WO2023230454A1 - Neurotrophic tyrosine receptor kinase inhibitors as therapeutics for liver disease - Google Patents

Abstract

The present invention discloses a method of treating or preventing a disorder associated with and/or caused by neurotrophic tyrosine receptor kinase wherein the method comprises administering at least one neurotrophic tyrosine receptor kinase inhibitor to a subject in need thereof.

Description

NEUROTROPHIC TYROSINE RECEPTOR KINASE INHIBITORS AS THERAPEUTICS FOR LIVER DISEASE

RELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/345,236, filed May 24, 2022, the content of which is hereby incorporated by reference in its entirety into this disclosure.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant no. DK109287 and grant no. 1R03DK124742-01 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

[0003] Liver fibrosis involves the replacement of healthy liver tissue with scar tissue and is a leading cause of liver failure and liver-related mortality. In the United States, liver fibrosis is highly prevalent due to high rates of obesity and type II diabetes. The typical disease progression of nonalcoholic fatty liver disease starts with accumulation of fat in hepatocytes, which can lead to a condition called non-alcoholic steatohepatitis (NASH) which is characterized by inflammation and fibrosis of the liver. NASH affects up to 12% of adults in the United States, and fibrosis associated with NASH, potentially triggered by liver injury and inflammation, is currently the strongest predictor of patient mortality.

[0004] There is no FDA-approved therapy for NASH or for liver fibrosis. The current standard of care involves treating the underlying causes of disease, such as obesity or inflammation. These methods typically fail to reverse fibrosis and associated liver damage, creating a great unmet need for novel treatments. In recent years, there have been several attempts to develop new drugs, but so far, the clinical efficacy of these compounds has proved to be underwhelming. BRIEF SUMMARY

[0005] In one aspect, the disclosure provides a method of treating or preventing a disease or disorder associated with and/or caused by neurotrophic tyrosine receptor kinase, comprising administering at least one neurotrophic tyrosine receptor kinase (Trk) inhibitor, a salt thereof, or a composition comprising the Trk inhibitor or salt thereof to a subject in need thereof.

[0006] In an aspect, the at least one neurotrophic tyrosine receptor kinase inhibitor targets Neurotrophic Tyrosine Receptor Kinase 1 (NTRK1), Neurotrophic Tyrosine Receptor Kinase 2 (NTRK2), and/or Neurotrophic Tyrosine Receptor Kinase 3 (NTRK3).

[0007] In another aspect, the disease is a liver disease. In a further aspect, the liver disease is selected from the group including liver fibrosis, liver cirrhosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, Hepatocellular carcinoma (HCC), and NASH fibrosis. [0008] In an aspect, the tyrosine receptor kinase (Trk) inhibitor is selected from the group including LOXO-195, entrectinib, RXDX-102, altiratinib, larotrectinib, sitravatinib, cabozantinib, merestinib, dovitinib, crizotinib, TSR-011, DS-6051, PLX7486, lestaurtinib, danusertib, F17752, AZD6918, AZD7451, and AZ-23.

[0009] In an aspect, the at least one neurotrophic tyrosine receptor kinase inhibitor targets scar tissue in the liver and/or fibrosis on the liver.

[0010] In another aspect, the Trk inhibitor is LOXO-195.

[0011] In an aspect, the subject is administered a pharmaceutically effective amount or therapeutically effective amount of the Trk inhibitor or the composition. In an aspect, the Trk inhibitor or the composition is administered orally, parenterally, intradermally, subcutaneously, topically, rectally, nasally, bucccally, vaginally, subdermally, or ophthalmically. In another aspect, the Trk inhibitor or the composition is orally administered in an orally acceptable dosage form selected from the group including of capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In an aspect, the subject is a mammal, preferably a human.

[0012] In an aspect, the salt is selected from the group consisting of an acetate, L-aspartate, besylate, bicarbonate, carbonate, D-camsylate, L-camsylate, citrate, edisylate, formate, fumarate, gluconate, hydrobromide/bromide, hydrochloride/chloride, D-lactate, L-lactate, D,L-lactate, D,L- malate, L-malate, mesylate, pamoate, phosphate, succinate, sulfate, bisulfate, D-tartrate, L-tartrate, D,L-tartrate, meso-tartrate, benzoate, gluceptate, D-glucuronate, hybenzate, isethionate, malonate, mcthylsulfatc, 2-napsylatc, nicotinate, nitrate, orotatc, stearate, tosylatc, thiocyanate, accfyllinatc, aceturate, aminosalicylate, ascorbate, borate, butyrate, camphorate, camphocarbonate, decanoate, hexanoate, cholate, cypionate, dichloroacetate, edentate, ethyl sulfate, furate, fusidate, galactarate, galacturonate, gallate, gentisate, glutamate, glutarate, glycerophosphate, heptanoate, hydroxybenzoate, hippurate, phenylpropionate, iodide, xinafoate, lactobionate, laurate, maleate, mandelate, methanesulfonate, myristate, napadisilate, oleate, oxalate, palmitate, picrate, pivalate, propionate, pyrophosphate, salicylate, salicylsulfate, sulfosalicylate, tannate, terephthalate, thiosalicylate, tribrophenate, valerate, valproate, adipate, 4-acetamidobenzoate, camsylate, octanoate, estolate, esylate, glycolate, thiocyanate, undecylenate, sodium, potassium, calcium, magnesium, zinc, aluminum, lithium, cholinate, lysinium, ammonium, troethamine, and a mixture thereof.

[0013] In an aspect, the composition further comprises one or more excipients, wherein the excipients are selected from the group consisting of anti-adherents, binders, coatings, disintegrants, fillers, flavors, dyes, colors, glidants, lubricants, preservatives, sorbents, sweeteners, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0015] FIGs. 1A-1G show single nuclei RNA-seq of NASH patient uncovers autocrine signaling loop in NASH-associated hepatic stellate cells according to an aspect of the disclosure. Hematoxylin and Eosin (FIG. 1A) and Sirius Red staining of control and NASH patient liver samples used for snRNA-seq. FIG. IB is UMAP visualization of all nuclei derived from control and NASH livers colored by cell type. FIG. 1C shows the top two marker gene expression for each cell type. FIG. ID is UMAP visualization of all nuclei derived from control and NASH livers colored by sample of origin. FIG. IE shows differentially expressed genes between HSCs from control vs NASH patient (red dots indicate DEG with Padj. < 0.05 and log2FC > 0.5). FIG. IF shows that all significantly enriched PANTHER pathways (FDR < 0.05) in genes up-regulated in NASH-associated HSCs. FIG. 1G shows the total number of significant interactions between different cell types as predicted using CellphoneDB. Thickness of the connecting line is proportional to the total number of interactions, with interactions > 100 highlighted. [0016] FIGs. 2A-2F show single nuclei RNA-seq results of patients with NASH according to an aspect of the disclosure. FIG. 2A is UMAP visualization of all nuclei derived from control (n = 3) and NASH (n = 9) livers colored by cell type. FIG. 2B is UMAP visualization of all nuclei derived from control and NASH livers colored by sample of origin. FIG. 2C shows the top eight marker gene expression for each cell type. FIG. 2D shows differentially expressed genes between HSCs from controls vs patients with NASH (red dots indicate DEG with Padj. < 0.05 and log2FC > 1). FIG. 2E shows that all significantly enriched PANTHER pathways (FDR < 0.05) in genes up- and down-regulated in NASH-associated HSCs. GO, Gene Ontology. FDR, false discovery rate. FIG. 2F shows that the total number of significant interactions between different cell types as predicted using CellphoneDB. Thickness of the connecting line is proportional to the total number of interactions, with interactions > 65 highlighted.

[0017] FIGs. A-3D show additional analyses from control and NASH snRNAseq data from patients according to an aspect of this disclosure. UMAP of merged datasets before batch correction colored by cell type (FIG. 3A) or patient of origin (FIG. 3B). FIG 3C shows the percentage distribution by cell type for each sample. FIG. 3D shows the total number of significant interactions between different cell types in control (CTRL) patient livers as predicted using CellphoneDB. Thickness of the connecting line is proportional to the total number of interactions, with interactions > 65 highlighted.

[0018] FIGs. 4A-4I show autocrine signaling in NASH-associated hepatic stellate cells is conserved in FAT NASH mice. FIG. 4A is a schematic of control (n=2), NASH (n=3), and NASH- HCC (n=3) mice used according to one aspect of the disclosure. FIG. 4B shows UMAP visualization of nuclei combined from all liver samples colored by cell type. FIG. 4C shows the top two marker gene expression for each cell type. FIG. 4D shows UMAP visualization of hepatic stellate cell (HSC) clusters colored by sample origin. FIG. 4E shows a dot plot depicting canonical quiescence and activation marker gene expression for each HSC cluster. FIG. 4F shows differentially expressed genes between HSC1 and HSC2 (red dots indicate DEG with Padj- < 0.05 and logzFC > 1). FIG. 4G shows that all significantly enriched PANTHER pathways (FDR < 0.05) in genes up-regulated in NASH-associated HSCs. FIG. 4H shows the total number of significant interactions between different cell types as predicted using CellphoneDB. Thickness of the connecting line is proportional to the total number of interactions, with interactions > 70 highlighted in red. FIG. 41 shows HSC autocrine interactions classified as short-range or long- range. FIG. 41 shows that all significantly enriched PANTHER pathways (FDR < 0.05) in genes up- and down-regulated in NASH-associated HSCs. FIG. 4K shows the total number of significant interactions between different cell types from NASH/NASH-HCC mice as predicted using CellphoneDB (P < 0.05).

