Therapeutic targeting of gastrointestinal stromal tumors (GISTs) by disrupting the menin-MLL epigenetic complex

Abstract

Methods and inhibitors for treating gastrointestinal stromal tumors (GIST) in a subject with active agents that inhibit the menin-MLL complex are disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 289,943, filed December 15, 2021, which is incorporated by reference in its entirety.

[0002] Government support This invention was made with Government support under Grant Nos. K08 CA245235 and UL 1TR002541 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. Said XML copy, created on December 14, 2022, is named 52095-752001WO_SL.xml and is 51 KB in size. [Background technology]

[0004] Gastrointestinal stromal tumors (GISTs) are soft tissue sarcomas that can be located in any part of the digestive system, most commonly the stomach and small intestine. GISTs are characterized by recurrent activating mutations in or around the tyrosine kinase KIT gene, receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). (Corless et al., Annu. Rev. Pathol. Mech. Dis. 3:557-86 (2008), Hemming et al., Annals of Oncology. 3:557-9 (2018)).

[0005] Mutations in or around KIT and / or PDGFRA account for more than 85% of GIST cases. The majority of primary KIT mutations respond to treatment with the tyrosine kinase inhibitor (TKI) imatinib. However, secondary kinase mutations arise over time, resulting in imatinib-resistant GIST. Sunitinib, regorafenib, and zinzamidine are approved for the treatment of imatinib-resistant GIST in later lines of therapy, but resistance to these drugs also develops over time (Demetri et al., N. Engl. J. Med. 347(7):472-80 (2002), Blay et al., Lancet Oncol. 21(7):923-34 (2020)), Voss and Hager, Nat. Rev. Genet. 15(2):69-81 (2014), Chen and Dent, Nat. Rev. Genet. 15(2):93-106 (2014)).

[0006] Therefore, there is an urgent need for treatments for multidrug-resistant GIST. Summary of the Invention [Means for solving the problem]

[0007] A first aspect of the present disclosure relates to a method for treating gastrointestinal stromal tumor (GIST), comprising administering to a subject a therapeutically effective amount of a Menin inhibitor. In some embodiments, the method also involves administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor (TKI) and / or a therapeutically effective amount of a MOZ inhibitor.

[0008] Another aspect of the present disclosure is a method for reducing KIT activity in vitro or in vivo.The method involves contacting a cell having an activating mutation in or around the KIT gene with a menin inhibitor.In some embodiments, the method involves administering to the subject a therapeutically effective amount of a TKI and / or a therapeutically effective amount of a MOZ inhibitor.

[0009] Yet another aspect of the present disclosure relates to a kit comprising a therapeutically effective amount of a menin inhibitor, a pharma- ceutically acceptable carrier disposed in a suitable container, and a printed instruction for using the menin inhibitor in the treatment of GIST in a subject.In some embodiments, the kit also contains a therapeutically effective amount of a TKI and a printed instruction for using the TKI in the treatment of GIST in a subject, and the menin inhibitor and the TKI are contained in the same dosage form or different dosage forms disposed in the same container or different containers.In some embodiments, the kit also contains a therapeutically effective amount of a MOZ inhibitor and a printed instruction for using the MOZ inhibitor in the treatment of GIST in a subject, and the menin inhibitor and the MOZ inhibitor are contained in the same dosage form or different dosage forms disposed in the same or different containers.

[0010] As shown in the Examples, the inventors demonstrated that menin-MLL and MOZ chromatin regulatory complexes were enriched in GIST-related genes and regulated their transcription and that of the GIST epigenome. Inhibition of menin-MLL complexes, alone or in combination with MOZ complex inhibition, reduced GIST cell proliferation by disrupting the interaction with transcription and chromatin regulators such as DOT1L. Menin and MOZ inhibition caused a significant reduction in tumor burden in vivo, and an even greater effect was observed with the combination of menin and KIT inhibition. [Brief description of the drawings]

[0011] [Figure 1]1A-1G are a series of scatter plots, bar graphs, and stipple diagrams showing the identification of GIST epigenetic dependencies by genome-wide CRISPR-dependent screening. 1A and 1B are scatter plots showing correlation β scores. 1A shows the correlation between H1 and H2 sgRNA libraries, each targeting 18,436 genes with five sgRNAs per library. 1B shows the correlation between GIST430 and GIST-T1 cell lines. 1C is a scatter plot showing β scores merging H1 and H2 libraries and GIST cell lines as well as ranks during screening. 1D and 1E are bar graphs showing relative reads for individual sgRNAs comparing baseline plasmid library sequencing to screening end results. 1D is a bar graph showing KIT sgRNA and 1E is a bar graph showing MTOR sgRNA. 1F is a stipple diagram comparing β scores of pan-essential and non-essential genes. FIG. 1G is a bar graph showing 8 of the top 18 significantly enriched Gene Ontology entries among GIST-specific essential genes.

[0012] [Diagram 2]Figures 2A-F are a series of scatter plots, Circos plots, line graphs, and bar graphs showing the unique codependence of MOZ and the menin-MLL complex. Figure 2A is a merged scatter plot of the beta scores in GIST-T1 and GIST430 for chromatin-modifying enzymes, as well as the average CERES score of all cell lines in the DepMap. Figure 2B is a Circos plot showing the overlap of the top 50 DepMap correlated dependencies of seven chromatin-modifying enzymes with enriched dependencies in GIST. Figures 2C and 2D are scatter plots showing ranked susceptibility scores from Project Drive cell lines for menin-MLL complex members KMT2A and ASH2L, with GIST-T1 highlighted in red. Figure 2E is a line graph showing a time course proliferation assay following transduction of the indicated sgRNAs targeting menin-MLL complex members in GIST-T1. FIG. 2F is a bar graph showing cell numbers at day 21 in a time-course proliferation assay comparing GIST-T1 with GIST48B.

[0013] [Diagram 3]3A-3I are a series of heat maps, Venn diagrams, scatter plots, and tracks showing the genomic localization of MOZ and menin-MLL complexes in GIST. FIG. 3A is a series of heat maps demonstrating the genomic localization of H3K27ac, H3K9ac, H3K4me3, BRPF1, and KAT6A by ChIP-seq, and menin and MLL1n by CUT&Tag in GIST-T1. FIG. 3B-3D are diagrams showing overlapping peaks defined by MACS. FIG. 3B is a diagram showing BRPF1 and KAT6A. FIG. 3C is a diagram showing menin and BRPF1. FIG. 3D is a Venn diagram showing menin and MLL1n. FIG. 3E is a scatter plot showing enriched genomic regions of BRPF1 binding, with TFs shown in red. FIG. 3F is a scatter plot showing enriched genomic regions of menin binding, with TFs shown in red. Figures 3G-I are tracks showing regions of genomic occupancy of the TF HAND1, the MOZ complex members BRPF1 and KAT6A, the menin-MLL complex members menin and MLL1n, and the histone markers H3K4me3, H3K9ac, and H3K27ac at various loci, with Figure 3G showing the FOXF1 locus, Figure 3H showing the DUSP6 locus, and Figure 3I showing the USP1 locus.

[0014] [Figure 4]Figures 4A-4F are a series of line and bar graphs demonstrating that inhibition of the menin-MLL complex with and without MOZ complex inhibition results in cell cycle arrest. Figure 4A is a line graph showing a time course proliferation assay in GIST-T1 with the menin inhibitor VTP-50469 at the indicated concentrations. Figure 4B is a line graph showing a time course proliferation assay in GIST-T1 treated with VTP-50469 with or without WM-1119, where each inhibitor was used at 0.1 μM. Figure 4C is a bar graph showing cell numbers at day 21 normalized to DMSO following treatment of GIST48B, GIST-T1 or the KIT enhancer-independent cell line GIST-T1 / KITΔe11 (endogenous KIT knocked out by rescue of CMV promoter-driven mutant KIT) with VTP-50469 with or without WM-1119. Figure 4D is a bar graph showing a time course proliferation assay in GIST430, showing relative cell numbers shown at day 42 after treatment with 0.5 μM VTP-50469 with or without VTP-50469; this combination was used with 0.1 μM of each drug. Figure 4E is a bar graph showing cell cycle analysis showing the percentage of cells in G0 / G1, S or G2 / M comparing DMSO with or without VTP-50469, imatinib for 72 hours or VTP-50469 at 0.5 μM for 8 days; this combination was used with 0.1 μM of each drug. Figure 4F is a bar graph showing the fold change in cells in early or late apoptosis and cell death compared to DMSO controls after treatment with 0.5 μM imatinib for 72 hours, or with 0.5 μM VTP-50469 or VTP-50469 and WM-1119 (0.1 μM of each drug) for 8 days.

[0015] [Diagram 5]Figure 5A-5W are a series of scatter plots and bar graphs showing the transcriptional effects of menin inhibition with and without MOZ inhibition. Figure 5A is a scatter plot showing the ratio of expression between inhibitor and DMSO treatment for all expressed genes after 5 days of inhibitor treatment in GIST-T1 cells. Figure 5B is a butterfly plot of the all Hallmark gene set showing the NES and FDR q-values ​​comparing VTP-50469 (blue) to the DMSO control at day 5. Figure 5C is a scatter plot showing the Hallmark MTORC1 signaling and EMT gene set comparing DMSO to VTP-50469. Figure 5D is a bar graph showing the relative expression normalized to the DMSO control of all expressed genes, essential genes, genes downregulated >2.5-fold by 6 hours of imatinib treatment, and the Hallmark EMT gene set in GIST-T1 cells treated with VTP-50469 for 5 days. Figure 5E is a bar graph showing the relative expression of all expressed genes normalized to DMSO control comparing genes enriched or not enriched for menin binding. Figure 5F is a bar graph showing the relative expression of core GIST TFs bound by menin. Figure 5G and Figure 5H are bar graphs showing the relative mRNA levels by qRT-PCR of the negative regulator of KIT signaling DUSP6, SE-associated NPR3, and essential gene USP1 in cells treated with 0.5 μM VTP-50469 or WM-1119 and VTP-50469 (each drug at 0.1 μM) for 5 days. Figure 5I is a heat map showing unsupervised hierarchical clustering of RNA-seq data comparing GIST-T1 and VTP-50469 treatment. Figure 5J is a heat map showing unsupervised hierarchical clustering of RNA-seq data comparing sgRNA-transduced GIST-T1 / Cas9 cells. Figure 5K is a heatmap showing Pearson correlation of control-normalized RNA-seq data. Figures 5L-N are correlation plots of gene expression changes in the top 5,000 expressed transcripts comparing control-normalized sgRNA or combination drug treatments. Figure 5O is a heatmap showing normalized normalized enrichment scores (NES) from the GSEA gene set.Figures 5P-S are GSEA plots showing changes in menin / BRPF1 enriched genes, SE associated genes, and HAND1 regulated genes. Figure 5T is a box plot showing control normalized expression of genes upregulated by HAND1. Figures 5U-W are stipple plots showing expression of select genes associated with GIST lineage, TFs, or HAND1 regulation across drug and sgRNA treatment conditions.

[0016] [Figure 6]Figure 6A-6Q are a series of photographic images, scatters, bars, heatmaps, stipple plots, and tracks showing changes in protein interactions following menin inhibition. Figure 6A is a western blot of parental GIST-T1 cells or cells after sgRNA deletion and rescue with a codon-optimized MEAF6 construct fused to BirA*(R118G). Figure 6B is a scatter plot of PSMs and log2 signal intensities of proximal proteins identified by MEAF6 BioID. Figure 6C is a bar graph showing GO term enrichment of MEAF6 proximal proteins. Figure 6D is a scatter plot of the log2 ratio of VTP-50469 / DMSO signal intensities of MEAF6 enriched proteins after 2 days of pretreatment with inhibitor and an additional 24 h treatment during biotin labeling. Figure 6E is a heatmap showing unsupervised hierarchical clustering of DMSO-normalized signal intensities of 67 genes significantly changed in at least one condition in response to VTP-50469 or combined VTP-50469 and WM-1119 treatment. Figures 6F-6G are stipple plots of DMSO-normalized signal intensities for protein interactors enriched with VTP-50469 or VTP-50469 in combination with WM-1119. Figure 6H is a series of heatmaps showing spike-in normalized signal of DOT1L at MACS-defined peaks in GIST-T1 cells treated with DMSO or VTP-50469. Figures 6I and 6J are boxplots showing spike-in normalized DOT1L (Figure 6I) or MEAF6 (Figure 6J) signals at MACS-defined peaks. Figure 6K is a plot of tracks showing regions of spike-in normalized DOT1L genomic occupancy under the indicated treatments, H3K79me2, MEAF6, and H3K27ac at the HAND1 locus. Figure 6L shows cell counts at day 21 normalized to DMSO after treatment of GIST-T1 or GIST48B with the indicated concentrations of EPZ-5676. Figure 6M is a heatmap showing Pearson correlation of control-normalized RNA-seq data from cells treated for 5 days with the indicated inhibitors.Figure 6N is a Pearson correlation of gene expression changes in expressed transcripts (n=5,000) comparing control normalized drug treatment with EPZ-5676 and VTP-50469. Figure 6O is a GSEA plot showing changes in HAND1 regulated genes resulting from EPZ-5676 treatment. Figures 6P and 6Q are plots showing expression of select genes associated with GIST lineage and TFs (n=4 per condition).

[0017] [Figure 7] Figures 7A-7F are a line graph and a series of photomicrographs showing the effect of menin inhibition on GIST in vivo. Figure 7A is a line graph showing GIST-T1 cell line xenografts treated with imatinib, VTP-50469, a combination of imatinib and VTP-50469, or a vehicle control for 28 days. Figure 7B is a line graph showing PG27 PDXs treated with imatinib, VTP-50469, a combination of imatinib and VTP-50469, or a vehicle control for 18 days. Figure 7C is a series of photomicrographs showing tissue sections from PG27 tumors harvested, fixed, sectioned, and stained with H&E at the end of the treatment period. Figure 7D is a line graph showing GIST-T1 cell line xenografts treated for 28 days. Figure 7E is a heat map showing data from RNA-seq performed on GIST-T1 cell line xenografts treated for 5 or 10 days. Figure 7F is a plot showing expression in FPKM of select genes associated with GIST lineage, imatinib regulation, or cell proliferation.

[0018] [Figure 8]8A-8N are a series of bar graphs, line graphs, and scatter plots showing unique GIST dependency. FIG. 8A is a bar graph showing the top 18 significantly enriched gene ontology terms among genes uniquely essential for GIST. FIG. 8B is a line graph of rank and β score in the screen highlighting menin-MLL complex members. FIG. 8C is a line graph of rank and β score in the screen highlighting INO80 complex members. FIG. 8D is a line graph of rank and β score in the screen highlighting NuA4 histone acetyltransferase complex members. FIG. 8E-8G are scatter plots of ranked susceptibility scores from project driven cell lines for selected members of the INO80 and NuA4 complexes. FIG. 8H is a line graph of rank in the screen and β score highlighting FACT complex members. FIG. 8I-8J are scatter plots of ranked susceptibility scores from project driven cell lines for members of the FACT complex. Figure 8K is a line graph of rank and beta scores in the screen highlighting PAF1 complex members. Figures 8L-8M are scatter plots of ranked sensitivity scores from project driven cell lines for select members of the PAF1 complex. Figure 8N is a bar graph showing relative reads for the top 8 sgRNAs targeting the indicated genes in GIST-T1 or GIST430 normalized to the baseline plasmid library (n=2 per sgRNA).

