Targeted metabolic therapies for g34r gliomas

Abstract

Described herein are method of treating G34R mutant tumors in a subject in need thereof; for example, in a pediatric subject. Further disclosed are method of selecting treatment for a patient having a tumor, such as glioma. Non-human animal models and methods for drug screening are also described.

Description

TARGETED METABOLIC THERAPIES FOR G34R GLIOMASCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority to U.S. provisional patent application No. 63 / 604,084, filed November 29, 2023, the entirety of which is hereby incorporated by reference.FIELD OF INVENTION

[0002] This invention relates to the treatment of tumors, drug screening, and patient stratification and treatment selection.BACKGROUND

[0003] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Brain tumors are the most common solid tumors in children, having surpassed leukemia as the leading cause of cancer-related mortality in children. Pediatric high-grade gliomas (pHGGs) account for 15-20% of all brain tumors in children and are associated with a poor prognosis. Specific studies on these pediatric pathologies have led to the conclusion that gliomas in children differ from those that arise in adults. While commonly, adult cancer accumulates multiple genomic events that induce turn ori genesis, childhood cancers present a different tumorigenic pathway, namely epigenetic disruption, which causes a massive dysregulation of gene expression. Indeed, the value of molecular diagnostics in pHGGs tumor classification has been highlighted in the fifth edition of the WHO classification of CNS tumors. This new classification differentiates “Pediatric-type diffuse high-grade gliomas,” which are further subdivided into four subgroups based on their distinct molecular features, namely: diffusemidline glioma, H3K27-altered; diffuse hemispheric glioma, H3G34-mutant; diffuse pediatric- type high-grade glioma, H3-wildtype and IDH-wildtype; and infant-type hemispheric glioma.

[0005] Despite the remarkable similarities between the mutational landscape of K27M and G34R driven pediatric diffuse brain tumors, both tumors present striking differences in their development, location, and etiology. K27M gliomas appear in young children and are characterized as midline gliomas, which strongly complicate to completely remove them and accelerate the fatal outcome. On the other and, G34R gliomas are exclusively hemispheric tumors that usually appear in teenagers and young adults with a slow growth ratio.

[0006] Additionally, while the tumors occur most commonly in pediatric patients, the G34R mutations can occur in young adults and as well as in adults. (See e.g., Histone H3.3 G34- mutant Diffuse Gliomas in Adults, The American Journal of Surgical Pathology, 46(2):p 249- 257, February 2022; Characteristics of diffuse hemispheric gliomas, H3 G34-mutant in adults, Neuro-Oncology Advances, Volume 3, Issue 1, January-December 2021, vdab061).

[0007] Accordingly, there remains a need in the art for drug discovery, treatment selection and targeted treatment for gliomas, including these pediatric gliomas, which also occur in young adults and adults.SUMMARY OF THE INVENTION

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0009] Various embodiments of the invention provide for a method of treating a G34R mutant tumor, comprising: administering an arginine depleting agent, proline depleting agent, or both to a subject in need thereof.

[0010] In various embodiments, the subject can be a pediatric subject. In various embodiments, the subject can be an adult subject. In various embodiments, the subject can be a subject who is up to 35 years of age. In various embodiments, the subject can be a subject who is up to 33 years of age.

[0011] In various embodiments, the subject has been detected to have a G34R mutation.

[0012] In various embodiments, the subject has been detected to have a G34R mutation by a method comprising performing DNA or RNA sequencing of a biological sample obtained from the subject to detect the presence of the G34R gene mutation, or using an antibody to detect the presence of a protein or peptide translated from the G34R mutation in a biological sample obtained from the subject. In various embodiments, the biological sample can comprise tumor cells.

[0013] Examples of DNA and RNA sequencing include but are not limited to Sanger sequencing, fragment analysis, next-generation sequencing (NGS), RNA-Seq with nextgeneration sequencing (NGS), whole genome sequencing, nanopore-based DNA sequencing, shotgun sequencing, and high-throughput sequencing, total RNA Whole transcriptome, mRNA sequencing, smRNA sequencing, and Targeted RNA sequencing.

[0014] In various embodiments, the arginine depleting agent can comprise pegargiminase. In various embodiments, the arginine depleting agent can comprise arginine deiminase or human arginase I.

[0015] In various embodiments, the proline depleting agent can comprise LP-403812 (1142050-84-7) or a proline transport inhibitor.

[0016] In various embodiments, the G34R mutant tumor can be a glioma, giant cell tumor of the bone, or a chondroblastoma.

[0017] In various embodiment of the method, the G34R mutant tumor can be a G34R mutant glioma, the arginine depleting agent can comprise pegargiminase, and the subject can be a pediatric subject. In various embodiment of the method, the G34R mutant tumor can be a G34R mutant glioma, the arginine depleting agent can comprise pegargiminase, and the subject can be an adult subject. In various embodiment of the method, the G34R mutant tumor can be a G34R mutant glioma, the arginine depleting agent can comprise pegargiminase, and The subject can be up to 35 years of age. In various embodiment of the method, the G34R mutant tumor can be a G34R mutant glioma, the arginine depleting agent can comprise pegargiminase, and the subject can be up to 33 years of age.

[0018] In various embodiments, the method can further comprise administering a standard-of-care therapy to the subject. In various embodiments, the standard-of-care therapy can comprise tumor resection, chemotherapy, radiation, or combinations thereof. In variousembodiments, standard-of-care therapy can comprise temozolomide (TMZ), combination of lomustine (CCNU) and TMZ, combination of TMZ and radiation therapy, or combination of CCNU, TMZ and radiation therapy.

[0019] Various embodiments of the invention provide for a method of selecting a tumor therapy for a subject in need thereof, comprising detecting a G34R mutation in a biological sample from the subject; selecting a tumor therapy comprising an arginine depleting agent, a proline depleting agent, or both for the subject.

[0020] In various embodiments, the biological sample can comprise tumor cells.

[0021] In various embodiments, detecting the G34R mutation can comprise performingDNA or RNA sequencing of the biological sample to detect the presence of the G34R gene mutation, or using an antibody to detect the presence of a protein or peptide translated from the G34R mutation.

[0022] Examples of DNA and RNA sequencing include but are not limited to Sanger sequencing, fragment analysis, next-generation sequencing (NGS), RNA-Seq with nextgeneration sequencing (NGS), whole genome sequencing, nanopore-based DNA sequencing, shotgun sequencing, and high-throughput sequencing, total RNA Whole transcriptome, mRNA sequencing, smRNA sequencing, and Targeted RNA sequencing.

[0023] For example, the G34R mutation can be identified by pathology from a tumor tissue sample or cells obtained from the tumor. For example, G34R antibodies can be used to detect the presence of the mutation in tissue samples or cells obtained from the tumor.

[0024] In various embodiments, the subject can be a pediatric subject. In various embodiments, the subject can be an adult subject. In various embodiments, the subject can be a subject who is up to 35 years of age. In various embodiments, the subject can be a subject who is up to 33 years of age.

[0025] In various embodiments, the method can further comprise administering the arginine depleting agent, the proline depleting agent, or both to the subject. In various embodiments, the arginine depleting agent can comprise pegargiminase. In various embodiments, the arginine depleting agent can comprise arginine deiminase or human arginase I. In various embodiments, the proline depleting agent can comprise LP-403812 (1142050-84-7) or a proline transport inhibitor.

[0026] In various embodiments, the G34R mutant tumor can be a glioma, giant cell tumor of the bone, or a chondroblastoma.

[0027] In various embodiments, can further comprise administering the standard-of-care therapy comprising tumor resection, chemotherapy, radiation, or combinations thereof In various embodiments, standard-of-care therapy can comprise TMZ, combination of CCNU and TMZ, combination of TMZ and radiation therapy, or combination of CCNU, TMZ and radiation therapy.

[0028] Various embodiments of the invention provide for a system, comprising:(i) a single promoter-less donor vector, comprising: a polyadenylation signal or transcription stop element upstream from a nucleic acid, the nucleic acid, wherein the nucleic acid encodes a K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominant-active PdgfraD842V, dominant-negative Trp53R270H, or a combination thereof, and paired recombinase recognition sites, OR one or more promoter-less donor vectors, each promoter-less donor vector independently comprising a polyadenylation signal or transcription stop element upstream from a nucleic acid, the nucleic acid, wherein the nucleic acid encodes one or more of K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominant-active Pdgfral)X42V, dominantnegative Trp53R270H; and(ii) one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites, OR two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.

[0029] In various embodiments, the promoter-less donor vector can further comprise a post-transcriptional regulatory element, a polyadenylation signal downstream from the nucleic acid, or both.

[0030] In various embodiments of the system, the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp, the paired recombinase recognition sites are modified loxP and / or modified flippase recognition target (FRT), and the recombinases are ere and flp, the paired recombinase recognition sites are VloxP and flippase recognition target (FRT), and the recombinases are VCre and flp, the pairedrecombinase recognition sites are SloxP and flippase recognition target (FRT), and the recombinases are SCre and flp, the recombinase is PhiC31 recombinase and the recombinase recognition sites are attB and attP, the recombinase is Nigri, Panto, or Vika and recombinase recognition sites are nox, pox, and vox, respectively, or one or both of the paired recombinase recognition sites comprise a mutation.

[0031] In various embodiments of the system, the pA signal is PGKpA and trimerized SV40pA, the pA downstream from the nucleic acid post is a rabbit beta-globin pA, or the post- transcriptional regulatory element (WPRE) is a woodchuck hepatitis virus.