[0019] FIGs. 5A- 5D show additional analyses from CTRL and NASH mouse snRNAseq data. UMAP of merged datasets before batch correction colored by cell type (FIG. 5A) or sample of origin (FIG. 5B). FIG.5C shows the percentage distribution by cell type for each sample. FIG. 5D shows the total number of significant interactions between different cell types in chow control livers as predicted using CellphoneDB. Thickness of the connecting line is proportional to the total number of interactions, with interactions > 100 highlighted.

[0020] FIGs. 6A-6J show increased stellate cell-stellate cell contact in FAT NASH mice revealed by tissue-clearing and 3D imaging. FIG. 6A shows livers from FAT-NASH mice that were perfused and cleared at the depicted timepoints. FIG. 6B are representative images of perfused liver pieces before and after clearing. FIG. 6C are representative images from Sirius red staining of FAT-NASH livers from different timepoints in the model. FIG. 6D shows Al-based quantification of Sirius red collagen staining from multiple mice per timepoint. FIG. 6E shows composite fibrosis scores calculated based on FIG. 6D. FIG. 6F shows confocal imaging and 3D reconstruction of DESMIN staining in cleared livers. FIG. 6G shows IMARIS surface and spot segmentation of DESMIN and nuclear staining, respectively, of the same images from FIG. 6C. FIG. 6H shows an enlarged image of a single segmented DEMSIN+ surface object highlighted in yellow in FIG. 6G along with nuclear staining in red. FIG. 61 shows the quantification of the percentage of nuclei that arc found as a single nucleus per surface object or multiple nuclei per surface object. FIG. 6J is an illustration of a proposed model according to one aspect of the disclosure.

[0021] FIGs. 7A- 7C show an overview of the liver clearing and 3D imaging pipeline. FIG. 7A is a schematic overview of the clearing, staining, imaging, and quantification process. FIG. 7B is a 3D reconstructed images from lightsheet microscopy. FIG. 7C is a 3D reconstructed images from confocal microscopy.

[0022] FIGs. 8A-8P show NTRK3 as HSC autocrine drug target in NASH according to one aspect of the disclosure. FIG. 8A shows an overlap of autocrine interactions identified in human and mouse NASH. Dot plot depicting NTRK3 gene expression across different cell types in human (FIG. 8B) and mouse (FIG. 8C) snRNAseq data. NTRK3 and aSMA protein expression in human control (FIG. 8D) and NASH (FIG. 8E) liver. NTRK3 and DESMIN protein expression in mouse control (FIG. 8F) and NASH (FIG. 8G) liver. FIG. 8H shows knocking down NTRK3 in LX2- Cas9 cells using two different gRNAs reduced ERK phosphorylation. Knocking down NTRK3 in LX2-Cas9 cells reduced cell migration in scratch assay (FIG. 81), representative images shown in FIG. 8J. FIG. 8K shows the pharmacological inhibition of NTRK3 using LOXO-195 dose- dependently reduced LX2 cell migration in a scratch assay. FIG. 8L shows a schematic depiction of the LOXO-195 in vivo study design. FIG. 8M shows a representative Sirius red staining from FAT-NASH mice with LOXO-195 or vehicle control. FIG. 8N shows an Al-based quantification of Sirius red collagen staining from multiple mice per timepoint. FIG. 80 shows composite fibrosis scores calculated based on FIG. 8N. FIG. 8P shows a cartoon illustration of a proposed model according to an aspect of the disclosure.

[0023] FIGs. 9A - 9H show that NTRK3 is expressed on cellular projections of NASH HSCs. FIG. 9A shows an overlap of significant (P < 0.05) autocrine interactions identified in human and mouse NASH by CellphoneDB. Dot plot depicting NTRK3 gene expression across different cell types in human (FIG. 9B) and mouse (FIG. 9C) snRNA-seq data. (FIG. 9D) NTRK3 and aSMA protein expression in whole-liver lysates of control or 24-week FAT-NASH mice (n = 5 mice per group, P value calculated using Student's t test), as detected by Western blot and quantified by densitometry normalized to CALNEXIN as loading control. NTRK3 and aSMA protein expression in human control (FIG. 9E) and NASH (FIG. 9F) liver as detected by immunofluorescence. NTRK3 and DESMIN protein expression in mouse control (FIG. 9G) and NASH (FIG. 9H) liver as detected by immunofluorescence.

[0024] FIGs. 10A-10L show NTRK3 is an HSC autocrine drug target in NASH. FIG. 10A shows ERK phosphorylation and aSMA protein expression in LX-2 cells with NTRK3 knockdown by CRISPR or siRNA, quantification ofWestern blot by densitometry using CALNEXIN as loading control (P value calculated using Student's t test, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P< 0.0001). ns, not significant. FIG. 10B shows gene ontology analysis of the top 500 most significantly down-regulated genes from RNA-seq analyses of NTRK3 knockdown cells (FDR < 0.05, fold enrichment score > 5), with biological processes related to HSC fibrogenicity highlighted in red and genes from these categories shown in FIG. 10C. FIG. 10D shows the expression of an established fibrogenic gene panel with NTRK3 knockdown from RNA-seq analyses (*Padj. < 0.05 compared with control). FIG. 10E shows a representative migration of NTRK3 CRISPR knockdown LX-2 cells compared with control LX-2 cells in scratch assay (FIG. 10F, average over four biological replicates, P value calculated by Student's t test). FIG. 10G shows pharmacological inhibition of NTRK3 using LOXO-195 dose-dependently reduced LX-2 cell migration in scratch assay (averaged over three biological replicates, representative shown in FIG. 10H. P value represents effect of treatment calculated using one-way ANOVA. FIG. 101 shows the expression of an established fibrogenic gene panel in LX-2 cells treated with varying concentrations of LOXO-195 for 24 hours (averaged over three replicates in a single experiment reproduced in a separate experiment, P values represent effect of drug treatment compared with vehicle by two-way ANOVA). FIG. 10J shows a schematic depiction of the LOXO-195 in vivo study design. FIG. 10K shows a representative Sirius red staining from FAT-NASH mice with LOXO-195 or vehicle control. FIG. 10L shows an Al-based quantification of Sirius red collagen staining from n = 7 or 8 mice per treatment. FIG. 10M shows composite fibrosis scores calculated on the basis of FIG. 10L, and P values calculated by Student's t test.

[0025] FIG. 11A-11D show the effect of NTRK3 knockdown or pharmacological inhibition on LX2 cell viability or proliferation. FIG. 11A shows the effect of CRISPR knockdown with 2 different gRNAs against NTRK3 on LX2 cell viability measured using MTS assay. FIG. 11B shows the effect of LOXO-195 treatment on LX2 cell viability measured using MTS assay. FIG. 11C shows the effect of CRISPR knockdown with 2 different gRNAs against NTRK3 on LX2 cell proliferation measured using BrdU assay. FIG. 1 ID shows the effect of LOXO-195 treatment on LX2 cell proliferation measured using BrdU assay.

[0026] FIGs. 12A and 12B show data from LOXO-195 treated FAT-NASH mice. FIG. 12A shows Sirius red stained liver sections from vehicle or LOXO-195 treated FAT-NASH mice. Whole slides were scanned and quantified with FibroNest - only one representative 50x image from each mouse is shown here. FIG. 12B shows mouse weight tracked over 4 weeks of LOXO-195 or vehicle treatment in the FAT-NASH model. [0027] FIG. 13 shows liver clearing and 3D imaging of DESMIN staining in vehicle or LOXO- 195 treated FAT-NASH mice. 6 mice were cleared per group with all data shown.

[0028] FIG. 14 shows an illustration of a control sample and a sample with NASH.

[0029] FIGs. 15A-15D show the results of a LOXO-195 mechanistic studies in FAT-NASH mouse livers. FIG. 15A shows Cd68 staining for a vehicle. FIG. 15B shows Cd68 staining for a LOXO- 195 treated mouse. FIG. 15C shows Cd68 staining for OCA treated mouse. FIG. 15D is a comparison of treated mice along with Cd68 quantification across N-9-1 1 mice per group.

[0030] FIG. 16 is a comparison of treatments of NTRK3 antagonism in NASH-HCC in FAT- NASH mouse model.

[0031] FIG. 17 is a graph showing plasma and tissue pharmacokinetics of LOXO-195 in mice.

[0032] FIGs. 18A-18D show the results of a LOXO-195 validation studies in human precision cut liver slices. FIG. 18A shows alpha smooth muscle actin staining for a vehicle. FIG. 18B shows alpha smooth muscle actin staining for a human liver slice treated with 250nM of LOXO-195. FIG. 18C shows alpha smooth muscle actin staining for a human liver slice treated with 25pM of LOXO-195. FIG.18D shows the quantification of alpha smooth muscle actin staining for each treatment.

DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0035] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. [0036] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0037] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0038] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0039] By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0040] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and/or" unless the content clearly dictates otherwise. The term "and/or" means any one or more of the items in the list joined by "and/or". As an example, "x and/or y" means any element of the three -element set {(x), (y), (x, y) } . In other words, "x and/or y" means "one or both of x and y". As another example, "x, y, and/or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and/or z" means "one or more of x, y and z".

[0041] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0042] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0043] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is +/-10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0044] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. [0045] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0046] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0047] The invention is defined in the claims. However, below is a non-exhaustive listing of nonlimiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0048] Fibrosis across diverse tissues, including liver, can persist or worsen even if the injury- driven inflammation is attenuated by treating the underlying disease. For example, in patients with cirrhosis due to hepatitis C fibrosis progresses in -15-25% even after antiviral cure. Similarly, in advanced NASH, fibrosis regression was not observed in patients with cirrhosis after bariatric surgery, even though earlier fibrosis stages may regress. The mechanisms underlying fibrosis persistence in advanced disease, and specifically how activated hepatic stellate cells (aHSCs), contribute to this response, are not known.