[0019] [Figure 9]Figures 9A-9E are a series of line graphs and scatter plots showing PRC2 complex dependency in GIST. Figure 9A is a line graph showing a plot of rank in the screen and beta score highlighting core PRC2 complex members. Figures 9B-9C are ranked susceptibility scores from project driven cell lines for select members of the PRC2 complex. Figure 9D is a plot of rank and CERES dependency scores for EZH2 complex members across DepMap cell lines (n=726), with a dotted line at -1 indicating significant dependency. Figure 9E shows epistatic gene dependency correlations for EZH2 in the DepMap. Co-dependent chromatin modifying enzymes and complex members are labeled.

[0020] [Figure 10] Figure 10A-H are a series of diagrams and tracks showing the localization of menin-MLL complex in GIST. Figure 10A is a diagram showing the overlap in enriched regions between menin and BRPF1, and selected GIST-associated genes are shown. Figure 10B-H are tracks showing the genomic occupancy of the TF HAND1, menin-MLL complex members menin and MLL1n, and histone marks H3K4me3, H3K9ac, and H3K27ac; Figure 10B shows the OSR1 locus, Figure 10C shows the PDGFRA locus; Figure 10D shows the KIT locus, Figure 10E shows the KDR locus, Figure 10F shows the MEIS1 locus, Figure 10G shows the HAND1 locus, and Figure 10H shows the NPR3 locus.

[0021] [Figure 11] FIG. 11 is a bar graph showing DMSO-normalized cell numbers after the first passage of the slowly growing GIST cell lines GIST430 and GIST882 inhibited by VTP-50469 or VTP-50469 in combination with WM-1119.

[0022] [Figure 12]12A-C are a series of scatter plots and bar graphs showing the transcriptional effects of menin inhibition. FIG. 12A is a scatter plot showing the ratio of expression between inhibitor and DMSO treatment for the top 500 essential genes after 5 days of inhibitor treatment. FIG. 12B is a bar graph showing the relative expression of the negative regulators of KIT signaling, SPRY2, SPRY4, and DUSP6, upon treatment with VTP-50469 for 1 or 5 days. FIG. 12C is a bar graph showing the relative expression of KIT upon treatment with VTP-50469 for 1 or 5 days.

[0023] [Figure 13]13A-13J are a series of heatmaps, scatter plots, and tracks showing ChIP-seq results of DOT1L, H3K79me2, and MEAF6, as well as the effect of VTP-50469. FIG. 13A is a heatmap showing spike-in normalized signal of MEAF6 at MACS-defined peaks in GIST-T1 cells treated with DMSO or VTP-50469. FIG. 13B-13C are scatter plots showing enriched genomic regions of DOT1L and H3K79me2 binding. FIG. 13D is a series of heatmaps showing genomic localization of DOT1L, H3K79me2, and MEAF6 in GIST-T1 by ChIP-seq. FIG. 13E is a series of tracks showing regions of spike-in normalized DOT1L genomic occupancy under the indicated treatments, H3K79me2, and H3K27ac at the GPR20 locus. Figure 13F is a plot showing the top 70 gene-dependent correlations of DOT1L in the DepMap, with members of menin-MLL, MOZ and PRC2 complexes shown. Figure 13G is a box plot showing DMSO-normalized signals for DOT1L in regions with enriched (n=1,343) or typical (n=45,256) signals for DOT1L. Figure 13H is a photograph of a Western blot showing DOT1L signals after 5 days of treatment with the indicated inhibitors. Figure 13I is a plot showing the levels of DOT1L expression by RNA-seq after 5 days of treatment with the indicated drugs. Figure 13J is a bar graph showing GIST-T1 cell numbers at day 21 in a time-course proliferation assay comparing sgRNAs targeting two DOT1L exons or Luc or RPS19 as a control.

[0024] [Figure 14]Figures 14A-F are a line graph and a series of photomicrographs showing the effect of menin inhibition in vivo. Figure 14A is a line graph showing the body weight of mice implanted with GIST-T1 cell line and treated with imatinib, VTP-50469, a combination of imatinib and VTP-50469, and vehicle control for 28 days. Figure 14B is a line graph showing the body weight of mice implanted with PG27 PDX and treated with imatinib, VTP-50469, a combination of imatinib and VTP-50469, and vehicle control for 18 days. Figure 14C is a series of photomicrographs showing tissue sections from PG27 tumors, where fixed tissues were harvested at the end of the treatment period, sectioned, and assessed for Ki-67 (top row) and cleaved caspase-3 (bottom row); scale bar = 25 μm. Figure 14D is a line graph showing tumor size after mice were implanted with GIST-T1 cell lines and treated with sgRNA. Figure 14E is a line graph showing the weight of mice implanted with GIST-T1 cell lines and treated with VTP-50469, WM-1119, a combination of VTP-50469 and WM-1119, or vehicle control for 28 days. Figure 14F is a box plot showing the control-normalized expression of all expressed genes (n=7,434) or genes whose expression is upregulated by HAND1 (n=438) in each treatment group.

[0025] [Figure 15] Figures 15A-C are a set of line graphs and bar graphs showing KAT6A, menin and BRPF1 inhibition in GIST cell lines. Figure 15A is a line graph showing a time course proliferation assay after treatment of GIST-T1 or GIST48B with 50 nM imatinib. Figure 15B is a bar graph showing DMSO normalized cell counts after the first passage of the slow growing GIST cell lines GIST430 (day 6), GIST882 (day 12) and GIST48 (day 12) compared to GIST-T1 (day 4). Figure 15C is a line graph showing a time course proliferation assay of GIST-T1 or GIST48B cells treated with selective BRPF1 inhibitors GSK6853 or PFI-4.

[0026] [Figure 16] Figures 16A-H are a set of bar graphs, box plots, and heat maps showing the transcriptional effects of MOZ and menin disruption. Figure 16A is a heat map of control-normalized expression of 10 GIST-associated TFs in response to drug or sgRNA treatment. Figure 16B is a box plot showing DMSO-normalized expression of 18 GIST-associated TFs in the indicated drug treatments (n=4 per condition). Figures 16C-F are a set of bar graphs showing relative mRNA levels of the negative regulator of KIT signaling DUSP6 and HAND1 and SE-associated gene NPR3 in GIST cell lines. Figure 16G is a heat map showing GSEA data showing the NES of the Reactome translation-associated gene set in each drug or sgRNA treatment condition. Figure 16H is a box plot showing control-normalized expression of all translation-associated genes (n=48) in each sgRNA and drug treatment condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] definition Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0028] By "agent" is meant any small chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. As used herein, unless otherwise stated or clear from the context, the term "or" is understood to be inclusive. As used herein, unless otherwise stated or clear from the context, the terms "a," "an," and "the" are understood to be singular or plural.

[0029] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0030] The transitional phrase "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unrecited elements or method steps. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the claim to "particular materials or steps" of the claimed disclosure and those that do not materially affect the basic and novel characteristics.

[0031] Other features and advantages of the present disclosure will be apparent from the following description of its preferred embodiment and claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0032] How to use In some embodiments, the present disclosure relates to treating GIST in a subject. The method involves administering an effective amount or a therapeutically effective amount of a menin inhibitor to a subject in need thereof.

[0033] GIST is a soft tissue sarcoma that is often characterized by recurrent activating mutations in or around the tyrosine kinase KIT gene and / or PDGFRA gene. The phrase "in or around" as used herein refers to mutations in the coding region of a gene or in the 5' or 3' proximal region of a gene that contributes to gene function (e.g., regulatory regions that affect gene transcription). GIST lacks oncogene amplification and depends on an established network of transcription factors. Intrinsic chromatin modifying enzymes are shown in the examples as essential for organizing the GIST epigenome, for example, KMT2A / MLL1 is established herein as a previously unknown dependency of GIST, and more broadly, it has been found to show similar regulation across selected cancer subtypes. KMT2A / MLL1 is a member of the menin-MLL complex and is responsible for H3K4 methylation and transcriptional activation (Ruthenburg et al., Molecular Cell 25:15-30 (2007), Krivtsov et al., Nat. Rev. Cancer 7:823-33 (2007)). In some embodiments, the subject has been diagnosed with a GIST with a mutation in or around the KIT gene. In some embodiments, the mutation is an activating mutation. Activating mutations cause the mutant protein to remain in a dysregulated state compared to the non-mutated protein. Activating mutations in the kinase domain most often result in ligand-independent activation of the kinase domain, thus targeting phosphorylation. In some embodiments, the subject has metastatic GIST.

[0034] The term "subject" (or "patient") as used herein includes all members of the animal kingdom that are susceptible to (or susceptible to) or afflicted with GIST. In some embodiments, the subject is a human. Thus, a subject that "has GIST" or "needs treatment" according to the present disclosure broadly encompasses positively diagnosed subjects, including subjects with active disease that may have been previously treated with one or more treatments, and subjects that are not currently being treated (e.g., in remission) but may still be at risk of recurrence, and subjects that have not been positively diagnosed but are predisposed to cancer or autoimmune disease (e.g., because of previous medical history and / or family history, or otherwise exhibit one or more risk factors that allow a medical professional to reasonably suspect that the subject is predisposed to GIST).

[0035] As used herein, the terms "treat," "treating," and "treatment" refer to any type of intervention, process, or administration of an effective or therapeutically effective amount of a menin inhibitor, TKI, and / or MOZ inhibitor to a subject in need thereof for the therapeutic purpose of reversing, mitigating, ameliorating, inhibiting, reducing, slowing, halting, stabilizing, or preventing the onset, progression, occurrence, severity, or recurrence of symptoms, complications, or pathologies, or biochemical manifestations associated with GIST (a "therapeutic effect").

[0036] The active agent used in the implementation of the present disclosure is a menin inhibitor.As disclosed herein, in some embodiments, one or more additional active agents may be used, including the inhibitor of tyrosine kinase (TKI) and the inhibitor of MOZ (monocytic leukemia zinc finger; also known as lysine (K) acetyltransferase 6A (KAT6A), which is a histone acetyltransferase (HAT).

[0037] The term "inhibitor" is used in its broadest sense and includes any agent, such as a small molecule, a nucleic acid (e.g., a ribozyme, an antisense nucleic acid, an siRNA), an antibody or a functional fragment thereof, a peptide, a peptidomimetic or an aptamer, that acts directly or indirectly to disrupt, reduce, or even eliminate the function of a target.

[0038] Menin inhibitors The terms "menin inhibitor", "menin inhibitors" and "menin-MLL complex inhibitor" are used interchangeably herein and may be understood in their broadest sense. Menin inhibitors include any agent (e.g., small molecule, nucleic acid (e.g., siRNA), or one or a combination of antibodies, peptides, peptidomimetics, or aptamers) that acts directly or indirectly to disrupt, reduce, or even eliminate the function or expression of menin protein, multiple endocrine neoplasia 1 (MEN1) gene, or menin-MLL complex. Protein disruption may include direct activity blocking, protein-protein interaction blocking, and the like. Menin, the protein product of the MEN1 (multiple endocrine neoplasia syndrome type 1) gene, interacts with mixed lineage leukemia (MLL) family proteins in a histone methyltransferase complex that includes MLL1 (also known as lysine (K)-specific methyltransferase 2A (KMT2A)), Ash2, Rbbp5, and WDR5. As a result of chromosomal rearrangements of the MLL gene, MLL fuses with one of over 60 different protein partners, leading to upregulated expression of the HOXA9 and MEIS1 genes, which are important in leukemogenesis. Unlike AML or ALL, MLL fusion proteins are absent in GISTs.

[0039] Representative small molecule menin inhibitors include VTP-50469 (5-fluoro-N,N-diisopropyl-2-((4-(7-(((1r,4r)-4-(methylsulfonamido)cyclohexyl)methyl)-2,7-diazaspiro[3.5]non-2-yl)pyrimidin-5-yl)oxy)benzamide), KO-539 ((R)-4-methyl-5-((4-((2-(methylamino)-6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)methyl)-1-(2-(4 (R)-N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-2,6-diazaspiro[3.4]octan-6-yl)-1,2,4-triazin-6-yl)oxy)benzamide, also used as NCT04811560; SNDX-5613 (N-ethyl-5-fluoro-N-isopropyl-2-((5-(2-(6-((2-methoxyethyl)(methyl)amino)-2-methylhexan-3-yl)-1,2,4-triazin-6-yl)oxy)benzamide, also used as NCT04811560; ethyl-2-((4-(7-(((1r,4r)-4-(ethylsulfonamido)cyclohexyl)methyl)-2,7-diazaspiro[3.5]non-2-yl)pyrimidin-5-yl)oxy)-5-fluoro-N-isopropylbenzamide, also used as NCT04065399), DS-1594 ((1R,2S,4R)-4-((4-(5,6-dimethoxypyridazin-3-yl)benzyl)amino)-2-(methyl(6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)cyclopentyl also used as NCT04752163), BMF-219 ((R)-N-(1-(2-(2-((4-(4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-6-yl)phenyl)amino)-2-oxoethyl)pyridin-4-yl)piperidin-3-yl)but-2-ynamide), DSP-5336 (N-ethyl-5-fluoro-N-isopropyl-2-((5-(7-((1S,3S,4R)-5-methylene-2-azabicyclo[2.2.2]octane-3-carbonyl)-2,7-diazaspiro[3.5]nonan-2-yl)-1,2,4-triazin-6-yl)oxy)benzamide, also used as NCT04988555), antibody A300-105A (commercially available from Bethyl Laboratories), MI-3453 (N-(3-((2-cyano-4-methyl-5-((4-((2-(methylamino)-6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)methyl)-1H-indol-1-yl)methyl)bicyclo[1.1.1]pentan-1-yl)formamide, M-808 (methyl((1S,2R)-2-((S)-2-(azetidin-1-yl)-1- (3-fluorophenyl)-1-(1-((1-(4-((1-((E)-4-(piperidin-1-yl)but-2-enoyl)azetidin-3-yl)sulfonyl)phenyl)azetidin-3-yl)methyl)piperidin-4-yl)ethyl)cyclopentyl)carbamate), MI-0202 (4-(4-(5,5-dimethyl-4,5-dihydrothiazol-2-yl)piperazin-1-yl)-6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyri MI-503 (1-((1H-pyrazol-4-yl)methyl)-4-methyl-5-((4-((6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)methyl)-1H-indole-2-carbonitrile), MI-463 (4-methyl-5-((4-((6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)methyl)-1H-indole-2-carbonitrile), These small molecule inhibitors include 5-((4-((6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)methyl)-1H-indole-2-carbonitrile), MI-136 (5-((4-((6-(2,2,2-trifluoroethyl)thieno[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)methyl)-1H-indole-2-carbonitrile), and ML-227 (4-(3-(4-(cyclopentyl(hydroxy)(phenyl)methyl)piperidin-1-yl)propoxy)benzonitrile). The structures of these small molecule inhibitors are as follows: .