[0032] Various embodiments of the invention provide for a non-human animal made by administering any one of the systems of the present invention, and catalyzing a dual recombinase mediated cassette exchange in the non-human animal.

[0033] Various embodiments of the invention provide for a method of screening a test agent for its effect on a G34R mutant tumor, comprising: administering the test agent to a non- human model of the present invention; and measuring a parameter in the non-human animal model. In various embodiments, the method can further comprise identifying the test agent as a drug candidate or not a drug candidate for G34R mutant tumor based on the measured parameter. In various embodiments, the parameter comprises inhibition of tumor growth, promotion of tumor growth, induction of apoptosis of tumor cells, arginine metabolism, proline metabolism, upregulation or downregulation of arginine metabolism related genes, upregulation or downregulation of proline metabolism related genes, survival, immunophenotype changes, epigenome changes, transcriptome changes, or a combination thereof.

[0034] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0035] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0036] Figures 1A-1D depict MADR system in H3.3 derived pediatric glioma models overview. 1A) MADR pDonors schematic: the general structure is identical for the 3 plasmids, only differing in the specific mutation at the histone 3. IB) MADR reaction diagram, mTmG mouse cells express membrane-tdTomato in normal conditions; in the presence of Cre recombinase, floxed tdTomato is removed inducing the expression of membrane-GFP (Cre reporter normal behavior); by the addition of FlpO together with Cre recombinase, pDonor transgene can be integrated in a directional fashion starting the expression of the transgene under the control of the endogenous ROSA26 locus CAG promoter, this expression renders cells with nuclear-EGFP 1C) H3.3 pediatric tumor model variant generation and differential phenotype. Each pDonor is electroporated in a mixture 1 :0.1 with an expression vector of Cre and FlpO in the ventricles of pl-p2 mTmG heterozygous pups. Each tumor variant develops with their expected spatial ID) and temporal characteristic.

[0037] Figures 2A-2D show the analysis and integration of the H3.3 tumor variants scRNAseq datasets. 2A) UMAP embedding of all the samples integrated (G34R n=3, K27M n=6, H3.3 WT n=4). Cell types were identified based in their DGE profiles, the tumor cells are classified based in the transcriptional programs identified in (Filbin et al., Science. 2018). 2B) Tumor and non-tumor cells are clearly identified by the transgene or endogenous mRNAs expressions. 2C) UMAPs showing: first row, the expression of each transcriptional program from Filbin, M.G., et al., Developmental and oncogenic programs in H3K27M gliomas dissected by single-cell RNA-seq. Science, 2018. 360(6386): p. 331-335; second and third rows, representative markers for each cell type identified within the tumor stroma. 2D) Distribution of the cells within each tumor subtype

[0038] Figures 3A-3E show the tumor mass comparative at scRNAseq level. 3A) UMAP showing the overlapping of the 3 H3.3 tumor subtypes. 3B) RNA velocity analysis showing the theoretical transcriptional pathways of the cells within the tumor heterogeneity. 3C) RNA velocity-derived Latent time define poles within the UMAP embedding determining differential transcriptional profiles. 3D) percentages of cells within each transcriptional profile in the different H3.3 tumor variants. 3E) scRNAseq dataset derived clonality analysis with inferCNV and uPhiloplot2 for each of the scRNAseq datasets.

[0039] Figures 4A-4E show 4A) UMAP embedding of the snATACseq dataset of the 3 tumor datasets with inputted clustering based in their scRNAseq counterpart (up) and cell distribution of each tumor subtype in the UMAP embedding. 4B) Inputted genescore values of representative markers of each of the cell types inputted from the scRNAseq datasets. 4C) Transcription factor regulons differentially enriched by subtype in the snATACseq datasets. 4D) Correlation of TF Motif Delta vs Gene Score (left) and Gene Expression (right). 4E) SCENIC derived Transcript Factor regulons Heatmap of the scRNAseq datasets with correlated regulons with snATACseq analysis highlighted.

[0040] Figures 5A-5G show Methylation analysis of H3.3 variants tumor. 5A) Heatmap of the differentially methylated probes of each sample, probes and samples are unserpisedly clustered. 5B) 3D PCA plot showing each sample colored by tumor subtype. 5C) Hypomethylated gene signatures of each tumor subtype were used to generate DotPlot over the scRNAseq datasets, showing the percentage of cells that express the hypomethylation signature (size of the circles) and the level of expression (color of each circle from blue to yellow to red). 5D) Hypomethylation signature expression on each subtype at single cell levels in the UMAP embedding. 5E) KEGG pathways correlated with the hypomethylation signature of each tumor. 5F) DotPlot showing in each tumor subtype the percentage of cell and expression level of the genes composing the “Arginine and Proline Metabolism” KEGG pathway. 5G) In vitro measure of cell death in 2D cultures derived from MADR H3.3 tumors. * denote significant difference against the cell culture in GBO media (*, pvalue<0.05, **; pvalue<0.01). f denote significant differences against cell culture in Hank Basal salt solution supplemented with amino acids (f, pvalue<0.05; ff, pvalue<0.01)

[0041] Figures 6A-6E show MADR derived H3.3 pediatric brain tumor models vs Human datasets. 6A) Human K27M, G34R and H3.3 WT tumor scRNAseq integrated and harmony-dependent batch corrected in a UMAP embedding (left), Human and Mouse scRNAseq datasets integration at UMAP embedding (right). 6B) Human and mouse tumor subtypes integrated individually in a UMAP embedding (up). Expression of the transcriptional program determined in mouse scRNAseq datasets. 6C) HeatMap of the transcriptional programs adapted to include the new G34R and WT human datasets as well as the MADR derived models. 6D) Expression in the human H3.3 pediatric brain tumor datasets of the Hypomethylation signaturesderived from our MADR models. 6E) Expression levels of the genes that comprise the “arginine and proline metabolism” KEGG pathway by tumor subtype.

[0042] Figures 7A-7B show 7A) Root cells determined by the RNA velocity analysis with the package scVelo. 7B) Left: UMAP of the cell cycle state of each cell based in the cell cycle score module in the package Seurat. Right: Tumor cell types subdivisions based in the Root cells clusters as well as the cell cycles core and their differential expression.

[0043] Figures 8A-8B shows 8A) Heatmap of the differentially expressed genes by cell type in the tumor population. 8B) DotPlot showing the expression levels of module scores generated with the first 50 differentially expressed genes in each cell type.

[0044] Figures 9A-9C show 9A) Heatmap top 50 DGE by Fold change comparing H3.3 tumor subtypes. 9B) DotPlot of the Module Scores derived from the tumor subtypes DGEs in each subtype and 9C) in the different cell types.

[0045] Figures 10A-10C show 10A) DotPlot showing the cell types where each methylation module is more prevalent in all the tumor cells together and by tumor subtype. 10B) Asparaginase induced cell death measured in monolayer cultures of the different tumor subtypes (n=5). * pvalue<0.05, ** pvalue<0.01 10C) Cell death induced by amino acid starvation (n=6). t, ** pvalue<0.01. f Contrast of cell culture with and without amino acids; *Contrast of cell culture in complete media and HBSS with or without amino acids.

[0046] Figures 11A-11B show the survival curve of mice treated with or without pegargiminase. 11 A) Represent a consolidated image of all the different treatments together, including saline, radiotherapy and chemotherapy alone (control) or in combination with pegargiminase (ADI-PEG20). 11B) shows the different treatments with or without ADI-PEG20 treatment individually. The ongoing results shows a trend of increased survival on the groups treated with pegargiminase, especially prevalent in combination with radiotherapy alone or with the chemotherapeutics temozolomide and lomustine.DESCRIPTION OF THE INVENTION

[0047] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which thisinvention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0048] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0049] As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0050] “Promoterdess” as used herein with respect to vectors, refers to a vector that does not contain a eukaryotic promotor.

[0051] Treatment” and “treating,” as used herein refer to both therapeutic treatment wherein the object is to slow down and / or lessen the disease even if the treatment is ultimately unsuccessful.

[0052] The term “biological sample” as used herein denotes a sample taken or isolated from a biological organism. Exemplary biological samples include, but are not limited to cheek swab; mucus; whole blood, blood, serum; plasma; urine; saliva; semen; lymph; fecal extract; sputum; other body fluid or biofluid; cell sample; and tissue sample etc. The term also includes a mixture of the above-mentioned samples. In some embodiments, a biological sample is a tissue or tissue sample. In some embodiments, the biological sample is a tumor, tumor sample, tumor tissue, or tumor cells.

[0053] As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. The terms, “patient”, “individual” and “subject” are used interchangeably herein. In an embodiment, the subject is mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In some embodiments, the subject is a human. In some embodiments, the subject is a pediatric subject (e g., human child). In some embodiments, the subject is an adult subject. In some embodiments, the subject has a cancer. In some embodiments, the subject has a tumor.

[0054] “Pediatric” as used herein refers to a subject who is up to 21 years of age.

[0055] ‘Adult” as used herein refers to a subject who is 21 or more years of age.

[0056] Tumor heterogeneity must be addressed in order to completely understand the disease evolution and response to treatment. In this regard, application of single-cell based -omic technologies in pediatric brain tumor studies have allowed for a level of resolution never achieved before.