[0049] It was previously hypothesized that the persistence of fibrosis in advanced tissue injury is driven by cell-circuitry that establishes a "cold fibrosis" state, in which myofibroblasts activate self-perpetuating autocrine signaling loops that maintain their fibrogenic state, even in the absence of inflammation; however, this model has not yet been validated experimentally. Based on this framework, the inventors speculated that in liver, persistent hepatic fibrosis in advanced disease represents a state of cold fibrosis wherein myofibroblasts derived from activated hepatic stellate cells (aHSC) establish self-sustaining autocrine signaling loops. If true, aHSC autocrine signaling might engage a unique repertoire of ligands and receptors to sustain advanced fibrosis that do not contribute to fibrogcncsis at earlier stages. [0050] Single cell RNA-seq (scRNA-seq) technologies have transformed the understanding of human liver disease by uncovering remarkable cellular heterogeneity that clarifies disease mechanisms. However, scRNA-seq preferentially captures immune populations at the expense of liver epithelial and fibrogenic cells (HSCs). Here, using single nuclei RNA-seq (snRNA-seq), the inventors have profiled gene expression changes in HSCs from both human and murine NASH, the latter using a rodent model that faithfully recapitulates features of advanced disease, in particular, fibrosis and tumors (i.e., 'FAT-NASH' model) . The disclosure describes the emergence in late-stage disease of a conserved autocrine signaling circuit in NASH-associated HSCs that comprises of -100 predicted ligand-receptor pairs, over half of which involve short-range interactions requiring physical cell-cell proximity.

[0051] Like neurons, HSCs, which express many neuronal markers, display elaborate cellular projections in vivo that could serve important signaling functions yet their contributions have been overlooked. Using tissue clearing and 3D imaging based on iDISCO, the inventors generated high- resolution 3D images of HSCs that enabled characterization of these cellular projections in situ in NASH. This approach has unveiled a marked amplification of HSC-HSC contacts linked to progressive disease severity that could support short-range HSC autocrine interactions. To establish proof of principle, the inventors interrogated the predicted HSC-specific receptor-ligand autocrine loop mediated by NTRK3-NTF3 in both human and murine NASH. NTRK3 protein was localized to HSC projections in NASH, but absent from normal liver. Finally, the inventors functionally validated this interaction through antagonism of NTRK3, which inhibited HSC migration in cultured human HSCs, and attenuated fibrosis in vivo in murine NASH. This work uncovers a novel HSC physical interactome that establishes the basis for 'cold fibrosis' in advanced hepatic fibrosis through an autocrine circuitry.

[0052] In this study, the inventors have leveraged recent technological advances in snRNA-seq to capture HSC single-cell transcriptomes coupled with tissue clearing of mouse models to enable high resolution 3D tissue imaging. Using this strategy, the inventors have uncovered a novel autocrine signaling circuitry in HSCs supported by markedly amplified cell-cell contacts that underlie HSC activation in advanced NASH fibrosis.

[0053] A "disease", as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. In contrast, a "disorder" is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health. A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0054] As used herein, the terms "subject", "individual", and "patient" are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term "animal" as used herein includes humans. The term "subject" may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0055] The terms "treat", "treating", or "treatment" refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and/or decrease the number, frequency, or severity of clinical symptoms and/or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder. In particular, the terms "treatment of a disease" and "treating a disease" include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.

[0056] The terms "prophylactic", "preventive", "preventing", and "prevention" refer to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention can be complete, e.g., the total absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods.

[0057] As used herein, the term "preventing a disease" in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.

[0058] An aspect of this disclosure includes a method of treating a disease or disorder associated with and/or caused by neurotrophic tyrosine receptor kinase, comprising administering at least one neurotrophic tyrosine receptor kinase (Trk) inhibitor or salt thereof. The salt may be any suitable salt, including suitable pharmaceutically acceptable salts. Non-limiting examples include acetate, L-aspartate, besylate, bicarbonate, carbonate, D-camsylate, L-camsylate, citrate, edisylate, formate, fumarate, gluconate, hydrobromide/bromide, hydrochloride/chloride, D-lactate, L- lactate, D,L-lactate, D,L-malate, L-malate, mesylate, pamoate, phosphate, succinate, sulfate, bisulfate, D-tartrate, L-tartrate, D,L-tartrate, meso-tartrate, benzoate, gluceptate, D-glucuronate, hybenzate, isethionate, malonate, methylsulfate, 2-napsylate, nicotinate, nitrate, orotate, stearate, tosylate, thiocyanate, acefyllinate, aceturate, aminosalicylate, ascorbate, borate, butyrate, camphorate, camphocarbonate, decanoate, hexanoate, cholate, cypionate, dichloroacetate, edentate, ethyl sulfate, furatc, fusidate, galactaratc, galacturonate, gallate, gcntisatc, glutamate, glutarate, glycerophosphate, heptanoate, hydroxybenzoate, hippurate, phenylpropionate, iodide, xinafoate, lactobionate, laurate, maleate, mandelate, methanesulfonate, myristate, napadisilate, oleate, oxalate, palmitate, picrate, pivalate, propionate, pyrophosphate, salicylate, salicylsulfate, sulfosalicylate, tannate, terephthalate, thio salicylate, tribrophenate, valerate, valproate, adipate, 4- acetamidobenzoate, camsylate, octanoate, estolate, esylate, glycolate, thiocyanate, undecylenate, sodium, potassium, calcium, magnesium, zinc, aluminum, lithium, cholinate, lysinium, ammonium, troethamine, and a mixture thereof.

[0059] In some aspects, the Trk inhibitor or salt thereof is administered as a composition. The Trk inhibitor or composition may be administered to a subject in need thereof, for example a human. The disease may be liver disease, for example, but not limited to, liver fibrosis, liver cirrhosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, Hepatocellular carcinoma (HCC), or NASH fibrosis. FIG. 14 shows an illustration of a control sample and a sample with NASH.

[0060] In these aspects, the at least one neurotrophic tyrosine receptor kinase inhibitor targets Neurotrophic Tyrosine Receptor Kinase 1 (NTRK1), Neurotrophic Tyrosine Receptor Kinase 2 (NTRK2), and/or Neurotrophic Tyrosine Receptor Kinase 3 (NTRK3). The tyrosine receptor kinase (Trk) inhibitor may be any suitable Trk inhibitor including those listed in Table 1.

[0061] Table 1. Exemplary Trk inhibitors

[0062] Other suitable Trk inhibitors include RXDX-102, PLX7486, and F17752.

[0063] In a non-limiting example, analysis of this autocrine signaling pathway led to the identification of Neurotrophic Tyrosine Receptor Kinase 3 (TrkC or NTRK3) as a potential druggable target that is strongly implicated in advanced fibrosis or cirrhosis. In one aspect, it was observed that only four weeks of treatment with the Trk inhibitor LOXO-195 at a modest dosage was sufficient to reverse fibrosis in a mouse model of advanced NASH.

[0064] In an aspect, the at least one neurotrophic tyrosine receptor kinase inhibitor targets scar tissue in the liver and/or fibrosis on the liver. In a non-limiting example, LOXO-195 directly targets fibrosis via hepatic stellate cells, which provides unexpected advantages over other treatments of liver diseases or disorder that attempt treatment by reducing inflammation in the liver or fat accumulation in hepatocytes.

[0065] Trk inhibitor or the composition may be administered orally, parenterally, intradermally, subcutaneously, topically, rectally, nasally, bucccally, vaginally, subdermally, or ophthalmically. If the Trk inhibitor or the composition is orally administered, an orally acceptable dosage form may be selected from the group consisting of capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. The composition may also include one or more excipients. Non-limiting examples include anti- adherents, binders, coatings, disintegrants, fillers, flavors, dyes, colors, glidants, lubricants, preservatives, sorbents, sweeteners, and combinations thereof. [0066] The Trk inhibitor or composition may be administered in an effective amount. An "effective amount" includes a "therapeutically effective amount", a "pharmaceutically effective amount", and a "prophylactically effective amount". The term "therapeutically effective amount" and/or "pharmaceutically effective amount", refers to an amount effective in treating and/or ameliorating a disease or condition in a subject. The term "prophylactically effective amount" refers to an amount effective in preventing and/or substantially lessening the chances of a disease or condition in a subject.

[0067] The exact amount required to achieve a therapeutically effective outcome will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.

[0068] Using snRNA-seq, the inventors successfully captured transcripts from all liver cell types including HSCs at frequencies mirroring those found in situ. NASH associated HSCs not only activate well-established fibrogenic pathways Pdgfrb and Integrin signaling, but also a previously uncharacterized CCKR signaling pathway, providing a mechanistic explanation for the observed anti-fibrotic effects of an CCKR antagonist in a murine NASH model. The inventors also uncovered an intercellular communication hub that consists of HSCs, endothelial cells, cholangiocytes, and their autocrine interactions that emerges in NASH.

[0069] Mathematical modeling of growth factor exchange between different cell types during homeostasis and perturbation (e.g., pathological conditions such as tissue injury) implicated a self- sustaining autocrine signaling circuit in fibroblasts that maintains fibrosis even in the absence of inflammation. This state of "cold fibrosis" has been predicted in different tissues, including liver. Using snRNA-seq to profile HSCs in human and mouse livers, the inventors provide direct experimental evidence for the emergence of such an autocrine signaling loop in NASH fibrosis.

[0070] More than half of the HSC autocrine receptor-ligand interactions are short-range and require close physical proximity between interacting HSCs. To establish an anatomic basis for this signaling, the inventors cleared liver tissue from mice at various stages of the FAT-NASH model using iDISCO, establishing that with worsening NASH-fibrosis, HSCs increase physical contact with each other through their cellular projections. In advanced disease (i.e., 24 weeks on the FAT- NASH model), almost all HSCs are enmeshed within a densely interconnected network or "physical interactome" of HSCs that can support short range cell-cell signaling.