[0040] [ka] [ka] [ka]

[0041] Other menin inhibitors that may be useful in the practice of the present disclosure are known in the art. See, for example, WO2017 / 112768, WO2017 / 214367, WO2018 / 053267, WO2020 / 069027 A1, WO2021 / 207335 A1, US2021 / 0115018 A1, US2019 / 0307750, US20160339035 (compounds of formula (I) therein), and Borkin et al., Cancer Cell 27(4):589-602(2015).

[0042] Additional menin inhibitors that may be useful in the practice of the present disclosure include MI-2-2, which inhibits the interaction between menin and MLL, Grembecka et al., Nat. Chem. Biol. 8:277-284 (2012); Shi et al., Blood 120:4461-4469 (2102)), and N,N'-bis(4-aminophenyl)-N,N'-dimethylethylenediamine (also known as ISC-30, which inhibits the interaction of MLL enzymes with menin), and Krivtsov et al., Cancer Cell. 36(6):660-673 (2019), Klossowski et al., J. Clin. Invest. 130:981-97 (2020), Xu et al., J. Med. Chem. 63:4997-5010 (2020).

[0043] In some embodiments, menin inhibitors are used as active agents to reduce the levels of MEN1 or another menin-MLL complex member, such as interfering RNA, e.g., small interfering RNA (siRNA). RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes the degradation of complementary mRNA. Soutschek et al., 432:173-178 (2004) describes chemical modifications to siRNA that aid in intravenous systemic delivery. Optimization of siRNA includes consideration of overall G / C content, C / T content at the termini, melting temperature (Tm) and nucleotide content of 3' overhangs. See, e.g., Schwartz et al., Cell 115:199-208 (2003) and Khvorova et al., Cell 115:209-216 (2003). Thus, the present disclosure also includes methods of reducing levels of MEN1, MOZ, or other target proteins using RNAi technology. Representative siRNA nucleic acid sequences that bind to members of the menin-MLL complex are shown in Table 1.

[0044] [Table 1]

[0045] Menin inhibitors may be administered to patients as monotherapy or in combination with, for example, TKIs and / or MOZ inhibitors. Both monotherapy and combination therapy may be "front / first line" i.e., as initial treatment in patients who have not previously received an anti-GIST cancer treatment regimen, alone or in combination with other treatments; or "second line" i.e., as treatment in patients who have previously received an anti-cancer treatment regimen, alone or in combination with other treatments; or "third line", "fourth line", etc. treatment, alone or in combination with other treatments. Therapy may also be given to patients who have previously received unsuccessful or partially successful treatments but have become intolerant to a particular treatment. Therapy may also be given as adjuvant treatment i.e., to prevent recurrence of GIST in patients who currently have no detectable disease or after surgical removal of the tumor. Thus, in some embodiments, the inhibitor(s) may be administered to patients who have received another therapy, such as chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy, radiation therapy, targeted therapy, or any combination thereof.

[0046] Combination therapy with tyrosine kinase inhibitors (TKIs) and / or MOZ inhibitors In some embodiments, the subject is treated with menin inhibitor therapy in combination with or simultaneously with an effective or therapeutically effective amount of TKI and / or MOZ inhibitor. Blocking KIT or MOZ can provide an additional means of enhancing the therapeutic effect of menin inhibitor.

[0047] The terms "in combination" and "simultaneously" used in the context of combination therapy mean that the active agents are administered simultaneously, including substantially simultaneous administration, by the same or separate dosage forms, and by the same or different modes of administration, or sequentially, for example, as part of the same treatment regimen, or sequential treatment regimen. Thus, when given sequentially, at the start of administration of the second inhibitor, the first inhibitor is in some cases still detectable at effective concentrations at the site of treatment. The order and time intervals can be determined so that they can act together, for example, synergistically, to provide a greater benefit than if they were administered otherwise. For example, the therapeutic agents can be administered sequentially in any order at the same time or at different times, but if not administered simultaneously, they can be administered close enough in time to provide the desired therapeutic effect, which can be in a synergistic manner. Thus, these terms are not limited to administering the active agents at exactly the same time.

[0048] Tyrosine Kinase Inhibitors TKIs include any one or combination of small molecules, nucleic acids, such as siRNA, or agents such as peptides, peptidomimetics, or aptamers that act directly or indirectly to disrupt, reduce, or even eliminate the function or expression of KIT protein or KIT. In some embodiments, the TKI is imatinib, sunitinib, regorafenib, avapritinib, ripretinib, or nilotinib. The TKI can be an antibody, such as anti-KIT antibodies monoclonal anti-D4 and anti-D5. See Shi et al., Proc. Natl. Acad. Sci. USA 113(33):E4784-93 (2016). In some embodiments, the KIT inhibitor is an antibody fragment, such as the bivalent antibody fragments 2D1-Fc and 3G1-Fc. See Gall et al., Mol. Cancer. Ther. 14(11):2595-605 (2015). Combinations of two or more TKI inhibitors can be used.

[0049] In some embodiments, the TKI is administered subsequent to administration of the menin inhibitor. In some embodiments, the TKI is administered substantially simultaneously (i.e., concurrently) with administration of the menin inhibitor. In some embodiments, the TKI is administered prior to administration of the menin inhibitor.

[0050] MOZ inhibitors In some embodiments, the additional active agent may be an effective amount of a MOZ inhibitor. In some embodiments, the additional active agent may be a therapeutically effective amount of a MOZ inhibitor. The MOZ inhibitor includes any agent, such as a small molecule, a nucleic acid (e.g., siRNA), or an antibody, a peptide, a peptidomimetic, or an aptamer, that acts to directly or indirectly disrupt, reduce, or even eliminate the function or expression of the MOZ protein or MOZ gene. In some embodiments, the MOZ inhibitor is administered subsequent to the administration of the menin inhibitor. In some embodiments, the MOZ inhibitor is administered substantially simultaneously with the administration of the menin inhibitor. In some embodiments, the menin inhibitor is combined with both the TKI and the MOZ inhibitor. In some embodiments, the TKI is administered subsequent to the administration of the MOZ inhibitor. In some embodiments, the TKI is administered substantially simultaneously with the administration of the MOZ inhibitor.

[0051] Representative examples of MOZ inhibitors that may be useful in the practice of the present disclosure include WM-1119 (2-fluoro-N'-(3-fluoro-5-(pyridin-2-yl)benzoyl)benzenesulfonohydrazide, WM-8014 (N'-(4-fluoro-5-methyl-[1,1'-biphenyl]-3-carbonyl)benzenesulfonohydrazide), PF-9363 (N'-(4-fluoro-5-methyl-[1,1'-biphenyl]-3-carbonyl)benzenesulfonohydrazide), and antibody 21620002 (commercially available from Novus Biologicals).

[0052] The structures of these representative small molecule MOZ inhibitors are as follows:

[0053] [ka]

[0054] In some embodiments, the MOZ inhibitor is an interfering RNA (e.g., siRNA) that is used as an active agent to reduce the level of MOZ or another MOZ complex member. The nucleic acid sequences of representative siRNAs that bind to members of the MOZ complex are shown in Table 2.

[0055] [Table 2]

[0056] In some embodiments, the MOZ inhibitor is administered subsequent to the administration of the menin inhibitor. In some embodiments, the MOZ inhibitor is administered substantially simultaneously (i.e., concurrently) with the administration of the menin inhibitor. In some embodiments, the MOZ inhibitor is administered prior to the administration of the menin inhibitor.

[0057] For embodiments involving administration of a TKI and a MOZ inhibitor (in addition to a menin inhibitor), the MOZ inhibitor may be administered prior to, substantially simultaneously with, or following administration of the TKI.

[0058] In some embodiments, the MOZ inhibitor is administered subsequent to the administration of the TKI and the menin inhibitor. In some embodiments, the MOZ inhibitor is administered substantially simultaneously (i.e., concurrently) with the administration of the TKI and the menin inhibitor. In some embodiments, the menin inhibitor is administered subsequent to the administration of the MOZ inhibitor and the TKI.

[0059] Compositions and Formulations The active agents described herein can be formulated into pharmaceutical compositions according to known techniques. The pharmaceutical compositions of the present disclosure include an effective amount of a menin inhibitor, alone or in combination with an effective amount of a TKI and a MOZ inhibitor. In some embodiments, the pharmaceutical compositions of the present disclosure include an effective amount or a therapeutically effective amount of a menin inhibitor, alone or in combination with an effective amount or a therapeutically effective amount of a TKI and a MOZ inhibitor. The active agent may be in the form of a pharmaceutically acceptable salt, or its isomer (e.g., a stereoisomer). Salts and stereoisomers are encompassed by the terms "inhibitor" and "active agent". As used herein, "pharmaceutically acceptable salt" refers to any non-toxic salt that can provide, either directly or indirectly, a compound of the present disclosure or a prodrug of a compound upon administration to a recipient. Pharmaceutically acceptable salts may be formed with acids, representative examples of which include hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, and succinic acid.

[0060] The active agents disclosed herein and their pharma- ceutically acceptable salts and stereoisomers can be formulated individually or together in combinations of two or more into a given type of composition according to conventional pharmaceutical practices such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping and compression processes (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), ed. AR Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York). The type of formulation will depend on the mode of administration, which may include enteral (e.g., oral, buccal, sublingual and rectal), parenteral (e.g., subcutaneous (sc), intravenous (iv), intramuscular (im) and intrasternal injection or infusion techniques, intraocular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, intradermal, intravaginal, intraperitoneal, mucosal, nasal, intratracheal instillation, bronchial instillation and inhalation) and topical (e.g., transdermal). In general, the most appropriate route of administration will depend on a variety of factors, including, for example, the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). For example, parenteral (e.g., intravenous) administration may also be advantageous in that the inhibitor may be administered relatively quickly, such as in the case of single dose treatments and / or acute conditions.

[0061] In some embodiments, the active agent is formulated for oral or intravenous administration (eg, systemic intravenous injection).

[0062] Thus, the active agents may be formulated into solid compositions (e.g., powders, tablets, dispersible granules, capsules, cachets, and suppositories), liquid compositions (e.g., solutions in which the inhibitor is dissolved, suspensions in which solid particles of the inhibitor are dispersed, emulsions, and solutions containing liposomes, micelles, or nanoparticles, syrups and elixirs), semi-solid compositions (e.g., gels, suspensions, and creams), and gases (e.g., propellants for aerosol compositions). The inhibitors may also be formulated for immediate, intermediate, or sustained release.

[0063] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active inhibitor is mixed with a carrier such as sodium citrate or dicalcium phosphate, and a) a filler or extender such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) a binder such as, for example, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) a wetting agent such as glycerol, d) a cross-linked polymer (e.g., cross-linked polyvinylpyrrolidone (crospovidone), cross-linked sodium carboxymethylcellulose, (croscarmellose sodium), sodium starch glycolate, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) disintegrating agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include buffering agents. Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings, and may further contain opacifying agents.

[0064] In some embodiments, the inhibitors of the present disclosure can be formulated in hard or soft capsules, such as gelatin capsules.Representative excipients that can be used include pregelatinized starch, magnesium stearate, mannitol, sodium stearyl fumarate, lactose anhydrous, microcrystalline cellulose and croscarmellose sodium.Gelatin shells can include gelatin, titanium dioxide, iron oxide and coloring agents.

[0065] Liquid dosage forms for oral administration include solutions, suspensions, emulsions, microemulsions, syrups and elixirs.In addition to inhibitors, liquid dosage forms can contain aqueous or non-aqueous carriers (depending on the solubility of inhibitors) commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.Oral compositions can also contain excipients, representative examples of which include wetting agents, suspending agents, coloring agents, sweeteners, flavoring agents and aromatic agents.

[0066] Injectable preparations may include sterile aqueous or oily suspensions. They may be formulated according to standard techniques using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in injectable preparations. Injectable preparations may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium before use. The effect of the compound can be prolonged by slowing its absorption, which can be accomplished by the use of a liquid suspension of poor water solubility or of crystalline or amorphous material. Prolonged absorption of the inhibitor from a parenterally administered formulation can also be accomplished by suspending the inhibitor in an oil vehicle.

[0067] Dosage As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount of the active agent disclosed herein (e.g., menin inhibitor, TKI, or MOZ inhibitor) or its pharma- ceutically acceptable salt or isomer that is effective in producing a desired response in GIST patients. Thus, the terms "effective amount" and "therapeutically effective amount" include the amount of active agent that, when administered, induces positive alterations in GIST, or inhibits the development or progression of GIST, or is sufficient to alleviate to some extent one or more of the symptoms of GIST, or simply kills or inhibits the growth of GIST, or otherwise blocks or reduces the activity of menin-MLL complex in affected cells. The effective amount of active agent may vary depending on several factors, which may include the severity and stage of GIST, the mode of administration, the age, weight, and general health of the subject, and similar factors well known in the medical field. See, e.g., Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 10th Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001. Ultimately, the attending physician or veterinarian will decide the appropriate amount and administration regimen.

[0068] Active agents useful in the practice of the present disclosure may be effective over a wide dosage range. In some embodiments, the total daily dosage of a given active agent may range, for example, from about 0.001 to about 1600 mg, 0.01 to about 1600 mg, 0.01 to about 500 mg, about 0.01 to about 100 mg, about 0.5 to about 100 mg, 1 to about 100 to about 400 mg per day, about 1 to about 50 mg per day, and about 5 to about 40 mg per day for an adult human, and in yet other embodiments, from about 10 to about 30 mg per day. Individual dosages may be formulated to contain the desired dosage, depending on the number of times the active agent is administered per day. By way of example, capsules may be formulated with about 1 to about 200 mg of active agent, e.g., 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, and 200 mg. In some embodiments, individual dosages may be formulated to contain the desired dosage depending on the number of times the active agent is to be administered per day.

[0069] In some embodiments, a suitable daily dosage of the menin inhibitor may range from 1 ng / kg to about 200 mg / kg, from about 1 μg / kg to about 100 mg / kg, or from about 1 mg / kg to about 50 mg / kg body weight. Other dosages of menin inhibitors are disclosed in the art. See, for example, International Publication Nos. WO2017 / 112768, WO2017 / 214367, WO2018 / 053267, WO2020 / 069027 A1, WO2021 / 207335 A1, and U.S. Patent Publication Nos. 2021 / 0115018 A1 and 2019 / 0307750.

[0070] In some embodiments, the daily dosage of the TKI imatinib is about 100 mg / day. In some embodiments, the KIT inhibitor is administered at a daily dosage of about 300 mg / day, about 340 mg / day, about 400 mg / day, about 600 mg / day, or about 800 mg / day.

