Aneuploidy-associated drug targets in lung cancer

Abstract

Provided are compositions and methods that relate to cells that exhibit aneuploidy. Methods for promoting aneuploidy in mammalian cells are provided. Methods for treating cancers that exhibit high aneuploidy are provided. The compositions and methods include a fusion protein that contains an auroraB protein segment and a dCas9 segment. Polynucleotides and expression vector encoding the fusion protein are also provided. Single-guide RNAs that are functional with the fusion protein and can be used to promote aneuploidy are provided.

Description

[0001] Attorney Docket No.: 058636.00829

[0002] ANEUPLOIDY-ASSOCIATED DRUG TARGETS IN LUNG CANCER

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. provisional patent application no. 63 / 687,886, filed August 28, 2024, the entire disclosure of which is incorporated herein by reference.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under R37 CA248631 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] SEQUENCE LISTING

[0008] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy was created on August 28, 2025, is named “058636.00829.xml” and is 13,256 bytes in size.

[0009] RELATED INFORMATION

[0010] Globally, cancer incidences and related deaths are on the rise, with lung cancer being the most commonly diagnosed form of cancers. Lung cancer remains the leading cause of global cancer mortalities. Several anti-cancer strategies like surgery, chemotherapy, irradiation and immunotherapy are used to treat lung cancer, there is an urgent need for more effective strategies to cure or manage lung cancer. Among these strategies, immunotherapy possesses most potential efficacy irrespective of the histology and driver mutational status, leading to sustained remission. However, in the past few years, there are still several drawbacks in immunotherapy for lung cancer like suppression of the T-cell mediated cytotoxicity, secretion of immune-suppressive cytokines, and loss of major histocompatibility complex (MHC) expression. Furthermore, in the clinic many patients do not respond to immunotherapy (including anti-PDl and others) well. Thus, there is an ongoing and unmet need for improved approaches to treating lung cancer and other cancers, particularly those where aneuploidy of cancer cells is involved, and non-human animal models that can be used to identify such approaches. The present disclosure is related to these needs.

[0011] BRIEF SUMMARY

[0012] In an example, the disclosure provides a method comprising selecting an individual for a cancer treatment based on a determination that the individual has the cancer and that cancer cells exhibit high aneuploidy. The method may include administering to the individual an agent to treat the cancer. In an example, high aneuploidy means a chromosome number of 55 or greater, such as for human cancer cells, or five whole chromosome arm gains or losses, or a combination thereof. In examples, an individual diagnosed with a cancer with high aneuploidy is treated with at least one agent that targets any of any of CCL2, C3, CXCL1, CXCL5, IL1RN, TFF1, TFF2, TFF3, CCL17, CD36, CDCP1. In an example, a combination of agents targeting CCL2 and C3 are administered. Any combination of agents can be administered sequentially or concurrently. In examples, an agent is targeted to the CCL2 and a comprises an antibody or antigen binding fragment thereof that specifically binds to the CCL2. In examples, the agent is targeted to C3 and comprises compstatin, or a compstatin derivative selected from the group consisting of AMY-101, Cp40, or PEGylated Cp40.

[0013] In another aspect, the disclosure provides a fusion protein comprising and auroraB protein segment and a dCas9 segment. Polynucleotides and expression vector encoding the fusion protein are also provided. The fusion protein functions in a single-guide (sg) RNA targeted manner. The disclosure includes combinations, such as systems, that include a described fusion protein and a sgRNA that targets the fusion protein to a centromere. The disclosure comprises introducing such a system into non-human mammalian cells to promote development of aneuploidy. In an example, the non-human mammalian cells may be mouse cells. In examples, the cells may be lung cancer cells, but the approach is extendable to any type of cancer cells. The disclosure provides non-human mammals comprising the cells made according to the described methods.

[0014] The disclosure provides kits. In examples, the kit includes a fusion protein comprising an auroraB protein and a dCas9, or an expression vector encoding the fusion protein. The kit may include a single guide sgRNA that targets the fusion protein to a centromere, or a polynucleotide that encode the sgRNA.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] The figures that accompany and are part of this disclosure illustrate approaches to making and using aneuploidy lung cancer models, reveal genes and their protein products that can be targeted when high levels of aneuploidy are detected in cancer cells, and present representative demonstrations of targeting such genes by way of their protein products, such as CCL2 and C3.

