Therapeutic approaches to target KIAA1549-BRAF fusion-driven cancers

Abstract

A method of identifying therapeutic agent for the treatment of a cancer with a KIAA1549-BRAF fusion is disclosed. The method includes contacting a cell expressing a KIAA1549-BRAF fusion protein with a test agent and determining if the test agent inhibits O-mannosylation of the KIAA1549-BRAF fusion protein, inhibits heterodimerization of protein O-mannosyltransferase 1 (POMT1) and protein O-mannosyltransferase 2 (POMT2), and / or inhibits activity of the protein O-mannosyl transferase (POMT) complex in the cell expressing a KIAA1549-BRAF fusion protein. A therapeutic agent and its use for the treatment of a cancer comprising a KIAA1549-BRAF fusion is also disclosed.

Description

[0001] THERAPEUTIC APPROACHES TO TARGET KIAA1549-BRAF FUSION-DRIVEN

[0002] CANCERS

[0003] GOVERNMENT FUNDING This invention was made with government support under Grant No. 1F32CA284834, awarded by National Institutes of Health. The Government has certain rights in the invention.

[0004] BACKGROUND

[0005] Over 70% of pediatric low-grade gliomas (pLGGs) are driven by a single oncogenic rearrangement between KIAA1549 and BRAF (KIAA1549::BRAF), resulting in activation of the MAPK pathway (Bandopadhayay et al., 2016, Nat Genet,' 48(3):273-82; and Jones et al., 2008, Cancer Res,' 68(21 ): 8673-7). MAPK pathway inhibitors are efficacious against a subset of fusion-driven pLGGs (Fangusaro et al., 2019, Lancet Oncol,' 20(7): 1011-1022), although obstacles persist. Approximately one third of patients have primary resistance to MAPK pathway inhibitors. Those that do respond often experience dose-limiting toxicity and relapse upon treatment cessation. The MAPK pathway is necessary for development, especially neural development. Prolonged MAPK inhibitor treatment subjects children to extended therapy with unknown long-term effects on development. This underscores the need to develop orthologous approaches to therapeutically target KIAA1549:BRAF independent of the MAPK pathway.

[0006] SUMMARY

[0007] Provided herein are methods for identifying a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion, the method including: i) contacting a cell expressing a KIAA1549-BRAF fusion protein with a test agent; and ii) determining if the test agent inhibits O-mannosylation of the KIAA1549-BRAF fusion protein, inhibits heterodimerization of protein O-mannosyl transferase 1 (POMT1) and protein O- mannosyltransferase 2 (P0MT2), and / or inhibits activity of the protein O-mannosyl transferase (POMT) complex in the cell expressing a KIAA1549-BRAF fusion protein, wherein determining that the test agent inhibits the O-mannosylation of the KIAA1549-BRAF fusion protein, inhibits heterodimerization of POMT 1 and P0MT2, and / or inhibits activity of the POMT complex in the cell expressing a KIAA1549-BRAF fusion protein identifies the test agent as a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion.

[0008] In some aspects of the methods, the cell expressing a KIAA1549-BRAF fusion protein comprises a murine neural stem cell expressing a KIAA1549-BRAF fusion protein or a human neural stem cell expressing a KIAA1549-BRAF fusion protein.

[0009] Also provided are methods of treating a subject with a cancer associated with a KIAA1549-BRAF fusion, the method including administering an effective amount of an agent identified by a method for identifying a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion as described herein. In some aspects, the cancer includes a pediatric low-grade glioma (pLGG). In some aspects, the cancer includes a cancer refractory to treatment with a mitogen-activated protein kinase (MAPK) inhibitor.

[0010] Also provided herein are methods of treating a subject with a cancer associated with a KIAA1549-BRAF fusion, the method including administering an effective amount of a therapeutic agent identified by a method for identifying a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion as described herein and administering one or more additional therapeutic interventions. In some aspects, the one or more additional therapeutic intervention includes chemotherapy, radiation, and / or surgical intervention. The administration of the therapeutic agent and the additional therapeutic intervention may demonstrate synergy.

[0011] Also provided herein are methods of treating a subject with a cancer associated with a KIAA1549-BRAF fusion, the method including administering an effective amount of a therapeutic agent identified by the described method and administering one or more mitogen- activated protein kinase (MAPK) inhibitors. In some aspects, the MAPK inhibitor comprises trametinib, selumetinib, cobometinib, ulxertinib, tovorafenib, vemurafenib, and / or dabrafenib. Administration of the therapeutic agent and the MAPK inhibitor may demonstrate synergy. In some aspects, the therapeutic agent comprises R3A-5a or a derivative thereof.

[0012] Also provided herein are therapeutic agents for the treatment of a cancer identified by the described method for identifying a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion. Tn some aspects, the cancer comprises a pediatric low-grade glioma (pLGG). In some aspects, a cancer includes a cancer refractory to treatment with a mitogen- activated protein kinase (MAPK) inhibitor.

[0013] Also provided herein are therapeutic agents for the treatment of a cancer comprising a KIAA1549-BRAF fusion, the therapeutic agent comprising an inhibitor of i) O-mannosylation of the KIAA1549-BRAF fusion protein; ii) heterodimerization of protein O-mannosyltransferase 1 (P0MT1) and / or iii) protein O-mannosyltransferase 2 (P0MT2); and / or iv) activity of the protein O-mannosyl transferase (POMT) complex. In some aspects, the cancer comprises a pediatric low-grade glioma (pLGG). In some aspects, the cancer includes a cancer refractory to treatment with a mitogen-activated protein kinase (MAPK) inhibitor.

[0014] Also provided herein are therapeutic agents for the treatment of a cancer comprising a KIAA1549-BRAF fusion, wherein the therapeutic agent comprises R3A-5a or a derivative thereof. In some aspects, the cancer comprises a pediatric low-grade glioma (pLGG). In some aspects, the cancer includes a cancer refractory to treatment with a mitogen-activated protein kinase (MAPK) inhibitor.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1. Growth of isogenic mNSCs is dependent on overexpression of common pLGG drivers. Incucyte sphere growth experiment growth curve showing normalized largest brightfield area over time. Mouse neural stem cells with pLGG oncogenic driver overexpression are viable and proliferate in growth factor depleted culturing conditions. The same cells do not grow under those conditions when expressing a vector control (Luciferase and GFP).

[0017] FIG. 2. Transplantation of isogenic mNSCs generate tumors in mice. mNSCs were engineered to express KIAA1549::BRAF, BRAFV600E, or LacZ (vector control) and orthotopically transplanted into the right hemisphere of Fox Chase SCID mice. These data indicate that ectopic expression of KIAA1549::BRAF or BRAFV600Eis sufficient to induce tumor formation in mice. n=10 mice / cell line.

[0018] FIG. 3. Schematic overview of a genome-wide CRISPR / Cas9-screen.

[0019] FIG. 4. KIAA1549::BRAF-dependent mNSCs are selectively dependent on genes that regulate O-mannosylation. FIG. 5. Isogenic hNSCs are oncogene addicted. Viability of hNSCs with expression of IKZF3-degron-tagged oncogenic pLGG drivers (isogenic) was assessed using CellTiter-Glo. The cells were grown with or without EGF / bFGF and with pomalidomide or DMSO as control for 72 hours to determine the dependence on oncogenic MAPK signaling with and without extracellular growth stimulation.

[0020] FIG. 6. Impact of P0MT1 / 2 knockout on cell viability. Oncogene-transformed or luciferase-expressing h9-NSCs were transduced with CRISPR / Cas9 guides targeting either intergenic control regions or POLR2B (positive control), P0MT1, or P0MT2. Relative guide cell proportions were assessed with flow cytometry. The fraction of cells expressing POLR2B, POMT1, or POMT2 guides (co-expressing mCherry) was quantified.

[0021] FIG. 7. The Pomt complex inhibitor R3A-5a selectively decreases viability of KIAA1549::BRAF-driven mNSCs compared to control. Viability of isogenic mNSCs after 5 days treatment with nine increasing concentrations of R3A-5a was assessed with CellTiter-Glo to determine IC50 for cells expressing Luciferase (vector control), BRAF WT, KIAA1549 WT, BRAFV600Eand KIAA1549::BRAF.

[0022] FIG. 8. hNSCs with expression of IKZF3-degron-tagged oncogenic pLGG drivers (isogenic) transform growth-factor independently.

[0023] FIG. 9A represents viability of mNSC KIAA1549::BRAF, BRAFV600E, and vector control (luciferase) cells treated with R3A-5a and Belvarafenib. FIG. 9B represents synergy presented as a BLISS score for mNSC KIAA1549::BRAF,

[0024] BRAFV600E, and vector control (luciferase) cells treated with R3A-5a and Belvarafenib.

[0025] FIG. 10A is a schematic of the KIAA1549-BRAF fusion event resulting from a tandem duplication on chromosome 7. Figure adapted from Larsen et al., 2017, Proc Natl Acad Set U S A; 114(42):11163-11168.

[0026] FIG. 10B is a schematic of the KIAA1549-BRAF rearrangement truncating the negative regulatory domains. Figure adapted from Larsen et al., 2017, Proc Natl Acad Set USA,' 114(42):11163-11168.

[0027] FIG. 10C is a schematic indicating KIAA1549 is one of the most heavily mannosylated proteins. Figure adapted from Larsen et al., 2017, Proc Natl Acad Set USA,' 114(42): 11163- 11168. FIG. 10D is a schematic indicating that O-mannosylation is essential for the oncogenicity. Figure adapted from Larsen et al., 2017, Proc Natl Acad Sci USA 114(42):11163-11168.

[0028] FIG. 11 A is a bar graph of the number of FDA-approved drugs directly targeting either the fusion proto-oncogene or fusion partner.

[0029] FIG. 1 IB depicts a bar graph showing the number of unique fusion species in tumors from 4,329 pediatric patients. Bars represent the number of unique fusion species identified (x- axis) for each fusion gene (y-axis).

[0030] FIG. 11C is a graphical representation of intronic breakpoint loci for 55 unique BRAF fusion species identified in an analysis of 4,329 tumors from pediatric patients. The x-axis depicts the first complete exon downstream of the fusion breakpoint, and the y-axis depicts the total number of unique fusion species with a breakpoint in the given intron.

[0031] FIG. 1 ID shows representative images and quantification of three independent colony formation assays of h9-NSCs expressing Luciferase (control), KIAA1549::BRAF, KIAA1549: (exons 1-15), and :BRAF (exons 9-18). Quantification of the percentage of plate area with cells is shown as bars in the bar graph.

[0032] FIG. 1 IE is a representative immunoblot showing protein levels of phosphorylated and total ERK in h9-hNSCs expressing the same constructs as in FIG. 1 ID.

[0033] FIG. 1 IF shows representative images and quantification of two independent colony formation assays of h9-hNSCs expressing Luciferase (control), FAM131B::BRAF, and FAM131B::BRAFG2A.

[0034] FIG. 11G is a representative V5, pERK, ERK, and Actin immunoblot from h9-NSCs expressing the same constructs as in FIG. 1 IF.

[0035] FIG. 11H shows representative Airyscan images of h9-hNSCs expressing V5-tagged FAM 131 B : :BRAF and FAM 131 B : BRAFG2 A.

[0036] FIG. I ll shows the heatmap from an analysis of n = 28 (of a total of 59) BRAF fusion partners identified in an analysis of 4,329 pediatric cancers.

[0037] FIG. 12A depicts a schematic showing wildtype human KIAA1549 (top panel), wildtype human BRAF (middle panel), and KIAA1549::BRAF (15 : :9, lower panel). N- glycosylation (n = 5) and O-glycosylation (n = 64) sites are annotated as forks or small circles, respectively. SP: signal peptide; TM: transmembrane domain; CR1 : conserved region 1 (RAS- binding domain and cysteine-rich domain); CR2: conserved region 2 (autoinhibitory hinge region).

[0038] FIG. 12B shows representative Airy scan immunofluorescence images of h9-hNSCs expressing C-terminal V5-tagged KIAA1549::BRAF or KIAA1549::BRAFdel™ labelled with V5-tag and PDI (marker of endoplasmic reticulum) antibodies. Quantification of V5 and PDI colocalization of V5 and PDI (right).

[0039] FIG. 12C shows representative images and quantification of colony formation assays of h9-hNSCs expressing Luciferase (control), KIAA1549::BRAF, and KIAA1549::BRAFdel™. Percent area covered: Luciferase = 5.87%, K::B = 89.2%, K::Bdel™ = 8.78%.

[0040] FIG. 12D shows a representative immunoblot of pERK, ERK, and Actin in h9-hNSCs expressing the same constructs as in FIGS. 2B and 2C.

[0041] FIG. 12E shows representative images and quantification of colony formation assays of h9-hNSCs expressing Luciferase (control), KIAA1549::BRAF, and KIAA1549::BRAFASP. Percent area covered: Luciferase = 1.2%, K::B = 61.8%, K::Bdelsp= 0.89%.

[0042] FIG. 13 A is a schematic depicting the generation of isogenic cell models from embryonic (E14.5) mouse neural stem cells with ectopic expression of human KIAA1549::BRAF (15:9), BRAFV600E, and Luciferase (Control).

[0043] FIG. 13B is a scatter plot showing log2 fold change (day 18 vs early timepoint) in gene-level CRISPR guide abundance scores. Screen from mouse neural stem cells (mNSCs) expressing KIAA1549::BRAF (in the absence of EGF / bFGF) is represented on the y-axis. Screen from mNSCs expressing Luciferase (in the presence of EGF / bFGF) is represented on the x-axis. Each dot represents a single gene, selected genes are highlighted.

[0044] FIG. 13C depicts a graphical representation of POMT1 / POMT2 specific O- Mannosylation in mammals. DPM1 / 2 / 3 : dolichol-phosphate mannosyltransferase-1 / 2 / 3 of the dolichol phosphate mannose (Dol-P-Man) complex; GDP / GTP: guanosine diphosphate / triphosphate; POMT1 / 2: protein O-mannosyltransferase 1 and 2 of the POMT complex.

[0045] FIG. 13D shows a bar plot of CRISPR competition assay in h9-NSCs. The fraction of mCherry-positive cells, normalized to the initial time point, is displayed on the y-axis. The sgRNA pair conditions are displayed on the x-axis. Statistically significant p-values (p < 0.05) are indicated with * on the plot.

[0046] FIG. 13E shows a dot plot with Chronos (Bandopadhayay et al., 2016, Nat. Genet., 48:273-282) gene dependency scores for POMT1, POMT2, DPMI, DPM2, and DPM3 across 1150 cancer cell lines profiled in The Cancer Dependency Map (DepMap) (Bandopadhayay et al., 2016, Nat. Genet., ' 48:273-282). A Chronos score of 0 or -1 is the median of all genes classified in the DepMap 24q2 release as non-essential or common essential, respectively.

[0047] FIG. 13F shows a best-fit curve of mNSC isogenic models expressing Luciferase (control), KIAA1549::BRAF, and BRAFV600Etreated with increasing concentrations of the POMT complex inhibitor R3A-5a. AUC luciferase control = 220.8; KIAA1549::BRAF = 140.8; BRAFV600E= 233).

[0048] FIG. 14A shows the workflow for quantitative O-Man glycoproteomics of stable isotope labelled total cell digests enriched for O-Man glycopeptides by BC2L-A lectin chromatography.

[0049] FIG. 14B depicts the relative quantification of the total proteomes as a boxplot with whiskers for which the interquartile range calculations were used to determine the boundaries (dashed lines) for peptide spectral matches (PSMs) derived from Luciferase- or human KIAA1549::BRAF-expressing mNSCs. The dot-plot shows heavy -to-light (H / L) ratios of individual O-Man PSMs from KIAA1549:BRAF, endogenous mouse KIAA1549, and other C- and O-Man glycoproteins.

[0050] FIG. 14C is a schematic illustration of human KIAA1549::BRAF showing the mapped O-Man glycans (spheres) to residues 84-849 of the N-terminal domain.

[0051] FIG. 14D shows representative PSMs used for identification and site mapping of O- Man glycans on human KIAA1549::BRAF (top) and endogenous mouse KIAA1549 (bottom). The insert shows precursor isotope envelopes for heavy and light labelled glycopeptides selected for MS2 fragmentation.

[0052] FIG. 15A shows representative images depicting EGFP+ transfected cells within EGFP control and KIAA1549::BRAF expressing brain sections (left). High magnification representative images of the upper cortical layer region co-stained for EGFP (middle left), EGFP / Gfap (middle right, or NeuN (right). Scale bars: tiled section images on the left side = 500 micrometers; high magnification images = 100 micrometers.

[0053] FIG. 15B shows representative images depicting EGFP+ transfected cells within K::B + Rosa26sgRNAand K::B + PomtlsgRNAexpressing brain sections. High magnification representative images of the upper cortical layer region depicting EGFP+ cell morphology (middle) and 3D reconstruction of example EGFP+ astrocytes in each condition (right). Scale bars: tiled section images on left side = 500 micrometers; high magnification images = 100 micrometers; 3D reconstruction images = 5 micrometers for K::B + Rosa26sgRNAand 7 micrometers for K::B + PomtlsgRNA. FIG. 16 shows a Kaplan-Meier plot of SCID mice with KIAA1549::BRAF allografts treated with R3A-5a or vehicle.

[0054] DETAILED DESCRIPTION

[0055] Over 70% of pediatric low-grade gliomas (pLGGs) are driven by a single oncogenic rearrangement between KIAA1549 and BRAF, resulting in a KIAA1549::BRAF fusion. This fusion truncates the BRAF N-terminal regulatory domains and results in activation of the mitogen activated protein kinase (MAPK) signaling pathway (Bandopadhayay et al., 2016, Nat Genet, 48(3):273-82; and Jones et al., 2008, Cancer Res,' 68(21):8673-7). This rearrangement fuses the C-terminus of BRAF to the large transmembrane protein KIAA1549 (FIG. 10A) and truncates the negative regulatory domains of wildtype BRAF (FIG. 10B).