[0071] In some embodiments, the daily dosage of the TKI sunitinib is about 50 mg, e.g., orally once daily for four weeks followed by two weeks off treatment, typically in the form of a hard gelatin capsule containing 12.5 mg, 25 mg, or 50 mg of sunitinib.

[0072] In some embodiments, the daily dosage of the TKI regorafenib is about 160 mg, for example orally for 21 days followed by a one week drug holiday, typically in the form of a 40 mg film coated tablet.

[0073] In some embodiments, the daily dosage of the TKI avapritinib is about 300 mg, e.g., orally once daily, typically in the form of a film-coated capsule containing 25, 50, 100, 200, or 300 mg.

[0074] In some embodiments, the daily dosage of the TKI ripretinib is about 150 mg, eg, orally once daily, typically in the form of a 50 mg tablet.

[0075] In some embodiments, the daily dosage of the TKI nilotinib is about 300-400 mg, e.g., in hard capsules of 150 mg and 200 mg, typically administered on an empty stomach. It is taken twice a day, 12 hours apart.

[0076] In some embodiments, the daily dosage of the MOZ inhibitor can range from about 0.5 μg to about 50 mg per kilogram of the subject's body weight. In some embodiments, the daily dosage of the MOZ inhibitor can range from about 1 μg to about 10 mg per kilogram of the subject's body weight, and in some embodiments, from about 3 μg to about 1 mg per kilogram of the subject's body weight.

[0077] The method may involve administration of a menin inhibitor, and optionally one or more additional active agents or pharmaceutical compositions thereof, to a patient in a single dose or multiple doses, for example, 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more doses. For example, the dosing frequency may range from once a day to about once every 8 weeks. In some embodiments, the dosing frequency ranges from about once a day to 1, 2, 3, 4, 5, or 6 weeks, and in other embodiments involves a 28-day cycle that includes daily dosing for 3 weeks (21 days) followed by a 7-day "off" period, or 4 weeks of dosing followed by a 14-day "off" period. In other embodiments, the active agent may be administered twice a day (BID) for 2 1 / 2 days (5 doses total), or once a day (QD) for 2 days (2 doses total). In other embodiments, the active agent(s) may be administered once a day (QD) for 5 days.

[0078] Further combination therapy The methods of the present invention may require the administration of at least one other active anti-cancer agent. Representative anti-cancer agents are disclosed in U.S. Patent No. 9,101,622 (Section 5.2 thereof).

[0079] Still other treatments include immunotherapy, chemotherapy and radiation.

[0080] Immunotherapy, including immune checkpoint inhibitors, can be used to treat diagnosed cancer. Immune checkpoint molecules include, for example, PD1, CTLA4, KIR, TIGIT, TIM-3, LAG-3, BTLA, VISTA, CD47, and NKG2A. Clinically available examples of immune checkpoint inhibitors include durvalumab (Imfinzi®), atezolizumab (Tecentriq®), and avelumab (Bavencio®). Clinically available examples of immune checkpoint inhibitors include durvalumab (Imfinzi®), atezolizumab (Tecentriq®), and avelumab (Bavencio®).

[0081] Combination chemotherapy includes, for example, Abraxane®, altretamine, docetaxel, Herceptin®, methotrexate, Novantrone®, Zoladex®, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, Taxol®, gemcitabine, Navelbine®, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, or any analog or derivative variants of the foregoing, and combinations thereof.

[0082] Combination radiation therapy involves the directed delivery of gamma rays, commonly known as X-rays, and / or radioisotopes to tumor cells that cause widespread damage to DNA, DNA replication and repair, and chromosome assembly and maintenance. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells, and are determined by the attending physician.

[0083] Radiation therapy can include external or internal radiation therapy. External radiation therapy involves a source of radiation outside the subject's body and directing radiation toward the area of ​​the cancer inside the body. Internal radiation therapy uses radioactive material enclosed in needles, seeds, wires, or catheters that are placed directly into or near the cancer.

[0084] kit The pharmaceutical composition can be assembled into a kit or pharmaceutical system for use in the treatment of GIST. The kit or pharmaceutical system may include one or more dosage formulations comprising a menin inhibitor and a pharma- ceutically acceptable carrier, disposed in a suitable container, for example, a tube, vial, ampoule, bottle, syringe, or bag. In some embodiments, the kit or pharmaceutical system may also include one or more dosage formulations of a TKI. In some embodiments, the kit or pharmaceutical system may also include one or more dosage formulations of a MOZ inhibitor. In some embodiments, the kit or pharmaceutical system may also include one or more dosage formulations of a TKI inhibitor and one or more dosage formulations of a MOZ inhibitor. The additional active agents may be formulated separately or together and may be disposed in the same or separate containers. The kit or pharmaceutical system of the present disclosure may also include printed instructions for using the additional active agent(s) contained therein.

[0085] In some embodiments, the kit comprises the menin inhibitor and the TKI in the same dosage form, hi other embodiments, the menin inhibitor and the TKI are in different dosage forms.

[0086] These and other aspects of the present disclosure will be further understood by consideration of the following examples, which are intended to illustrate certain embodiments of the present disclosure, but are not intended to limit the scope of the disclosure as defined by the claims. EXAMPLES

[0087] Example 1: Materials and Methods Cell culture and virus production. All cell lines were tested negative for mycoplasma infection by routine surveillance (MilliporeSigma Cat#MP0025-1KT). Human embryonic kidney (HEK) 293FT (Thermo Fisher Scientific catalog number R70007, RRID: CVCL_6911) and GIST cell lines GIST-T1 (Cosmo Bio catalog number PMC-GIST01-COS, RRID: CVCL_4976; KIT mutation in exon 11 Δ560-578), GIST430 (RRID: CVCL_7040; KIT mutation in exon 11 Δ560-576), GIST48B (RRID: CVCL_M441; KIT-independent), and GIST882 (RRID: CVCL_7044; KIT mutation in exon 13 K642E) were cultured in Dulbecco's modified Eagle's medium containing 10% FBS, 2 mM L-glutamine, 100 mg / ml penicillin, and 100 mg / ml streptomycin. KIT-rescue cell lines, independent of the KIT enhancer, were generated as previously described (Hemming et al., Cancer Research 79:994-1009 (2019)). Non-commercial cell lines were obtained from Jonathan Fletcher's laboratory between 2014 and 2016. KIT exons were sequenced to confirm the expected coding mutations and cellular identity of GIST cell lines, and cells were thawed from original or derived stocks and used in the described experiments within approximately 3 months. Transfections were performed using X-tremeGene (Roche, Cat. No. 6365809001). Lentiviral production was performed as previously described (Hemming et al., PLoS Biol. 6:e2571-15(2008)). Briefly, 293FT cells were co-transfected with pMD2.G (Addgene#12259), psPAX2 (Addgene#12260) and lentivirus expression plasmids. Viral supernatants were harvested at approximately 72 hours and debris was removed by centrifugation at 1,000 g for 5 minutes.Cells were transduced with viral supernatant and 8 μg / mL polybrene by spinoculation at 680 g for 60 min. Drugs were used at the indicated concentrations and included imatinib (LC Laboratories Catalog No. I-5508), WM-1119 (Selleck Chemicals Catalog No. S8776), VTP-50469 (a gift of Syndax Pharmaceuticals), tazemetostat (Selleck Chemicals Catalog No. S7128), and EPZ-5676 (Selleck Chemicals Catalog No. S7062). For time course proliferation assays, 15 × 10 3 Cells were dispensed per well into 96-well plates, transduced with virus or treated with drugs, and cell counts were performed approximately twice a week on a Guava easyCyte flow cytometer (Luminex Corporation) with cell counts normalized to control conditions.

[0088] Genome-wide CRISPR screening. The Liu human CRISPR knockout library (Addgene#1000000132; Fei et al., Proc. Natl. Acad. Sci. USA 116:25186-95(2019)), targeting 18,436 genes with 185,634 sgRNAs, is split into two pooled libraries H1 and H2, containing approximately five sgRNAs per gene in each library. Each virion contained sgRNA, Cas9, and a puromycin resistance gene derived from lentiCRISPRv2. The cell lines GIST-T1 and GIST430 were transduced twice with each library (n=8 total). For each library transduction, 44.64×10 6 Cells were transduced at a target MOI of 0.3 with an estimated library coverage of 134×. Puromycin was applied for selection at 72 hours. Cells were passaged at confluence for approximately 30 days to maintain library coverage >134×. At the end of the experiment, genomic DNA was cultured at 30×10 per library. 6The genomic DNA was extracted from cells. The region of the sgRNA between U6 and EF-1α was amplified from 200 μg of genomic DNA from each experimental replicate in 32 separate 100 μL reactions. The products were pooled and a second PCR reaction was performed to incorporate Illumina adapters and 6 bp barcodes. A third PCR reaction was performed to enrich for full-length amplicons (primers are detailed in Tables 1-3). The final amplicon library was purified by agarose gel electrophoresis and extraction with the QIAquick Gel Extraction Kit (Qiagen catalog no. 28704). Next-generation sequencing was performed on a NovaSeq 6000 (Illumina). Screen data was analyzed using MAGeCK software (version 0.5.8) (Wang et al. Protoc. 14(3):756-780 (2019)). The "count" command was used to generate read counts for all libraries (n=8), with the initial plasmid library (n=2) used as a baseline control. Total counts were normalized across samples to minimize the effect of sequencing depth. The maximum likelihood estimation command was used to generate β scores for each screen, and data were normalized to the control AAVS1 sgRNA contained within the H1 and H2 libraries. Metascape was used for gene ontology enrichment analysis (Zhou et al. Commun. 10(1):1523(2019)).

[0089] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0090] Cloning and CRISPR. Cell lines stably expressing human codon-optimized Streptococcus pyogenes Cas9 (Addgene #73310) were generated by viral transduction. CRISPR single guide RNAs (sgRNAs) were designed using CHOPCHOP (Labun et al., Nucleic Acids Research 44:W272-6 (2016)) (chopchop.cbu.uib.no)) and cloned into Lenti-sgRNA-EFS-GFP (LRG, Addgene #65656) modified with GFP replaced by copGFP linked to a puromycin resistance gene by a 2A peptide, as detailed in Tables 1-3. BioID expression vectors were synthesized using codon optimization to modify the sgRNA binding sequence (Twist Bioscience). Dependency Map (DepMap) portal data was accessed via DepMap.org (Barretina et al., Nature 483:603-7 (2012)) using CCRISPR (Avana) releases 20Q3 to 20Q4.

[0091] Cell cycle and apoptosis. Cell cycle analysis was performed after drug treatment for 72 hours (imatinib) or 8 days (VTP-50469, WM-1119). Cells were trypsinized, washed with PBS, and fixed with 70% ethanol. Nuclear DNA was stained using 25 μg / mL propidium iodide (Life Technologies Catalog No. P1304MP) and 0.2 mg / mL RNAse A (Thermo Fischer Scientific Catalog No. EN0531). Analysis was performed on a Guava easyCyte flow cytometer (Luminex Corporation) and single cells were assessed for nuclear content using Guava InCyte software. Apoptosis and cell death were measured 72 hours after drug treatment using Guava Nexin reagent (Luminex Corporation Catalog No. 4500-0450) according to the manufacturer's recommendations. Non-apoptotic cells stain negatively for Annexin V and 7-AAD, early apoptotic cells stain positively for Annexin V but negative for 7-AAD, and late apoptotic and dead cells stain positively for both Annexin V and 7-AAD. Staining was assayed on a Guava easyCyte flow cytometer and data were analyzed using Guava InCyte software.

[0092] Quantitative RT-PCR. Cells were trypsinized and washed in PBS for RNA extraction using RNeasy Mini Kit (Qiagen Cat. No. 74106). A library of cDNA was generated using SuperScript IV VILO cDNA Synthesis Kit (Invitrogen Cat. No. 11766050). RT-PCR was performed using Power SYBR Green PCR Master Mix (Life Technologies Cat. No. 4367659) on a QuantStudio6 Flex Real-Time PCR System (Thermo Fischer Scientific). Relative mRNA levels were calculated by the ΔΔCt method using GAPDH expression as a reference. Primers are listed in Tables 1-3.

[0093] Total RNA was isolated using the RNeasy Plus kit (Qiagen catalog no. 74136), concentration was measured by Nanodrop (Thermo Fisher Scientific), and quality was measured by TapeStation 4200 (Agilent). Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs catalog no. E7645S). Paired-end 150 bp sequencing was performed on a NovaSeq 6000 (Illumina). RNA-seq data were aligned to hg19 using STAR (Dobin et al., Bioinformatics 29:15-21 (2012)) and expression was quantified using Cufflinks (Trapnell et al., Nat. Biotechnol. 28:511-5 (2010)). Gene expression values ​​generated in fragments per kilobase of transcript per million mapped reads (FPKM) Gene Set Enrichment Analysis (GSEA, RRID:SCR_003199) (Subramanian et al., Proc. Natl. Acad. Sci. USA 102:15545-50(2005)) was performed using the Hallmark gene list in the Molecular Signatures Database.

[0094] ChIP-seq and Cut&Tag. For ChIP-seq, approximately 20 × 10 6Cells were incubated in 1% formaldehyde for 10 min. After fixation, excess formaldehyde was quenched with 0.125 M glycine for 5 min. Samples were washed with PBS and intact nuclei were suspended in SDS buffer (0.5% SDS, 50 mM Tris, 100 mM NaCl, 5 mM EDTA, containing protease inhibitor cocktail (Roche catalog number 11873580001)) and sonicated with an E220 Focused-ultrasonicator (Covaris, Inc.). Sonicated samples were centrifuged at 20,000g for clarification, and the supernatant was diluted to <0.1% SDS and then incubated with antibodies (H3K9ac, Active Motif catalog number 39137, RRID:AB_2561017; H3K4me3, Abcam catalog number ab8580, RRID:AB_306649; BRPF1, Thermo Fisher Scientific catalog number PA5-27783, RRID:AB_2545259; KAT6A, Cell Signaling Technology catalog number 78462; HA, Cell Signaling Technology catalog number 3724, RRID:AB_1549585; DOT1L, Cell Signaling Technology catalog number 77087, RRID:AB_2799889; H3K79me2, Cell Signaling Technology catalog number 3724, RRID:AB_1549585; The antibody was incubated overnight with Dynabeads Protein A (Life Technologies catalog number 10002D) prebound with IgG (Life Technologies catalog number 5427, RRID:AB_10693787). Samples were washed successively with buffer A (150 mM NaCl, 5 mM EDTA, 5% sucrose, 1% Triton X-100, 0.2% SDS, 20 mM Tris), buffer B (5 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 20 mM Tris), buffer C (250 mM LiCl, 1 mM EDTA, 0.5% NP40, 0.5% deoxycholate, 10 mM Tris) and TE, after which the beads were resuspended in elution buffer (200 mM NaCl, 100 mM NaHCO3, 1% SDS) and incubated at 65 °C to reverse cross-linking for 12-15 h.DNA was purified using AMPureXP beads (Beckman Coulter catalog no. A63881) according to the manufacturer's recommendations and quality was assessed by Qubit dsDNA HS Assay Kit (Life Technologies catalog no. Q32854) and TapeStation 4200 (Agilent). Sequencing libraries were prepared using ThruPLEX DNA-seq kit (Takara Bio catalog no. R400675) and sequenced on a NextSeq 500 or 550 system (Illumina). ChIP-seq spike-in normalization was performed by pre-binding spike-in antibodies (Active Motif catalog no. 61686) to Dynabeads together with the IP antibody of interest. Equal amounts of Drosophila melanogaster chromatin (Active Motif catalog no. 53083) were added to the prepared GIST cell chromatin according to the manufacturer's recommendations. The resulting sequenced samples were aligned against the Drosophila genome and total Drosophila read counts were used to normalize Homo sapiens read counts across samples.