[0017] FIG. 1. Generation of low and high aneuploidy cell lines -METHODI. Panel A. Graphical depiction of aneuploidy mouse model production using single cell cloning starting from KP lung tumors (Kras / TP53 induced by Cre recombinase). Panel B. Results obtained from single cell-derived clone by FACS. Panel C. Results obtained from Propidium Iodide (PI) staining. Panel D and E. Single cell-derived clones were obtained by FACS sorting into single wells and analyzed by FACS, by metaphase spreads (to count N of chromosomes) and by Whole-genome sequencing (WGS). KPE1 and 9 are near diploid and KPE5 and 6 are high aneuploid clones.

[0018] FIG. 2. Panels showing schematics used for generation of low and high aneuploidy cell lines using METH0D2 - mKaryoScramble.

[0019] FIG. 3. Schematic (top) showing 10 sgRNAs targeting mouse centromeres designed for screening efficient candidate sgRNA, 5 targeting Major Satellite region and 5 targeting Minor Satellite region in mouse centromeres. Panel A. Graphs showing percentage of cells expressing mCherry using the guide RNAs as indicated. Panel B. FACS analysis of cells edited using a control sgRNA (sgRosa26) and sgRNAMinSat5. sgRNAMinSat5 is the designated sgRNA used to induce aneuploidy and Rosa26 is used as a control. Panel C. Induction of aneuploidy by KaryoScramble by counting the number of chromosomes using metaphase spreads (representative images shown and quantification shown) and FACS analysis of DNA content. The parental cells are the starting KP cell line that is near diploid.

[0020] FIG. 4. Results showing aneuploidy promotes tumor formation in wild type (WT) but not immune deficient mice. Panel A. Graphs and Magnetic Resonance Imaging (MRI) results from immune competent mice (BC). Panel B. Graphs and Magnetic MRI from immunodeficient mice (NSG). Panel C. Images from low aneuploidy staining. Panel D. Images from high aneuploidy staining. Panel E. Graphs, image staining, and MRI imaging showing tumor data for immune competent mice. Panel F. Graphs and MRI imaging showing tumor data for immune deficient mice. Panel G. Graphs showing tumor by MRI data from immune deficient mice (nude mice).

[0021] FIG. 5. Panel A. Results from single-cell RNA sequencing (ScRNAseq) showing less effector T cell infiltration and lower M1 / M2 macrophage ratio in aneuploid tumor microenvironment. Panel B. ScRNAseq results showing less effector T cell infiltration and lower M1 / M2 macrophage ratio in aneuploid tumor microenvironment with T cell, NK cells (top) and macrophage (bottom) data.

[0022] FIG. 6. Graph of ScRNAseq data showing less effector T cell infiltration and lower M1 / M2 macrophage ratio in aneuploid tumor microenvironment. FIG. 7. Colorized graphs from LUMINEX analysis shows that CCL2 is increased in aneuploid versus diploid tumors. Panel A. Cytokine level of Bronchoalveolar Lavage (Bal) (pg / ml). Panel B. Cytokine level of Conditioned medium (pg / ml).

[0023] FIG. 8. Graphs showing RNAseq analysis (comparing high vs low aneuploidy KP cell lines) as indicated in graph titles.

[0024] FIG. 9. Graphs showing RNAseq analyses revealing three pathways upregulated in high versus low aneuploid tumors as indicated for extracellular matrix and collagen (left); immunoregulatory interactions (middle) and initial triggering of Complement (right).

[0025] FIG. 10. Results from FACS analysis showing CCL2 expression difference between high and low aneuploidy mouse cells demonstrating in high aneuploidy cells CCL2 expression was upregulated.

[0026] FIG. 11. Results from FACS analysis showing C3 expression difference between high or low aneuploidy mouse cells, demonstrating in high aneuploidy cells, C3 expression was upregulated.

[0027] FIG. 12. Results from FACS analysis showing CCL2 expression is upregulated in human aneuploid cells compared to low aneuploidy human cells. Similar results were obtained using LUMINEX and RNAseq. Human high and low aneuploidy generated after treating human colon epithelial cells (hCEC) with reversine and deriving clones (tested then by WGS) show results obtained using cells modified in a manner to METHOD 1.

[0028] FIG. 13. Results from FACS analysis showing C3 expression in high and low human aneuploidy cells. Similar results were obtained using RNAseq.

[0029] FIG. 14. Flowchart summarizing results showing that conditioned media (CM) derived from tumor cells modulates macrophage (M ) phenotypes with using a known anti- CCL2 antibody (BE0185) or a C3 inhibitor (Cp40).

[0030] FIG. 15. Graphs showing conditioned medium from high aneuploid cells lead to downregulation of anti-tumor markers (left) on macrophages and upregulation of pro-tumor markers (right) on macrophages. sgRNAs used for inducing aneuploidy are as indicated. The analysis shows that the anti-CCL2 and C3 inhibitor decrease expression of pro-tumor markers and promote expression of anti-tumor markers.