[0056] The resultant protein product of the KIAA1549::BRAF rearrangement may also be reflected as “KIAA1549::BRAF fusion,” “KIAA1549::BRAF fusion protein,” “KIAA1549::BRAF protein,” “KIAA1549:BRAF,” “KIAA1549BRAF fusion,” “KIAA1549:BRAF fusion protein,” “KIAA1549:BRAF protein,” “KIAA1549-BRAF,” “KIAA1549-BRAF fusion,” “KIAA1549-BRAF fusion protein,” “KIAA1549-BRAF protein,” “KIAA1549-BRAF mutant,” “BRAF -KIAA1549 fusion,” “BRAF -KI AA 1549 fusion protein,” “BRAF -KIAA1549 protein,” and “BRAF -KIAA1549.” These terms may be used interchangeably herein. As used herein, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.

[0057] The term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements.

[0058] The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0059] The terms “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

[0060] Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.

[0061] Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0062] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0063] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0064] In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0065] All headings throughout are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. The terms “treating” and “treatment” as used herein refer to the administration of an agent or formulation to a clinically symptomatic individual afflicted with an adverse condition, disorder, or disease, so as to effect a reduction in severity and / or frequency of symptoms, eliminate the symptoms and / or their underlying cause, and / or facilitate improvement or remediation of damage. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0066] By the terms “effective amount” and “therapeutically effective amount” of a formulation or formulation component is meant a sufficient amount of the formulation or component, alone or in a combination, to provide the desired effect. For example, by “an effective amount” is meant an amount of a compound, alone or in a combination, required to ameliorate the symptoms of a disease, e.g., cancer, relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.

[0067] There are no KIAA1549:BRAF-selective drugs and developing KIAA1549:BRAF fusion-selective inhibitors represents a major unmet medical need. Via the use of genome-scale loss of function CRISPR / Cas9 screens (Abid et al., 2023, Nat Protoc, 18(7):2014-2031) in mouse and human neural stem cells expressing KIAA1549:BRAF, the POMT1 / 2 complex has been discovered to be essential for the survival of KIAA1549: :BRAF-dependent models of pLGG. In particular, it has been discovered that the heterodimeric P0MT1 / 2 complex catalyzes the addition of mannose adducts to Ser and Thr residues to transmembrane and secreted proteins in the endoplasmic reticulum. This finding indicates that modification of KIAA1549:BRAF by POMT is necessary for protein stability and trafficking. The protein O-mannosyltransferase 1 (P0MT1) enzyme catalyzes the first step in O-mannosyl glycan synthesis, with the attachment of a mannose via an O-glycosyl linkage to the Ser / Thr of the protein (Wilier et al., 2003, Curr Opin Struct Biol,' 13(5):621 -30). The protein O-mannosyltransferase 2 (POMT2) enzyme is a second O-mannosyltransferase, which complexes with POMT1 to form the protein O- mannosyltransferase (POMT) enzyme complex for the O-mannosyltransferase activity (Manya et al., 2004 2004, Proc Natl Acad Sci U A; 101(2):500-5; and Akasaka-Manya et al., 2006, J Biol

[0068] Chem; 281(28): 19339-45).

[0069] Disclosed herein are methods of identifying genetic vulnerabilities in KIAA1549::BRAF- dependent cells, the method including performing a genome-scale CRISPR-Cas9 screen across isogenic neural stem cells transduced to express a pediatric low-grade glioma (pLGG)-associated oncogene, generating a dependency map of genetic vulnerabilities associated with the expression of the (pLGG)-associated oncogene, and identifying one or more genetic vulnerabilities. In some aspects, the pLGG-associated oncogene is KAII1549::BRAF, BRAFV600E, FGFR1, or a MYB family alteration. The isogenic neural stem cells transduced to express a pLGG-associated oncogene may be murine or human neural stem cells transduced to express a KIAA1549-BRAF fusion protein. The genetic vulnerability includes O-mannosyltransferase 1 (POMT1) and / or protein O-mannosyltransferase 2 (POMT2).

[0070] KIAA1549 is a very heavily mannosylated protein (FIG. 10C) and the results disclosed herein indicate that proper O-mannosylation of KIAA1549::BRAF is essential for oncogenicity (FIG. 10D). As described herein, both O-mannosyltransferase 1 (POMT1) and protein O- mannosyltransferase 2 (POMT2) are strong and selective dependencies in human models of KIAA1549:BRAF-dependent pLGG and these enzymes can serve as targets for the identification of therapeutic agents for the for the treatment of cancers associated comprising a KIAA1549- BRAF fusion.

[0071] Described herein are methods of identifying a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion, the methods including contacting a cell expressing a KIAA1549-BRAF fusion protein with a test agent and determining if the test agent inhibits one or more of i) O-mannosylation of the KIAA1549-BRAF fusion protein, ii) heterodimerization of protein O-mannosyltransferase 1 (P0MT1) and protein O- mannosyltransferase 2 (P0MT2), iii) and / or activity of the protein O-mannosyl transferase (POMT) complex in the cell expressing a KIAA1549-BRAF fusion protein,. In some aspects, determining that the test agent inhibits the O-mannosylation of the KIAA1549-BRAF fusion protein, inhibits heterodimerization of POMT 1 and P0MT2, and / or inhibits activity of the POMT complex in the cell expressing a KIAA1549-BRAF fusion protein identifies the test agent as a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion.

[0072] A cell expressing a KIAA1549-BRAF fusion protein includes, but is not limited to a murine neural stem cell expressing a KIAA1549:BRAF fusion protein or a human neural stem cell expressing a KIAA1549:BRAF fusion protein, including, but not limited to those described in the Examples included herewith.

[0073] The inhibition of O-mannosylation of the KIAA1549-BRAF fusion protein, the inhibition of the heterodimerization of POMT 1 and P0MT2, and the inhibition of the activity of the POMT complex may be determined by any of a variety of methods known to one of skill in the art, including, but not limited those described in the Examples section included herewith. Any of a variety of test agents may be screened, including, but not limited to test agents that are nucleic acids, antibodies, proteins, peptide, and low molecular weight organic or inorganic small molecules.

[0074] A therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion identified by the methods described herein may include any of a variety of agents, including, but not limited to, a nucleic acid, an antibody, a protein, a peptide, and a low molecular weight organic or inorganic small molecule. In some aspects, such a therapeutic agent is not a wildtype BRAF kinase domain inhibitor.

[0075] A therapeutic agent for the treatment of cancer associated with a KIAA1549-BRAF fusion may include the small molecule mannosyl transferase 1 (PMT1) inhibitor R3A-5a or a derivative thereof. R3A-5a is a Rhodanine-3-acetic acid derivative developed for inhibition of enzymatic activity of yeast PMT complexes which has been demonstrated to be an in vitro and in vivo highly specific inhibitor of both fungal and mammalian protein O-mannosyltransferases at micromolar concentrations (Orchard et al., 2004, Bioorg Med Chem / .c / Z; 14(15):3975- 3978;

[0076] Arroyo et al., 2011, Mol Microbiol, 79(6): 1529-1546; and Lommel et al., 2013, Proc Natl Acad Sci USA,' 110(52):21024-21029). As described in the examples, the small molecule POMT inhibitor R3A-5a (5-[[3-(l-phenylethoxy)-4-(2-phenylethoxy)phenyl]methylene]-4-oxo-2- thioxo-3-thiazolidineacetic acid (5a)) is a rhodanine-3 -acetic acid derivative (Orchard et al., 2004, Bioorg Med Chem Lett, 14(15):3975-3978) that selectively kills KIAA1549:BRAF dependent cells. R3A-5a was tested in the pLGG isogenic embryonic mouse neural stem cell models using a cell-titer gio viability assay. The compound had 2 to 20 times lower IC50 concentration in KIAA1549::BRAF-driven isogenic cells than BRAFV600E, BRAF WT, KIAA1549 WT overexpressing cells as well as vector control.

[0077] Provided herein is the use of compounds and derivatives of formula (I) or a pharmaceutically acceptable salt or prodrug thereof wherein each of R1, R2, R3, R4,and R5independently comprises a hydrogen atom, a Cl- C6 alkyl group, a hydroxy group, a C1-C6 acyloxy group, a C1-C6 alkoxy group, an oxyalkyl acid group with 1 to 4 C-atoms, a benzyloxy group, or a combination thereof. In one or more embodiments, wherein one or more of R1, R2, R3, R4,and R5may be a benzyloxy group, the benzyloxy group may be substituted by a group X. Group X may be a hydrogen atom, a hydroxy group, a C1-C4 alkyl group, or a C1-C4 alkoxy group. The phenyl group of the benzyloxy group may be condensed with a methylenedioxy or ethylenedioxy group, or R2and R3together may form a methylenedi oxy or ethylenedi oxy group, R6may be a hydrogen atom, or a C1-C4 alkyl group. Group A may be a straight-chain or branched-chain C1-C8 alkyl group, an aryl group an arylalkyl group, a heteroalkyl group, or a carboxyalkyl group. When one or more of R1, R2, R3, R4’ and R5is a benzyloxy group, the benzyloxy group may be substituted by a second benzyloxy group. R1, R2, R3, R4,and R may independently be one or more of a phenethoxy group, a 1- phenylethoxy group, or both. The rhodamine-3 -acetic acid derivative is (Z)-2-(4-oxo-5-(4-phenethoxy-3-(l- phenylethoxy)benzylidene)-2-thioxothiazolidin-3-yl)acetic acid (Ila), or (£)-2-(4-oxo-5-(4- phenethoxy-3-(l-phenylethoxy)benzylidene)-2-thioxothiazolidin-3-yl)acetic acid (lib), or a mixture thereof.

[0078] The term "alkyl" as used herein, includes both straight and branched chain radicals.

[0079] As described herein, reference to compounds of formula (I) or formula (II) encompasses the pharmaceutically acceptable salts and prodrugs thereof. Suitable, pharmaceutically acceptable salts of the compounds include those derived from inorganic and organic bases. Examples of suitable inorganic bases for the formation of salts of compounds described in this disclosure include the hydroxides, carbonates, and bicarbonates of ammonia, lithium, sodium, calcium, potassium, aluminum, iron, magnesium, zinc, and the like. Salts can also be formed with suitable organic bases. Such organic bases are already well known in the art and may include amino acids such as arginine and lysine, mono-, di-, or trihydroxy alkylamines such as mono-, di-, and triethanolamine, choline, mono-, di-, and trialkyl amines, such as methylamine, dimethylamine, and trimethylamine, guanidine, N-methyl glucosamine, N- methyl piperazine, morpholine, ethylenediamine, N- benzyl phenethylamine, tris(hydroxymethyl) aminomethane, meglumine and the like. Salts may be prepared in a conventional manner using methods well-known in the art.

[0080] The rhodamine-3 -acetic acid derivatives may be crystallized or recrystallized from solvents such as aqueous and organic solvents. In such cases, solvates may be formed. Also described are stoichiometric solvates, including hydrates, as well as compounds containing variable amounts of water that may be produced by processes such as lyophilization. Prodrugs of the aforementioned compounds are contemplated, as are derivatives. A prodrug is commonly described as an inactive or protected derivative of an active ingredient or a drug, which is converted to the active ingredient or drug in the body. Examples of prodrugs include pharmaceutically acceptable esters, including -C6 alkyl esters, and pharmaceutically acceptable amides, including secondary C1-C3 alkylamides.

[0081] The present rhodamine-3 -acetic acid derivative compounds may exist in the form of optical isomers, for example, diastereoisomers, and mixtures of isomers in all ratios, for example, racemic mixtures. The isomeric forms (R or S) are contemplated. The different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses. The compounds may be provided in racemic form or in a form containing any relative proportions of the enantiomers recited below. When the enantiomeric forms are provided substantially free of the other enantiomer, they will preferably contain less than 5% w / w, more preferably less than 2% w / w, and especially less than 1% w / w of the other enantiomer.

[0082] Since the rhodamine-3 -acetic acid derivative compounds are intended for use in pharmaceutical compositions, it is understood that they are each preferably provided in substantially pure form, for example, at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis). Impure preparations may be used for preparing the purer forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1%, more suitably at least 5%, for example, 10 to 59% of the compound.

[0083] A therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion identified by the methods described herein may be administered to a subject in need thereof in an amount effective for the treatment of a cancer associated with a KIAA1549-BRAF fusion. A cancer associated with a KIAA1549-BRAF fusion includes, but is not limited to, pancreatic adenocarcinoma, invasive breast carcinoma, glioblastoma, melanoma, gallbladder carcinoma, esophageal squamous carcinoma, endometrial mixed adenocarcinoma, colon adenocarcinoma, bladder urothelial cancer, papillary thyroid carcinoma (including papillary thyroid carcinoma arising from ovarian teratoma), ovarian serous tumors, colorectal carcinoma, gliomas, hepatobiliary carcinomas, hairy cell leukemia, pilocytic astrocytoma, low-grade glioma, NOS, astrocytoma, pilomyxoid astrocytoma, optic nerve glioma, or ganglioglioma. In some aspects, a cancer associated with a KIAA1549-BRAF fusion is a pediatric low-grade glioma (pLGG).

[0084] A subject suffering from a cancer and in need of treatment thereof has a sufficient number of risk factors or presents with a sufficient number or combination of signs or symptoms of the disease such that a competent individual would diagnose or suspect that the subject was suffering from the disease. Methods for identification of subjects suffering from a cancer is within the ability of those in the art. In some cases, a therapeutic agent for the treatment of a cancer comprising a KIAA1549-

[0085] BRAF fusion inhibits or reduces the size of the cancer. For example, a therapeutic agent may inhibit or reduce the size of a tumor by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some cases, administration of a therapeutic agent results in a reduction in the cancer cell population in the patient. In one example, the patient undergoing the therapeutic regimen is monitored to determine whether the regimen has resulted in a reduction in the cancer cell population in the patient. Typically, the monitoring of the cancer cell population is conducted by detecting the number or amount of cancer cells in a specimen extracted from the patient. Methods of detecting the number or amount of cancer cells in a specimen are known in the art. In other cases, the cancer cell population in the extracted specimen can be compared with a predetermined reference range. In a specific embodiment, the predetermined reference range is based on the number or amount of cancer cells obtained from a population(s) of patients suffering from the same type of cancer as the patient undergoing the therapy.

[0086] A therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion as described herein may be administered along with a pharmaceutically acceptable carrier. The phrase “pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition, or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0087] In some cases, a therapeutic agent or a composition thereof is administered orally or systemically. Other modes of administration include rectal, topical, intraocular, buccal, intravaginal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within / on implants, or parenteral routes. The term “parenteral” includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or infusion.

[0088] A therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion as described herein may be assembled into a kit or a pharmaceutical system for use in treating cancer. Kits or pharmaceutical systems may include a carrier means, such as a box, carton, tube, having in close confinement therein one or more container means, such as vials, tubes, ampoules, bottles, syringes, or bags. A kit or a pharmaceutical system may also include instructions for using the kit.

[0089] In some aspects, a cancer associated with a KIAA1549-BRAF fusion is a cancer that is a cancer refractory to treatment with an mitogen-activated protein kinase (MAPK) inhibitor. Despite excellent overall survival (OS) following surgery, conventional chemotherapeutics, radiotherapy and targeted therapy, many patients with a refractory cancer associated with a KIAA1549-BRAF fusion do not respond to treatment or get tumor regrowth after treatment cessation. This problem has become evident as patients are forced to continue targeted therapy for many years without curative effects and unknown long-term side-effects. A therapeutic agent for the treatment of a cancer comprising a KIA Al 549-BRAF fusion identified by the methods described herein may be administered to a subject in need thereof for the treatment of a cancer associated with a KIAA1549-BRAF fusion along with one or more additional therapeutic interventions. Such an additional therapeutic intervention may include, for example, chemotherapy, radioimmunotherapy, toxin therapy, prodrug-activating enzyme therapy, antibody therapy, surgical intervention, immunotherapy, radiation therapy, targeted therapy, or any combination thereof. In some aspects, the prior therapy has failed in the patient. In some cases, the therapeutically effective regimen comprising administration of a therapeutic agent as described herein is administered to the patient immediately after patient has undergone the prior therapy. For instance, in certain cases, the outcome of the prior therapy may be unknown before the patient is administered a compound of the invention.

[0090] In some aspects, a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion as described may be administered in combination therapy, i.e., combined with other agents, e.g., therapeutic agents, that are useful for treating pathological conditions or disorders, such as various forms of cancer. The term “in combination” in this context means that the agents are given substantially contemporaneously, either simultaneously or sequentially. In some aspects, the dosages of the one or more additional anti cancer therapeutics used in the combination therapy is lower than those which have been or are currently being used to prevent, treat, and / or manage cancer when the additional anticancer therapeutic is administered alone.

[0091] In some aspects, the administration of a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion identified by the methods described herein along with an additional therapeutic agent may demonstrate synergy.