[0095] Cut&TAG was performed as previously described (Kaya-Okur et al., Nat. Commun. 10(1):1930 (2019)) using Protein A and Tn5 transposase fusion protein (Addgene#124601) for amplification. Briefly, 100,000 GIST-T1 cells were washed in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail) and bound to Concanavalin A beads (Bangs Laboratories catalog number BP531) for 15 min at room temperature. Bound cells were resuspended in 50 μL Dig-Wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail, 2 mM EDTA, 0.05% digitonin) and incubated with antibodies (Menin, Bethyl Cat. No. A300-105A, RRID:AB_2143306; MLL1n, Bethyl Cat. No. A300-086A, RRID:AB_242510) diluted 1:100 overnight at 4° C. Beads were collected using a magnet and cells were resuspended in 100 μL Dig-Wash buffer containing secondary antibodies diluted 1:100 and incubated at room temperature for 30 min. Cells were washed three times with Dig-Wash buffer, resuspended in Dig-Med buffer (0.05% digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail) containing 1:250 pA-Tn5 transposase and incubated for 1 hour at room temperature. Cells were washed three times in Dig-Med buffer, resuspended in 300 μL tagmentation buffer (10 mM MgCl2 in Dig-Med buffer) and incubated for 1 hour at 37°C. Tagmentation was stopped by adding 10 μL 0.5 M EDTA, 3 μL 10% SDS and 2.5 μL 20 mg / mL proteinase K (Invitrogen catalog number 25530049) and samples were incubated for 1 hour at 50°C. The tagmented DNA was purified by phenol:cholorphorm:isoamyl alcohol extraction, the aqueous layer was subjected to ethanol precipitation, and the DNA was resuspended in 30 μL of TE.For each sample, 21 μL of DNA was mixed with universal i5 and uniquely barcoded i7 primers and amplified using NEBNext High Fidelity 2× PCR Master Mix (New England Biolabs Catalog No. M0541S) in a thermocycler using the following conditions: 98° C. for 30 seconds; 14 cycles of 98° C. for 10 seconds, 63° C. for 10 seconds; 72° C. for 2 minutes. DNA was purified with AMPureXP beads according to the manufacturer's recommendations and quality was assessed by Qubit dsDNA HS Assay Kit and TapeStation 4200. Samples were sequenced on a NextSeq 550 System (Illumina).

[0096] All sequencing data were aligned to the human reference genome assembly hg19 using Bowtie2 (Langmead et al., Genome Biol. 10:R25.1-R25.10(2009)). Normalized read density was calculated using the Bamliquidator (version 1.0) read density calculator. Aligned reads were extended by 200 bp and the density of reads per base pair was calculated. For each region, the density of reads was normalized to the total number of mapped reads per million and read density was generated in units of reads per million mapped reads per bp (rpm / bp). Peak discovery was performed by model-based analysis of ChIP-seq (MACS, version 1.4.2, Fenge et al., Nature Protocols 7:1728-40 (2012)), and signal enriched regions were identified using ROSE2 (Lovenet al., Cell 153:320-34 (2013)). Individual ChIP-seq track displays were generated using bamplot (github.com / linlabbcm). Heatmap visualization of ChIP-seq data was generated using ChAsE (Younesy Barton et al., Bioinformatics 32:3324-6 (2016)).

[0097] Immunoblotting. Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche Cat. No. 11873580001) and centrifuged at 14,000 g for 10 min to remove genomic DNA and debris. Protein concentrations were determined using a bicinchoninic acid-based assay (Pierce Biotechnology Cat. No. 23225). Protein samples were subjected to SDS-PAGE and Western blotting with the following antibodies: HA (1:1,000, Cell Signaling Technology Catalog No. 2367, RRID:AB_10691311), MEAF6 (1:500, Proteintech Catalog No. 26465-1-AP, RRID:AB_2880524), actin (1:1,000, Cell Signaling Technology Catalog No. 4967, RRID:AB_330288), menin (1:10,000, Bethyl Catalog No. A300-105A, RRID:AB_2143306), or streptavidin-HRP (1:40,000, Abcam Catalog No. ab7403). Western blots were probed with anti-mouse or anti-rabbit secondary antibodies and detected using an Odyssey CLx infrared imaging system (LI-COR Biosciences) or using streptavidin-HRP by chemiluminescence (MilliporeSigma catalog no. WBKLS0500). Immunoblots shown are representative of at least three independent experiments.

[0098] Mass spectrometry and BioID. GIST-T1 cell lines stably expressing control or experimental mutant biotin ligase (BirA*R118G) tagged fusion proteins were generated. 24 h biotin-labeled whole cell lysates were subjected to affinity pull-down overnight at 4 °C using streptavidin-sepharose beads (GE Healthcare catalogue no. 17-5113-01). Beads were washed three times in 2% SDS in 50 mM Tris, two times in BioID buffer (50 mM Tris, 500 mM NaCl, 0.4% SDS), six times in 50 mM Tris, and resuspended in 100 μL ammonium bicarbonate. Samples were subjected to trypsin digestion, and beads and salts were removed in a reverse-phase clean-up step. Extracts were dried in a speed-vac and then reconstituted in 5–10 μl of 2.5% acetonitrile and 0.1% formic acid. Nanoscale reversed-phase HPLC capillary columns were made by packing 2.6 μm C18 spherical silica beads into a fused silica capillary (100 μm inner diameter × approx. 30 cm length) with a frame-stretched tip. After equilibrating the column, each sample was loaded via a Famos Auto Sampler (LC Packings). A gradient was formed and peptides were eluted with increasing concentrations of 97.5% acetonitrile and 0.1% formic acid. Once the peptides eluted, they were subjected to electrospray ionization and then entered into an LTQ Orbitrap Velos Pro ion trap mass spectrometer (Thermo Fisher Scientific). Peptides were detected, isolated, and fragmented to generate tandem mass spectra of specific fragment ions for each peptide. Peptide sequences (and therefore protein identities) were determined by matching protein databases with fragmentation patterns acquired by Sequest (Thermo Fisher Scientific). All databases contained reversed versions of all sequences, and the data were filtered to a peptide false discovery rate between 1% and 2%. For quantitative comparison, label-free quantification of signal intensity was used in replicate samples. Log of signal compared to DMSO 2Heatmaps of fold changes were generated using Morpheus (software.broadinstitute.org / morpheus / ).

[0099] Xenograft model. PG27 patient-derived xenografts were obtained from patients who underwent clinically indicated surgery and followed written informed consent to a Dana-Farber Cancer Institute IRB-approved research protocol. Cryopreserved tumors or GIST-T1 cell lines mixed 1:1 with Matrigel were implanted subcutaneously into 6-week-old female nude mice (NU / NU; Charles River Laboratories). GIST-T1 tested negative for mycoplasma and rodent pathogens (Charles River Laboratories). For in vivo assessment of growth of CRISPR / Cas9-modified cell lines, GIST-T1 / Cas9 left and right flanks were treated with the indicated sgRNAs for 14 days prior to cell implantation and selected with puromycin in vitro. For drug treatment studies, tumor volumes were measured by caliper and calculated according to the formula: volume = major axis. 2 The tumor is approximately 100-200 mm in diameter, as determined by x minor axis x 0.5. 3Single engrafted mice were enrolled in treatment groups when they reached a tumor size of 1000 μg / mL. Mice were randomly assigned to treatment groups receiving imatinib (50 mg / kg daily gavage, 5 days per week), WM-1119 (50 mg / kg three times daily gavage, 7 days per week), VTP-50469 (0.1% in chow) or combination treatment. Imatinib was administered at less than the maximum tolerated dose to facilitate testing of combination therapy. No statistical methods were used to predetermine sample size, and no animals died during drug treatment. Two GIST-T1 cell line xenograft mice in the control group were excluded from analysis because the subcutaneous grafts measured initially did not grow. One outlier tumor-bearing mouse in the VTP-50469 arm in Figure 7D was excluded due to early termination from rapid tumor growth. Tumors were dissected and fixed in 10% formalin for the resulting studies, including H&E staining and immunohistochemistry of sectioned tumors. Four-μm sections were cut from fixed and embedded tumors and stained with Ki-67 (1:400, Cell Signaling Technology Catalog No. 9027) and cleaved caspase-3 (1:250, Cell Signaling Technology Catalog No. 9579). Reactions were developed using DAB (Cell Signaling Technology Catalog No. 8059) or NovaRed (Vector Laboratories Catalog No. SK-4800) substrate kits according to the manufacturers' recommendations. All procedures were performed under protocols approved by the Institutional Animal Care and Use Committee at Dana-Farber Cancer Institute.

[0100] Statistical Analysis. Median, error bars, P-value cutoff, number of replicates and statistical tests are specified in the corresponding figure legends. For box plots, the box extends from the 25th to the 75th percentile, the center line indicates the median, and whiskers are drawn to the 10th and 90th percentiles. Sample sizes were not predetermined.

[0101] Availability of data and materials. De novo sequencing data are available through GEO publication reference number GSE172154. Additional RNA-seq, ATAC-seq and ChIP-seq datasets analyzed in this study include GSE95864 (HemMing et al., Proc. Natl. Acad. Sci. USA 115(25):E5746-E5755 (2018)), GSE113207 and GSE113217 (HemMing et al., Cancer Res. 79(5):994-1009(2019)).

[0102] Example 2: Genome-wide screening identifies GIST epigenetic dependencies. The global transcription and enhancer landscape of GISTs was characterized using RNA-seq, chromatin immunoprecipitation with sequencing (ChIP-seq), and assay for transposase-accessible chromatin using sequencing (ATAC-seq). These studies focused on TFs related to GIST biology, including core TFs such as ETV1, FOXF1, HIC1, and OSR1 present across GIST samples, as well as auxiliary TFs BARX1 and HAND1, which are expressed in disease state-specific patterns (HemMing et al., Cancer Res. 79(5):994-1009(2019)). However, it was unknown how these TFs integrate with other epigenetic regulators to establish GIST-associated gene expression programs. A genome-wide CRISPR / Cas9-based dropout screen in two KIT mutant GIST cell lines, GIST-T1 and GIST430, was performed to establish which genes are essential in GIST biology. A split library approach utilizing paired human genome-wide sgRNA libraries (denoted H1 and H2) was used, with approximately 5 sgRNAs per gene in each library targeting 18,436 genes with a total of 185,634 sgRNAs in the screen. Significant correlations of dependency (β) scores were observed between the H1 and H2 libraries, as shown in Figure 1A, and between the two GIST cell lines, as shown in Figure 1B (n=4 libraries per cell line). The data sets were then merged for subsequent analysis (n=4 per library) to improve statistical power. P values ​​and r as shown in Figures 1A and 1B. 2Pearson correlation was performed using the . Genes were stratified as "pan-essential", previously determined to be universally essential for cell survival (Blomen et al., Science 350:1092-6 (2015), Wang et al., Science 350:1096-101 (2015)), "GIST essential", or "non-essential", with FDR<0.05 in the screen but absent from the pan-essential list (see Figure 1C). Pan-essential genes (Blomen et al., Science 350:1092-6(2015)) are shown in blue, genes significantly depleted in GIST but not pan-essential are shown in red, and non-essential genes lacking significant depletion are shown in grey. Selected GIST-associated genes are labeled.

[0103] As expected, KIT was one of the strongest detected dependencies, and as shown in Figure 1D, sgRNA-level data showed a near-complete dropout of most (9 / 10) sgRNAs during the screen (n=2 per library). Other canonical downstream signaling mediators of the KIT pathway, such as mTOR, showed significant dropout in the screen, but less so compared to KIT (see Figure 1E (n=4)). As shown in Figure 1F, we focused on the "GIST-essential" subset of genes among pan-essential (n=1,702) and non-pan-essential (n=16,757) to identify biological processes that may be specifically enriched for GIST. Conditions in Figure 1D, 1E, and 1F were compared by t-test (compared to non-essential genes or baseline sgRNAs; **, P<0.01; ***, P<0.001). These intrinsic dependencies in GISTs were assessed by gene ontology enrichment analysis, revealing that 8 of the top 18 terms are associated with epigenetic control mechanisms, including chromatin and chromosome organization (see Figures 1G and 8A). Collectively, data from these unbiased dependency screens characterize GISTs as prominently dependent on epigenetic mechanisms to maintain their oncogenic programs (Tabone et al. Biophys. Acta. 1741(1-2):165-72(2005)).

[0104] To better define which chromatin regulatory complexes may be most relevant and unique to GIST biology, the β scores for all chromatin modifying enzymes in GIST cell lines were compared to similar CERES dependency scores averaged across all cell lines in the DepMap project (Barretina et al., Nature 483:603-7(2012)). Only 7 of the 77 assessed chromatin modifying enzymes were unique and essential for GIST, with β scores <-0.7 and CERES scores >-0.25 (Figure 2A and Figure 8N), with dependency score cutoffs and interpersonal correlation coefficient labels chosen to select chromatin regulators likely to be uniquely dependent. Enriched enzymes included members of the lysine acetyltransferase (KAT), MYST, lysine demethylase (KDM), and lysine methyltransferase (KMT) families. The dotted lines divide the plot into quadrants, with the upper quadrant containing the seven genes that were GIST dependent but not common dependent across the DepMap cell lines. To establish which modifying enzymes may function collaboratively to maintain the epigenome, gene-level codependency data was analyzed within DepMap. Comparative analysis of the top 50 codependencies of each chromatin modifying enzyme showed the highest interactions at the gene and ontology term level between KMT2A, EZH2, and KAT6A, as shown in Figure 2B, suggesting their genetic codependency. In Figure 2B, red lines connect genes shared on multiple codependency lists. Blue lines connect genes that fall into the same ontology term. KMT2A, a catalytic member of the menin-MLL complex, also had multiple recognized complex members with significant dependency scores, including MEN1 / menin and ASH2L, as shown in Figure 8B. Because the DepMap CRISPR-dependent screening effort has not profiled GIST, we took advantage of comparative screening results available from Project DRIVE (McDonald et al., Cell 170:577-592 (2017)), which included GIST-T1 among nearly 400 cell lines profiled by RNAi.Among all cell lines profiled, KMT2A and ASH2L were in the top 5% highest sensitivity with GIST-T1, as shown in Figures 2C-2F, further demonstrating the essential and co-dependent nature of MOZ and menin-MLL complexes in GISTs through an independent comparative screening approach. As shown in Figures 8C-8M, several other chromatin regulatory complexes were found to have multiple members with significant dependency in the screen and also showed enrichment of GIST-T1 in project-driven, including members of the INO80 complex, NuA4 histone acetyltransferase complex, FACT complex, and PAF1 complex. Figures 8I-8J show ranked sensitivity scores from project-driven cell lines (n=387) for members of the FACT complex, with GIST-T1 highlighted in red. Figures 8L-8M show ranked sensitivity scores from project-driven cell lines (n=387) for select members of the PAF1 complex, with GIST-T1 highlighted in red. EZH2, SUZ12 and EED, core members of the PRC2 complex (Laugesen et al., Cold Spring Harb. Perspect. Med. 6(9):a026575(2016)), were all dependent in the screen, with GIST-T1 having the highest sensitivity scores for EZH2 and EED in Project DRIVE, as shown in Figures 9A-9C. Figures 9B-9C show ranked sensitivity scores from Project Driven cell lines (n=387) for select members of the PRC2 complex, with GIST-T1 highlighted in red. Although a few cell lines in the DepMap had significant dependence on core PRC2 complex members, the epistatic codependency of EZH2 with DOT1L, EP300 and MEN1 showed overlap with MOZ and menin-MLL complex codependency, as shown in Figure 9E, indicating complementary functions of the transcriptionally repressive PRC2 complex.