[0071] To establish HSC autocrine signaling as a driver of NASH-fibrosis, the inventors focused subsequent studies on the NTRK3-NTF3 interaction, which is 1 of the 51 predicted HSC autocrine interactions that is conserved between NASH patients and mice. In both patients and mice, NTRK3 protein is not expressed in the healthy liver but is induced in HSCs in NASH, where its protein expression overlaps with HSC cellular projections. Using gRNA against NTRK3 in LX2-Cas9 cells, the inventors generated NTRK3 CRISPR knockouts that displayed marked reduction in cell migration in a wound closure assay, and an effect that was phenocopied by pharmacological inhibition using LOXO-195, a second generation NTRK small molecule inhibitor that is used clinically to treat tumors expressing NTRK fusion proteins. To validate HSC autocrine signaling as a potential therapeutic target in NASH, fibrosis was improved in FAT-NASH mice with advanced fibrosis (24 weeks) by only 4 weeks of treatment with LOXO-195.This finding not only establishes proof of principle for an autocrine pathway in advanced NASH fibrosis, but more importantly suggests that treatment of advanced fibrosis may be more effective if it leverages autocrine targets that dominate in late-stage disease.

[0072] The basis for the morphological transition of HSCs in NASH is not well understood. HSCs express a neuronal gene signature including neurotrophins and their receptors. In fact, the low affinity neurotrophin receptor P75NTR has been described as a marker of HSC precursors in the fetal liver and drives in culture HSC cytoskeletal remodeling through Rho signaling. Future studies will investigate the potential link between increased expression of neuronal genes and morphological evolution of HSCs in vivo in NASH. In other tissues, skin fibroblasts also undergo membrane extension following injury, and a recent single cell transcriptomics revealed a similar neuronal gene signature in these cells. These findings are in line with recent pan-tissue scRNAseq efforts to characterize global transcriptomic signatures shared among tissue fibroblasts and point to conserved injury response mechanisms that extend from the fibroblast transcriptome to morphology. Thus it would be highly interesting to test whether the principles the inventors uncover here extend to other fibrotic tissues such as skin, lung, and heart, leveraging the same snRNA-seq and iDISCO based tissue clearing techniques that in theory can be applied to other tissues as well.

[0073] This study revealed several novel elements regarding HSC responses in vivo. The inventors identified novel genes and pathways that are selectively expressed in NASH-associated HSC and uncovered a role for autocrine signaling supported by an expanded physical interactome in latestage disease, unveiling a new repertoire of antifibrotic drug targets that may be uniquely effective for treating advanced "cold fibrosis".

[0074] It was found that the single nuclei RNA-seq uncovers the stellate cell autocrine signaling network in NASH patients. The inventors performed snRNAseq of snap-frozen liver samples from two control and seven NASH patients that captured RNA expression from hepatocytes and HSCs more efficiently than those previously reported using single-cell RNAscq. Representative hematoxylin and eosin, and Sirius red staining of control and NASH patient livers revealed steatosis and fibrosis in NASH not found in control (FIG. 1A). By informatically merging datasets from these samples and clustering using UMAP, gene expression from all major cell types was tracked at the expected proportions in both control and disease samples, based on cell-specific marker genes (FIGs. IB- ID). While no new cell type emerged in NASH (FIG. IB), HSCs from NASH patients, termed 'NASH-associated HSCs', expressed an altered transcriptomic profile with significantly over-expressed and repressed genes compared to HSCs from control patients (FIG. IE). Top enriched pathways in NASH-associated HSCs include PDGF, integrin, and endothelin signaling as expected, but also CCKR signaling, which has not previously been implicated in HSC activation in vivo. Using CellphoneDB software to predict cell-cell interactions in NASH livers revealed a tripartite intercellular communication network comprised of HSCs, cholangiocytes, endothelial cells, and their autocrine interactions (FIG. 1G).

[0075] In another aspect, snRNA-seq of snap-frozen liver samples from three controls (nontumor tissue from liver metastasis resections) and nine patients with NASH (NAFLD Activity Score, or NAS and fibrosis scores provided in Table 2) that captured RNA expression from hepatocytes and HSCs more efficiently than those previously reported using scRNA-seq.

[0076] Table 2. Information on patients with NASH.

[0077] Human tissues from three distinct sources were analyzed to ensure reproducibility, yielding snRNA-seq data from 128,851 total nuclei and 3783 HSC nuclei after quality control, the largest compilation of human HSC transcriptomes to date. By informatically merging datasets from these samples and clustering using a UMAP (Uniform Manifold Approximation and Projection)-based approach, we recovered all major liver cell types at the expected proportions in both control and disease samples based on cell-specific marker genes (FIGs. 2A - 2D, and FIGs. 3A-3D). Although no new cell types emerged in NASH (FIG. 2B), HSCs from patients with NASH, termed “NASH- associated HSCs,” expressed an altered transcriptomic profile with 169 significantly overexpressed and 291 significantly repressed genes compared with HSCs from control patients (FIG. 2D). The most highly enriched pathways in NASH-associated HSCs included cellmatrix adhesion and integrin signaling, as expected (FIG. 2E). Several genes belonging to the category “extracellular matrix organization” were down-regulated, supporting HSCs not only as a secretor of extracellular matrix (ECM) components but also as a key contributor to overall ECM remodeling. Using CellphoneDB software to predict cell-cell interactions in NASH livers revealed a tripartite intercellular communication network composed of HSCs, cholangiocytes, endothelial cells, and their autocrine interactions (FIG. 2F). These results reinforce recent observations highlighting HSCs as a hub of cell-cell communication in liver disease. They further establish in vivo evidence of a myofibroblast autocrine signaling circuit in fibrosis, consistent with an autocrine signaling loop underlying cold fibrosis that was predicted using mathematical modelling. [0078] Also shown is analysis of snRNA-seq of snap-frozen liver samples from three controls (nontumor tissue from liver metastasis resections) and nine patients with NASH (NAFLD Activity Score) (FIG. 2A). Human tissues from three distinct sources were analyzed to ensure reproducibility, yielding snRNA-seq data from 128,851 total nuclei and 3783 HSC nuclei after quality control. By informatically merging datasets from these samples and clustering using UMAP, gene expression from all major cell types was tracked at the expected proportions in both control and disease samples, based on cell-specific marker genes (FIGs. 2A-2C). While no new cell type emerged in NASH (FIG. 2B), HSCs from NASH patients, termed 'NASH-associated HSCs', expressed an altered transcriptomic profile with significantly over-expressed and repressed genes compared to HSCs from control patients FIG. 2D. The most highly enriched pathways in NASH-associated HSCs included cell matrix adhesion and integrin signaling (Fig. 2E). Several genes belonging to the category “extracellular matrix organization” were down-regulated. These results support HSCs not only as a secretor of extracellular matrix (ECM) components but also as a key contributor to overall ECM remodeling. Using CellphoneDB software to predict cell-cell interactions in NASH livers revealed a tripartite intercellular communication network composed of HSCs, cholangiocytes, endothelial cells, and their autocrine interactions (FIG. 2F).

[0079] It was also found that stellate cell autocrine signaling is conserved in FAT -NASH mice. The inventors tested whether the FAT-NASH murine model of NASH faithfully recapitulates the cell-cell communication networks uncovered in human NASH. To do so, the inventors profiled HSC transcriptomes from control and 24 weeks FAT-NASH livers using snRNA-seq (FIG. 4A and FIGs. 5A-5D). Two or three livers each from control and 24 week-FAT-NASH mice (with or without HCC) were pooled into a single sample and the data were informatically merged using Seurat-LIGER algorithm to generate a single UMAP of 19,249 nuclei from all samples (Fig. 4B). Following annotation of different nuclei clusters as different liver cell types using canonical marker genes for mice, the inventors recovered all known liver cell types represented at the expected proportions based on existing literature (Fig. 4C). Two distinct populations of HSCs were apparent from the data, termed 'HSC1' and 'HSC2'. HSC1 was mostly comprised of nuclei from control mice and expresses classical markers of HSC quiescence, including Lrat and Des, whereas HSC2 was primarily comprised of nuclei from FAT-NASH mice and expresses markers of activation such as Pdgfrb, Acta2, Coll al, Col3al, Mmp2, Timpl, and Timp2 (FIGs. 4D and 4E), which represent NASH-associated HSCs. Differential gene expression analysis between HSC1 and HSC2 revealed 996 significantly up-regulated genes in HSC2 compared with HSC 1, including cell adhesion and integrin signaling pathways present in patients with NASH, among other pathways that are known drivers of fibrosis, including transforming growth factor-^ signaling, actin cytoskeleton remodeling, and cell migration (FIG. 4F and 4G). As in human NASH, CellphoneDB analysis of FAT-NASH mice established a prominent autocrine signaling circuit in HSCs, with HSC2 cells harboring the greatest number of predicted ligand-receptor interactions, followed by interactions between HSC2 and cholangiocytes, and then between HSC2 with a subset of endothelial cells (FIG. 4H). More than half of the 120 HSC2 autocrine ligandreceptor pairs involved short-range interactions (defined as either nonsecreted or collagen/integrin interactions) that require cell-cell proximity between the ligand and its receptor (FIG. 41), highlighting an autocrine circuitry that emerges in activated HSCs in advanced fibrosis.