[0092] Mitogen-activated protein kinase (MAPK) pathway inhibitors are efficacious against a subset of fusion-driven pLGGs (Fangusaro et al., 2019, Lancet Oncol, 20(7): 1011-1022). In some aspects, an additional therapeutic intervention may include, for example, the administration of a MAPK inhibitor, including, but not limited to, belvarafenib, trametinib, selumetinib, cobometinib, ulxertinib, tovorafenib, vemurafenib, abrafenib, encorafenib, and / or dabrafenib. In some aspects, the additional therapeutic intervention is the administration of belvarafenib, a small molecule RAF dimer (type II) inhibitor which shows anti-tumor clinical activity in cancer patients with BRAFV600E- and NRAS- mutations (Awada et al., 2022, Current Opinion in Oncology, 34 (2): 115-122; and Kim et al., 2019, Journal of Clinical Oncology. 37 (15 suppl): 3000). In some aspects, the administration of a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion identified by the methods described herein along with a MAPK inhibitor may demonstrate synergy.

[0093] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0094] EXAMPLES

[0095] Example 1 Therapeutic Approaches to Target KIAA1549-BRAF

[0096] With this example, through genome-scale CRISPR / Cas9 screens, the POMT1 / 2 complex was identified as being essential for the survival of KIAA1549::BRAF-dependent models of pLGG. The POMT1 / 2 complex catalyzes the addition of mannose to transmembrane and secreted proteins, suggesting that modification of KIAA1549:BRAF by POMT is necessary for protein stability and trafficking.

[0097] Isogenic pLGG cell models were generated with overexpression of common pLGG drivers such as KIAA1549::BRAF and BRAFV600E, as well as vector controls (Luciferase). These oncogenes were transduced into CD1 E14.5 mouse neural stem cells. After blasticidin selection, the cells were propagated to establish stable cell models (mNSC KIAA1549::BRAF, mNSC BRAFV600E, mNSC Luciferase). The two BRAF cell models were then moved to media depleted of EGF and FGF to have proliferation and growth fully dependent on the MAPK signaling from the oncogenic drivers and not extracellular growth factor signaling. However, the vector control could not grow under such conditions and was continuously cultured with growth factors. These cell models were also able to generate grafted tumors in vivo. To explore genetic dependencies in MAPK -driven brain tumors, a whole-genome CRISPR / Cas9 screen was designed in the isogenic cell model described above. To achieve one guide per cell, the cell models were infected with a whole-genome CRISPR / Cas9 library at a MOI of 30%. An early time point reference sample was taken one day after antibiotic selection and cultured the cells for a total of 18 days. Sequencing of the reference and end point samples revealed guide depletion or abundance, depending on if the target gene was a dependency or not. The vast majority of target genes were not dependencies. By plotting our BRAF-driven models against each other and the vector control we could identify genes with model specific dependency. Protein O-mannosyltransferase l(Pomtl) and 2 (Pomt2) were the top two specific dependencies in the mNSC KIAA1549::BRAF model identified.

[0098] In order to validate pomtl and 2 as dependencies in a human model system, human isogenic cell models were generated based on the same drivers as the mouse isogenic cell model. Human neural stem cells, H9, were transduced with lentivirus carrying IKZF3-degron tagged KIAA1549::BRAF or BRAFV600E (and vector control, Luciferase) constructs (H9 KIAA1549::BRAF, H9 BRAFV600E). Similarly to the mouse isogenic cells, the human cell models could be cultured in growth-factor independent conditions and were fully dependent on oncogenic signaling.

[0099] These human cell models were used in a CRISPR / Cas9 competition assay. CRISPR / Cas9 guides were designed against human Pomtl and Pomt2 and these guides cloned into all-in-one Cas9 / sgRNA vectors, also expressing mCherry. sgRNA against POLR2B, a pan-essential gene, was used as positive control and was cloned into the all-in-one mCherry vector. Intergenic DNA was used for negative control. Negative control vectors are identical to Pomtl, Pomt2 and positive control vectors, but with an eGFP reporter. Lentivirus with vectors expressing mCherry, Cas9, and sgRNA (against Pomtl, Pomt2 or Polr2b), and vectors expressing eGFP, Cas9 and sgRNA against intergenic DNA were mixed 1 : 1 and transduced into the H9 KIAA1549::BRAF and H9 BRAFV600E and vector controls. This way sgRNA against the gene of interest and positive control (mCherry) was competed against the negative control (eGFP). The mCherry:eGFP ratio was measured with flow cytometry every 3-4 days (when confluent) and at 18 days post-transduction (end point). mCherry signal from vectors with sgRNA against Pomtl and Pomt2 was depleted to the same extent as the pan-essential positive control at the end point. This result validated initial findings from the whole-genome CRISPR / Cas9 screen in the mouse cell model and the human isogenic cell models of MAPK-driven pLGG.

[0100] METHODS

[0101] Engineering mouse neural stem cells

[0102] Embryonic (E14.5) neural stem cell neurospheres from CD1 mice were engineered to express Luciferase (vector control), BRAFV600E, KIAA1549::BRAF (15:9 fusion), BRAF WT, and KIAA1549 WT using lentiviral delivery. Following 10 days of blasticidin selection (4 ug / ml), KIAA1549:BRAF- and BRAFV600E-expressing cells were grown in TSM without exogenous EGF / bFGF to create oncogene-dependent cell lines.

[0103] Orthotopic transplantation of isogenic mNSC to assess in vivo transformation

[0104] For orthotopic injections, MAPK oncogene dependent (BRAFV600Eand KIAA1549::BRAF) mNSCs and LacZ expressing mNSCs were used. 300,000 cells in 3 ul PBS were injected into the right hemisphere (from Bregma, 0 mm AP, -2 mm ML, -3 mm DV slowly pull back to 2.5 mm in depth) of 6-week of Fox Chase SCID mice (n=10 mice / cell line, male:female). The endpoint was reached when any of the following conditions were fulfilled: 15% loss in body weight from peak weight, poor body condition (BCS 2), if there are signs the animal is in morbid condition, neurological symptoms. Brains were collected at the endpoint and fixed in 10% formalin for 24 hours, then transfer samples to 70% ethanol. Brains where then paraffin embedded.

[0105] Engineering human neural stem cell s

[0106] Human neural stem cells (H9 hESC-Derived, #N7800-100, Gibco) were engineered to express IKZF3 degron-tagged Luciferase, BRAFV600E, or KIAA1549::BRAF. Following puromycin selection, KIAA 1549:: BRAF- and BRAFV600E-expressing cells were grown in TSM without exogenous EGF / bFGF to create oncogene-dependent cell lines.

[0107] Pomalidomide treatment response Human neural stem cells expressing luciferase, BRAFy600E(oncogene-dependent), or KIAA1549:BRAF (oncogene-dependent) were seeded into geltrex-coated 96-well plates at a density of 10,000 cells / well. The next day, growth medium was changed to TSM or TSM without growth factors and cells were treated with the indicated pomalidomide concentrations. After 72 hours, 50 uL of CellTiter-Glo was added to each well and luminescence was measured on an EnVision plate reader.

[0108] Genome-scale CRISPR / Cas9 screen

[0109] The genome-wide CRISPR / Cas9 screen was performed as previously described (Abid et al., 2023, NatProtoc, 18(7):2014-2031).

[0110] R3A-5a concentration response

[0111] Mouse neural stem cells expressing luciferase, BRAFV600E(oncogene-dependent), or KIAA1549:BRAF (oncogene-dependent), BRAF WT and KIAA1549 WT were seeded in whitewall 96-well plates at a density of 2,000 cells / well in 100 ul TSM with or without (oncogenedependent) growth factors. The same day, R3A-5a was added in decreasing concentrations (50, 25, 15, 12.5, 10, 7.5, 3.75, 0.9375, 0.234375 and 0.234 uM) with equivalent DMSO controls. After 5 days, 100 ul of CellTiter-Glo was added to each well and luminescence was measured on a SpectraMax M5 plate reader. IC50 was determined with GraphPad Prism 10.

[0112] Colony formation assays

[0113] Human neural stem cells were transduced with lentivirus encoding Luciferase, KIAA1549:BRAF, or KIAA1549:BRAFdelsp. Following puromycin selection, cells were seeded into geltrex-coated 6-well plates at a density of 25,000 cells / well. Cells were cultured in TSM without exogenous EGF / bFGF for 21 days, with growth medium being changed every 2-3 days. Cells were fixed with 3.7% paraformaldehyde and stained with crystal violet.

[0114] RESULTS

[0115] Experimental Models With this example, a series of isogenic mouse neural stem cell models overexpressing pLGG-relevant oncogenes, including KIAA1549::BRAF and BRAFV600E, were generated. Expression of activated BRAF is sufficient to drive proliferation in the absence of exogenous growth factor supplementation in vitro (FIG. 1 and are sufficient to form tumors in vivo (FIG. 2).

[0116] Identification of POMT as a vulnerability in KIAA1549:BRAF-dependent cells

[0117] To identify selective vulnerabilities in KIAA1549:BRAF-dependent cells, a series of genome-scale CRISPR / Cas9 screens was performed in isogenic mouse neural stem cell models expressing KIAA1549:BRAF (FIG. 3). The top two KIAA1549:BRAF-selective dependencies were Pomtl and Pomt2 (FIG. 4). Both Pomtl and Pomt2 were not dependencies in luciferaseexpressing (control) cells, or in cells expressing BRAFV600E. Pomtl and Pomt22 form a heterocomplex (Pomt complex) that catalyzes the transfer of mannose adducts from a Dol-P-Man donor peptide to Ser and Thr residues on acceptor proteins in the Endoplasmic Reticulum (ER) (Manya et al., 2004, Proc Natl Acad Sci USA,' 101(2):500-5). The heterotrimeric dolicholphosphate — mannose (DPM) synthase complex is critical for biosynthesis of the mannose (Orchard et al., 2004, Bioorg Med Chem Lett, 14(15):3975-8). All three donor peptide (Manya et al., 2004, Proc Natl Acad Sci USA,' 101 (2): 500-5). All three members of this complex were selective vulnerabilities in KIAA1549:BRAF-dependent cells (FIG. 4).

[0118] To validate POMT1 and POMT2 as dependencies in a human model system, human isogenic cell models were generated ectopically expressing IKZF3-degron tagged KIAA1549::BRAF, BRAFX 600E, or Luciferase. These models, when grown in the absence of exogenous growth factor supplementation are dependent on the exogenous oncogene for survival (FIG. 5 and FIG. 8). Using CRISPR / Cas9 competition assay, it was demonstrated that both POMT1 and POMT2 dependence is equal to that of a pan-essential positive control gene (POLR2B) (FIG. 6). Consistently, both POMT1 and POMT2 were not dependencies in BRAFV600E-expressing or Luciferase-expressing cells. This data validated initial CRISPR / Cas9 screen findings in human isogenic cell models of MAPK-driven pLGG.

[0119] Pharmacological POMT inhibition in KIAA1549:BRAF-dependent cells A small molecule POMT inhibitor (R3A-5a) (Orchard et al., 2004, Bioorg Med Chem Lett; 14(15):3975-8) was used to leverage an orthologous approach to validate POMT as a vulnerability in KIAA1549:BRAF-dependent cells. Consistently, R3A-5a selectively reduced the viability of KIAA1549:BRAF-dependent cells, and had an IC50 2 to 20 times lower KIAA1549::BRAF-driven cells than in control cells expressing BRAFV600E, wildtype BRAF, wildtype KIAA1549, or Luciferase (FIG. 7). This example shows that:

[0120] POMT complex is a genetic dependency in KIAA1549::BRAF-driven isogenic cell models of pLGG.

[0121] The fusion partner KIAA1549 is necessary for oncogenicity of the pLGG fusion protein KIAA1549::BRAF.

[0122] The POMT complex is targetable in KIAA 1549:: BRAF -driven isogenic cell models highlighting POMT inhibition as a novel therapeutic strategy for treatment of KIAA1549::BRAF-driven cancers.

[0123] Example 2

[0124] Pomt inhibition synergizes with RAF inhibition at low concentrations

[0125] METHODS

[0126] Synergy assay. Cells were plated as 1,000 cells / well in 384-well plates with 25 ul of media per well. Belvarafenib and R3A-5a were added 30 mins after cell plating. Each concentration of drug was added in triplicates. Viability was measured with CellTiter-Glo after 72 hours of drug treatment and analyzed in a plate reader. Synergy was assessed using SynergyFinder+ and presented as a BLISS score. mNSC KIAA1549::BRAF, BRAFV600E, and vector control (luciferase) cells were used to assess potential synergistic effects between Pomt inhibition with R3A-5a and RAF inhibition with Belvarafenib in isogenic fusion-driven mouse cells. R3A-5a had a dose-dependent negative effect on the viability of KIAA1549::BRAF-driven mNSCs treated with low concentrations of Belvarafenib (FIG. 9A). Only very high concentrations had any added effect on control cell lines (BRAFV600E and luciferase). There was no general synergy in any cell line found using BLISS score (FIG. 9B). However, looking at specific drug concentration combinations synergy is likely to happen between these two drugs at low concentrations. Synergy score higher than 10 and simultaneous negative effect on viability is however only seen in KIAA1549::BRAF-driven cells (FIG. 9B). This indicate that the doses of Belvarafenib and R3A-5a can be decreased when using them in combination.

[0127] Example 3

[0128] A glycosylation checkpoint is essential for KIAA1549::BRAF maturation and oncogenesis Fusions between protein-coding genes are among the most common oncogenic drivers across cancers. These typically pair a single proto-oncogene with one of a set of partners that do not independently drive cancerthemselves. In all cases where the fusion oncoprotein is therapeutically actionable, the protein sequence of the proto-oncogene is the drug target; the contributions to oncogenicity of the encoded portion of the fusion partner have largely been ignored. The role of KIAA1549 in KIAA1549::BRAF fusions, which are among the most common genetic drivers of pediatric gliomas, is presented. KIAA1549 is indeed necessary for the oncogenicity of KIAA1549::BRAF by guiding the intracellular trafficking and membrane localization of the fusion protein. This aberrant trafficking engenders pharmacologically actionable, fusion-specific genetic dependencies. For example, genetic silencing or pharmacologic inhibition of the protein O-mannosyltransferase 1 and 2 (POMT1 / 2) complex reverses fusion-induced transformation, and POMT1 / 2 is required to glycosylate and stabilize KIAA1549 during maturation of KIAA1549::BRAF. These findings represent a proof-of-concept for targeting the roles of proto-oncogene partners in oncogenic fusions as a potential cancer therapeutic strategy.

[0129] Since the introduction of imatinib as an inhibitor of BCR-ABL1 in patients with chronic myelogenous leukemia (O’Brien et al., 2003, N. Engl. J. Med. 348:994-1004), oncogenic fusion proteins have served as targets for the development of small molecule therapeutics. Typically, oncogenic fusions pair a single proto-oncogene (e.g., ALK, NTRK, ROS1, and RET) with any of a number of partner loci that do not encode known proto-oncogenes but nevertheless contribute to the amino acid sequence of the resulting protein (Gao et al., 2018, Cell Rep. 23:227 -238 ,e3) . An example is the proto-oncogene BRAF, which is fused in pediatric gliomas to one of multiple partners including KIAA1549 (Sievert et al., 2009, Brain Pathol., - 19:449-458; Jones et al., 2009, Oncogene,’ 28:2119-2123; Forshew et al., 2009, J. Pathol.,' 218: 172-181), FAM131B (Cin et al., 2011, Acta Neuropathol.-, 121 :763-774), RNFJ30 (Jones et al., 2013, Nat. Genet.,- 45:927-932), and GNAI1 (Jones et al., 2013, Nat. Genet.,- 45:927-932). The constancy of the proto-oncogene and variability in its partner have directed most attention to understanding the role of the proto-oncogene component, with much less attention to the component encoded by the fusion partner. For example, BRAF fusions are thought to activate BRAF by truncating and removing autoinhibitory domains on its N-terminus, with reports suggesting that the fusion partners do not contribute to the oncogenicity of the protein product (Shin et al., 2015, Genes Cancer, 6:9-18; Ross et al., 2016, Int. J. Cancer, 138:881-890).

[0130] KIAA1549::BRAF (K::B) rearrangements are particularly interesting as the sole and pathognomonic genetic alteration underlying most pilocytic astrocytomas (PAs) — the most common brain tumor in children (Collins et al., 2015, Acta Neuropathol., ' 129:775-788). K::B is challenging to therapeutically target since it retains a wildtype BRAF kinase domain, thus precluding the use of BRAF inhibitors developed to target BRAFV600Ein its monomeric state (Hanrahan et al., 2024, Nat. Rev. Clin. Oncol. , 21 :224-247; Usta et al., 2020, Mol. Cancer Ther. , ' 19: 1736-1750). Recent clinical trials have evaluated on-target MAPK pathway inhibition in PAs and other pediatric low-grade gliomas (pLGGs) using either RAF or MEK inhibitors (Fangusaro et al., 2021, Neuro. Oncol.,- 23: 1777-1788; Kilburn et al., 2024, Nat. Med.,- 30:207-217). The type II BRAF inhibitor tovorafenib is approved for pLGG, but primary resistance, dose-limiting toxicity, and re-growth upon inhibitor withdrawal limit the utility of on-target MAPK inhibition in many patients (Fangusaro et al., 2021, Neuro. Oncol.,' 23: 1777-1788; Kilburn et al., 2024, Nat. Med., - 30:207-217).

[0131] In this example, whether proto-oncogene fusion partners are truly dispensable for oncogenicity, focusing on both common and rare BRAF fusion partners, is assessed. Specific structural and functional elements of the BRAF fusion partners are necessary for transformation, in many cases, by enhancing BRAF membrane localization, is demonstrated. Genome-scale antitransformation screens to demonstrate that BRAF fusion partners engender fusion-specific, therapeutically tractable, genetic dependencies are also leveraged. Most notably, a necessary step for K::B oncogenicity is glycosylation of its KIAA1549 component by the protein O- mannosyltransferase 1 and 2 complex (P0MT1 / 2) is demonstrated, thereby suggesting this complex as a target for therapeutic development. This disclosure highlights an underexplored approach to inhibit oncogenic fusions across pediatric and adult cancers.