[0031] FIG. 16. Graphs showing inhibiting CCL2 or C3 decreases tumor growth only in high aneuploidy tumors.

[0032] FIG. 17. Graphs showing increase of survival of mice injected with high aneuploidy cells compared to low aneuploidy cells using BE0185 administration. sgRNAs used for inducing aneuploidy are as indicated. FIG. 18. Graphs showing a significant increase of survival of mice injected with high aneuploidy cells compared to low aneuploidy cells under Cp40 or both drugs. Double refers to Cp40+BE0185.

[0033] FIG. 19. Graph showing survival of mice after anti- Colony stimulating factor 1 receptor (CSF1R) / AFS98 administration.

[0034] FIG. 20. Graphs demonstrating that CCL2 plays an important role in the growth of aneuploid tumors. KPE9 clone is nearly diploid and KPE5 is highly aneuploid.

[0035] FIG. 21. Depiction of a method used to derive human cell lines shown in FIG 12 and 13. Shown is the use of human colon epithelial cells (hCEC) to derive clones with low or high aneuploidy. We treated parental diploid cells with reversine (an MPS1 inhibitor) that drives chromosome mis segregation, Single cell clones are derived after the treament. Each clone is then characterized by whole genome sequencing to identify chromosome gains and losses.

[0036] FIG. 22. Examples of low and high aneuploid clones are shown with copy number profiles. AY value of zero corresponds to no gain or loss, copy number higher or lower than zero corresponds to gain or loss respectively. Near-diploid or low aneuploid cells those with 5 or fewer than five chromosome arm gains or losses. High aneuploid cells those with more than five whole chromosome arm gains or losses.

[0037] DETAILED DESCRIPTION

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0039] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0040] As used in the specification and the appended claims, the singular forms “a” “and” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / -10%. This disclosure includes every amino acid sequence described herein and all nucleotide sequences encoding the amino acid sequences. Every sequence having from 80- 99% similarity, inclusive, and including and all numbers and ranges of numbers there between, with the sequences provided here are included in the invention. All of the amino acid sequences described herein can include amino acid substitutions, such as conservative substitutions, that do not adversely affect the function of the protein that comprises the amino acid sequences. All amino acid sequences encoded by the described polynucleotides are expressly included within this disclosure. The disclosure includes all DNA sequences described herein, and all RNA equivalents of the DNA sequences where T is substituted with U, all complementary sequences as DNA and RNA, and all reverse complementary sequences as DNA and RNA.

[0041] All sequences that are described by reference to a database are incorporated herein by reference as the sequences exist in the database as of the effective filing date of this application or patent. All sequences referred to in publications are incorporated herein by reference.

[0042] The present disclosure provides in one aspect a new model of aneuploidy for lung cancer, but is applicable to other tumor types as well. The models are generated to be applicable to mouse cells in some aspects by adapting an vivo model of aneuploidy described in PCT publication 2024 / 055002, from which the disclosure is incorporated herein by reference. In examples, the presently provided mKaryoScramble approach includes expressing in mouse cells a fusion protein comprising AuroraB and dCas9 with a single guide RNA (sgRNA) targeting centromeres of mouse chromosomes. AuroraB is also known as AURKB. Representative sequences of sgRNA / sgRNA targeting sequences are provided.

[0043] In arriving at this approach, as illustrated by the Figures as Method 2, the disclosure includes designing and testing 10 sgRNAs targeting mouse centromeres (5 targeting MinorSatellite sequence and 5 targeting Major Satellite sequence) and identified one sgRNA (referred to herein as MinSat5) that is highly efficient in targeting mouse centromeres. Since all mouse centromeres contain the MinSat5 target site, the described mKaryoScramble method induces gains and losses across all mouse chromosomes. Having generated and validated this system in mouse lung adenocarcinoma lines, we injected these cells in mice and found that high aneuploid lines formed larger tumors than low aneuploid ones. Further, through RNAseq analyses we found CCL2 and C3 (among other genes as described in the figures and a table of this disclosure) as increased in high versus low aneuploid tumors. In vivo experiments revealed that highly aneuploid lung tumors treated with antibodies targeting CCL2, or by using a representative C3 inhibitor, either alone or in combination, lived longer than untreated mice. The disclosure thus demonstrates that administration of CCL2 antibody or a C3 inhibitor alone or in combination prolongs survival of aneuploid cell-injected mice. Without intending to be constrained by any particular theory, it is considered that administration of a CCL2 antibody or a C3 inhibitor, alone or in combination, may regulate polarization of macrophages, leading to higher ratio of M1 / M2 macrophage, promote infiltration of CD8+ T cells and reduce exhausted CD8+ T cells. The disclosure thus demonstrates the use of CCL2 antibody or C3 inhibitors or combinations thereof as new immunotherapies for lung cancer or other cancers where high aneuploidy is present. It is considered that the presently provided mouse models are an improvement over previous mouse models as relates to the influence of complement and C3 on cancer cells and their growth.