[0080] It was also found that hepatic stellate cell-cell contacts increase progressively in livers of FAT-NASH mice. In the resting liver, HSCs localize to the space of Disse, interposed between hepatocytes and endothelial cells. To clarify how HSCs interact with each other within this anatomical milieu, a pipeline for iDISCO tissue clearing and confocal imaging was established to generate high-resolution 2D images of HSCs using staining against DESMIN, a marker of HSCs in the murine liver (FIGs. 6A and 6B and FIGs. 7A-7C). Stacks of images were then 3D reconstructed and segmented using the IMARIS software (FIGs. 6F and 6G). With these high- resolution 3D reconstructed images, detailed morphology of HSCs was visualized, including their cellular projections. Remarkably, these projections closely resemble those of neurons. Individual HSCs (each represented by a different pseudo color in FIG. 6G) were classified by IMARIS as separate DESMIN⁺ surface objects that overlaid a single nucleus marked by propidium iodide (moceled by spheres in FIG. 6G) that visualized nuclear DNA (FIG. 6G). HSCs appeared uniform and regularly spaced in livers of healthy mice fed a chow diet.

[0081] To characterize potential morphological changes in HSCs in NASH, the inventors cleared and imaged livers from 6, 12, and 24 weeks on the FAT-NASH model and correlated with levels of fibrosis measured at the same time-points. To comprehensively quantify tissue fibrosis, the inventors leveraged an Al based platform, FibroNest, which extensively characterizes not only the absolute amount of collagen content, but also various features of the collagen fiber architecture. The inventors found that the severity of fibrosis increases progressively in our FAT-NASH model from 6 to 24 weeks as quantified using Fibronest (FIGs. 6C-6E). Correspondingly, HSCs undergo dramatic, progressive morphological transition in NASH, in which their projections appear more elongated and initiate physical contacts with one another. The inventors quantified these direct interactions as the number of DESMIN⁴" objects (as detected by IMARS) that were multinucleated, since HSCs arc mononuclcatcd in vivo. One such DESMIN⁴" object from each timepoint is highlighted yellow in FIG. 6G with original staining visualized in FIG. 6H. Using this method, the inventors detected an increase in HSC-HSC contacts, i.e., an increasing fraction of DESMIN⁺ objects that arbor multiple nuclei, as the FAT-NASH model progresses that mirrors the increase in fibrosis severity detected using Fibronest (FIG. 61). At 24 weeks in the FAT-NASH model, almost all HSCs made physical contact with each other, establishing a previously unrecognized HSC physical interactome in advanced NASH that could mediate short-range autocrine signaling (FIG. 6J). Propidium iodide nuclear staining demonstrated a loss of granularity with larger nuclei during NASH progression, which most likely represents hepatocytes, suggesting a loss of heterochromatic foci in NASH-associated hepatocytes (Fig. 6H).

[0082] It was also found that the HSC autocrine signaling factor NTRK3 may be used as an anti- fibrotic drug target in NASH. The data demonstrates that HSCs create a physical interactome in NASH that parallels increased autocrine signaling predicted by snRNA-scq. To provide a proof- of-principle that HSC autocrine signaling may be mediated by HSC physical interactions in the FAT-NASH model, in one aspect, the inventors localized autocrine receptors to HSC projections in vivo. The inventors overlapped autocrine interactions identified in human NASH patients and FAT-NASH mice, which led to 51 conserved autocrine receptor-ligand pairs (FIG. 8A). Among these candidate receptor-ligand pairs, the inventors focused on NTRK3-NTF3 because this pathway has not been described in HSCs, and interrogation of our snRNA-seq datasets confirmed the selective expression of NTRK3 and its predicted ligand NTF3 in NASH patients and FAT- NASH mice, but not control livers (FIGs. 8B and 8C). Consistent with these data, NTRK3 protein expression increased in NASH patients (FIGs. 8D and 8E) and FAT-NASH mice (FIGs. 8F and 8G) compared to controls. Furthermore, NTRK3 protein could be traced to HSC projections in NASH, visualized as an overlap with HSC-specific markers aSMA in humans (FIG. 8E) and DESMIN in mice (FIG. 8G).

[0083] To establish HSC NTRK3-NTF3 autocrine signaling as a driver of NASH-fibrosis, NTRK3 was knocked down in the immortalized human stellate cell line, LX-2, using CRISPR-Cas9. As expected, NTRK3 knockdown reduced ERK phosphorylation by Western blot and inhibited LX-2 cell migration based on the wound-closure assay. In parallel, NTRK3 activity was also blocked pharmacologically in LX-2 cells using LOXO-195, a second-generation, highly specific NTRK3 kinase domain inhibitor currently in clinical trials for the treatment of TRK fusion-positive cancers. As with CRISPR-Cas9 knockdown, LOXO-195 in LX-2 cells dose-dependently inhibited LX-2 cell migration at nanomolar concentrations (FIG. 8J) without affecting cell viability (data not shown).

[0084] To provide a proof of principle that inhibition of this HSC autocrine signaling circuit could reverse fibrosis in vivo, FAT-NASH mice at 24 weeks on the model were treated with LOXO-195 or vehicle twice daily, 5 days a week, for 4 weeks while continuing the model (FIG. 8L). Fibronest- based fibrosis quantification of Sirius red stained liver sections revealed significant reduction of all major features of fibrosis in LOXO-195 treated mice compared to vehicle treated controls (FIGs. 8N-8O).

[0085] To provide another proof of principle that HSC autocrine signaling may be mediated by HSC physical interactions in the FATNASH model, in another aspect, the inventors sought to localize a specific autocrine receptor, NTRK3, to HSC projections in vivo. The inventors informatically overlapped autocrine interactions identified in human NASH patients with FATNASH mice, yielding 68 autocrine receptor-ligand pairs conserved in both species (FIG. 9A). Among these candidate receptor ligand pairs, the inventors focused on NTRK3-NTF3 because this pathway has not been described in HSCs. Interrogation of snRNA-seq datasets confirmed the selective expression of NTRK3 and its predicted ligand NTF3 in HSCs in both patients and mice (FIG. 9B and 9C). Whereas the amount of NTRK3 and NTF3 gene expression diverged between mice and patients with NASH, both genes increased in FAT-NASH mice compared with chow control. In patients with NASH, NTRK3 gene expression was reduced compared with control patients. Using whole-liver lysates, NTRK3 protein was present in mouse livers and strongly induced in NASH (FIG. 9D) but not detected in human livers, likely because the signal was diluted by other cell types. These species differences may reflect the inherent heterogeneity of the human samples, in which patients are of diverse ages, sexes, and backgrounds, whereas mice are inbred and all exposed to identical conditions before analysis. Thus, the inventors suggest that NTRK3 expression likely increases in NASH, but further verification is necessary in a more standardized cohort of patients. NTRK3 protein was localized by immunofluorescence to HSC projections in NASH in both species, which were visualized as an overlap with HSC-specific markers alphasmooth muscle actin (aSMA) in humans (FIG. 9E and 9F) and DESMIN in mice (FIG. 9G and 9H). These key findings reinforce the proposed model that "effective signaling" between HSCs is increased through expanded physical autocrine contacts between HSCs in NASH.

[0086] In another aspect, to establish HSC NTRK3-NTF3 autocrine signaling as a driver of NASH fibrosis, the inventors knocked down NTRK3 in the immortalized human stellate cell line, LX-2, using CRISPR-Cas9 and small interfering RNA (siRNA). Whereas stable NTRK3 knockdown by CRISPR reduced extracellular signal-regulated kinase (ERK) phosphorylation and ccSMA expression by Western blot, transient NTRK3 knockdown by siRNA only reduced aSMA expression (FIG. 10A). Unbiased RNA-seq analyses of NTRK3 CRISPR- and NTRK3 siRNA- treated LX-2 documented a decrease in fibrogenic pathways, including "regulation of extracellular matrix organization," "extracellular matrix assembly," "collagen fibril organization," "integrin activation," and "regulation of fibroblast migration" (FIG. 10B), with genes captured in these pathways shown in FIG. 10C. The inventors also probed a panel of established fibrogenic genes in the bulk RNA-seq datasets, which demonstrated an overall decrease in both NTRK3 CRISPR- and NTRK3 siRNA-treated LX-2 cells (FIG. 10D). Consistent with RNA-seq analyses indicating a decrease in regulation of fibroblast migration, knocking out NTRK3 inhibited LX-2 cell migration based on the wound-closure assay (FIG. 10E and 10F). In parallel, NTRK3 activity was also pharmacologically blocked in LX-2 cells using LOXO-195, a second-generation, highly specific NTRK3 kinase domain inhibitor currently in clinical trials for the treatment of neurotrophic receptor tyrosine kinase (TRK) fusion-positive cancers. As with CRISPR-Cas9 knockdown, LOXO-195 in LX-2 cells dose-dependently inhibited LX-2 cell migration at nanomolar concentrations (FIGs. 10G and 10H) without affecting cell viability or proliferation (FIGs. 11A-11D). Concurrent with decreased migration, LOXO-195 treatment reduced LX-2 fibrogenicity as demonstrated by lower expression of an established panel of fibrogenic genes compared with vehicle control (FIG. 101).

[0087] To provide a proof of principle that inhibition of this HSC autocrine signaling circuit could reverse fibrosis in vivo, the inventors treated FATNASH mice at 24 weeks on the model with advanced fibrosis with LOXO-195 or vehicle twice daily, 5 days per week, for 4 weeks while continuing the model (FIG. 10J and FIG. 12A). There was no observed toxicity of LOXO-195 in mice compared with vehicle-treated controls; the only notable difference was greater weight gain in a subset of LOXO-195-treated animals (FIG. 12B). FibroNest-based phenotypic fibrosis quantification of Sirius red-stained liver sections documented significant reduction of all major features of fibrosis in mice treated with LOXO-195 compared with vehicle-treated controls (P < 0.05), which was coupled with a pruning of stellate cell processes in cleared livers (FIG. 10K-10M and FIG. 13), establishing NTRK3 as a potential antifibrotic target in NASH.

