WO2025174751A1 - Methods of treating cancers dependent upon the alternative lengthening of telomeres (alt) pathway - Google Patents

Abstract

Provided are methods (including computer-implemented methods) of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex. Such compounds may be used to treat a cancer characterized by alternative lengthening of telomeres (ALT). Accordingly, methods of treating ALT-dependent cancers are also provided.

Description

METHODS OF TREATING CANCERS DEPENDENT UPON THE ALTERNATIVE LENGTHENING OF TELOMERES (ALT) PATHWAY

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/552,875, filed February 13, 2024, and U.S. Provisional Application No. 63/554,275, filed February 16, 2024, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING

[2] The present specification makes reference to a Sequence Listing, submitted electronically as an .xml file name “3360W01WO_Sequence listing” on February 10, 2025. The .xml file was generated on January 20, 2025, and is 12,288 kilobytes in size. The entire contents of the Sequence Listing are herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[3] This invention was made with government support under grant number HL052725 awarded by the National Institutes of Health. The government has certain rights to the invention.

FIELD

[4] Provided are methods (including computer-implemented methods) of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP 1) complex. Such compounds may be used to treat a cancer characterized by alternative lengthening of telomeres (ALT). Accordingly, methods of treating ALT-dependent cancers are also provided.

BACKGROUND

[5] The eukaryotic cell replicative lifespan is linked to the gradual shortening of telomeres, the protective caps at the ends of chromosomes. When telomeres shrink below a critical length, cell proliferative checkpoints are activated, ultimately leading to cellular senescence. [6] To avoid replicative senescence, cancer cells acquire mechanisms to prevent telomere attrition during DNA replication, essentially rendering themselves immortal. Most cancers upregulate telomerase to extend and maintain telomeres; however, approximately 10- 15% of cancers use a unique homologous recombination (HR) pathway called alternative lengthening of telomeres (ALT). ALT tumor cells display several unique cellular features including increased telomeric HR repair activity and enhanced H3K9Me3 deposition. Heterochromatin is densely packed, and many DNA repair pathways are inhibited or inactive in this setting. How heterochromatin formation and H3K9Me3 upregulation in telomeres contribute to ALT is unknown.

[7] The proteins Chromosome alignment-maintaining phosphoprotein 1 (CHAMP 1), Pogo transposable element with zinc finger domain (POGZ), and Heterochromatin Protein la (HP la) have been identified as forming part of a complex that is capable of binding to histone H3 methylated at the 9^th lysine residue (H3K9Me3) in heterochromatin. In addition, CHAMP 1 and POGZ have been shown to play a role in homologous recombination (HR) repair. The relationship between this DNA repair function and the heterochromatin localization of the complex is not known.

[8] ALT is prevalent in cancers of mesenchymal origin, such as osteosarcomas and liposarcomas, and usually is associated with a poor clinical outcome. Therefore, an urgent need exists to provide new therapeutic modalities that provide an effective treatment option for subjects suffering from ALT-dependent cancers.

SUMMARY

[9] It has been discovered that the protein-protein interactions between CHAMP 1 , POGZ, SETDB 1 and HP 1 a are important for the functioning of the ALT pathway. Thus, compounds that have the capability of reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex may therefore be effective in treating cancers that are dependent on ALT to extend or maintain their telomeres. Such inhibitors can be identified by screening existing compound libraries using the assays described herein.

[10] Accordingly, in one aspect, a method of identifying one or more compounds for treating a cancer characterized by alternative lengthening of telomeres (ALT) is provided, comprising: (i) providing a first polypeptide comprising all or a complex-forming portion of a protein of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP 1) complex; contacting said first polypeptide or protein with an interaction partner of said first polypeptide or protein of the CHAMP 1 complex, and (ii) determining formation of a complex between the first polypeptide and the interaction partner in the presence or absence of the one or more compound(s), wherein the one or more compounds may be capable of treating the cancer if they reduce, attenuate or inhibit formation of the complex.

[11] In another aspect, computer-implemented methods of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignmentmaintaining phosphoprotein 1 (CHAMP 1) complex are provided. Such methods may comprises: (a) receiving a first data set representing a three-dimensional structural model of at least amino acids 1-87 of CHAMP1; and/or at least amino acids 1021-1410 of POGZ; (b) receiving a second data set representing the one or more compound(s); (c) modeling one or more molecular interactions between CHAMP 1 or POGZ and the one or more compound(s); and (d) determining (i) a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q56, A59, L61, H63, F68, T70 and/or K72 of CHAMP 1 and amino acids K1223, L1235, LI 240, SI 244, S1247, L1249, P1250 and/or V1252 of POGZ; and/or (ii) a likelihood that the one or more compound(s) is/are capable of specifically binding to a region of CHAMP 1 comprising amino acids Q56, A59, L61, H63, F68, T70 and K72 or a region of POGZ comprising amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252.

[12] Alternatively, such methods may comprise: (a) receiving a first data set representing a three-dimensional structural model of at least amino acids 694-812 of CHAMP 1; and/or at least amino acids 1-80 of HP la; (b) receiving a second data set representing the one or more compound(s); (c) modeling one or more molecular interactions between CHAMP 1 or HP la and the one or more compound(s); and (d) determining (i) a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids C715 and/or R717 of CHAMP 1 and amino acids Y20 and/or V22 of HPla; and/or (ii) a likelihood that the one or more compound(s) is/are capable of specifically binding a region of CHAMP 1 comprising amino acids C715 and R717 or a region of HPla comprising amino acids Y20 and V22.

[13] In some instances, such methods may comprises: (a) receiving a first data set representing a three-dimensional structural model of at least amino acids 791-850 of POGZ; and/or at least amino acids 109-180 of HPla; (b) receiving a second data set representing the one or more compound(s); (c) modeling one or more molecular interactions between POGZ or HP la and the one or more compound(s); and (d) determining (i) a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and/or 1845 of POGZ and amino acids 1127, L139 and/or L150 ofHPla; and/or (ii) a likelihood that the one or more compound(s) is/are capable of specifically binding a region of POGZ comprising amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and 1845 or a region ofHPla comprising amino acids 1127, L139 and L150.

[14] In other instances, such methods may comprise: (a) receiving a first data set representing a three-dimensional structural model of at least amino acids 560-1291 of SETDB1; and/or at least amino acids 468-693 of POGZ; (b) receiving a second data set representing the one or more compound(s); (c) modeling one or more molecular interactions between SETDB 1 or POGZ and the one or more compound(s); and (d) determining (i) a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q811, N812, K813, and/or E1260 of SETDB 1 and amino acids C532, H534, C535, N546, H548, E550, N551 and/or H553 of POGZ; and/or (ii) a likelihood that the one or more compound(s) is/are capable of specifically binding a region of SETDB1 comprising amino acids Q811, N812, K813 and E1260 or a region of POGZ comprising amino acids C532, H534, C535, N546, H548, E550, N551 and H553.

[15] It has further been discovered that ALT-dependent tumors, in particular those harboring ATRX mutations, depend at least in part on the upregulation of the CHAMP 1 complex to preserve telomere heterochromatin integrity and promote the ALT pathway. Accordingly, reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex may impair the functioning of the ALT pathway.

[16] Accordingly, in a further aspect, there are provided methods of treating a cancer dependent upon the ALT pathway, in a subj ect in need thereof. The method comprises reducing, attenuating, or inhibiting the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex in the subject, e.g., by decreasing the levels of one or more proteins of the CHAMP 1 complex. BRIEF DESCRIPTION OF THE DRAWINGS

[17] Further description, by way of example, is provided with reference to the following drawings.

[18] FIG. 1 A shows a model of the POGZ C-terminus (POGZ Cter) complexed to the CHAMP 1 N-terminal zinc finger domain (CHAMP I NZnF), generated using AlphaFol d2_multimer, including a magnification of the binding interface showing key residues.

[19] FIG. IB shows a model of the POGZ Cter complexed to the CHAMPI NZnF, generated using AlphaFol d2_multimer, shaded to indicate the level of model confidence.

[20] FIG. 1C shows a predicted aligned error plot (PAE) matrix, which corresponds to the model confidence shown in FIG. IB.

[21] FIG. 2 A shows a model of the CHAMP 1 C-terminal zinc finger domain (CHAMPI CZnF) complexed to the HPlcr N-terminus (HPlcr Nter), generated using AlphaFol d2_multimer, including a magnification of the binding interface showing key residues.

[22] FIG. 2B shows a model of the CHAMP I CZnF complexed to the HP 1 cr Nter, generated using AlphaFold2_multimer, shaded to indicate the level of model confidence.

[23] FIG. 2C shows a predicted aligned error plot (PAE) matrix, which corresponds to the model confidence shown in FIG. 2B.

[24] FIG. 3 A shows a model of a homodimer formed by two HPlcr C-terminal domains, generated using AlphaFol d2_multimer, including a magnification of the binding interface showing key residues.

[25] FIG. 3B shows a model of a homodimer formed by two HPlcr C-terminal domains, generated using AlphaFol d2_multimer, shaded to indicate the level of model confidence.

[26] FIG. 3C shows a predicted aligned error plot (PAE) matrix, which corresponds to the model confidence shown in FIG. 3B. [27] FIG. 4A shows a model of the portion of POGZ including the HPZ motif complexed to the C-terminal portion of HPlcr (HP I a Cter), generated using AlphaFol d2_multimer, including a magnification of the binding interface showing key residues.

[28] FIG. 4B shows a model of the portion of POGZ including the HPZ motif complexed to the HPlcr Cter, generated using AlphaFol d2_multimer, shaded to indicate the level of model confidence.

[29] FIG. 4C shows a predicted aligned error plot (PAE) matrix, which corresponds to the model confidence shown in FIG. 4B.

[30] FIG. 5 schematically illustrates the predicted model of the CHAMP 1 complex comprising CHAMP 1, POGZ, and HP la, interacting with H3K9Me3 of chromatin.

[31] FIG. 6A schematically shows the domain structure of CHAMP 1, with the N- terminal Zinc finger domains (N-ZNFs), the SPE domain, the WK domain, the FPE domain, and the C-terminal Zinc finger domains shown in darker grey. The interaction regions for POGZ, REV7 and HPlcr are also indicated. The three horizontal bars below illustrate the length of the full length (FL) CHAMP 1 and CHAMP 1 mutants tested in the examples.

[32] FIG. 6B shows representative western blots showing that POGZ does not coimmunoprecipitate with CHAMP 1 N-terminal deletion mutants and HPlcr does not coimmunoprecipitate with CHAMP 1 N-and C-terminal deletion mutants.

[33] FIG. 6C shows representative western blots showing that substitution of three amino acids Q56, L61, and T70 in the N-terminal of CHAMP 1 with arginine (resulting in a mutant CHAMP 1 labelled “QLT/RRR”) almost completely ablates coimmunoprecipitation with POGZ.

[34] FIG. 6D shows representative western blots showing that the I165E in HPla- reduces coimmunoprecipitation with POGZ and CHAMP 1.

[35] FIG. 7 A shows representative immunofluorescence images showing increased colocalization of H3K9me3 and CHAMP 1 in human U2OS cells after irradiation. The boundary of each nucleus is shown with a dashed line. [36] FIG. 7B shows representative immunofluorescence images showing loss or reduction of H3K9me3 foci in CHAMP 1 or POGZ knockout cells. The boundary of each nucleus is shown with a dashed line.

[37] FIG. 7C shows a representative bar graph quantifying the reduction of H3K9me3 foci in CHAMP1 or POGZ knockout cells.

[38] FIG. 7D shows representative immunofluorescence images showing loss or reduction of HP la foci in CHAMP 1 or POGZ knockout cells.

[39] FIG. 7E shows a representative bar graph quantifying the reduction of HP la foci in CHAMP 1 or POGZ knockout cells.

[40] FIG. 7F shows representative western blots showing a reduction of POGZ and CHAMP 1 in the soluble nuclear (S-Nuc) and chromatin fractions in CHAMP 1 knockout (sgCHAMPl) U2OS and HeLa cells.

[41] FIG. 7G shows representative western blots showing a reduction of CHAMP 1 in the soluble nuclear (S-Nuc) and chromatin fractions in POGZ knockout cells (sgPOGZ).

[42] FIG. 7H shows representative bar graphs showing increased colocalization of H3K9me3 and yH2AX foci in irradiated CHAMP1 knockout (sgCHAMPl) cells.

[43] FIG. 8 shows representative box and whisker plots showing increased protein levels of CHAMP 1, HP la, POGZ, and SETDB1 in ALT positive neuroblastomas compared to ATRX-wild type tumors.

[44] FIG. 9A shows representative bar graphs showing a reduction in relative telomere length in U2OS CHAMP 1 or POGZ knockout cells.

[45] FIG. 9B shows representative telomere restriction fragment assays showing decreased telomere lengths in U2OS CHAMP 1 or POGZ knockout cells. Grey bars show the average telomere lengths.

[46] FIG. 9C shows representative telomere restriction fragment assays showing rescue of telomere length in U2OS cells with CHAMP 1 knockout ectopically expressing wild-type CHAMP 1 (“sgC+C” in the figure). U2OS cells with CHAMP 1 knockout are referred to as “sgC” in the figure. Grey bars show the average telomere lengths. [47] FIG. 9D shows representative line graphs showing rescue of relative telomere length in U2OS CHAMP 1 knockout cells that ectopically express wild-type CHAMP 1, as detected by telomeric FISH analysis.

[48] FIG. 9E shows representative immunofluorescent metaphase spread images and violin plots showing increased telomere loss in metaphase spread in U2OS CHAMP 1 or POGZ knockout cells. The arrow in the right image above the violin plot illustrates telomere loss in these knockout cells. The image on the left shows a normal control cell with intact telomeres.

[49] FIG. 10A shows a schematic representation of an experiment to induce DNA double-strand breaks (DSBs) at telomeres and measure telomere clustering. Cells generated for use in this assay were U2OS-TRF1-FOKI cells.

[50] FIG. 10B shows representative immunofluorescent images showing telomere clustering (TelC) in WT or CHAMP 1 and POGZ knockout U2OS-TRF1-FOKI cells. As shown in FIG. 10 A, cells were either administered doxycycline (+Dox) to induce DSBs, or not (-Dox). The boundary of each nucleus is shown with a dashed line.

[51] FIG. 10C shows a representative bar graph showing a reduction of average telomere foci size per nucleus in U2OS-TRF1-FOKI CHAMP 1 or POGZ knockout cells.

[52] FIG. 11A shows a representative bar graph showing a reduction of ALT- associated promyelocytic leukemia bodies (APBs) in U2OS-TRF1-FOKI CHAMP 1 or POGZ knockout cells.