[0044] The sgRNA binding sequences used to create examples of aneuploid cells are provided in a table as DNA sequences. The disclosure expressly includes each DNA sequence in the form of RNA wherein each T is replaced by a U, and all RNA sequences targeted to the sequences in the table.

[0045] In an example, the disclosure provides selecting an individual based on an analysis of a biological sample from the individual to determine that the individual has cancer cells that have high aneuploidy. In examples, high aneuploidy means the sample has a chromosome number of 55 or more in at least some of the cells tested, or more than five whole chromosome arm gains or losses, as supported by FIGs. 12, 13, 21, and 22. In examples, an average number of chromosomes determined from the biological sample is more than 55 chromosomes per cell and / or has more than five whole chromosome arm gains or losses per cell. The number of chromosomes and gains and losses of chromosome arms in a biological sample can be determined using any suitable technique, such as whole genome sequencing, karyotyping, and / or determination of the total amount of DNA in the cells. In examples, the disclosure includes testing a sample obtained from an individual to determine cancer cells in the sample have high aneuploidy, based at least in part on that determination, selecting the individual for treatment as described herein, and optionally treating the individual. In examples, the individual is selected for a treatment that comprises using an agent that targets one or more of CCL2, C3, CXCL1, CXCL5, IL1RN, TFF1, TFF2, TFF3, CCL17, CD36, or CDCP1, or a combination thereof. In examples the individual is treated. In examples, the treatment comprises administering one or more agents that target the described genes and / or their protein targets. In examples, the agent is an inhibitor of the target. In examples, the agent is an antibody or antigen binding fragment thereof. The antibody or antigen binding fragment thereof can be provided in any format, including but not necessarily limited to intact immunoglobulins or as fragments of immunoglobulins, including but not necessarily limited to antigen-binding (Fab) fragments, Fab’ fragments, (Fab’)2 fragments, Fd (N-terminal part of the heavy chain) fragments, Fv fragments (two variable domains), diabodies (Dbs), dAb fragments, single domain fragments or single monomeric variable antibody domains, singlechain Diabodies (scDbs), isolated complementary determining regions (CDRs), single-chain variable fragment (scFv), and other antibody fragments that retain antigen binding function. In examples, antibody format is a multivalent format.

[0046] For targeting complement, the agent may comprise a peptide or modified peptide that selectively binds to native C3, and / or to C3 bioactive fragments selected from C3b, iC3b and C3c. In examples, the C3 targeting agent is compstatin, or a compstatin derivative, such as Cp40, PEGylated Cp40, or AMY-101.

[0047] In examples, methods of the disclosure further comprise administering to the individual an immune checkpoint inhibitor. The disclosure includes sensitizing a cancer to an immune checkpoint inhibitor.

[0048] In examples, an effective amount of one or described agent is administered to an individual in need thereof. In examples, an effective amount is an amount that reduces one or more signs or symptoms of a disease and / or reduces the severity of the disease. An effective amount may also inhibit or prevent the onset of a disease or a disease relapse. A precise dosage can be selected by the individual physician in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of the agent to maintain the desired effect. Additional factors that may be taken into account include the severity and type of the disease state, age, weight, and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and / or tolerance / response to therapy.

[0049] In examples, compositions comprising one or more of the described agents can be administered to an individual in need thereof using any suitable route, examples of which include intravenous, intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, oral, or inhalation routes, depending on the particular condition being treated. The compositions may be administered parenterally or enterically. The compositions may be introduced as a single administration or as multiple administrations or may be introduced in a continuous manner over a period of time. For example, the administration(s) can be a pre- specified number of administrations or daily, weekly, or monthly administrations, which may be continuous or intermittent, as may be therapeutically indicated.

[0050] In examples, the individual in need of a treatment who has been identified by a method of this disclosure has been diagnosed with or is suspected of having cancer. In examples, the cancer is a solid tumor or a hematologic malignancy. In embodiments, the cancer is any form of lung cancer, including but not necessarily limited non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) of any stage. In examples, the lung cancer is a lung adenocarcinoma, a lung squamous cell carcinoma, or a lung large cell carcinoma. In examples, the cancer is any of liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma or another brain cancer, stomach cancer, bladder cancer, testicular cancer, head and neck cancer, melanoma or another skin cancer, any sarcoma, including but not limited to fibrosarcoma, angiosarcoma, osteosarcoma, and rhabdomyosarcoma, or any blood cancer, including all types of leukemia, lymphoma, and myeloma. The following tables are pertinent to the disclosure.