[0057] Brain cancer research has advanced significantly as a result of the development of novel platforms in vivo and in vitro. Genetically engineered mouse models (GEMMs) are excellent systems for accurately simulating tumor formation and development in gliomas. GEMMs generation, however, is costly and time-consuming; additionally, the intrinsic heterogeneity of brain tumors and the growing list of tumor gene drivers cannot be modeled in acceptable time frames by GEMMs. Platforms for direct delivery of tumor-driver genes based on genetically engineered viruses or electroporation (EP) have also quickly adapted, bringing efficient and faster methods to create highly personalized brain tumor models (e.g., EP with transposons). Unfortunately, viral and transposon-derived methods are vulnerable to random genomic integration, transgene silencing, and transgene copy number variability. CRISPR / Cas9 systems, on the other hand, allow for multiple knockouts in mice with the risk of unintended off- target genomic alterations.

[0058] We developed mosaic analysis using dual recombinase-mediated cassette exchange (MADR) technology to overcome these limitations. With genetic labeling of recombined cells, the MADR platform enables precise control of single-copy transgenesis. Furthermore, any cell line (human or murine) can be easily modified to function as a MADR acceptor cell line, allowing for more personalized and fine-tuned genetic engineering. MADR technology enables the precise generation of different types of murine gliomas that resemble and behave like their human counterparts by combining gain-of-function (GOF) and loss-of-function (LOF) genetic alternations in immunocompetent rodents. MADR tumor models have the potential to become a high-throughput preclinical platform for functionalizing many tumor driver mutations, allowing for a rapid pipeline for preclinical drug testing both in vivo and in vitro.

[0059] Described herein, we explored by comparing the differential characteristic of diffuse pediatric brain tumor based in their H3.3 genotype (H3f3aWT, H3f3aK27M, and H3f3aG34R). The three models generated by MADR technologies shared driver oncogenes signatures (PdgfraD842V, Trp53R270H) only varying in the mutations carried by the introduced H3f3a. This precise modelling allows to explore the differential effect of just a single nucleotide in the development and characteristics of the tumors. K27M harboring tumors appeared as fast-growing midline gliomas, developing a diffuse mass mainly in the Striatum; On the other hand, G34R tumors emerged as slower cortical tumor firstly invading the corpus callosum and then developing masses in the cortex; Finally, H3f3a WT tumors presented a greatly retarded development with survival curves twice as long as those observed in K27M and G34R tumors, these tumors invaded the corpus callosum as G34R tumors and from there slowly invaded mainly the striatum with some tumor presence in the cortex.

[0060] Described herein we analyze at single cell resolution the transcriptional and epigenomic particularities that cause the differential phenotype of H3.3 G34R-derived pediatric brain tumor in contrast with K27M and H3.3 WT tumors in our MADR derived murine models contrasting these results in human datasets as a tool to undercover the differential pathways and developmental processes implicated in their divergent tumor evolution.

[0061] The development of more efficient therapeutic approaches to pediatric brain tumors has been hindered by the lack of precise, faithful models. MADR technology have proven to be a reliable, fast and easily customizable method to model oncogenic process based in theintroduction of cancer-driver genomic alterations. Describe herein we explored multi-OMIC facets of pediatric diffuse gliomas such as H3.3 -dependent pediatric diffuse gliomas. scRNAseq and snATACseq analysis revealed a striking overlap of the 3 tumor variants despise their differential spatiotemporal phenotypes (Fig.l, panels C-D).

[0062] RNA velocity analysis undercover specific poles of differentiation within the tumor mass including transcriptional program described before, but also defining novel transcriptional profiles (Fig.3). The different tumors subtypes presented similar compositions of cells within each transcriptional profiles aside of a higher representation of oligo-like and cycling cells in the K27M tumors which goes in agreement with their more malignant phenotype. On the other hand, G34R tumors presented a higher percentage of cells within the Myeloid-like and High Hsp / Rbp transcriptional programs. The Myeloid program is highly relevant for the differential expression of markers of stem cells such as I16a / e which could reflect a different metabolic pathways and possible avenues of treatment for G34R tumor compared with K27M and H3.3 WT tumors. Additionally, H3.3 mutations promote a higher degree of copy number variations (figure 8) on our tumors compared to H3.3 WT which resulted in a higher clonality (Fig.3E). This genomic instability has been observed before in patients highlighting the value of these models in faithfully replicating the disease development.

[0063] While snATACseq datasets showed a less granular clustering of the tumor cells (Fig.4A), the analysis of the differential motif presence in each of the tumor subtypes undercover a set of transcription factors and regulons which are significantly enriched in G34R cells, interestingly this regulons where most apparent in the Myeloid-like transcriptional program cells (Fig.4E), highlighting the relevance of this program in the G34R tumor transcriptional regulation.

[0064] K27M mutant pHGGs display a global decrease of the repressive posttranslational histone modification H3K27me3, which under physiological conditions is mainly established by the H3K27-specific histone methyltransferase enhancer of zeste 2 (EZH2) within the Polycomb Repressive Complex 2 (PRC2). Mechanistically, reduction of H3K27me3 levels is caused by an inhibitory effect of the K27M mutant H3.3 protein.

[0065] The analysis of our samples showed a higher general hypomethylation state of K27M tumor in correlation what observed in human patients. Genome wide alterations of DNA methylation due to mutations in H3.3 in cancer have been reported before.

[0066] The Hypomethylation signatures extracted from this analysis clearly separate the scRNAseq datasets by tumor subtypes showing preferential expression in different transcriptional programs which highlighted the relevance of a promoted cell cycle in K27M tumors and the defining role of the Myeloid-like transcriptional program in G34R tumors.

[0067] KEGG pathway analysis of the 3 tumor variants highlighted an enrichment in the Hippo signaling pathway within the hypomethylation signature of K27M tumors. Hippo signaling pathway enrichment correlate with an enhanced proliferation and migration capacity which goes in agreement with the more malignant phenotype and higher cycling population of K27M tumors compared with G34R and H3.3 WT tumors (Fig.2A). On the other hand, G34R tumors hypomethylation signature was enriched in genes within the “arginine and proline metabolism” KEGG pathway. Arginine involvement in tumor biology have been explored. On one side several tumors show arginine addition phenotypes and the suppression of this pathway have been correlated with higher malignancy due to the relationship with increase proliferation. Moreover, this pathway has been also associated with the induction of immunosuppressive pathways through the recruitment and manipulation of myeloid suppressive cells. On the other hand, amino acid starvation coadjuvant therapies are being studied for several tumor pathologies. In this regard, the special sensitivity of G34R cells (Fig.5G) to amino acid starvation could be high jacked together with the current or novel therapeutic approaches to enhance the antitumor activity.

[0068] As such, various embodiments of the invention are based, at least in part, on these findings.Methods of Treatment

[0069] Various embodiments of the present invention provide for a method of treating a G34R mutant tumor, comprising: administering an arginine depleting agent, proline depleting agent, or both to a subject in need thereof.

[0070] In various embodiments, the subject is a pediatric subject. In other embodiments, the subject is an adult subject. In various embodiments, the subject can be a subject who is up to 35 years of age. In various embodiments, the subject can be a subject who is up to 33 years of age.

[0071] In various embodiments, the subject has been detected to have a G34R mutation (e.g., G34R mutant tumor). In various embodiments, the subject is a pediatric subject who has been detected to have a G34R mutation. In various embodiments, the subject is an adult subject who has been detected to have a G34R mutation. In various embodiments, the subject is a subject who is up to 35 years of age who has been detected to have a G34R mutation. In various embodiments, the subject is a subject who is up to 33 years of age who has been detected to have a G34R mutation. In various embodiments, the subject has been detected to have a G34R mutation by a method comprising performing DNA or RNA sequencing of a biological sample obtained from the subject to detect the presence of the G34R gene mutation, or using an antibody to detect the presence of a protein or peptide translated from the G34R mutation in a biological sample obtained from the subject. For example, the subject was previously detected to have the G34R mutation prior to administering the arginine depleting agent, proline depleting agent, or both, and thus, would be a subject who can benefit from or be susceptible the treatment.

[0072] Examples of DNA and RNA sequencing include but are not limited to Sanger sequencing, fragment analysis, next-generation sequencing (NGS), RNA-Seq with nextgeneration sequencing (NGS), whole genome sequencing, nanopore-based DNA sequencing, shotgun sequencing, and high-throughput sequencing, total RNA Whole transcriptome, mRNA sequencing, smRNA sequencing, and Targeted RNA sequencing.

[0073] The G34R mutation can be identified by pathology from a tumor tissue sample or cells obtained from the tumor. For example, G34R antibodies can be used to detect the presence of the mutation in tissue samples or cells obtained from the tumor.

[0074] In various embodiments, the biological sample comprises tumor cells from the subject.

[0075] Various embodiments of the present invention provide for a method of treating a G34R mutant tumor, comprising: identifying a subject having a G34R mutant tumor; and administering an arginine depleting agent, proline depleting agent, or both to the subject.

[0076] In various embodiments, the arginine depleting agent comprises pegargiminase. In various embodiments, the arginine depleting agent comprises arginine deiminase. In various embodiments, the arginine depleting agent comprises human arginase I.

[0077] In various embodiments, the proline depleting agent comprises LP-403812 (1142050-84-7) or a proline transport inhibitor.

[0078] In various embodiments, the G34R mutant tumor is a glioma. In various embodiments, the G34R mutant tumor is a giant cell tumor of the bone, or a chondroblastoma.