[0132] RESULTS

[0133] The role of BRAF fusion partners in pediatric low-grade gliomagenesis

[0134] Direct therapeutic targeting of oncogenic fusions has relied almost exclusively on inhibitors of the proto-oncogene component of the fusion. An analysis of FDA-approved compounds for fusion-driven cancers was performed to determine whether these target the protooncogene or fusion partner (FIG. 11A) and identified 21 FDA-approved compounds. These inhibitors include crizotinib, ceritinib, alectinib, brigatinib, lorlatinib, crizotinib, entrectinib, larotrectinib, entrectinib, selpercatinib, pralsetinib, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, pemigatinib, infigratinib, futibatinib, tovorafenib, and ATRA (All-Trans Retinoic Acid). In every case, the inhibitor targets the proto-oncogene product and not its fusion partner. This suggests that strategies to target the fusion partners remain largely underexplored. Reasoning that fusion partners may provide additional targets for fusion-driven cancers, invokes an interest in understanding the heterogeneity of fusion partners for the most common protooncogenes across adult cancers, and all those involving kinases in pediatric tumors (FIG. 1 IB. Across pediatric tumors with kinase fusions, 770 unique fusion species were identified, with ALK having the most (n = 86) followed by BRAF (n = 60). Interestingly, in a second dataset of predominantly adult tumors (FusionGDB (Kim et al., 2022, Nucleic Acids Res. ' 50:D1221- D1230)), BRAF also had the second greatest number of unique fusions, with 37 identified across 8101 tumor samples with at least one gene fusion. This heterogeneity in BRAF fusion partners is consistent with previous analyses (Chen et al., 2024, Clin. Cancer Res. ; 30:3812-3823). Given the frequency of BRAF fusions across pediatric and adult cancers, and the large number of fusion variants, whether fusion partners may present unexplored therapeutic potential was evaluated, using BRAF as an example.

[0135] BRAF fusions have largely been thought to induce oncogenesis through truncation and removal of critical negative regulatory domains on the N-terminus of BRAF, thereby rendering the kinase domain constitutively active (Shin et al., 2015, Genes Cancer, 6:9-18; Lavoie et al., 2015, Nat. Rev. Mol. Cell Biol., ' 16:281-298) BRAF is composed of N-terminal regulatory domains (CR1 and CR2) and a C-terminal kinase domain (CR3). CR1 and CR2 act as autoinhibitory regions and help recruit BRAF to the plasma membrane (Park et al., 2023, Nat. Commun., ' 14:4580). In every BRAF fusion present in the pediatric cohort of this example, the rearrangement disrupts at least CR1, resulting in deletion of these N-terminal auto-inhibitory domains (FIG. 11C). However, the loss of these residues also necessarily includes loss of the regions in BRAF that mediate physical interactions with RAS and the localization of BRAF at the plasma membrane, which are necessary for placing it in position for activation (Hanrahan et al., 2024, Nat. Rev. Clin. Oncol. , ' 21 :224-247; Ghosh et al., 1994, J. Biol. Chem. , 269:10000- 10007). The loss of these BRAF domains suggests a role for the BRAF fusion partners in localizing truncated BRAF to the plasma membrane to enable its activation. Consistent with this hypothesis, every BRAF fusion adds non-BRAF amino acids to the truncated BRAF kinase. This is in contrast to enhancer-hijacking fusions, in which upstream, non-coding regulatory elements of the 5’ gene partner are hijacked to drive overexpression of the 3’ effector (Jones et al., 2013, Nat. Genet.,' 45:927-932; ICGC / TCGA Pan-Cancer Analysis of Whole Genomes Consortium, 2020, Nature,' 578:82-93; Northcott et al., 2014, Nature'. 511 :428-434). The contributions of BRAF fusion partners to the cellular localization of the fusion oncogene are disclosed.

[0136] BRAF fusions are pathognomonic for pilocytic astrocytoma (PA), the most common subtype of pediatric low-grade glioma (pLGG) (ICGC / TCGA Pan-Cancer Analysis of Whole Genomes Consortium, 2020, Nature,' 578:82-93; Northcott et al., 2014, Nature'. 511:428-434). BRAF fusions are typically the only genetic alteration across a PA’s genome. The vast majority (>90%) fuse C-terminal BRAF to N-terminal KIAA1549 (Northcott et al., 2014, Nature. 511 :428-434; Selt et al., 2017, Oncotarget, 8:11460-11479). The role of KIAA1549 was studied.

[0137] A major challenge to the study of PAs is a lack of patient-derived model systems; PAs cannot be propagated in vitro or as patient-derived xenografts using standard methods. SV40-immortalized models have been generated (Selt et al., 2017, Oncotarget, 8: 11460-11479; Selt et al., 2023, J. Neurooncol.,' 165:467-478), but these do not retain dependency on K::B. As an alternative strategy to generate oncogene-relevant model systems, both murine and human neural stem cell (NSC)-derived PA models by exogenously expressing K::B were engineered. h9 embryonic stem cell-derived human neural stem cells (h9-NSCs) were engineered to express IKZF3 degron-tagged K::B, BRAFV600E, and luciferase controls. In this system, pomalidomide induces the interaction between the IKZF3 tag and the CRBN-CRL4 E3 ligase, resulting in rapid titratable degradation of tagged proteins (Koduri et al., 2019, Proc. Natl. Acad. Sci. U. S. A.,' 116:2539-2544).

[0138] Expression of K: :B or BRAFV600Eusing this system is sufficient to transform hNSCs as measured by in vitro growth in the absence of exogenous EGF and bFGF supplementation. In contrast, the luciferase controls remained dependent on exogenous EGF and bFGF. Growth of oncogene-transformed h9-NSCs was blocked by pomalidomide-induced degradation of K::B or BRAFV600E, confirming specificity of this phenotype to oncogene expression. Similarly, degradation of K::B or BRAFV600Eprevented induction of ERK phosphorylation and proliferation. These data indicate that expression of either form of activated BRAF is sufficient for transformation of human neural stem cells.

[0139] These models were used to determine whether KIAA1549 is necessary for K::B- induced transformation, using proliferation in the absence of exogenous EGF / bFGF as the transformation phenotype. To this end, truncated variants of K::B with only the KIAA1549 portion of the fusion (K:) or the BRAF portion of the fusion (:B) were expressed and their transforming potential was compared to full-length K::B or the luciferase control. Expression of full-length K::B, but not the K: or :B truncation mutants, significantly induced two-dimensional colony formation (FIG. 1 ID; p < 0.0001) and MAPK pathway activation (FIG. 1 IE). FIG. 1 ID shows representative images and quantification of three independent colony formation assays of h9-NSCs expressing Luciferase (control), KIAA1549::BRAF, KIAA1549: (exons 1-15), and :BRAF (exons 9-18), and cultured in the absence of exogenous EGF and bFGF supplementation. Quantification of the percentage of plate area with cells is shown as bars in the bar graph. Error bars represent Standard Error of the Mean (SEM) across biological replicates, one-way ANOVA p < 0.0001***. FIG. 1 IE is a representative immunoblot showing protein levels of phosphorylated and total ERK in h9-hNSCs expressing the same constructs as in FIG. 1 ID. Lysates were collected 24 hours after withdrawal of exogenous EGF and bFGF supplementation. These observations indicate that KIAA1549 is indispensable for K::B activation. Furthermore, this may be true for all BRAF fusion partners in oncogenic fusions. A meta-analysis of kinase-driven fusions in pediatric solid tumors was performed and cataloged characteristics of fusions described in 531 papers published between 2013 and 2022. This analysis identified 31 unique protein coding BRAF fusion partners in pediatric brain tumors including PAs. Among these fusion partners, the partner with the smallest contribution to the fusion protein was FAM13 IB, which appends as few as nine FAM13 IB residues to the N- terminus of truncated BRAF (Cin et al., 2011, Acta Nenropathol. ; 121 :763-774). Strikingly, adding these nine amino acids to truncated BRAF was sufficient to transform h9-NSCs (FIG.

[0140] 1 IF) and activate the MAPK pathway (FIG. 11G; p < 0.0001). FIG. 1 IF shows representative images and quantification of two independent colony formation assays of h9-hNSCs expressing Luciferase (control), FAM131B::BRAF, and FAM131B::BRAFG2A and cultured in the absence of exogenous EGF and bFGF supplementation. FIG. 11G is a representative V5, pERK, ERK, and Actin immunoblot from h9-NSCs expressing the same constructs as in FIG. 1 IF. Luciferase (control), FAM131B::BRAF, and FAM131B::BRAFG2A constructs all have C-terminal V5 tag. Data are from two independent replicates.

[0141] Intriguingly, the first six residues of the FAM131B::BRAF fusion encode a canonical MGXXXS myristoylation motif (Moriya et al., 2012, Biosci. Biotechnol. Biochem.,' 76: 1201- 1209; Sievert et al., 2013, Proc. Natl. Acad. Set. U. S. A. 110:5957-5962). Myristoylation is a post-translational modification that adds a lipid to the N-terminus of a protein, thereby facilitating attachment of the protein to membranes (Meinnel et al ., 2020, Trends Biochem. Sci.,' 45:619-632). This observation indicated that myristoylation of the fusion protein mediates oncogenesis by attaching it to membranes, resulting in increased local concentrations and the dimerization required for aberrant activation (Lavoie et al., 2015, Nat. Rev. Mol. Cell Biol.,' 16:281-298; Terrell et al., 2019, Cold Spring Harb. Perspect. Med. , ' 9:a033746. Park et al., 2019, Nature,' 575:545-550; Martinez Fiesco et al., 2022, Nat. Commun. 13:486; Rajakulendran et al., 2009, Nature,' 461 :542-545). The necessity of myristoylation to the oncogenicity of FAM131B::BRAF was tested by mutating the second FAM131B amino acid, glycine, to alanine, abolishing the myristoylation site. This completely prevented FAM131B::BRAF-induced transformation (22-fold decrease relative to FAM131B::BRAF) (FIG. 1 IF) and MAPK pathway activation (4.17-fold change, p < 0.0001) (FIG. 11G. Since myristoylation tethers proteins to intracellular membranes, subcellular localization of FAM131B::BRAF and FAM131B::BRAFG2Awere also assessed using immunofluorescence microscopy. FAM131B::BRAF predominantly localized to the plasma membrane (FIG. 11H), whereas FAM131B::BRAFG2Adid not, consistent with the idea that myristoylation of FAM131B::BRAF enhances membrane localization. FIG. 11H shows representative Airyscan images of h9-hNSCs expressing V5-tagged FAM131B::BRAF and FAM131B::BRAFG2A. FAM131B::BRAF expression was detected with an antibody against the C-terminal V5 tag.

[0142] Whether additional BRAF fusion partners harbored domains that may similarly facilitate recruitment of truncated BRAF to the membrane, allowing activation of downstream MAPK signaling, was determined. A computational analysis of 59 BRAF fusion partners identified in pediatric cancer was performed to evaluate whether they harbor domains that might facilitate membrane tethering (FIG. I ll), including transmembrane domains and myristoylation sites. FIG. 1 II shows the heatmap from an analysis of n = 28 (of a total of 59) BRAF fusion partners identified in an analysis of 4,329 pediatric cancers. Heatmap displays those with evidence for membrane localization (on Uniprotl 14), an N-terminal myristoylation motif, or one or more transmembrane domains. Across all 59 fusion partners, seven contained myristoylation sites, and an additional nine, including KIAA1549, were predicted to contain at least one transmembrane domain for at least one isoform. A variety of lipid modifications in addition to myristoylation can also localize proteins to membranes, and a UniProt subcellular localization survey of all 59 BRAF fusion partners identified an additional 10 that localized to a membrane, for 26 (44%) overall (FIG. 1 II).

[0143] Wildtype KIAA1549 is predicted to contain transmembrane domains, which are retained in K::B. Their necessity for oncogenic activity was assessed. Initial modeling suggested that KIAA1549 has an N-terminal signal peptide (residues 1-61) and two transmembrane domains (residues 998-1018 and 1299-1319), but this is inconsistent with oncogenic activity of K::B since BRAF would not be exposed to the cytoplasm and its downstream effector MEK.

[0144] The pattern of post-translational modifications to wildtype KIAA1549 supports a model of a single transmembrane domain. Phosphorylation occurs primarily within the cytoplasm, with few exceptions (Klement et al., 2017, Mol. Cell. Proteomics, 16: 1-7), whereas glycosylation tends to be extracellular. A single transmembrane domain model would predict that all residues that are C-terminal to the TM domain (residues 1299-1319) would be intracellular and thus more likely to be phosphorylated. Publicly available datasets (Ochoa et al., 2020, Nat. BiotechnoL, 38:365-373) and databases (Hornbeck et al., 2015, Nucleic Acids Res. , ' 43:D512-20), show that wildtype KIAA1549 is primarily phosphorylated within residues 1388- 1914 of the C-terminal domain (FIG. 12A). Conversely, N-linked glycans have been identified at residues of mouse Kiaal549 (Potel et al., 2025, Nat. Struct. Mol. Biol. doi: 10.1038 / s41594-025- 01485-w) that correspond to 1088, 1095, 1124 and 1226 in human KIAA1549 and residue 1063 exclusively in human KIAA1549 (FIG. 12A).

[0145] Human KIAA1549 is heavily glycosylated (>60 sites) by O-linked mannose (O-Man) glycans within residues 73-864 (Vester-Christensen et al., 2013, Proc. Natl. Acad. Sci. U. S. A. 110:21018-21023) (FIG. 12A). FIG. 12A depicts a schematic showing wildtype human KIAA1549 (top panel), wildtype human BRAF (middle panel), and KIAA1549::BRAF (15::9, lower panel), annotated to show the presence of glycosylation or phosphorylation modifications. Approximate locations of n = 15 phosphorylation sites are annotated according to Uniprotl 14. N-glycosylation (n = 5) and O-glycosylation (n = 64) sites are annotated as forks or small circles, respectively. The KIAA1549 ectodomain (residues 62-1299) is annotated as all residues preceding the KIAA1549 transmembrane domain (residues 1299-1319), excluding the ER signal peptide (residues 1-61). SP: signal peptide; TM: transmembrane domain; CR1 : conserved region 1 (RAS-binding domain and cysteine-rich domain); CR2: conserved region 2 (autoinhibitory hinge region).

[0146] Both N- and O-Man glycosylation is initiated within the lumen of the endoplasmic reticulum prior to trafficking of transmembrane proteins to the cell surface. The identification of ER-signal peptide (residues 1-61) and glycosylation within residues 73-1212 of KIAA1549 thus shows extracellular orientation for these residues. Taken together, these data demonstrate that KIAA1549 is a type I transmembrane protein with a predicted transmembrane domain at residues 1299-1319 (FIG. 12A). The observation that KIAA1549 harbors a TM domain suggests that oncogenic activity may result from KIAA1549 tethering the BRAF kinase domain to the cytoplasmic face of membranes. Therefore, the subcellular localization of K::B using immunofluorescence microscopy was examined. Surprisingly, K::B appeared to be distributed throughout the cell, with expression detected in the cytoplasm and at cellular membranes (FIG. 12B). In contrast, K::B in which the putative KIAA1549 transmembrane domain had been deleted (K::Bde1™) co-localized with the endoplasmic reticulum (ER) marker PD I, indicating that loss of the transmembrane domain results accumulation in the ER (FIG. 12B, Pearson correlation coefficient K::B = 0.19, K::Bdel™ = 0.57, p < IxlO'10). FIG. 12B shows representative Airyscan immunofluorescence images of h9- hNSCs expressing C-terminal V5-tagged KIAA1549::BRAF or KIAA1549::BRAFdel™ labelled with V5-tag and PDI (marker of endoplasmic reticulum) antibodies. Data are from three independent biological replicates. Quantification was performed by segmenting individual cells, then computing the Pearson correlation coefficient between the V5 and PDI channels for each individual cell. Quantification of V5 and PDI colocalization of V5 and PDI (right). Median Pearson correlation coefficient (K::B = 0.19, K::Bdel™ = 0.57). Statistical significance was computed with a two-tailed t-test (p < IxlO'10).

[0147] To test whether ER-localized K::B remains sufficient for oncogenic signaling, two- dimensional colony formation in the absence of exogenous EGF and bFGF was assessed (FIG. 12C). FIG. 12C shows representative images and quantification of colony formation assays of h9-hNSCs expressing Luciferase (control), KIAA1549::BRAF, and KIAA1549::BRAFdel™. Data are from three independent biological replicates. Percent area covered: Luciferase = 5.87%, K::B = 89.2%, K::Bdel™ = 8.78%. Statistical significance was computed with a one-way ANOVA test with Tukey’s multiple comparison test (Luc vs K::B p < 0.0001****, Luc vs K::Bdel™ p > 0.05). While K::B was sufficient to activate ERK (p = 0.0051, K::B vs K::Bde1™) and induce growth under these conditions, K::Bdel™ was not (FIG. 12D, p = 0.616). FIG. 12D shows a representative immunoblot of pERK, ERK, and Actin in h9-hNSCs expressing the same constructs as in FIGS. 12B and 12C.