[53] FIG. 1 IB shows a representative bar graph showing decreased incorporation of 5-ethynyl-2’-deoxyuridine (EdU) in G2 phase U2OS-TRF1-FOKI CHAMP1 or POGZ knockout cells.

[54] FIG. 11C shows a representative bar graph showing restoration of ALT- associated promyelocytic leukemia bodies (APBs) in U2OS CHAMP 1 knockout cells ectopically expressing wild-type (WT) CHAMP 1, but not CHAMP 1 with an N or C terminal deletion. An empty vector (EV) served as negative control.

[55] FIG. 1 ID shows a representative bar graph showing the absence of Telomere Dysfunction Induced Foci (TIFs) in U2OS CHAMP1 knockout cells ectopically expressing wild-type (WT) CHAMP 1, but not CHAMP 1 with an N or C terminal deletion. An empty vector (EV) served as negative control.

[56] FIG. 12 A shows a representative bar graph showing that average telomere foci size per nucleus was reduced when U2OS-TRFl-FokI cells were treated with HPla siRNA.

[57] FIG. 12B shows a representative bar graph showing that the number of ALT- associated promyelocytic leukemia bodies (APBs) was reduced when U2OS-TRFl-FokI cells were treated with HPla siRNA.

[58] FIG. 12C shows a representative bar graph showing the restoration of telomere foci size in U2OS-TRFl-FokI HPla knockout cells ectopically expressing wildtype (WT) HPla, but not I165E mutant HPla. An empty vector (EV) served as negative control.

[59] FIG. 12D shows a representative bar graph showing restoration of the number of ALT-associated promyelocytic leukemia bodies (APBs) in U2OS-TRFl-FokI HPla knockout cells ectopically expressing wild-type (WT) HPla, but not I165E mutant HPla. An empty vector (EV) served as negative control.

[60] FIG. 13 A shows a representative bar graph showing that knockout of CHAMP 1 or POGZ reduces the number of foci in which SETDB 1 colocalized with mCherry in TRFl-Fokl-incuded double-strand breaks (DSBs) in U2OS cells.

[61] FIG. 13B shows a model of a portion of POGZ including the zinc finger core (POGZ ZnFcore) complexed to the C-terminus of SETDB 1 (SETDB I Cter), generated using AlphaFol d2_multimer, including a magnification of the binding interface showing key residues.

[62] FIG. 13C shows a model of a portion of POGZ including the zinc finger core complexed to the SETDB I Cter, generated using AlphaFol d2_multimer, shaded to indicate the level of model confidence.

[63] FIG. 13D shows a predicted aligned error plot (PAE) matrix, which corresponds to the model confidence shown in FIG. 13C.

[64] FIG. 13E shows representative western blots showing a reduction in co- immunoprecipitating of POGZ and SETDB 1 with CHAMP 1 when the N-terminal region required for POGZ binding is deleted from CHAMP 1. [65] FIG. 14 schematically illustrates an exemplary computer system configured to perform a method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP 1) complex according to one or more aspects described herein.

[66] FIG. 15A shows representative immunofluorescence images showing presence of micronuclei, a marker of replication stress, in U2OS CHAMP 1 knockout cells, but not U2OS cells.

[67] FIG. 15B shows a representative bar graph showing increased micronuclei in U2OS CHAMP 1 knockout cells, but not U2OS cells.

[68] FIG. 15C shows a representative scatterplot representing the telomere-specific proteome profile of wild-type U2OS cells and U2OS CHAMP 1 knockout cells in response to the induction of double-strand breaks (DSBs) with TRFl-FokI as determined by mass spectroscopy. FANCM (circled) was the most differentially enriched protein in the telomeric proteome.

[69] FIG. 16 shows a representative graph comparing survival of U2OS CHAMP 1 knockout cells and U2OS POGZ knockout cells relative to untreated wild-type (WT) U2OS cells in the presence of the FANCM inhibitor PIP- 199.

[70] FIG. 17 shows a representative graph comparing survival of U2OS CHAMP 1 knockout cells and U2OS POGZ knockout cells relative to untreated wild-type (WT) U2OS cells in the presence of the ATR inhibitor VE822.

DETAILED DESCRIPTION

[71] In order for the following description to be more readily understood, certain terms are first defined below. Additional definitions may be set forth throughout the specification.

[72] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Thus, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a ribonucleotide” is understood to represent one or more ribonucleotides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. [73] Throughout this specification, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[74] Unless specifically stated or obvious from context, term “or” is understood to be inclusive and covers both “or” and “and.” Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to include “A and/or B and/or C” and to thus encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[75] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided. In other words, if an aspect is described as comprising A, B and C, aspects consisting essentially of A, B and C are also contemplated, as are aspects consisting of A, B and C.

[76] The term “about” refers to an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term indicates a deviation from the indicated numerical value of ±10%, ±5%, or ±1%.

[77] The terms “therapy,” “treatment” and “treating” include both preventative and curative treatment of a condition, disease or disorder. It also includes slowing, interrupting, controlling or stopping the progression of a condition, disease or disorder. It also includes preventing, curing, slowing, interrupting, controlling or stopping the symptoms of a condition, disease or disorder.

[78] The term “interface” or “binding interface” refers to a region of a first protein that is contacted by a second protein, e.g., to form a complex or sub-complex as described herein. The interface may comprise one or more amino acid residues that aid in complex formation between the first and second proteins.

[79] The term “portion” as used in reference to a protein, refers to a functional fragment of a full-length protein. A functional fragment has the capability to perform a function associated with the full-length protein. For example, it may retain the capability to fold correctly and/or interact with binding partners in the same fashion as the full-length protein. A portion of a full-length protein may be sufficient to computationally model a molecular interaction with an acceptable degree of confidence.

[80] The terms “specifically binding” and “selectively binding” are used interchangeably herein and refer to the preferential association of a binding moiety (e.g., a compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex) to a target entity (e.g., a protein of the CHAMP 1 complex) as compared to a non-target (e.g., other cellular proteins). A certain degree of non-specific binding may occur between a binding moiety and a non-target entity. In some instances, a binding moiety selectively binds a target entity if binding between the binding moiety and the target entity is greater than 2- fold, greater than 5-fold, greater than 10-fold, or greater than 100-fold as compared with binding of the binding moiety to a non-target entity. Typically, the binding between the binding moiety and the target entity is at least 100-fold greater as compared with the binding of the binding moiety to a non-target entity. In some instances, a binding moiety selectively binds a target entity if the binding affinity is less than about 10'⁵ M, less than about 10'⁶ M, less than about 10'⁷ M, less than about 10'⁸ M, less than about 10'⁹ M, or less than about 10’ ¹² M. In particular instances, the binding affinity of the binding moiety to the target is in the nanomolar or picomolar range. In some instances, binding can be assessed by a suitable assay system known in the art, e.g., BIACORE.

[81] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly used and/or understood by one of ordinary skill in the art to which this application belongs. In case of conflict, the present specification, including definitions, will control.

[82] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Chromosome alignment-maintaining phosphoprotein 1 (CHAMP1)

[83] Chromosome alignment-maintaining phosphoprotein 1 (CHAMP 1) is an 812 amino acid protein comprising zinc-finger domains at each end and several repeat motifs in the middle region. CDK1 (cyclin-dependent kinase 1) has been shown to phosphorylate CHAMP 1 at various serine residues, especially during mitosis.

[84] CHAMP 1 localizes to the nucleus in interphase and to chromosomes and the spindle during mitosis. Most of the mutations identified in CHAMP 1 are either nonsense or frameshift mutations that result in the formation of truncated proteins lacking the C-terminal portion to varying degrees. These C-terminally truncated CHAMP 1 mutant proteins cannot localize to the chromatin in interphase nor chromosomes during mitosis.

Pogo transposable element with zinc finger domain (POGZ)

[85] Pogo transposable element with zinc finger domain (POGZ) is a known interacting partner of CHAMP 1. POGZ mutations are either nonsense or frameshift mutations and, similar to CHAMP 1 mutations, typically result in the C-terminal truncation of the protein. Subjects with mutations in the genes encoding CHAMP 1 and POGZ share similar phenotypes.

[86] POGZ contains eight canonical C2H2-like zinc finger domains that are implicated in protein-protein and DNA interactions. The C-terminus of the protein contains a B-like centromere binding domain and a DDE domain. POGZ is known to interact with Heterochromatin Protein la (HP la) via a zinc finger domain.

Heterochromatin Protein la (HPla)

[87] Heterochromatin Protein la (HPla) is a major component of heterochromatin. HPla typically forms a homodimer that binds to both DNA and to histone H3 methylated at the 9^th lysine residue (H3K9Me). The direct association of HPla with H3K9Me heterochromatin and its direct binding to Suv39hl/2, a histone methyltransferase that deposits H3K9Me, has led to reports that HPla is necessary for either maintenance or establishment of histone methylation.

SET domain bifurcated histone methyltransferase 1 (SETDB1)

[88] SET domain bifurcated histone methyltransferase 1 (SETDB1) is a histone lysine methyltransferase. It catalyzes di- and tri-methylation of the 9^th lysine of histone H3 (H3K9Me2 and H3K9Me3) on euchromatin. SETDB1 amplification and abnormal activation have been linked to an unfavorable prognosis in multiple malignant tumors.

CHAMP1 complex

[89] It has now been discovered that the CHAMP 1 complex comprises one or more sub-complexes, including, e.g., a CHAMP1-P0GZ sub-complex, a POGZ-HPla subcomplex, a CHAMP 1 -HP la sub-complex, and a SETDB1-POGZ sub-complex. The CHAMP 1 complex includes two or more HP la monomers and SETDB1. The two or more HP la monomers are believed to form sub-complexes with POGZ and CHAMP 1, respectively. These sub-complexes are also referred to herein as “POGZ-HPla sub-complex” and “CHAMPl-HPla sub-complex”. It is shown for the first time herein that SETDB 1 forms a sub-complex with POGZ, also referred to as “SETDB 1-POGZ sub-complex”.

[90] Without wishing to be bound by any particular theory, HP la (e.g., as a homodimer) may facilitate interactions between the CHAMP 1-POGZ sub-complex and heterochromatin. It is believed that this sub-complex comprising CHAMP 1, POGZ and two or more HPla monomers (e.g., a monomer and a homodimer) recruits SETDB 1 to heterochromatin, thereby facilitating the transfer of methyl groups to the 9^th lysine of histone H3 (H3K9Me2 and H3K9Me3) on euchromatin. As shown herein, POGZ directly interacts with SETDB 1 forming a SETDB 1-POGZ sub-complex within the larger CHAMP 1 complex. Accordingly, without wishing to be bound by any particular theory, it is believed that the fully assembled CHAMP 1 complex comprises a CHAMP 1-POGZ sub-complex, a POGZ- HPla sub-complex, a CHAMPl-HPla sub-complex, and a SETDB 1-POGZ sub-complex.

[91] Typically, the CHAMP 1 complex comprises more than two HPla monomers. In some instances, the CHAMP 1 complex comprises a first HPla protein, a second HPla protein, and a third HPla protein. For example, the CHAMP 1 complex may comprise a first HPla protein in the POGZ-HPla sub-complex and a second HPla protein in the CHAMPl- HPla sub-complex. The second HPla protein in the CHAMPl-HPla sub-complex may be part of a HPla homodimer, wherein the HPla homodimer further comprises the second HPla protein and a third HPla protein.

[92] The first HPla protein in the POGZ-HPla sub-complex may interact with histone H3 modification H3K9Me3 on a first chromatid. The third HPla protein in the HPla homodimer may interact with histone H3 modification H3K9Me3 on a second chromatid. For example, the first HP la protein in the POGZ-HPla sub-complex may interact with histone H3 modification H3K9Me3 on a first chromatid and the third HPla protein in the HP la homodimer interacts with histone H3 modification H3K9Me3 on a second chromatid. Such interactions may facilitate heterochromatin clustering and subsequent homologous recombination repair, e.g., through the recruitment of additional enzymes or enzyme complexes that mediate the repair.

[93] Typically, the CHAMP1 complex comprises CHAMP1, POGZ, SETDB1 and

HPla. The amino acid sequences of each of CHAMP 1, POGZ, SETDB1 and HPla are shown in Table 1.

Table 1. Amino acid sequences

CHAMP 1 -POGZ sub-complex formation

[94] Without wishing to be bound by any particular theory, formation of a

CHAMP 1 -POGZ sub-complex is believed to be important for the assembly of the larger CHAMP 1 complex. It has now been discovered that at least amino acids 1-87 of CHAMP 1 and at least amino acids 1021-1410 of POGZ can interact with each other to form a stable sub-complex.

[95] As subjects having inactivating gene mutations in either CHAMP 1 or POGZ survive to adulthood, specifically targeting one or both of these proteins may provide a particularly attractive treatment option, as these proteins do not seem essential for the survival of healthy cells. Reducing the levels of CHAMP 1 and/or POGZ therefore may have minimal side effects while being highly effective in killing ALT-dependent tumor cells.

[96] Formation of the CHAMP 1 -POGZ sub-complex may comprise one or more intermolecular interactions between amino acids Q56, A59, L61, H63, F68, T70 and/or K72 of CHAMP1 and amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and/or V1252 of POGZ. Formation of the CHAMP1-POGZ sub-complex may comprise intermolecular interactions between at least two amino acids of CHAMP 1 selected from Q56, A59, L61, H63, F68, T70 and K72 and at least two (e.g., three or more, five or more, or six or more) amino acids of POGZ selected from K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252. Formation of the CHAMP1-POGZ sub-complex may comprise intermolecular interactions between amino acids Q56, L61, and T70 of CHAMP 1 and two or more (e.g., three, five or six, or more) of amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252 of POGZ. Compounds that interfere with one or more (e.g., two or more, or three or more) of these interactions may reduce, attenuate, or inhibit the formation of the CHAMP 1 complex.

[97] The interface between CHAMP 1 and POGZ may comprise a region of CHAMP1 comprising amino acids Q56, L61, and T70 of CHAMP1 and a region of POGZ comprising amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252. The interface between CHAMP 1 and POGZ may comprise a region of CHAMP 1 comprising amino acids Q56, A59, L61, H63, F68, T70 and K72 of CHAMP1 and a region of POGZ comprising amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252. Compounds that bind to one or both of these regions may reduce, attenuate, or inhibit the formation of the CHAMP 1 complex. SETDB1-P0GZ sub -complex formation

[98] It is demonstrated herein that at least amino acids 560-1291 of SETDB1 and at least amino acids 468-693 of POGZ can form a stable complex. The formation of the SETDB1-POGZ sub-complex may comprise one or more intermolecular interactions between amino acids Q811, N812, K813 and/or E1260 of SETDB1 and amino acids C532, H534, C535, Q546, H548, E550, N551 and/or H553 of POGZ.