[0079] In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11- 20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / m2of arginine depleting agent or proline depleting agent or a combination thereof. In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21- 50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901- 1000 mg / m2of arginine depleting agent or proline depleting agent or a combination thereof. Here, “pg / m2” or “mg / m2” refers to pg or mg of agent per m2body surface area of the subject.

[0080] In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 1-5, 6-10, 11-20, 21-30, 31,-40, 41-50, 51- 60, 61-70, 71-80, 81-90, 91-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221- 240, 241-260, 261-280, 281-300, 301-350, 351-400, 401-450, 451-500, 501-600, 601-700, 701- 800, 801-900, or 901-1000 IU / m2of arginine depleting agent or proline depleting agent or a combination thereof. Here, “IU / m2” refers to IU of agent per m2body surface area of the subject. An International Unit (IU) is the amount of a substance that has a certain biological effect. There is an international agreement on the biological effect that is expected for 1 International Unit.

[0081] In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11- 20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / kg of arginine depleting agent or proline depleting agent or a combination thereof. In various embodiments, the effective amount of arginine depleting agent or prolinedepleting agent is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11 -20, 21 - 50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901- 1000 mg / kg of arginine depleting agent or proline depleting agent or a combination thereof. Here, “pg / kg” or “mg / kg” refers to pg or mg of agent per kg body weight of the subject.

[0082] These dosages can be given once per day, once every other day, once every 3 days, once per week, once every other week, once per month, once every other month, once every 3 months, once every 4 months, once every 5 months or once every 6 months.

[0083] In various embodiments, the duration of the treatment can be 1 day, 1 week, 2 weeks, 4 weeks, 6 week, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 15 weeks, 20 weeks, 24 weeks, 30 weeks, 36 weeks, 40 weeks, or 50 weeks. In various embodiments, the duration of treatment can be more than 50 weeks. In various embodiments, the duration of the treatment can be 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months 12 months, 15 months, 18 months, or 24 months. In various embodiments, the duration of treatment can be more than 24 months.

[0084] In various embodiments, a method of treating a G34R mutant tumor, comprises: administering pegargiminase (e.g., ADI-PEG20) to a subject in need thereof. In various embodiments, a method of treating a G34R mutant tumor, comprises: administering pegargiminase to a pediatric subject in need thereof. In various embodiments, a method of treating a G34R mutant tumor, comprises: administering pegargiminase to an adult subject in need thereof. In various embodiments, a method of treating a G34R mutant tumor, comprises: administering pegargiminase to a subject who is up to 35 years of age. In various embodiments, a method of treating a G34R mutant tumor, comprises: administering pegargiminase to a subject who is up to 33 years of age.

[0085] In various embodiments, a method of treating a G34R mutant tumor, comprises: identifying a subject having a G34R mutant tumor; and administering pegargiminase (ADI- PEG20) to the subject. In various embodiments, a method of treating a G34R mutant tumor, comprises: identifying a pediatric subject having a G34R mutant tumor; and administering pegargiminase (ADI-PEG20) to the pediatric subject.

[0086] In various embodiments, a method of treating a G34R mutant tumor, comprises: identifying a subject having a G34R mutant tumor who is up to 35 years of age; and administering pegargiminase (ADI-PEG20) to the subject. In various embodiments, a method of treating a G34R mutant tumor, comprises: identifying a subject having a G34R mutant tumor who is up to 33 years of age; and administering pegargiminase (ADI-PEG20) to the subject.

[0087] In various embodiments, the G34R mutant tumor is a glioma. In various embodiments, the G34R mutant tumor is a giant cell tumor of the bone, or a chondroblastoma.

[0088] In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / m2of pegargiminase. In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 mg / m2of pegargiminase. Here, “pg / m2” or “mg / m2” refers to pg or mg of pegargiminase per m2body surface area of the subject.

[0089] In various embodiments, the effective amount of pegargiminase is any one or more of about 1-5, 6-10, 11-20, 21-30, 31,-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101- 120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301- 350, 351-400, 401-450, 451-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 IU / m2pegargiminase. Here, “IU / m2” refers to IU of pegargiminase per m2body surface area of the subject. An International Unit (IU) is the amount of a substance that has a certain biological effect. There is an international agreement on the biological effect that is expected for 1 International Unit.

[0090] In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / kg, or a combination thereof. In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 mg / kg ofpegargiminase. Here, “pg / kg” or “mg / kg” refers to pg or mg of pegargiminase per kg body weight of the subject.

[0091] These dosages can be given once per day, once every other day, once every 3 days, once per week, once every other week, once per month, once every other month, once every 3 months, once every 4 months, once every 5 months or once every 6 months.

[0092] In various embodiments, the duration of the treatment can be 1 day, 1 week, 2 weeks, 4 weeks, 6 week, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 15 weeks, 20 weeks, 24 weeks, 30 weeks, 36 weeks, 40 weeks, or 50 weeks. In various embodiments, the duration of treatment can be more than 50 weeks. In various embodiments, the duration of the treatment can be 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months 12 months, 15 months, 18 months, or 24 months. In various embodiments, the duration of treatment can be more than 24 months.

[0093] In various embodiments, the method further comprises administering a standard- of-care therapy to the subject. In various embodiments, the standard-of-care therapy comprises tumor resection, chemotherapy (e.g., temozolomide, CCNU), radiation, or combinations thereof.

[0094] Additional examples of chemotherapeutic agents include cytotoxic agents e.g., 5- fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin (Adriamycin®), vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic akylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis-platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, di anhydrogal actitol, fluorodopan, hepsulfam, hycanthone, iphosphamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxirone, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic agents (e.g., allocolchicine, HalichondrinM, colchicine, colchicine derivatives, dolastatin 10, maytansine, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mitramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine and taxotere), biologicals (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2), topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantron, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26 and VP-16), and synthetics (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis- diamminedichloroplatimun, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel and porfimer sodium).

[0095] In various embodiments, the method further comprises administering TMZ to the subject. In various embodiments, the method further comprises administering CCNU and TMZ to the subject. In various embodiments, the method further comprises administering radiation therapy to the subject in need thereof. In various embodiments, the method further comprises administering TMZ and radiation therapy to the subject in need thereof. In various embodiments, the method further comprises administering CCNU, TMZ and radiation therapy to the subject in need thereof.

[0096] In various embodiments, the subject does not have prostate cancer, metastatic prostate cancer, sarcoma, soft tissue sarcoma, leukemia, acute myeloid leukemia, non-small cell lung cancer, metastatic non-small cell lung cancer, small cell lung cancer, extensive stage, small cell lung cancer, Non-Hodgkin’s lymphoma, or leiomyosarcoma. In various embodiments, the subject does not have bladder cancer, breast cancer, gastrointestinal cancer, head and neck cancer (i.e. mouth, sinuses, nose, or throat), lymphoma, non-small cell lung cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, sarcoma, or uveal melanoma.

[0097] In various embodiments, the subject is not a subject who has not undergone identification for a G34R mutation. In various embodiments, the subject does not have glioblastoma. In various embodiments, if the subject has glioblastoma, the method does notinclude treating glioblastoma that has not been genetically profiled (e.g., assayed for possible mutations). In various embodiments, the subject does not have argininosuccinate synthetase (ASSl)-deficient high-grade glioma (HGGs) or recurrent AS SI -deficient HGG. In various embodiments, the subject does not have epimutations of argininosuccinate lyase (AS ) highgrade glioma (HGG).

[0098] Various embodiments of the invention provide for a method of selecting a tumor therapy for a subject in need thereof, comprising detecting a G34R mutation in a biological sample from the subject; selecting a tumor therapy comprising an arginine depleting agent, a proline depleting agent, or both for the subject.

[0099] In various embodiments, the biological sample comprises tumor cells. In various embodiments, the biological sample comprises glioma cells. In various embodiments, the biological sample comprises cells from a giant cell tumor of the bone or chondroblastoma cells. In various embodiments, the biological sample can be any biological sample as provided herein.

[0100] In various embodiments, detecting the G34R mutation comprises performing DNA or RNA sequencing of biological sample to detect the presence of the G34R gene mutation. Examples of DNA and RNA sequencing include but are not limited to Sanger sequencing, fragment analysis, next-generation sequencing (NGS), RNA-Seq with nextgeneration sequencing (NGS), whole genome sequencing, nanopore-based DNA sequencing, shotgun sequencing, and high-throughput sequencing, total RNA Whole transcriptome, mRNA sequencing, smRNA sequencing, and Targeted RNA sequencing.

[0101] In various embodiments, detecting the G34R mutation comprises using an antibody to detect the presence of a protein or peptide translated from the G34R mutation. For example, the G34R mutation can be identified by pathology from a tumor tissue sample or cells obtained from the tumor. In various embodiments, detecting the G34R mutation comprises using an antibody to detect the presence of a protein or peptide translated from the G34R mutation. For example, G34R antibodies can be used to detect the presence of the mutation in tissue samples or cells obtained from the tumor.

[0102] In various embodiments, the method further comprises administering the arginine depleting agent, the proline depleting agent, or both to the subject.

[0103] In various embodiments, the arginine depleting agent comprises pegargiminase. In various embodiments, the arginine depleting agent comprises arginine deiminase. In various embodiments, the arginine depleting agent comprises human arginase I. In various embodiments, the proline depleting agent comprises UP -403812 (1142050-84-7) or a proline transport inhibitor.