[0051] Table 1. Guide RNA / target sequence table Representative auroraB-dCas9 fusion protein amino acid sequence:

[0052] MAQKENSYPWPYGRQTAPSGLSTLPQRVLRKEPVTPSALVLMSRSNVQPTAAPGQKVME NSSGTPDILTRHFTIDDFEIGRPLGKGKFGNVYLAREKKSHFIVALKVLFKSQIEKEGVEHQLR REIEIQAHLHHPNILRLYNYFYDRRRIYLILEYAPRGELYKELQKSCTFDEQRTATIMEELADAL MYCHGKKVIHRDIKPENLLLGLKGELKIADFGWSVHAPSLRRKTMCGTLDYLPPEMIEGRM HNEKVDLWCIGVLCYELLVGNPPFESASHNETYRRIVKVDLKFPASVPMGAQDLISKLLRHN PSERLPLAQVSAHPWVRANSRRVLPPSALQSVAGGSGGGSLEDKKYSIGLAIGTNSVGWA VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDS TDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLL KALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRF AWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQK NSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD VDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFD NLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLS MPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRML ASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEF SKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTK EVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO:12)

[0053] A fusion protein of this disclosure may include linking amino acids positioned between the auroraB and dCas9 segments. The auroraB and dCas9 segments may be respectively configured in an N-C terminal orientation, or a C-N terminal orientation. The fusion protein may comprise additional amino acids other than the linker.

[0054] In examples, the disclosure provides an article of manufacture, which may comprise a kit. In examples, the article of manufacture may comprise a described fusion protein, or a polynucleotide encoding the fusion protein. The kit can comprise a described sgRNA or a polynucleotide encoding the sgRNA. An article of manufacture may include one or more sealed containers that contain any of the aforementioned components, and may further comprise packaging and / or printed material. The printed material may provide information on the contents of the article and may provide instructions or other indication of how the contents of the article may be used. In an example, the printed material provides an indication as to how to use the contents of the kit to produce aneuploid cells.

[0055] Examples of this disclosure are depicted in the figures and are not intended to be limiting.

[0056] EXAMPLES

[0057] FIG. 1 shows generation of low and high aneuploidy cell lines -METHODI. Panel A provides a graphical depiction of aneuploidy mouse model production using single cell cloning. Mouse adenocarcinoma cell lines were derived from K-rasG12D / +Tp53fl / fl mice treated with intra-nasal Aden-Cre (10-12 weeks). Panel B provides results obtained from single cell-derived clone by FACS. Panel C provides results obtained from PI staining. Panel C shows how single cell-derived clones were obtained by FACS sorting into single wells and analyzed by FACS and by metaphase spreads to assess their chromosome content. Panels D and E show how cell-derived clones were obtained by FACS sorting into single wells and analyzed by FACS, by metaphase spreads and by Whole-genome sequencing (WGS).

[0058] FIG. 2 shows schematics used for generation of low and high aneuploidy cell lines using METHOD2 - mKaryoScramble. The method involves the co-expression of two constructs. The first construct is dCas9 fused to AurkB mediated by inducible lentiviral vector pIND20. The second is a sgRNA targeting the Minor-Satellite mouse centromeric region (MinSat5 having the sequence of SEQ ID NO:5); as a control a sgRNA targeting the Rosa26 locus was used. The schematics also show the procedure to derive single cell-derived clones with different levels of aneuploidy using m-KaryoScramble (METHOD 2). Single cell-derived mouse lung cancer clones from the left panel with low level of aneuploidy like KPE-9 are treated with m-KaryoScramble constructs. After two weeks of doxycycline induction, cells are harvested and processed for metaphase spreads and FACS analysis.

[0059] FIG. 3 provides a schematic (top) showing 10 sgRNAs targeting mouse centromeres designed for screening efficient candidate sgRNA, 5 targeting Major Satellite region and 5 targeting Minor Satellite region in mouse centromeres. Panel A. Graphs showing percentage of cells expressing mCherry using the guide RNAs as indicated. Panel B. FACS analysis of cells edited using a control sgRNA (sgRosa26) and sgRNAMinSat5. Panel C. Results showing effects on mCherry expression using the sgRNAs as indicated. For Panel C, mCherry-positive cells rapidly decreased (-50% to 5%) for centromere-targeting sgRNAs as compared to an sgRNA targeting a unique site in the mouse genome at the Rosa26 locus (Rosa26 sgRNA), with the most prominent effect for MinSat5 sgRNA (sgMinSat5). This sgRNA was selected for the experiments that include generation of low and high aneuploidy cell lines.