[0088] Using snRNA-seq, the inventors successfully captured transcripts from all liver cell types, including HSCs, at frequencies mirroring those found in situ (77). NASH-associated HSCs not only engaged the well-established fibrogenic pathways platelet derived growth factor receptor beta (PDGFRB) and integrin signaling but also a previously uncharacterized cholecystokinin receptor (CCKR) signaling pathway, providing a mechanistic explanation for the observed antifibrotic effects of an CCKR antagonist in a murine NASH model. The inventors uncovered an intercellular communication hub that emerges in NASH that consists of HSCs, endothelial cells, cholangiocytes, and their autocrine interactions cataloged in the resource for future investigations into NASH therapeutics.

[0089] Mathematical modeling of growth factor exchange between different cell types during homeostasis and perturbation (for example, during pathological conditions such as tissue injury) predicted the development of a self-sustaining autocrine signaling circuit in fibroblasts that maintains fibrosis in different tissues, including liver.

[0090] Using snRNA-seq to profile HSCs in human and mouse livers, the inventors provide direct functional and morphologic evidence for the emergence of such an autocrine signaling loop in NASH fibrosis. More than half of the HSC autocrine receptor-ligand interactions are short range and require close physical proximity between interacting HSCs. To establish an anatomic basis for this signaling network, the inventors cleared liver tissue from mice at various stages of the FAT- NASH model using iDISCO, establishing that with worsening NASH-fibrosis, HSCs increased physical contact with each other through their cellular projections. In advanced disease (24 weeks on the FAT-NASH model), almost all HSCs were enmeshed within a dense interconnected network or "physical interactome" of HSCs that can support short-range cell-cell signaling. The results support a paradigm of autocrine HSC activation in disease in which physical proximity between these cells increases the effectiveness of autocrine ligand-receptor interactions.

[0091] To establish HSC autocrine signaling as a driver of NASH-fibrosis, the inventors focused on the NTRK3-NTF3 interaction, one among the 68 predicted HSC autocrine interactions that were conserved between patients with NASH and the murine FAT-NASH model. In both patients and mice, NTRK3 protein was not expressed in the healthy liver but was induced in HSCs in NASH, where its protein expression localized to HSC cellular projections. Using CRISPR and siRNA against NTRK3 in LX-2 cells, the inventors generated NTRK3 knockdowns, which displayed markedly reduced fibrogenicity and cell migration. These effects were phenocopied by pharmacological inhibition using LOXO-195, a second-generation NTRK small-molecule inhibitor that is used clinically to treat tumors expressing NTRK fusion proteins. Migration was assessed as a key readout in culture because it most directly underlies expansion of cell-cell contacts. To validate HSC autocrine signaling as a potential therapeutic target in NASH in vivo, fibrosis was improved in FAT-NASH mice with advanced fibrosis (24 weeks) by only 4 weeks of treatment with LOXO-195. This finding not only establishes proof of principle for an autocrine pathway in advanced NASH fibrosis but also more importantly suggests that treatment of advanced fibrosis may be more effective if it leverages autocrine targets that dominate in late-stage disease. [0092] The basis for the morphological transition of HSCs in NASH is not well understood. HSCs express a neuronal gene signature including neurotrophins and their receptors. The low-affinity neurotrophin receptor P75NTR has been described as a marker of HSC precursors in the fetal liver and drives cytoskeletal remodeling in cultured HSCs through Rho signaling. In other tissues, skin fibroblasts also undergo membrane extension after injury, and a recent single-cell transcriptomics study revealed a similar neuronal gene signature in these cells. These findings are in line with recent pan-tissue scRNA-seq efforts to characterize global transcriptomic signatures shared among tissue fibroblasts and point to conserved injury response mechanisms that incorporate both the fibroblast transcriptome and morphology. Thus, the principle of enhanced autocrine interactions during advanced fibrosis may extend to other fibrotic tissues, including skin, lung, kidney, and heart, but requires direct validation.

[0093] This study reveals several features of HSC responses in vivo. The inventors have identified genes and pathways that are selectively expressed in NASH-associated HSCs and uncover a role for autocrine ligand-receptor interactions supported by an expanded physical interactome in late- stage disease, revealing a new repertoire of antifibrotic drug targets that may be uniquely effective for treating advanced fibrosis.

[0094] EXAMPLES

[0095] Study design

[0096] In this study, the inventors leveraged recent developments in snRNA-seq and tissue clearing techniques to uncover signaling circuits that emerge in NASH that may serve as therapeutic targets for advanced fibrosis. The inventors profiled HSC transcriptomes from both human and mouse NASH livers and identified a conserved autocrine signaling circuit that consists of 68 ligand-receptor pairs. Targeting one of these HSC autocrine receptors, NTRK3, reduced HSC activation in culture and reversed advanced fibrosis in vivo in the FATNASH model. All results in this study have been reproduced in independent cohorts of mice and in biological replicates for cell culture. For quantifications that can be subjected to human bias, samples were randomly coded and quantification was done in a blinded fashion. Use of human tissues for snRNA-seq was Institutional Review Board-exempt in accordance with guidelines of the Mount Sinai Institutional

Review Board, because there were no patient identifiers and the tissue was otherwise intended to be discarded.

[0097] .Patient and clinical specimens

[0098] Samples collected at Mount Sinai Hospital, New York, were freshly resected liver samples collected at time of surgery with informed consent. Samples collected at University of California, San Diego were deidentified livers declined for transplantation obtained via Lifesharing OPO. Samples collected at Gordian Biotechnology were livers not suitable for transplantation from research-consented donors and were perfused and transported on ice to Gordian Biotechnology from regional hospitals after transplantable organ removal. Sample sections suitably sized for snRNA-seq (-4 mm by 4 mm by 4 mm) were removed from at least 5 mm below the outer surface of the liversnap frozen in liquid nitrogen, and stored at -80°C until nuclei extraction.

[0099] Mice [0100] The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the Icahn School of Medicine at Mount Sinai, NY (IACUC-2018-0060). 6-week-old male and female C57BL/6J mice (housed separately) were purchased from Jackson Laboratories (Farmington, CT). Five mice per cage were housed in a Helicobacter-free room for 12 hours light - 12 hours dark cycle and weighed once weekly.

[0101] Carbon tetrachloride (CC14) was purchased from Sigma-Aldrich, MO. CC14 was freshly dissolved in corn oil at final concentration of 5% before injection. The final dose of pure CC14 was 0.2 pl/g of body weight of mice, delivered intra-peritoneally once/week starting from initiation of the western diet/sugar water feeding and continued for a total period of 6, 12, or 24 weeks. Western diet containing 21.2% fat (42% Kcal), 41% sucrose and 1.25% cholesterol by weight was purchased from Envigo, WI (Teklad Custom diet). Sugar water solution contained 18.9 g/L D-(+)- Glucosc (Sigma- Aldrich, MO) and 23.1 g/L D-(-)-Fructosc (Sigma- Aldrich, MO) dissolved in autoclaved water and filter sterilized. The diet and sugar water were replaced once weekly.

[0102] For LOXO-195 in vivo studies, LOXO-195 (TargetMol) was first dissolved in dimethyl sulfoxide (DMSO) to 41.6 mg/ml before com oil was added to a final LOXO-195 concentration of 12.5 mg/ml; 100 pl of this drug solution or vehicle control was given to a 25-g mouse to achieve a dosage of 50 mg/kg (weight adjusted daily). The dosing regimen was oral gavage twice a day (separated by 8 hours), 5 days a week for a total of four consecutive weeks. All mice were euthanized at 72 hours after the last dose of LOXO-195 or vehicle control.

[0103] At termination of study, pieces of liver (unless used for tissue clearing) were formalin- fixed for paraffin embedding and directly frozen in Tissue-Tek OCT compound (Thermo Fisher Scientific catalog no. 4583) over dry ice and then stored at -80°C for subsequent immunohistochemistry. The remaining mouse livers were chopped into ~2-cm³ pieces, flash- frozen in liquid nitrogen, and stored at -80°C until nuclei extraction..

[0104] Tissue clearing, staining, confocal imaging, and 3D image reconstruction

[0105] Mice were anesthetized and perfused through the portal vein with 20 mL of PBS followed by 20 mL of 4% PFA/PBS, liver tissue were trimmed to -2 mm3, cleared and stained as described in the iDISCO protocol (D01:https://doi.org/10.1016/j.cell.2014.10.010). A detailed protocol that accompanies the original iDISCO publication along with an updated list of validated antibodies for this method can be found at https://idisco.info/. Antibodies used in this study are DESMIN (Abeam catalog no. ab 15200, 1:400 dilution) and CD31 (R&D Systems catalog no. #AF3628, 1:400 dilution). Propidium iodide (Sigma catalog no. #P4170, 0.02 mg/mL final concentration) is used to stain DNA. Imaging is carried out on the Leica SP5 DMI confocal microscope and subsequently reconstructed using the IMARIS software at the Sinai Microscope CoRE. The IMARIS "surface" tool is used for segmenting the DESMIN staining using Surface Grain Size of 0.303 pm, diameter of largest sphere of 1.14 pm, and manual Threshold range from 13.4 to 168.4. The IMARIS "spot" tool is used to segment propidium iodide DNA staining into nuclei by setting the diameter of spots to 5pm, these nuclei are then manually curated as belonging to HSC if it is surrounded by DESMIN signal in the overlay channel.

