventing CCL2+ immune cell infiltration or by re-expressing HNF4α mitigated systemic manifestations of tumorigenesis, such as weight loss and changes in body composition.

[0272] Example 7: Biochemical liver scores based on conventional tests can predict outcomes in BC and PDAC patients, including cachexia

[0273] To understand the translational relevance of our findings on liver metabolism in cancer patients, we analyzed the Clalit health maintenance organization (HMO) dataset, which contains 18 years of digital health data from 5 million Israeli subjects. We found that patients with non-metastatic BC and pancreatic ductal adenocarcinoma (PDAC) with abnormal liver parameters on the day of diagnosis had shorter survival than those with normal liver parameters ( Fig. 7A , Fig. 7E ). Notably, some of these liver changes (such as LDH and ALP) were clearly detectable in the plasma of BC patients even one year before diagnosis and predicted outcome ( Figure 7F ).

[0274] In addition, we specifically analyzed a dataset of PDAC patients from two independent medical centers in Israel, Sheba and Souraski Medical Center (two of the largest oncology centers in Israel). We first confirmed that the performance of this cohort was consistent with that published in the literature and showed a correlation between shortened survival and weight loss ( Figure 7G Similar to our previous score for UC enzyme expression (see Lee et al., "Urea cycle dysregulation generates clinically relevant genomic and biochemical signatures", Cell. 2018 Sep 6; 174(6): 1559–1570.e22, which is incorporated herein by reference in its entirety), we now developed a "liver score" - which is based on measured levels of liver biochemical and functional parameters (AST, ALT, ALP, albumin, INR) - and trained it with a small dataset ( Figure 7H We next tested the potential of our score to correlate with survival in the Clalit and these hospital datasets. We found that in all three cohorts, our “liver score” was associated with shortened survival in PDAC patients ( Figure 7B Interestingly, liver score was not associated with disease stage but was associated with weight loss ( Figure 7C-D , Fig.7I ).

[0275] Depletion of HNF4α can trigger a cascade of events that leads to muscle protein breakdown in CAC by reducing albumin levels. Analysis of data from Sheba Medical Center and Souraski Medical Center showed that changes in BMI in PC patients were significantly associated with liver enzyme scores after controlling for tumor stage and patient age. Therefore, the shortened survival predicted by our liver score is consistent with systemic manifestations induced by cancer development, including cachexia.

[0276] In conclusion, we demonstrate that molecular and functional systemic metabolic changes occur in the liver early in extrahepatic carcinogenesis, even before clinical manifestations. These metabolic changes are mediated by innate immune cells, leading to activation of pERK signaling and depletion of HNF4α, a master metabolic regulator, in hepatocytes. Perturbations in multiple liver metabolic pathways contribute to carcinogenesis, immune evasion, and ultimately to the development of CAC ( Figure 8 ).

[0277] Goldman et al., “Early infiltration of innate immune cells to the liver depletes HNF4αand promotes extra-hepatic carcinogenesis”, 2023, Cancer Discov. 2023 Mar 27; CD-22-1062 (which is incorporated herein by reference in its entirety) also provides further support.

[0278] Example 8. LNPs deliver HNF4α specifically to liver cells to both treat and prevent CAC

[0279] Although AAV delivery of HNF4α can prevent the development of CAC symptoms, it is not feasible to deliver AAV directly to humans. Thus, lipid nanoparticles (LNPs) are designed to deliver HNF4αmRNA to the liver. The first LNP, referred to herein as SM-LNP, has the following composition: 50mol% SM-102, 38.5mol% cholesterol, 10mol% DOPE, and 1.5mol% DMG-PEG200. The second LNP, referred to herein as H4-LNP, has the same composition as the first LNP, but uses different ionizable lipids (not SM-102). Lipids are dissolved in ethanol and LNPs are generated by ethanol injection. Ethanol is mixed with an aqueous solution (pH 5.2) containing mRNA at a ratio of about 8 (N:P) of nitrogen (in the lipid head group) to phosphoric acid (in RNA) (ratio 1:3). LNPs, which are generated with mRNA encoding from emGFP, are used to test the biodistribution of LNPs. Although both LNPs were predicted to target the liver, only SM-LNP produced fluorescence in the liver, while H4-LNP did not ( Fig.9A ). Neither LNP composition resulted in detectable expression in the spleen. SM-LNP was chosen for further experiments due to its high selectivity for the liver.