[0060] FIG. 4 provides results showing aneuploidy promotes tumor formation in wild type (WT) but not immune deficient mice. Panel A shows graphs and Magnetic Resonance Imaging (MRI) results from immune competent mice (BC). Panel B shows graphs and MRI results from immunodeficient mice (NSG). Panel C shows images from low aneuploidy staining. Panel D shows images from high aneuploidy staining. Panel E shows graphs, image staining, and MRI imaging showing tumor data for immune competent mice. Panel F shows graphs and MRI imaging showing tumor data for immune deficient mice. Panel G shows graphs showing tumor by MRI data from immune deficient mice (nude mice). The results obtained in Fig. 4 panels A-D were generated using the described aneuploidy model based on single cell cloning as discussed herein. The disclosure accordingly provides determining if high levels of aneuploidy leads to increased tumor formation in vivo. We injected KP lung cancer (lung adenocarcinoma) clones with low or high level of aneuploidy through tail vein injection in syngeneic mice. By monitoring tumor burden using MRI imaging, we found a significant increase in the kinetics of tumor growth of high aneuploidy cells compared to low aneuploidy cells in wild type mice. The same experiment performed in immune deficient mice did not lead to a significant difference in the tumor growth between high and low aneuploidy tumors. Panels E, F and G of FIG. 4 show results obtained using a model of aneuploidy obtained using m-KaryoScramble (METHOD 2). Similar in vivo results were found using this model of aneuploidy as in the single cell cloning approach (METHOD 1).

[0061] FIG. 5 panel A shows results from ScRNAseq and demonstrate less effector T cell infiltration and lower M1 / M2 macrophage ratio in aneuploid tumor microenvironment. The Panel B shows ScRNAseq results showing less effector T cell infiltration and lower M1 / M2 macrophage ratio in aneuploid tumor microenvironment with T cell, NK cells (top) and macrophage (bottom). The results were generated using scRNAseq of CD45+cells isolated from the lung lesions. This revealed a profound reshaping of the immune microenvironment. First, in high aneuploidy tumors there was a significant decrease in T cells compared to low aneuploidy tumors. More specifically both CD8+and CD4+T cells decreased in high aneuploidy tumors. Moreover, tumor promoting macrophages significantly increased while anti-tumor macrophages significantly decreased in high aneuploidy tumor compared to low aneuploidy tumor. Panel B shows ScRNAseq shows less effector T cell infiltration and lower M1 / M2 macrophage ratio in aneuploid tumor microenvironment in T cells and NK cells (left) and in macrophages (right).

[0062] FIG. 6 provides a graph of ScRNAseq data showing less effector T cell infiltration and lower M1 / M2 macrophage ratio in aneuploid tumor microenvironment. The results revealed that anti-tumor macrophages / tumor promoting macrophages ratio significantly decreased in high aneuploidy tumor compared to low aneuploidy tumor.

[0063] FIG. 7 provides colorized graphs from LUMINEX analysis and shows that CCL2 is increased in aneuploid versus diploid tumors. Panel A shows cytokine level of Bronchoalveolar Lavage (Bal) (pg / ml). Panel B shows cytokine level of Conditioned medium (pg / ml). Collectively the results show that in high aneuploidy cells or tumors there was a significant increase of secreted CCL2 compared to low aneuploidy cells or tumors.

[0064] FIG. 8 provides graphs showing RNAseq analysis as indicated in graph titles. To obtain the results, RNAseq was conducted to detect gene expression profiles or cell pathway differences in high and low aneuploidy cells. This method revealed in high aneuploidy cells there were three pathways upregulated including extra cellular matrix and collagen, immunoregulatory interactions, and initial triggering of complement pathways

[0065] FIG. 9 also provides graphs showing RNAseq analyses revealing three pathways upregulated in high versus low aneuploid tumors as indicated for extracellular matrix and collagen (left); immunoregulatory interactions (middle) and initial triggering of Complement (right). The main pathways upregulated in RNAseq comparing high versus low aneuploid cells (MinSat5 vs Rosa26) include representative genes for each pathway which may be targeted as cancer therapies.

[0066] FIG. 10 provides results from FACS analysis showing CCL2 expression differences between high and low aneuploidy mouse cells. The results demonstrated that CCL2 expression was upregulated in high aneuploidy cells.