[0104] In various embodiments, the subject is a pediatric subject. In various embodiments, the subject is an adult subject. In various embodiments, the subject can be a subject who is up to 35 years of age. In various embodiments, the subject can be a subject who is up to 33 years of age.

[0105] In various embodiments, the G34R mutant tumor is a glioma, giant cell tumor of the bone, or a chondroblastoma.

[0106] In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11- 20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / m2of arginine depleting agent or proline depleting agent or a combination thereof. In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21- 50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901- 1000 mg / m2of arginine depleting agent or proline depleting agent or a combination thereof. Here, “pg / m2” or “mg / m2” refers to pg or mg of agent per m2body surface area of the subject.

[0107] In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 1-5, 6-10, 11-20, 21-30, 31,-40, 41-50, 51- 60, 61-70, 71-80, 81-90, 91-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221- 240, 241-260, 261-280, 281-300, 301-350, 351-400, 401-450, 451-500, 501-600, 601-700, 701- 800, 801-900, or 901-1000 IU / m2of arginine depleting agent or proline depleting agent or a combination thereof. Here, “IU / m2” refers to IU of agent per m2body surface area of the subject. An International Unit (IU) is the amount of a substance that has a certain biological effect. There is an international agreement on the biological effect that is expected for 1 International Unit.

[0108] In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201-300, 301-400, 401 -500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / kg, or a combination thereof. In various embodiments, the effective amount of arginine depleting agent or proline depleting agent is any one or more of about 0.001-0.01, 0.02- 0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 mg / kg, or a combination thereof. Here, “pg / kg” or “mg / kg” refers to pg or mg of agent per kg body weight of the subject.

[0109] In embodiments wherein the arginine depleting agent is pegargiminase, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6- 5, 6-10, 11-20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / m2, or a combination thereof. In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801- 900, or 901-1000 mg / m2, or a combination thereof. Here, “pg / m2” or “mg / m2” refers to pg or mg of pegargiminase per m2body surface area of the subject.

[0110] In embodiments wherein the arginine depleting agent is pegargiminase, the effective amount of pegargiminase is any one or more of about 1-5, 6-10, 11-20, 21-30, 31,-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201- 220, 221-240, 241-260, 261-280, 281-300, 301-350, 351-400, 401-450, 451-500, 501-600, 601- 700, 701-800, 801-900, or 901-1000 IU / m2of pegargiminase. Here, “IU / m2” refers to IU of pegargiminase per m2body surface area of the subject. An International Unit (IU) is the amount of a substance that has a certain biological effect. There is an international agreement on the biological effect that is expected for 1 International Unit.[0U1] In embodiments wherein the arginine depleting agent is pegargiminase, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6- 5, 6-10, 11-20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / kg, or a combination thereof. In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801- 900, or 901-1000 mg / kg, or a combination thereof. Here, “pg / kg” or “mg / kg” refers to pg or mg of pegargiminase per kg body weight of the subject.

[0112] These dosages can be given once per day, once every other day, once every 3 days, once per week, once every other week, once per month, once every other month, once every 3 months, once every 4 months, once every 5 months or once every 6 months. In various embodiments, the duration of the treatment can be 1 day, 1 week, 2 weeks, 4 weeks, 6 week, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 15 weeks, 20 weeks, 24 weeks, 30 weeks, 36 weeks, 40 weeks, or 50 weeks.

[0113] In various embodiments, the duration of treatment can be more than 50 weeks. In various embodiments, the duration of the treatment can be 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months 12 months, 15 months, 18 months, or 24 months. In various embodiments, the duration of treatment can be more than 24 months.

[0114] Various embodiments of the invention provide for a method of selecting pegargiminase for a subject in need thereof, comprising detecting a G34R mutation in a biological sample from the subject; selecting pegargiminase for the subject. In various embodiments, the method further comprises administering the pegargiminase to the subject.

[0115] In various embodiments, the biological sample comprises tumor cells. In various embodiments, the biological sample comprises glioma cells. In various embodiments, the biological sample comprises cells from a giant cell tumor of the bone or chondroblastoma cells. In various embodiments, the biological sample can be any biological sample as provided herein.

[0116] In various embodiments, the subject is a pediatric subject. In various embodiments, the subject is an adult subject. In various embodiments, the subject can be a subject who is up to 35 years of age. In various embodiments, the subject can be a subject who is up to 33 years of age.

[0117] In various embodiments, detecting the G34R mutation comprises performing DNA or RNA sequencing of biological sample to detect the presence of the G34R gene mutation. Examples of DNA and RNA sequencing include but are not limited to Sanger sequencing, fragment analysis, next-generation sequencing (NGS), RNA-Seq with nextgeneration sequencing (NGS), whole genome sequencing, nanopore-based DNA sequencing, shotgun sequencing, and high-throughput sequencing, total RNA Whole transcriptome, mRNA sequencing, smRNA sequencing, and Targeted RNA sequencing.

[0118] In various embodiments, detecting the G34R mutation comprises using an antibody to detect the presence of a protein or peptide translated from the G34R mutation. For example, the G34R mutation can be identified by pathology from a tumor tissue sample or cells obtained from the tumor. In various embodiments, detecting the G34R mutation comprises using an antibody to detect the presence of a protein or peptide translated from the G34R mutation. For example, the G34R mutation can be identified by pathology from a tumor tissue sample or cells obtained from the tumor. For example, G34R antibodies can be used to detect the presence of the mutation in tissue samples or cells obtained from the tumor.

[0119] In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 pg / m2, or a combination thereof. In various embodiments, the effective amount of pegargiminase is any one or more of about 0.001-0.01, 0.02-0.1, 0.2-0.5, 0.6-5, 6-10, 11-20, 21-50, 51-100, 101-200, 201- 300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 mg / m2, or a combination thereof. Here, “pg / m2” or “mg / m2” refers to pg or mg of pegargiminase per m2body surface area of the subject.

[0120] In various embodiments, the effective amount of pegargiminase is any one or more of about 1-5, 6-10, 11-20, 21-30, 31,-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101- 120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301- 350, 351-400, 401-450, 451-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 IU / m2, or a combination thereof. Here, “IU / m2” IU of agent per m2body surface area of the subject. An International Unit (IU) is the amount of a substance that has a certain biological effect. There is an international agreement on the biological effect that is expected for 1 International Unit.

[0121] These dosages can be given once per day, once every other day, once every 3 days, once per week, once every other week, once per month, once every other month, once every 3 months, once every 4 months, once every 5 months or once every 6 months.

[0122] In various embodiments, the duration of the treatment can be 1 day, 1 week, 2 weeks, 4 weeks, 6 week, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 15 weeks, 20 weeks, 24 weeks, 30 weeks, 36 weeks, 40 weeks, or 50 weeks. In various embodiments, the duration of treatment can be more than 50 weeks. In various embodiments, the duration of the treatment can be 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 9 months 12 months, 15 months, 18 months, or 24 months. In various embodiments, the duration of treatment can be more than 24 months.

[0123] In various embodiments, the method further comprises administering a standard- of-care therapy to the subject. In various embodiments, the standard-of-care therapy comprises tumor resection, chemotherapy (e.g., temozolomide, CCNU), radiation, or combinations thereof.

[0124] In various embodiments, the method further comprises administering TMZ to the subject. In various embodiments, the method further comprises administering CCNU and TMZ to the subject. In various embodiments, the method further comprises administering radiation therapy to the subject in need thereof. In various embodiments, the method further comprises administering TMZ and radiation therapy to the subject in need thereof. In various embodiments, the method further comprises administering CCNU, TMZ and radiation therapy to the subject in need thereof.

[0125] Various embodiments of the invention provide for a system, comprising: (i) a single promoter-less donor vector, comprising: a polyadenylation signal or transcription stop element upstream from a nucleic acid, the nucleic acid, wherein the nucleic acid encodes a K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominant-active PdgfraD842V, dominant-negative Trp53R270H, or a combination thereof, and paired recombinase recognition sites, and (ii) one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites.

[0126] Various embodiments of the invention provide for a system, comprising: (i) a single promoter-less donor vector, comprising: a polyadenylation signal or transcription stop element upstream from a nucleic acid, the nucleic acid, wherein the nucleic acid encodes a K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominant-active PdgfraD842V, dominant-negative Trp53R270H, or a combination thereof, and paired recombinase recognition sites, and (ii) two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.

[0127] Various embodiments of the invention provide for a system, comprising: (i) one or more promoter-less donor vectors, each promoter-less donor vector independently comprising a polyadenylation signal or transcription stop element upstream from a nucleic acid, the nucleic acid, wherein the nucleic acid encodes one or more of K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominant-active PdgfraD842V, dominant-negative Trp53R270H; and (ii) one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites,

[0128] Various embodiments of the invention provide for a system, comprising: (i) one or more promoter-less donor vectors, each promoter-less donor vector independently comprising a polyadenylation signal or transcription stop element upstream from a nucleic acid, the nucleic acid, wherein the nucleic acid encodes one or more of K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominant-active PdgfraD842V, dominant-negative Trp53R270H; and (ii) two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.

[0129] These systems are combinations of components that can be used together in accordance with various embodiments of the invention.

[0130] In various embodiments, the promoter-less donor vector further comprises a post- transcriptional regulatory element. In various embodiments, the promoter-less donor vector further comprises a polyadenylation signal downstream from the nucleic acid.