[0148] These data indicate that K::B is trafficked through the ER, in contrast to wildtype BRAF, which is translated directly into the cytoplasm (Diedrich et al., 2017, EMBOJ:, 36:646- 663). Proteins that traffic through the ER often have an N-terminal signal peptide that directs the nascent ribosome-bound peptide to the translocon channel, where it is subsequently translated directly into the ER lumen. To test the necessity of trafficking into the ER, the KIAA1549 signal peptide was deleted (K::Bdelsp), and the ability of K::Bdelspto transform cells was measured. Again, K::B was sufficient to transform cells, whereas K::Bdelspwas not (FIG. 12E, 69-fold decrease relative to K::B). FIG. 12E shows representative images and quantification of colony formation assays of h9-hNSCs expressing Luciferase (control), KIAA1549::BRAF, and KIAA1549::BRAFA SP. Data are from two independent biological replicates. Percent area covered: Luciferase = 1.2%, K::B = 61.8%, K::Bdelsp= 0.89%. The findings that both K::Bdel™ and K::Bdelspare non-functional suggest that K::B trafficking through the ER lumen is required for its activity. It also suggests that proteins that facilitate this trafficking may represent dependencies in K::B-driven tumors.

[0149] Anti -transformation screens identify additional dependencies in KIAA1549::BRAF- vs BRAFV600E-transformed cells

[0150] Given that KIAA1549 is necessary for the oncogenicity of the K: :B fusion, additional genes and pathways beyond the canonical MAPK pathway may also be required. These would not be shared by activating point mutations like BRAFV600E. To test this, genome-scale loss of function CRISPR / Cas9 screens in embryonic murine neural stem cells (mNSCs) expressing human K::B (the 15 : :9 fusion breakpoint variant), human BRAFV600E, or luciferase were performed (FIG. 13A). FIG. 13A is a schematic depicting the generation of isogenic cell models from embryonic (E14.5) mouse neural stem cells with ectopic expression of human KIAA1549::BRAF (15:9), BRAF600E, and Luciferase (Control). Isogenic lines with expression of KIAA1549::BRAF and BRAFV600Ewere cultured without EGF / bFGF, rendering them oncogene dependent. Luciferase-expressing cells were cultured with EGF / bFGF. These models were subsequently leveraged to perform genome-scale loss of function CRISPR / Cas9 screens). mNSCs were chosen because conditions for growing mNSCs sufficiently to conduct genomescale screens were optimized previously (Khadka et al., 2022, Nat. Commun:, 13:604; Abid et al., 2023, Nat. Protoc. 18:2014-2031).

[0151] As with hNSCs, expression of K::B or BRAFV600Ewas sufficient to transform mNSCs as measured by anchorage-independent in vitro growth as neurospheres in the absence of exogenous EGF and bFGF supplementation (p<0.001), and by glioma formation in vivo when injected as intracranial allografts (Morin et al., 2024, bioRxiv 2024.08.27.609922, doi: 10.1101 / 2024.08.27.609922), further indicating that both K::B fusions and BRAFV600Eare sufficient for transformation. In lieu of a standard synthetic lethal screen, this screen was designed to also assess cofactors that contribute to growth factor independence, the transformation phenotype of K::B, and BRAFV600E. Specifically, the K::B- and BRAFV600E- expressing cells were grown in the absence of the growth factors EGF and bFGF, rendering these cell lines oncogene-dependent. Both synthetic lethalities and transformation cofactors will appear as dependencies in this screen. The luciferase controls were grown in standard media containing EGF and bFGF (FIG. 13A).

[0152] Strikingly, K::B expressing cells exhibited strong and selective dependencies that were not observed across the other mNSC models (BRAFX 600Eor luciferase), with dependencies on Pomtl and Pomt2 standing out as the most significant (Pomtl differential logfc = -2.82, Pomt2 differential logfc = -3.07, Pomtl p = 0.0006, Pomt2 p = 0.0004) (FIG. 13B). FIG. 13B is a scatter plot showing log2 fold change (day 18 vs early timepoint) in gene-level CRISPR guide abundance scores. Screen from mouse neural stem cells (mNSCs) expressing KIAA1549::BRAF (in the absence of EGF / bFGF) is represented on the y-axis. Screen from mNSCs expressing Luciferase (in the presence of EGF / bFGF) is represented on the x-axis. Each dot represents a single gene, selected genes are highlighted. Each screen was performed with three independent biological replicates.. Pomtl and Pomt2 heterodimerize to form the POMT1 / 2 complex (Manya et al., 2004, Proc. Natl. Acad. Set. U. S. A.; 101 :500-505). POMT1 / 2 catalyzes O- mannosylation, transferring mannose from the Dol-P-Man donor to Ser and Thr residues on acceptor protein substrates (Vester-Christensen et al., 2013, Proc. Natl. Acad. Set. U. S. A.; 110:21018-21023; Povolo et al., 2024, Mol. Cell. Proteomics; 23 A 00296) (YIG. 13C). FIG. 13C depicts a graphical representation of POMT1 / POMT2 specific O-Mannosylation in mammals.

[0153] The dolichol phosphate mannose (Dol-P-Man) donor is synthesized from Dol-P and GDP -Man by the DPMI-3 complex. Dol-P-Man flips and is utilized by the POMT1 / POMT2 heterodimer for co / post-translational modification of Ser / Thr residues with O-linked mannose within unstructured protein domains in the ER lumen. DPM1 / 2 / 3 : dolichol-phosphate mannosyltransferase- 1 / 2 / 3 of the dolichol phosphate mannose (Dol-P-Man) complex; GDP / GTP: guanosine diphosphate / triphosphate; POMT1 / 2: protein O-mannosyltransferase 1 and 2 of the POMT complex. KIAA1549 is the most heavily O-mannosylated protein across the human proteome (Manya et al., 2004, Proc. Natl. Acad. Sci. U. S. A.; 101:500-505; Povolo et al., 2024, Mol. Cell. Proteomics; 23: 100796) and KIAA1549 O-mannosylation is specifically dependent on P0MT1 / 2 activity (Povolo et al., 2024, Mol. Cell. Proteomics,' 23: 100796; Larsen et al., 2017, J. Biol. Chem. , ' 292: 11586-11598). Thus, the finding ofPomtl / 2 dependency in K::B -transformed cells was biologically plausible.

[0154] The dependency on Pomtl / 2 was further supported by highly selective K::B cell dependencies on all three members of the heterotrimeric DPM complex (Dpml, Dpm2, and DpmS) (FIG. 13B). Pomtl, Pomt2, and Dpm 1-3 all exhibited greater than 65% depletions (log2 change <-1.5; Dpml p = 0.021, Dpm2 p = 0.0024, Dpm3 p = 0.0023) over the 18-day screen. These findings indicate that K::B expressing NSCs harbor a striking dependency on Dol-P-Man donor availability, POMT1 / 2 activity, and O-mannosylation of the extracellular K::B domain. Expression of POMT1 and POMT2 in cancer and normal brain

[0155] To assess whether O-mannosylation by the POMT1 / 2 complex might have a role in human pLGGs with K::B fusions, expression of both P0MT1 / 2 and DPM complex members across a panel of 87 human K::B-positive pLGGs profiled with bulk RNA-Seq was analyzed. P0MT1, P0MT2, and all three members of the DPM complex were detected in every tumor profiled, with read counts of at least 8 per million reads (average across all genes = 67.3; range 11.9-261.1). Tumors from pLGG patients typically have high non-malignant cell infiltration (Reitman et al., 2019, Nat. Commun., 10:3731), confounding bulk RNA-seq analysis since many sequencing reads are derived from non-malignant cells. Single-cell RNA-seq data from 10 K::B- driven pLGGs comprising 60,131 cells (average per tumor: 6013 cells; range 912-16,633) was interrogated. Because these tumors have few genetic alterations, it can be difficult to distinguish between cancer and normal cells in these data. However, analyses of single-cell RNA-seq data from K::B-driven pLGGs indicated that the vast majority of glia exhibited the fusion (Reitman et al., 2019, Nat. Commun., ' 10:3731). Therefore, Leiden clustering and manual annotation of each cluster according to 10 prior gene sets was performed. Nine different cell types were detected. POMT1 / 2 and DPM complex genes were preferentially expressed across glia and neurons relative to immune infiltrates (myeloid cells and T-cells) and endothelial cells (p < 0.001).

[0156] K::B-driven pLGGs predominantly arise in the cerebellum (Reitman et al., 2019, Nat. Commun.,' 10:3731), and the high expression of POMT1 and POMT2 seen in K::B driven pLGGs appears to be consistent with expression patterns in normal brain. Expression of P0MT1, P0MT2, DPMI, DPM2, and DPM3 in bulk RNA-seq (GTEx (GTEx Consortium, 2020, Science,' 369: 1318-1330)) data spanning 54 normal tissue types, including 13 brain regions was analyzed. Both P0MT1 and P0MT2 exhibited preferential expression in the brain, with highest expression in the cerebellum. DPM complex genes were more uniformly distributed across tissue types, reflecting their pleiotropic functions beyond O-mannosylation (Colussi et al., 1997, Proc. Natl. Acad. Sci. U. S. A. ; 94:7873-7878; Gandini et al., 2017, Nat. Commune, 8: 120; Takahashi et al., 2014, Handbook of Glycosyltransferases and Related Genes,' Springer Japan, Tokyo: 1637- 1647). These observations suggest that PAs with K::B fusions arise in brain locations that express high levels of POMT1 / 2 complex genes relative to other tissues, and that the PAs retain this expression.

[0157] POMT1 and POMT2 are necessary for KIAA1549::BRAF-induced transformation

[0158] Next, Pomtl and Pomt2 as genetic dependencies were validated across a panel of isogenic mNSCs and hNSCs. CRISPR / Cas9 was used to silence Pomtl or Pomt2 in K: :B- expressing or control mNSCs and assessed proliferation over 18 days. Cells expressing guides against GFP were included as a non-targeting negative control. Loss of Pomtl or Pomt 2 significantly reduced proliferation of K::B-dependent mNSCs (p= 0.007 and, p= 0.02 respectively), but not luciferase-expressing controls (p=0.92 and p=0.95). CRISPR cut-site sequencing of K::B-expressing mNSCs that survived to day 18 provided further evidence of a POMT dependency: almost all reads (91% and 94%, respectively) had either no editing or in- frame indels at the targeted loci, indicating that these surviving K::B-expressing mNSCs retained functional Pomt 1 / 2. In contrast, 74% and 79% of luciferase control mNSCs exhibited frameshift mutations at the Pomtl and Pomt2 cut sites, respectively. Indeed, whereas the proportion of reads with frameshift mutations at the targeted loci increased over time in these control mNSCs, they decreased in K::B-expressing mNSCs, suggesting that K::B cells with frameshift mutations were selectively lost.

[0159] Similarly, CRISPR / Cas9 ablation of P0MT1 or P0MT2 was sufficient to impede growth of K::B-expressing hNSCs relative to the non-targeting vector controls (FIG. 13D, p = 0.0403) to a similar degree as ablation of the pan-essential gene POLR2B luciferase p = 0.002, BRAFV600Ep = 0.001, K::B p = 0.034). No differences following suppression of either P0MT1 or P0MT2 in control hNSCs engineered to express BRAFV600Eor luciferase were observed (FIG. 13D, luciferase p = 0.98, BRAFV600Ep = 0.98). FIG. 13D shows a bar plot of CRISPR competition assay in h9-NSCs. Human neural stem cells were engineered to express KIAA1549::BRAF, BRAFy600E, or Luciferase and cultured as described for FIG. 13A. Cells were transduced with either intergenic control guides (EGFP) or guides targeting POLR2B, POMT1, POMT2, or the same intergenic control regions (mCherry). Three independent guide sequences were used for each condition. Cells were pooled 1 : 1. The fraction of mCherry- or EGFP -positive cells was assessed with flow cytometry three days post-transduction (initial timepoint) and 18 days post-transduction (final timepoint). The fraction of mCherry-positive cells, normalized to the initial time point, is displayed on the y-axis. The sgRNA pair conditions are displayed on the x-axis. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparison test (vs sgControl). Statistically significant p-values (p < 0.05) are indicated with * on the plot.

[0160] Indeed, dependence on POMT1 / 2 complex members is extremely specific to K::B- dependent cells. None of the 1150 cell lines profiled with genome-scale CRISPR screens in the Cancer Dependency Map (Tshemiak et al., 2017, Cell,' 170:564-576. el6) contain a K::B fusion, and none exhibited a strong dependency on either P0MT1 or P0MT2 (Chronos (Dempster et al., 2021, Genome Biol. , 22:343) score <-l) (FIG. 13E). A subset of cell lines (n = 22 / 1150, 2%) did exhibit dependency on one or more members of the DPM complex (FIG. 13E), potentially due to pleiotropic functions of Dol-P-Man that are unrelated to the POMT1 / 2 complex51-53. These observations suggest that the POMT1 / 2 complex is specifically essential for the proliferation of K::B-dependent NSCs. FIG. 13E shows a dot plot with Chronos (Bandopadhayay et al., 2016, Nat. Genet.,' 48:273-282) gene dependency scores for POMT1, POMT2, DPMI, DPM2, and DPM3 across 1150 cancer cell lines profiled in The Cancer Dependency Map (DepMap) (Bandopadhayay et al., 2016, Nat. Genet., ' 48:273-282). A Chronos score of 0 or -1 is the median of all genes classified in the DepMap 24q2 release as non-essential or common essential, respectively.

[0161] This dependence on POMT1 / 2 appears to relate to its enzymatic activity. Isogenic mNSC models were treated with R3 A-5a, a competitive inhibitor of the active site of the yeast ortholog of POMT1 / 2, Pmtl / 2 (Bai et al., 2019, Nat. Struct. Mol. Biol., ' 26:704-711; Orchard et al., 2004, Bioorg. Med. Chem. Lett.,' 14:3975-3978). R3A-5a impeded growth of mNSCs expressing K::B, but not BRAFV600Eor the luciferase vector control (AUC K::B: 141, BRAFV600E: 233, Luciferase control: 221; p = 0.0009 and p = 0.7) (FIG. 13F). FIG. 13F shows a best-fit curve of mNSC isogenic models expressing Luciferase (control), KIAA1549::BRAF, and BRAFV600Etreated with increasing concentrations of the POMT complex inhibitor R3A-5a. Viability was measured with CellTiterGlo after 72 hours of treatment. Cell viability was normalized to each equivalent DMSO control. Viability was assessed using the area under the curve (AUC) of each experimental replicate (n = 3), and then compared using one-way ANOVA with Sidak’s multiple comparisons test (luciferase vs KIAA1549::BRAF p = 0.0009***, luciferase vs BRAFV600Ep = 0.7). AUC luciferase control = 220.8; KIAA1549::BRAF = 140.8; BRAFV600E= 233).

[0162] The POMT1 / 2 complex O-mannosylates K::B in the ER

[0163] K::B expressing cells being exquisitely dependent on the POMT1 / 2 complex suggests that, like wildtype KIAA1549, the K::B fusion is also heavily glycosylated and that this is necessary for its oncogenic function. First, K::B is glycosylated by performing enzymatic digestion of the fusion protein was confirmed. Treatment of lysates from K::B-expressing cells with PNGase F, which removes all N-linked glycans, induced an apparent mass shift of K::B of at least 20 kDa in immunoblots. This magnitude of difference in the molecular mass corresponds to approximately 8-10 occupied N-glycosylation sites, consistent with the 10 potential N- glycosylation sites N-terminal to the TM domain of KIAA1549 (residues 1299-1319).

[0164] To further confirm that K::B undergoes O-mannosylation in the mNSC model described herein, lectin enrichment and differential O-glycoproteomics were combined (FIG. 14A) (Bai et al., 2019, Nat. Struct. Mol. Biol. 26:704-711). FIG. 14A shows the workflow for quantitative O-Man glycoproteomics of stable isotope labelled total cell digests enriched for O- Man glycopeptides by BC2L-A lectin chromatography. Total cell extracts of control and K::B expressing mNSC cells were differentially labeled with diethyl stable isotopes and enriched for O-Man glycosylated peptides for identification and relative quantification by mass spectrometry (FIG. 14 A).

[0165] 28 O-Man glycosylated proteins in mNSCs were identified, including O-Man glycosylation of endogenous mouse Kiaal549 and the stably expressed human K: :B fusion protein (FIGS. 4B, FIG. 14C, and FIG. 14D). FIG. 14B depicts the relative quantification of the total proteomes as a boxplot with whiskers for which the interquartile range calculations were used to determine the boundaries (dashed lines) for peptide spectral matches (PSMs) derived from Luciferase- or human KIAA1549::BRAF-expressing mNSCs. The dot-plot shows heavy-to- light (H / L) ratios of individual O-Man PSMs from KIAA1549:BRAF, endogenous mouse KIAA1549, and other C- and O-Man glycoproteins, demonstrating that O-Man of endogenous mouse KIAA1549 is equally abundant while O-Man glycopeptides of human KIAA1549::BRAF are exclusively derived from the mNSCs engineered to express human KIAA1549::BRAF. FIG. 14C is a schematic illustration of human KIAA1549::BRAF showing the mapped O-Man glycans (spheres) to residues 84-849 of the N-terminal domain. FIG. 14D shows representative PSMs used for identification and site mapping of O-Man glycans on human KIAA1549::BRAF (left) and endogenous mouse KIAA1549 (right). The insert shows precursor isotope envelopes for heavy and light labelled glycopeptides selected for MS2 fragmentation.

[0166] O-Man glycopeptides derived from endogenous Kiaal549 were present at about 1: 1 heavy-to-light ratios. These findings confirm that wildtype mouse Kiaal549 substrate is expressed and acquires O-Man glycans in comparable amounts in control and K::B-expressing mNSCs (Orchard et al., 2004, Bioorg. Med. Chem. Lett. , ' 14:3975-3978). In contrast, human K::B O-Man glycopeptides were identified exclusively with heavy stable isotope labels. This showed that human K::B is expressed and O-Man glycosylated only in the human K::B- expressing mNSC cells (FIG. 14B). 32 O-Man glycosites on the K::B fusion protein were identified, all of which were N-terminal to the TM domain (K::B residues 1299-1319). Collectively, these results demonstrate that K::B is expressed as a type I transmembrane protein in mNSCs and that the N-terminal domain acquires O-Man glycans in the ER lumen, where the Pomtl / 2 complex is active (Neubert et al., 2016, Curr. Opin. Cell Biol. 41 : 100-108). Together, these data confirm that the K::B fusion protein is heavily mannosylated by the POMT complex.