[99] Formation of the SETDB1-POGZ complex may comprise one or more intermolecular interactions between amino acids Q811, N812, K813 and/or E1260 of SETDB1 and amino acids C532, H534, C535, Q546, H548, E550, N551 and/or H553 of POGZ. Formation of the SETDB1-POGZ complex may comprise interactions between at least two amino acids of SETDB1 selected from Q811, N812, K813 and E1260 and at least two (e.g., three or more, five or more, or six or more) amino acids of POGZ selected from C532, H534, C535, Q546, H548, E550, N551 and H553. Compounds that interfere with one or more (e.g., two or more, or three or more) of these interactions may reduce, attenuate, or inhibit the formation of the CHAMP 1 complex.

POGZ-HPla sub-complex formation

[100] As demonstrated herein, at least amino acids 791-850 of POGZ and at least amino acids 109-180 of HPla can form a stable complex. Formation of the POGZ-HPla complex may comprise one or more intermolecular interactions between amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and/or 1845 of POGZ and amino acids 1127, L139 and/or L150 of HPla.

[101] Formation of the CHAMP 1 -POGZ complex may comprise one or more intermolecular interactions between amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and/or 1845 of POGZ and amino acids 1127, L139 and/or L150 of HPla. Formation of the POGZ-HPla complex may comprise interactions between at least two amino acids (e.g., three or more, five or more, or six or more) of POGZ selected from 1813, L815, C817, C820, D828, H833, L834, H840 and 1845 and at least two amino acids of HPla selected from 1127, L139 and L150. Compounds that interfere with one or more (e.g., two or more, or three or more) of these interactions may reduce, attenuate, or inhibit the formation of the CHAMP 1 complex. CHAMP 1 -HP la sub-complex formation

[102] As demonstrated herein, at least amino acids 694-812 of CHAMP 1 and at least amino acids 1-80 of HP la can form a stable complex.

[103] Formation of the CHAMP 1 -HP la complex may comprise one or more intermolecular interactions between amino acids C715 and/or R717 of CHAMP 1 and amino acids Y20 and/or V22 of HPla. Formation of the CHAMPl-HPla complex may comprise intermolecular interactions between amino acid C715 of CHAMP 1 and V22 of HPla. Formation of the CHAMPl-HPla complex may comprise intermolecular interactions between amino acid R717 of CHAMP1 and Y20 of HPla. Formation of the CHAMPl-HPla complex may comprise intermolecular interactions between amino acid C715 of CHAMP 1 and V22 of HPla and between amino acid R717 of CHAMP 1 and Y20 of HPla. Compounds that interfere with one or more (e.g., both) of these interactions may reduce, attenuate, or inhibit the formation of the CHAMP 1 complex.

HPla homodimer formation

[104] As demonstrated herein, at least amino acids 109-180 of a first HPla and at least amino acids 109-180 of a second HPla protein can form a stable complex.

[105] Formation of the HPla homodimer may comprise one or more intermolecular interactions between amino acids N157, QI 62, VI 64, 1165 and/or El 69 of the first HP la and amino acids N157, Q162, V164, 1165 and/or E169 of the second HPla.

Alternative lengthening of telomeres (ALT) pathway

[106] The alternative lengthening of telomeres (ALT) is a telomerase-independent mechanism that relies on telomere heterochromatin clustering, homologous recombination, and telomeric DNA amplification.

[107] ALT telomeres are associated with nuclear bodies formed by the promyelocytic leukemia (PML) protein, known as ALT-associated PML bodies. ALT emerges as a latent reaction to the inactivation of chromatin modifiers, including the ATRX (alpha-thalassemia/mental retardation, X-linked) and DAXX (death domain associated protein) chromatin remodeling and histone deposition complex, which governs the deposition of histone H3.3 at telomeres. ALT-dependent cancers

[108] In most human cancers, the process of maintaining sufficiently long telomeres is achieved through reactivation of the enzyme telomerase, allowing for the lengthening of the shortest telomeres. Approximately 15% of cancers, including, but not limited to, many sarcomas, gliomas, glioblastomas, and neuroblastomas, employ a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). The cancers typically are of mesenchymal and neuroepithelial origins.

[109] The majority of ALT-positive tumors exhibit inactivating mutations of the histone chaperone protein a thalassemia/mental retardation syndrome X-linked (ATRX) gene. Mutations in DAXX and H3.3 mutations have also been identified in ALT-positive cancers.

[110] Accordingly, the methods described herein may be particularly suitable for the identification of compounds that may have therapeutic activity in ALT-positive cancers. The ALT-positive cancer may be of mesenchymal or neuroepithelial origins. For example, the ALT-positive cancer is a sarcoma, a glioblastoma, or a neuroblastoma.

[Hl] In genome-wide sequencing of human glioblastoma, mutations in the ATRX gene are seen in approximately 30% of younger patients. ATRX is mutated only rarely in adult primary glioblastoma, but is commonly found in younger adults with lower grade (WHO grade II/III) glioma.

[112] Accordingly, the subject to be treated with a compound identified with a method described may be a pediatric patient (e.g., less than 18 years of age) or a young adult (e.g., less than 30 years of age).

Methods of treatment

[113] Provided herein are methods of treating a cancer in a subject in need thereof, wherein the method comprises reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex in the subject, e.g., by decreasing the levels of one or more proteins of the CHAMP 1 complex or preventing the formation of one or more sub-complexes. The cancer may be characterized by alternative lengthening of telomeres (ALT).

[114] For example, provided herein are methods of treating a cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a means of reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex in the subject. Such methods may comprise inhibiting CHAMP 1 complex formation, e.g., via administration of small molecule inhibitors, RNA-based interference, nucleic acid therapies, targeted protein degradation, etc.

Cancer type

[115] The cancer may comprise a sarcoma. For example, the sarcoma may be selected from osteosarcoma, leiomyosarcoma, liposarcoma, and an undifferentiated pleomorphic sarcoma.

[116] The cancer may comprise a glioma. The glioma may be selected from glioblastoma or gliosarcoma. The glioblastoma may be glioblastoma multiforme. The cancer may comprise a neuroblastoma.

[117] The cancer may comprise a neuroblastoma, a breast cancer, a colorectal cancer, a hematopoietic cancer, a kidney cancer, a liver cancer, a lung cancer, a pancreatic cancer, an ovarian cancer, a prostate cancer, a skin cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urinary bladder cancer, or a uterine cancer.

[118] The pancreatic cancer may comprise neuroendocrine pancreatic cancer.

RNA interference-based therapy

[119] A method of treating cancer as disclosed herein may comprise reducing, attenuating, or inhibiting the expression of one or more proteins of the CHAMP 1 complex. Reducing, attenuating, or inhibiting the expression of one or more proteins of the CHAMP 1 complex may comprise RNA interference (RNAi). The one or more proteins may be CHAMP 1 and/or POGZ.

[120] Accordingly, the therapeutic methods described herein may comprise administering to the subject one or more small interfering RNAs (siRNA) or one or more antisense RNAs that specifically hybridize to an mRNA encoding a protein of the CHAMP 1 complexes (e.g., a CHAMP 1- or POGZ-encoding mRNA). RNAi can be used efficiently to reduce, attenuate or inhibit the expression of a protein of the CHAMP 1 complex in vivo. As shown herein, the effectiveness of RNAi can be assessed in cell-based in vitro assays by determining damage-induced telomere clustering and/or by quantifying the number of ALT- associated PML bodies (APBs), e.g., using an ALT-dependent tumor cell line. [121] Also provided herein are nucleic acids (e.g., siRNAs or antisense RNAs) that specifically hybridize to an mRNA encoding a protein of the CHAMP 1 complexes and reduce, attenuate, or inhibit the expression of the proteins for use in a method of treating cancer. Moreover, the use of nucleic acids in the manufacture of a medicament for use in treating cancer is also provided.

Other nucleic acid therapies

[122] As shown herein, the N-terminus and the C-terminus of CHAMP 1 comprise binding domains that allow CHAMP 1 to interact with POGZ and HP la, respectively. Similarly, the C-terminus of POGZ comprises a binding domain that allows POGZ to interact with CHAMP 1.

[123] Recent advances in nucleic acid therapy have made it possible to efficiently deliver one or more protein-encoding nucleic acid(s) (e.g., DNA or RNA) to a subject in vivo, e.g., by encapsulating the nucleic acid(s) in one or more lipid nanoparticles. Such lipid nanoparticles can be configured to be tissue-specific, e.g., to specifically localize in a subject to a tumor or a tissue comprising the tumor. The protein-encoding nucleic acid may be an mRNA.

[124] Therefore, a method for reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex may comprise administering a nucleic acid encoding a CHAMP 1 protein (e.g., an mRNA) comprising an N-terminal truncation to the subject. The N-terminal truncation may remove the POGZ binding domain of the CHAMP 1 protein. The N-terminal truncation may remove amino acids 1-85 of the CHAMP 1 protein.

[125] A method for reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex may comprise administering a nucleic acid (e.g., an mRNA) encoding a CHAMP 1 protein comprising a C-terminal truncation to the subject. The C-terminal truncation may remove the HP la binding domain of the CHAMP 1 protein. The C-terminal truncation may remove amino acids 628-812 of the CHAMP 1 protein.

[126] A method for reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex comprises administering a nucleic acid (e.g., an mRNA) encoding a POGZ protein comprising a C-terminal truncation to the subject. The C-terminal truncation may remove the CHAMP 1 binding domain of the POGZ protein. The C-terminal truncation may remove amino acids 1021-1410 of the POGZ protein. [127] Also provided herein are nucleic acids (e.g., mRNAs) that encode a truncated CHAMP 1 or POGZ protein capable of reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex for use in a method of treating cancer. Moreover, the use of such nucleic acids in the manufacture of a medicament for use in treating cancer is also provided.

Targeted protein degradation

[128] The method may comprise targeted protein degradation of one or more proteins of the CHAMP1 complex. Targeted protein degradation may comprise a compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s). The one or more ubiquitin ligase(s) typically are E3 ubiquitin ligase. E3 ubiquitin ligases that are localized to the nucleus are particularly suitable for targeted degradation of proteins of the CHAMP 1 complex.

[129] The compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s) may be a molecular glue. The compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s) may be a Proteolysis-Targeting Chimera (PROTAC). The compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s) may be a Chaperone-mediated Protein Degrader (CHAMP).

[130] Compounds that selectively interact with various E3 ubiquitin ligases are well known. Such compounds are also referred to as E3 ligands. E3 ubiquitin ligases with known ligands include CRBN, VHL, MDM, IAP, RNF114, DCAF15, DCAF16, and FEM1B. Representative E3 ligands include the compounds shown in Table 2A.

Table 2A. E3 ligands

[131] For example, targeted protein degradation of nuclear proteins can be accomplished by compounds that specifically interact with DCAF16. DCAF16 is the substrate recognition component of the CUL4-DDB1 E3 ubiquitin ligase. Similarly, targeted protein degradation of nuclear proteins can be achieved with compounds that specifically interact with the E3 ligase complex CRL4-DCAF15.

[132] Interaction with one or more protein ligases can also be accomplished via the chaperone protein HSP90. Suitable HSP90-binding moieties are well known and include geldanamycin analogs, resorcinol analogs, and novobiocin analogs (Li et al., J. Med. Chem. 2023, 66:733-751), as well as the compounds shown in Table 2B. Table 2B: HSP90-binding moieties [133] Targeted protein degradation can be achieved by linking a compound that specifically binds to a protein of the CHAMP 1 complex to a compound that selectively interacts with an E3 ubiquitin ligase or HSP90. For example, various compounds that specifically bind a protein of the CHAMP 1 complex are known (see Table 3). These include the SETDB1 inhibitors VH01 and VH06 (Park et al., J Comput Aided Mol Des. 2017; 31(10):877-889), (R, R)-59 (Guo et al., Angew Chem Int Ed Engl. 2021; 60(16):8760-876), and 5-allyloxy-2-(pyrrolidin-l-yl)quinoline (Hwang et al., JEnzyme Inhib Med Chem. 2021; 36(l):856-868).

Table 3. Compounds that specifically bind to a protein of the CHAMP1 complex

[134] Various known linker moieties have been used to couple a compound that specifically binds to a target protein to an E3 ligand or an HSP90-binding moiety. These include the linkers disclosed in, e.g., WO 2020/206608, WO 2021/194878, WO 2021/011913, WO 2022/078470, and WO2023/081476. For example, the linker may be selected from an alkane linker, an alkyne-alkane linker, a piperidine/benzene linker, a triazole linker, a triazole-PEG linker, a PEG linker, a PEG-alkane, a benzene linker, an amide linker, and an azobenzene linker. The linker moiety may be flexible. Flexible linkers include alkane linkers, alkyne-alkane linkers, PEG linkers, PEG-alkane linkers, and amide linkers. The linker moiety may be rigid. Rigid linkers include piperidine/benzene linkers, benzene linkers, and azobenzene linkers. Exemplary flexible linker moieties are shown in Table 4A and exemplary rigid linker moieties are shown in Table 4B.

Table 4A: Flexible linkers

Table 4B: Rigid linkers

[135] A method of treating a cancer characterized by alternative lengthening of telomeres (ALT) may comprise administering to the subject a molecular glue, a Proteolysis- Targeting Chimera (PROTAC) or a Chaperone-mediated Protein Degrader (CHAMP), wherein the molecular glue, PROTAC or CHAMP specifically binds to and targets SETDB1 for degradation by a E3 ubiquitin ligase.

[136] A method of treating a cancer characterized by alternative lengthening of telomeres (ALT) may comprise administering to the subject a molecular glue, a Proteolysis- Targeting Chimera (PROTAC) or a Chaperone-mediated Protein Degrader (CHAMP), wherein the molecular glue, PROTAC or CHAMP specifically binds to and targets CHAMP 1 or POGZ for degradation by an E3 ubiquitin ligase.

[137] Also provided herein are compounds that specifically bind to one or more proteins of the CHAMP 1 complex and interact with one or more ubiquitin ligase(s) (either directly or via HSP90) for use in a method of treating cancer. Moreover, the use of such compounds in the manufacture of a medicament for use in treating cancer is also provided.

[138] The compound that specifically binds to a protein of the CHAMP 1 complex may be a first antibody (e.g., a single-chain Fv or a single-domain antibody). The compound that selectively interacts with or binds to a ubiquitin ligase may be a second antibody (e.g., a single-chain Fv or a single-domain antibody).