[0131] In various embodiments, the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp. In various embodiments, the paired recombinase recognition sites are modified loxP and / or modified flippase recognition target (FRT), and the recombinases are ere and flp. In various embodiments, the paired recombinase recognition sites are VloxP and flippase recognition target (FRT), and the recombinases are VCre and flp. In various embodiments, the paired recombinase recognition sites are SloxP and flippase recognition target (FRT), and the recombinases are SCre and flp. In various embodiments, the recombinase is PhiC31 recombinase and the recombinase recognition sites are attB and attP. In various embodiments, the recombinase is Nigri, Panto, or Vika andrecombinase recognition sites are nox, pox, and vox, respectively. In various embodiments, one or both of the paired recombinase recognition sites comprise a mutation.

[0132] In various embodiments, the pA signal is PGKpA and trimerized SV40pA. In various embodiments, the pA downstream from the nucleic acid post is a rabbit beta-globin pA. In various embodiments, the post-transcriptional regulatory element (WPRE) is a woodchuck hepatitis virus.

[0133] Various embodiments of the invention provide for a non-human animal made by administering the system of the present invention as described herein, and catalyzing a dual recombinase mediated cassette exchange in the non-human animal.

[0134] In various embodiments, the non-human animal is a mouse or rat.

[0135] Various embodiments of the invention provide for a method of screening a test agent for its effect on a G34R mutant tumor, comprising: administering the test agent to a non- human model of the present invention as described herein; and measuring a parameter in the non-human animal model.

[0136] In various embodiments, the non-human animal is a mouse or rat.

[0137] In various embodiments, the method further comprises identifying the test agent as a drug candidate or not a drug candidate for G34R mutant tumor based on the measured parameter.

[0138] In various embodiments, the parameter comprises inhibition of tumor growth, promotion of tumor growth, induction of apoptosis of tumor cells, arginine metabolism, proline metabolism, upregulation or downregulation of arginine metabolism related genes, upregulation or downregulation of proline metabolism related genes, survival, immunophenotype changes, epigenome changes, transcriptome changes, or a combination thereof.

[0139] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of arginine depleting agent, the proline depleting agent, or both. In various embodiments, the present invention provides pharmaceutical compositions including apharmaceutically acceptable excipient along with a therapeutically effective amount of pegargiminase. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous.

[0140] In certain embodiments, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts, esters, amides, and prodrugs” as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use of the compounds of the invention. The term “salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. These may include cations based on the alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylanunonium, tetraethyl ammonium, methyl amine, dimethyl amine, trimethylamine, triethylamine, ethylamine, and the like (see, e.g., Berge S. M., et al. (1977) J. Pharm. Sci. 66, 1, which is incorporated herein by reference).

[0141] The term “pharmaceutically acceptable esters” refers to the relatively nontoxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. Theterm is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters.

[0142] As used herein, “pharmaceutically acceptable salts or prodrugs” are salts or prodrugs that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0143] The term “prodrug” refers to compounds that are rapidly transformed in vivo to yield the functionally active arginine depleting agent or the proline depleting agent as disclosed herein. As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. A prodrug of the one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof can be designed to alter the metabolic stability or the transport characteristics of one or more peptides as disclosed herein or a mutant, variant, analog or derivative thereof, to mask side effects or toxicity, to improve the flavor of a compound or to alter other characteristics or properties of a compound.

[0144] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral.

[0145] Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch.

[0146] “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrastemal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0147] Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlledrelease. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection.

[0148] Via the topical route, the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication.

[0149] Via the ocular route, they may be in the form of eye drops.

[0150] The pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0151] The pharmaceutical compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0152] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or nonaqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0153] The pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject’s response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).EXAMPLES

[0154] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.Example 1Material & Methods

[0155] Mice: The Cedars-Sinai Institutional Animal Care and Use Committee approved the use of all mice. Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J mice (JAX Mice) were bred with C57BL / 6J mice to generate heterozygous mice. Pups between postnatal day (P)0 and P3 were used for the postnatal experiments. Pregnant dams were kept in single cages and pups were kept with their mothers until P21, in the institutional animal facility under standard 12: 12 h light / dark cycles. IVF process

[0156] Mice and electroporation: Postnatal lateral ventricle EPs were performed. Before the procedure Pl -3 pups were placed on ice for ~5 min, to induce an hypotermic state to reduce pain and damage to the pups. All DNA mixtures contained Img / ml of Flp-Cre expression vector and MADR donor plasmid diluted in Tris-EDTA buffer, unless noted otherwise. Fast green dye was added (10%v / v) to the mixture, which was injected into the lateral ventricle. 5 pulses of 120 V (50ms; separated by 950 ms) from the ECM 830 System (Harvard Apparatus) were delivered by using Platinum Tweezertrodes. SignaGel was used to boost conductance. Mice were returned to their cages after being warmed by a heat lamp.

[0157] Tumor microdissection and dissociation: Mice that reach endpoint in their brain tumor development as stated by Cedars-Sinai approved protocols were euthanized and brain dissected. Brains were sliced to determine the presence and location of the tumors and, aided by the green fluorescent marking of the tumor against the red fluorescent background of the normal tissue, tumor tissue was microdissected from the brain.

[0158] Microdissected tumor tissues were chopped to pieces >lmm2of size and erythrocytes were removed by ACK lysis buffer incubation. A portion of the tumor pieces were immediately used to generate organoids and the rest of it was further dissociated to a single cell suspension for 2D cell cultures, scRNAseq and / or snATACseq.

[0159] Organoids generation and culture: Microdissected tumor pieces were culture at 37C in ultralow attachment 6-well plates in agitation in a organoids media (Neurobal-A media [Life Technologies 10888-022]: DMEM-F12 1 : 1, B27 supplement without Vitamin A [Life Technologies 12587-010], N2 supplement, GlutaMAX [Life Technologies 35050], Antibiotic- Antimycotic [Life Technologies 15240], Insulin, b-Mercaptoethanol). Pieces of tumor were observed for organoid generation for 1-2 weeks until spheres structures were formed considering this spherical cultures mature organoids. Organoid’s media was changed every other day andorganoids size was observed to chop excessively big organoids (>1.5mm diameter) to avoid central necrosis.

[0160] Cell lines generation and culture: Microdissected pieces of tumors were incubated at 37C in collagenase cocktail (collagenase IV, DNAse I and Trypsin inhibitor) for 45 minutes. Single cell suspensions were filtered through a 40mm cell strainer and culture in CELLstar CTS (Thermo Fisher Scientific, Waltham, MA) coated plates in organoid’s media supplemented with hEGF (Sigma E9644), bFGF (Millipore GF003), PDGF-AA (Shenandoah Biotechnology, Warwick, PA) and heparin (StemCell Technologies, Cambridge, MA).

[0161] Amino acid starvation assay: Cell lines derived from our MADR G34R, K27M and WT murine models were culture in 96 well plates to full confluency before start the assay, then media was removed and cells were culture in Hank’s Balanced Salt Solution (references) with or without amino acid, apoptosis was measure with the Incucyte Cas3 / 7 red reagent (references), cell necrosis was observed with a green necrosis reagent.

[0162] Cut&Tag: Tumor dissociation maintenance in culture for one to 5 passages were used to do Cut&Tag. To this end 100,000 cells of each dissociation was used per run and antibody. To analyze the epigenetic changes induced by the presence of the mutated histones a panel of histones marks were analyzed including H3K4me3, H3K27Ac, H3K27me3, H3K36me3 and H2aKl 19-Ubi. To compare the differential epigenetic targets of the mutated histones K27M and G34R antibodies were used in contrast with the other H3 modifications. Following EpiCypher protocol (version 1.7, revised February 16, 2022). Cut&Tag libraries were generated and quality check analysis on a tapestation confirmed positive reactions in contrast with the negative controls. ~5 million reads per library were used for sequencing and resultant datasets were analyzed through Partek software.

[0163] scRNAseq / snATACseq: Single cell suspensions of freshly dissociated tumors as well as freshly dissociated organoids were processed in the Chromium 10X platform following manufacturer's instructions to generate scRNAseq and or snATACseq. Library preps were sequence with a minimum of 400M reads depth.

[0164] DNA Methylation array: Mature organoids cultures were used to extract the DNA using the DNAease extraction kit (ref). DNA samples were used for a Methylation array assay.

[0165] scISO-Seq: Mature organoids of G34R, K27M and H3.3 WT mice tumors were dissociated to a single cell suspension and strained through a 40mm cell strainer and Chromium 10X protocol for scRNAseq was followed until step 3. cDNA libraries generated until this point, just before fragmentation were split in half continuing with one half with the scRNAseq protocol and using the other half to generate libraries for scISO-SeqResultsMADR technology allows to faithfully model H3.3 mutations derived Pediatric brain tumors

[0166] In order to mimic these pediatric brain tumors, 3 MADR plasmids donors (pDonor) were build carrying a different mutation of the H3.3. Alongside of the mutated / wt histone sequence in fusion with a eGFP protein another three oncogenes normally co-present in the human tumors were introduced, namely dominant-active PdgfraD842Vand dominant-negative Trp53R270H(Fig.lA). Following MADR procedure pl-2 mTmG heterozygous pups where electroporated with each pDonor to generate single-copy driven tumors (Fig.lB). Appropriate tumor spatial and temporal development was observed in concordance with the human modeled tumor variants (Fig.lC). Specifically, K27M tumor rapidly developed in the striatum with low or absent invasion of the cortex, while G34R tumors showed a delayed development with a primary invasion of the corpus callosum and cortex followed by a full brain invasion. H3.3 WT tumors were strongly delayed in comparison with both K27M and G34R carrying tumors, with a primary invasion of the corpus callosum followed with the invasion of the striatum but not the cortex (Fig.lC-D).K27M, G34R and H3.3 WT tumor single cell profiles strongly overlap

[0167] To explore the cellular heterogeneity in these three types of pediatric diffuse gliomas we analyze their transcriptomic and epigenomic profiles at single cell resolution (Fig.2A-C). Single-cell RNA sequencing and single-nucleus ATAC sequencing showed a striking alignment of the G34R, K27M and WT tumors, with similar profiles and internal heterogeneity (Fig.2D). Taking advantage of the MADR technology, which swaps the expression of the endogenous tdTomato for our transgene, we used a customized genome during the alignment of the scRNAseq datasets where the transcripts expressed by the ROSA26 locus were included along with the expected transcript after the recombination. This customized genomeallowed us to easily separate between recombined and non-recombined cells which clearly matched the tumor and stromal populations, respectively (Fig.2B).