[0067] FIG. 11 provides results from FACS analysis showing C3 expression difference between high or low aneuploidy mouse cells, demonstrating in high aneuploidy cells, C3 expression was upregulated.

[0068] FIG. 12 provides results from FACS analysis showing CCL2 expression is upregulated in human aneuploid cells compared to low aneuploidy human cells. Similar results were obtained using LUMINEX and RNAseq. Human high and low aneuploidy generated after treating human colon epithelial cells (hCEC) with reversine and deriving clones (tested then by WGS) show results obtained using cells modified in a manner to METHOD 1.

[0069] FIG. 13 provides results from FACS analysis showing C3 expression in high and low human aneuploidy cells. Similar results were obtained using RNAseq.

[0070] FIG. 14 provides a flowchart summarizing results showing that conditioned media (CM) derived from tumor cells modulates macrophage (M) phenotypes with using a known anti-CCL2 antibody (BE0185) or a C3 inhibitor (Cp40). Generation of conditioned media (CM) was performed with treatment using an anti-CCL2 antibody (BE0185) or C3 inhibitor (Cp40). 20ng / ml IgG (BE0091) or anti-CCL2 antibody was added in CM after CM is collected (control or anti-CCL2 group). Vehicle or 5pM Cp40 was added in vehicle or C3 inhibitor treatment group, held for 24h and then the medium was replaced with fresh medium. After 24h, the medium+20ng / ml IgG or + 20ng / ml anti-CCL2 was collected for co-culture.

[0071] FIG. 15 provides graphs showing conditioned medium from high aneuploid cells lead to downregulation of anti-tumor markers (left) on macrophages and upregulation of protumor markers (right) on macrophages. sgRNAs used for inducing aneuploidy are as indicated. The results summarized in the graphs show that conditioned medium from high aneuploid cells led to downregulation of anti-tumor markers on macrophages and upregulation of pro-tumor markers on macrophages. This effect was rescued by inhibition of CCL2 (antibody: BE0185) or C3 (inhibitor: Cp40).

[0072] FIG. 16 provides graphs showing inhibiting CCL2 or C3 decreases tumor growth only in high aneuploidy. To generate the data shown the graphs, the model of aneuploidy produced using m-KaryoScramble (METHOD 2) was used. The data were based on a determination of whether administration of CCL2 antibody (BE0185) or a C3 inhibitor (Cp40) could decrease tumor formation compared with IgG (BE0091) group in vivo. We injected KP lung cancer clones with low or high level of aneuploidy through tail vein injection in syngeneic mice. By monitoring tumor burden using MRI imaging, we found a significant decrease in the kinetics of tumor growth of high aneuploidy cells compared to low aneuploidy cells under the treatment of two drugs, respectively.

[0073] FIG. 17 provides graphs showing increase of survival of mice injected with high aneuploidy cells compared to low aneuploidy cells using BE0185 administration. sgRNAs used for inducing aneuploidy are as indicated. By monitoring the survival of the mice, we also found a significant increase of survival of the mice injected with high aneuploidy cells compared to low aneuploidy cells under the treatment of BE0185.

[0074] FIG. 18 provides graphs showing a significant increase of survival of mice injected with high aneuploidy cells compared to low aneuploidy cells under Cp40 or both drugs. Double refers to Cp40+BE0185.

[0075] FIG. 19 provides graphs showing survival of mice after anti-Colony stimulating factor 1 receptor (CSF1R) / AFS98 administration. By monitoring the survival of the mice, we also found a significant increase of survival of the mice injected with high aneuploidy cells while there was a significant decrease of survival of the mice injected with low aneuploidy cells under the treatment of the anti-CSFIR agent AFS98.

[0076] FIG. 20 provides graphs demonstrating that CCL2 plays an important role in the growth of aneuploid tumors. Using the aneuploidy model based on single cell cloning (METHOD 1), we analyzed whether administration of CCL2 antibody (BE0185) could decrease tumor formation in vivo. We injected KP lung cancer clones with low or high level of aneuploidy through tail vein injection in syngeneic mice. By monitoring tumor burden using MRI imaging, we found a significant decrease in the kinetics of tumor growth of high aneuploidy cells compared to low aneuploidy cells using the BE0185 treatment. The data also show there is a significant increase of survival of the mice injected with high aneuploidy cells using BE0185.

[0077] FIG. 21 shows a representative method used to derive human cell lines shown in Figure 12 and 13. Here we used human colon epithelial cells (hCEC) to derive clones with low or high aneuploidy. We treated parental diploid cells with reversine (MPS1 inhibitor) that drives chromosome missegregation and then derive single cell clones. Each clone is then characterized by whole genome sequencing to identify chromosome gains and losses.