[0280] An mRNA encoding HNF4A was designed to be included in the LNP. The mouse spliced ​​mRNA coding sequence (SEQ ID NO: 2) was used, but the human sequence (SEQ ID NO: 5) can also be used. The capping domain and ribosome binding domain are included in the 5'UTR (SEQ ID NO: 7) and the polyadenylated 3'UTR (SEQ ID NO: 8). The 5'UTR was designed to achieve high expression and it is at the 5' end that the T7 RNA promoter sequence (SEQ ID NO: 46) is included, as well as a sequence including the Kozak consensus sequence of the 5'UTR from human alpha globin mRNA (HBA1). The 3'UTR (SEQ ID NO: 8) from the mitochondrial rRNA sequence was selected because it has a highly thermodynamically stable secondary structure under the LNP formation temperature conditions (ΔG = -85.3 kcal / mole - calculated using UNAFold for two-state folding). After loading the mRNA into SM-LNP, its ability to induce HNF4A protein expression in hepatocytes was tested. Cells of the THLE-2 human hepatocyte cell line were incubated with increasing concentrations of LNPs and after 16 h the cells were lysed and subjected to western blotting for HNF4A. Robust dose-dependent HNF4A expression was observed in the cells ( Fig. 9B ), indicating that the mRNA has a function in hepatocytes.

[0281] The therapeutic and preventive potential of LNPs for cachexia was tested in a PC mouse model. KPC cells were injected orthotopically into the pancreas of mice on day 0 and tumors were allowed to develop. Without intervention, by day 21, symptoms of CAC were clearly observed in mice, including weight loss, fat loss, and increased free fluid ( Figure 9C-9E , control group). In the treated group, 6.5 mcg of LNPs were administered intravenously into the tail vein on day 21, and cessation / slowdown of cachectic changes was observed already by day 24 ( Figure 9C-9E , HNF4A group). As described above, metabolic changes can be detected early in cancer progression, before cachexia develops. This allows for preventive restoration of HNF4A expression in the liver, which would preempt the development of cachexia. In the preventive group, before symptoms of cachexia could be observed (but after metabolic changes had already begun, see Figure 1A and Figure 5A ) LNP was administered on day 7. This preventive treatment completely abolished the development of cachexia symptoms, as there was no significant loss in weight, fat, or free fluid by day 25 after cancer administration ( Figure 9F-9H ). These results demonstrate that delivery of HNF4A to the liver has therapeutic and preventive efficacy.

[0282] Although the present invention has been described in conjunction with the specific embodiments of the present invention, it is apparent that various alternatives, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

1. A method for predicting a clinical outcome in a subject having cancer, wherein the cancer is a non-liver cancer, the method comprising measuring the function of a urea cycle in the subject, wherein a reduction in urea cycle function compared to urea cycle function in a healthy control indicates a worse clinical outcome compared to a subject in which the urea cycle function is not reduced, thereby predicting the clinical outcome of the subject.

2. The method of claim 1, wherein the non-liver cancer is selected from breast cancer and pancreatic cancer, excluding detectable liver metastasis or both.

3. The method according to claim 1 or 2, wherein measuring the function of the urea cycle comprises at least one of the following: a. measuring the expression of at least one urea cycle enzyme selected from the group consisting of argininosuccinate synthetase 1 (ASS1), ornithine transcarbamylase (OTC), argininosuccinate lyase (ASL), carbamyl phosphate synthetase-1 (CPS1), and ornithine translocase (ORNT1 / SLC25A15) in the liver of the subject; b. measuring the ratio of urea to glutamine or the ratio of urea to glutamate in the blood or liver of the subject; c. measuring the level of glutamate, aspartate or fumarate in the blood or liver of the subject; d. measuring ammonia levels in the subject's blood; e. measuring the urea level in the urine of the subject; f. measuring the expression of hepatocyte nuclear factor 4α (HNF4A) in the liver of the subject; g. measuring the level of at least one liver enzyme selected from the following in the blood of the subject: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALK-P / ALP) and lactate dehydrogenase (LDH); h. measuring the prothrombin time international normalized ratio (INR) of blood from the subject; and i. measuring albumin in the blood or liver of the subject.

4. The method according to claim 3, wherein a. reducing the expression of at least one urea cycle enzyme; b. The ratio of urea to glutamine or the ratio of urea to glutamate is reduced; c. Increased glutamate or aspartate; d. Fumaric acid reduction; e. Increased ammonia; f. Decrease of urea; g.HNF4A decreased; h. an elevated level of at least one liver enzyme; i. Decreased INR; j. Decreased albumin; or k. Any combination thereof indicating decreased urea cycle function in the subject.

5. The method of any one of claims 1 to 4, wherein measuring function comprises generating a liver function score, and wherein a liver function score exceeding a predetermined threshold indicates reduced urea cycle function.

6. The method of claim 5, wherein the liver function score is a weighted sum of normalized levels of AST, ALT, ALP, and albumin and INR in a blood sample from the subject.

7. The method of claim 6, wherein the score is normalized from 0 to 1, the predetermined threshold is 0.6, and wherein a score above the predetermined threshold indicates reduced urea cycle function.