[0168] The tumor stroma (determined by the expression of tdTomato or eGFP) was composed mainly by cells from the immune system (microglia, peripheral macrophages, Dendritic and Plasmocytoid dendritic cells, B-Cells and T-Cells), blood and blood vessel components (Erythroblasts, Endothelial cells and Pericytes) and cells from the Choroid Plexus. On the other hand, the tumor cells (determined by the detection of the transgene, more specifically the WPRE sequence in the 3’UTR ) were readily separated in cycling, Oligo-like, Astro-like and OPC like cells based on the transcriptional profiles. (Fig.2C).

[0169] Interestingly, the 3 tumor variants, despise their differential genotypic and phenotypic differences, completely overlapped on the UMAP embedding (Fig.2D and Fig.3A). To further understand the tumor development and cellular states we performed RNA Velocity analysis to determine the different transcriptomic pathway in their tumor heterogeneity (Fig.3B). From this analysis the tumor mass can be separated in 7 different transcriptomic programs which are determined as 6 regions of root cells at RNA velocity (Figure 7A) and the Cycling cells, cell undergoing mitosis can by split in phase S, and G2M (figure 7B). These 6 transcriptional profiles are: Oligo-like program, characterized for a transcriptomic profile with abundance of oligodendrocytes-related genes (e.g., Mog, Mag, Mbp); Astro-like program, with high expression of astrocytes-marker genes (e.g., Gfap, Aqp4, A2m); Neuron-like program, with a higher expression of Neuron differentiation-related genes (e g., Tubb3, Celf4, Cd24a, Igfbpll); Myeloid-like program, this cells present a high expression of immune related genes (e.g., Ly6a / e, Wfdcl8, Socsl); High Rb / Hsp program, the cells within this transcriptional profile present high expression of ribosomal and Heat shock proteins (e.g., Hspala / b, Hspbl, Rps3al, Dnajbl, RpllO); and a small cluster presenting a Cell cycle-arrested program (e.g. Cdknla, Txnip, Cirbp, Bbc3) these cells appear split in 2 group of cells at the UMAP embedding, one of them next to the Cycling cells and the other near the Oligo-like cells (Figure 8). Root points from the latent time analysis appeared as part of non-differentiated cells within the tumor cluster as well as Cycling cells (Fig.3C). To analyze the differential expression of the tumor subtypes we generate DGE tables (DGE within the different tumors) and explored the expression levels of these “tumor signatures”, generated by the Module Score tool of the Seurat Package, withinthe different transcriptomic programs delineated above. G34R and, specially, K27M transcriptomic signatures were enriched among the High Hsp / Rbp cluster which showed a strongly reduced enrichment of the H3.3 WT tumor signature. G34R signature was highly enriched in the Myeloid and in a portion of the OPC-like cells which showed a closer transcriptomic profile to the Myeloid like program. On the other hand, H3.3 WT tumor signature was highly enriched in the remaining OPC-like cells non-similar to the Myeloid program as well as in the Astro-like cells compared with the G34R and K27M signatures (Figure 9).

[0170] While similar percentages of tumor mass fall within each transcriptomic program when comparing the different tumor variants, some differences arise. H3.3 WT tumor cells present a lower percentage of cells within the transcriptional programs defined above, which could represent a lower differentiation state. Moreover, both G34R and K27M present higher proportion of cells within the High Rb / Hsp program. Additionally, K27M cells present the higher proportion of cells cycling which goes in agreement with its higher malignancy and faster development (Fig.3D). These percentages go in agreement with the differential enrichment of the tumor subtypes module scores which suggest that these subtype specific DGE signatures are, at least in part, derived from the subtle differences in cell type compositions.

[0171] To explore the origin of the tumor internal heterogeneity delineated above we analyzed the clonality of these datasets by inferring the chromosomic copy number variation (CNV) from their scRNAseq profiles. The CNV analysis showed a strong alteration of the expression patterns suggesting an altered genome where a number of chromosomes expression levels were greatly downregulated in G34R and K27M tumors and in one of the H3.3 WT tumor sample. We further explored the clonality of this tumor samples finding a higher clonal divergency within G34R samples compared to K27M samples which likewise presented a higher average number of clones than WT tumors. Taking in to account that WT tumor takes twice as long to develop compared to both G34R and K27M tumor the lower presence of clonal divergency could suggest a possible role of these mutations in the observed genomic instability in G34R and K27M tumors (Fig.3E).

[0172] The epigenomic landscape of these tumors further resembles the incredible similarities between these phenotypically and genotypically different tumors (Fig.4A). snATACseq analysis showed an absolute overlapping of both, the stroma cell types (Microgliaand Peripheral Macrophages, T-Cells, Endothelial cells and Pericytes), and the tumor cells (Fig.4A-B) Within the later we could distinguish a general Tumor Cluster with two subdivisions corresponding to mitotic cells and tumor cells differentiated to the Oligo-like program. However, this cluster were just subdivisions of the same general Tumor cluster with no visual separation or branching from the general mass. Other transcriptional programs that arise in the scRNAseq analysis were not detected by gene imputation. While the 3 tumor variants clearly overlapped in the UMAP embedding, the analysis of the differential enrichment for transcription factors regulons showed a strong enrichment of differential motif in G34R samples compared with K27M and H3.3 WT (Fig.4C). These differentially enriched regulons were contrasted with their inferred expression in the snATACseq datasets and their detected expression at scRNAseq (Fig.4D) In parallel we analyzed the transcription factor network in the scRNAseq datasets through SCENIC were each of the transcriptional profiles were easily separated. Interestingly, the cells within the OPC-like program were split in 2 subgroups: A first part maintained a more OPC-like profile while the second showed to be more similar to the Myeloid-like cells (Fig.4E). These splitting could represent and axis in the tumor cell differentiation which correlate with the observed trajectories in RNA velocity where cells that follow a path through the Myeloid-like cluster can progress to Astro-like cells, but not to Oligo-like cells. On the other hand, cells that progress through the upper side of the UMAP can reach the Oligo-like transcriptional program but not transdifferentiate to Astro-like or Myeloid-like cells (Fig.3B). Moreover, the differentially enriched regulons determined in the snATACseq which correlate with the gene expression, mainly find as differentially enriched motifs in G34R cells, were primarily enriched in the scRNAseq in the Myeloid-like cells, reinforcing a higher relevance of this transcriptional profile in G34R cells compared with K27M and H3.3 WT tumors.DNA Methylation, splicing and histone marks distinguish each tumor variant

[0173] Mutations in H3.3 are linked to genome wide changes in the methylation pattern, to explore the differential effect of K27M and G34R mutations in the tumor cells epigenome we carried out a genome wise DNA methylation array covering 285K CpG sites. Interestingly, the results of this assay showed clearly separated clustering of the 3 tumor models in PCA with no overlapping and clear differential signatures (Fig. 5A-B). Talk about the biological pathways altered by K27M and G34R mutations in contrast with H3.3 WT. We analyzed the differentiallymethylated probes to correlate the genomics region with the genes encoded generating a list of differentially methylated genes (. Since DNA methylation mainly correlate with gene expression silencing, we used differentially hypomethylated genes in each tumor subtype as an inferred higher expression level and applied those lists of genes to build Module Scores over the scRNAseq datasets using Seurat packages utilities. This Tumor subtype-derived Methylation Module Scores clearly distinguish between the different tumor subtypes in our scRNAseq datasets verifying that, even in this greatly overlapping embedding, each tumor subtype carries differences due to differential methylation states(Fig.5C).

[0174] These Methylation Modules were not evenly present among the different tumor cell programs. G34R Methylation Module was highly expressed in cells within the Myeloid-like program but low in OPC-like cells; K27M Hypomethylation Module was more widely expressed with a higher core in Cycling and High Hsp / Rbp programs; meanwhile, WT Hypomethylation Module was mainly expressed at high levels by OPC-like cells (Fig.5D).

[0175] While these modules showed clear correlations with different tumor programs, they were also more prevalent within its tumor subtype (e.g., G34R Methylation module was expressed in a higher percentage of cells in G34R samples as compare with K27M or WT samples). This indicate that the modules are also subtype-dependent and not based merely in the number of cells within each module in each tumor sample (figure 9).