[0105] To validate the dependency on menin-MLL complex members, a time course proliferation assay was performed utilizing unique sgRNAs targeting menin-MLL complex members KMT2A and MEN1. For each gene, using two independent sgRNAs per gene, sgRNA treatment significantly reduced cell proliferation as shown in Figure 2E, where sgRNAs targeting Luc and RPS19 are indicated by open boxes and circles, respectively (n=3 per sgRNA). The relative toxicity of these sgRNAs was compared to a control cell line using GIST48B. GIST48B has a similar proliferation rate to GIST-T1, but loses KIT expression and GIST-associated epigenetic and transcriptional programs through in vitro selection (Hemming et al., Proc. Natl. Acad. Sci. USA 115(25):E5746-E5755 (2018), Hemming et al., Cancer Res. 79(5):994-1009 (2019)). All sgRNAs targeting the menin-MLL complex significantly reduce GIST-T1 cell proliferation, whereas GIST48B shows little or no change in cell proliferation, as shown in Figure 2F, where n=6 per gene from two sgRNAs. Data were analyzed by two-way ANOVA with Tukey's multiple comparison test compared to GIST48B in the same treatment condition; ***, P<0.001; **, P<0.01. Collectively, these data demonstrate a dependency on the menin-MLL complex in maintaining the GIST epigenome, both in GIST and across selected cell lines in the DepMap.

[0106] ChIP-seq of histone marks H3K4me3, BRPF1, and KATA6 was performed to define where in the GIST genome the menin-MLL complex binds and acetylates histones. Genomic regions of menin and MLL1 binding were identified using a similar method CUT&Tag (Kayo-Okur et al., Nat. ComMun. 10(1):1930 (2019)). Menin-MLL complex members were found to localize to the transcription start sequences (TSS) of active genes as determined by their co-occupancy with H3K27ac and H3K9ac (see top row in Figure 3A). In contrast, little occupancy of these chromatin complex members at H3K27ac-defined enhancers was observed (see middle row in Figure 3A). ATAC peaks containing DNA sites accessible at both TSSs and enhancers showed intermediate levels of menin-MLL complex binding (see bottom row in Figure 3A). In Figure 3A, rows show scaled read density ±10 kb from TSSs, H3K27ac-defined super-enhancers or ATAC-defined peaks.

[0107] We next analyzed genomic regions that showed strong enrichment for menin in the ChIP-seq and CUT&Tag datasets and reasoned that these factors represent the menin-MLL complex. Although this protein binds to thousands of sites throughout the genome, disproportionate enrichment was seen in 3-5% of these genomic regions, many of which have clear association with GISTs, as shown in Figures 3E and 10A. The percent of all enriched regions associated with TF genes and the top quartile percent are shown in Figures 3E and 3F. Transcription factor sites (TFs), particularly those within the group of core and auxiliary GIST TFs (Hemming et al., Proc. Natl. Acad. Sci. USA 115(25):E5746-E5755(2018), Hemming et al., Cancer Res. 79(5):994-1009(2019)), were included in these enriched regions, as were negative regulators of KIT signaling from the DUSP and sprouty families, and genes used as biomarkers for GIST (e.g., GPR20, CD34 (Corless et al., J. Clin. Oncol. 22(18):3813-25(2004)). ChIP-seq and CUT&Tag tracks show binding of menin-MLL complex members at the TSS and gene body of these enriched genes similar to H3K4me3, such as the core TF member DUSP6 and the essential gene USP1, as shown in Figures 3F-3H and 10B; in contrast, H3K27ac and H3K9ac are enriched in both enhancer regions and gene bodies, and the GIST accessory TF HAND1 binds exclusively to enhancers. Menin-MLL complex members were not significantly enriched at the KIT locus, but there was evidence that these regulators bind to regions downstream of the TSS and gene body, see Figure 10C. Maximum binding of menin-MLL complex members was seen within and immediately downstream of the TSS of other enriched regions, with detectable signal evident at the enhancers of several of these highly regulated genes (see Figures 10D-10H).These data indicate that the menin-MLL complex is globally present at active genes, with enrichment at a subset of genes associated with the GIST transcription program.

[0108] Example 3: Menin inhibition disrupts GIST cell proliferation without apoptosis. Based on the genetic data and genomic colocalization of the menin-MLL complex, it was reasoned that small molecule inhibitors targeting the GIST junction complex would be a viable therapeutic approach. To investigate the functional consequences of menin-MLL disruption, GIST-T1 cells were treated with the menin inhibitor VTP-50469 (Krivtsov et al., Cancer Cell 36:660-673(2019)) alone or in combination with the selective KAT6A inhibitor WM-1119 (Baell et al., Nature 560(7717):253-257(2018)). As shown in Figure 4A-4B, at submicromolar concentrations, VTP-50469 reduced GIST cell proliferation in a time-course proliferation assay, with a greater effect seen with the combination of the two drugs (Figure 4B). Demonstrating the selective toxicity of these inhibitors in KIT-dependent GIST cell lines, the KIT-independent GIST48B cell line showed moderate or no changes in proliferation after 21 days of drug treatment (Figure 4C), consistent with the genetic data from the CRISPR experiments (Figure 2I). Previous studies of the KIT enhancer used sgRNAs directed at the KIT TSS to ablate endogenous KIT expression while simultaneously rescuing cell viability by expressing a viral promoter-driven KIT construct with the same activating mutation (Hemming et al., Cancer Res. 79(5):994-1009(2019)). This KIT-dependent KIT-rescued cell line was similarly sensitive to VTP-50469 alone or in combination with WM-1119, as shown in Figure 4C, indicating that regulation of the endogenous KIT locus is not the primary mechanism of toxicity of these compounds. A statistical comparison to the reference GIST48B under identical treatment is shown in Figure 4C. To confirm the proliferative effects of these inhibitors in additional GIST cell lines, the slower growing KIT mutant cell lines GIST430 (HemMing et al., Cancer Res. 79(5):994-1009(2019)) and GIST882 were treated with VTP-50469 alone or in combination with WM-1119 and observed similar antiproliferative effects resulting from drug treatment, as shown in Figures 4D and 11A.Data in FIG. 11 were analyzed by one-way ANOVA with Dunnett's multiple comparison test; ***, P<0.001; **, P<0.01; *, P<0.05 compared to DMSO control.

[0109] To evaluate the cellular phenotypic consequences of VTP-50469 treatment, cell cycle and apoptosis assays were performed utilizing VTP-50469 and the TKI imatinib as a comparator. Imatinib acutely and potently caused G0 / G1 phase cell cycle arrest within 72 h, whereas 8 days of treatment with VTP-50469 resulted in a moderate increase in the fraction of cells in G0 / G1 phase, as shown in Figure 4E; the combination of VTP-50469 with WM-1119 resulted in a more pronounced disruption of the cell cycle after 8 days of treatment (Figure 4E). Imatinib treatment for 72 h produced a significant increase in early and late apoptosis and cell death, whereas 8 days of treatment with VTP-50469 alone or in combination with WM-1119 did not significantly increase apoptosis or cell death compared to DMSO controls, as shown in Figure 4F (where n=3-5 per condition). Data were analyzed by two-way or one-way ANOVA with Tukey's post-hoc test, where appropriate, compared with DMSO or the indicated condition; ***, P<0.001; **, P<0.01. Collectively, these data indicate that the menin-MLL complex is targetable, exhibits unique vulnerability in GISTs, and, consistent with its distribution across the genome at the TSS of active genes, plays a more important role in gene regulation than simply KIT gene expression. Disruption of this complex, alone or in combination with disruption of the MOZ complex, causes alterations in the cell cycle but not programmed cell death.

[0110] Example 4: Menin-MLL inhibition induces global gene expression changes. Observing genome-wide occupancy of menin-MLL complexes with select enriched regions, we next probed for selective changes in gene expression resulting from VTP-50469, WM-1119, and combined VTP-50469 and WM-1119 treatment to further delineate the growth inhibitory phenotype resulting from their targeted disruption (Figure 5A, with Person correlations shown). RNA-seq was performed on GIST-T1 cells treated with VTP-50469 or WM-1119 for 1 and 5 days and compared to DMSO treatment as a control. VTP-50469 and WM-1119 treatments modestly altered the expression of numerous genes (n=5,095 with FPKM>10), with significant correlation of gene expression changes between these inhibitors, with VTP-50469 and combined groups showing the greatest deviation from control (Figure 5A and Figure 5I). Figure 5I shows unsupervised hierarchical clustering of RNA-seq data comparing 5-day treatment of GIST-T1 with 0.5 μmol / L VTP-50469, 1 μmol / L WM-1119, or combinations of 0.1 μmol / L of each drug (n=4 per condition) for all expressed genes (>10 fragments per kilobase of transcript per million mapped reads (FPKM), n=7,106).

[0111] We also assessed gene expression changes following genetic disruption of MOZ and the menin-MLL complex using sgRNAs targeting the two complex members, respectively. Using this genetic system, sgRNAs targeting MEN1 led to global changes in gene expression, with disruption of the other MOZ and menin-MLL1 complex members showing less dramatic changes (Figure 5J). This shows unsupervised hierarchical clustering of RNA-seq data comparing GIST-T1 / Cas9 cells transduced with sgRNAs targeting KAT6A, BRPF1, KMT2A, MEN1, or luciferase as a control (n=3 per condition) and collected on day 5.

[0112] To integrate and compare transcriptional changes resulting from either pharmacological or genetic disruptions, we assessed correlation of gene expression changes from controls across the transcriptome. For comparison, we also assessed transcriptional changes from sgRNA-mediated disruption of the GIST TFs HAND1 and ETV1. sgRNAs targeting the MOZ complex members KAT6A and BRPF1 showed the highest degree of correlation and further induced similar global transcriptional changes as did disruption of HAND1, ETV1, or KMT2A (Figure 5K-Figure 5S). Pharmacological inhibition of MOZ and / or menin-MLL complex had relatively weak but positive correlations with sgRNAs targeting GIST TFs, MOZ complex members, and KMT2A, whereas genetic disruption of MEN1 showed the least correlation with other conditions (Figure 5K and Figure 5N). Figure 5K shows a Pearson correlation of the control normalized RNA-seq data from Figure 5I and Figure 5J, including control normalized RNA-seq data from GIST-T1 / Cas9 cells transduced with sgRNAs targeting HAND1 and ETV1. These data demonstrate the disparate global transcriptional outcomes resulting from genetic or chemical disruption of these epigenetic regulators.

[0113] Gene set enrichment analysis (GSEA) was used to assess the expression of genes enriched for MOZ and the menin-MLL complex, H3K27ac-defined super-enhancer (SE)-associated genes, and genes regulated by the TFs HAND1 and ETV1. Among these GIST-associated gene lists, genes regulated by TFs were most significantly affected, with drug or sgRNA treatment causing a decrease in expression of genes upregulated by HAND1 and an increase in expression of genes normally downregulated by HAND1 or ETV1 function (Figure 5O), which shows normalized enrichment scores (NES) from a GSEA gene set that includes genes showing enrichment for menin and BRPF1 (n=385), GIST-T1 SE-associated genes (n=366) defined by H3K27ac, genes upregulated (n=421) or downregulated (n=165) by HAND1, and genes upregulated (n=438) or downregulated (n=31) by ETV1.

[0114] Only gene sets with significant FDR are displayed using a color scale, those with non-significant FDR are shown in grey. Genetic or pharmacological MOZ disruption showed the greatest effect on genes bound by menin or BRPF1 (Figure 5H), whereas only targeting HAND1 or a combination of WM-1119 and VTP-50469 reduced expression of SE-associated genes (Figure 5Q). However, common to all conditions is the disruption of expression of HAND1-associated genes, and expression values ​​of disruption of MOZ and menin-MLL complex using either inhibitors or sgRNAs directly phenocopy HAND1 knockout (Figure 5R-T).

[0115] KIT gene expression was most prominently affected by pharmacological or genetic disruption of menin, although there was a common loss of DUSP6, a negative regulator of KIT signaling, and the GIST biomarker CD34 (Figure 5U). Expression of several core GIST TFs was altered by genetic or pharmacological MOZ or menin-MLL complex disruption, most prominently FOXF1, HAND2, and PITX1, with WM-1119 exerting the greatest overall reduction in TF expression (Figure 5V, Figures S16A-S16B). Several other genes highly regulated by HAND1 expression, including NPR3, ITGA4, and RASL11A, showed a similar loss of expression with disruption of MOZ and the menin-MLL complex (Figure 5W). Comparable decreases in DUSP6 and NPR3 gene expression were seen by qRT-PCR in the KIT-dependent GIST cell lines GIST430, GIST882, and GIST48 (Figures 16C-F).

[0116] Among all Reactome gene sets, processes related to protein translation were the most recurrently altered gene sets across treatment conditions, with most drug and sgRNA datasets showing reduced gene expression (Figures S16G-S16H). Collectively, these results indicate that both genetic and pharmacological means of MOZ and menin-MLL complex disruption result in selective alterations of transcriptional programs associated with GIST TFs and most notably HAND1. Furthermore, dual inhibition of menin and MOZ by small molecules induces complementary effects on global gene expression, decreasing the expression of GIST SE-associated genes.