8. The method of any one of claims 1 to 7, wherein the clinical outcome is development of cancer-related cachexia, and wherein reduced urea cycle function predicts an increased risk of developing the cancer-related cachexia.

9. The method according to any one of claims 5 to 7, wherein the clinical outcome is overall survival, and wherein a liver function score exceeding a predetermined threshold is indicative of a shortened overall survival.

10. A method for detecting a non-liver cancer in a subject in need thereof, the method comprising receiving a blood sample from the subject and measuring a function of a urea cycle in the subject based on the blood sample, wherein a decrease in urea cycle function compared to a urea cycle function in a healthy control indicates that the subject suffers from a non-liver cancer, thereby detecting a non-liver cancer in the subject.

11. The method of claim 10, wherein the non-liver cancer is selected from breast cancer and pancreatic cancer, excluding detectable liver metastasis, or both.

12. The method according to claim 10 or 11, wherein measuring the function of the urea cycle comprises at least one of the following: a. measuring the ratio of glutamine to glutamate in the blood sample; b. measuring the level of glutamate, aspartate or fumarate in the blood sample; c. measuring the ammonia level in the blood sample; d. measuring the level of aspartate aminotransferase (AST) in the blood sample; and e. measuring the albumin level in the blood sample.

13. The method according to claim 12, wherein a. The ratio of glutamine to glutamate decreases; b. Increased glutamate or aspartate; c. Fumaric acid reduction; d. Increased ammonia; e. Elevated AST levels; f. Decreased albumin levels; or g. Any combination thereof indicating decreased urea cycle function in the subject.

14. The method according to any one of claims 1 to 13, further comprising administering to the subject having a worse clinical outcome or determined to have non-liver cancer at least one therapeutic agent selected from the group consisting of an anti-IL6 blocking antibody, an ERK inhibitor, a STAT3 inhibitor, and an agent capable of increasing the expression of HNF4A in the liver of the subject.

15. A synthetic lipid nanoparticle (LNP) comprising mRNA encoding HNF4A encapsulated therein, wherein: a. The lipid nanoparticles include SM-102 lipids, cholesterol, DOPE and DMG-PEG; b. The mRNA comprises or consists of: a sequence of SEQ ID NO: 10 or 12 or a sequence having at least 85% identity thereto and encoding HNF4A and a poly A tail; or c. Both.

16. The synthetic LNP of claim 15, wherein the mRNA comprises a 5' cap and a poly A tail.

17. The synthetic LNP according to claim 15 or 16, wherein the mRNA encoding HNF4A comprises the mRNA coding sequence of SEQ ID NO: 2 or SEQ ID NO: 5 or a sequence encoding HNF4A having at least 85% identity thereto.

18. The synthetic LNP according to any one of claims 15 to 17, wherein the lipid nanoparticles target liver cells.

19. The synthetic LNP according to any one of claims 15 to 18, wherein the lipid nanoparticles comprise about 50 mol% SM-102, 38.5 mol% cholesterol, 10 mol% DOPE and 1.5 mol% DMG-PEG200.

20. A pharmaceutical composition comprising the synthetic LNP of any one of claims 15 to 19 and a pharmaceutically acceptable carrier, excipient or adjuvant.

21. A method of treating a non-liver cancer in a subject in need thereof, the method comprising administering to the subject an agent capable of increasing expression of HNF4A in the liver of the subject, thereby treating the non-liver cancer in the subject.

22. A method for treating or preventing cancer-related cachexia in a subject in need thereof, the method comprising administering to the subject a composition comprising at least one agent selected from the group consisting of an anti-IL6 blocking antibody, an ERK inhibitor, a STAT3 inhibitor, and an agent capable of increasing HNF4A expression in the subject's liver, thereby treating or preventing cancer-related cachexia in the subject.

23. The method of claim 21 or 22, wherein the agent comprises a nucleic acid molecule encoding the HNF4A.

24. The method of claim 23, wherein the nucleic acid molecule is contained within an adeno-associated virus (AAV).

25. The method of claim 24, wherein the nucleic acid molecule is mRNA.

26. The method of claim 25, wherein the mRNA comprises a 5' cap and a poly A tail.

27. The method of claim 25 or 26, wherein the nucleic acid molecule comprises or consists of the sequence of SEQ ID NO: 10 or 12 or a sequence comprising at least 85% identity thereto and encoding HNF4A.

28. The method of any one of claims 14 and 21 to 27, wherein the agent is a synthetic LNP of any one of claims 15 to 19.

29. The method of any one of claims 22 to 28, wherein the subject has an early stage cancer, a precancerous lesion, or is at risk of developing cancer.

30. The method of any one of claims 21 and 23 to 28, wherein the subject is determined to have the non-liver cancer by the method of any one of claims 11 to 14.

31. The method of any one of claims 22 to 29, wherein the subject is determined to be at increased risk for developing the cancer-related cachexia by the method of claim 8.

Images