[0106] Generation of LX2-Cas9 cells and cell culture

[0107] LX-2 cells are an immortalized human HSC line previously generated in the Friedman Laboratory. Stable Cas9-expressing LX-2 cells were generated through lentiviral transduction of a Cas9-expressing plasmid (lentiCas9-Blast, Addgene 52962-LV). Cells were then selected based on blasticidine resistance and homogenous Cas9-expressing LX-2 cells were isolated by single cell dilution cloning (available as LX-2 Cas9 line from MilliporeSigma, SCC613). Scratch assay was performed in a 24-well plate by using a P200 pipette tip to scrape the LX-2 cell monolayer in a straight line. Debris were removed from the edge of the scratch by washing and replacing with fresh media. To obtain consistent field of view in imaging, we made markings on the plate using a fine-tip marker. Plates were then imaged immediately after the scratch with a microscope using the phase-contrast objective at xlO magnification. Cells were returned to incubation and imaged again 24, 48, and 72 hours after initial scratch. Images acquired were analyzed quantitatively using Photoshop. Images obtained at each time point and treatment condition were visually overlaid on top of each other and cropped to the same size. The perimeters and areas of the scratches were then delineated and quantified. For all cell culture assays, LX-2 cells were serum-starved overnight to quiesce and synchronize metabolic activity in serum-free Dulbecco's modified Eagle's medium

(Thermo Fisher Scientific) supplemented with 0.1% bovine serum albumin (BSA), without antibiotics at 37°C.. [0108] Immunoblotting

[0109] Flash-frozen livers were homogenized using TissueLyser (Qiagen) in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors [Pierce complete protease and phosphatase inhibitors (Thermo Fisher Scientific, A32963 and 78420), 1 tablet per 10 ml of RIPA buffer]. Bradford protein quantification was performed (Bio-Rad, 5000006) for liver lysates. LX-2 cells were directly collected in 2x Laemmli buffer (Bio-Rad, 1610737). Fifty micrograms of liver protein or 10 pl of LX-2 lysate were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with antibodies to hNTRK3 (Biotechne, AF373), mNTRK3 (Biotechne AF1404), aSMA (Abeam, ab5694), pERK (Cell Signaling Technology, 4370S) and CALNEXIN (Abeam, ab75801).

[0110] Picrosirius red staining and artificial intelligence-based collagen quantification in liver sections

[0111] Liver was fixed in 10% formalin buffer and paraffin embedded liver tissues were sectioned using a 4 pm microtome. Slides containing tissue sections were baked at 60°C for 1 hour and rehydrated through xylene followed by graded ethanol (100%, 95%, 85% and 70%) into distilled water and processed for picrosirius red/Fast green staining. For collagen staining, rehydrated slides were stained for one hour in saturated picric acid with 0.1% Sirius Red (Direct Red-80; Sigma- Aldrich catalog no., MO) followed by counterstain with 0.01% Fast Green (Sigma- Aldrich catalog no., MO) for another hour. The slides were removed from the stain, rinsed in water and rapidly dehydrated through graded ethanol (70%, 85%, 95% and 100%) followed by xylene and finally placed on cover slips in Permount (ThermoFisher Scientific catalog no., NJ). Whole slides with stained sections were digitally scanned in an Aperio AT2 digital scanner (Leica Biosystems Inc., IL) at 40x (0.221 pm per pixel).

[0112] FibroNest was used for the quantification of Sirius red-positive collagen fibers from each image, where Sirus red-positive pixels are used to detect collagen. The whole-tissue fibrosis phenotypewas described for its collagen content and structure (12 traits), the morphometric traits of the segmented collagen fibers, and fibrosis architecture traits. The collective distribution of each trait was quantified with seven statistical parameters [quantitative Fibrosis Traits (qFTs)] to account for severity, progression, distortion, and variance for both fine and assembled collagens, resulting in a total of 315 qFTs. Principal qFTs were automatically detected using the mouse FAT- NASH fibrosis progression cohort consisting of 15 liver tissues (chow mice, n = 5; 6-week FAT- NASH mice, n = 2; 12-week FAT-NASH mice, n = 5; 24-week FAT-NASH mice, n = 5; FIG. 6D and 6E). Automatically selected principal qFTs were normalized and combined to form a continuous composite score for fibrosis severity in each phenotypic layer (collagen content, fibers morphometry, and fibrosis architecture) and for the whole fibrosis phenotype (Ph-CFS). In addition, collagen fibers were classified as "fine" or "assembled" on the basis of the complexity of their skeleton, and the morphometric phenotypes can be quantified for each subgroup. The relative changes of each qFT between animals and groups were visualized in the form of a heatmap.

[0113] Single nuclei RNA-seq and data processing

[0114] For snRNA-seq of mouse samples and human samples collected at Mount Sinai (CTRL 1- 3, NASH 6-9), 40 to 60 mg of total liver tissue were chopped on ice in 1 ml of 0.03% TST buffer [146mMNaCl, 10 mM Tris-HCl (pH 7.5), 1 mM CaC12, and 21 mM MgC12 with freshly added 0.03% Tween 20, 0.01% BSA, and RiboLock RNase Inhibitor (0.4 U/ml; Thermo Fisher Scientific FEREO0382)] with Tungsten Noyes Spring Scissors (FST 15514-12). The resulting nuclei suspension was then filtered through 40-pm cell strainers (Thermo Fisher Scientific 352340) into a fresh 50-ml conical tube on ice. One more milliliter of 0.03% TST was used to rinse the cell strainer, and 3 ml of ST buffer were added to the nuclei suspension, followed by briefly mixing by gentle flicking. The nuclei suspensions were spun at 500g for 5 min at 4°C in a swing-bucket centrifuge. The final nuclei pellet was suspended in 200 pl of 0.4% BSA in PBS by pipetting 30 times with a regular p 1000 tip. snRNA-seq datasets from two control mouse livers (“Chow_l”and “Chow_2”) can be found at GEO accession no. GSE212327. For “NASH” and “NASH_HCC” samples, 20 mg of liver from three FAT-NASH mice were pooled for each sample and processed together in an attempt to average out heterogeneity associated with the diseased liver.For snRNA- seq of human samples collected at Gordian (NASH 1-5), frozen human liver tissue (~60mm3) was lysed by Dounce homogenization (Kimble 2 ml: 20 times with Pestle A over ~60 s) in 2 ml of lysis buffer [10 mM tris (pH 7.0), 10 mM NaCl, 3 mM MgC12, 0.05% Triton X-100, 0.13% RNAse Inhibitor (Enzymatics Y9240L), and 0.25% Superase RNAse Inhibitor (Thermo Fisher Scientific AM2694)] with 45 pM Actinomycin D and then dripped through a 40-pm cell strainer (Celltreat 229481) into a protein low-binding 15-ml centrifuge tube, and the filter was washed into the collection tube with another 2 ml of lysis buffer. After 5-min total exposure time to lysis buffer, samples were spun for 2 min at 300g at 4°C; the supernatant was aspirated from the top first to remove fat, and then the miclei-containing pellet was resuspended in 5 ml of wash buffer (Dulbecco's phosphate buffered saline + 2% BSA + 0.13% Enzymatics RNAse Inhibitor + 0.25% Superase RNAse Inhibitor +3 (1M actinomycin D) and filtered again through a 35-pm strainer into a 5-ml fluorescence-activated cell sorting (FACS) tube. Nuclei were sorted into a 1.7-ml protein low-binding microcentrifuge tube precoated with 900 pl of wash buffer on a Bio-Rad S3e FACS using FSC/SSC > FSC-H versus FSC-W > FL4 versus SSC gating to eliminate debris. Nuclei were pelleted at 300g for 3 min at 4°C in a swing-bucket centrifuge and resuspended in wash buffer for lOx capture using an LT200 pipettor.

[01 15] Nuclei preparations were processed by the Chromium 3' Gene Expression V2 Kit (for mouse samples) or Chromium 3' Gene Expression V3 Kit (for human samples) according to the manufacturer’s guidelines. Qubit 3 (Fisher Scientific) and 2100 Bioanalyzer (Agilent Technologies) were used for quality check of complementary DNA. Libraries were sequenced on the NovaSeq at Sinai or NextSeq 550 at Gordian. To generate a count matrix, the inventors processed the sequenced files from each independent sample through 10X Genomics Cell Ranger software v6. The raw base call files were demultiplexed using Cell Ranger mkfastq pipeline to generate FASTQ files. Cellranger count pipeline was applied to the FASTQs to perform alignment against modified transcriptomes based on the mmlO and GRCh38 reference builds for mice and humans, respectively, that contain introns to increase mapping efficiency and the number of genes detected.

[0116] Filtered feature-barcode matrices from Cell Ranger were subsequently run through a standard Seurat pipeline for QC. Different QC parameters were used for snRNA-seq data generated using Chromium 3 ' Gene Expression V2 (mouse samples) and V3 (human samples) Kits using Seurat. For snRNA-seq generated from mouse samples, we included nuclei that fit the following criteria: (i) total number of expressed genes greater than 300 and less than 6000, (ii) <5% of which annotated as mitochondrial, and (iii) total counts greater than 500 and less than 15000.

[01 17] Bulk RNA-seq and data processing [0118] Bulk RNA-seq from LX-2 cells was carried out by a commercial vendor (Novogene). Briefly, RNA-seq libraries from LX-2 cells were prepared with polyA capture. RNA-seq libraries were prepared according to the Illumina NovaSeq RNA sample preparation protocol. RNA pooled from two wells on 24-well plates was used to generate libraries, which were analyzed on an Agilent 2100 Bioanalyzer. One hundred fifty base-pair paired-end reads that passed QC were aligned to reference genome GRCh38. After alignment, read counts were generated and analyzed using the DESeq2 for differential gene expression. Three biological replicates were adopted for all experiments.