[139] A fusion protein is provided that may comprise the first antibody, a linker sequence, and the second antibody. A nucleic acid (e.g., an mRNA) is provided that may encode the fusion protein. Methods of making such fusion proteins and nucleic acids encoding the same are described in, e.g., W02021/081058. The linker sequence may comprise the amino acid sequence GS, GGS, GGGGS (SEQ ID NO: 5), and/or GGGGGS (SEQ ID NO: 6), or 2, 3, 4, or 5 repeats of the amino acid sequence GS, GGS, GGGGS (SEQ ID NO: 5), or GGGGGS (SEQ ID NO: 6).

[140] The first antibody may specifically bind to CHAMP 1. The first antibody may specifically bind to POGZ. The first antibody may specifically bind to SETDB1.

[141] The second antibody may specifically bind to an E3 ubiquitin ligase.

[142] A fusion protein or a nucleic acid (e.g., an mRNA) encoding the fusion protein as described above may be used in a method of treating cancer. The fusion protein or nucleic acid may also be used in the manufacture of a medicament for use in treating cancer.

[143] A method of treating a cancer characterized by alternative lengthening of telomeres (ALT) may comprise administering to the subject a fusion protein or a nucleic acid (e.g., an mRNA) encoding the fusion protein, wherein the fusion protein specifically binds to and targets SETDB1 for degradation by a E3 ubiquitin ligase.

[144] Alternatively, a method of treating a cancer characterized by alternative lengthening of telomeres (ALT) may comprise administering to the subject a fusion protein or a nucleic acid (e.g., an mRNA) encoding the fusion protein, wherein the fusion protein specifically binds to and targets CHAMP 1 or POGZ for degradation by an E3 ubiquitin ligase.

Combination therapy

[145] The therapies disclosed herein that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex may be used in a combination therapy to treat a cancer in a subject in need of such treatment. The cancer may be characterized by alternative lengthening of telomeres (ALT).

[146] A therapy that reduces, attenuates, or inhibits the formation of CHAMP 1 complex may be combined with a FANCM inhibitor to treat a cancer in a subject in need of such treatment.

[147] Fanconi anemia complementation group M protein (FANCM) and its ATPase activity is elevated in ALT telomeres and may function to alleviate replication stress and to maintain tumor cell viability (Pan, X. et al., 2017, Proc Natl Acad Set USA 114, E5940- E5949; Lu, R. et al., 2019, Nat Commun 10, 2252; Silva, B. et al., 2019, Nat Commun 10, 2253). As shown herein, FANCM is enriched in the telomeric proteome of ALT tumor cells without a functioning CHAMP 1 complex. Therefore, combining a compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex with a FANCM inhibitor may be beneficial in the treatment of ALT-dependent cancers.

[148] Accordingly, a method of treating a cancer characterized by ALT described herein may further comprise administering to a subject a FANCM inhibitor. A compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex and the FANCM inhibitor may be administered simultaneously, separately, or sequentially to the subject.

[149] Methods of inhibiting FANCM are described in WO 2021/262898, which is incorporated herein by reference in its entirety. The FANCM inhibitor may reduce, attenuate, or inhibit one or more of ATP -binding, nucleotide-binding, DNA-binding, DNA remodeling, DNA strand separation, DNA-RNA strand separation, and hydrolysis activity of FANCM.

[150] The FANCM inhibitor may be an ATPase inhibitor. Suitable ATPase inhibitors are developed by MOMA Therapeutics, Inc. [151] The FANC pathway requires interaction between FANCM and the RecQ- mediated genome instability protein (RMI) complex. Specifically, RMI interacts with an amino acid motif in FANCM termed MM2. Methods of identifying inhibitors that disrupt the formation of the FANCM-RMI complex are described, e.g., in Voter et al. J Biomol Screen (2016) 21(6): 626-633, which is incorporated herein by reference in entirety. Accordingly, the FANCM inhibitor may reduce, attenuate, or inhibit interaction between FANCM and the RecQ-mediated genome instability protein (RMI complex). The FANCM inhibitor may interact with the FANCM MM2 domain. The FANCM inhibitor may be PIP- 199, which has the following formula:

[152] Alternatively, the FANCM inhibitor can be FANCM-BTR PPI-IN-1, which has the following formula:

FANCM-BTR PPI-IN-1 is a disruptor of the interaction between FANCM and BLM- TOP3A-RMI (BTR) which hampers localization of FANCM to telomeres.

[153] Other suitable FANCM inhibitors are developed by Tessellate Bio B.V. and are disclosed in United Kingdom patent application no. GB2407221.7, which was filed on 21 May 2024. [154] As demonstrated herein, treatment of ALT-dependent cancer cells with a compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex can be rendered more effective when it is combined with a FANCM inhibitor. Inhibiting CHAMP 1 complex formation alleviates heterochromatin formation. It has also been shown herein that a similar reduction in heterochromatin may be achieved with a SETDB1 inhibitor by decreasing H3K9me3 levels. Therefore, also provided herein is a method of treating a cancer characterized by ALT in a subject in need thereof, wherein the method comprises administering a FANCM inhibitor and a SETDB 1 inhibitor to the subj ect. SETDB 1 inhibitors include, but are not limited to, VH01, VH06, (R,R)-59, and 5-allyloxy-2-(pyrrolidin-l- yljquinoline. Their structural formulae are shown in Table 3 above.

[155] For example, a FANCM inhibitor may be used in a method of treating a cancer characterized by ALT in a subject in need thereof that comprises administering simultaneously, concomitantly, or sequentially a SETDB 1 inhibitor to the subject. In some instances, simultaneous or concomitant administration of a FANCM inhibitor and a SETDB 1 inhibitor renders treatment of the cancer more effective than administration of each inhibitor separately. For instance, simultaneous or concomitant administration may reduce the emergence of cancer cells that are resistant to one or both types of inhibitors.

[156] In further aspects, a method of treating a cancer characterized by alternative lengthening of telomeres (ALT) in a subject in need thereof, is provided wherein the method comprises administering a FANCM inhibitor and a therapy that inhibits CHAMP 1 complex formation, e.g., by RNA interference or another nucleic acid therapy, or by targeted protein degradation.

[157] A therapy that reduces, attenuates, or inhibits the formation of CHAMP 1 complex may be combined with an Ataxia telangiectasia and Rad-3 related kinase (ATR) inhibitor to treat a cancer in a subject in need of such treatment. Accordingly, a method of treating a cancer characterized by ALT described herein may further comprise administering to a subject an ATR inhibitor. A compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex and the ATR inhibitor may be administered simultaneously, separately, or sequentially to the subject. Typically, the ATR inhibitor is a selective ATR inhibitor. [158] Exemplary ATR inhibitors include, but are not limited to, the following compounds: Berzosertib (e.g., VE822 (Vertex) or M6620 (VX-970; Merck)), Ceralasertib (AZD6738; AstraZeneca), AZ20 (AstraZeneca), Elimusertib (BAY1895344; Bayer), IMP9064 (Impact Therapeutics), ATG-018 (Antengene), ATRN-119 (Aprea), ART0380 (Artios), Gartisertib (M4344, VX-803), Tuvusertib (Ml 774; Merck), and Camonsertib (RP-3500; Repare Therapeutics).

[159] Also provided herein is a method of treating a cancer characterized by ALT in a subject in need thereof, wherein the method comprises administering an ATR inhibitor and a SETDB1 inhibitor to the subject. In further aspects, a method of treating a cancer characterized by alternative lengthening of telomeres (ALT) in a subject in need thereof, is provided wherein the method comprises administering an ATR inhibitor and a therapy that inhibits CHAMP 1 complex formation, e.g., by RNA interference or another nucleic acid therapy, or by targeted protein degradation.

In vitro screening methods

[160] Compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex can be determined using in vitro, in vivo or in silico methods.

[161] An in vitro method may comprise (i) providing a first polypeptide or protein comprising all or a complex -forming portion of a protein of the CHAMP 1 complex; contacting said first polypeptide or protein with an interaction partner of said first polypeptide or protein of the CHAMP 1 complex, and (ii) determining formation of a complex between the first polypeptide or protein and the interaction partner in the presence or absence of the one or more compound(s). The term “interaction partner” as used herein refers to a second polypeptide or protein that is demonstrated herein as forming a sub-complex with the first polypeptide or protein of the CHAMP 1 complex.

[162] The first protein of the CHAMP 1 complex may be CHAMP 1 and the interaction partner may be POGZ. The CHAMP 1 polypeptide or protein may comprise or consist of a portion of CHAMP1 comprising amino acids Q56, A59, L61, H63, F68, T70 and K72 (e.g., amino acids 1-87 of CHAMP1). The POGZ polypeptide or protein may comprise or consist of a portion of POGZ comprising amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252 (e.g., amino acids 1021-1410 of POGZ). [163] Or, the first protein of the CHAMP 1 complex may be CHAMP 1 and the interaction partner may be HP la. In such cases, the CHAMP 1 polypeptide or protein may comprise or consist of a portion of CHAMP1 comprising amino acids C715 and R717 (e.g., amino acids 694-812 of CHAMP1). The HPla polypeptide or protein may comprise or consist of a portion of HPla comprising amino acids Y20 and V22 (e.g., amino acids 1-80 of HP la).

[164] The first protein of the CHAMP 1 complex may be POGZ and the interaction partner may be SETDB1. The SETDB1 polypeptide or protein may comprise or consist of a portion of SETDB1 comprising amino acids Q811, N812, K813 and E1260 (e.g., amino acids 560-1291 of SETDB1). The POGZ polypeptide or protein may comprise or consist of a portion of POGZ comprising amino acids C532, H534, C535, Q546, H548, E550, N551 and H553 (e.g., amino acids 468-693 of POGZ).

[165] Or, the first protein of the CHAMP 1 complex may be POGZ and the interaction partner may be HPla. The POGZ polypeptide or protein may comprise or consist of a portion of POGZ comprising amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and 1845 (e.g., amino acids 791-850 of POGZ). The HPla polypeptide or protein may comprise or consist of a portion of HPla comprising amino acids 1127, L139 and L150 (e.g., amino acids 109-180 of HPla).

[166] The step of determining formation of a complex may employ Surface Plasmon Resonance (e.g., BIACORE) or Bio-Layer Interferometry. Alternatively, the step of determining formation of a complex may employ an immunoassay, e.g., an ELISA. Alternatively, the step of determining formation of a complex may employ Fluorescence resonance energy transfer (FRET).

[167] The first polypeptide or protein, or the second polypeptide or protein, may be immobilized to a surface. The surface may be a detector (e.g., the surface of a BIACORE chip). Alternatively, the surface may form part of a receptacle (e.g., an ELISA plate).

[168] The immobilization may be either covalently via a linker moiety or non- covalently via a tag. The tag may be streptavidin or biotin. The tag may be fused to the first polypeptide or protein, or the second polypeptide or protein, optionally via a linker peptide. The first polypeptide or protein and/or the second polypeptide or protein may be fusion proteins. In vivo screening methods

[169] In vivo methods may also be used to identify one or more compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex.

[170] Suitable in vivo screening methods may comprise: (a) fusing a first protein (e.g., a first protein of the CHAMP 1 complex, or a complex-forming fragment thereof) to a DNA binding domain and fusing a second protein (e.g., a second protein of the CHAMP 1 complex, or a complex-forming fragment thereof) to an activation domain, wherein binding of the first protein to the second protein brings the DNA binding domain and the activation domain into close proximity, thereby allowing expression of a reporter gene, (b) expressing the first and second proteins in a cell, (c) contacting the cell with the one or more compound(s), (d) determining expression of the reporter gene in the presence or absence of the one or more compound(s). The reporter gene may encode a reporter protein. For example, a screening methods may comprise: (a) contacting a cell with first nucleic acid encoding a first protein of the CHAMP 1 complex, or a complex-forming fragment thereof, fused to a DNA binding domain and a second nucleic acid encoding a second protein of the CHAMP 1 complex, or a complex-forming fragment thereof, fused to an activation domain, wherein binding of the first protein to the second protein brings the DNA binding domain and the activation domain into close proximity, thereby allowing expression of a reporter protein (e.g., a luciferase or a fluorescent protein), (b) incubating the cells under conditions suitable for the expression the first and second proteins from the first and second nucleic acids, (c) contacting the cell with the one or more compound(s), and (d) determining expression of the reporter protein in the presence or absence of the one or more compound(s). In some instances, step (a) further comprises contacting the cell with a third nucleic acid encoding the reporter protein.

[171] The cell may be a bacterial cell (e.g., Escherichia coll). The cell may be a fungal cell (e.g., a yeast cell). The cell may be a mammalian cell.

[172] Alternatively, fluorescence resonance energy transfer (FRET) may be employed in an in vivo screening method. Such a methods may comprise: (a) providing a first protein of the CHAMP 1 complex, or a complex-forming fragment thereof, linked to a donor fluorescent molecule and a second protein of the CHAMP 1 complex, or a complex-forming fragment thereof, linked to an acceptor fluorescent molecule, wherein binding of the first protein to the second protein brings the donor fluorescent molecule into close proximity with the acceptor fluorescent molecule for FRET to occur, thereby allowing detection of complex formation, (b) expressing the first and second proteins in a cell, (c) contacting the cell with the one or more compound(s), (d) determining FRET in the presence or absence of the one or more compound(s). In some instances, the donor fluorescent molecule is a first fluorescent protein fused to the first protein, and the acceptor fluorescent molecule is a second fluorescent protein fused to the second protein.

Computational implementation

[173] In some aspects, a computer program is provided that comprises computer program code configured to cause one or more physical computing devices to perform a method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP 1) complex when the code is run. The computer program may be stored on a computer readable storage medium. The computer readable storage medium may be a non-transitory computer readable storage medium.

[174] FIG. 14 of the accompanying drawings schematically illustrates an exemplary computer system 100 upon which such a computer program may run. The exemplary computer system 100 comprises a computer-readable storage medium 102, a memory 104, a processor 106 and one or more interfaces 108, which are all linked together over one or more communication busses 110. The exemplary computer system 100 may take the form of a conventional computer system, such as, for example, a desktop computer, a personal computer, a laptop, a tablet, a smartphone, a server, a mainframe computer, and so on.

[175] The computer-readable storage medium 102 and/or the memory 104 may store one or more computer programs (or software or code) and/or data (including but not limited to: a first data set representing a three-dimensional structural model of a set of amino acids as specified above (for example: at least amino acids 1-87 of CHAMP 1; and/or at least amino acids 1021-1410 of POGZ) and a second data set representing one or more compound(s) as specified above (for example: one or more compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex). The computer programs stored in the computer-readable storage medium 102 and/or the memory 104 may include computer programs that, when executed by the processor 106, cause the processor 106 to carry out a method provided herein. The computer-readable storage medium 102 and/or the memory 104 may be a non-transitory computer readable storage medium. The computer-readable storage medium 102 and/or the memory 104 may be one or more memory chips or memory blocks implemented within the processor, magnetic media such as hard disk or floppy disks, or optical media such as for example DVD and the data variants thereof, CD.