[0078] FIG. 22 shows of low and high aneuploid clones are with their copy number profile. AY value of zero corresponds to no gain or loss, copy number higher or lower than zero corresponds to gain or loss respectively. In an example, near-diploid or low aneuploid cells are those with 5 fewer than 5 chromosome arm gains or losses. In an example, high aneuploid cells are those with more than 5 whole chromosome arm gains or losses.

[0079] The following table includes results from comparison of gene expression profiles using RNA sequencing analyses comparing high versus low aneuploidy mouse cells (METHOD 2) KaryoScramble, in mouse cells) and is related to the RNSseq data presented in the figures. Any gene or the protein product of the gene may be targeted in the method of treating cancer patients described herein. Table 2. KaryoScramble Table Gene Targets

[0080]

[0081]

[0082] It will be apparent from the foregoing description that the present disclosure provides in examples development of KaryoScramble as a model of aneuploidy in mice using MinSat5 sgRNA and AuroraB-dCas9, which is shown to be a useful model of lung adenocarcinoma. RNASeq showed upregulation of 3 main pathways: extracellular matrix and collagen, immunoregulatory interactions, and initial triggering of complement. Genes upregulated in high versus low aneuploidy cells included but are not limited to CCL2, C3, CXCL1, CXCL5, IL1RN, TFF1, TFF2, TFF3, CCL17, CD36, CDCP1. These are considered targets for therapeutic intervention for cancers associate with associated with high aneuploidy. CCL2 and C3 upregulation in high versus low aneuploidy cells were confirmed by LUMINEX (CCL2) and FACS (CCL2 and C3). CCL2 and C3 upregulation in high versus low aneuploidy cells were confirmed by FACS (CCL2 and C3) in human cells. Conditioned medium from high aneuploid mouse cells leads to downregulation of anti-tumor markers (Tnfa) on macrophages and upregulation of pro-tumor markers (Argl) on macrophages; This effect is rescued by inhibition of CCL2 (shown with an antibody) or C3 (shown with a C3 inhibitor). Retreatment of mice injected with high aneuploid lung cancer cells with anti-CCL2 antibody (IgG anti-mouse CCL2) or C3 inhibitor (AMY-101) slowed down tumor formation and increased survival. This effect was seen only in the context of high aneuploidy. Thus, this phenotype for cancer cells is shown to aneuploidy-dependent.

[0083] Data presented on the figures and as described herein is not meant to be limiting. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. It is intended that the specification and examples be considered as exemplary only.

Claims

What is claimed is:

1. A method comprising selecting an individual for a cancer treatment based on a determination that the individual has the cancer, wherein cancer cells exhibit high aneuploidy, the method optionally further comprising administering to the individual an agent to treat the cancer.

2. The method of claim 1, wherein the high aneuploidy comprises a chromosome number of 55 or greater in the cancer cells of the individual.

3. The method of claim 2, wherein the individual is treated with an agent that targets any of CCL2, C3, CXCL1, CXCL5, IL1RN, TFF1, TFF2, TFF3, CCL17, CD36, CDCP1, or a combination of said agents.

4. The method of claim 3, wherein the agent is targeted to the CCL2, CCR2, C3, or wherein a combination of agents targeting CCL2 and C3 are administered, and wherein the combination of agents is administered sequentially or concurrently.

5. The method of claim 4, wherein the agent is targeted to the CCL2 and a comprises an antibody or antigen binding fragment thereof that specifically binds to the CCL2.

6. The method of claim 4 or claim 5, wherein the agent is targeted to the C3 and comprises compstatin, or a compstatin derivative selected from the group consisting of AMY-101, Cp40, or PEGylated Cp40.

7. A fusion protein comprising auroraB protein and a dCas9, or an expression vector encoding the fusion protein.

8. The fusion protein of claim 7, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 12.

9. A system comprising the fusion protein of claim 8, and a single guide RNA (sgRNA) that targets the fusion protein to a centromere.

10. The system of claim 9, wherein the sgRNA targets SEQ ID NO:5.

11. A method comprising introducing the system of claim 9 into non-human mammalian cells such that the cells develop aneuploidy.

12. The method of claim 11, wherein the non-human mammalian cells are mouse cells.

13. The method of claim 12, wherein the mouse cells are lung cancer cells.

14. A non-human mammal comprising the cells made according to claim 11, wherein the cells are optionally lung cancer cells.

15. A kit comprising a fusion protein comprising an auroraB protein and a dCas9 or an expression vector encoding the fusion protein, and a single guide sgRNA that targets the fusion protein to a centromere, or a polynucleotide that encode the sgRNA.

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