[0167] Pomtl is required for KIAA1549::BRAF fusion activity in vivo

[0168] The finding of POMT1 / 2 dependency across the in vitro models raises the possibility that it may represent a potential therapeutic target for children with K::B-driven pLGGs. Whether Pomtl suppression reverses in vivo phenotypes associated with K::B expression in the murine brain was evaluated. To generate in vivo models of K::B expression, in-utero- electroporation (IUE) of embryonic mouse brains was leveraged. IUE of the 16: :9 variant of the K::B fusion into the embryonic day 14.5 (E14.5) cortex resulted in abnormal migration of GFP- positive transfected glutamatergic neurons and increased glial reactivity seen by Gfap expression in both transfected and non-transfected cells compared to mice subjected to IUE of vector control (FIG. 15 A).

[0169] FIG. 15A shows representative images depicting EGFP+ transfected cells within EGFP control and KIAA1549::BRAF expressing brain sections (left). High magnification representative images of the upper cortical layer region co-stained for EGFP (middle left), EGFP / Gfap (middle right), or NeuN (right). Scale bars: tiled section images on the left side = 500 micrometers; high magnification images = 100 micrometers. The increased glial reactivity in

[0170] K::B-expressing cells resulted in striking differences in astrocytic morphology. While vector control transfected cortical gray matter astrocytes displayed classic, finely branched protoplasmic morphologies, those in the K::B condition showed dense and ramified morphologies associated with fibrous and reactive astrocytes (FIG. 15 A). These observations suggest that expression of K::B in embryonic neural stem cells induces developmental and morphological changes in subsequently produced neurons and glia that persists into adulthood.

[0171] With this model, whether Pomtl suppression reverses K::B-associated in vivo phenotypes was evaluated. First, CRISPR / Cas9 components targeting Rosa26 (control G« / 26sRX) or Pomtl PomtPsRNA') were co-electroporated with plasmids expressing the K::B fusion and EGFP for visualization, and the efficiency of Rosa26sgRNAand Pomtr^Awas validated by isolating and culturing neural stem cells 24 hours post-IUE with EGFP and sgRNA / Cas9 components. Rosa26i&VNA- and Po / MZ7S8RNA-electroporated cells showed indel rates above 70% and 90%, respectively. K::B + Rosa26sgRNA-electroporated brains displayed phenotypes similar to prior K::B fusion samples, including mislocalization of transfected neurons and glial reactivity within the electroporated region, with gray matter cortical astrocytes displaying reactive and fibrous morphology (FIG. 15B).

[0172] FIG. 15B shows representative images depicting EGFP+ transfected cells within K::B + Rosa26sgRNAand K::B + PomtlsgRNAexpressing brain sections. High magnification representative images of the upper cortical layer region depicting EGFP+ cell morphology (middle) and 3D reconstruction of example EGFP+ astrocytes in each condition (right). Scale bars: tiled section images on left side = 500 micrometers; high magnification images = 100 micrometers; 3D reconstruction images = 5 micrometers for K::B + Rosa26sgRNAand 7 micrometers for K::B + PomtlsgRNA. These reactive astrocytes displayed little to moderate branching and had clearly distinguishable thickened truncal processes. However, brains harvested from K::B + PomtP^^ contained transfected cells that more closely resembled those found in EGFP control-electroporated brains. Specifically, most EGFP-positive transfected K::B + / Vw? / / sgRX' astrocytes displayed classic protoplasmic, spongy astrocyte morphology, with many fine branches and branchlets, similar to EGFP-control brains and in stark contrast to those found in K::B + Rosa26sgRNAbrains (FIG. 15B). Taken together, these observations suggest that expression of K::B induces developmental and morphological changes in the brain that require Pomtl / 2 for their manifestation.

[0173] DISCUSSION

[0174] K::B fusions undergo a series of post-translational modifications that contribute to the intracellular trafficking and activation of the K::B proto-oncogene, which are required for oncogenesis, thereby engendering novel vulnerabilities. The results of the experiments with KIAA1549 and FAM131B disclosed herein indicate that these BRAF fusion partners are essential for the oncogenic function of their BRAF fusions and suggest that this property extends to other BRAF fusion partners. It is likely that other fusion oncogenes also undergo post- translational modifications and intracellular trafficking that promote oncogenesis, and that these may afford novel dependencies in other cancer types. For example, N-glycosylation of wildtype BCR mediates several of its protein-protein interactions (Ubelhart et al., 2010, Nat. Immunol. , 11 :759-765; Mortales et al., 2020, iScience,' 23:101380. The contribution of N-glycosylation of BCR::ABL1 to its oncogenicity is not well understood. Other oncogenic fusions, including FGFR fusions, likely also traffic through the secretory pathway, given they retain the FGFR transmembrane domain; the role of glycosylation in these contexts is similarly unclear.

[0175] The findings disclosed herein stand in contrast to a prevalent view that truncated BRAF kinase alone is sufficient for transformation, based on experiments that have focused on the BRAF kinase (Shin et al., 2015 Genes Cancer,' 6:9-18; Pritchard et al., 1995, Mol. Cell. Biol.,' 15:6430-6442; Liu et al., 2007, Oncogene,' 26: 1954-1958; Woods et al., 1997, Mol. Cell. Biol., ' 17:5598-5611 ; Bosch et al., 1997, Oncogene, 15: 1021-1033). However, all prior studies that showed in vitro transformation phenotypes due to expressing the truncated BRAF kinase included additional N-terminal amino acids that are not in the wildtype protein, or were performed in immortalized cells (Pritchard et al., 1995, Mol. Cell. Biol., 15:6430-6442; Woods et al., 997, Mol. Cell. Biol., ' 17:5598-5611; Liu et al., 2007, Oncogene,' 26: 1954-1958; Bosch et al., 1997, Oncogene, 15: 1021-1033). The only prior in vivo report in wildtype mice found that truncated BRAF alone is insufficient to induce pilocytic astrocytoma formation (Gronych et al., 2011, J. Clin. Invest. , 121: 1344-1348).

[0176] It is possible that anti -transformation screens of the sort performed with K::B might help identify cofactors that enable necessary post- translational modifications and trafficking of other fusion oncoproteins. Extensive efforts have been directed towards leveraging synthetic lethal screens to detect potential therapeutic targets whose modification is synthetic lethal with common genetic alterations in cancer. These include prominent successes such as PARP1 inhibition in the context of BRCA1 / 2 loss (Farmer et al., 2005, Nature,' 434:917-921), PRMT5 suppression in the context of MTAP deletions (Kryukov et al., 2016, Science,' 351 :1214-1218; Mavrakis et al., 2016, Science,' 351: 1208-1213; Marjon et al., 2016, Cell Rep.,' 15:574-587), and WRN suppression in the context of microsatellite instability (Chan et al., 2019, Nature,' 568:551- 556; van Wietmarschen et al., 2020, Nature,' 586:292-298 (2020). The screening approach described herein was capable of identifying genes whose loss is synthetic lethal with K::B expression, but also assessed for reversal of a transformation phenotype (growth in the absence of EGF / bFGF). The design of this screen also lends itself to the use of isogenic controls, which can limit the effects of confounding variables.

[0177] The function of KIAA1549 remains unknown, but its properties of heavy mannosylation and cleavage suggest similarities to signaling mucins. Signaling mucins are a diverse class of proteins that share many common architectural characteristics (densely O- glycosylated extracellular domain and a cytoplasmic signaling domain), but differ in the number and nature of key details such as the type of glycosylation found on the extracellular domain, the presence of SEA domains, and the presence / effect of extracellular cleavage (Cullen et al., 2007, Crit. Rev. Eukaryot. Gene Expr. , ' 17:241-257; Cullen et al., 2011, Curr. Opin. Struct. Biol., ' 21 :590-596). An example of a prototypical animal signaling mucin is MUC1, whose extracellular mucin domain carries O-GalNAc-type glycosylation, possesses an autoproteolytically cleaved SEA domain, and has a cytoplasmic tail for signaling (Carson et al., 2008, Sci. Signal., - I:e35). In contrast to this mucin, baker’s yeast (Saccharomyces cerevisiae), encodes for two bona fide O- Man signaling mucins - Msb2 and Hkrl, both of which regulate MAPK signaling (Cullen et al., 2004, Genes Dev.,- 18: 1695-1708). Moreover, and similarly to K::B, knockout of the O- mannosyltransferase Pmt4 regulates MAPK signaling (Yang et al., 2009, EMBO J:, 28: 1380- 1391). Both Pmt4 and POMT1 belong to the PMT4 subfamily, sharing similar capacity for O- glycosylation of substrates (Bausewein et al., 2016, J. Biol. Chem., ' 291 : 18006-18015. Furthermore, Msb2 possesses a cleavage domain between residues 1045 and 1145, whose cleavage by Ypsl is required for MAPK activation (Vadaie et al., 2008, J. Cell Biol. 181 : 1073- 1081), and overlaps with an unannotated SEA domain.

[0178] KIAA1549 and K::B fusions share features with the classical POMT1 / 2 substrate DAG1, whose functions also depend on O-mannosylation for functions (Yoshida-Mori guchi et al., 2015, Glycobiology,- 25:702-713). Autoproteolysis in the SEA domain of DAG1 facilitates maturation into alpha-DG and beta-DG chains. Mutants that disrupt DAG1 SEA domain cleavage traffic to the cell surface, acquire functional O-mannosylation, and retain laminin binding properties (Akhavan et al., 2008, FASEB J. 22:612-621) — indicating that SEA domain cleavage is dispensable for DAG1 functions. However, whether wildtype KIAA1549 or K::B depend upon SEA domain cleavage for function is not yet known.

[0179] The findings described herein harbor particular significance for therapeutic approaches for children with K::B driven pLGGs and the occasional extracranial tumors with K::B fusions (Ross et al., 2016, Int. J. Cancer, 138:881-890; Vega et al., 2024, Cancer Res. , - 84:2857-2857). The last decade has seen unprecedented progress with the use of MAPK pathway inhibitors as a therapeutic approach for these children, culminating in the recent FDA approval of the pan-RAF inhibitor tovorafenib (Fangusaro et al., 2021, Neuro. Oncol.,' F. V T1- 1788; Vadaie et al., 2008, J. Cell Biol , 181 : 1073-1081). However, approximately 30% of K::B driven gliomas are innately resistant. Those that respond often exhibit rebound growth upon cessation of therapy, necessitating continuous long-term treatment. Long-term treatment can be intolerable: the MAPK pathway is essential for the development of many tissues and organs, and children have experienced severe reductions in growth velocity while on treatment (Fangusaro et al., 2021, Neuro. Oncol.,- 23: 1777-1788; Milde et al., 2023, Neuro. Oncol., 25:1920-1931).

[0180] These considerations increase the value of alternative strategies to target K::B fusions, such as inhibiting POMT1 / 2, the DPM complex, or PCSK proteases. Indeed, whereas MAPK inhibitors may only induce cell cycle arrest or senescence (O’Hare et al., 2024, Neuro. Oncol., ' 26: 1357-1366), targeting K::B trafficking may have the additional benefit of causing toxic accumulation of K: :B within the secretory pathway. This would recall the sensitivity of transformed B-cells (which produce high levels of immunoglobulin) to proteosome inhibitors, which lead to the toxic accumulation of immunoglobulin in the ER (Schenkeinet al., 2002, Clin. Lymphoma, 3:49-55). Targeting POMT1 / 2 may result in its own toxicities: congenital homozygous loss-of-function mutations have been associated with several syndromes, most prominently muscular dystrophies (Manya et al., 2008, Neuromuscul. Disord., - 18:45-51; Haines et al., 2007, Mol. Biol. Cell,- 18:4721-4730; Ichimiya et al., 2004, J. Biol. Chem., 279:42638- 42647; Lommel et al., 2008, Glycobiology,- 18:615-625; Lommel et al., 2010, Neurology, 74: 157-164; Beltran- Valero de Bernabe et al., 2002, Am. J. Hum. Genet., ' 71 : 1033-1043). This likely reflects effects on DAG1 (Vester-Christensen et al., 2013, Proc. Natl. Acad. Sci. U. S. Ay 110:21018-21023; Povolo et al., 2024, Mol. Cell. Proteomics,' 23: 100796; Larsen et al., 2017, J. Biol. Chem., - 292: 11586-11598; Ichimiya et al., 2004, J. Biol. Chem.,- 279:42638-42647). However, heterozygous carriers of these mutations exhibit few symptoms (Ichimiya et al., 2004, J. Biol. Chem.,' 279:42638-42647; Beltran-Valero de Bernabe et al., 2002, Am. J. Hum. Genet. , ' 71 : 1033-1043), raising the possibility that partial inhibition of the POMT complex in postnatal tissue may be tolerated, perhaps in combination with MAPK pathway inhibition.

[0181] Finally, the observation that the N terminus of K::B is heavily mannosylated and likely secreted raises an intriguing possibility that this is the mechanism causing the formation of tumor-associated mucinous cysts, which are pathognomonic to PAs and currently only treatable through surgery (Akhavan et al., 2008, FASEB J:, 22:612-621). Mucinous proteins have a preponderance of O-GalNAc adducts; while different from O-mannosylation, it raises the possibility that the heavily glycosylated KIAA1549 ectodomain may be secreted, contributing to cyst formation. Disrupting K::B trafficking and thus cleavage would thereby be expected to reduce KIAA1549 ectodomain shedding and cyst formation. The identification of the O- mannosylation complexes as potential MAPK -independent therapeutic targets is therefore of substantial clinical importance.

[0182] METHODS

[0183] Patient sample collection

[0184] Patient-derived pediatric low-grade glioma samples were obtained from the Dana- Farber / Boston Children’s Cancer Center Brain Tumor Bank, governed by the Dana-Farber IRB approved protocol DFCI 10417. Informed consent for tumor banking was provided by patients and / or their guardians. Additional samples were obtained from McGill University and collected under an institute approved protocol with full informed consent.

[0185] Analysis of Dana-Farber Cancer Institute (DFCI) sequencing cohort

[0186] An initial group of adult and pediatric neuro-oncology tumors was identified from a DFCI institutional database (OncDRS), followed by a query from two CNS-tumor specific REDCap databases with IRB approval (10-043 and 10-417 REDCaps). A cohort of >5000 tumors was identified, all of which had targeted exome sequencing via OncoPanel performed by the Brigham and Women’s Hospital Center for Advanced Molecular Diagnostics (BWH CAMD) for at least one of their brain tumor samples (N = 5115 tumors). Patients were deemed eligible if their first pathologic brain tumor diagnosis was at the age of 21 years or younger and they had a pathologically confirmed pediatric low grade glioma (pLGG) diagnosis (N = 435 tumors). Due to the diversity of pediatric low grade glioma diagnoses (pLGG) and the increasing reliance on molecular characterization to finalize a pLGG diagnosis, tumor diagnoses were given in a stepwise manner. Following the WHO 2021 Classification of Central Nervous System Tumors, tumors with a corresponding top line diagnosis and molecular characteristic (e.g., pilocytic astrocytoma with BRAF rearrangement or ganglioglioma with BRAFV600Emutation) were classified as such. Similarly, tumors with a top line diagnosis of low-grade glioma, not otherwise specified (LGG, NOS) with a single MAPK alteration were manually reviewed for histologic features of a corresponding pLGG diagnosis. These tumors were classified more specifically if the necessary histologic features were mentioned in the microscopic description of the pathology report. Any tumors that did not meet the criteria for more specific classification were grouped into the LGG, NOS diagnosis. All deidentified clinical and molecular information was coded in an Excel sheet and converted to a text file for the finalized OncoPrint.

[0187] Single-cell RNA-sequencing analysis scRNAseq tissue collection and processing. Tumor samples were obtained from DFCI and McGill University Health Centre. Glioma biopsies were dissociated using Miltenyi (Catalog No. 130-095-942). Cells were loaded Chromium Controller to generate single-cell gel beads in emulsion, before RT-PCR amplification followed by indexed library preparation. An Agilent 2100 Bioanalyzer was used to check the quality control of cDNA library concentration and fragment size before proceeding to NextSeq sequencing, aiming for a minimum depth of about 20,000 reads per cell. During preprocessing, cells in which fewer than 200 genes were detected, were removed, and genes with poor coverage (that is they were detected in fewer than three cells) were filtered out. Finally, a threshold of 30% for mitochondrial genes was applied to exclude low-quality cells and genes, including MALAT1, mitochondrial genes (MT-), and hemoglobin genes (HB excluding HBP). Reads were normalized and log transformed. Principal component analysis (PCA) using the arpack solver was performed to retain the top 30 components. Harmony integration was used to correct for batch effects, following which UMAP dimensional reduction to visualize cellular heterogeneity was applied.

[0188] Bulk RNA-sequencing analysis, Bulk RNA sequencing data of primary human low-grade glioma samples were generated as previously described (Jones et al., 2013, Nat. Genet. ; 45:927-932;

[0189] Perez-Riverol et al., 2025, Nucleic Acids Res. ; 53:D543-D553), with transcript count matrices used for downstream analyses, prior to evaluating expression of protein O-mannosyltransferase 1 / 2 (POMT1 and POMT 2) dolichol-phosphate mannosyltransferase polypeptide 1-3 (DPMI, DPM2, and DPM3), across the dataset.