[176] The processor 106 may be any data processing unit suitable for executing one or more computer readable program instructions, such as those belonging to computer programs stored in the computer-readable storage medium 102 and/or the memory 104. The processor 106 may include one or more of: general purpose computers, special purpose computers, microprocessors, digital signal processors (DSPs), application specific integrated circuits (ASIC), gate level circuits and processors based on multi-core processor architecture, as non-limiting examples. As part of the execution of one or more computer-readable program instructions, the processor 106 may store data to and/or read data from the computer-readable storage medium 102 and/or the memory 104. The processor 106 may comprise a single data processing unit or multiple data processing units operating in parallel or in cooperation with each other. The processor 106 may, as part of the execution of one or more computer readable program instructions, store data to and/or read data from the computer-readable storage medium 102 and/or the memory 104.

[177] The one or more interfaces 108 may comprise a network interface enabling the computer system 100 to communicate with other computer systems across a network. In some examples, the computer system 100 may obtain the first data set and/or the second data set via the network. The network may be any kind of network suitable for transmitting or communicating data from one computer system to another. For example, the network could comprise one or more of a local area network, a wide area network, a metropolitan area network, the internet, a wireless communications network, and so on. The computer system 100 may communicate with other computer systems over the network via any suitable communication mechanism/protocol. The processor 106 may communicate with the network interface via the one or more communication busses 110 to cause the network interface to send data and/or commands to another computer system over the network. Similarly, the one or more communication busses 110 enable the processor 106 to operate on data and/or commands received by the computer system 100 via the network interface from other computer systems over the network. [178] The interface 108 may alternatively or additionally comprise a user input interface and/or a user output interface. The user input interface may be arranged to receive input from a user, or operator, of the system 100. The user may provide this input via one or more user input devices (not shown), such as a mouse (or other pointing device, track-ball or keyboard. The user output interface may be arranged to provide a graphical/visual output to a user or operator of the system 100 on a display (or monitor or screen) (not shown). The processor 106 may instruct the user output interface to form an image/video signal which causes the display to show a desired graphical output. The display may be touch-sensitive enabling the user to provide an input by touching or pressing the display.

[179] In general, the various examples may be implemented in hardware or special purpose circuits, software, logic or any combination thereof. For example, some aspects may be implemented in hardware, while other aspects may be implemented in firmware or software which may be executed by a controller, microprocessor or other computing device, although these are not limiting examples. While various aspects described herein may be illustrated and described as block diagrams, it is well understood that these blocks may be implemented in, as non-limiting examples, hardware, software, firmware, special purpose circuits or logic, general purpose hardware or controller or other computing devices, or some combination thereof.

[180] A single processor or other unit may fulfill the functions of several items recited in the claims. The functions may be performed in a single integrated electronic device, or the functions may be distributed across different discrete devices. For example, some functions may be performed by a remote service accessed via a wired or wireless network connection. A computer program may be stored/distributed on a suitable medium, such as an optical storage medium or a solid-state medium supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless telecommunication systems.

Exemplary in silico methods

[181] Provided herein are computer programs comprising computer program code that is configured to cause one or more physical computing devices to perform a method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex when the code is run. [182] The Protein Preparation Wizard feature of the Schrodinger Small Molecule Suite 31 may be used to provide a first data set representing a three-dimensional structural model of one or both complex-forming proteins described herein. Alternatively, open-source software such as AutoDock or SwissDock may be used for this purpose.

[183] The structural model of the protein(s) may be processed by deleting crystallographic water molecules with less than three H-bonds. This can be done manually in a text editor by editing the .PDB file comprising the structural model of the protein(s). Hydrogen atoms corresponding to neutral pH may be added to represent the ionization states of amino acids. The free energy of the structural model(s) may be minimized using software such as OPLS 2005 force field or AutoDock.

[184] Similarly, Schrodinger Small Molecule Suite 31 can be used to provide a second data set representing one or more compounds. Alternatively, the three-dimensional conformations of one or more compounds can be modeled with AutoDock. The free energy of the one or more compound(s) can be minimized using software such as OPLS 2005 force field or AutoDock.

[185] The interface region between two complex-forming proteins disclosed here can be defined by the key residues that contribute to the protein-protein interaction between the two proteins, e.g., by using a 10A radius around each residue. The molecular interactions between one or both complex-forming proteins and the one or more compounds may be performed using Glide v7.8 (Schrodinger) or AutoDock.

[186] The likelihood that a compound is capable of specifically binding to the interface region of one or both complex-forming proteins may be determined by calculating the minimum energy required for binding, e.g., using Glide energy and/or E model.

[187] In an aspect, a computer program is provided comprising computer program code configured to cause one or more physical computing devices to perform a method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex when the code is run. The method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) at least amino acids 1-87 of CHAMP 1; and/or

(ii) at least amino acids 1021-1410 of POGZ; b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between CHAMP 1 or POGZ and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q56, A59, L61, H63, F68, T70 and/or K72 of CHAMP1 (e.g., amino acids Q56, L61, and T70 of CHAMP1), and amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and/or V1252 of POGZ.

[188] The method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) CHAMP1, or a portion thereof comprising amino acids Q56, A59, L61, H63, F68, T70 and K72; and/or

(ii) POGZ, or a portion thereof comprising K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252; b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between CHAMP 1 or POGZ and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q56, A59, L61, H63, F68, T70 and/or K72 of CHAMP1 (e.g., amino acids Q56, L61, and T70 of CHAMP1), and amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and/or V1252 of POGZ.

[189] In another aspect, a computer implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignmentmaintaining phosphoprotein 1 (CHAMP1) complex is provided, the method comprising: a) receiving a first data set representing a three-dimensional structural model of:

(i) CHAMP1 or a portion thereof comprising amino acids Q56, A59, L61, H63, F68, T70 and K72 (e.g., amino acids 1-87 of CHAMP1); and/or

(ii) POGZ, or a portion thereof comprising K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252 (e.g., amino acids 1021-1410 of POGZ); b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between CHAMP 1 or POGZ and the one or more compound(s); and d) determining:

(i) a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q56, A59, L61, H63, F68, T70 and/or K72 (e.g., amino acids Q56, L61, and T70), of CHAMP1 and amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and/or VI 252 of POGZ; and/or

(ii) a likelihood that the one or more compound(s) is/are capable of specifically binding to a region of CHAMP 1 comprising amino acids Q56, L61, and T70, (e.g., a region of CHAMP1 comprising amino acids Q56, A59, L61, H63, F68, T70 and optionally K72), or a region of POGZ comprising amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and/or V1252.

[190] In a further aspect, a computer program is provided comprising computer program code configured to cause one or more physical computing devices to perform a method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex when the code is run. The method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) at least amino acids 694-812 of CHAMP1; and/or

(ii) at least amino acids 1-80 of HP la; b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between CHAMP 1 or HP la and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids C715 and/or R717 of CHAMP 1 and amino acids Y20 and/or V22 of HP la.

[191] Or, the method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) CHAMP1, or a portion thereof comprising amino acids C715 and R717; and/or

(ii) HP la, or a portion thereof comprising amino acids Y20 and V22; b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between CHAMP 1 or HP la and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids C715 and/or R717 of CHAMP 1 and amino acids Y20 and/or V22 of HP la.

[192] The interface between CHAMP 1 and HP la may comprise a region of CHAMP 1 comprising amino acids C715 and R717 and a region of HP la comprising amino acids Y20 and V22. Compounds that bind to one or both of these regions may reduce, attenuate, or inhibit the formation of the CHAMP 1 complex.

[193] The three-dimensional structural model of HP la may be a homodimer.

[194] In a further aspect, a computer program is provided comprising computer program code configured to cause one or more physical computing devices to perform a method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex when the code is run. The method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) at least amino acids 560-1291 of SETDB1; and/or

(ii) at least amino acids 468-693 of POGZ; b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between SETDB1 or POGZ and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q811, N812, K813 and/or E1260 of SETDB1 and amino acids C532, H534, C535, Q546, H548, E550, N551 and/or H553 of POGZ.

[195] The method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) SETDB1, or a portion thereof comprising Q811, N812, K813 and E1260; and/or

(ii) POGZ, or a portion thereof comprising C532, H534, C535, Q546, H548, E550, N551 and H553; b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between SETDB1 or POGZ and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q811 , N812, K813 and/or E1260 of SETDB1 and amino acids C532, H534, C535, Q546, H548, E550, N551 and/or H553 of POGZ.

[196] The interface between SETDB1 and POGZ may comprise a region of SETDB1 comprising amino acids Q811, N812, K813 and E1260 and a region of POGZ comprising amino acids C532, H534, C535, Q546, H548, E550, N551 and H553. Compounds that bind to one or both of these regions may reduce, attenuate, or inhibit the formation of the CHAMP 1 complex.

[197] In another aspect, a computer program is provided comprising computer program code configured to cause one or more physical computing devices to perform a method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the CHAMP 1 complex when the code is run. The method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) a first HP la, or a portion thereof comprising amino acids N157, QI 62, VI 64, 1165 and E169 (e.g., amino acids 109-180 of a first HPla); and

(ii) a second HPla, or a portion thereof comprising amino acids N157, QI 62, VI 64, 1165 and E169 (e.g., amino acids 109-180 of a second HPla); b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between the first HPla and/or the second HPla and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of specifically binding to a region of the first and/or second HPla comprising amino acids N157, Q162, V164, 1165 and E169.

[198] Or, the method may comprise: a) receiving a first data set representing a three-dimensional structural model of:

(i) a first HPla, or a portion thereof comprising amino acids N157, QI 62, VI 64,

1165 and El 69; and/or (ii) a second HP la, or a portion thereof comprising amino acids N157, QI 62, VI 64, 1165 and E169; b) receiving a second data set representing the one or more compound(s); c) modelling one or more molecular interactions between the first HP la and/or the second HP la and the one or more compound(s); and d) determining a likelihood that the one or more compound(s) is/are capable of specifically binding to a region of the first and/or second HP la comprising amino acids N157, Q162, V164, 1165 and E169.

[199] Output of step (d) of the computer-implemented method may be used to generate a third data set representing one or more compounds that are optimized for interference and/or binding.

[200] In the above sections, receiving the first data set and/or the second data set in the context of a computer program may comprise obtaining the first data set and/or the second data from a memory device. Alternatively, receiving the first data set and/or the second data set may comprise obtaining the first data set and/or the second data set from another physical computing device.

[201] The above computer programs may be stored on a computer-readable storage medium, e.g., a non-transitory computer-readable storage medium.

NUMBERED EMBODIMENTS

[202] The following numbered embodiments are provided:

1. A method of treating a cancer in a subject in need thereof, wherein the cancer is characterized by alternative lengthening of telomeres (ALT), and the method comprises reducing, attenuating, or inhibiting the formation of the chromosome alignmentmaintaining phosphoprotein 1 (CHAMP 1) complex in the subject.

2. The method of embodiment 1, wherein the method comprises reducing, attenuating, or inhibiting the expression of one or more proteins of the CHAMP 1 complex.

3. The method of embodiment 2, wherein reducing, attenuating, or inhibiting the expression of one or more proteins of the CHAMP 1 complex comprises RNA interference (RNAi). The method of embodiment 2 or 3, wherein the one or more proteins is/are CHAMP1 and/or POGZ. The method of embodiment 1, wherein the method comprises administering a nucleic acid encoding a CHAMP 1 protein comprising an N-terminal truncation to the subject. The method of embodiment 4, wherein the N-terminal truncation removes the POGZ binding domain of the CHAMP 1 protein. The method of embodiment 4 or 5, wherein the N-terminal truncation removes amino acids 1-85 of the CHAMP 1 protein. The method of embodiment 1, wherein the method comprises administering a nucleic acid encoding a CHAMP 1 protein comprising a C-terminal truncation to the subject. The method of embodiment 8, wherein the C-terminal truncation removes the HP la binding domain of the CHAMP 1 protein. The method of embodiment 8 or 9, wherein the C-terminal truncation removes amino acids 628-812. The method of embodiment 1, wherein the method comprises targeted protein degradation of one or more proteins of the CHAMP 1 complex. The method of embodiment 11, wherein targeted protein degradation comprises a compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s). The method of embodiment 12, wherein the compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s) is a molecular glue, a Proteolysis-Targeting Chimera (PROTAC) or a Chaperone-mediated Protein Degrader (CHAMP). The method of embodiment 1, wherein the method comprises reducing or inhibiting the association of two or more proteins of the CHAMP 1 complex. The method of embodiment 14, wherein the method comprises administering to the subject one or more compound(s) that interfere(s) with interm olecular interact! on(s) between: a. CHAMP 1 and POGZ; b. CHAMP 1 and HP la; c. POGZ and HP la; and/or d. POGZ and SETDB1. The method of any one of embodiments 1-15, wherein the cancer comprises an inactivating mutation of the ATRX gene. The method of embodiment 16, wherein the cancer is a sarcoma, a glioblastoma, or a neuroblastoma. The method of embodiment 17, wherein the sarcoma is an osteosarcoma or a liposarcoma. The method of any one of embodiments 1-15, wherein the cancer is leukemia. The method of any one of embodiments 1-19, wherein the subject is an adult. The method of any one of embodiments 1-20, further comprising administering a Fanconi anemia complementation group M protein (FANCM) inhibitor to the subject. The method of any one of embodiments 1-20, further comprising administering an Ataxia telangiectasia and Rad-3 related kinase (ATR) inhibitor to the subject. A computer-implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 1-87 of CHAMP1; and/or ii. at least amino acids 1021-1410 of POGZ; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between CHAMP 1 or POGZ and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q56, A59, L61, H63, F68, T70 and/or K72 of CHAMP1 and amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and/or V1252 of POGZ; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding to a region of CHAMP 1 comprising amino acids Q56, A59, L61, H63, F68, T70 and K72 or a region of POGZ comprising amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252. A computer-implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 694-812 of CHAMP1; and/or ii. at least amino acids 1-80 of HP la; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between CHAMP 1 or HP la and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids C715 and/or R717 of CHAMP 1 and amino acids Y20 and/or V22 of HP la; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding a region of CHAMP 1 comprising amino acids C715 and R717 or a region of HP la comprising amino acids Y20 and V22. A computer-implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 791-850 of POGZ; and/or ii. at least amino acids 109-180 of HP la; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between POGZ or HP la and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and/or 1845 of POGZ and amino acids 1127, L139 and/or L150 of HP la; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding a region of POGZ comprising amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and 1845 or a region of HPla comprising amino acids 1127, L139 and LI 50. A computer-implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 560-1291 of SETDB1; and/or ii. at least amino acids 468-693 of POGZ; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between SETDB1 or POGZ and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q811, N812, K813, and/or El 260 of SETDB1 and amino acids C532, H534, C535, Q546, H548, E550, N551 and/or H553 of POGZ; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding a region of SETDB1 comprising amino acids Q811, N812, K813 and E1260 or a region of POGZ comprising amino acids C532, H534, C535, Q546, H548, E550, N551 and H553.