[0117] GSEA (Subramanian et al., Proc. Natl. Acad. Sci. USA 102:15545-50(2005)) was used to explore pathway changes associated with drug treatment. VTP-50469 treatment resulted in similar changes in the Hallmark gene set, with significant upregulation of gene sets associated with myogenesis and epithelial-mesenchymal transition (EMT); drug treatment also resulted in decreased expression of gene sets associated with cell cycle and mitogenic signaling, as shown in Figures 5B and 5C, with MTORC1 signaling, G2M checkpoint, myogenesis and EMT gene sets shown in the figures for each condition. There was minimal change with drug treatment to the overall mean of gene expression, but GIST-related gene sets showed significant changes. Genes identified as essential for GIST (see Figures 1C and 12A) and downregulated after 6 hours of imatinib treatment (Hemming et al., Cancer Res. 79(5):994-1009(2019)) showed a corresponding decrease in expression resulting from VTP-50469 treatment and had a Hallmark EMT signature showing upregulation in comparison, as shown in Figure 5D (all expressed genes n=5,093, essential genes n=1,507, genes downregulated >2.5-fold by 6 hours of imatinib treatment n=544, and Hallmark EMT genes n=63). Data were analyzed by one-way ANOVA with Dunnett's multiple comparison test compared to DMSO; ***, P<0.001; **, P<0.01; *, P<0.05). Figure 12A shows the ratio of expression between inhibitor and DMSO treatment for the top 500 essential genes after 5 days of inhibitor treatment, with Pearson correlation shown. As shown in Figure 5E, genes with disproportionately high menin loading (n=294) showed a larger decrease in gene expression after inhibitor treatment at both days 1 and 5 (compared to genes lacking enrichment, n=4,799).As shown in Figures 12B-12C, among the DUSP and sprouty family members highly expressed in GIST, DUSP6 was most significantly decreased, whereas KIT expression only showed a trend toward decreased expression by VTP-50469 by day 5. These decreases were confirmed in select transcripts DUSP6, NPR3 and USP1 in GIST-T1 and GIST430 resulting from VTP-50469 alone or in combination with WM-1119 using qRT-PCR, as shown in Figures 5G-5H, where it is notable that the combination treatment does not show a strong additive effect on the gene expression of these targets. For Figures 5G-5H and 12B-12C, n=3-4 per group and data were analyzed by one-way ANOVA with Dunnett's multiple comparison test compared to DMSO; ***, P<0.001; **, P<0.01; *, P<0.05. Together with chromatin studies, these data highlight pathway-selective changes in gene expression associated with pharmacological menin inhibition (alone or in combination with MOZ inhibition) and identify changes in genes related to cell cycle, viability and trophic KIT signaling, potentially altering the differentiation state by activating the mesenchymal developmental program.

[0118] Example 5: Disruption of chromatin and transcriptional regulatory protein interactions by menin inhibition. Because MOZ and the menin-MLL complex function cooperatively at highly regulated genomic regions along with other chromatin regulators, the effect on local protein interactions was assessed in the presence or absence of VTP-50469 alone or in combination with WM-1119. Using the BioID system (Lambert et al., J. Proteomics 118:81-94(2015)), we added the biotin ligase BirA* to the N-terminus of the MOZ complex member MEAF6, which allows for the covalent labeling of proteins localized within 10 nm with a biotin moiety. To ensure proper incorporation of BirA*-tagged MEAF6 into the MOZ complex, we used CRISPR / Cas9 and MEAF6-targeting sgRNA to disrupt endogenous MEAF6, which is otherwise lethal if not functionally replaced by a stably expressed MEAF6-BirA construct (Figure 2E). Stable expression of N-terminally tagged MEAF6-BirA in GIST-T1 resulted in high levels of protein production, with evidence of N-terminal degradation products including MEAF6 and HA observed by Western blot, indicating endogenous and full-length rescue constructs, or actin as a loading control (Figure 6A). A construct in which BirA* was fused to the DNA-binding domain of IKZF1 (which retained its nuclear localization signal) was used as a nuclear background BioID control. After labeling cells for 24 h with biotin, streptavidin pulldown followed by mass spectrometry identified 243 proteins that were labeled by MEAF6-BirA and enriched over the control (Figure 6B, Table 4). In Figure 6B, MEAF6-enriched proteins shown in blue show >2-fold intensity enrichment compared to the background control (n=243). Selected interactors are labeled. Labeled interactors included chromatin regulatory proteins such as KMT2A / MLL1, KMT2B / MLL2, JADE3, and RUVBL1, in addition to MOZ complex members, enhancer-associated proteins such as BRD4, and the core GIST TF HIC1.These MOZ-proximal proteins were ordered by gene ontology and enriched for cellular processes including DNA repair, mRNA processing and chromatin complex regulation (Figure 6C). These data demonstrate the integrated cellular functions of these transcriptional regulatory proteins and their complex interactions among splicing factors, enhancers and chromatin complexes.

[0119] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0120] To assess changes in the MEAF6 proximal proteome as a result of menin inhibition (alone or in combination with MOZ inhibition), MEAF6-BirA-expressing GIST-T1 cells were pretreated for 3 days with VTP-50469 alone or in combination with WM-1119, then labeled with biotin followed by label-free quantification using mass spectrometry. While the majority of MEAF6 proximal proteins remained the same, a subset of proteins showed significant changes in abundance with drug treatment, significantly correlating with the changes seen with VTP-50469 and WM-1119 (Figure 6D). MLL family members KMT2A / MLL1, KMT2B / MLL2 and the DNA- and RNA-binding anti-apoptotic protein GPATCH4 (Lambert et al., J. Proteomics 118:81-94 (2015)) interactions were decreased by VTP-50469 treatment alone, and combined treatment with VTP-50469 and WM-1119 had no further effect (Figure 6F). Proximity of selected chromatin regulators, splicing factors and polymerase regulator proteins was similarly altered by VTP-50469 alone or in combination with WM-1119, most notably DOT1L was significantly decreased in all treatment conditions (Figure 6G).

[0121] To determine how menin inhibition altered chromatin association of DOT1L, spike-in normalized ChIP-seq in GIST-T1 cells treated with VTP-50469 alone or in combination, which significantly reduced DOT1L association with chromatin at all DOT1L binding sites and reduced genome-wide average DOT1L signal, as shown in Figures 6G-6I and Figures 13G-13I. Figure 6G has a heatmap showing the spike-in normalized signal of DOT1L at MACS-defined peaks (n=67,769) in GIST-T1 cells treated for 3 days with DMSO, VTP-50469. Scaled read densities ±1.25 kb from the peak center are shown in the rows. Figures 6I-6J have an n of 67,769 for DOT1L signal and an n of 22,581 for MEAF6 signal. ***, P<0.001;**, P<0.01;*, P<0.05.

[0122] Similar to other menin-MLL complex members, DOT1L, H3K79me2, a histone mark deposited by DOT1L, and MEAF6 all showed genome-wide enrichment at the TSS and gene body of active genes, with enrichment at loci relevant for GIST biology and reduction of DOT1L signal upon VTP-50469 treatment, as shown in Figures 6K and 13B-13E. Figure 13D shows heatmaps demonstrating genomic localization of DOT1L and H3K79me2 in GIST-T1 by ChIP-seq. Scaled read density ±10 kb from TSS, H3K27ac-defined super-enhancers or ATAC-defined peaks is shown in the rows. Figure 13E includes tracks showing regions of genomic occupancy of spike-in normalized DOT1L under the indicated treatments, H3K79me2, and H3K27ac at the GPR20 locus. DOT1L TE vs SE. SE=1343, TE=45256. Welch's T-test P<0.001 but absolute difference ∼1%. To further investigate whether loss of DOT1L function may constitute a mechanism of cytotoxicity downstream of menin inhibition, GIST-T1 cells or GIST48B as a control were treated with various doses of the selective DOT1L inhibitor EPZ-5676 in a time-course proliferation assay (Daigle et al., Cancer Cell 20:53-65(2011)). At all doses tested, GIST-T1 showed significantly reduced cell proliferation compared to DMSO control or GIST48B (5 per condition), indicating selective toxicity of DOT1L inhibition similar to menin inhibition, as shown in Figure 6L.

[0123] DOT1L-targeting sgRNAs resulted in a significant reduction in GIST-T1 cell proliferation, but more moderate than that seen with menin-MLL and MOZ complex-targeting sgRNAs, consistent with findings from genome-wide CRISPR screening (Figure 2A and Figure 13I). To better characterize the transcriptional consequences of DOT1L inhibition in GIST, GIST-T1 cells were treated with EPZ-5676 for 5 days followed by RNA-seq. Overall, transcriptional changes associated with EPZ-5676 treatment were highly correlated with those resulting from menin inhibition with VTP-50469 (Figure 6M-6N), a phenomenon previously observed in MLL-rearranged leukemias. Similar to menin-MLL and MOZ inhibition, DOT1L inhibition resulted in significant disruption of the HAND1-regulated transcriptional program (Figure 6O), accompanied by altered expression of KIT, CD34, NPR3, and GIST TFs (Figure 6P-6Q). Taken together, these data demonstrate the complexity of proximal protein interactions between these epigenetic complexes, the changes in protein and chromatin association of multiple transcription factors with MOZ or menin inhibition, and DOT1L function is dependent in KIT-dependent GISTs, with loss of DOT1L chromatin association serving as a downstream consequence of MOZ or menin inhibition. The above time-course growth experiments were analyzed by two-way ANOVA with Tukey's post-hoc test compared to GIST48B; ***, P<0.001; *, P<0.05. Taken together, these data demonstrate the complexity of proximal protein interactions between these epigenetic complexes, the changes in protein and chromatin association of multiple transcription factors with menin inhibition, and DOT1L function is dependent in KIT-dependent GISTs, with loss of DOT1L chromatin association serving as a downstream consequence of menin inhibition.

[0124] Example 6: Therapeutic effect of menin inhibition in vivo alone or in combination with TKIs. To evaluate the effect of genetic loss of KAT6A, menin, or DOT1L on tumor growth in vivo, cells expressing sgRNAs directed against KAT6A (sgKAT6A), menin (sgMEN1), or DOT1L (sgDOT1L), or a luciferase control (sgLuc), were prepared in GIST-T1 cells co-expressing Cas9. After transplantation of an equal number of modified cells, mice were monitored for tumor formation and growth. Although all grafts generated tumors, grafts derived from cells treated with sgKAT6A or sgMEN1 showed significantly reduced proliferation compared to the sgLuc control, while expression of sgDOT1L led to a non-significant trend toward reduced proliferation (Figure 14D). Although the growth restriction was milder than in similar in vitro experiments (Figure 2H and Figure S13I), the sgKAT6A and sgMEN1 conditions required 2 weeks of selection and expansion to generate sufficient cells for transplantation in vitro, likely selecting for cells with less deleterious genetic alterations.

[0125] Several menin inhibitors have progressed into early stage clinical trials for the treatment of leukemia, including National Clinical Trials (NCT) numbers NCT04067336 (study of compound KO539), NCT04811560 (study of compound JNJ-75276617), and NCT04065399 (study of compound SNDX-5613). KO539 is also known as Unii-4mod1F4enc and diftomenib.

[0126] To evaluate the in vivo effects of menin inhibition alone or in combination with WM-1119 or imatinib, mice were implanted with GIST-T1 cells and treated with imatinib (n=5), VTP-50469 (fed continuously in chow; n=4), WM-1119 (dosed three times a day, 7 days / week; n=6), a combination of VTP-50469 and WM-1119 (n=6), a combination of imatinib and VTP-50469 (n=5), or vehicle control (n=5). At the end of the 28-day treatment period, the monotherapy treatment groups showed a similar significant reduction in tumor growth compared to vehicle, while the combination group showed a complete cessation of tumor growth. Monitoring of tumor recovery continued without further drug treatment, and tumors from the imatinib and VTP-50469 monotherapy groups regained a similar tumor growth trajectory as the vehicle group, while the combination of imatinib and VTP-50469 maintained a 3-4 fold reduced slope of tumor recovery, as shown in Figure 7A. Tumor volumes relative to baseline are detailed in Table 5 for vehicle control, Table 6 for VTP-50469, Table 7 for WM-1119, and Table 8 for the combination of VTP-50469 and VM-1119. During continued monitoring of tumor recovery without further drug treatment, all conditions showed comparable tumor growth rates similar to the vehicle control. The combination therapy of VTP-50469 and WM-1119 had a similar tumor growth trajectory as the imatinib and VTP-50469 therapy, as shown in Figure 7D. Mice tolerated treatment with WM-1119, VTP-50469, imatinib, and their combinations without evidence of overt toxicity or weight loss (FIGS. 14E-F).

[0127] [Table 5]

[0128] [Table 6]

[0129] [Table 7]

[0130] [Table 8]

[0131] RNA-seq on GIST-T1 xenografts 5 and 10 days after imatinib and / or VTP-50469 treatment was performed to evaluate changes in the GIST transcriptional program resulting from menin and / or KIT inhibition in vivo. All treatment conditions resulted in global transcriptional changes compared to vehicle controls, but greater changes were seen at both time points after treatment with VTP-50469 and the combination of imatinib and VTP-50469, with the gene expression profile of imatinib treatment more closely correlating with vehicle-treated tumors (Figure 7E). Figure 7E shows the Pearson correlation of group-averaged fragments per kilobase of transcript per million mapped reads (FPKM) of all expressed genes (FPKM>10, n=7,434). Imatinib, VTP-50469, and combination treatment all resulted in a decrease in expression of genes regulated by HAND1 (Figure 14G). GIST-associated transcripts, including KIT, CD34, and NPR3, were preferentially decreased by VTP-50469 treatment, whereas other KIT signaling-dependent transcripts, including TMEM100 and SPRY2, were preferentially decreased by imatinib treatment. PCNA, a marker of cell proliferation, was only decreased by the combination of imatinib and VTP-50469 at both days 5 and 10 (Figure 7F), consistent with a greater effect of the combination treatment on tumor growth.