[0190] Analysis of POMT1 / 2 and DPMI-3 expression in normal adult tissues:

[0191] Bulk tissue gene expression data (RNA-sequencing) for POMT1, POMT2, and DPMI-3 were obtained from the GTEx Portal (GTEx Consortium, 2020, Science,' 369: 1318- 1330) on 12 / 13 / 2024. The data covered all available tissues from both sexes and was presented as transcripts per million (TPM). Fusion partner analysis in pediatric and adult tumors

[0192] Pediatric kinase fusion partners were collected as previously described (Vega et al., 2024, Cancer Res. ; 84:2857-2857). In-frame fusions from adult tumors were downloaded from FusionGDB 2.0 (Milde et al., 2023, Neuro. Oncol., 25: 1920-1931). Fusions were filtered to include only in-frame fusions with annotated breakpoints. The number of unique fusion species, including fusions with different partner genes and those with different intronic breakpoints for the same partner gene, was computed for each fusion partner gene. To map BRAF breakpoint loci, fusions in the pediatric kinase dataset were filtered to include only those in which BRAF is a fusion partner.

[0193] Protein topology, hydrophobicity, myristoylation, and cleavage site analysis:

[0194] Protein topology prediction of signal peptides, intracellular domains, extracellular domains, and transmembrane domains was done with DeepTMHMM (Lommel et al., 2008, Glycobiology, 18:615-625). Proteins with an MGXXX(S / T) sequence at positions 1-5 in their coding sequence were annotated as having a consensus myristoylation site. Local hydrophobicity was computed as described in (Jumper et al., 2021, Nature', 596:583-589; Lommel et al., 2010, Neurology, 74: 157-164) by binning the KIAA1549 amino acid sequence into 20-mers, then computing the hydrophobicity score using previously described weights (Kyte et al., 1982, J. Mol. Biol., - 157: 105-132)

[0195] Alphafold prediction of KIAA1549 structure

[0196] The predicted structure for human KIAA1549 (accession AF-Q9HCM3-Fl-v4) was retrieved from the AlphaFold database (Jumper et al., 2021, Nature,' 596:583-589; Duckert et al., 2004, Protein Eng. Des. Sei. -, 17: 107-112), and paucimannose N-glycans were attached to the structure as illustrative of N-glycosylation using the Re-Glyco tool (Varadi et al., 2024, Nucleic Acids Res.- 52:D368-D375). Genome-scale anti -transformation screens

[0197] Genome-scale CRISPR / Cas9 screens were performed in mouse neural stem cells (mNSCs) transduced to express K::B, BRAFV600E, or luciferase controls as previously described (Hardin et al., 2023, Neuro. Oncol., ' 25:2087-2097; Duckert et al., 2004, Protein Eng. Des. Sei. , ' 17: 107-112; Varadi et al., 2024, Nucleic Acids Res., ' 52:D368-D375; Ives et al., 2024, Nat. Methods,' 21 :2117-2127). The KIAA1549-BRAF and BRAFV600Escreens were performed in the absence of exogenous EGF / bFGF supplementation, thereby rendering cells oncogene dependent and allowing the identification of genes / pathways that are necessary for transformation. Screen analysis was performed by first computing the differential in fold change in CRISPR guide abundance between the early (day 3) and final (day 18) timepoint samples. Single-gene scores were computed by first averaging all four sgRNAs for a given gene, then averaging across biological replicates. To identify statistically significant differences in guide depletion, guidelevel depletion scores were collapsed into mean gene-level depletion scores across all four guides, then statistically significant differences across replicates were computed with two-tailed t-tests with Benjamini -Hochberg (Ives et al., 2024, Nat. Methods,' 21 :2117-2127) multiple hypothesis correction. Depletion scores were compared between the luciferase (control) screen and either the BRAFV600Eor KIAA1549::BRAF screens.

[0198] Analysis of gene essentiality across cancer cell lines Using gene essentiality scores for 1150 cell lines profiled in The Dependency Map

[0199] 24q2 release (Tshemiak et al., 2017, Cell,' 170:564-576. el6), chronos (Dempster et al., 2021, Genome Biol., ' 22:343) gene essentiality scores were plotted for each of six genes, POMT1, POMT2, DPMI, DPM2, DPM3, and POLR2B (RNA polymerase II subunit B), for all 1150 cell lines.

[0200] Chemical compounds CRISPR guides, and plasmid constructs

[0201] CRISPR guides were designed using CRISPick (Kim et al., 2018, Nat. Biotechnol.,' 36:239-241; DeWeirdt et al., 2021, Nat. Biotechnol. , ' 39:94-104; Doench et al., 2016, Nat. Biotechnol.,' 34: 184-191; Sanson et al., 2018, Nat. Commune, 9:5416). Plasmid constructs were synthesized by Epoch Life Science (Houston, USA) or Genscript (Piscataway, USA). R3A-5a (2-[(5Z)-4-oxo-5-[[3-(l -phenyl ethoxy)-4-(2-phenylethoxy)phenyl]-methylene]-2-thioxo- thiazolidin-3-yl]acetic acid) (Manya et al., 2008, Neuromuscul. Disord. , 18:45-51) was synthesized by Genesis Drug Discovery & Development (Montreal, Canada).

[0202] Tissue Culture

[0203] Culture of h9-NSCs. h9-NSCs (Invitrogen, N7800-100) were cultured on geltrex- coated (ThermoFisher, Catalog No. A1413302) cell culture plates in growth medium containing 500 mL NeuroBasal A (Gibco, Catalog No. 0888022), 500 mb DMEM / F-12 (Gibco, Catalog No. 1 1330057), 20 mL B-27 (Gibco, Catalog No. 17504044), 10 mL HEPES (ThermoFisher, Catalog No.15630080), 10 mL glutamax (ThermoFisher, Catalog No. 35050061), 10 mL sodium pyruvate (ThermoFisher, Catalog No. 11360070), 10 mL MEM NEAA (ThermoFisher, Catalog No. 11140050), 10 mL Pen-Strep (ThermoFisher, Catalog No. 15140122), 20 nanograms / mL EGF (STEMCELL Technologies, Catalog No. 78006.2), 20 nanograms / mL bFGF (STEMCELL Technologies, Catalog No. 78003.2), and 1 mL of heparin (STEMCELL Technologies, Catalog No. 07980). This growth medium is herein referred to as TSM. For growth factor withdrawal experiments, cells were cultured in TSM without the addition of EGF, bFGF, and Heparin. Cells were detached with Accutase, pelleted, and resuspended in TSM to remove residual Accutase, which disrupts re-attachment of cells when subculturing. Cells were subcultured at a ratio of 1 :5 - 1 :2 into freshly coated cell culture plates and split when cells reached a confluence of 75-90%. To generate growth factor-independent models, cells were transduced with pDEG- KIAA1549::BRAF or pDEG-BRAFV600Eand were cultured in TSM without EGF / bFGF for at least three weeks.

[0204] Culture of mNSCs. Mouse neural stem cells (mNSCs) were established from the subventricular zone of E14.5 mouse embryos (CD-I) and cultured as neurospheres in TSM in ultra-low attachment treated plates and flasks as previously described (Sanson et al., 2018, Nat. Communr, 9:5416). Neurospheres were dissociated with Accutase at 37 degrees C for 5-10 minutes when passaging or seeding for experiments. Mouse NSCs were cultured in TSM media. For growth factor withdrawal experiments, cells were cultured in TSM without addition of EGF, bFGF, and Heparin. Neurospheres were dissociated with Accutase, pelleted, and resuspended in TSM to remove residual Accutase. Cells were seeded at a density of 120,000 cells / mL into ultra- low attachment cell culture flasks and split two times per week. To generate growth factorindependent models, cells were transduced with KIAA1549::BRAF or BRAFV600Eand were cultured in TSM without EGF / bFGF for at least three weeks.

[0205] Culture of HEK-293T cells. HEK-293T cells were grown in DMEM + 10% FBS + 1% Pen / Strep. Cells were subcultured at a 1 : 10 ratio and were split when cells reached a confluence of 75-90%.

[0206] All cell lines were routinely screened for mycoplasma contamination with a commercially available kit (Lonza, Catalog No. LT07-318). Quantitative PCR (qPCR)

[0207] Quantitative PCR was used to validate expression of KIAA1549::BRAF or BRAFV600Ein isogenic mouse neural stem cell lines. RNA was isolated using either Qiagen RNeasy Mini kit (Qiagen, Catalog No. 74106) or Qiagen RNeasy Micro kit (Qiagen, Catalog No. 74004). cDNA was made using i Script™ Reverse Transcription Supermix for RT-qPCR (BioRad, Catalog No. 1708840), then used to quantify gene expression with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Catalog No. 1725272) on a Quantstudio flex7. All gene expression values were normalized to B2M expression, and fold changes were analyzed using the AACt method. Lentivirus production and transduction

[0208] Viral production. HEK293T cells cultured in Dulbecco's Modified Eagle Medium (Gibco, Catalog No. 11965118) with 10% fetal bovine serum and with or without 1% Penicillin- Streptomycin (ThermoFisher, Catalog No. 15070063). HEK-293T cells were seeded into 10-cm tissue culture plates at a density of 3-5.38xl06cells / plate. The following day, cells were transduced with 1 microgram of either VSV-G (Addgene Plasmid, Catalog No. 14888) or pMD2.G (Addgene Plasmid, Catalog No. 12259), 10 micrograms of psPAX (Addgene Plasmid, Catalog No. 12260), and 10 micrograms of lentiviral plasmid using Lipofectamine 3000 (ThermoFisher, Waltham, USA, Catalog No. L3OOOO15) according to the manufacturer's protocol. The following day, the growth medium was changed to 10 mb of TSM (Haines et al., 2007, Mol. Biol. Cell,' 18:4721-4730) growth medium. Approximately 24 hours later, the growth medium was collected, filtered through a 0.45-micron syringe filter. Viral supernatant was concentrated using LentiX Concentrator (Takara, Catalog No. PT4421-2) according to the manufacturer’s protocol. Alternatively, lentiviral supernatant was concentrated to a final volume of 200 mL with an Amicon 10 kDa centrifugal filter (Sigma-Aldrich, St. Louis, USA, Catalog No. UFC9010). The virus was stored at -80 degrees C and thawed on ice prior to use.

[0209] Viral transduction. h9-NSC cells were seeded into geltrex-coated 10-cm plates at a density of 2-3x106cells / plate. Plates were supplemented with 5-20 mL of concentrated virus per 1x106cells. The following day, the growth medium was changed to 10 mL of TSM (Ichimiya et al., 2004, J. Biol. Chem. , 279:42638-42647) growth medium and, if applicable, selection antibiotics (2 micrograms / mL of puromycin or 10-20 micrograms / mL of blasticidin) were added. Cells were cultured for 5-7 days prior to being used in experiments. Mouse neural stem cells were infected with lentiviral and incubated with centrifugation at 700 grams at 30 degrees C for two hours. Cells were selected with either 0.5 micrograms / mL puromycin (for three days) or 10 micrograms / mL blasticidin (for 10 days).

[0210] CellTiterGlo Viability assays h9-NSCs were seeded into geltrex-coated 96-well plates (Corning, Catalog No. 3903) at a density of 1,000-5,000 cells / well in a final volume of 100 mL of TSM growth medium. The next day, cells were treated with the indicated concentrations of pomalidomide diluted in an additional 100 mL of TSM growth medium. Cells were grown for the indicated time, then 50 mL of CellTiterGlo (Promega, Catalog No. G7572) was added to each well. Plates were incubated for 10 minutes at room temperature, then luminescence was measured on an Envision plate reader or a SpectraMax M5 plate reader. Luminescence signal in compound-treated wells was normalized to vehicle control.

[0211] Mouse NSCs were seeded into 96-well plates (Thermo Scientific, Catalog No.

[0212] 165306) at a density of 2,000 cells / well in 90 microliters of TSM growth medium. Cells were left for at least 30 minutes before being treated with 10 microliters of medium containing R3A-5a at the indicated concentration (final concentrations: 50, 25, 15, 12, 10, 7.5, 3.75, 0.9375, or 0.234 micromoles) or equivalent DMSO controls. Cells were grown for 5 days, then 100 microliters of CellTiterGlo (Promega, Catalog No. G7572) was added to each well. Plates were incubated on a shaker at room temperature for 10 minutes, then luminescence was measured on an Envision plate reader or a SpectraMax M5 plate reader. Luminescence signal in compound-treated wells was normalized to vehicle control. Area under the curve (AUC) was calculated for each experimental replicate (n = 3) and compared using one-way ANOVA with Tukey’s multiple comparison test in Graphpad Prism Version 10.4.1.

[0213] Colony formation assays

[0214] Cells were plated in geltrex-coated 6-well plates at a density of S-lOxlO3cells / well in a final volume of 2 mL TSM without growth factors. Growth medium was changed every 2-3 days. After 21 days, cells were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature and then stained with a 1% Crystal Violet solution (w / v) for 1 hour at room temperature. The 6-well plates were rinsed in ultrapure water, then dried for 24 hours prior to imaging on either a flatbed scanner or the LiCOR Odyssey M.

[0215] Incucyte growth experiments

[0216] Human h9-hNSCs were seeded at a density of 20,000 cells / well in geltrex coated 48- well plate (Coming, Catalog No. 3548) in TSM growth media with or without EGF / bFGF supplementation and with or without pomalidomide (1 microgram / mL). Plates were placed in the IncuCyte S3, set for imaging every six hours, four images / well with a lOx objective. Confluence was then analyzed with IncuCyte Basic Analyzer, Al Confluence with segmentation set to default, and normalized to the 0-hour timepoint.

[0217] Mouse NSCs were seeded at a density of 1,000 cells / well in u-bottom ultra-low attachment 96- well plates (Corning, Catalog No. 3548) in TSM growth media with or without EGF / bFGF supplementation. Cell plates were centrifuged at 200 grams for 10 minutes at room temperature and then placed in the IncuCyte S3, set for imaging every six hours, 4x objective. The largest brightfield object area was then analyzed with IncuCyte Spheroid, with segmentation: Sensitivity: 10, Area > 2,000 square micrometers, Eccentricity < 0.75. Pomtl and Pomt2 knockout and cumulative doublings assays

[0218] Mouse NSCs were plated as 2xl06cells / well in 2 mL TSM growth media with (luciferase control) or without (KIAA1549::BRAF) EGF / bFGF supplementation in 12-well plates. Concentrated lentiCRISPRv2 lentivirus (15 microliters) encoding both Cas9 and sgRNA targeting mouse Pomtl / 2, two guides per target (sgRNA sequences in), as well as EGFP, was added to each well in four technical reps. The plates were centrifuged for two hours at 700g at 30 degrees C and then left overnight in an incubator. The day after the cells were centrifuged at 340g for 4 mins, virus media was aspirated, and cells were replated in 20 mL fresh TSM growth media with / without EGF / bFGF. 1 microgram / mL Puromycin was used for selection over 72 hours, after which cells were counted on a Countess 3 FL Automated Cell Counter (Thermo Fisher Scientific). Cells were replated in fresh growth media (500,000 in 5 mL media) in T25 ultra-low attachment flasks. After this, cells were passaged and counted every 3-4 days until the 18-day post-transduction endpoint. Each time, the same reseeding conditions were used. Cumulative doublings were calculated using the end of selection as the starting point and with the formula log2(total viable 811 cells / seeded viable cells).

[0219] CRISPR / Cas9 competition assays

[0220] Cas9 guide sequences were used in CRISPR competition assays. Negative control (intergenic) guides were cloned into an EGFP-expressing vector. Negative control (intergenic), positive control (POLR2B), POMT1, and POMT2 guides were cloned into an mCherry- expressing vector. Luciferase-expressing, growth factor-independent BRAFV600E-expressing, or growth factor-independent KIAA1549::BRAF-expressing h9-NSCs were seeded into geltrex- coated 6-well plates at a density of 5xlO5cells / well. Each well was supplemented with 15 mL of concentrated virus. After 16-24 hours, the growth medium was changed to 2 mL of fresh TSM (luciferase-expressing cells) or TSM-EGF / bFGF (BRAFV600Eand KIAA1549::BRAF-expressing cells) per well. After 72 hours, cells were detached with accutase, and EGFP- and mCherry- expressing cells were pooled 1 :1. The fraction of EGFP- and mCherry-expressing cells was analyzed on a Cytoflex flow cytometer. Cells were cultured for an additional 18 days, and the fraction of EGFP- and mCherry-expressing cells was assessed with flow cytometry. Immunoblotting

[0221] Cells were lysed in RIPA buffer supplemented with protease / phosphatase inhibitors according to the manufacturer’s protocol. Lysates were clarified at 20,000g for 10-20 minutes, then NuPAGE sample buffer with 5% beta-mercaptoethanol was added and samples were boiled at 95 degrees C for 10 minutes. Samples were loaded onto a 4-12% bis-tris gradient gel and run at 150V for 2-3 hours. The gel was wet transferred to PVDF for 16-24 hours at 150 mA at room temperature. Membranes were blocked using the appropriate blocking solution. Membranes were incubated in primary antibody overnight for 16-24 hours at 4 degrees C. The next day, membranes were washed 3x in TBS-T for 5 minutes per wash. Membranes were incubated in the appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Membranes were washed 3x in TBS-T for 5 minutes per wash. TBS-T was removed, and membranes were incubated with 1 mL of each SignalFire Elite (Cell Signaling Technology, Catalog No. 12757), SuperSignal West Pico PLUS (Thermo Fisher Scientific, Catalog No. 34580), or SuperSignal West Femto Maximum Sensitivity (Thermo Fisher Scientific, Catalog No. 34094) reagent for 1 minute with shaking before being imaged on the Li-COR Odyssey M.