27. The method of any one of embodiments 23-26, wherein the output of step (d) is used to generate a third data set representing one or more compounds that are optimized for interference and/or binding.

28. A non-transitory computer readable storage medium having stored thereon a computer program comprising computer program code configured to cause one or more physical computing devices to perform the method of any one of embodiments 23-27.

EXAMPLES

[203] Although methods and materials similar or equivalent to those described herein can be used, suitable methods and materials are described below. The following examples are included for illustrative purposes only and are not intended to be limiting. Example 1. Model of the CHAMPl-POGZ-HPla complex

[204] This example demonstrates that CHAMP 1 functions as a bridge to assemble a multi-subunit CHAMP 1 complex additionally comprising POGZ and HP la.

[205] The CHAMPl-POGZ-HPla complex was originally identified as a heterochromatin complex which binds to H3K9Me3. AlphaFol d2_multimer_v3 (AF2) was used to predict individual interactions between CHAMP 1, POGZ, and HP la. Table 1 provides the amino acid sequences that were used in this analysis. The residue numbers in the text refer to the amino acids of the sequences shown in this table.

[206] Briefly, amino acid sequences for CHAMP 1 (accession# Q96JM3), POGZ (accession# Q7Z3K3), HP la (accession# P45973) and SETDB1 (accession# QI 5047) were retrieved from UniProtKB. Initially, full-length structural models of CHAMP 1 -POGZ, CHAMP 1 -HP la, homodimer of HP la and POGZ-SETDB1 complexes were predicted using a locally installed ColabFold (DOI: 10.1101/2021.10.04.463034, PMID:34265844, PMID:35637307) on a GPU machine. For each prediction, AF2 with the default parameters for the run including num_recycles=20, num_models=5 and amber relaxation were used.

[207] After analyzing the resulting structural models of the full-length protein complexes for the most likely interface from each pair, a more focused prediction was performed using truncated protein sequences involving only protein domains that were part of the interface. For CHAMP 1 -POGZ complex structure prediction, CHAMP 1 N-terminus Zn-finger domain (amino acids 1-87) and POGZ C-terminus domain (amino acids 1021- 1410) were used as input. For CHAMPl-HPla complex structure prediction, CHAMP1 C- terminus Zn-finger domain (amino acids 694-812) and HP la N-terminus domain (amino acids 1-80) were used as input. For HP la homodimerization, the HP la C-terminal domain (amino acids 109-180) were used as input sequence. For POGZ-HPla complex prediction, the POGZ HPZ-domain (amino acids 791-850) and the HP la C-terminal domain (amino acids 109-180) sequences were used as input.

[208] For each prediction, the resulting models were ranked based on the interface- predicted template modeling (ipTM) score. Only the top models from each run were used for further analyses. All structural analyses and figures were generated using Pymol (Schrodinger, Inc) and grey scale PAE metric figure were prepared using PAE viewer (PAE viewer is described in Elfmann and Stiilke Nucleic Acids Research 2023, 51(W1)W4O4- W410) for the top AF2 model from each prediction using corresponding pdb file and PAE score JSON file as input.

[209] For each PAE figure, the x and y axes denote residues for each of the two proteins, or protein domains, or motifs, for which a structural model is shown in the preceding figure. In each PAE figure, the shading at (x, y) indicates AlphaFold’s expected position error at residue x if the predicted and true structures were aligned on residue y. If the PAE is low (e.g., less than 5 as indicated in the expected position error bar) for a given residue pair x, y from two different proteins, protein domains, or motifs, it indicates that AlphaFold predicts well-defined relative positions and orientations for the residue pair.

Interaction between CHAMP 1 and POGZ

[210] The N-terminal of CHAMP 1 was predicted to interact with the C-terminal of POGZ. FIG. 1A shows the AF2-predicted structural model of the C-terminal portion of POGZ comprising amino acids 1021-1410 (POGZ-Cter) and CHAMPl’s N-terminal zinc finger domain comprising amino acids 1-87 (CHAMP I NZnF). The key residues (shown in sticks) that contribute to the formation of extended a-helix interactions between POGZ and CHAMP1 are highlighted in the inset. Amino acids Q56, A59, L61, H63, F68, T70 and K72 of CHAMP1 and amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and VI 252 of POGZ are predicted to contribute to the interm olecular interactions between CHAMP 1 and POGZ.

[211] FIG. IB shows the AF2-predicted model of the POGZ-Cter-CHAMPI NZnF complex shaded to indicate confidence in the model prediction (100-high and 50-low). The corresponding predicted aligned error plot (PAE) matrix is shown in FIG. 1C, which shows the confidence in the predicted interaction between POGZ-Cter and CHAMP I NZnF at various residue pairs, along with ipTM scores for the AF2 prediction.

Interaction between CHAMP 1 and HP la

[212] The C-terminus of CHAMP 1 was predicted to interact with the N-terminus of HP la. FIG. 2 A shows the AF2-predicted structural model of CHAMPl’s C-terminal zinc finger domain comprising amino acids 694-812 (CHAMPI CZnF) and the N-terminal portion of HPlcr comprising amino acids 1-80 (HPlcr Nter). The key residues (shown in sticks) that contribute to the formation of extended ?-sheet interactions between CHAMP 1 and HPlcr are highlighted in the inset. Amino acids C715 and R717 of CHAMP 1 and amino acids Y20 and V22 of HP la are predicted to contribute to the intermolecular interactions between CHAMP 1 and HP la.

[213] FIG. 2B shows the AF2-predicted model of the CHMPA I CZnF-HP I a Nter complex shaded to indicate confidence in the model prediction (100-high and 50-low). The corresponding PAE matrix is shown in FIG. 2C, which shows the confidence in the predicted interaction between CHAMP I CZnF and HP I a Nter at various residue pairs, along with ipTM score for the AF2 prediction.

HPla homodimer formation

[214] Furthermore, the C-terminal of HP la was predicted to form a homodimer. FIG. 3 A shows the AF2-predicted structural model of homodimerization of HP la’s C- terminus comprising amino acids 109-180 (HPla Cter). The key residues (shown in sticks) that contribute to the protein-protein interactions are highlighted in the inset. Amino acids N157, Q162, V164, 1165 and E169 are predicted to contribute to homodimer formation.

[215] FIG. 3B shows the AF2-predicted model of the HPla Cter dimer shaded to indicate confidence in the model prediction (100-high and 50-low). The corresponding PAE matrix is shown in FIG. 3C, which shows the confidence in the predicted interaction between two HPla Cter domains at various residue pairs, along with ipTM score for the AF2 prediction.

Interaction between POGZ and HPla

[216] In addition, the HPZ domain of POGZ was predicted to interact with the C- terminal of HP la. FIG. 4 A shows the AF2-predicted structural model of a portion comprising amino acids 791-850 of POGZ including the HPZ motif (POGZ HPZ) and the C-terminal portion of HP l a comprising amino acids 109-180 (HPla Cter). The key residues (shown in sticks) that contribute to the protein-protein interactions are highlighted in the inset. Amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and 1845 of POGZ and amino acids 1127, L139 and L150 of HPla are predicted to contribute to the intermolecular interactions between POGZ and HPla. Residues C817, C820, H833 and H840 of POGZ form a C2H2- type Zn finger which was shown to be critical to maintain interactions with HPla.

[217] FIG. 4B shows the AF2-predicted model of the POGZ HPZ- HPla Cter complex shaded to indicate confidence in the model prediction (100-high and 50-low). The corresponding PAE matrix is shown in FIG. 4C, which shows the confidence in the predicted interaction between POGZ HPZ motif and HP 1 cr Cter domain at various residue pairs, along with ipTM score for the AF2 prediction.

Co-immunoprecipitation studies

[218] The results of the AF2 predictions were used to model the CHAMP 1 complex comprising CHAMP 1, POGZ, and HP la, which can interact with H3K9Me3 (FIG. 5). In this model, the N-terminus of CHAMP 1 interacts with POGZ, while the C-terminus of CHAMP 1 directly engages with an HP la dimer. Additionally, HP la is known to have a direct interaction with H3K9Me3. Based on this model, a series of CHAMP 1 mutants were generated (FIG. 6A).

[219] Expression plasmids encoding the CHAMP 1 mutants shown in FIG. 6A fused to GFP were used to transfect 293T cells. After transfection for 48 hours, 293T cells were collected and subjected to lysis using NETN lysis buffer containing a proteinase and phosphatase inhibitor cocktail (ThermoFisher, 1 : 100) for 30 minutes on ice. The lysed samples were incubated overnight at 4°C with an antibody-bead conjugate (GFP-Trap_A; Chromotek). The beads were then thoroughly washed four times with NETN lysis buffer, and the immunoprecipitated materials were eluted by boiling. Western blot analysis was conducted to detect the immunoprecipitates, and the intensities of the resulting bands were quantified using ImageJ. The results of these experiments are summarized in FIG. 6B, FIG. 6C and FIG. 6D.

[220] Consistent with the model shown in FIG. 5, an N-and C-terminal mutant (CHAMP 1 - ANAC), but not an N-terminal-only mutant (CHAMP 1 -AN), failed to bind HP 1 a (FIG. 6B, lanes 8 and 7, respectively). Furthermore, the N-terminal truncations of CHAMP 1 (CHAMP 1 -AN and CHAMP 1 -ANAC) failed to bind to POGZ (FIG. 6B, lanes 7 and 8). The model was further validated by performing coimmunoprecipitation with a GFP-fused CHAMP1 mutant, in which three amino acids Q56, L61, T70 in the CHAMP1 N-terminus were substituted with arginines (GFP-CHAMP1^QLT/RRR), that showed loss of binding to POGZ (FIG. 6C, lane 6). Instead, this CHAMP 1 triple mutant had increased binding to REV7 (FIG. 6C, lane 6).

[221] Notably, a known dimerization mutant of HP la, HPla^I165E, failed to coimmunoprecipitate with CHAMP 1 but still coimmunoprecipitated with H3K9Me3 and POGZ (FIG. 6D, lane 6). This indicates that POGZ and CHAMP 1 each independently interact with HP 1 a and H3K9me3. The direct interaction between POGZ and HP 1 a observed in CHAMP 1 knockout cells further validated the model shown in FIG. 5.

[222] Taken together, the results of these coimmunoprecipitation experiments support a conclusion that CHAMP 1 functions as a bridge to assemble a multi-subunit CHAMP 1 complex comprising POGZ and HP la. This complex may facilitate the bridging of two distinct heterochromatin regions.

Example 2. CHAMP1 and POGZ promote the formation of heterochromatin clusters

[223] This example illustrates the role that CHAMP 1 and POGZ play in promoting the formation of heterochromatin clusters.

[224] The localization of CHAMP 1 and POGZ within heterochromatin foci was assessed using an immunofluorescent-fluorescence in situ hybridization (IF -FISH) assay in human U2OS cells. U2OS cells are derived from a moderately differentiated sarcoma of the tibia.

[225] Cells were seeded onto glass coverslips placed in 24-well plates. Subsequently, they were either left untreated or exposed to 5 Gy of ionizing radiation (IR). After 6 hours, the cells were collected by pre-extraction with 0.5% Triton X-100 for 5 minutes, followed by fixation with 4% paraformaldehyde for 10 minutes at 4°C. After three PBS washes, a blocking step was carried out using 3% BSA in PBS for 1 hour at room temperature. This was followed by consecutive incubations with primary and secondary antibodies, conducted overnight at 4°C and 1 hour at room temperature, respectively.

[226] The coverslips were first stained with the primary and secondary antibodies, fixed for 10 minutes at room temperature and dehydrated in ethanol series. After denaturation at 85°C for 5 minutes, coverslips were incubated with TelG-Cy3 or TelC-Alexa488 PNA probe (PNAbio) overnight at 37°C, then washed. Finally, the coverslips were mounted with DAPI (Vector Laboratories) and imaged using a Zeiss AX10 fluorescence microscope and Zen software. Foci were then counted, with a minimum of 100 cells assessed for each sample.

[227] Colocalization of CHAMP 1 was observed within heterochromatin foci, marked by H3K9me3, in human U2OS cells. This colocalization was further enhanced by the induction of DNA double-strand breaks using irradiation at 5 Gy (FIG. 7 A). [228] The gene for either protein was knocked out in U2OS cells using CRISPR. sgRNAs targeting CHAMP 1 and POGZ, respectively, were either cloned into the pSpCas9 BB-2A-GFP (PX458) vector (GenScript) or introduced into the cell together with the Cas9 protein via electroporation (Lonza) according to the manufacturer’s protocol. After Cas9- gRNA PX458 plasmid transfection, GFP-positive cells were sorted using a BD FACSAria II cell sorter 48 hours post-transfection. A GFP-positive pool or single cells were screened for knockouts by western blotting.

[229] The CRISPR-knockout of CHAMP 1 or POGZ resulted in a significant reduction in the formation of H3K9me3 foci (FIG. 7B and FIG. 7C) and HPla foci (FIG. 7D and FIG. 7E). Neither knockout affected the overall expression level and chromatin binding activity of H3K9me3 and HPla, as shown in lane 10 of FIG. 7F for the CHAMP 1 knockout and in lane 10 of FIG. 7G for the POGZ knockout. Furthermore, CHAMP 1 depletion resulted in more heterochromatin damage, as indicated by the colocalization of H3K9me3 and yH2AX foci (FIG. 7H).

[230] Taken together, these findings indicated that CHAMP 1 and POGZ play a role in promoting the formation of heterochromatin clusters.

Example 3. The CHAM Pl complex is upregulated in ATRX-mutated ALT tumors

[231] This example illustrates that ALT tumors harboring ATRX mutations require the upregulation of the CHAMP 1 complex to preserve telomere heterochromatin integrity and promote the ALT pathway.

[232] ALT tumor cells display several unique cellular features including increased telomeric homologous recombination (HR) repair activity and enhanced H3K9Me3 deposition. ALT activity is linked to the alpha thalassemia/mental retardation syndrome X- linked (ATRX) gene mutations across various tumor types. ATRX functions as an inhibitor of ALT. The overexpression of ATRX in ATRX-mutant cells suppresses ALT markers. ATRX gene mutations result in the impairment of heterochromatin, and this dysfunctional heterochromatin is associated with the activation of the ALT pathway.