[0132] We next evaluated the effect of imatinib and VTP-50469 treatment on PG27 (HemMing et al., Cancer Res. 79(5):994-1009(2019)), a KIT-mutated patient-derived xenograft (PDX) model of GIST. Imatinib administration below the maximum tolerated dose had a significant, but modest, growth inhibitory effect compared to GIST-T1 cell line xenografts, whereas treatment with VTP-50469 alone (n=5) or in combination with imatinib (n=5) resulted in a significant reduction in tumor growth, as shown in Figure 7B. Data were analyzed by two-way ANOVA and compared to vehicle. ***, P<0.001; compared to imatinib; #, P<0.01. Mice treated with VTP-50469 in chow diet (n=5) did not show weight loss in the GIST-T1 xenograft experiment, whereas PG27 mice treated with different batches of VTP-50469 at the same concentration showed moderate weight loss (see Figures 14A-14B, data analyzed by two-way ANOVA compared to vehicle; *, P<0.05). At the end of the treatment period, PG27 tumors were harvested, fixed, sectioned, and tumor histology was evaluated. Vehicle and imatinib-treated tumors had monomorphic sheets of tumor cells, whereas xenografts treated with VTP-50469 or drug combinations showed areas of tumor necrosis, as shown in Figure 7C. Representative images in Figure 7C are shown from treatment groups at 4x magnification (upper panel, scale bar=250 μm) and 40x magnification (lower panel, scale bar=25 μm). Despite the restriction of tumor growth by VTP-50469 treatment, viable areas of the tumors showed similar levels of Ki-67 and cleaved caspase-3 across conditions (see FIG. 14C, where PG27 tumors were harvested at the end of the treatment period, fixed tissues were sectioned, and assessed for Ki-67 (top row) and cleaved caspase-3 (bottom row); scale bar = 25 μm). Mice treated with VTP-50469 in chow did not show weight loss in the GIST-T1 xenograft experiment, whereas PG27-implanted mice treated with the same concentration of VTP-50469 showed slight weight loss, possibly related to the systemic effects of tumor necrosis observed (FIG. 14B).Despite the limitation of tumor growth by VTP-50469 treatment, viable areas of tumors displayed similar levels of Ki-67 and cleaved caspase-3 across conditions (Figure 14B). These data demonstrate the therapeutic activity of VTP-50469 alone or in combination with imatinib, where menin inhibition reduced GIST xenograft growth, produced tumor necrosis, and when combined with imatinib, produced durable antitumor responses after cessation of treatment.

[0133] These embodiments demonstrate that chromatin organization and remodeling are essential for cell lineage, identity and function. Histone post-translational modifications serve as epigenetic regulatory nexuses that control the binding of TFs and chromatin regulators, ultimately governing gene expression and chromosome structure. Chromatin modifications are dynamic and reversible, and they require active maintenance by cell type and state-specific chromatin modifying enzymes. Cancers exploit or adapt the chromatin state of their progenitors to maintain their malignant phenotype through the maintenance of an environment permissive for oncogene activation or by gain-of-function changes in chromatin regulators such as MLL gene fusions (Krivtsovet et al., Nat. Rev. Cancer 7:823-33 (2007)). Here, the present disclosure shows that specific chromatin regulators are essential for maintaining the GIST epigenome, and that the menin-MLL complex binds to actively expressed genes throughout the genome, controls GIST-associated gene expression programs, coordinates protein-protein interactions between multiple regulators of gene expression, and ultimately controls cell proliferation and tumor growth.

[0134] Menin, encoded by the MEN1 gene, has been classically described as a tumor suppressor with mutations in MEN1 driving endocrine tumorigenesis. However, in other tissues, multiple functions have been ascribed to menin resulting from its ability to positively or negatively regulate gene expression, associate with different chromatin complexes, integrate inputs from upstream signaling pathways, and coordinate DNA replication and repair (Matkar et al., Trends Biochem. Sci. 38(8):394-402(2013)). Menin has been best studied as an oncogenic dependency in the context of MLL-rearranged leukemias, where it binds to MLL fusion proteins and, together with the recruitment of DOT1L, executes the expression program of leukemic genes (Krivtsovet al., Cancer Cell36:660-673(2019); Yokoyama et al., Cell123:207-18(2005); Dafflon et al., Leukemia31:1269-77(2017)). In GISTs, menin-MLL complex members are essential for global chromatin control and ultimately tumor cell proliferation. Compared to hundreds of other cell types profiled in Project DRIVE and DepMap, GISTs have exceptional sensitivity to targeted disruption of menin-MLL complex members. Consistent with the preservation of TFs and transcriptional and chromatin landscapes in KIT-dependent GISTs (HemMing et al., Proc. Natl. Acad. Sci. USA115:E5746-55(2018); Dafflon et al., Leukemia31:1269-77(2017)), sensitivity to genetic or pharmacological menin-MLL complex disruption was lost in KIT-independent GIST cell lines. These data indicate that, unlike the oncogenic hijacking seen in MLL-rearranged leukemias, GISTs rely on the native function of menin-MLL complexes and their associated dependencies to maintain the chromatin landscape that provides the basis for malignant gene expression programs.

[0135] Multiple lines of evidence suggest cooperation between menin-MLL, MOZ, and other complexes in transcriptional regulation. Here, the present disclosure shows genome-wide co-localization of menin-MLL and MOZ complex members at the TSS of actively expressed genes, similar changes in gene expression resulting from inhibition of either complex, proximal protein interactions between these two complexes, coordinated regulation of DOT1L and other transcription-related proteins, and shows that effects on cell cycle and cell proliferation were more pronounced when inhibiting menin-MLL complex was combined with MOZ complex inhibition. Consistent with the findings disclosed herein, interactions between MOZ and MLL complexes that promote gene expression have been previously described at the HOXA locus in hematopoietic progenitor cells (Paggetti et al., Oncogene 29:5019-31(2010)). Leveraging DepMap data, the present disclosure highlights the previously poorly understood complementary genetic codependency of these chromatin regulatory complexes in a minority of cancer cell lines. Dependence on the PRC2 complex was also seen in GISTs and a similar co-dependence was observed across the DepMap data, suggesting contrasting yet complementary roles for PRC2 in chromatin silencing that balances the activating function of the menin-MLL complex.

[0136] These data also suggest that menin-MLL and PRC2 complexes function cooperatively genome-wide to control chromatin state and transcriptional output. This disclosure highlights the superior activity of simultaneous inhibition of menin-MLL and MOZ complexes by VTP-50469 alone or in combination with WM-1119 on cell cycle and cell proliferation assays, while the expression of selected GIST-related genes and disruption of protein-protein interactions were largely similar between monotherapy and combination treatment. Although the mechanism of combination toxicity requires further investigation, these results suggest that disruption of one complex may maximally deregulate both at specific target loci, and that non-redundant functions of menin-MLL and MOZ complexes are likely to exist.

[0137] Consistent with its association with the TSSs of genome-wide active genes, disruption of the menin-MLL complex resulted in widespread changes in transcription that were both moderate and enriched in specific pathways. Genes with the greatest enrichment of these chromatin complexes had significantly reduced gene expression with menin inhibition. This disclosure also shows a disproportionate reduction in the transcription of genes essential for GIST, as well as genes downregulated by imatinib treatment, suggesting a fundamental role that the menin-MLL complex plays in supporting transcription downstream of KIT signaling. Using GSEA, transcriptional changes resulting from menin inhibition were significantly associated with gene sets indicative of reduced cell cycle and mitogenic signaling, as well as activation of developmental and EMT programs. Previous studies have observed upregulation of EMT signatures in less aggressive forms of GIST or after disruption of the oncogenic TF HAND1 (Hemming et al., Clin. Cancer Res. 27:1706-19(2021)), indicating convergence of transcriptional pathways by either TF disruption or pharmacological chromatin regulator inhibition. Consistent with menin-MLL disruption causing modest changes in gene expression, effects on proliferation and cell cycle were observed only after several days of drug treatment, in contrast to the acute toxic effects of imatinib.

[0138] Downstream consequences of menin inhibition include the disruption of proximal interactions between multiple transcription factors disclosed herein, including the loss of DOT1L from chromatin. DOT1L methylates H3K79 to support an active transcriptional state, and has been investigated in leukemia, where its recruitment by MLL fusion proteins is essential for leukemogenesis (Okada et al., Cell. 121:167-78(2005)). In solid tumors, DOT1L has been found to cooperate with oncogenic transcription factors (Wong et al., Cancer Research 77:2522-33(2017); Vatapalliet al., Nat. ComMun. 11(1):4153(2020)), but DOT1L inhibitors have not been evaluated in clinical trials in solid tumors to date. Previous studies demonstrating TF dependency in GIST, as well as the current studies showing the vulnerability of GIST cells to both genetic and pharmacological disruption of DOT1L, suggest that DOT1L may function as a downstream integrator of TF and menin-MLL complex activity in establishing the transcriptionally active state of select cancer-associated genes.

[0139] Taken together, these data demonstrate an essential function of the menin-MLL complex in GISTs, serving as an essential component of chromatin regulation and oncogenic gene expression programs.

[0140] Several menin inhibitors that disrupt the association between menin and MLL have been developed (Krivtsov et al., Cancer Cell. 36(6):660-673 (2019), Klossowski et al., J. Clin. Invest. 130:981-97 (2020), Xu et al., J. Med. Chem. 63:4997-5010 (2020)) and are currently in clinical trials for leukemia. To evaluate the in vivo effects of menin inhibition on xenograft models of GIST, the present disclosure describes treatment of cell lines and patient-derived xenografts with TKI, menin inhibition, or combination treatment, which demonstrated activity of menin inhibition as a monotherapy and even greater activity with the combination of TKI and menin inhibition. After the treatment period, tumors in both monotherapy arms resumed their growth trajectory, while tumors treated with menin inhibition and TKI combination therapy maintained long-term tumor suppression effects observed for several weeks after treatment cessation. In the above disclosure, PDX models of GIST demonstrated the potent antitumor activity of menin inhibition, and histology showed areas of necrosis interspersed with viable tumors. These results support the clinical development of menin inhibitors for GIST patients, either alone or ideally in combination with a TKI.

[0141] As TKIs are the only active therapeutic strategy in GISTs that have natural resistance to cytotoxic chemotherapy (Maki et al., Oncologist 20(7):823-30(2015)), targeting menin and other essential components of the GIST epigenome may prove therapeutically advantageous. The conserved transcriptional and enhancer landscape found in GIST tumors and cell lines, together with oncogenic KIT gene expression controlled by disease-specific TFs and enhancer elements, dictates the dependency of this disease on epigenetic mechanisms of disease control. As described herein, the cooperating chromatin regulators responsible for the maintenance of the GIST epigenome, and how their disruption at multiple different nodes by small molecule inhibitors (e.g., VTP-50469, EPZ-5676) show promising and selective anticancer activity; each member of these inhibitor classes has reached clinical trials (e.g., NCT04606446, NCT02141828). Compared to leukemias, which harbor oncogenic alterations in chromatin regulators, GISTs may be outliers among solid tumors in their dependency on these pathways and their susceptibility to disruption.

[0142] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications, including any specific portions thereof referenced, are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0143] Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. It is thus to be understood that numerous modifications can be made to the illustrative embodiments and other configurations can be devised without departing from the spirit and scope of the disclosure as defined by the appended claims.

Claims

1. 1. A menin inhibitor for use in the treatment of gastrointestinal stromal tumor (GIST) in a subject, wherein the menin inhibitor is JNJ-75276617, KO-539, SNDX-5613, DS-1594, or DSP-5336, MI-3454, M-808, BMF-219, A300-105A, VTP-50469, small interfering RNA (siRNA), or a combination of two or more thereof.

2. The menin inhibitor of claim 1, wherein the menin inhibitor is for oral, intramuscular, subcutaneous, or intravenous administration.

3. The menin inhibitor described in claim 1, wherein the menin inhibitor is administered in combination therapy with a tyrosine kinase inhibitor (TKI).

4. The menin inhibitor of claim 3 , wherein the TKI is administered subsequent to administration of the menin inhibitor.

5. The menin inhibitor of claim 3 , wherein the TKI is administered substantially simultaneously with the administration of the menin inhibitor.

6. The menin inhibitor of claim 3 , wherein the TKI is administered prior to administration of the menin inhibitor.

7. The menin inhibitor of any one of claims 1 to 6, administered as a combination therapy with a MOZ inhibitor.

8. 8. The menin inhibitor of claim 7, wherein the MOZ inhibitor is administered orally, intramuscularly, subcutaneously, or intravenously.

9. The menin inhibitor of claim 7 , wherein the MOZ inhibitor is administered subsequent to administration of the menin inhibitor.

10. The menin inhibitor of claim 7 , wherein the MOZ inhibitor is administered simultaneously with the administration of the menin inhibitor.

11. The menin inhibitor of claim 7, wherein the MOZ inhibitor is administered prior to administration of the menin inhibitor.

12. The menin inhibitor of claim 9, wherein the MOZ inhibitor is administered subsequent to administration of the TKI.

13. The menin inhibitor of claim 10, wherein the MOZ inhibitor is administered substantially simultaneously with the administration of the TKI.

14. The menin inhibitor of claim 11 , wherein the MOZ inhibitor is administered prior to administration of the TKI.

15. The menin inhibitor of any one of claims 1 to 6, wherein the subject is diagnosed with an activating mutation in or around the receptor tyrosine kinase (KIT) gene.

16. The menin inhibitor of any one of claims 1 to 6, wherein the GIST is metastatic.

17. 1. A method for reducing KIT activity in vitro, comprising: contacting a cell having an activating mutation in or around the KIT gene with a menin inhibitor; wherein the menin inhibitor is JNJ-75276617, KO-539, SNDX-5613, DS-1594, or DSP-5336, MI-3454, M-808, BMF-219, A300-105A, VTP-50469, a small interfering RNA (siRNA), or a combination of two or more thereof.

18. The method described in claim 17, further comprising the step of contacting the cells with a TKI and / or MOZ inhibitor.

19. The menin inhibitor of claim 1 or the method of claim 17 or 18, wherein the menin inhibitor is SNDX-5613 or VTP-50469.

20. The menin inhibitor of claim 1 or the method of claim 19, wherein the menin inhibitor is SNDX-5613.

21. 19. The menin inhibitor of claim 3 or the method of claim 18, wherein the TKI is imatinib, sunitinib, regorafenib, ripretinib, nilotinib, pazopanib, cabozantinib, avapritinib, or a combination of two or more thereof.

22. 22. The menin inhibitor of claim 3 or the method of claim 21, wherein the TKI is imatinib.

23. The menin inhibitor of claim 7 or the method of claim 18, wherein the MOZ inhibitor is WM-1119, WM-8014, PF-9363, siRNA, or a combination of two or more thereof.

24. The menin inhibitor of claim 7 or the method of claim 23, wherein the MOZ inhibitor is WM-1119.

25. A kit comprising a suitable container having disposed therein a menin inhibitor and a pharmaceutically acceptable carrier, and printed instructions for using the menin inhibitor in the treatment of GIST in a subject, comprising:

10. The kit, wherein the menin inhibitor is JNJ-75276617, KO-539, SNDX-5613, DS-1594, or DSP-5336, MI-3454, M-808, BMF-219, A300-105A, VTP-50469, a small interfering RNA (siRNA), or a combination of two or more thereof.

26. 26. The kit of claim 25, wherein the menin inhibitor is SNDX-5613 or VTP-5613.

27. ​​The kit described in claim 25, wherein the menin inhibitor is administered in combination therapy with a TKI, the printed instructions further include instructions for use of the TKI in treating GIST in a subject, and the menin inhibitor and the TKI are contained in the same dosage form or different dosage forms disposed in the same container or different containers.

28. 28. The kit of claim 27, wherein the TKI is imatinib.

29. A kit described in any one of claims 25 to 28, wherein the menin inhibitor is administered as combination therapy with a MOZ inhibitor, the printed instructions further comprising instructions for use of the MOZ inhibitor in treating GIST in a subject, and the menin inhibitor and the MOZ inhibitor are contained in the same dosage form or different dosage forms located in the same or different containers.

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