[0119] Dimensionality reduction, clustering, and visualization

[0120] For mouse snRNA-seq datasets, the inventors normalized and transformed the filtered count matrix as per standard Seurat pipeline. The inventors performed dimension reduction using the top 2000 highly variable genes, performed principal components analysis on the top 30 principal components and clustered using Louvain community detection algorithm with resolution of 0.3. Mouse samples from different conditions were merged using SeuratWrappers/LIGER into a single dataset for downstream data analysis. Processing of human snRNA-seq datasets is shown in accompanying scripts from Jupyter notebook. The inventors identified cluster- specific gene expression by differential gene expression of nuclei in the cluster versus all other nuclei using Wilcoxon rank sum test and manually assigned cell types based on top differentially expressed genes. The inventors visualized the reduced dimensionality data using the same principal components as previously used for clustering and UMAP. For Gene Ontology analysis, differentially expressed genes between cell clusters (Wilcoxon rank sum test, Padj. < 0.05) were input into the AmiG02 web server (http://amigo.geneontology.org/amigo) as well as all significantly enriched “PANTHER GO-Slim Biological Processes”) pathways (FDR < 0.05) with more than one gene reported. Predicting and visualizing cell-cell interactions

[0121] Filtered and normalized feature-barcode matrices of mouse and human snRNAseq datasets along with cell type annotation files were exported from Seurat/R and input into CellphoneDB/Python. Mouse gene names were converted to orthologous human gene names based on the Ensembl database, only genes with unique human orthologues were kept for downstream analysis. Default in put parameters were used for statistical analysis. For mouse datasets, significant autocrine receptor-ligand interactions (pvalue < 0.05) for all cell clusters (table not shown). For human datasets, significant autocrine ligand-receptor interactions (pvalue < 0.05) for all cell clusters from NASH or CTRL patient (tables not shown). Cytoscape is used visualize the number of ligand-receptor interactions between cell types, with the thickness of the connecting line directly proportional to the number of significant interactions between each pair of interacting cell types.

[0122] Statistical analysis

[0123] Results are shown as means ± standard deviation unless indicated otherwise. Statistical analysis was performed using Prism unless otherwise specified. Analysis involving multiple groups was performed using one-way or two-way ANOVA, followed by post hoc t test.

[0124] LOXO-195 mechanistic studies in FAT-NASH mouse livers

[0125] Male mice on the FAT-NASH model for 24 weeks were treated with LOXO-195 (50mg/kg twice daily), OCA (obeticholic acid, 30mg/kg once daily), or vehicle control (twice daily), 5 days/week for 4 weeks while continuing the FAT-NASH model. Mice treated with LOXO-195 display increased Cd68 staining for macrophages in the liver compared to vehicle treated mice. OCA treated mice which is not expected to restore ‘hot fibrosis’ showed reduced Cd68 staining compared to vehicle control. Typical staining is shown for vehicle (FIG. 15A), LOXO-195 (FIG. 15B), and OCA (FIG. 15C) treated mice along with quantification across N=9-ll mice per group (FIG. 15D).

[0126] Increased Cd68 staining in LOXO-195 treated male mice compared to vehicle treated controls at 28 weeks of FAT-NASH model, consistent with restoration of ‘hot fibrosis’ that is predicted to increase matrix degradation and fibrosis resolution.

[0127] NTRK3 antagonism in NASH-HCC in FAT-NASH mouse model

[0128] Male mice on the FAT-NASH model for 24 weeks were treated with LOXO-195 (50mg/kg twice daily), OCA (obeticholic acid, 30mg/kg once daily), or vehicle control (twice daily), 5 days/week for 4 weeks while continuing the NASH model. Mice treated with LOXO-195 led to a non-significant decrease in liver tumors compared to vehicle treated mice. OCA treated mice display significantly reduced overall tumor burden compared to vehicle control. n=9-l 1 mice per group. * P < 0.05 by one-way ANOVA followed by Dunnett’s multiple comparisons test.

[0129] As shown in FIG. 16, there is a towards decreased tumor burden in LOXO-195 treated male mice compared to vehicle treated controls following only 4 weeks of treatment in very advanced stage of FAT-NASH model.

[0130] LOXO-195 PK studies in mice

[0131] Male mice were treated with a single dose of LOXO-195 (lOmg/kg PO). Blood and tissue samples were collected from a set of three mice at 0.25, 1, 2, 4, 8 and 24 h (average values potted in (FIG. 17)). LOXO-195 in plasma, brain, liver, kidney, heart and lung is quantified by protein precipitation and LC-MS/MS. Peak LOXO-195 concentration (Cmax) measured in plasma and various tissues are summarized in Table 3.

[0132] Table 3.

[0133] High levels of LOXO-195 were found in the liver, kidney, heart, and lungs. The plasma half-life is 2.78 hrs, so newer NTRk3 inhibitors that have longer half-life and are liver-targeted are under development.

[0134] LOXO-195 validation studies in human precision cut liver slices

[0135] Precision cut liver slice (PCLS) from a control human patient were treated with vehicle (FIG. 18A), 250nM (FIG. 18B), or 25mM (FIG. 18C) of LOXO-195 for 48hrs. PCLS samples were OCT embedded, cryo- sectioned, and stained with aSMA antibody. Reduction of aSMA-i- area observed with PCLS treated with 25mM of LOXO-195 (N=l, FIG. 18D).

[0136] Reduced HSC activation as measured using aSMA staining in human PCLS (N=l). There was no overt toxicity as measured by albumin and LDH in media.

[0137] References

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[0138] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0139] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method of treating or preventing a disease or disorder associated with and/or caused by neurotrophic tyrosine receptor kinase, comprising administering at least one neurotrophic tyrosine receptor kinase (Trk) inhibitor, salt thereof, or a composition comprising the Trk inhibitor or salt thereof to a subject in need thereof.

2. The method of claim 1, wherein the at least one neurotrophic tyrosine receptor kinase inhibitor targets Neurotrophic Tyrosine Receptor Kinase 1 (NTRK1), Neurotrophic Tyrosine Receptor Kinase 2 (NTRK2), and/or Neurotrophic Tyrosine Receptor Kinase 3 (NTRK3).

3. The method of claim 1 or 2, wherein the disease is a liver disease.

4. The method of claim 3, wherein the liver disease is selected from the group consisting of liver fibrosis, liver cirrhosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease, Hepatocellular carcinoma (HCC), and NASH fibrosis.

5. The method of any one of the preceding claims, wherein the tyrosine receptor kinase (Trk) inhibitor is selected from the group consisting of LOXO-195, entrectinib, RXDX-102, altiratinib, larotrectinib, sitravatinib, cabozantinib, merestinib, dovitinib, crizotinib, TSR-011, DS-6051, PLX7486, lestaurtinib, danusertib, F17752, AZD6918, AZD7451, and AZ-23.

6. The method of any one of the preceding claims, wherein the at least one neurotrophic tyrosine receptor kinase inhibitor targets scar tissue in the liver and/or fibrosis on the liver.

7. The method of any one of the preceding claims, wherein the Trk inhibitor is LOXO-195.

8. The method of any one of the preceding claims, wherein the subject is administered a pharmaceutically effective amount or therapeutically effective amount of the Trk inhibitor or the composition.

9. The method of any one of the preceding claims, wherein the Trk inhibitor or the composition is administered orally, parenterally, intradermally, subcutaneously, topically, rectally, nasally, bucccally, vaginally, subdermally, or ophthalmically.

10. The method of any one of the preceding claims, wherein the Trk inhibitor or the composition is orally administered in an orally acceptable dosage form selected from the group consisting of capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.

11. The method of any one the preceding claims, wherein the subject is a mammal, preferably a human.

12. The method of any one of the preceding claims, wherein the salt is selected from the group consisting of an acetate, L-aspartatc, bcsylatc, bicarbonate, carbonate, D-camsylatc, L- camsylate, citrate, edisylate, formate, fumarate, gluconate, hydrobromide/bromide, hydrochloride/chloride, D-lactate, L-lactate, D,L-lactate, D,L-malate, L-malate, mesylate, pamoate, phosphate, succinate, sulfate, bisulfate, D-tartrate, L-tartrate, D,L-tartrate, mesotartrate, benzoate, gluceptate, D-glucuronate, hybenzate, isethionate, malonate, methylsulfate, 2- napsylate, nicotinate, nitrate, orotate, stearate, tosylate, thiocyanate, acefyllinate, aceturate, aminosalicylate, ascorbate, borate, butyrate, camphorate, camphocarbonate, decanoate, hexanoate, cholate, cypionate, dichloroacetate, edentate, ethyl sulfate, furate, fusidate, galactarate, galacturonate, gallate, gentisate, glutamate, glutarate, glycerophosphate, heptanoate, hydroxybenzoate, hippurate, phenylpropionate, iodide, xinafoate, lactobionate, laurate, maleate, mandelate, methanesulfonate, myristate, napadisilate, oleate, oxalate, palmitate, picrate, pivalate, propionate, pyrophosphate, salicylate, salicylsulfate, sulfosalicylate, tannate, terephthalate, thiosalicylate, tribrophenate, valerate, valproate, adipate, 4-acetamidobenzoate, camsylate, octanoate, estolate, esylate, glycolate, thiocyanate, undecylenate, sodium, potassium, calcium, magnesium, zinc, aluminum, lithium, cholinate, lysinium, ammonium, troethamine, and a mixture thereof.

13. The method of any one of the preceding claims, wherein the composition further comprises one or more excipients, wherein the excipients are selected from the group consisting of anti-adherents, binders, coatings, disintegrants, fillers, flavors, dyes, colors, glidants, lubricants, preservatives, sorbents, sweeteners, and combinations thereof.