[233] To investigate the relationship between ATRX and the CHAMP 1 complex, neuroblastomas were examined which had been classified as ALT positive. The cells were lysed using RIPA buffer supplemented with a cocktail of phosphatase and protease inhibitors from Roche. Cell lysates were separated by electrophoresis using NuPAGE 4-12% Bis-Tris gels (Invitrogen) and transferred onto nitrocellulose membranes. These membranes were then blocked with 5% BSA in TBST (Tris-buffered saline with Tween) and subsequently incubated with primary and secondary antibodies. The detection was accomplished using either chemiluminescence or fluorescence (LI-COR Biosciences). In comparison to ALT ATRX-wild type tumors, the protein levels of the CHAMP 1 complex were upregulated in these ALT ATRX-mutated tumors (FIG. 8). Specifically, FIG. 8 shows decreased CHAMP 1, HP la, POGZ and SETDB1 protein expression in ALT ATRX- wild-type (WT) compared to mutated (Mut) tumors from ALT-positive neuroblastoma patients.

[234] These results indicate that ALT tumors harboring ATRX mutations require the upregulation of the CHAMP 1 complex to preserve telomere heterochromatin integrity and promote the ALT pathway. Accordingly, reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex may impair the functioning of the ALT pathway. Impairment of this pathway may be useful in treating cancers that are dependent on ALT to maintain their telomeres. Such cancers may be identifiable by the presence of one or more inactivating mutations in the ATRX gene.

Example 4. The CHAMP1 complex maintains the ALT pathway

[235] This example illustrates that the CHAMP 1 complex plays a central role in maintaining the ALT pathway in ALT tumor cells.

[236] The human sarcoma cell line U2OS cells, in the presence or absence of the intact CHAMP 1 complex.

[237] Quantitative telomere fluorescence in situ hybridization (Q-FISH) and Telomere Restriction Fragment (TRF) assays were used to determine telomere length as described by Dilley et al. (Nature 2016; 539(7627):54-5). Q-FISH experiments were performed as previously described (Li et al., Mol Cell. 2018; 70(3):395-407.e4). Cell synchronization was achieved through 4-6 hours nocodazole treatment. Cells were collected and suspended in 0.075M KC1 for 25 minutes before fixation in methanol/acetic acid (3: 1) for subsequent spreading. Fluorescence in situ hybridization (FISH) was conducted following established procedures with TelG-Cy3 PNA probe (PNAbio).

[238] Telomere restriction fragment (TRF) analysis was used to measure telomere length. Genomic DNA was purified with DNeasy Blood & Tissue Kit (Qiagen, 69506), and measured (Qubit 3.0 Fluorometer, Thermo Fisher Scientific). Five pg gDNA was treated with Hinf I (New England Biolabs, R0155S) and Rsa I (New England Biolabs, R0167L). Electrophoresis was performed with the CHEF-DRII system (Bio-Rad). DNA samples were loaded in 1% pulse field (PFGE) certified agarose (1620137, BioRad) in 0.5* TBE buffer using the following parameters: 4 V/cm; initial switch time 5 seconds, final switch time 5 seconds, 20 hours for ALT cell lines; or 10 hours for non -ALT cell lines at 14 °C. The gels were dried for 4 hours at 42 °C, stained with Ethidium Bromide (Sigma-Aldrich, 2375), and subjected to denatured in-gel hybridization with P-32 labeled telomeric C-probe prepared as described by Zhao et al. (Methods Mol Biol. 2011; 735:47-54).

[239] CRISPR knockout of CHAMP 1 or POGZ in these cells significantly reduced telomere length (FIG. 9A and FIG. 9B). Specifically, sgCHAMPl and sgPOGZ U2OS cells were analyzed by Q-FISH using a Cy5-labeled telomere probe. sgRNA targeting CHAMP 1 and POGZ were transfected in U2OS cells for more than 6 months. Subsequent correction with wildtype CHAMP 1 restored telomere length in CHAMP 1 knockout cells (FIG. 9C and FIG. 9D). Additionally, a substantial increase in telomere loss was observed in U2OS cells with knockout of CHAMP 1 or POGZ (FIG. 9E), further demonstrating the importance of CHAMP 1 and POGZ in telomere maintenance of ALT tumor cells. The telomere shortening from CHAMP 1 downregulation did not occur in telomerase-positive cell lines, 293 T and RPEl-hTERT.

[240] DNA double-strand breaks (DSB) at telomeres initiate long-range movements and clustering of telomeres, which is crucial for homology-directed telomere synthesis. TRFl-FokI was used to induce DSBs specifically at telomeres, as shown in FIG. 10A. The resultant telomere clustering was examined. Telomere foci size and clustering was measured by Imaged using a consistent threshold to images followed by binarization as described by Cho et al. (Cell. 2014; 159: 108-121). The sizes of the foci were quantified in square pixels for each telomeric focus within a nucleus, and the average size was computed for each analyzed nucleus.

[241] TRFl-Fokl-induced telomere clustering, as indicated by an increase in the average size of telomere foci and a reduction in the number of telomeres involved, was significantly diminished in CHAMP 1 or POGZ knockout U2OS cells. Representative images are shown in FIG. 10B and quantification obtained from such images is shown in FIG. 10C. [242] ALT telomeres are associated with nuclear bodies formed by the promyelocytic leukemia (PML) protein, and when so associated are referred to as ALT- associated PML bodies (APBs). Depletion of either CHAMP1 or POGZ through gene deletion using CRISPR led to a decrease in multiple hallmarks of ALT recombination, including a significant reduction in APBs (FIG. 11 A) and a decrease in telomere-associated DNA synthesis, as shown by the incorporation of the thymidine analog 5-ethynyl-2’- deoxyuridine (EdU) in G2 phase cells (FIG. 11B). To visualize telomeric DNA synthesis, U2OS cells were synchronized in G2 by treatment with 15 pM CDKli (RO-3306) for 16 hours with or without the addition of doxycycline. Cells were incubated with 20 pM EdU for an additional 2 hours, with or without 4-OH tamoxifen, and then fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes at room temperature, blocked with 10% normal goat serum for 1 hour at 4°C then incubated overnight with anti-TRF2 antibody. Cells were then washed with 0.1% Triton X-100 in PBS, incubated with fluorescently-labeled secondary antibody. EdU was labeled with fluorescent dye using Click-iT EdU kit (Invitrogen) according to the manufacturer’s protocol. DNA with stained with DAPI and z-stack images were acquired using Zeiss AxioObserver microscope at 63x magnification. Images were analyzed using Imaged software and the number of EdU+ TRF2 foci was assessed in at least 150 cells from 3 independent experiments.

[243] sgCHAMPl U2OS-TRFl-FokI cells were transfected with empty vector (EV; negative control), CHAMP 1 -wild-type (WT), a CHAMP 1 N -terminal deletion mutant (AN) or a CHAMP 1 C-terminal deletion mutant (AC), followed by PML immunostaining combined with telomere FISH. Wild-type U2OS cells served as a positive control. Quantification of PML-TelC colocalizations (APBs) were shown as indicated in FIG. 11C, where error bars indicate SEM. An N-terminal deletion mutant of CHAMP 1 which loses the POGZ binding activity (see Example 1) also exhibited a defect in APB formation (FIG. 11C).

[244] Consistent with this result, the expression of wild-type CHAMP 1 but not AN CHAMP 1 in U2OS cells with a CRISPR-induced knockout of CHAMP 1 was able to prevent telomere-specific damage (FIG. 11D). Specifically, sgCHAMPl U2OS cells were transfected with empty vector (EV; negative control), CHAMP 1 -wild-type (WT), a CHAMP 1 N -terminal deletion mutant (AN), or a CHAMP 1 C-terminal deletion mutant (AC), followed by TRF2 and yH2AX immunostaining. TRF2-yH2AX colocalizations were quantified. Wild-type U2OS cells served as a positive control.

[245] Taken together, these results indicate that the CHAMP 1 complex plays a central role in facilitating telomere clustering and maintaining the ALT pathway, in particular in ALT tumor cells. The results provide further support that reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex can be used to impair the functioning of the ALT pathway in tumor cells that depend on this pathway for maintaining the length of their telomeres. Since the interaction between CHAMP 1 and POGZ is crucial for formation of the complex, small molecule inhibitors that interfere with this interaction may therefore be effective in treating ALT-dependent tumors.

Example 5. CHAMPl-HPla interaction is required for ALT pathway function

[246] This example demonstrates that the CHAMPl-HPla interaction plays a central role the functioning of the ALT pathway.

[247] Specifically, for the experiments summarized in FIG. 12A, U2OS-TRFl-FokI cells were treated with siREV7 or siHPla, followed by examination by FISH using a FITC- labeled telomere probe after D0X/4-0HT treatment. Average telomere foci size per nucleus was calculated using Imaged. Error bars indicate SEM. For the experiments summarized in FIG. 12B, U2OS-TRFl-FokI cells were treated with siREV7 or siHPla, followed by examination of PML immunostaining combined with telomeric FISH after D0X/4-0HT treatment. Quantification of PML-TelC colocalizations (APBs) were shown. Error bars indicate SEM. siRNA knockdowns were performed using RNAiMax (Invitrogen) as the transfection reagent, following the manufacturer’s guidelines.

[248] A C-terminal deletion mutant of CHAMP 1 (see Example 1), which loses the HP la binding activity, could not rescue the decrease of APB formation caused by CRISPR- mediated CHAMP 1 depletion (FIG. 11C). Consistent with this observation, the expression of AC CHAMP 1 in U2OS cells with a CRISPR-induced knockout of CHAMP 1 led to telomere-specific damage (FIG. 11D). These results highlight the role of interaction of CHAMP1 and HPla in the ALT pathway. HPla was found to promote ALT, as evidenced by the decrease of damage-induced telomere clustering and decrease in ALT-associated PML bodies (APBs) in U2OS cells following an siRNA-induced knockdown of HPla expression (FIG. 12 A, FIG. 12B). [249] Through the detection of telomere clustering and APB formation, it was confirmed that the ALT pathway could be restored by expression of wild-type HP la in U2OS cells in which HP la had been knocked out using CRISPR. In contrast, expression of the HPla I165E mutant was unable to restore ALT pathway function (FIG. 12C and FIG. 12D). Specifically, for the experiments summarized FIG. 12C, sgHPla U2OS-TRFl-FokI cells were transfected with empty vector (EV; negative control), HP la- wild-type (WT), or a HPla-H65E mutant (I165E), followed by examination by FISH using a FITC-labeled telomere probe after D0X/4-0HT treatment. Wild-type U2OS cells served as a positive control. Average telomere foci size per nucleus was calculated using Imaged. Error bars indicate SEM. For FIG. 12D, sgHPla U2OS-TRFl-FokI cells were transfected with empty vector (EV; negative control), HP la- wild-type (WT), or HPla-I165E mutant (I165E), followed by examination by PML immunostaining combined with telomeric FISH after D0X/4-0HT treatment. Wild-type U2OS cells served as a positive control. Quantification of PML-TelC colocalizations (APBs) were shown. Error bars indicate SEM.

[250] Taken together, these results indicate that the CHAMP 1 -HP la interaction is important for the functioning of the ALT pathway. Small molecule inhibitors that interfere with this interaction may therefore be effective in treating ALT-dependent tumors.

Example 6. The CHAM Pl complex recruits SETDB1

[251] This example demonstrates that the CHAMP 1 complex recruits SETDB1 to mediate telomere heterochromatin formation.

[252] To further examine the regulation of telomere heterochromatin, additional experiments focused on the H3K9 methyltransferases, specifically SETDB 1. Double-strand breaks were induced in U2OS cells or U2OS cells treated with sgCHAMPl (sgC) or sgPOGZ (sgP) using TRFl-FokI for 2 h. TRF-FokI was fused to mCherry and therefore could be used to identify telomeres with double-strand breaks. DAPI was used to stain the nuclei. Immunofluorescence was used to detect SETDB 1. Colocalization events of SETDB 1 and telomeres with double-strand breaks were quantified. The results are shown in FIG. 13 A. Error bars indicate SEM. The number of colocalization events were reduced in CHAMP 1 or POGZ knockout U2OS cells following TRFl-FokI induction. Therefore, it was hypothesized that the CHAMP 1 complex promotes SETDB 1 recruitment to telomeres. [253] A direct interaction of POGZ with SETDB1 was investigated using AlphaFol d2_multimer_v3 (AF2) using the same methodology described in Example 1. The amino acid sequences used for this analysis are shown in Table 1. The residue numbers in the text refer to the amino acids of the sequences shown in this table. The POGZ Zn-finger core (amino acids 468-693) and a C-terminal portion of SETDB1 which incorporates the methyl -CpG-binding domain (MBD) and two SET domains (amino acids 560-1291) were used as input.

[254] A strong interaction between SETDB1 and POGZ was predicted. FIG. 13B shows an AF2-predicted complex of the core portion of POGZ’s zinc finger domain comprising amino acids 468-693 (POGZ ZnFcore) and the C-terminal portion of SETDB1 comprising amino acids 560-1291 (SETDBI Cter). The key residues (shown in sticks) that contribute to the protein-protein interactions are highlighted in the inset. They include Q811 , N812, K813, and E1260 of SETDB1 and amino acids C532, H534, C535, Q546, H548, E550, N551 and H553 from POGZ. Amino acids C532, C535, H534, H548 and H553 of POGZ may form a C2H2-type zinc finger that could be important in maintaining interaction between POGZ and SETDB1.

[255] FIG. 13C shows the AF2-predicted model of POGZ ZnFcore-SETDBI Cter complex shaded to indicate confidence in the model prediction (100-high and 50-low (red)). The corresponding PAE matrix shown in FIG. 13D shows the confidence in the predicted interaction between POGZ ZnFcore and SETDBI Cter domains along with interface- predicted template modeling (ipTM) scores for the AF2 prediction. The ipTM score was 0.85.

[256] Based on the AF2 prediction, the previously generated CHAMP 1 mutants (see Example 1) were used in co-immunoprecipitation experiments probing for SETDB1. The results of these experiments are summarized in FIG. 13E. FIG. 13E shows western blots showing GFP-immunoprecipitation of an empty vector control, full-length GFP-CHAMP1 (GFP-C-FL), a GFP-CHAMP1 N-terminal deletion mutant (GFP-C-AN), a GFP-CHAMP1 C-terminal deletion mutant (GFP-C-AC), or GFP-CHAMP1 N-and C-terminal deletion mutant (GFP-C-ANAC). The co-immunoprecipitation of endogenous SETDB1 and POGZ was assessed by probing blots with SETDB1- and POGZ-specific antibodies. GAPDH acts as a negative control for immunoprecipitation (IP). The N-terminal deletion of CHAMP 1, which drastically reduces the interaction with POGZ, also substantially reduced the interaction of CHAMP 1 and SETDB1. [257] Collectively, these findings indicate that the CHAMP 1 complex recruits SETDB 1 via direct interactions with POGZ to mediate telomere heterochromatin formation. Various small molecule SETDB 1 inhibitors that either target its catalytic domain or its tandem Tudor domain were previously identified. These may find utility in the treatment of ALT-dependent cancers. Alternatively, small molecule inhibitors that interfere with the interaction between POGZ and SETDB 1 may provide a new therapeutic modality for such cancers.