[0222] Immunofluorescence staining

[0223] Cells were plated onto geltrex-coated p-Slide 8 Well chambered coverslip (Ibidi, Catalog No. 80807). After 24 hours of plating, cells were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature. Cells were rinsed once with PBS, then permeabilized with ice- cold methanol for 15 minutes at 4 degrees C. Cells were rinsed with PBS, then blocked in 2% BSA diluted in PBS for one hour at room temperature. Cells were incubated in primary antibody diluted in blocking buffer for 16-24 hours at 4 degrees C. The next day, cells were washed 3x in PBS (5-15 minutes per wash), and incubated in secondary antibody for 1 hour at room temperature, shielded from light. Cells were again washed 3x in PBS (5-15 minutes per wash) before being mounted in Ibidi Mounting Media with DAPI (Ibidi, Catalog No. NCI 943852), cured at room temperature for 15 minutes, and stored shielded from light at 4 degrees C prior to imaging. Confocal microscopy

[0224] Images taken for fusion protein localization analysis were acquired on a Zeiss LSM microscope equipped with an Airyscan 2 detector (Carl Zeiss) using a LD LCI Plan-Apochromat 25x / 0.8 Imm Corr DIC M27 objective. Bidirectional scanning with 2.05pseg pixel dwell time and averaging of 2 was used. Pixel size was set to 0.1657 micrometers in compliance with Nyquist sampling, and pixel intensities were encoded using 16-bit depth. Pinhole sizes were set to 0.8-1.4 Airy units. Excitation (detailing nominal laser power) and emission wavelengths were as follows: DAPI, Xex405nm (at 15mV) -em408-501nm; Alexa Flour 488, Xex488nm (at 13mV) - em 491-588nm; Alexa Fluor 594, Xex594nm (at 4mV) -em 597-694nm. Airyscan post processing was performed using Zeiss ZEN software.

[0225] Image analysis pipeline

[0226] Localization analysis of fusion proteins and the endoplasmic reticulum (labeled with PDI) was performed using CellProfiler 4.2.8109. Briefly, cells were segmented using the PDI channel, and colocalization was assessed in the cytoplasmic region using Perason’s Correlation Coefficient.

[0227] Intracellular flow cytometry

[0228] Cells were detached with Accutase, and 300,000 cells were seeded into each well of a round-bottom 96-well plate. Cells were pelleted by centrifuging at 300g for 3 minutes, then the supernatant was removed. Cells were washed 2x more in PBS. Cells were fixed in 3.7% paraformaldehyde for 15 minutes at room temperature and were washed 3x in PBS. Cells were permeabilized and blocked in 5% BSA-TBST + 0.1% Triton-XlOO for 1 hour at room temperature. Cells were incubated with primary antibody for 2 hours at room temperature with agitation every 30 minutes. Cells were pelleted and washed 3x in PBS. Cells were incubated in PE-conjugated secondary antibody for 1 hour at room temperature, with agitation every 30 minutes. Cells were washed 3x in PBS, then resuspended in 200 mL of PBS + 2% BSA and were analyzed on a Cytoflex flow cytometer. Glycoproteomics

[0229] Sample preparation and analysis were performed as previously described (Lommel et al., 2008, Glycobiology, 18:615-625) with minor modifications. Briefly, mNSC cell pellets were extracted in 0.5% SDS, 40 mM Tris-HCl, pH 8.0, 150 mM NaCl before probe sonication for disruption of cellular debris and DNA. Each extract was reduced (10 mM DTT, 72 degrees C, 15 minutes) and alkylated (25 mM IAA, room temperature, 15 minutes) before adding Triton X-100 to a final concentration of 1% (v / v). N-glycans were digested with 10U PNGase F enzyme (Roche), 37 degrees C, 18 hours. N-deglycosylated protein extracts were subsequently acetone precipitated (-20 degrees C, 16 hours) and resolubilized in 40 mM Tris-HCl, pH 8.0, 150 mM NaCl buffer for digestion with 50 micrograms trypsin or 25 micrograms GluC at 37 degrees C and 18 hours. Peptides were desalted on Sep-Pak C18 columns (50 mg) and labeled with diethyl stable isotopes (Lommel et al., 2010, Neurology, 74: 157-164) before mixing the samples in a 1 : 1 (v / v) ratio. C- and O-linked mannose glycopeptides were enriched in batch-mode with 200 microliters agarose slurry conjugated to BC2L-A lectin, prepared as previously described (Beltran-Valero de Bernabe et al., 2002, Am. J. Hum. Genet. , ' 71: 1033-1043), in 1 mL 40 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM CaCL buffer. Following 3x2 mL washes of the agarose beads, the glycopeptides were eluted with 100 mL 40 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM CaCh, 40 mM EDTA solution. Enriched glycopeptides were desalted on Stage-tip C18 columns and analyzed by mass spectrometry as previously described (Benjamini et al., 1995, J. R. Slat. Soc., ' 57:289-300). Data processing, identification, and relative quantification of C- and O-Man glycopeptide abundances were performed with the Proteome Discoverer 1.4 software as previously described (Kim et al., 2018, Nat. Biotechnol. ; 36:239-241).

[0230] TAILS mass spectrometry

[0231] HEK-293T cells were transfected with pMIBerry KIAA1549::BRAF (16: :9) plasmid and lysed in NLB 48 hours later. 30 mL of anti -HA affinity matrix (Roche) was added to 900 microliters of cleared lysate and incubated with end-over-end rotation at 4 degrees C for 4 hours. Sample workup was performed as previously described (Stirling et al., 2021, BMC Bioinformatics, 22:433). Briefly, proteins were denatured with 6 M guanidine hydrochloride in HEPES (pH 7.8), reduced with 1 mM DTT, and alkylated with 5.5 mM IAA. N-terminal and lysine side chain amines were blocked by demethylation using formaldehyde and sodium cyanoborohydride. After acetone precipitation, proteins were resuspended and digested overnight with trypsin. Newly formed peptides with unblocked N-termini were bound overnight to HPG- ALDII polymer in the presence of sodium cyanoborohydride. Unbound blocked peptides were recovered with 30 kDa molecular weight cut-off spin filters. Filtrates were desalted with STAGE tips (Kleifeld et al., 2011, Nat. Protoc., 6: 1578-1611). LC-MS / MS measurements were performed on an Exploris 480 mass spectrometer, coupled to an EasyLC 1200 nanoflow-HPLC (ThermoFisher Scientific). Peptides were separated on a fused silica HPLC column tip (I.D. 75 micrometers, New Objective, self-packed with ReproSil-Pur 120 C18-AQ, 1.9 micrometers to a length of 20 cm) using a gradient of A (0.1% formic acid in water) and B (0.1% formic acid in

[0232] 80% acetonitrile in water). The mass spectrometer was operated in data-dependent mode; after each MS scan (mass range m / z = 370 - 1750; resolution: 120’000) a maximum of twenty MS / MS scans were performed using an isolation window of 1.3, a normalized collision energy of 28%, a target AGC of 50% and a resolution of 15’000. Spray voltage was set to 2.3 kV, and the ion-transfer tube temperature to 250 degrees C; no sheath and auxiliary gas were used.

[0233] Raw mass spectrometry files were analyzed using MaxQuant (version 2.0.1.0) (Rappsilber et al., 2007, Nat. Protoc. , 2: 1896-1906) using either a customized database containing the KIAA1549::BRAF (16::9) protein sequence and common contaminants, or together with a UniProt full-length human database with common contaminants including keratins and digestion enzymes using semispecific ArgC as enzyme specificity. Carbamidomethylcysteine and dimethylation of lysine were set as fixed modifications, and protein amino-terminal acetylation, oxidation of methionine, and dimethylation of peptide N-termini were set as variable modifications. The MS / MS tolerance was set to 20 parts per million. Peptide, site, and protein FDR based on a forward-reverse database were set to 0.01, and the minimum number of peptides for identification of proteins was set to one, which must be unique. The “match-between-run” option was used with a time window of 0.7 minutes.

[0234] Protease inhibitor screen

[0235] Growth factor-independent KIAA1549::BRAF-expressing h9-NSCs were seeded into geltrex-coated 6-well plates and were allowed to attach overnight. The next day cells were treated with 1 micromole of each protease inhibitor available within a commercially available library (Enzo, Catalog No. BML-2833-0100), pomalidomide, or DMSO (vehicle control). Cells were treated for 48 hours before being lysed and immunoblotted as previously described.

[0236] Animal studies

[0237] In Utero Electroporation (IUE) Mouse Models. All IUE mouse work was conducted according to institutional guidelines and standards from the Institutional Animal Care and Use Committee review board (University of Cincinnati, IACUC No. 22-05-15-01). CD1-ICR (Charles River code No. 022) mice were used for all IUE experiments. Briefly, IUE models were created by lateral ventricle injection of nucleic acid mixtures followed by electroporation. All DNA plasmids were used at a final concentration of 1 microgram / microliter. KIAA1549::BRAF and BRAFX 600Eplasmids were co-electr operated with PBCAG-EGFP and pCAG-PBase. Cas9 was used at a final concentration of 33.3 micromoles. sgRNAs were used at a final concentration of 20 micromoles.

[0238] Plasmid construction and CRISPR-Cas9 reagents. Piggybac DNA plasmids were constructed as previously described (Cox et al., 2008, Nat. Biotechnol.,' 26: 1367-1372). PBCAG-KIAA1549::BRAF plasmid were generated by PCR amplification of KIAA1549::BRAF or BRAFV600Eand insertion into linearized PBCAG-EGFP, which was digested with EcoRl and Notl to remove the EGFP coding sequence. Ligations were performed with InFusion Snap assembly (Takara), and plasmid sequence verified by whole plasmid sequencing (Plasmidsaurus). All plasmid stocks were prepared using NucleoBond Xtra Maxi or Midi EF endotoxin-free kits (Machery Nagel). For CRISPR / Cas9: Cas9 was purchased from Trilink (Catalog No. L-7206-100). Positive control Rosa26sgRNAand multi-guide Pomtl gene KO kit were purchased from Synthego (now Editco).

[0239] Tissue collection, processing, and immunostaining. Following euthanasia by CO2 inhalation, brains were dissected into ice-cold Dulbecco’s phosphate-buffered saline (DPBS). Samples were drop-fixed in freshly prepared 4% paraformaldehyde overnight at 4 degrees C. The following day, samples were washed in cold PBS and then transferred into a 30% sucrose solution for cryopreservation. Samples were embedded in optimal cutting temperature (OCT) embedding medium and stored at -80 degrees C until sectioned. 45 micrometers thick free- floating sections were cut on a cryostat (Leica) and stored in PBS + 0.05% sodium azide until processed for staining. For immunofluorescent staining, sections were first transferred to blocking solution (PBS + 0.5% Triton X-100 + 10% normal donkey serum) prior to the addition of primary antibodies and incubated at 4 degrees C overnight. The next day, sections were washed in PBS and then placed into blocking solution containing corresponding fluorescent secondary antibodies for overnight incubation at 4 degrees C. Finally, sections were counterstained with Hoechst (1 : 1,000) and washed in PBS prior to mounting on slides and coverslipping (Fisherbrand, Superfrost Plus slides, Thermo Fisher ProLong Gold Antifade Mountant; Fisherbrand Microscope Cover Glass 24x50). Slides were imaged using a Nikon 953 Al confocal microscope. Image processing and analysis were performed in NIH Fiji / ImageJ and Imaris.

[0240] Targeted sequencing for CRISPR / Cas9

[0241] Genomic DNA was harvested from mouse neural stem cell cultures generated from electroporated brains. Briefly, 24 hours post IUE, brains were harvested and dissociated into single-cell suspensions by papain enzyme digestion. Neurosphere cell lines were established in NeuroCult media with mouse neural stem cell proliferation supplement and bFGF / EGF. Transfected cells were isolated by FACS sorting, and gDNA was subsequently isolated from this purified transfected population. Appropriate PCR primers were used to amplify the Rosa locus. PCR products were isolated by gel purification and submitted for Sanger sequencing at the CCHMC DNA sequencing core with the sequencing primer. Appropriate PCR primers were used to amplify and sequence the Pomtl locus.

[0242] Example 4

[0243] Treatment of SCID mice with KIAA1549::BRAF allografts treated with R3A-5a

[0244] METHODS

[0245] 18 Fox Chase SCID mice (female:male, 1 :1; 6 weeks old) were stereotactically injected in the right hemisphere with mNSCs engineered to express KIAA1549::BRAF (15:9 variant). Mice were monitored for tumor growth with magnetic resonance imaging (MRI). When the first mouse showed signs of tumor on MRI, treatment was started (day 51 post cell injections). Mice were treated with R3A-5a at a saturated concentration in 10 %DMSO (6ul) which was injected using convection-enhanced delivery (CED) to the same site as cell injections. DMSO 10% was used as vehicle treatment. Each group had n=9 mice. CED was repeated once, two weeks after the first dose. Mice were followed for survival over 6 months at which living mice were censored. Data was analyzed using Kaplan-Meier survival plots for survival probability, with shaded regions showing standard error. Differences between groups (R3A-5a and vehicle) were tested using Mantel-Cox (log-rank test). p-value= 0.287, non-significant. RESULTS

[0246] FIG. 16 is a Kaplan-Meier plot of data of SCID mice with KIAA1549::BRAF allografts treated with R3A-5a or vehicle by utilizing convection-enhanced delivery to the tumor site, as the bioavailability and blood-brain-barrier penetrance of R3A-5a is unknown. Two doses of the compound resulted in a trend (p=0.287, non-significant) of prolonged survival for the treatment group compared to vehicle. No mice showed any signs of acute toxicity from the treatment.

[0247] DISCUSSION

[0248] This example demonstrates a trend but no statistical significance. KIAA1549::BRAF expression in isogenic mNSCs leads to tumor formation in SCID mouse brains but with varying penetrance. Morin et al. ((bioRxiv 2024.08.27.609922; doi: https: / / doi.org / 10.1101 / 2024.08.27.609922) explains the low power of this approach. This experiment is being repeated with double the number of mice to increase power.

[0249] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

What is claimed is:

1. A method of identifying a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion, the method comprising: contacting a cell expressing a KIAA1549-BRAF fusion protein with a test agent; and determining if the test agent inhibits O-mannosylation of the KIAA1549-BRAF fusion protein; inhibits heterodimerization of protein O-mannosyltransferase 1 (P0MT1) and protein O-mannosyltransferase 2 (P0MT2); and / or inhibits activity of the protein O-mannosyl transferase (POMT) complex in the cell expressing a KIAA1549-BRAF fusion protein; wherein determining that the test agent inhibits the O-mannosylation of the KIAA1549- BRAF fusion protein, inhibits heterodimerization of P0MT1 and P0MT2, and / or inhibits activity of the POMT complex in the cell expressing a KIAA1549-BRAF fusion protein identifies the test agent as a therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion.

2. The method of claim 1, wherein the cell expressing a KIAA1549-BRAF fusion protein comprises a murine neural stem cell expressing a KIAA1549-BRAF fusion protein or a human neural stem cell expressing a KIAA1549-BRAF fusion protein.

3. A method of treating a subject with a cancer comprising a KIAA1549-BRAF fusion, the method comprising administering an effective amount of an agent identified by the method of claim 1 or 2 to the subject.

4. The method of claim 3, wherein the cancer comprises a pediatric low-grade glioma (pLGG).

5. The method of claim 3 or 4, wherein the cancer comprises a cancer refractory to treatment with a mitogen-activated protein kinase (MAPK) inhibitor.

6. A method of treating a subject with a cancer comprising a KIAA1549-BRAF fusion, the method comprising administering an effective amount of a therapeutic agent identified by the method of claim 1 or 2 and an additional therapeutic intervention.

7. The method of claim 6, wherein the additional therapeutic intervention comprises chemotherapy, radiation, and / or surgical intervention.

8. The method of claim 6 or 7, wherein the administration the agent and the additional therapeutic intervention demonstrate synergy.

9. A method of treating a subject with a cancer comprising a KIAA1549-BRAF fusion, the method comprising administering an effective amount of a therapeutic agent identified by the method of claim 1 or 2 and a mitogen-activated protein kinase (MAPK) inhibitor.

10. The method of claim 9, wherein the MAPK inhibitor comprises trametinib, selumetinib, cobometinib, ulxertinib, tovorafenib, vemurafenib, and / or dabrafenib.

11. The method of any one of claims 9 to 10, wherein the administration the therapeutic agent and the MAPK inhibitor demonstrate synergy.

12. The method of any one of claims 3-11, wherein the therapeutic agent comprises R3A-5a or a derivative thereof.

13. A therapeutic agent for the treatment of a cancer identified by the method of any one of claims 1 to 2.

14. A therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion, the therapeutic agent comprising an inhibitor of O-mannosylation of the KIAA1549-BRAF fusion protein; heterodimerization of protein O-mannosyltransferase 1 (POMT1) and / or proteinO-mannosyltransferase 2 (P0MT2); and / or activity of the protein O-mannosyl transferase (POMT) complex.

15. A therapeutic agent for the treatment of a cancer comprising a KIAA1549-BRAF fusion, wherein the therapeutic agent comprises R3A-5a or a derivative thereof.

16. The use of a therapeutic agent of any one of claims 13 to 15 for use in the treatment of a cancer comprising a KIAA1549-BRAF fusion.

17. The use of a therapeutic agent of claim 16, wherein the cancer comprises a pediatric low- grade glioma (pLGG).

18. The use of a therapeutic agent of any one of claims 16 or 17, wherein the cancer comprises a cancer refractory to treatment with a mitogen-activated protein kinase (MAPK) inhibitor.

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