[0176] KEGG pathway analysis of the methylation modules showed differential biological implications of the genes differentially methylated in each tumor (Fig.5E). G34R methylation module was significantly enriched in genes related with the “proline and arginine metabolism”, K27M methylation module was significantly enriched for the “Hippo signaling pathway” and WT methylation module was enriched in genes of the “JAK-STAT signaling pathway”. G34R tumor altered proline and arginine metabolism was also show by a clear dysregulation of most of the genes in this pathway compared with K27M and WT tumors (Fig.5F). Amino acid metabolism has become a novel and widespread target for cancer treatment. In fact, several types of cancer show altered amino acid metabolism and are auxotrophic for specific amino acids. To confirm the susceptibility of G34R tumors to this venue of treatment, in vitro studies were performed in amino acid starvation conditions. These studies showed an inverse dose-response cell death in G34R cells compared with WT and specially withK27M cells which showed an opposite behavior presenting a trend for a higher toxicity driven by the amino acids presence (Fig.5G). This increased cell death in both G34R and K27M, was not due a differential induction of apoptosis as concomitant measures showed (figure 9).

[0177] To further explore the epigenetic differences between our tumor variants, we employed Cut&Tag to uncover the changes in methylation state on histone 3 and its effect in the genomic binding of the histone.Mice models mimic human tumor counterpart heterogeneity at single cell level

[0178] While the K27M murine model have been already compared to human datasets proving a great fidelity. Human G34R and H3f3a WT pediatric glioblastoma datasets were not available to compare until recently. To this end we compared our datasets of G34R, K27M and H3f3a WT murine glioblastomas to other human datasets for G34R and H3F3a WT pediatric glioblastomas datasets for K27M, GBM, IDH-A and IDH-0 datasets from Filbin, M.G., et al. The integrated embedding of the human datasets showed a strong overlapping of G34R, K27M and H3.3 WT gliomas as found in our murine models (Fig.6A). Moreover, by using orthologous gene swapping we “humanized” the murine datasets and merged them with the human datasets finding an almost complete overlapping of all the samples (Fig.6B). In this general mass the underlying tumor programs derived from the murine models were still prevalent and defined the tumor cluster subdivisions in both mouse and human datasets. Interestingly the cellular programs defined in Filbin, M.G., et al. were also present in the human G34R and WT datasets highlighting that the transcriptomic similarities observed in our murine models matched the human data (Fig.6C).

[0179] The methylation modules generated in mouse organoids were explored in these human datasets finding that while it did not fit as well as in the mouse datasets it still showed a reasonable correlation (Fig.6D) Moreover, human G34R datasets showed an almost identical downregulation of the genes in the proline and arginine metabolism KEGG pathway as observed in the mouse datasets (Fig.6E),Example 2

[0180] Neonatal electroporation was employed to create MADR-derived G34R pediatric diffuse gliomas. Mice were assigned to different experimental groups randomly. Pegargiminasetreatment consisted in weekly intramuscular injections of 5IU (30mg / kg) of ADT-PEG20 over 4 weeks, alone or in combination with the standard of care.

[0181] Kaplan-Meier Survival Curve analysis: Figure 11 shows the survival curve of mice treated with or without pegargiminase. A) Represent a consolidated image of all the different treatments together, including saline, radiotherapy and chemotherapy alone (control) or in combination with pegargiminase (ADI-PEG20). B) shows the different treatments with or without ADI-PEG20 treatment individually. The ongoing results shows a trend of increased survival on the groups treated with pegargiminase, especially prevalent in combination with radiotherapy alone or with the chemotherapeutics temozolomide and lomustine.

[0182] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. ETnless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0183] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0184] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broaderaspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”

[0185] Unless stated otherwise, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) may be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0186] “Optional” or “optionally” means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0187] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a G34R mutant tumor, comprising: administering an arginine depleting agent, proline depleting agent, or both to a subject in need thereof.

2. The method of claim 1, wherein the subject is a pediatric subject.

3. The method of claim 1 or claim 2, wherein the subject has been detected to have a G34R mutation.

4. The method of claim 1 or claim 2, wherein the subject has been detected to have a G34R mutation by a method comprising performing DNA or RNA sequencing of a biological sample obtained from the subject to detect the presence of the G34R gene mutation, or using an antibody to detect the presence of a protein or peptide translated from the G34R mutation in a biological sample obtained from the subject.

5. The method of any one of claims 1-4, wherein the arginine depleting agent comprises pegargiminase.

6. The method of any one of claims 1-4, wherein the arginine depleting agent comprises arginine deiminase or human arginase I.

7. The method of any one of claims 1-4, wherein the proline depleting agent comprises LP- 403812 (1142050-84-7) or a proline transport inhibitor.

8. The method of any one of claims 1-7, wherein the G34R mutant tumor is a glioma, giant cell tumor of the bone, or a chondroblastoma.

9. The method of claim 1, wherein G34R mutant tumor is a G34R mutant glioma, wherein the arginine depleting agent comprises pegargiminase, and wherein the subject is a pediatric subject.

10. The method of any one of claims 1-9, further comprising administering a standard-of- care therapy to the subject.

11. The method of claim 10, wherein the standard-of-care therapy comprises tumor resection, chemotherapy, radiation, or combinations thereof.

12. The method of claim 10, wherein standard-of-care therapy comprises TMZ, combination of CCNU and TMZ, combination of TMZ and radiation therapy, or combination of CCNU, TMZ and radiation therapy.

13. A method of selecting a tumor therapy for a subject in need thereof, comprising detecting a G34R mutation in a biological sample from the subject; and selecting a tumor therapy comprising an arginine depleting agent, a proline depleting agent, or both for the subject.

14. The method of claim 13, wherein the biological sample comprises tumor cells.

15. The method of claim 13 or claim 14, wherein detecting the G34R mutation comprises performing DNA or RNA sequencing of biological sample to detect the presence of the G34R gene mutation, or using an antibody to detect the presence of a protein or peptide translated from the G34R mutation.

16. The method of any one of claims 13-15, further comprising administering the arginine depleting agent, the proline depleting agent, or both to the subject.

17. The method of any one of claims 13-16, wherein the subject is a pediatric subject.

18. The method of any one of claims 13-17, wherein the arginine depleting agent comprises pegargiminase.

19. The method of any one of claims 13-17, wherein the arginine depleting agent comprises arginine deiminase or human arginase I.

20. The method of The method of any one of claims 13-17, wherein the proline depleting agent comprises LP-403812 (1142050-84-7) or a proline transport inhibitor.

21. The method of any one of claims 13-20, wherein the G34R mutant tumor is a glioma, giant cell tumor of the bone, or a chondroblastoma.

22. The method of any one of claims 13-21, further comprising administering the standard- of-care therapy comprising tumor resection, chemotherapy, radiation, or combinations thereof23. The method of claim 22, wherein standard-of-care therapy comprises TMZ, combination of CCNU and TMZ, combination of TMZ and radiation therapy, or combination of CCNU, TMZ and radiation therapy.

24. A system, comprising:(i) a single promoter-less donor vector, comprising: a polyadenylation signal or transcription stop element upstream from a nucleic acid the nucleic acid, wherein the nucleic acid encodes a K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominantactive PdgfraD842\ dominant-negative Trp53R270H, or a combination thereof, and paired recombinase recognition sites,OR one or more promoter-less donor vectors, each promoter-less donor vector independently comprising a polyadenylation signal or transcription stop element upstream from a nucleic acid, the nucleic acid, wherein the nucleic acid encodes one or more of K27M mutation of H3.3, G34R mutation of H3.3, wildtype of H3.3, dominant-active PdgfraD842V, dominant-negative Trp53R270H; and(ii) one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites,OR two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.

25. The system of claim 24, wherein the promoter-less donor vector further comprises a post- transcriptional regulatory element, a polyadenylation signal downstream from the nucleic acid, or both.

26. The system of any one of claims 24-25, wherein the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are ere and flp,wherein the paired recombinase recognition sites are modified loxP and / or modified flippase recognition target (FRT), and the recombinases are ere and flp, wherein the paired recombinase recognition sites are VloxP and flippase recognition target (FRT), and the recombinases are VCre and flp, wherein the paired recombinase recognition sites are SloxP and flippase recognition target (FRT), and the recombinases are SCre and flp, wherein the recombinase is PhiC31 recombinase and the recombinase recognition sites are attB and attP, wherein the recombinase is Nigri, Panto, or Vika and recombinase recognition sites are nox, pox, and vox, respectively, or wherein one or both of the paired recombinase recognition sites comprise a mutation.

27. The system of any one of claims 24-26, wherein the pA signal is PGKpA and trimerized SV40pA, wherein the pA downstream from the nucleic acid post is a rabbit beta-globin pA, or wherein the post-transcriptional regulatory element (WPRE) is a woodchuck hepatitis virus.

28. A non-human animal made by administering the system of any one of claims 24-27, and catalyzing a dual recombinase mediated cassette exchange in the non-human animal.

29. A method of screening a test agent for its effect on a G34R mutant tumor, comprising: administering the test agent to a non-human model of claim 28; and measuring a parameter in the non-human animal model;30. The method of claim 29, further comprising identifying the test agent as a drug candidate or not a drug candidate for G34R mutant tumor based on the measured parameter.

31. The method of claim 30, wherein the parameter comprises inhibition of tumor growth, promotion of tumor growth, induction of apoptosis of tumor cells, arginine metabolism, proline metabolism, upregulation or downregulation of arginine metabolism related genes, upregulation or downregulation of proline metabolism related genes, survival,immunophenotype changes, epigenome changes, transcriptome changes, or a combination thereof.

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