Example 7. FANCM expression increases when CHAMP1 is knocked out

[258] This example demonstrates that reduction of the CHAMP 1 complex results in an increase of Fanconi anemia complementation group M protein (FANCM) expression in ALT-dependent cancer cells.

[259] ALT telomeres have elevated replication stress, and this enhancement of replication stress is required for the ALT mechanism of telomere maintenance. Recent studies (Pan, X. etal., 2017 , Proc Natl Acad Sci USA 114, E5940-E5949; Lu, R. etal., 2019, Nat Commun 10, 2252; Silva, B. et al., 2019, Nat Commun 10, 2253) indicate that FANCM protein and its ATPase activity are elevated in ALT telomeres and may function to alleviate replication stress and to maintain tumor cell viability. As shown herein, the CHAMP 1 complex also functions, at least in part, to reduce replication stress. A reduction, attenuation, or inhibition of FANCM activity may be lethal in ALT tumor cells in which formation of the CHAMP 1 complex is reduced, attenuated, or inhibited as a result of excessive replication stress.

[260] To test a possible interaction between CHAMP 1 and FANCM, CHAMP 1 was knocked out in U2OS cells as described in Example 5. The presence of micronuclei, a marker of replication stress, was assessed using immunofluorescence microscopy (FIG. 15 A). Compared to U2OS control cells, significantly more micronuclei were observed in U2OS CHAMP 1 knockout cells (FIG. 15B).

[261] TRFl-FokI induction was performed in U2OS wildtype and CHAMP 1 knockout cells as described in Example 6. The telomeric proteome of the knockout cells was assessed relative to wild-type control cells. FANCM was found to be the most differentially enriched protein in the telomeric proteome (FIG. 15C). [262] This example shows that reducing, attenuating, or inhibiting the activity of FANCM in addition to reducing, attenuating, or inhibiting the formation of the CHAMP 1 complex may result in enhanced anti-tumor activity against ALT-dependent cancers.

Example 8. FANCM inhibition in CHAMP1 complex-depleted ALT cancer cells

[263] This example demonstrates that inhibition of the FANC pathway reduces the survival of ALT-dependent cancer cells in which CHAMP 1 complex formation is reduced, attenuated, or inhibited.

[264] Example 7 demonstrates that reducing, attenuating, or inhibiting formation of the CHAMP1 complex in ALT-dependent cancer cells results in an increase in FANCM expression. Therefore, it was hypothesized that combining a compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex with a FANCM inhibitor may be lethal for ALT-dependent cancer cells.

[265] To test this hypothesis, CHAMP 1 or POGZ was knocked out in U2OS cells with sgRNAs targeting CHAMP 1 and POGZ, respectively, using CRISPR as described in Example 2. Unmodified U2OS cells (“WT”) were used as a control. Specifically, clonogenic assays were performed as described previously (Li, F. et al. Mol Cell 80, 410-422 e416 (2020)). Briefly, each of the three cell populations was seeded separately at 500 cells/well in 6-well plates. After 24 hours, the FANCM inhibitor PIP-199 was added at the specified concentrations, and the cells were permitted to grow for 8 days. Colony formation was scored by fixing and staining with 0.5% (w/v) crystal violet in 20% methanol. The results of this assay are summarized in FIG. 16.

[266] Fewer CHAMP 1 and POGZ knockout U2OS cells survived compared to WT U2OS cells in the presence of PIP-199. Less than 30% of the U2OS POGZ knockout cells survived in the presence of 1 pM PIP- 199. The effect was even more pronounced in U2OS CHAMP 1 knockout cells. Only about 20% of the U2OS CHAMP 1 knockout cells survived in the presence of 0.5 pM PIP- 199. This is in stark comparison to WT U2OS cells. Administration of 0.5 pM PIP-199 had no impact on the survival of the control cells. Even in the presence of 1 pM PIP-199 only about a 10% decrease in survival was observed.

[267] This example shows that treatment of ALT-dependent cancer cells with a compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex can be rendered more effective when it is combined with a FANCM inhibitor. Example 9. ATR inhibition in CHAMP1 complex-depleted ALT cancer cells

[268] This example demonstrates that inhibition of Ataxia telangiectasia and Rad-3 related kinase (ATR) reduces the survival of ALT-dependent cancer cells in which the CHAMP 1 complex formation is reduced, attenuated, or inhibited.

[269] ATR is activated by replication stress and is known for its role in homologous recombination-mediated repair of double-strand breaks. Therefore, it was hypothesized that combining a compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex with an ATR inhibitor may be lethal for ALT-dependent cancer cells.

[270] To test this hypothesis, CHAMP 1 or POGZ was knocked out in U2OS cells with sgRNAs targeting CHAMP 1 and POGZ, respectively, using CRISPR as described in Example 2. Unmodified U2OS cells (“WT”) were used as a control. Clonogenic assays were performed as described in Example 8. Each of the three cell populations was seeded at 500 cells/well in 6-well plates. After 24 hours, the ATR inhibitor VE822 was added at the specific concentrations, and the cells were permitted to grow for 8 days. Colony formation was scored by fixing and staining with 0.5% (w/v) crystal violet in 20% methanol. The results of this assay are summarized in FIG. 17.

[271] Fewer CHAMP 1 and POGZ knockout U2OS cells survived compared to WT U2OS cells in the presence of VE822. Less than 30% of the U2OS POGZ knockout cells survived in the presence of 25 nM VE822. The effect was even more pronounced in U2OS CHAMP 1 knockout cells. Only about 20% of the U2OS CHAMP 1 knockout cells survived in the presence of 25 nM VE822. This is in stark comparison to WT U2OS cells, where even in the presence of 25 nM VE822 only about a 15% decrease in survival was observed.

[272] This example demonstrates that knockout of the CHAMP 1 complex increases telomere replication stress and sensitizes the ALT cells to drugs which further increase replication stress. The example also shows that treatment of ALT-dependent cancer cells with a compound that reduces, attenuates, or inhibits the formation of the CHAMP 1 complex can be rendered more effective when it is combined with an ATR inhibitor.

Example 10. Targeted protein degradation of a protein of the CHAMP1 complex

[273] This example demonstrates targeted protein degradation of a protein of the CHAMP1 complex (SETDB1). [274] As demonstrated in Example 6, the CHAMP 1 complex recruits SETDB1 via direct interactions with POGZ to mediate telomere heterochromatin formation. In particular, SETDB1 catalyzes di- and tri -methylation of the 9^th lysine of histone H3 (H3K9me2 and H3K9me3, respectively) on euchromatin.

[275] (R,R)-59 was previously identified as SETDB1 inhibitor (see Guo et al., supra). A SETDB1 degrader was prepared by coupling (R,R)-59 via a flexible linker to an E3 ligand. To determine whether the degrader can specifically bind to SETDB1 and target it for degradation through interactions with a ubiquitin ligase, U2OS cells were treated with 1 pM or 10 pM of the degrader for 72 hours. In addition, to determine whether inhibition of SETDB1 resulted in a reduction of histone H3 methylation comparable to that observed with the degrader, U2OS cells were separately treated with 1 pM or 10 pM of (R,R)-59. Untreated U2OS cells served as a negative control.

[276] At the end of the 72 hours, the cells were lysed, and western blot analysis was performed using the cell lysates. The blots were probed with antibodies directed against GAPDH, SETDB1, histone H3, and H3K9me3. GAPDH served as a loading control.

[277] After 72 hours of treatment with 10 pM of the SETDB1 degrader, a marked reduction in SETDB1 levels compared to the negative control was observed. Notably, this treatment also resulted in a marked reduction in H3K9me3 compared to the negative control. Treatment with 1 pM of the SETDB1 degrader resulted in a lesser reduction of SETDB1 levels and had no appreciable effect on histone H3 methylation.

[278] 72 hours of treatment with 1 pM (R,R)-59 did not result in a detectable reduction of H3K9me3 levels. However, 72 hours of treatment with 10 pM (R,R)-59 resulted in a marked reduction of H3K9me3 levels, but the reduction was slightly less pronounced than after treatment with the corresponding concentration of the SETDB1 degrader.

[279] This example demonstrates that reducing the formation of the CHAMP 1 complex by targeted degradation of a protein of the CHAMP 1 complex decreases H3K9me3 levels and therefore alleviates heterochromatin formation. Accordingly, such inhibitors or degraders may be used in treating a cancer characterized by ALT. [280] It should be understood that the particular embodiments described herein are given by way of illustration only, not limitation. Other features, objects, and advantages are apparent from the above detailed description, drawings and examples. Various changes and modifications will be apparent to those skilled in the art.

[281] All patents patent publication, and non-patent publications referenced herein are indicative of the level of skill of those skilled in the art to which this invention pertains. All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

Claims

1. A method of identifying one or more compounds for treating a cancer characterized by alternative lengthening of telomeres (ALT), comprising: (i) providing a first polypeptide comprising all or a complex-forming portion of a protein of the chromosome alignmentmaintaining phosphoprotein 1 (CHAMP 1) complex; contacting said first polypeptide or protein with an interaction partner of said first polypeptide or protein of the CHAMP 1 complex, and (ii) determining formation of a complex between the first polypeptide and the interaction partner in the presence or absence of the one or more compound(s), wherein the one or more compounds may be capable of treating the cancer if they reduce, attenuate or inhibit formation of the complex.

2. The method of claim 1, wherein: a. the first polypeptide comprises amino acids 1-87 of chromosome alignmentmaintaining phosphoprotein 1 (CHAMP 1) and the interaction partner is a second polypeptide comprising amino acids 1021-1410 of Pogo transposable element with zinc finger domain (POGZ); b. the first polypeptide comprises amino acids 694-812 of CHAMP 1 and the interaction partner is a second polypeptide comprising amino acids 1-80 of Heterochromatin Protein la (HP la); c. the first polypeptide comprises 791-850 of POGZ and the interaction partner is a second polypeptide comprising amino acids amino acids 109-180 of HP la; or d. the first polypeptide comprises 560-1291 of SET domain bifurcated histone methyltransferase 1 (SETDB1) and the interaction partner is a second polypeptide comprising amino acids 468-693 of POGZ.

3. A computer-implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 1-87 of CHAMP1; and/or ii. at least amino acids 1021-1410 of POGZ; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between CHAMP 1 or POGZ and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q56, A59, L61, H63, F68, T70 and/or K72 of CHAMPl and amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and/or V1252 of POGZ; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding to a region of CHAMP1 comprising amino acids Q56, A59, L61, H63, F68, T70 and K72 or a region of POGZ comprising amino acids K1223, L1235, L1240, S1244, S1247, L1249, P1250 and V1252.

4. A computer-implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP 1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 694-812 of CHAMP 1; and/or ii. at least amino acids 1-80 of HP la; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between CHAMP 1 or HP la and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids C715 and/or R717 of CHAMP 1 and amino acids Y20 and/or V22 of HP la; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding a region of CHAMP 1 comprising amino acids C715 and R717 or a region of HPla comprising amino acids Y20 and V22.

5. A computer implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 791-850 of POGZ; and/or ii. at least amino acids 109-180 of HP la; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between POGZ or HP la and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and/or 1845 of POGZ and amino acids 1127, L139 and/or L150 ofHPla; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding a region of POGZ comprising amino acids 1813, L815, C817, C820, D828, H833, L834, H840 and 1845 or a region ofHPla comprising amino acids 1127, L139 and L150.

6. A computer-implemented method of identifying one or more compound(s) that reduce, attenuate, or inhibit the formation of the chromosome alignment-maintaining phosphoprotein 1 (CHAMP1) complex, wherein the method comprises: a. receiving a first data set representing a three-dimensional structural model of: i. at least amino acids 560-1291 of SETDB1; and/or ii. at least amino acids 468-693 of POGZ; b. receiving a second data set representing the one or more compound(s); c. modelling one or more molecular interactions between SETDB1 or POGZ and the one or more compound(s); and d. determining: i. a likelihood that the one or more compound(s) is/are capable of interfering with one or more intermolecular interactions between amino acids Q811, N812, K813, and/or E1260 of SETDB1 and amino acids C532, H534, C535, Q546, H548, E550, N551 and/or H553 of POGZ; and/or ii. a likelihood that the one or more compound(s) is/are capable of specifically binding a region of SETDB1 comprising amino acids Q811, N812, K813 and E1260 or a region of POGZ comprising amino acids C532, H534, C535, Q546, H548, E550, N551 and H553.

7. The method of any one of claims 3-6, wherein the output of step (d) is used to generate a third data set representing one or more compounds that are optimized for interference and/or binding.

8. A non-transitory computer readable storage medium having stored thereon a computer program comprising computer program code configured to cause one or more physical computing devices to perform the method of any one of claims 3-7.

9. A method of treating a cancer in a subject in need thereof, wherein the cancer is characterized by alternative lengthening of telomeres (ALT), and the method comprises reducing, attenuating, or inhibiting the formation of the chromosome alignmentmaintaining phosphoprotein 1 (CHAMP1) complex in the subject.

10. The method of claim 9, wherein the method comprises targeted protein degradation of one or more proteins of the CHAMP 1 complex.

11. The method of claim 10, wherein targeted protein degradation comprises a compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s).

12. The method of claim 11, wherein the compound that specifically binds to one or more proteins of the CHAMP 1 complex and interacts with one or more ubiquitin ligase(s) is a molecular glue, a Proteolysis-Targeting Chimera (PROTAC) or a Chaperone-mediated Protein Degrader (CHAMP).

'll

13. The method of any one of claims 9-12, wherein the cancer comprises an inactivating mutation of the ATRX gene.

14. The method of claim 13, wherein the cancer is a sarcoma, a glioblastoma, or a neuroblastoma.

15. The method of claim 14, wherein the sarcoma is an osteosarcoma or a liposarcoma.

16. The method of any one of claims 9-12, wherein the cancer is leukemia.

17. The method of any one of claims 9-16, wherein the subject is an adult.

18. The method of any one of claims 9-17, further comprising administering a Fanconi anemia complementation group M protein (FANCM) inhibitor to the subject.

19. The method of any one of claims 9-18, further comprising administering an Ataxia telangiectasia and Rad-3 related kinase (ATR) inhibitor to the subject.